Present Position: Professor Department of Medical Microbiology and Immunobiology Faculty of Medicine University of Szeged Dóm tér 10, H-6720 Szeged, Hungary Tel: (36-62) 312-438, 455-114, 455-115 Fax: (36-62) 312-438, 455-113
Education: D.Sc. Hungarian Academy of Sciences, Budapest, (1986) “Drug Design in Chemotherapy” M.S. University Medical School, Szeged, Microbiology (1980) Ph.D. University Medical School, Szeged, Bacteriology (1974) M.S. University Medical School, Szeged, Medical Laboratory (1965) M.D. University Medical School, Szeged, Physician (1961)
Professional Positions: 1988- Professor 1985-1991 Vice Dean for Faculty of Medicine, Albert Szent-Györgyi Medical University 1976-1987 Associate Professor 1965-1976 Assistant Professor 1961-1965 Research Assistance, Institute of Microbiology
Member of European Commission Cost Action Directory and European Science Foundation 1993-Present.
Chairperson-Cost B16 1999-2006 Rapporteur Cost BM0701 2008-present. Member of Domain Committee of Bio-medicine and Bio-science 2007-present.
Membership in Professional Societies.
2003 Management Committe Member of COST 927 Thermally Processed Foods: Possible Health Implications. 2003 Management Committe Member of Cost 926 Physiological Responses to Biologically active plant compounds. 2004 Management Committee Member Cost B21,Physiological modelling of MR Image Formation. 2000 Chairman of Management of Committee of Corporation in Science and Technology,COST B16 Action of European Commission. Reversal of multidrug resistance from Bacteria to cancer cells. 1999 Management of Committee of COST 821 1996 Management of Committee of COST 916 1995- Chair of Szeged Foundation for Cancer Research 1995 Management of Committee of COST 821 1994- Member of the New York Academy of Sciences 1993- International Endotoxin Society European Society of Chemotherapy Infectious Diseases 1993- European Society for Biomodulation and Chemotherapy 1992-1996 Management Committee of COST Acrival Program of European Educational Commission s (EEC) 1989- Committee for Microbiology, Epidemiology and Vaccination of the Hungarian Academy of Sciences 1984- Hungarian Association for Medical Education 1979- Biology Section, Szeged Committee of Hungarian Academy of Sciences 1976- Hungarian Association for Biochemistry 1962- Hungarian Association for Microbiology
Research Grants: National Science Foundation (NSF) Corporation in Science and Technology European Educational Commissions (COST-EEC) Pays d'Europe Centrale et Orientale (PECO) Hungarian Ministry of Health and Ministry of Higher Education Hungarian Committee of Technical Development Hungarian Academy of Sciences Szeged Foundation of Cancer Research Cancure, Minnesota, USA. Foundation of Australian Resourches, Hungarian Chinese-Bilateral Research Cooperation
Publications: Over 400 publications in: International Journals of Cancer Research, Pharmacology, Medicinal Chemistry., Medical Microbiololgy and Chemotherapy, Immunology.
Abstract: Multidrug-resistant and extensively drug-resistant tuberculosis (MDR/XDR-TB) are problematic to manage, especially in patients with acquired immune deficiency syndrome (AIDS). There is therefore a dire need for effective anti-MDR/XDR-TB agents. We have previously shown that agents that inhibit the efflux pumps of MDR bacteria and cancer cells also enhance killing of intracellular mycobacteria, possibly by increasing the availability of K(+) and Ca(2+) needed for the activation of lysosomal enzymes of the phagolysosomal unit. In this study, the newly synthesised and recently patented SILA 409 and 421 were tested for in vitro and ex vivo activity against XDR-TB. The minimum inhibitory concentration (MIC) of SILA compounds was determined by the BACTEC 460 TB system. The effect of each compound on the killing activity of human macrophages infected with XDR-TB was determined by exposing the macrophage that had phagocytosed the bacterium to the compounds and assessing the killing activity by colony-forming unit counting. Amongst the two compounds tested, SILA 421 was shown to have in vitro activity against XDR-TB (MIC<3.5mg/L) and to transform non-killing macrophages into effective killers of phagocytosed bacteria, without any cytotoxic activity. Because SILA 421 revealed good in vitro and ex vivo activities and is devoid of any cytotoxic activity, it is a potential candidate as an anti-MDR/XDR-TB drug.
Abstract: Phospho-glycoprotein (P-gp) is a polytopic plasma membrane protein whose overexpression causes multidrug resistance (MDR) responsible for the failure of cancer chemotherapy. P-gp 170 is a member of the ATP-binding cassette (ABC) transporter superfamily and has two potentially interesting regions for drugs interfering with its efflux function, namely the oligosaccharides on the first extracellular loop with unknown function and the two intracellular ATP-binding regions providing the energy for drug efflux function. The polylactoseamine oligosaccharides on the first loop can specifically bind the tomato lectin (TL). The P-gp efflux activities of TL-pre-treated MDR resistant cells were measured in the presence of structurally unrelated resistance modifiers such as phenothiazines, terpenoids and carotenoids. The inhibition of efflux activity was measured via the increased rhodamine uptake by mouse lymphoma cells transfected in human MDR1 gene and in human brain capillary endothelial cells. The tested resistance modifiers inhibit the function of ABC transporter resulting in increased R123 accumulation in MDR1 expressing cells. TL prevented the inhibitory action of phenothiazine and verapamil on brain capillary endothelial and MDR1-lymphoma cells, presumably due to the stabilization of the functional active conformation of P-gp. Our results indicate that the polylactosamine chains of P-gp are part of the functionally active protein conformation.
Abstract: The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.
Abstract: PURPOSE: Novel multidrug resistance (mdr) modulators have been proved as inhibitors of P-glycoprotein (P-gp). We first investigated the in vitro effects of selected compounds in human cancer cells on multidrug resistance reversal effects compared to drug standards and on P-gp induction to characterize the potential of the compounds as clinical candidates. METHODS: The uptake of daunorubicin into a parental cancer cell line and P-gp expressing subcell line in presence of the modulators was characterized by flow cytometry. Induction of P-gp was investigated in P-gp expressing and non-expressing cancer cell lines on the RNA level by real-time quantitative polymerase chain reaction (RTQ-PCR) and protein quantification. Results were additionally confirmed by northern blot techniques and functionality assays in selected cell lines. RESULTS: The novel modulators showed activities as mdr reversers in a P-gp specific human cancer cell model with mainly increased uptake rates of daunorubicin into the drug-resistant cell line. H17 proved to be more active than cyclosporine A as a known strong mdr modulator. The induction studies revealed practically no induction potential of the compounds in usual short-time drug application regimes in all cell lines. Furthermore, the novel modulators did not increase the efflux of a P-gp model substrate in the functionality model assay. This confirmed the results of non-P-gp induction which was observed on both the RNA and the protein levels. CONCLUSIONS: The novel mdr modulators proved as perspective candidates for further clinical studies because they turned out to be highly active in human cancer cell models. Furthermore, they showed no potential to induce the transmembrane efflux pump P-gp. This is a significant advantage compared to modulators which failed in clinical trials because of induction-effects that increase cellular resistances and, moreover, side effects in normal cells.
Abstract: The antiretroviral activities of extracts of Euphorbia hirta were investigated in vitro on the MT4 human T lymphocyte cell line. The cytotoxicities of the extracts were tested by means of the MTT cell proliferation assay, and then the direct effects of the aqueous extract on HIV-1, HIV-2 and SIV(mac251) reverse transcriptase (RT) activity were determined. A dose-dependent inhibition of RT activity was observed for all three viruses. The HIV-1 inhibitory potency of E. hirta was studied further, and the activities of the aqueous and 50% methanolic extracts were compared. The 50% methanolic extract was found to exert a higher antiretroviral effect than that of the aqueous extract. The 50% MeOH extract was subjected to liquid-liquid partition with dichloromethane, ethyl acetate and water. Only the remaining aqueous phase exhibited significant antiviral activity; all the lipophilic extracts appeared to be inactive. After removal of the tannins from the aqueous extract, the viral replication inhibitory effect was markedly decreased, and it was therefore concluded that tannins are most probably responsible for the high antiretroviral activity.
Abstract: Three novel beta-carboline indole alkaloids (1-3) have been isolated from a MeOH extract of the leaves of Tabernaemontana elegans. The structures were established by means of spectroscopic techniques including 2D NMR experiments. Compounds 1 and 2 contain a two-carbon unit, attached to a structurally related beta-carboline skeleton, as part of an additional six-membered ring in 1 and a seven-membered ring in 2. To the best of our knowledge, this is the first report of beta-carboline indole alkaloids from the genus Tabernaemontana. Compounds 1-3 were evaluated for their ability to modulate multidrug resistance in mouse lymphoma cell lines. Compounds 1 and 3 exhibited a weak activity.
Abstract: The aim of the present study was to investigate the anticancer properties of five alkaloids isolated from Amaryllidaceae, including the inhibitory effect on P-glycoprotein (P-gp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for their multidrug resistance (MDR)-reversing activity on human MDR1-gene-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. Trisphaeridine and pretazettine increased the intracellular Rh-123 concentration 30- and 50-fold, respectively, as compared to the non-treated cells, and 2-O-acetyllycorine and trisphaeridine were demonstrated by means of the checkerboard method to enhance the antiproliferative activity of doxorubicin on L5178 MDR mouse lymphoma cells. The MTT assay revealed that pretazettine, trisphaeridine and 2-O-acetyllycorine displayed excellent antiproliferative effects on both the human and the mouse cell lines. The apoptosis-inducing activities of selected agents (2-O-acetyllycorine and trisphaeridine) were measured via acridine orange and ethidium bromide dual staining and flow cytometry of the subG1 population.
Abstract: Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant.
Abstract: A series of 2-(3-aryl-2-propenoyl)-3-methylquinoxaline-1,4-dioxides 3a-l were prepared by condensation of various aryl aldehydes with 2-acetyl-3-methylquinoxaline-1,4-dioxide 2. These compounds inhibit the growth of human Molt 4/C8 and CEM T-lymphocytes and the IC(50) values are mainly in the 5-30 microM range. The quinoxaline 1,4-dioxide 3j inhibited the growth of 58 human tumor cell lines, particularly leukemic and breast cancer neoplasms. All of the compounds 3a-l reversed the multidrug resistance (MDR) properties of murine L-5178Y leukemic cells which were transfected with the human MDR1 gene. The MDR-reversing effect may be due to the conjugated pi-electron system forming a weak electron charge transfer complex with the P-glycoprotein-mediated efflux pump. The compounds in series 2 and 3 were assessed against HL-60, HSC-2, HSC-3 and HSC-4 malignant cells as well as HGF, HPC and HPLF normal cell lines which revealed that the majority of the compounds displayed a greater toxicity to neoplastic than normal cells. Various ways in which the project may be expanded are presented.
Abstract: DNA methylation acts in early tumorigenesis. Its detection is possible either from tissue, stool or peripheral blood. Septin 9 is a sensitive methylation marker, which has been studied in several cancers such as breast and ovarian tumors and in neurological or hematological diseases. Septin proteins have an important role from cytoskeleton organisation to development of embryonal pattern. Nowadays intensive researches are going on about the relation between the septin 9 gene hypermethylation and colorectal cancer development.
Abstract: The ability of 11 chalcones with 3,4,5-trimethoxy substitution on ring A to inhibit the transport activity of P-glycoprotein was studied. Flow cytometry was applied in multidrug-resistant human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The reversal of multidrug resistance (MDR) was investigated by measuring the accumulation of rhodamine-123 in cancer cells. Verapamil was applied as a positive control. The majority of the tested compounds were proved to be effective inhibitors of the outward transport of rhodamine-123. In the MTT test, chalcones 2, 3, 5 and 7 exhibited the strongest antiproliferative effects, with 50% inhibitory dose (ID50) =0.19, 0.19, 0.29 and 0.14 microg/mL, respectively. The least effective compounds were 1, 4, 8 and 11, with ID50 values in the range of 1.5-3.5 microg/mL. The antiproliferative effect was shown to be affected by the type of substitution at the p-position on ring B. Chalcone 7, with a p-chloro group on ring B, was the most effective in MDR reversal, causing a marked increase in drug accumulation from 0.4 to 40 microg/mL. In combination with epirubicin, compound 7 displayed synergistic properties while compound 3 exhibited an additive effect. The data presented here indicated that some calcone derivatives can be regarded as effective compounds for reversal of MDR.
Abstract: BACKGROUND: Ineffectiveness of anticancer drugs is frequently observed in cancer chemotherapy. The resistance of tumor cells to various cytotoxic drugs is defined as multidrug resistance (MDR). The purpose of this study is to investigate the potential reversal effect of some synthetic and natural chemicals on drug-resistant MCF-7 cell lines. The effects of potential MDR modulators combined with some anticancer drugs were also studied. METHODS: Flow cytometry, MTT cytotoxicity assays and checkerboard combination assays were performed to study the reversal of drug resistance and to investigate the antiproliferative effects of the combination of anticancer drugs and the potential modulators. The results indicated that verapamil, capsanthin, zeaxanthin and promethazine inhibited P-gp effectively, but chrysin was not effective at reversing the resistance in MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and 4 effective MDR modulators (verapamil, capsanthin, zeaxanthin and promethazine) were applied to the sublines in combination. RESULTS AND CONCLUSION: Fractional inhibitory indices show that verapamil and zeaxanthin seem to be the most effective MDR reversal agents that may be used together with paclitaxel, docetaxel, vincristine and doxorubicin in drug-resistant mammary carcinoma sublines. In conclusion, this report represents the importance to find out active and efficient drug resistance modulators for improving the efficacy of chemotherapy.
Abstract: The 1-[4-(2-aminoethoxy)phenylcarbonyl]-3,5-bis-(benzylidene)-4-oxopiperidines 5-8 are a novel cluster of highly potent P-glycoprotein dependent multidrug resistance (MDR) revertants. Using a concentration of 4mug/mL, these compounds possess 11-43 times the potency of verapamil in reversing MDR in murine L-5178 lymphoma cells transfected with the human MDR1 gene. Structure-activity relationships reveal that the attachment of the N-aroyl group to various 3,5-bis(benzylidene)-4-piperidones is essential for MDR reversal to occur. In terms of potencies, the 1-piperidinyl group is the preferred terminal amine while the 4-methyl and 4-chloro substituents are the optimal groups for placement in the benzylidene aryl rings.
Abstract: PURPOSE: The aim of this study was to investigate the effects of two novel multidrug-resistance reverting agents, ALIS 409 [1,3-dimethyl-1,3-p-fluorophenyl-1,3(3-morfolinopropyl)-1,3-disiloxan dihydrochloride] and ALIS 421 [1,3-dimethyl-1,3-(4-fluorophenyl)-1,3[3(4-buthyl)-(1-piperazinyl)-propyl]-1,3-disiloxan tetrahydrochloride], on vascular functions in vitro. EXPERIMENTAL DESIGN: A comparison of their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, was performed. RESULTS: In endothelium-denuded rat aorta rings, ALIS 409 and ALIS 421 antagonized 60 mM K(+)-induced contraction in a concentration-dependent manner with IC(50) values of 52.2 and 15.5 microM, respectively. ALIS 409 and ALIS 421 inhibited L-type Ca(2+) current recorded in artery myocytes in a concentration-dependent manner with IC(50) values of 6.4 and 5.6 microM, respectively. In rat aorta, ALIS 409 and ALIS 421 antagonized the sustained tonic contraction induced by phenylephrine with IC(50) values of 58.0 and 13.7 microM (endothelium-denuded rings) and of 73.9 and 31.9 microM (endothelium-intact rings), respectively. In endothelium-denuded rings, ryanodine reduced significantly the response to phenylephrine in the absence of extracellular Ca(2+) whereas nifedipine, ALIS 409 or ALIS 421 did not affect it. Phenylephrine-stimulated influx of extracellular Ca(2+) was markedly reduced when tissues were pretreated with ALIS 409, ALIS 421 or nifedipine, and stimulated when they were pretreated with ryanodine. Application of ALIS 409 (up to 100 microM) to intact rat aorta rings failed to induce mechanical responses. CONCLUSIONS: Our results provide functional evidence that the myorelaxing effect elicited either by ALIS 409 or by ALIS 421 involved mainly the direct blockade of extracellular Ca(2+) influx. This effect, however, took place at concentrations much higher than those effective as modifiers of multidrug resistance in cancer cells.
Abstract: Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.
Abstract: Multidrug resistance in Gram-negative bacteria is now known to be primarily caused by overexpression of efflux pumps that extrude unrelated antibiotics from the periplasm or cytoplasm of the bacterium prior to their reaching their intended target. This review focuses on a variety of agents that have been shown to be efflux pump inhibitors (EPIs) and which, if used as 'helper compounds' in combination with antibiotics to which the organism is initially resistant, may produce the required cure. Although not all of the EPIs may serve a helper role owing to their toxicity, they may nevertheless serve as lead compounds.
Abstract: The macrocyclic lathyrane diterpene latilagascene B, previously isolated from Euphorbia lagascae, was acylated to afford three new diterpene esters, latilagascenes G (1), H (2), and I (3), whose structures were assigned by spectroscopic methods. These acyl derivatives, and the macrocyclic diterpenes of the jatrophane-type, tuckeyanols A (4) and B (5), and euphotuckeyanol (6), isolated from Euphorbia tuckeyana, were tested for P-gp modulating properties on human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines. All the compounds displayed very strong activity. The molecular orbital energies (HOMO and LUMO) of diterpenes 1-6 and 7-13, previously isolated, have also been calculated in order to estimate their probable charge transfer interactions with P-gp. Structure-activity relationships (SAR) are discussed. Furthermore, compounds (1-6) were assayed, in vitro, for their antiproliferative effects in combination with epirubicine and all of them synergistically enhance the effect of the antitumor drug.
Abstract: The aim of the present study was to investigate the anticancer properties of a set of furanoacridone alkaloids, arborinine and evoxanthine, including the inhibitory effect of P-glycoprotein (Pgp) and the apoptosis-inducing capacity. The tested alkaloids were evaluated for multidrug resistance (MDR)-reversing activity on human Pgp-transfected L5178 mouse lymphoma cells, using the rhodamine-123 (Rh-123) assay. The antiproliferative effects of natural compounds and their interactions with doxorubicin were determined in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Apoptosis-inducing activity was additionally measured by means of dual annexin V and propidium iodide staining. RT-PCR was used to test the expression of Pgp mRNA after acridone treatment. All of the acridones investigated increased the accumulation of Rh-123. Gravacridonetriol and gravacridonediol monomethyl ether increased the antiproliferative effect of doxorubicin on resistant L5178 cells. Treatment with these agents resulted in a decrease in Pgp mRNA levels. Naturally occurring acridone alkaloids exhibit a beneficial combination of anticancer effects and, accordingly, the acridone skeleton can be considered useful in the design of novel antiproliferative agents.
Abstract: The development of strategies intended to inhibit human cytomegalovirus (HCMV) immediate-early (IE) antigen expression is an important goal in research designed to prevent and treat certain forms of cancer. The aim of this study was to identify potent IE antigen-targeting natural compounds as antitumor promoters in an in vitro model of tumor promotion. Nineteen dihydro-beta-agarofuran sesquiterpenes isolated from Euonymus species were evaluated for their ability to inhibit HCMV IE antigen expression in human lung adenocarcinoma (A549) cells. Five esters of penta- and hexahydroxydihydro-beta-agarofuran proved to be active components in these Euonymus species, inhibiting the IE antigen expression of HCMV. The highest activity was displayed by 2beta,6alpha,15-triacetoxy1beta-benzoyloxy-9alpha-nicotinoyloxydihydro-beta-agarofuran. These effective compounds may be regarded as prototypes of antitumor promoters, as secondary chemopreventive agents which can modify or halt tumor promotion in general.
Abstract: Global rates of pulmonary tuberculosis (TB) continue to increase. Moreover, resistance of the causative organism Mycobacterium tuberculosis to the two most effective anti-TB medications continue to rise. Now, multi-drug resistant TB (MDR-TB) has progressed to extensively drug resistant TB (XDR-TB) - a M. tuberculosis organism that is resistant to the most effective second line drugs available for the treatment of TB. This review provides detailed, significant evidence that supports the use of an old neuroleptic compound, thioridazine (TZ), for the management of MDR-TB and XDR-TB infections and which has been shown to inhibit efflux pumps of bacteria. The argument has been previously presented but no one seems to be listening - and the disease continues unabated when there is a very good probability that the suggested drug will prove to be effective. When the prognosis is poor, available therapy predictably ineffective and death is inevitable, compassionate therapy with TZ should be contemplated. The risks are small and the rewards great.
Abstract: We have developed a number of methods that identify efflux pump mediated multi-drug resistant bacteria, characterize efflux systems and screen for inhibitors of efflux pumps. These approaches were complemented by the quantification of the expression of genes that regulate and code for constituents of efflux pumps. The methods described are easy to use, reproducible and for the most part, require instrumentation normally present in a clinical bacteriology laboratory. Because each method provides good reproducibility, they lend themselves for inter-laboratory use.
Abstract: Novel 3,9-diazatetraasteranes have been synthesized with varied aromatic substitution patterns and evaluated as P-glycoprotein (P-gp) inhibitors. Structure-activity relationships (SAR) are discussed in relation to determined physicochemical properties. The potential to induce P-gp expression has been evaluated in cancer cell lines. The bioanalytical results indicate favorable noninducing properties compared to P-gp inducing drug standard.
Abstract: This study revealed that various alicyclic and acyclic compounds containing the 3-(3,4,5-trimethoxyphenyl)-2-propenoyl group displayed potent MDR reversal properties. In particular, a concentration of 4 microg/ml of 2,5-bis(3,4,5-trimethoxyphenylmethylene)cyclopentanone was 31 times more potent than verapamil as a MDR revertant. In general, they were selectively toxic to malignant rather than normal cells. Two representative compounds induced apoptosis in human HL-60 cells and markedly activated caspase-3.
Abstract: Human cytomegalovirus (CMV) preferentially infects tumor tissues and the accumulated CMV immediate-early (IE) antigen may lead to tumor promotion and progression. The development of strategies to inhibit human CMV IE antigen expression and/or function is an important goal to prevent and treat certain forms of cancers associated with human CMV. The aim of this study was to search for antitumor promoters from plant sources. The effect of six macrocyclic lathyrane-type diterpenoids, latilagascenes A-E (1-5) and jolkinol B (6), isolated from the methanol extract of Euphorbia lagascae, on the expression of IE antigen in lung cancer cells (A549) infected by CMV was studied. All the compounds, except latilagascene D (4), decreased IE antigen expression of CMV.
Abstract: The macrocyclic lathyrane diterpenes, latilagascenes D-F (1-3) and jolkinol B (4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure-activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C (5-7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B (6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D-F (1-3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY).
Abstract: Novel heat shock protein 90 inhibitor peptide derivatives [D- Trp-Phe-D- Trp-Leu-AMB (1), p-HOPA-D- TrpPhe-D-Trp-Leu-psi(CH2NH)-Leu-NH2 (2), D-Trp-Phe-D-Trp-OH (3), Suc-D-Trp-Phe-D-Trp-Leu-AMB (4), D-Tyr-Phe-D-Trp-Leu-AMB (5), D-Arg-D-Trp-Phe-D-Trp-Leu-Leu-NH2 (6), Leu-psi(CH2NH)-Leu-NH2x2HCl (7), Phe-Trp-Phe-Trp-Leu-Leu-NH2 (8), Tyr-Trp-Phe-Trp-Leu-Leu-NH2 (9) and Tyr-D- Trp-Phe-D-Trp-Leu-Leu-NH2 (10)] were synthetized, and their ability to reverse multidrug resistance (MDR) was studied. Peptide derivatives 1, 4 and 5, with D-Trp or D-Tyr residues in the N-terminal position caused a marked inhibition of MDR in cancer cells. These MDR inhibitor compounds and epirubicin were demonstrated to have additive and synergistic antiproliferative effects in checkerboard experiments on human MDR1 gene-transfected mouse lymphoma cells in vitro. It is suggested that the MDR reversal effects of these anticancer peptide derivatives, together with their antiproliferative effects on lung cancer cells, may open up new horizons in cancer chemotherapy.
Abstract: Thioridazine (TZ) has previously been shown by us to have in vitro and ex vivo activity against antibiotic-susceptible and multidrug-resistant Mycobacterium tuberculosis (MDRTB). Because current therapy of MDRTB is highly problematic even when all five 'first line of defence' drugs are employed, there is a need for effective antituberculosis drugs. New derivatives of TZ were synthesised and their in vitro activity against a reference strain of M. tuberculosis was evaluated with the aid of the BACTEC 460 system. Derivatives that presented significant activity were evaluated by ex vivo studies and were shown to enhance the killing of intracellular M. tuberculosis.
Abstract: Cardiac effects of 3,5-dibenzoyl-4-(3-phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7), a novel multidrug resistance (MDR) inhibitor, in Langendorff-perfused rat heart have been investigated and compared to that of nifedipine. Nifedipine decreased concentration-dependently (IC50=8.89+/-1.09x10(-8) M) left ventricular pressure leaving unaltered coronary perfusion pressure, whereas DP7 did not affect both parameters. Nifedipine did not modify both QRS and QT intervals of electrocardiogram (ECG). Second-degree atrioventricular block or ventricular rhythm occurred in presence of nifedipine, however, in 4 out of 6 hearts. DP7, up to 30 microM, failed to alter ECG parameters. In conclusion, DP7, may represent a lead compound for the development of potent dihydropyridine MDR chemosensitizers devoid of cardiac effects.
Abstract: The survival data of patients with ductal pancreatic adenocarcinoma are rather poor, partly because the disease is frequently diagnosed at an advanced stage, partly because it is characterized by a chemoresistant phenotype. Even first-line chemotherapeutic drugs result in a modest objective response. This drug resistance is attributed to many different, unrelated mechanisms, including abnormal membrane receptor transport, ineffective metabolic drug conversion or enhanced metabolite inactivation, increased DNA repair and alterations in the apoptotic pathways. The role of NF-kappaB, cyclin D1 and stromal factors is also emphasized by many groups. The involvement of the ABC-transporters is not a universal feature, their alterations are important only in the resistance against specific cytostatics. Although several well-known molecular mechanisms have been elucidated, our understanding of drug insensitivity is still fragmentary, especially because recent microarray studies revealed that hundreds of genes are up- or down-regulated in resistant tumor cells, but their exact significance is still unclear. The reversal of the drug resistance is an area of intensive investigation, but to date, the compounds investigated are effective mainly in experimental systems and prospective studies are needed to validate their clinical applicability.
Abstract: BACKGROUND: The aim of the study was to evaluate the effectiveness of thioridazine (TZ) at different dose levels on mice that had been infected intraperitoneally (i.p.) with a high dose of the Mycobacterium tuberculosis ATCC H37Rv strain. SUBJECTS AND METHODS: Groups of five female BALB/C mice were infected i.p. with 10(6) colony forming units/mL. After thirty days, treatment with TZ was initiated, except for the control group. Mice were treated with TZ at equivalent concentrations to that used in the humans (1200 mg/day), ranging from 0.05 to 0.5 mg/day. RESULTS: The results demonstrated that a daily dose of 0.5 mg/day of TZ reduced the number of colony forming units retrieved from the lungs of infected mice within one month. CONCLUSION: By the end of 300 days of therapy, although mycobacteria were still retained their presence, in comparison to that of the control was 8 orders of magnitude lower.
Abstract: Patented SILA compounds 409 and 421, previously shown to inhibit the efflux pumps of bacteria and cancer cells, have been studied for their ability to reduce or eliminate the presence of plasmids from Escherichia coli strains that have been induced to high level resistance to tetracycline by gradual exposure to increasing concentrations of the antibiotic. The results demonstrate that SILA compound 421, which has greater efflux pump inhibitory activity than its parent SILA compound 409, can reduce plasmid loads by 5 logs, over that present in the absence of the drug. The ability of the SILA compound to eliminate much larger plasmids is substantially lower. Because in vivo studies have shown that these compounds are not toxic to the mouse, the results obtained in our study suggest a potential role for SILA compound 421 as an adjunct for the therapy of antibiotic-resistant E. coli infections whose resistance is plasmid-mediated. In addition, because plasmid-mediated resistance is often found in tetracycline-treated cattle, SILA compound 421 may have potential as an adjunct during the time that the cattle are maintained on tetracycline prior to slaughter.
Abstract: Several macrocyclic diterpenes with jatrophane or lathyrane skeletons were isolated from methanol extracts of Hungarian Euphorbia species and evaluated for multidrug resistance (MDR)-reversing activity on a human colon cancer cell line. MDR-reversing activity was tested by using a standard functional assay with Rhodamine 123 as a fluorescent substrate analogue of epirubicin. In the model of combination chemotherapy, the interactions between epirubicin and certain resistance modifiers were studied in vitro. Compound 8 proved to be the most active, exhibiting a synergistic interaction. The capacity of the most effective derivative to induce apoptosis was demonstrated by flow cytometric analysis and by staining with ethidium bromide and acridine orange, using human mdrl gene-transfected mouse lymphoma cells and a human cervical adenocarcinoma cell line. The selected diterpene was able to induce moderate apoptosis in the tested cell lines. The data presented here indicate that naturally occurring Euphorbia diterpenes can be regarded as effective lead compounds for the reversal of MDR.
Abstract: Diclofenac sodium (Dc), an anti-inflammatory agent, has remarkable inhibitory action both against drug-sensitive and drug-resistant clinical isolates of various Gram-positive and Gram-negative bacteria. The aim of this study was to determine the ability of Dc to protect mice from a virulent Salmonella infection. Dc injected at 1.5 microg/g and 3.0 microg/g mouse body weight significantly protected animals from the lethality of Salmonella infection. As was the case for the in vitro interaction, Dc in combination with streptomycin was even more effective. The non-antibiotic drug Dc has potential for the management of problematic antibiotic-resistant bacterial infections.
Abstract: Bacteria and cancer cells develop resistance to more than one agent as a consequence of being exposed to ineffective levels of the agent for a prolonged period of time. The resistance of these cells is mediated by over-expressed efflux pumps that have the ability to extrude a large variety of unrelated chemicals. This review discusses the main types of multidrug resistant (MDR) efflux systems of bacteria and cancer cells, and shows the similarity of specific efflux systems between them with respect to given agents that inhibit efflux, thus rendering these cells once more susceptible to agents to which they had developed MDR.
Abstract: The antimicrobial and antiplasmid activities of essential oils (orange oil, eucalyptus oil, fennel oil, geranium oil, juniper oil, peppermint oil, rosemary oil, purified turpentine oil, thyme oil, Australian tea tree oil) and of menthol, the main component of peppermint oil, were investigated. The antimicrobial activities were determined on the Gram (+) Staphylococcus epidermidis and the Gram (-) Escherichia coli F'lac K12 LE140, and on two yeast Saccharomyces cerevisiae 0425 delta/1 and 0425 52C strains. The antiplasmid activities were investigated on E. coli F'lac bacterial strain. Each of the oils exhibited antimicrobial activity and three of them antiplasmid action. The interaction of peppermint oil and menthol with the antibiotics was studied on the same bacterial strain with the checkerboard method. Peppermint oil and menthol displayed additive synergy with oxytetracycline. A new mechanism of plasmid curing was established for one of the oil components.
Abstract: New pentacyclic inhibitors of the P-glycoprotein carrying nitrogen-containing alkyl chains were synthesized and evaluated for MDR reverting activity on mouse lymphoma cells infected with pHa MDR1/A retrovirus. The activity of the compounds proved to be up to 5-fold higher than verapamil, used as reference compound.
Abstract: Previous studies suggested that the phenothiazine chlorpromazine (CPZ) could reverse or reduce the antibiotic resistance of bacteria. In some areas of the world, the majority of Staphylococcus aureus isolates are now resistant to methicillin, prompting this study to see whether such resistance can be altered by phenothiazine thioridazine (TZ), an agent with equal antibacterial activity, which is free of the severe side-effects associated with chronic administration of CPZ. The results indicated that, whereas methicillin-sensitive strains of Staphylococcus aureus (MSSA) were not rendered more susceptible to oxacillin, resistance to oxacillin by highly-resistant strains (MRSA) could be significantly reduced by sub-inhibitory concentrations of TZ. Reserpine, an inhibitor of efflux pumps, was also shown to reduce the resistance of MRSA strains to oxacillin in a concentration-dependent manner. The phenothiazines have been shown, by others, to inhibit the efflux pumps of bacteria and the mechanism by which MRSA are rendered more susceptible to oxacillin in the presence of TZ is believed to be due to a similar efflux pump.
Abstract: A series of N-substituted cage dimeric 1,4-dihydropyridines 3a-e was evaluated as inhibitors of membrane efflux pump P-glycoprotein (P-gp) in multidrug resistant (mdr) cancer cells. Structure-activity relationships (SAR) and cytotoxic properties are discussed. Effective concentrations for overcoming mdr have been demonstrated in competition studies with the P-gp substrate epirubicin.
Abstract: A proton pump-deleted mutant E. coli, AG100 A, had greater sensitivity to ampicillin, tetracycline and erythromycin than the wild-type parent E. coli AG100 containing the proton pump. This antibiotic sensitivity was further increased by resistance modifiers such as the Ca2+ channel blocker (+/-) verapamil (VP) and the calmodulin antagonist promethazine (PMZ). Whereas the newly-synthesized trifluoromethyl-ketone (TF) enhanced the activity of these antibiotics against the wild-type strain, it did not enhance the activity of ampicillin against the proton pump-deleted mutant. These results suggested that TF14 had an inhibitory effect on the proton pump. Elimination of plasmids from another strain of E. coli, K12, was promoted by PMZ and 9-amino-acridine (9-AA), but not by TF14 alone. However, combinations of TF14 with either PMZ or 9-AA enhanced the plasmid elimination capacity of the latter compounds. The combination of TF14, PMZ and VP proved that the Ca2+ channel blocker was not effective by itself These results collectively suggest that TF14 inhibited the proton pump of E. coli and that it was this pump which, when inhibited by TF14, allowed more PMZ to reach its plasmid elimination target.
Abstract: The development of multidrug resistance (MDR) causes difficulties in the chemotherapy of of human cancer. Investigation of the possibility of reversal of MDR has been greatly aided by the use of cell lines with acquired resitance to anticancer agents in vitro or transfected with the mdrl gene. The aim of this study was to examine new perspectives of chemotherapy focused on natural, carotenoid compounds, in connection with the modification of MDR. The function of the MDR protein was examined via the R123 drug accumulation of both cell lines in the presence of carotenoids. The fluorescence of the cell population was measured by flow cytometry. The most effective resistance modifiers Monoepoxy-beta-carotene, (SS, 8S)-capsochrome, (8'S) Luteoxanthin, (9Z)-Violaxanthin, (9Z)-Zeaxanthin, (13Z)-Zeaxanthin were assayed for their antiproliferative effects in combination with the anti-cancer drug epirubicin. (13Z)-Zeaxanthin was able to enhance the antiproliferative effect on human mdrl gene transfected mouse lymphoma and anthracycline resistant human breast cancer cell line MCF7. (8'S)-luteoxanthin, (5S, 8S)-capsochrome and (9Z)-zeaxanthin treatment revealed synergism with epirubicin on resistant mouse lymphoma. The enhanced antiproliferative activity of epirubicin combinated with (9Z)-Violaxanthin was more significant on MCF7 cells resistant to anthracycline.
Abstract: BACKGROUND: Failure of cancer chemotherapy is largely caused by multidrug resistance in tumor cells, mediated by ABC transporters that pump many cytostatics out from the cells. Thus, inhibition of the activity of P-glycoprotein efflux pumps can improve the therapeutic results. Disiloxanes are synthetic resistance modifiers that suppressed not only the multidrug resistance gene but also MRP in various cancer cell lines. Among these compounds, SILA-409 [1,3-dimethyl-1,3-bis(4-fluorophenyl)-1, 3-bis(3-morpholino-propyl)-disiloxanedihydro chloride] showed a remarkable antiproliferative effect and markedly inhibited the P-glycoprotein-mediated efflux mechanism in vitro. The efficacy of this organosilicon drug was investigated in vivo, in a xenograft system. MATERIALS AND METHODS: Human pancreatic cancer xenografts (PZX-40/19G) were treated s.c. with 10 mg/kg b.w. SILA-409 every second day for 34 days. Tumor volume changes were recorded every week. At the end of the experiment, a complete autopsy was performed and all the vital organs were evaluated histologically. The apoptotic and mitotic rates were counted and evaluated by morphometric methods, and the immunohistochemical expression of P-glycoprotein was determined using a monoclonal anti-p170 antibody. RESULTS: This large dose of the organosilicon compound did not result in histologically observable toxic effects, and some tumor growth delay was noted. SILA-409 did not affect the mitotic activity, but the number of apoptotic cells per mm2 was significantly increased. In the untreated tumors, 60% of the cells displayed p170-positivity, while in the treated group, P-glycoprotein was expressed in just 26% of the carcinoma cells. CONCLUSION: The multidrug reversal effect of SILA-409 was demonstrated in vivo without any apparent toxicity. In addition, it increased the apoptotic activity, exhibited some tumor growth delay, but did not affect the mitotic rate. This new organosilicon compound deserves further attention with a combination of multidrug-resistant substrate chemotherapeutic agents, especially in multidrug-resistant tumors.
Abstract: The resistance to chemotherapy of cancer cells is mediated by the overexpression of P-glycoprotein, as an ATP-dependent membrane efflux pump. Two families of compounds have been screened, the cinnamylidenecycloalkanones and cinnamylidenebenzocycloalkanones, as promising multidrug resistance (MDR) reversal agents on mouse lymphoma and human colon cancer (COL0320) cell lines. The antiproliferative effects of the cinnamylidene derivatives were tested with the MTT method The MDR effect on drug accumulation was tested by flow cytometry. Combinations of resistance modifiers and cytostatics were tested on the two cell lines to obtain evidence for additive or synergistic interactions. Verapamil was applied as a resistance-modifying positive control. The best effects in the reversal of MDR in both cell lines were exhibited by the methoxy derivatives 2-(2-methaoxycinnamylidene)indan-1-one, 2-(2-methoxycinnamylidene)-3,4-dihydro-2H-naphthalen-1-one, 6-(2-methoxycinnamylidene)-6,7,8,9-tetrahydrocyclohepten-5-one), 2-cinnamylidene-3,4-dihydro-2H-naphthalen-1-one and 6-cinnamylidene-6,7,8,9-tetrahydrobenzocyclohepten-5-one. 2-(2-methoxycinnamylidene) indan-1-one and 2-(2-methoxy-cinnamylidene)-3,4-dihydro-2H-naphthalen-1-one were able to enhance the antiproliferative activity of doxorubicin in a synergistic way.
Abstract: The multidrug resistance (MDR) proteins are member of the ATP-binding cassette superfamily and are present in a majority of human tumors. Their activity is a crucial factor leading to therapeutic failure. It is likely that compounds which inhibit the function of the MDR-efflux proteins such as MDR1 will improve the cytotoxic action of anticancer chemotherapy. Therefore, a search for MDR reversing compounds was conducted among three classes of plant derived compounds such as diterpenes, triterpenes and carotenoids in a hope to find inhibitors without adverse effects in these natural compounds. The inhibition of efflux activity was determined by measuring the accumulation of substrate analogues such as rhodamine in tumor cells in the presence of potential inhibitors. Thus we determined the effect of structurally unrelated diterpenes, triterpenes and carotenoids on reversal of multidrug resistance in MDR-1 gene-transfected L1210 mouse lymphoma cells and MDR mediated multidrug resistance of human breast cancer cells MDA-MB-231 (HTB-26) and MCF-7. The majority of diterpenes, cycloartane triterpenes and carotenoids isolated from vegetables and medicinal plants were able to enhance rhodamine 123 accumulations of MDR-cells. Synergistic interaction was found between epirubicine and resistance modifier terpenoids in vitro. It is supposed that these MDR modulators bind into transmembrane domains and the action of ABC transporters is inhibited by induced conformational changes.
Abstract: Two new tetracyclic diterpene polyesters, euphoportlandols A (1) and B (2), have been isolated along with 12 known tetracyclic triterpenes from an acetone extract of Euphorbia portlandica. Their structures were established as 5alpha,11alpha,14alpha,17-tetraacetoxy-3beta-benzoyloxy-6beta,15beta-dihydroxy-9-oxoseget-8(12)-ene (1) and 5alpha,11alpha,14alpha,17-tetraacetoxy-3beta-benzoyloxy-6beta,15beta-dihydroxy-9-oxosegetane (2), respectively, by spectroscopic data interpretation. Compounds 1 and 2 were evaluated for their ability to inhibit multidrug resistance in cancer cells. Both compounds were found to be inhibitors of P-glycoprotein activity.
Abstract: The new macrocyclic lathyrane diterpenes latilagascenes A and B ( 1 and 2), the diacetylated derivative of 2, latilagascene C ( 3), and the known diterpenes ent-16alpha,17-dihydroxyatisan-3-one ( 4) and ent-16alpha,17-dihydroxykauran-3-one ( 5), isolated from the methanol extract of Euphorbia lagascae, were examined for their effects on the reversal of multidrug resistance (MDR) on mouse lymphoma cells. Among the active lathyrane derivatives 1 - 3, compound 2 displayed the highest inhibition of rhodamine 123 efflux of human MDR1 gene transfected mouse lymphoma cells when compared to the untreated cells or the positive control verapamil. The new compounds are the first macrocyclic lathyrane diterpenes showing oxidation at C-16, whose structures were characterized by extensive spectroscopic methods, including 2D NMR experiments ( (1)H- (1)H COSY, HMQC, HMBC and NOESY). The known phenolic compounds vanillic acid ( 6), p-salicylic acid ( 7), isofraxidin ( 8) and cleomiscosin A ( 9) were also isolated from this species.
Abstract: Molecular orbital energies of energetically minimized series of extended aromatic and aminoalkyl side chain substituted phenothiazine compounds have been considered with respect to charge transfer (CT) binding properties to P-glycoprotein (P-gp) amino acids of the first P-gp loop. A dependency of decreasing energies of lowest unoccupied orbitals (E(lumo)) with reduced CT binding properties to an increasing P-gp mediated multidrug resistance (MDR) has been found for the extended aromatic compounds.
Abstract: The entropy production of tumorous cells is higher than that of normal cells, and entropy flow is therefore directed from tumorous toward healthy cells. This results in information concerning the cancer propagating into the surrounding normal tissue. However, ultrasound absorption results in additional entropy production in tissues. The entropy mechanism possibly provides a basis for a novel approach to anticancer therapy through the use of ultrasound irradiation. METHODS: Through the calculation of ultrasound-induced entropy production and comparison of the theoretical results with the experimental data on ultrasound absorption in biological tissues, we have demonstrated that ultrasound absorption will increase the entropy in normal tissue more efficiently than in tumorous tissue due to the more acidic nature of the latter. Consequently, the direction of entropy flow between these two kinds of cells may be reversed on exposure to ultrasound. CONCLUSION: The higher entropy accumulation of normal cells during ultrasound irradiation may possibly lead to a change in the original direction of entropy flow and avoid the propagation of information on the cancer into the normal tissues. We suggest that low-intensity, low-frequency ultrasound irradiation may be an efficient tool for the therapy of solid tumors.
Abstract: The aim of the study was to develop a simple, inexpensive, reproducible ethidium bromide (EB)-agar based method that is independent of any specialized instrumentation, for the demonstration of efflux pump activity, which is responsible for antibiotic resistance of bacteria. MATERIALS AND METHODS: A series of agar plates containing varying concentrations of EB were swabbed with strains of Escherichia coli or Staphylococcus aureus, which differed with respect to efflux pump activity. The plates were incubated at different temperatures and time periods and the measurements of fluorescence were used to evaluate the efflux activity of each culture. RESULTS: This simple assay allowed us to identify the efflux of EB in different bacteria following an overnight incubation. The minimal concentration of EB that produced fluorescence was significantly greater at 37 degrees C than at 4 degrees C, suggesting the presence of an energy-dependent pump. The method was shown to simultaneously identify strains of a mixed culture that differed from each other with respect to the activity of their efflux pumps. CONCLUSION: The method, in conjunction with the use of antibiotic-containing disks, provides an additional advantage for the easy identification and selection of colonies that differ with respect to antibiotic susceptibility and degree of efflux pump activity. Because the method is very reproducible it may form the basis for interlaboratory standardization of efflux pump activity of multi-drug resistant (MDR) clinical isolates.
Abstract: The hydroxystilbene trans-3,5,3',4'-tetrahydroxystilbene (piceatannol) (1), isolated from the methanol extract of Euphorbia lagascae defatted seeds, was methylated to yield the derivatives trans-3,5,3',4'-tetramethoxystilbene (2), (trans-3,5-dihydroxy-3',4'-dimethoxystilbene) (3) and trans-3,5,3'-trihydroxy-4'-methoxystilbene (4). The structures of the compounds were assigned by spectroscopic methods (IR, 1H-NMR, 13C-NMR and MS). The ability of piceatannol (1) and the three methylated derivatives to modulate the transport activity of P-glycoprotein (P-gp) and apoptosis induction on the L5178 mouse lymphoma cell line containing the human MDR1 gene was studied by flow cytometry. The reversal of multidrug-resistance (MDR) was investigated by measuring the accumulation of rhodamine-123, a fluorescent substrate analog of doxorubicin, in cancer cells. Verapamil was applied as a positive control. For the evaluation of the compounds as apoptosis inducers, tumor cells were stained with FITC-labelled annexin-V and propidium iodide. The tetramethylated derivative (2) was found to be a powerful inhibitor of P-gp activity. Compounds 1 and 2 showed an increased apoptotic effect in the MDR subline, the most active being piceatannol (1). Furthermore, in the combination chemotherapy model, the interaction between doxorubicin and the resistance modifier 2 was studied in vitro. The results of checkerboard experiments indicated that the type of interaction was additive between doxorubicin and compound 2 on the human MDR1 gene-transfected mouse lymphoma cells. However, in the MCF7/dox human breast cancer cells, the interaction was non-additive. The degree of additive and non-additive interactions were close to the borderline of the FIX values corresponding to the two types of interactions.
Abstract: BACKGROUND: There are many differences between healthy tissue and growing tumor tissue, including metabolic, structural and thermodynamic differences. Both structural and thermodynamic differences can be used to follow the entropy differences in cancerous and normal tissue. Entropy production is a bilinear form of the rates of irreversible processes and the corresponding "generalized forces". Entropy production due to various dissipation mechanisms based on temperature differences, chemical potential gradient, chemical affinity, viscous stress and exerted force is a promising tool for calculations relating to potential targets for tumor isolation and demarcation. METHODS: The relative importance of five forms of entropy production was assessed through mathematical estimation. Using our mathematical model we demonstrated that the rate of entropy production by a cancerous cell is always higher than that of a healthy cell apart from the case of the application of external energy. Different rates of entropy production by two kinds of cells influence the direction of entropy flow between the cells. Entropy flow from a cancerous cell to a healthy cell transfers information regarding the cancerous cell and propagates its invasive action to the healthy tissues. To change the direction of entropy flow, in addition to designing certain biochemical pathways to reduce the rate of entropy production by cancerous cells, we suggest supplying external energy to the tumor area, changing the relative rate of entropy production by the two kinds of cells and leading to a higher entropy accumulation in the surrounding normal cells than in the tumorous cells. CONCLUSION: Through the use of mathematical models it was quantitatively demonstrated that when no external force field is applied, the rate of entropy production of cancerous cells is always higher than that of healthy cells. However, when the external energy of square wave electric pulses is applied to tissues, the rate of entropy production of normal cells may exceed that of cancerous cells. Consequently, the application of external energy to the body can reverse the direction of the entropy current. The harmful effect brought about by the entropy flow from cancerous to healthy tissue can be blocked by the reversed direction of entropy current from the irradiated normal tissue around the tumor.
Abstract: BACKGROUND: [corrected] The effectiveness of chemotherapy is limited by the emergence of multidrug resistance (MDR). MDR is caused by the activity of various ATP binding cassette (ABC) transporters that pump anticancer drugs out of the cells in an ATP-dependent manner. Additionally some other cellular mechanisms of MDR have been reported. The purpose of this study was to investigate mechanisms of MDR in drug resistant MCF-7 cell lines and to modulate P-glycoprotein (P-gp) and MRP1-based MDR. MATERIALS AND METHODS: Paclitaxel (MCF-7/Pac), docetaxel (MCF-7/Doc), doxorubicin (MCF-7/Dox) and vincristine (MCF-7/Vinc) resistant sublines were developed from the parent MCF-7 cell line (MCF-7/S) by stepwise selection in dose increments over two years. Flow cytometry, MTT cytotoxicity assay, RT-PCR, caspase-3 activity assay and checkerboard combination assay were performed to investigate the degree of resistance developed in sublines and to reverse drug resistance phenotype. RESULTS: The flow cytometry histograms of drug accumulation assays demonstrated that the drug-resistant cell lines are P-gp and MRPI positive. RT-PCR results showed that the resistant sublines express both MDR1 and MRP1 genes. Resistance indices of each subline to each anticancer drug were determined using the MTT cytotoxicity assay and it was found that all the sublines were resistant to their respective agents. Caspase-3 activities of the cell lines were also determined. Caspase-3 activity is an important indicator of apoptosis in the cell. The reversal of MDR was attempted by two cinnamylidene ketone and two organosilicon compounds. The results indicated that these compounds modulated P-gp effectively, but they were not very effective at reversing MRP1 activity in the MCF-7 sublines. Four selective anticancer drugs (paclitaxel, docetaxel, doxorubicin and vincristine) and four synthetic MDR modulators [2-(2-methoxycinnamylidene) indan-1-one (cinnamylidene-1), 2-(2- methoxycinnamylidene)-3, 4-dihydro-2H-naphthelen-1-one) (cinnamylidene-2), ALIS 409 and ALIS 421] were applied to the sublines in combination. The fractional inhibitory indices illustrated that combined applications of cinnamylidene ketones and organosilicon compounds with paclitaxel, docetaxel or vincristine exerted significant antiproliferative effects on the resistant sublines. CONCLUSION: This report demonstrates the development of rational models for drug resistance MCF-7 cell lines and reversal of acquired drug resistance.
Abstract: In earlier experiments, the MDR (multidrug resistance)-reversal activities of Anastasia Black (Russian black sweet pepper) extracts had been analysed. Recently, the most effective MDR reversing extracts and fractions have been separated by HPLC (high-performance liquid chromatography, for carotenoids) and LC-MS-MS (HPLC combined with mass spectrometry, for phenolic compounds) methods. As a result of the analytical studies, the following flavonoids had been identified: feruloyl glucopyranoside, quercetin rhamnopyranoside glucopyranoside, luteolin glucopyranoside arabinopyranoside, apigenin glucopyranoside arabinopyranoside, quercetin rhamnopyranoside, luteolin arabinopyranoside diglucopy-ranoside, hesperidine and luteolin glucuronide. According to the literature, the aglycones of these phenolic compounds exhibit MDR-reversal activity in vitro, and the connection between the phenolic content of Anastasia Black and MDR-reversal action was therefore studied by different analytical methods. The results of this study revealed that the identified flavonoids of Anastasia Black may be only partially responsible for the modulation of the MDR of mouse lymphoma cells. Other lipophilic compounds, most probably carotenoids, present in Russian black sweet pepper may act as inhibitors of MDR reversal.
Abstract: Several new 3-formylchromone derivatives proved to be modifiers of multidrug resistance in mouse lymphoma cells and in human Colo320 colon cancer cells. There is apparently a structure-activity relationship between the antiproliferative multidrug resistance-reversing effect and the chemical structure of the 3-formylchromones. The total polar surface areas and the ground state dipole moments of the molecules are presumed to play a key role in the multidrug resistance-reversing effect. The log P values can provide an adequate explanation for the selective cytotoxicity against cancer cells.
Abstract: The mechanism of multidrug resistance (MDR) reversal is not fully understood yet. Interaction of MDR modifiers with lipid bilayer of cell membranes and alterations of fluidity or other biophysical properties of plasma membrane might be an important factor in mechanism of MDR modulation and reversal. In this review we focus on phenothiazines which belong to the group of drugs known to modify MDR in different types of cells, from cancer cells up to various kinds of microorganisms. First, the aggregation properties of phenothiazines and their interactions with lipid bilayers are described. The localization of phenothazine derivative molecules in bilayers and alteration of membrane properties are discussed. Apart from the influence on model bilayers also the interactions of phenothiazines with cellular membranes (especially of erythrocytes) are reviewed. In subsequent sections the anti-MDR activity of phenothiazine derivatives observed in microorganisms and in cancer cells is described. The possible molecular mechanisms involved in MDR reversal by these compounds are presented. The direct interactions of phenothiazines with multidrug transporters and other effects of these modulators on plasma membranes are discussed. Finally, the structural features of phenothiazine derivatives essential for their optimal MDR reversal activity are described.
Abstract: The ability of fifteen novel phenoxazine derivatives (four phenoxazines and eleven benzo[a]phenoxazines) to modulate multidrug resistance (MDR) in a P-gp-overexpressing mouse T lymphoma cell line (L5178 MDR) was studied. A flow cytometric functional test, based on the differential accumulation of rhodamine 123 by sensitive and multidrug-resistant cells, was employed. Seven benzo[a]phenoxazines were observed to increase the amount of rhodamine 123 accumulated by resistant cells, i.e. to be new effective MDR modulators. The results allowed us to draw preliminary conclusions about the structural features of benzo[a]phenoxazines which are important for MDR modulation.
Abstract: Cyclic ureas form a perspective class of non-peptidic HIV-1 protease inhibitors with major bioavailability problems. Low absorption rates of DMP 323 as one of the first representatives led to investigations whether transport efflux pump P-glycoprotein (P-gp) within the intestine may be partly responsible for the low absorption rates. DMP 323 proved to be a P-gp substrate in competition studies with P-gp inhibitor ritonavir and H17 as a representative of another class of non-peptidic HIV-1 protease inhibitors. Intestinal DMP 323 absorption was mainly increased under co-application of both ritonavir and H17. DMP 323 used as a membrane efflux pump inhibitor itself showed little affinities to P-gp compared to H17 as strong P-gp inhibitor. So P-gp proved to play a decisive role in the low intestinal absorption of the cyclic urea representative DMP 323.
Abstract: The ability of 41 1,4-diphenyl-1,4-dihydropyridine derivatives to inhibit the transport activity of P-glycoprotein were studied by flow cytometry in a multidrug-resistant human colon cancer cell line (COLO320) and in human mdr1 gene-transfected mouse lymphoma cells (L 5178 Y). The cytotoxicities of these compounds were also examined against human normal and cancer cell lines. The majority of the tested compounds proved to be effective inhibitors of rhodamine 123 outward transport, but their cytotoxicities were not negligible. Some dihydropyridine derivatives displayed cytotoxic activity against four human oral tumour cell lines and against three normal human oral cell lines. There was no clear-cut relationship between the multidrug-resistance activity or cytotoxicity and the chemical structures of the compounds. New ring substituents could prevent the oxidation of the ring of the aromatic compound.
Abstract: Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli, Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical pi-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.
Abstract: Physicochemical characteristics of two structurally different cage dimeric 1,4-dihydropyridines HX (1) and CC (2) have been determined and compared to their P-glycoprotein inhibiting properties. While the weakly basic compound (1) showed pH-dependent apparent partition coefficients (log D), the neutral compound (2) proved to have almost identical log D values at varying pH-values. The subsequent determination of partition coefficients (log P) resulted in comparably low log P values revealing a less lipophilic compound character. Determined significantly differing P-glycoprotein (P-gp) inhibitory properties indicated that the lipophilicity of the compounds does not play a decisive role for the P-gp activity.
Abstract: Despite the clinical efficacy of the most thoroughly studied conventional neuroleptic agent haloperidol, and the atypical antipsychotic risperidone is well established, little information is available on their molecular effects. Recent advances in high-density DNA microarray techniques allow the possibility to analyze thousands of genes simultaneously for their differential gene expression patterns in various biological processes, and to determine mechanisms of drug action. The aim of this series of experiments was to gain experience in antipsychotic gene-expression profiling and characterize (in the parlance of genomics) the "antipsychotic transcriptome." In this prospective animal study, broad-scale gene expression profiles were characterized for brains of rats treated with antipsychotics and compared with those of sham controls. We used DNA microarrays containing 8000 sequences to measure the expression patterns of multiple genes in rat fronto-temporo-parietal cortex after intraperitoneal treatment with haloperidol or risperidone. A number of transcripts were differentially expressed between control and treated samples, of which only 36 and 89 were found to significantly differ in expression as a result of exposure to haloperidol or risperidone, respectively (P<0.05). Acutely, 13 genes were more highly expressed and 15 transcripts were found to be significantly less abundant, whereas chronically nine genes were up-regulated and none of them was repressed in haloperidol-treated cortices. Risperidone acutely induced 43 and repressed 46 genes, and chronically over-expressed 6 and down-regulated 11 transcripts. Selected genes were assayed by real-time PCR, then normalized to beta-actin. These assays confirmed the significance of the array results for all transcripts tested. Despite their differing receptor affinity and selectivity, our findings indicate that haloperidol and risperidone interfere with cell survival, neural plasticity, signal transduction, ionic homeostasis and metabolism in a similar manner.
Abstract: A preliminary exploration of coumarin derivatives as novel multidrug resistance (MDR) modulators was carried out to determine the basic features of the structure responsible for the MDR reversal activity. Among 44 coumarins, 14 compounds moderately induced the reversal of MDR (fluorescence activity ratio (FAR) values > 1). The most active compound, 6-hydroxy-3-(2-hydroxyethyl)-4-methyl- 7-methoxycoumarin [C34], was equally potent as a MDR modulator verapamil. These data show a relationship between the chemical structure and MDR-reversal effect on tumor cells. All coumarins tested were more cytotoxic against tumor cells than normal cells. Several compounds displayed potent cytotoxic activities (CC50 15 - 29 microg/mL in HSC cells), comparable with that of gallic acid (CC50 = 24 microg/mL). Both 6-hydroxy- 7-methoxy-4-methyl-3-isopropylcoumarin [C43] and 3-ethyl-6-hydroxy- 7-methoxy-4-methylcoumarin [C44] showed the highest tumor-specific cytotoxicity (SI value = 4.1 and 3.6, respectively). We conclude that coumarins are potentially potent new MDR modulators with low toxicity against normal cells. A deeper understanding of the relationship between their structures and their potency will contribute to the design of optimal agents.
Abstract: The effects of various flavonoids and carotenoids on Rhodamine 123 accumulation in multidrug-resistant Colo 320 human colon cancer cells expressing MDR1/LRP were studied. The Colo 205 cell line was used as a drug-sensitive control. Rotenon, Catechin, Neohesperidin, Naringin, Robinin, Phloridzin, Robinetin, Dihydrobinetin, Dihydrofisetin, Kampferol, Dihidroquercetin, Sakuranin and Sakuranetin were tested on Colo 320 cells: only Rotenon was found to be effective as regards multidrug resistance (MDR) reversal, while a majority of the flavonoids, such as Catechin, Neohesperidin, Naringin, Robinin, Phloridzin, Dihydrobinetin and Sakuranetin, had only marginal effects on Rhodamine 123 accumulation. The tested carotenoids (beta-Cryptoxanthin, Luteoxanthin, Anteroxanthin, Violeoxanthin, Apple peel fetoxanthin, Lutein, Violaxanthin and Neoxanthin) were able to increase Rhodamine 123 accumulation in Colo 320 cells. Verapamil was applied as a resistance-modifying positive control. The levels of apoptosis induction in drug-resistant and sensitive cell lines were also compared. The results indicated that the tested flavonoids were weak apoptosis inducers on MDR and parent cells, without significant differences. A majority of the carotenoids induced only early apoptosis, but apoptosis and cell death were not induced in MDR colon cancer cells.
Abstract: The treatment of prostate cancer in an advanced state is still unsatisfactory. In the event of the ineffectiveness of total androgen blockade (TAB) therapy, cytostatic administration may be attempted. In this study, we modelled the drugs used in practice on human prostate cancer cell lines. Studies aimed at decreasing multidrug resistance were performed on PC-3 cells. With the use of various cytostatics, the cell proliferation-inhibiting effects were measured under in vitro conditions on human prostate cancer cell lines LNCaP-FGC and PC-3. Under the given experimental conditions, the examined cytostatics exhibited antiproliferative effects on each of the investigated cell lines. Our results indicate that it is not necessary to wait until the development of a hormone-resistant state. In the studies on the PC-3 cell line, we did not find a multidrug-resistant efflux activity responsible for the resistance of the tumour.
Abstract: The Carpobrotus edulis methanol extract, inactive against the methicillin-resistant Staphylococcus aureus or the multidrug-resistant Mycobacterium tuberculosis, does inhibit the growth of these two bacteria once they are phagocytosed by monocyte derived human macrophages.
Abstract: Various resistance mechanisms such as complex formation with DNA, tRNA and MDR1 p-glycoprotein were modified in bacteria and cancer cells in presence of pregnane, pyridoquinoline, and aza-oxafluorene derivatives. Interaction between the compounds, plasmid DNA and tRNA was shown and compared to the interaction with calf thymus DNA. Complex formation with MDR1 p-glycoprotein and drug accumulation increased in cancer cells. Both plasmid DNA and p-gp complex formation were related to the chemical structures of the resistance modifiers.
Abstract: The incorporation of the 1-aryl-5-(4-nitrophenyl)-3-oxo-1,4-pentadienyl group into 3,4-dihydro-1H-naphthalenyl, cyclohexyl, 2,3-dihydro-1H-indenyl and cyclopentyl scaffolds led to the discovery of various compounds with selective toxicity for malignant cells and ability to reverse multidrug resistance.
Abstract: Patients presenting with Acquired Immune Deficiency Syndrome (AIDS) are predisposed to co-infection with Mycobacterium avium. The management of such patients is problematic due to underlying immuno-incompetence and the high resistance of M. avium to most non-toxic compounds. Therefore, the need for effective agents is obvious. Because phenothiazines, especially the relatively mild thioridazine, have significant activity against Mycobacterium tuberculosis, we investigated the in vitro activity of chlorpromazine, thioridazine, promazine, promethazine and desipramine against a reference and clinical strains of M. avium. The results obtained show that whereas all of the phenothiazines employed in this study had an minimum inhibitory concentration (MIC) against the strains studied that ranged from ca. 10 to > 50 mg/L, as was previously shown for M. tuberculosis, thioridazine was the most active of the group against M. avium.
Abstract: The drug accumulation of a human multidrug resistance 1 (mdr1) gene-transfected mouse lymphoma cell line and a multidrug resistance protein (MRP)-expressing human breast cancer cell line MDA-MB-231 was compared in the presence of sixteen flavonoids and five isoflavonoids. The expression of the 170-kDa P-glycoprotein (P-gp) (MDR1) and 190-kDa multidrug resistance protein (MRP) in both cell lines was confirmed by immunocytochemistry. The rhodamine 123 accumulation of the P-glycoprotein (P-gp)-expressing cells increased up to 46.4, while 2,7'-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM) accumulation of the MRP-expressing cells increased up to 1.6, in fluorescence activity ratio (FAR). Major P-gp-mediated efflux pump modifiers are formononetin, amorphigenin, rotenone and chrysin, while MRP-mediated efflux pump modifiers are formononetin, afrormosin, robinin, kaempferol and epigallocatechin. In antiproliferative assay, afrormosin, amorphigenin, chrysin and rotenone exhibited the strongest antiproliferative effects in L5178 (max. ID50: 19.70) and MDA-MB-231 cell lines (max. ID50: 55.47). In a checkerboard microplate method in vitro, furthermore, the most effective multidrug resistance (MDR) resistance modifiers, amorphigenin, formononetin, rotenone and chrysin, were assayed for their antiproliferative effects in combination with epirubicin. Rotenone and afrormosin showed additive effects. Chrysin and amorphigenin on the mouse lymphoma cell line and formononetin on the MDA-MB-231 cell line synergistically enhanced the effect of epirubicin.
Abstract: Anastasia Black (Russian sweet pepper) of Capsicum annuum L. var. angulosum Mill. (Solanaceae) was successively extracted with hexane, acetone, methanol and 70% methanol, and the extracts were further separated into a total of twenty-three fractions by silica gel or octadecylsilane (ODS; C18) column chromatography. These extracts and fractions were investigated for their cytotoxicity, anti-human immunodeficiency virus (HIV), anti-Helicobacter pylori (H. pylori), urease inhibition and multidrug resistance (MDR) reversal activity. Some fractions of hexane and acetone extracts showed higher cytotoxic activity against three human oral tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG) than against three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specific cytotoxic activity. No fractions displayed anti-HIV activity, but some hydrophobic fractions showed higher anti-H. pylori activity, urease inhibition activity and MDR reversal activity. The higher MDR activity of these fractions against MDR gene-transfected L5178 mouse lymphoma T cells may possibly be due to their higher content of carotene or polyphenol. These data suggest that Anastasia Black should be further investigated as a potent supplement for cancer chemotherapy.
Abstract: Multidrug resistance (MDR) is believed to be a major reason for the failure of cancer treatment. It is in most cases caused by the activity of the various ABC transporters, multidrug resistance (MDR) gene-encoded p-glycoproteins that pump anticancer drugs out of the cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) are the most important and widely studied members of the ABC superfamily of transporters. The ability of four diterpenic lactones isolated from Euphorbia species to modulate the transport activity of P-gp in mouse lymphoma cells was evaluated by flow cytometry. The reversion of MDR was investigated by using a standard functional assay with rhodamine 123 as a fluorescent substrate analogue of doxorubicin. Verapamil was applied as a positive control. All the compounds were able to reverse the MDR of the tested human MDR1 gene-transfected mouse lymphoma cells, in a concentration-dependent manner from 4 to 40 microg/mL, in a short-term experiment below the cytotoxic doses.
Abstract: Androgen ablation is palliative and does not cure advanced prostate cancer. The hormone-sensitive cells die and the hormone-resistant cells overgrow, resulting in disease progression. The drug of choice for secondary treatment is estramustine (Estracyt). The success of the therapy is followed by changes of the prostate-specific antigen level and Karnofsky scale. In the present study, the results of estramustine treatment of 79 patients with advanced prostate cancer in 12 hospitals were evaluated. The mean prostate-specific antigen level improved for 6 months, but rose from the ninth month on. The improvement in the subjective condition of the patients paralleled the change in the prostate-specific antigen level. The short time of improvement was a consequence of the very high prostate-specific antigen level and the poor general condition. Estramustine administration is recommended when the prostate-specific antigen level becomes more than doubled following primary treatment. At a starting prostate-specific antigen level of > 100 ng/ml, the treatment leads to total androgen blockade. If the prostate-specific antigen level has not decreased after treatment for 3 months, the secondary strategy is to apply chemotherapy.
Abstract: Eighteen new fused heterocyclic compounds of benzazoles and benzoxazines were investigated for induction and inhibition of apoptosis on tumor cells (L5718, mouse lymphoma cell line containing the human mdr-1 gene). For evaluation of apoptosis, the cells were stained with FITC-labelled Annexin-V and propidium iodide and the results were analysed by flow cytometry. Nine of these substances were also checked for reversal of multidrug resistance. The reversal of multidrug resistance was determined by measuring the rhodamine-123 accumulation in the cancer cells. Rhodamine-123 shows a green fluorescence and its intracellular concentration correlates well with the inhibition of efflux pump activity. Three of the tested compounds, 5-(p-nitrobenzamido)-2-benzylbenzoxazole (BD-3), 6-methyl-2-(o-chlorophenyl) benzoxazole (A-9) and 5-(p-nitrophenoxyacetamido)-2-phenylbenzoxazole (D-30), showed an increased apoptotic effect on mouse lymphoma cells. Moreover, compounds BD-3, A-9 and 5-(2-thienylcarboxyamido)-2-phenylbenzoxazole (D-24) also amplified the apoptosis effect of 12H-benzo(a)phenothiazines (M-627). However, D-24, alone was not effective. Additionally, 2-(p-nitrobenzyl)benzoxazole (B-11), was also found to increase the apoptotic effect of M-627. On the other hand, 5-(p-nitrophenylacetamido)-2-phenylbenzoxazole (D-7) showed an anti-apoptotic effect. No positive correlation was found between the increased drug accumulation effect and the programmed cell death induced by the compounds studied.
Abstract: Carotenoid fractions were extracted from red paprika, Valencia orange peel and the peel of Golden delicious apple. Thus, hypophasic carotenoids of paprika (PM1), orange (PM3) and apple (PM4), and epiphasic extractions of paprika (PM2) and apple (PM5) were obtained by extraction, saponification and partition between MeOH-H(2)O (9:1) (hypophasic) and hexane (epiphasic). A high content of capsanthin was quantified in hypophasic carotenoids (PM1) from red spice paprika, whereas the hypophasic fractions from orange (PM3) and apple (PM4) were mainly composed of violaxanthin, zeaxanthin and lutein. On the other hand, a high content of beta,beta-carotene and beta-cryptoxanthin was found in epiphasic fractions (PM2 and PM5). The extracts were studied for their anti-Helicobacter pylori (H. pylori), anti-human immunodeficiency virus (HIV), cytotoxic, multidrug resistance (MDR) reversal and radical scavenging activity. Among five PM extracts and beta,betacarotene, PM4 showed potent anti-H. pylori activity (MIC(50) = 36 microg/mL), comparable to metronidazole (MIC(50) = 45 microg/mL). The extracts were inactive against HIV. PM3 and PM4 showed slightly higher cytotoxic activity against three human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG) and human promyelocytic leukemic HL-60 cells than against three normal human oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specific cytotoxic activity. PM1, PM3 and PM4 displayed much higher MDR-reversing activity than (+/-)-verapamil. ESR spectroscopy demonstrated that PM1-5 and beta,beta-carotene produced little or no detectable radical under alkaline conditions and did not scavenge the O(2) (-) produced by the hypoxanthine and xanthine oxidase reaction. On the other hand, PM1 and PM2 scavenged efficiently 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, whereas singlet oxygen was also quenched efficiently by PM5 and PM2. The data suggest the potential importance of carotenoids as possible anti-H. pylori and MDR reversal agents. The active principles in the carotenoid extract might differ, depending upon the types of fruits and vegetables.
Abstract: Expression of eight transporter genes of Escherichia coli K-12 and its DeltaacrAB mutant prior to and after induction of both strains to tetracycline resistance and after reversal of induced resistance were analyzed by quantitative reverse transcriptase PCR. All transporter genes were overexpressed after induced resistance with acrF being 80-fold more expressed in the DeltaacrAB tetracycline-induced strain.
Abstract: Multidrug resistance (MDR) of cancer cells can be the result of a variety of mechanisms that are not completely understood. One of the most significant among them concerns altered membrane transport in tumor cells, often referred to as typical or classic MDR. This mechanism is related to the overexpression of a variety of proteins, that belong to the super family of ABC transporters. The aim or this study was to look for new effective modulators of MDR1 and multidrug resistance-associated protein (MRP) transporters. Ten diterpenes based on the jatrophane skeleton, including rearranged polycyclic derivatives, were studied on the MDA-MB-231 (HTB-26) human breast cancer cell line. The majority of those compounds were able to strongly enhance the rhodamine 123 accumulation of the human MDR1 gene transfected mouse lymphoma cell line, as previously described. In the present study, the MDR reversal of the same jatrophanes on MDR1- and MRP- mediated resistance of human breast cancer cells is reported. These cells simultaneously express MDR1 and MRP proteins when identified by monoclonal antibodies. However, in a functional assay, where rhodamine 123 accumulation was measured and verapamil was the traditional positive control, only MRP was active, while MDR1 was inactive. Carboxyfluorescein served as a substrate for MRP-mediated drug efflux, and indomethacine was the positive control used as an inhibitor of MRP in the flow cytometric experiments. The effectivity of various jatrophanes was different on the carboxyfluorescein efflux inhibition of the human breast cancer cells. These results may have importance in the planning of a new type of combination chemotherapy.
Abstract: Lung cancer is the leading cause of mortality worldwide. The median survival of advanced disease is in the range of 8 to 10 months. Intrinsic or acquired drug resistance pose major challenges to the success of chemotherapy. The HER2 gene, also known as c-erbB-2 or neu, is a proto-oncogene that encodes a membrane-bound receptor tyrosine kinase of the epithelial growth factor receptor (EGFR) family. It has a possible role in tumor cell proliferation, tumor invasion, tumor metastasis and drug resistance. We retrospectively investigated 88 samples of non-small cell lung cancer (NSCLC) and assessed the correlation between HER2 expression and tumor histology. The expression of HER2 protein was analyzed by immunohistochemical staining (IHC) and HER2 DNA amplification was detected by using fluorescence in situ hybridization (FISH). HER2 overexpression (2+, 3+) was detected in 5 (5.7%) out of 88 specimens. All of the HER2-overexpressing tumors histologically proved to be squamous cell carcinoma (SCC). Cases with 2+ HER2 immunoreactivity showed either no amplification (3.875 and 2.525), or borderline amplification (4.75). Cases with 3+ HER2 immunoreactivity showed moderate amplification (7.35) and strong amplification (15-20 - cluster), respectively. The HER2 expression in NSCLC was relatively low in the selected Hungarian population; consequently, there is no indication for introduction of trastuzumab for the treatment of lung cancer.
Abstract: The ability of phenothiazine derivatives to inhibit the transport activity of P-glycoprotein in resistant mouse lymphoma and MDR/COLO 320 cells was studied. A rhodamine 123 efflux from the above-mentioned neoplastic cells in the presence of tested compounds was examined by flow cytometry. Two of the phenothiazine derivatives, namely perphenazine and prochlorperazine dimaleate, proved to be effective inhibitors of the rhodamine efflux. Other tested phenothiazine derivatives (promethazine hydrochloride, oxomemazine, methotrimeprazine maleate, trifluoropromazine hydrochloride, trimeprazine) also modulated the intracellular drug accumulation in both resistant cell lines, however, they exerted additional cytotoxic effects. The differences observed between the effects of the test compounds on intracellular drug accumulation could be the outcome of differences in phenothiazine's chemical structure, which is crucial for drug-cell membrane interactions. The results of this study provide information about a new group of compounds that offer promise in multidrug resistance reversal in tumor cells.
Abstract: The effect of antidepressants is the culmination of a series of molecular actions occurring in the brain. These events are thought to lead to changes in the expression level of numerous, but as yet unknown genes that result in different cellular functions. In our present study we addressed this issue by establishing gene expression profiles of the rat brain after treatment with imipramine and citalopram at therapeutic doses. After 96 h and 4 wk, fronto-temporal cortices from controls and each treated strain were prepared and total RNA was isolated, and assessed using a cDNA microarray system containing 3200 clones. The expression of 6 genes was decreased and 8 were over-expressed by imipramine, whereas 27 were repressed and 7 were up-regulated by citalopram. Members of signal transduction (e.g. phosphatidylinositol transfer protein), structural elements (e.g. tubulin, fibronectin), factors related to protein metabolism in general (e.g. proteasomal subunits, ubiquitin-like proteins, polyadenylation sites), components involved in cell survival (e.g. midkine, stress-inducible protein), and determinants of membrane conductance and ion transport (e.g. vacuolar H+-ATPase), and basics of nuclear functions (e.g. translin, basal transcription factor 3), were some of the genes with altered expression. These data demonstrate that antidepressants interfere with the expression of a large array of genes involved in signalling, survival and protein metabolism. Our results demonstrate for the first time that antidepressants specifically regulate neuronal plasticity through induction of a highly specific transcriptional programme in brain cells.
Abstract: The efficiency of chemotherapy is often decreased by the development of resistance of cancer cells to cytostatic drugs. This phenomenon is in most cases caused by the activity of the various ABC transporters, multidrug-resistance (MDR) gene-encoded p-glycoproteins, that pump anticancer drugs out of the cells. The inhibition of the activities of the MDR proteins MDR1 and MRP was investigated via the administration of two new organosilicon compounds, alis-409 and alis-421. The study was focused on the inhibition of MDR by blocking the ADR1 gene expression and through the inhibition of the pump-function of mdr-p-glycoprotein, in human breast cancer cell lines expressing mrp and prostate cancer cell line (PC-3). Apoptosis induction and the interaction between epirubicin and the silicon-substituted compounds were studied in human MDR-1 gene-transfected mouse lymphoma and its parent cell line, Colo320/MDR-LRP and sensitive subline Colo205, by means of rhodamine 123 accumulation. The activity of MRP1 p-glycoprotein was studied in human breast cancer cell lines such as HTB-26/MRP1 and two MRP-negative breast cancer cell lines, T47D and MCF7, by carboxyfluorescein accumulation, and on a stomach cancer cell line. The activity of MRP in 257P/MDR and its drug-sensitive derivative were studied in human stomach cancer cells by daunorubicin accumulation in a flow cytometer. The two representative organosilicon derivatives, alis-409 and alis-421, showed antiproliferative effects without apoptosis induction. The drug accumulation in the human MDR1 gene-transfected mouse lymphoma cells was increased without down-regulation of the MDR1 gene expression tested by RT-PCR assay. The rhodamine uptake was increased in L5178/MDR1 and Colo320/MDR1-LRP, but not drug-sensitive human breast cancer MCF-7 and T47D, and L5178 mouse lymphoma parent cells in the presence of alis-409 and alis-421. The MRP-mediated carboxyfluorescein accumulation in HTB-26/MRP human breast cancer cells and daunorubicin accumulation in human stomach cancer cells 257P/MDR were not modified by these alis compounds. A synergistic interaction between epirubicin and the silicon-substituted resistance modifiers was found only in MDR1-mediated MDR in the case of colo-320/MDR1-LRP cells and mouse lymphoma cells transfected with the human MDR1 gene. The results indicate that the organosilyl derivatives specifically act on MDR1 p-glycoprotein 170. The alis compounds act on pgp170 in a way which is similar to verapamil isomers.
Abstract: Antidepressants are widely used in the treatment of mood disorders associated with dementia, however little information is available on their effect at the molecular level. In certain neurodegenerative disorders, such as in Alzheimer's disease, lymphocytes have been used to assess mirror changes that thought to occur in the brain. Gene expression profiles of lymphocytes from Alzheimer patients have been shown to differ from that seen with controls. To address this issue in light of antidepressant treatment, we used lymphocytes derived from Alzheimer's disease patients and control individuals to assess the impact of the selective serotonine reuptake inhibitor citalopram on gene expression using a cDNA microarray representing 3200 distinct human genes. Sequences that are differentially regulated after treatment with citalopram were identified and categorized based on similarities in biological functions. This analysis revealed that the overexpression of genes in control and Alzheimer white blood cells by citalopram are implicated in cell survival. Apart from this, citalopram did not markedly alter genes involved in other molecular functions in control cells. In contrast, alteration of genes implicated in ionic currents, cell-adhesion, immune mechanism, and adrenergic functions, were also observed in Alzheimer lymphocytes. The expression of genes of Alzheimer lymphocytes by citalopram is modulated differently which may correlate with the pathology.
Abstract: We previously reported that a trifluoromethyl ketone derivative, 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18), exhibited the potent antibacterial activity against Helicobacter pylori, but had no urease activity. In order to clarify the mechanism of anti-H. pylori action of TF18, we evaluated the growth and motility of TF18 on clarithromycin-susceptible H. pylori (CSHP) and -resistant H. pylori (CRHP). An effective proton pump inhibitor (TF18) had remarkable dose-dependent antibacterial activity and was able to inhibit the flagellar motor of both CSHP and CRHP isolates. The antimotility effect of TF18 was more pronounced at subinhibitory concentration in CRHP than in CSHP. The swimming (the forward motion) was more sensitive to the inhibition than the tumbling. Based on the results, it is supposed that TF18 works as an uncoupler similar to the 'clutch' in a biological motor, in which counterclockwise rotation is more sensitive to the effect of TF18 than the clockwise rotation.
Abstract: Antidepressants are widely used in the treatment of mood disorders associated with dementia, however little information is available on their effect at the molecular level. We have demonstrated that gene expression profiles of lymphocytes from patients with Alzheimer dementia differ from that seen with controls, with alpha(2)-adrenoceptor being the most highly repressed transcript. To address this issue in light of antidepressant treatment, we used lymphocytes derived from Alzheimer patients and control individuals to assess the impact of mirtazapine, the novel antidepressant with alpha(2)-adrenoceptor antagonistic activities, on gene expression using a cDNA microarray representing 3200 distinct human genes. Sequences that are differentially regulated after treatment with mirtazapine were identified and categorized based on similarities in biological functions. This analysis revealed that selected biological processes, including protein metabolism, cytoskeleton integrity, immune response, cellular plasticity, and neurotransmission, are involved in early phases of administration of this antidepressant. In addition, although it was possible to identify common targets, the expression profiles of Alzheimer lymphocytes differed mainly in their magnitude from those seen with controls. These results confirm the usefulness of the gene array approach for studying Alzheimer-specific changes in the periphery and suggest that the expression of genes of Alzheimer lymphocytes is modulated differently by mirtazapine, which correlates with the pathology.
Abstract: The ability of fifteen cycloartanes, isolated from Euphorbia species, to reverse multidrug resistance (MDR) and apoptosis induction in L5178Y mouse lymphoma cells, including its multidrug-resistant subline, was studied by flow cytometry. Reversion of MDR was investigated using a standard functional assay with rhodamine 123 as a fluorescent substrate analogue. For the evaluation of apoptosis, the cells were stained with FITC-labeled annexin V and propidium iodide. The majority of the compounds were able to reverse MDR of the tested human MDR1 gene-transfected mouse lymphoma cells. Some of the compounds were able to induce moderate apoptosis in the PAR cell line, but this effect was less effective on multidrug-resistant cells. The results indicate that cycloartanes can be substrates of ABC transporters, which might compete with certain anticancer chemotherapeutics.
Abstract: The aim of this study was to investigate the effects of 3,5-diacetyl- (DP1-DP5) and 3,5-dibenzoyl-1,4-dihydropyridines (DP6-DP11) on vascular functions in vitro, by comparing their mechanical and electrophysiological actions in rat aorta rings and single rat tail artery myocytes, respectively, and to quantify their multidrug resistance (MDR)-reversing activity in L5178 Y mouse T-lymphoma cells transfected with MDR1 gene. In rat aorta, the 11 compounds tested, but 3,5-dibenzoyl-4-(3-phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7), 3,5-dibenzoyl-4-(3-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (DP9), 3,5-dibenzoyl-4-(4-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (DP10) and 3,5-dibenzoyl-4-phenyl-1,4-dihydro-2,6-dimethylpyridine (DP11), antagonized 60 mm K+ (K60)-induced contraction in a concentration-dependent manner, with IC50 (m) values ranging between 5.65 x 10(-7) and 2.23 x 10(-5). The 11 dihydropyridines tested, but DP7, inhibited L-type Ca2+ current recorded in artery myocytes in a concentration-dependent manner, with IC50 (M) values ranging between 1.12 x 10(-6) and 6.90 x 10(-5). The K+ -channel opener cromakalim inhibited the Ca2+ -induced contraction in K30 but not that evoked in K60. On the contrary, DP7 was ineffective in both experimental conditions. When the rings were preincubated with 1 mm Ni2+ plus 1 microm nifedipine, the response to phenylephrine was significantly reduced by 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), a well-known endoplasmic reticulum Ca2+ -ATPase inhibitor. DP7 had no effects on this model system. In L5178 MDR cell line, the 11 dihydropyridines tested, but 3,5-diacetyl-4-(2-nitrophenyl)-1,4-dihydro-2,6-dimethylpyridine (DP1), 3,5-diacetyl-4-(3-phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine (DP2) and 3,5-diacetyl-4-(3-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (DP4), exhibited an MDR-reversing activity, with IC50 values ranging between 3.02 x 10(-7) and 4.27 x 10(-5), DP7 being the most potent. In conclusion, DP7 may represent a lead compound for the development of potent dihydropyridine MDR chemosensitizers devoid of vascular effects.British Journal of Pharmacology (2004) 141, 415-422. doi:10.1038/sj.bjp.0705635
Abstract: In the present study, we showed the inhibition of motility by trifluoromethyl ketone (TF) derivatives (1-8) in Proteus vulgaris (P. vulgaris) cultures. Among them, 1-(2-benzoxazoyl)-3,3,3-trifluoro-2-propanone (1) showed a much stronger inhibitory effect on the motility of P. vulgaris than other TF compounds at 10% MIC. Our results suggest the possibility of an inhibitory action of TF compounds on the proton motive forces by affecting the action of biological motor and proton efflux in the membranes, resulting in a reduction of the ratio of running and the increased number of tumbling and non-motile cells.
Abstract: The rearranged jatrophane-type diterpenes ( 1 - 3), isolated from the Me (2)CO extracts of Euphorbia portlandica and Euphorbia segetalis, were examined for their effects on multidrug resistance (MDR) in mouse lymphoma cells. Compounds 2 and 3 revealed to be active with the latter being more active than the positive control verapamil, a known resistance modifier. The new compound 1, named portlandicine, was isolated from Euphorbia portlandica and its structure characterised by high-field NMR spectroscopic methods including 2D NMR techniques: COSY, HMQC, HMBC and NOESY. The known diterpene 2, together with aleuritolic acid ( 4), oleanolic acid ( 5), and betulin diacetate ( 6), were also isolated from the same species.
Abstract: The macrocyclic jatrophane diterpene polyesters, pubescenes A - D ( 1 - 4) were isolated from the whole dried plant of Euphorbia pubescens, and evaluated for multidrug resistance (MDR) reversing activity on mouse lymphoma cells. All the compounds displayed very strong activity compared with the positive control verapamil. Pubescene D ( 4) is a new compound, whose structure was established as 3beta,9alpha,-diacetoxy-7beta-benzoyloxy-15beta-hydroxy-14-oxo-2beta H-jatropha-5 E,12 E-diene by spectroscopic methods, including (1)H- (1)H COSY, HMQC, HMBC and NOESY.
Abstract: Fractionation of barbados cherry (acerola fruit, a fruit of Malpighia emarginata DC.) extracts were performed by organic solvent extractions and column chromatographies, using two extraction methods. Higher cytotoxic activity was concentrated in fractions A4 and A6 (acetone extract), and H3 and HE3 (hexane extract). These four fractions showed higher cytotoxic activity against tumor cell lines such as human oral squamous cell carcinoma (HSC-2) and human submandibular gland carcinoma (HSG), when compared with that against normal cells such as human periodontal ligament fibroblasts (HPLF) and human gingival fibroblasts (HGF). HE2 (hexane extract), AE2 (ethyl acetate extract), AE3, AE4, AE5, A8, A9 and A10 showed some relatively higher anti-bacterial activity on the Gram-positive Staphylococcus epidermidis ATCC 1228 but were ineffective on the representative Gram-negative species E. coli and Ps. aeruginosa. The fractions were inactive against Helicobacter pylori, two representative Candida species, and human immunodeficiency virus (HIV). H3, H4 and HE3, which displayed higher tumor-specific cytotoxicity also showed higher multidrug resistance (MDR) reversal activity, than (+/-)-verapamil as positive control. ESR spectroscopy shows that the radical-mediated oxidation is not involved in the induction of tumor-specific cytotoxic activity. The tumor specific cytotoxic activity and MDR reversal activity of barbados cherry may suggest its possible application for cancer therapy.
Abstract: The multidrug resistance (MDR) proteins that belong to the ATP-binding casette superfamily are present in a majority of human tumors and are an important final cause of therapeutic failure. Therefore, compounds which inhibit the function of the MDR-efflux proteins may improve the cytotoxic action of anticancer chemotherapy. The effects of carotenoids were studied on the activity of the MDR-1 gene-encoded efflux pump system. The carotenoids, isolated from paprika and other vegetables, were tested on the rhodamine 123 accumulation of human MDR-1 gene-transfected L1210 mouse lymphoma cells and human breast cancer cells MDA-MB-231 (HTB-26). Capsanthin and capsorubin enhanced the rhodamine 123 accumulation 30-fold relative to nontreated lymphoma cells. Lycopene, lutein, antheraxanthin and violaxanthin had moderate effects, while alfa- and beta-carotene had no effect on the reversal of MDR in the tumor cells. Apoptosis was induced in human MDR1 transfected mouse lymphoma cells and human breast cancer MDA-MB-231 (HTB-26) cell lines in the presence of lycopene, zeaxanthin and capsanthin. The data suggest the potential of carotenoids as possible resistance modifiers in cancer chemotherapy.
Abstract: PURPOSE: P-glycoprotein (P-gp) is made responsible for the limited oral bioavailability of P-gp substrates like peptidic HIV protease inhibitors (PIs). With respect to combined application of two PIs in antiretroviral regimes, we first investigated the influences on intestinal saquinavir uptake using different PIs in in situ perfusion studies. METHODS: Perfusion experiments were carried out in three intestinal segments with P-gp substrates talinolol and saquinavir using fixed concentrations of PIs and with each varying concentrations in the jejunum and ileum. Furthermore, cellular uptake of fluorescent P-gp substrate rhodamine-123 and MRP-substrate carboxyfluorescein has each been quantified in P-gp and MRP-expressing cells by flow cytometry under co-administration of PIs. RESULTS: Increase of calculated permeabilities of P-gp-specific substrate talinolol was found under co-administration of both PIs, ritonavir and H17, with highest absorption rates in the ileal and colon segment. H17 proved to be a better P-gp inhibitor than ritonavir by resulting IC50 values and also in the cellular uptake of rhodamine. Similar increases of permeabilities in ileum and colon have also been found for saquinavir as P-gp as well as MRP-substrate with differences in the jejunal uptake, which was found higher for H17. Additional MRP-inhibitory activities of H17 were proved by increasing cellular uptake rates of carboxyfluorescein in MRP-expressing cells. CONCLUSIONS: The investigated PIs were characterized as effective P-gp inhibitors in the intestinal absorption of P-gp substrates. H17 showed MRP-inhibitory effects that also favor intestinal drug absorption of corresponding substrates. With respect to combined therapeutic application of PIs, compounds like H17 raise hopes for improved bioavailability of poorly absorbed compounds.
Abstract: Multidrug-resistant Mycobacterium tuberculosis (MDRTB) and antibiotic-resistant Plasmodium falciparum are the major global lethal infections accounting for over 4 million deaths per year. Methicillin-resistant Staphylococcus aureus (MRSA) is the major global nosocomial infection and resistance to vancomycin is evident and may become common. This review provides the scientific and medical basis that support the use of one particular group of compounds, the phenothiazines, and in particular thioridazine, for the management of the above antibiotic-resistant infections. Because thioridazine, a relatively mild neuroleptic as compared to its parental compound chlorpromazine, kills intracellular MDRTB and MRSA at clinical concentrations, its use for the management of these infections may be considered. The review also discusses the activity of phenothiazines against protozoa and parasites, the mechanisms by which phenothiazines promote their antimicrobial effects, their potential for regulating efflux pumps that are a cause for mono or multidrug resistance, as well as their potential for the therapy of problematic infections caused by bacteria that have acquired plasmid-antibiotic-resistant genes.
Abstract: The inhibition of bacterial motility was studied by a trifluoro methyl ketone derivative on two Escherichia coli strains (wild strain having a proton pump system and the proton pump-deficient mutant strain) and two Helicobacter pylori strains (clarithromycin susceptible and clarithromycin resistant). Evidence is presented of the inhibitory action of 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18) on the proton motive forces of the two bacterial strains by affecting the action of biological motor and proton efflux in the membranes. The swimming, the forward motion was more sensitive than the vibration or tumbling to the inhibition. We suppose that the inhibiton of bacterial motility is related to the virulence of bacteria: consequently the pathogenicity can be reduced in the presence of TF18.
Abstract: Chlorpromazine (CPZ) is concentrated by human macrophages where it kills intracellular mycobacteria when the concentration outside the macrophage is sub-clinical. We have previously demonstrated that thioridazine (TZ), a much milder phenothiazine, has similar activity and kills intracellular methicillin-susceptible S. aureus at sub-clinical concentrations. We have extended this latter study to include methicillin-resistant S. aureus (MRSA) and show that TZ kills intracellular MRSA at clinically relevant concentrations. The ultrastructure of MRSA exposed to in vitro concentrations of TZ just below its MIC and that of MRSA phagocytosed by macrophages previously exposed to a clinically relevant concentration of TZ was also studied. TZ inhibits the replication of phagocytosed MRSA, affecting the structure of the cell envelope, resulting in lysis of the bacterium 6 hours post-phagocytosis. These ultrastructural changes are identical to those produced in vitro by a TZ concentration that is just below the MIC. Because macrophage intracellular MRSA is not killed by the macrophage and its intracellular location protects it from antibiotics that are unable to reach that site, recurrent infections which result may be successfully managed with the use of TZ.
Abstract: Most of the species of Euphorbiaceae are known to be toxic and poisonous plants because their milky latex has strong skin irritant activity, and chronic exposure can result carcinogenic effect. The toxic constituents of Euphorbiaceae species are specific diterpenes, called in common as phorboids. These compounds (tigliane, ingenane and daphnane derivatives) possess extreme pro-inflammatory and tumour promoting effects due to the activation of protein kinase C enzyme. The present article gives a survey about the present estimation of Euphorbiaceae diterpenes on the basis of own experimental results and literature data. The study tries to answer the question whether these compounds can be regarded as plant toxins or they may have therapeutic relevance? It was concluded that one group of diterpenes, such as most of phorbol and ingenol esters can be considered exclusively as toxins without any possible medicinal use. The other group of diterpenes comprises compounds, which display toxicity, but in adequate dose they have therapeutic perspective (e.g. the resiniferatoxin with capsaicine-like effect). The third group of compounds such as diterpenes of non-phorboid type with macrocyclic or polycyclic structures do not have toxic effect or this property is markedly reduced, however demonstrate interesting biological activities (anti-MDR, antiproliferative and tubulin-interacting effects). Thus, these compounds may be promising lead compounds for natural product based drug developments.
Abstract: The tumor-inducing effects of Agrobacterium, Bartonella and Helicobacter bacterial species are compared step by step. An analogy for the existence of these individual steps is considered in connection with the development of cancer. The transformations of eukaryotic cells occur in particular in the type IV secretion system, i.e. involving the simultaneous transmission of DNA and protein from bacterial cells to eukaryotic cells. Thus, transfected cells facilitate the indefinite growth of tissue cells and additionally produce growth factors, triggering further bacterial multiplication. The higher numbers of bacteria then produce more transfection and the cycle repeats as long as the host lives. The main limiting factor is the frequency of bacterial infection, while the secondary rate-limiting factors are the levels of transforming growth factors and factors triggering bacteria growth. CONCLUSIONS: Analogous processes are probably responsible for the tumor induction by the three different bacterial species; however, the critical points for eradication are different. The early eradication or limitation of B. henselae or H. pylori can prevent hemangiomas, stomach cancer and malignant cell proliferation. The crown gall formation by A. tumefaciens can only be avoided by prevention of the transforming activity of a single bacterial infection. Questions arise as to what is common in the three processes, and the nature of the rate-limiting step in the three different models. The frequency of transformation is the rate-limiting step, but the co-transmission of the DNA-protein complex is common in the three systems.
Abstract: Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.
Abstract: A previous preclinical study revealed that the maximal additive effect between chemotherapy (CT) and irradiation (RT) occurred at a low level of CT. Therapy was therefore designed with an oral drug daily given in combination with RT in order to determine the efficacy and toxicity. Locoregionally advanced head and neck tumor patients were treated with simultaneous RT and CT. RT was administered 5 times per week at 2 Gy per fraction in a total dose of 70 Gy. Throughout the treatment 30 mg/kg Tegafur was given daily orally. In the period between 2000 and 2002, 50 patients were enrolled. Complete remission was attained in 60%, with an overall response rate of 94%. Acute mucositis of grade 2 or 3 was observed in 56% (28 patients), and gastrointestinal and hematologic toxicity of grade 2 or 3 occurred in 8% (4 patients). Because of side-effects, the duration of treatment was at most 2 weeks longer. Toxicity was eliminated quickly by careful supportive therapy. In conclusion, it is considered that oral low-dose CT in combination with RT is an efficient and simple mode of treatment for locally advanced head and neck tumor patients with a poor prognosis.
Abstract: A structure-activity relationship of dopamine and 3-benzazepine derivatives is discussed, using theoretically calculated results. In order to clearly divide dopamines and 3-benzazepines into a strongly active and a weakly active group, the CC50, two different dipole moments (microESP-G and microESP-W) and heat of formation (deltaHf) of dopamine [1-13] and 3-benzazepine derivatives [14-23] were separately calculated in two states of gas-phase and water-solution by the COSMO/PM3 method. It was found that ten derivatives [1-3, 9, 12-13 and 20-23] (CC50: 0.056 to 2.5 mM) showed the strongest cytotoxic activity with small delta(deltaHf) values, whereas thirteen derivatives [4-8, 10-11, 14-19] (CC50: > 3.6 mM) showed the weakest cytotoxic activity with large delta(deltaHf) values.
Abstract: We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone [TF18] exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E. coli). In the present study, the inhibition of E. coli motility by TF18 was investigated. TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E. coli strains. The wild-type E. coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations. These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system.
Abstract: A new sesquiterpene-coumarin ether (5'beta,9'alpha,10'alpha)-7-0-(3alpha-methoxy-8'(12')-drimen-11'-yl)-scopoletin, designated driportlandin (1) and a new abietane quinoid diterpene 16-hydroxy-abieta-8,12-diene-11,14-dione, named portlanquinol (2) together with lupeol, nepehinol, wrightial, formonetin and davidigenin were isolated and characterized from the Me2CO extract of whole dried plant of Euphorbia portlandica. The structures of the new compounds were elucidated from spectral data including 2D-NMR experiments of COSY, HMQC, HMBC and NOESY. When examined for their effects on the reversal of multidrug resistance(MDR) on mouse lymphoma cells, compound 1 proved to be more active than the positive control verapamil and compound 2 was found to be toxic. This is the first report on the isolation of a sesquiterpene-coumarin and a quinoid-type diterpenoid from Euphorbia.
Abstract: Three alkaloids, lycorine, homolycorine and 2- O-acetyllycorine, were isolated from the bulbs of Leucojum vernum (Amaryllidaceae) and identified by means of NMR analysis. The alkaloids obtained from L. vernum and from other Amaryllidaceae species were studied in vitro for HIV-1 replication inhibitory activity on MT4 cells. The cytotoxicity of the compounds in uninfected cells was evaluated by using the MTT assay and the [ (3)H]thymidine incorporation test. The antiviral activities were determined by means of the p24 antigen assay and solid-phase reverse transcriptase testing. The results demonstrate that trisphaeridine, lycorine, homolycorine, and haemanthamine possess high antiretroviral activities (IC (50) = 0.4 - 7.3 microg/mL), accompanied by low therapeutic indices (TI (50) = 1.3 - 1.9).
Abstract: The vegetable, Anastasia Red, Capsicum annuum L. var. angulosum Mill. (Solanaceae) was successively extracted with hexane, acetone, methanol and 70% methanol, and the extracts were further separated into a total of 21 fractions by silica gel or octadecylsilane (ODS) column chromatography. The biological activities of extracts and fractions were determined. These extracts showed relatively higher cytotoxic activity against two human oral tumor cell lines (HSC-2, HSG) than against normal human gingival fibroblasts (HGF), suggesting a tumor-specific cytotoxic activity. The cytotoxic activity of these extracts was enhanced by fractionation on silica gel [H2, A2, M1-M3] or ODS column chromatography [70M]. Several fractions [H2, H4, H5, A1, A2, A3, A5, A6, A7, M2] reversed the multidrug resistance (MDR) phenotype with L5178 mouse lymphoma T cells, more efficiently than (+/-)-verapamil. The extracts and fractions did not show any detectable anti-human immunodeficiency virus (HIV) or anti-Helicobacter pylori activity. Thus, this study suggests the effective and selective antitumor potential of 'Anastasia Red' of sweet pepper for further phytochemical and biological investigation.
Abstract: The phenothiazine multidrug resistance (MDR) modulators are chemically diversified but share the common feature to be hydrophobic cationic molecules. Molecular mechanisms of their action may involve interactions with either P-glycoprotein or membrane lipid matrix. In the present work we study the anti-MDR and biophysical membrane effects of new phenothiazine derivatives differing in the type of group substituting phenothiazine ring at position 2 (H-, Cl-, CF(3)-) and in the side chain group (NHCO(2)CH(3) or NHSO(2)CH(3)). Within each phenothiazine subset we found that anti-MDR activity (determined by P-glycoprotein inhibition assessed by flow cytometry) correlates with the theoretically calculated hydrophobicity value (logP) and experimental parameters (determined by calorimetry and fluorescence spectroscopy) of lipid bilayers. It is concluded that the biological and biophysical activity of phenothiazine derivatives depends more on the type of ring substitution than on the nature of the side chain group.
Abstract: Although phenothiazines are known as multidrug resistance modifiers, the molecular mechanism of their activity remains unclear. Since phenothiazine molecules are amphiphilic, the interactions with membrane lipids may be related, at least partially, to their biological effects. Using the set of phenothiazine maleates differing in the type of phenothiazine ring substitution at position 2 and/or in the length of the alkyl bridge-connecting ring system and side chain group, we investigated if their ability to modulate the multidrug resistance of cancer cells correlated with model membrane perturbing potency. The influence exerted on lipid bilayers was determined by liposome/buffer partition coefficient measurements (using the absorption spectra second-derivative method), fluorescence spectroscopy and calorimetry. Biological effects were assessed by a flow cytometric functional test based on differential accumulation of fluorescent probe DiOC(2)(3) by parental and drug-resistant cells. We found that all phenothiazine maleates were incorporated into lipid bilayers and altered their biophysical properties. With only few exceptions, the extent of membrane perturbation induced by phenothiazine maleates correlated with their lipophilicity. Within the group of studied derivatives, the compounds substituted with CF(3)- at position 2 of phenothiazine ring were the most active membrane perturbants. No clear relation was found between effects exerted by phenothiazine maleates on model membranes and their ability to modulate P-glycoprotein transport activity.
Abstract: Fractionated extracts of persimmon (Diospyros kaki) peels were studied for cytotoxic activity, multidrug resistance (MDR) reversal activity, anti-human immunodeficiency virus (HIV) activity and anti-Helicobacter pylori (H. pylori) activity. The potent cytotoxic activity against human oral squamous cell carcinoma cells (HSC-2) and human submandibular gland tumor (HSG) cells was found in the acetone fractions (A4 and A5) with IC(50) ranging from 21 to 59 micro g/mL. However, the cytotoxic activity was not correlated with the radical intensity of the fractions. Three 70% MeOH extract fractions (70M2-4) produced radical and efficiently scavenged the O(2)(-) produced by hypoxanthine and xanthine oxidase reaction. All of the fractions tested were not effective for anti-H. pylori and anti-HIV. Fractions H3 and H4 of hexane extract, and M2 and M3 of MeOH extract showed a remarkable MDR reversal activity comparable with that of (+/-)-verapamil (a positive control). These results indicate the therapeutic value of persimmon peel extracts as potential antitumor and MDR-reversing agents.
Abstract: In order to analyse the radiosensitivity of tumours and to evaluate the possibility of improving the treatment results with regard to genetic alterations, we examined 33 patients with advanced head and neck tumours after 66-70 Gy irradiation. Between 1998 and 2001, 33 patients with advanced head and neck squamous cell carcinoma (HN-SCC) were observed. They received 66-70 Gy to the primary tumour site and pathological lymph nodes. One month later, physical examination and CT were performed to verify the effect of radiotherapy, and the patients were followed until their death. The histological grading and Ki-67, cyclin D1, p53 and bcl-2 status were examined from the aspect of their potential prognostic value in all patients. The average survival was 13 months; 25% of the patients survived for at least 20 months. Seventy-two percent of the patients demonstrated Ki-67 positivity, 69% p53 positivity and 40% cyclin D1 positivity; there were only 12% bcl-2-positive cases. A significant correlation was not found between the tumour response or the duration of survival and the Ki-67, p53 or cyclin D1 positivity. Only bcl-2-positive cases exhibited significantly better outcome. These parameters indicate the proliferating (Ki-67 and cyclin D1) and apoptotic (p53 and bcl-2) activities of advanced HN-SCC cells. Our results proved that they proliferate rapidly and have impaired repair or apoptotic functions. The heterogeneity of our results did not allow us to conclude that the above parameters are of clinically reliable prognostic value, but the obviously high kinetic activity of HN-SCC underlines the potential efficacy and need for accelerated irradiation in these cases.
Abstract: The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.
Abstract: Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections.
Abstract: The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent.
Abstract: A set of 9,10-dihydro-9,10-ethano and ethenoanthracene derivatives was tested with the aim to quantify the effect observed on drug efflux. Structure activity relationships and molecular modeling studies allowed to define topological display of pharmacophoric groups for these reversal agents.
Abstract: Anti MDR activity of a series of acridine, pyridoquinoline, quinoline and pyridine analogous amines was evaluated. Interesting activity is displayed by tricyclic compounds. Besides ring size, influence of the side chain was studied.
Abstract: The neurotoxic beta-amyloid peptide of Alzheimer's disease is formed from the amyloid precursor protein (APP), which is a member of an evolutionarily highly conserved gene family with significant functional importance. Because behavioral and psychiatric symptoms treated with antipsychotics may influence the course of the disease, we have investigated traditional and atypical antipsychotic drugs, administered through the intraperitoneal route, for their effects on rat cortical APP. Western-immunoblotting was utilized for semi-quantitative evaluation of APP levels. Treatment with haloperidol resulted in an acute elevation of cortical APP both in therapeutic and toxic doses, however, it had no significant chronic impact on APP. Atypical antipsychotic risperidone did not change cortical APP concentration. These results indicate that both haloperidol and risperidone are considered to be relatively safe with respect to APP metabolism. Possible mechanisms, including involvement of calcium and APP itself as a receptor, are discussed.
Abstract: A previous preclinical study revealed that the maximum additive effect between chemotherapy (CT) and irradiation (RT) occurred at a low level of CT. Therapy was therefore designed with an oral drug daily given in combination with RT in order to determine the efficacy and toxicity. Locoregionally advanced head and neck tumor patients were treated with simultaneous RT and CT. RT was administered 5 times per week at 2 Gy per fraction to a total dose of 70 Gy. Throughout the treatment 30 mg/kg Tegafur was given daily orally. In the period between 2000 and 2002, 50 patients were enrolled. In 60%, complete remission was attained with an overall response rate of 94%. Acute mucositis of grade 2 or 3 was observed in 56% (28 patients), while gastrointestinal and hematological toxicity of grade 2 or 3 occured in 8% (4 patients). Because of side-effects, the duration of treatment was at most 2 weeks long. Toxicity was eliminated quickly by careful supportive therapy. In conclusion, it is considered that oral low-dose CT in combination with RT is an efficient and simple mode of treatment for locally advanced head and neck tumor patients with a poor prognosis.
Abstract: Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.
Abstract: Promethazine has been recognised as an effective antiplasmid agent in cultures containing a single bacterial species such as Escherichia coli, Yersinia enterocolitica, Staphylococcus aureus and Agrobacterium tumefaciens. The objective of this study was to examine the effect of heterogenity of the microbial flora on plasmid elimination by promethazine in a laboratory based model system of mixed bacterial infection. F'lac plasmid elimination of E. coli K12 LE140 was studied in the presence of a numerically predominant Gram-positive species (Bacillus cereus, Staphylococcus epidermidis) by promethazine (0-120 mg/l) at 23, 37 and 39 degrees C. Growth kinetics of different bacterial species were studied at various temperatures without drug treatment in mixed bacterial cultures and it was found that the small number of added bacteria overgrew the pre-existing flora during the incubation period. We observed that bacterial-bacterial interactions modified the growth rate of individual bacterial species and gave selective advantages to some bacterial species of the microbial community. Some interactions between coexisting bacterial species enhanced the frequency of plasmid curing by promethazine in mixed cultures with S. epidermidis. In our experiments the plasmid curing action of promethazine was more effective at elevated temperature than at lower temperatures.
Abstract: Plasmid-containing bacteria often cause serious therapeutic failure during the treatment of infectious diseases. The selection of resistant-mutant strains and the transfer of mobile genetic determinants (such as plasmids and transposons) of resistance promote increased antibiotic resistance. In the last 30 years the antiplasmid effect of acridine dyes, ethidium bromide, sodium dodecyl sulphate and phenothiazines was described. The main aim of this study was to test the mechanism of the antiplasmid effect of promethazine and 9-aminoacridine on doxycycline-resistant enteric bacteria. The antiplasmid effects of promethazine and 9-aminoacridine were studied on plasmid elimination of native plasmid DNA and plasmid DNA isolated from drug-treated cells of plasmid-containing Escherichia coli, Citrobacter freundii and Enterobacter cloacae. The effects of some phenothiazines on plasmid profiles of bacterial strains isolated from urinary tract infections were analysed by agarose gel electrophoresis. Various complex of plasmid DNA were identified in the presence of promethazine, trifluoperazine and 9-aminoacridine in the agarose gel electrophoresis. Doxycycline resistance of tested enteric bacteria was the target of "curing" in the presence of promethazine and trifluoperazine. The frequency of elimination of tetracycline resistance was low despite the formation of antiplasmid compounds complex with isolated plasmid DNA. Tetracycline resistance plasmid was isolated and re-transformed. The plasmid curing effects of promethazine, trifluoperazine and 9-aminoacridine were increased in the presence of a trifluoroketone proton pump inhibitor on E. coli K12 LE140 strain in a model experiment. We propose that the inefficient penetration of antiplasmid compounds could be responsible for the weak plasmid-curing effect in some clinical isolates and that membrane active, calmodulin- and proton pump inhibitors may be combined for plasmid curing in antibiotic-resistant bacteria.
Abstract: The antibacterial activity and influence on lipid model membranes and erythrocyte membranes of 24 N-acylphenothiazines and trifluoperazine were studied. (1) Among 24 phenothiazines, the antimicrobial activity of amino maleates was the highest. (2) The influence of phenothiazines on model liposome and erythrocyte membranes was studied using N-phenyl-1-naphthylamine (NPN) as fluorescence probe. From the three types of phenothiazine substitution (H, Cl, CF3) at position 2, CF3-phenothiazines were the most effective in the interaction with liposomal membranes. (3) As measured by the polarization degree of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence, the alteration of membrane fluidity induced by CF3-phenothiazines was the biggest. Surprisingly, phenothiazines induced stomatocytic shape alterations (invaginations) in erythrocytes and at higher concentrations, also hemolysis of erythrocytes was observed. (4) The microcalorimetic measurements of influence of phenothiazines on thermal behaviour of synthetic lipid systems confirmed the previously obtained results. The main transition temperature and enthalpy of transition of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) were significantly modified by CF3-phenothiazines, suggesting their penetration of the lipid bilayer. Above results show that phenothiazine maleates were generally more effective than other phenothiazines used in this study.
Abstract: The dried aerial parts of Euphorbia mongolica afforded three new acylated polyhydroxy diterpenoids based on the jatrophane framework. The structures were established by means of a combination of 1D and 2D NMR techniques and mass spectrometry as (2S,3S,4R,5R,7S,8R,13S,15R)-5alpha,7beta,8alpha-triacetoxy-3beta-benzoyloxy-15beta-hydroxyjatropha-6(17),11E-diene-9,14-dione (1), (2S,3S,4R,5R,7S,8S,9S13S,15R)-5alpha,7beta,8alpha,9alpha,15beta-pentaacetoxy-3beta-benzoyloxyjatropha-6(17),11E-dien-14-one (2), and (2S,3S,4R,5R,7S,8S,9S13S,15R)-3beta,7beta,8alpha,9alpha,15beta-pentaacetoxy-5alpha-benzoyloxyjatropha-6(17),11E-dien-14-one (3). When the isolates were assayed for multidrug resistance-reversing activity in a rhodamine 123 exclusion test using L5178 mouse lymphoma cells, all compounds demonstrated a concentration-dependent effect in inhibiting the efflux pump activity of these tumor cells in the range 11.2-112 microM.
Abstract: The antiproliferative effect and apoptosis-inducing action of 5-fluorouracil (5-FU) in combination with vitamin C were tested in vitro against the chemosensitive mouse lymphoma, the chemoresistant HEp-2 and a human lung fibroblast cell line. Vitamin C itself had no antiproliferative effect on the fibroblasts, but increased the anticancer effect of 5-FU dose-dependently. In the case of the chemoresistant cell line, only a high concentration of vitamin C increased the cytotoxicity of 5-FU. A combination of 5-FU and vitamin C exerted a significantly enhanced apoptotic effect on the mouse lymphoma cell line, whereas for the HEp-2 cell line this effect was less marked and was achieved only at a high concentration of vitamin C. These findings suggest that the administration of a high dose of vitamin C in combination with 5-FU chemotherapy enhances the chemoresponsiveness of cancer cells and serves as a potential sensitizer, especially in chemo-resistant cell lines. One of the mechanisms by which vitamin C potentiates cytostatics could be apoptosis induction.
Abstract: The potency of flavonoids (isoflavones, flavones, and flavanones) to inhibit efflux of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was investigated. Structure-activity relationship analysis showed that the strongest inhibitors were found among flavanones bearing a hydrophobic prenyl, geranyl, or lavandulyl group at position 8 (and hydroxyl groups at 5 and 7) in ring A. A prenyl group at position 5' or stilbene at positions 4'-5' in ring B further seemed to increase inhibitor potency. The most efficient flavanones, euchrestaflavanone A and sophoraflavanone H, were approximately 20 times more efficient than genistein, and induced 50% inhibition of BCPCF efflux (IC50) at 3 microM (60 min, 37 degrees C). This is comparable to IC50 of benzbromarone (4 microM) and lower than IC50 of indomethacin (10 microM), both known MRP1 (ABCC1) inhibitors. It is suggested that BCPCF efflux is mainly due to MRP1 activity. Our results indicate that flavonoid molecular structure provides a promising base for development of potent MRP1 inhibitors.
Abstract: The concomitant administration of chemotherapy and radiation is an alternative tool in cancer therapy. The antiproliferative and the radiosensitizing effect of Cisplatin and 5-Fluorouracil (5-FU) were studied on mouse lymphoma cells transfected with human MDR1 gene (mdr) and its parent cell line (par) combined with and without radiation. HEp2 radioresistant cell culture was used in our experiments as a model of radioresistance. The growth rate and antiproliferative effect was measured by the MTT method. Significant inhibition of tumor cell growth was observed at a low concentration of Cisplatin and 5-FU combined with radiation on the mouse lymphoma cell lines. However an extremely high dose of Cisplatin and 5-FU resulted in moderate growth inhibition in the case of the HEp2 cell line. We assume that the radiosensitizing effect of 5-FU and Cisplatin can be considered as a synergistic antitumor effect at low doses of chemotherapy and radiation in a radiosensitive cell line. In the case of a radio-and chemoresistant cell line, high doses of radiation and chemotherapeutic agent achieved moderate tumour growth inhibition without significant synergistic effect. In addition the simultaneous application of both treatments can result in remarkable toxicity.
Abstract: In screening cytotoxic agents in morphine alkaloids [TE1-10], codeinone [TE8] was cytotoxic against two human oral tumor cells lines (HSC-2 and HSG). The cytotoxic activity of codeinone (CC50=1.0-1.2 microg/mL) against HSC-2 or HSG cells was higher than that of doxorubicin (CC50=1.9-2.0 microg/mL). Human oral gingival fibroblasts (HGF) were relatively resistant to codeinone, as judged by higher SI ratio (3.7) suggesting the tumor-selective cytotoxicity of codeinone. The cytotoxic activity of morphine (CC50=221 microg/mL) against HSC-2 was slightly lower than that of codeine (CC50=186 microg/mL), thebaine (CC50=125 microg/mL), etorphine (CC50=94 microg/mL) or dihydroetorphine (CC50=60 microg/mL). A study of structurally-related compounds suggested that the alpha,beta-unsaturated ketone group of codeinone was responsible for its antitumor cytotoxicity. The cytotoxic activity of codeinone was significantly reduced by N-acetylcysteine, but not affected by FeCl3, CuCl2, CoCl2, sodium ascorbate or catalase. Neither codeinone nor morphine inhibited P-glycoprotein-mediated rhodamine-123 efflux in multidrug resistant mouse T lymphoma L5178 transfected with human MDR 1 gene. These data suggest that codeinone induces cytotoxicity in oral tumor cell lines, possibly by a Michael-like addition of a protein SH or of an amino group to the bouble bond of codeinone.
Abstract: Seven alkaloids were isolated from Sprekelia formosissima, and five from Hymenocallis x festalis. Tazettine, lycorine, haemanthidine and haemanthamine were evaluated for antiproliferative and multidrug resistance (mdr) reversing activity on mouse lymphoma cells. Lycorine, haemanthidine and haemanthamine displayed pronounced cell growth inhibitory activities against both drug-sensitive and drug-resistant cell lines, but did not significantly inhibit mdr-1 p-glycoprotein. Thus, the tested alkaloids are apparently not substrates for the mdr efflux pump. Assays for interactions with DNA and RNA revealed that the antiproliferative effects of lycorine and haemanthamine result from their complex formation with RNA.
Abstract: A series of neuroleptic protonated phenothiazine derivatives (promethazine, promazine, triflupromazine, methotrimeprazine, propiomazine, trifluoperazine and fluphenazine), some with known anticancer properties, were complexed with water-insoluble antineoplastice agents such as 5-fluorouracil (5FU), methotrexate (MTX) and sulindac, as well as with components of biomembrane and synthetic phospholipids as possible models of cancer and microbial cells. In all cases water-soluble micellar inclusion adducts were formed exhibiting electron charge transfer complex behaviour, with the appearance of thazine free radicals. The thiazines sequestered the drugs and phospholipids in well-defined molar ratios (MR) parabolically-dependent on the dipole moments (mu) of the protonated phenothiazine derivatives. pH comparisons showed that the inclusion adducts followed a model in which the compounds were enveloped in the lipophilic interior of the thiazine aggregates, while the side-chains of the latter faced the aqueous environment. In the model experiment, interaction of the thiazines and the 5FU adducts with E. coli F' lac was additive to marginally synergistic, confirmed by the checkerboard technique. The parabolic dependence of the molar minimum inhibitory concentration of the thiazines and thiazine/5FU adducts on the thiazine dipole moments suggests that their primary loci of interaction are the cell wall or membrane phospholipid components. Thiazines, especially those with dipole moments centered on about 6 D, near which the lowest MR occurs, can act as effective carriers for insoluble or sparsely soluble drugs. Any new thiazine drug for use alone or as a carrier in anticancer therapy should be designed with this criterion in mind.
Abstract: The effects of 15 jatrophane diterpene polyesters (1-3 and 5-16) isolated from lipophilic extracts of Euphorbia serrulata, E. esula, E. salicifolia, and E. peplus (Euphorbiaceae) on the reversion of multidrug resistance of mouse lymphoma cells were examined. The structures of five new compounds (1-5) were elucidated by spectroscopic methods, including HRFABMS, ESIMS, (1)H-(1)H homonuclear and (1)H-(13)C heteronuclear correlations, long-range correlation spectra, and NOESY experiments. The stereochemistry and absolute configuration of one compound (3) were determined by X-ray crystallography. The structure-activity relationship is discussed.
Abstract: Multidrug resistance (mdr) constitutes the major obstacle to successful cancer treatment. The ability of fifteen newly-synthesised N-acylphenothiazine derivatives to inhibit the transport activity of P-glycoprotein was studied by flow cytometry in a resistant mouse lymphoma cell line. A standard functional assay with rhodamine 123 as a fluorescent substrate analogue was used. All derivatives proved to be effective inhibitors of rhodamine 123 outward transport, however their toxicity to the cells was not negligible. Phenothiazine maleates probably interact with transporter proteins of cancer cells by a different mechanism than other phenothiazine derivatives studied. The mdr reversal mechanism of phenothiazine acetylamides, methoxycarbonylamides and methylsulfonylamides is likely to involve modulator-cell membrane interactions since a connection between the compounds' hydrophobicity and their P-glycoprotein inhibition potency was observed. As a result of the present study a new group of mdr reversal agents was identified.
Abstract: The effect of some resistance modifiers on apoptosis induction by a benzo[alpha]phenothiazine derivative was studied on the L5178Y mouse lymphoma cells (parent) and its multidrug resistant (MDR) subline. For evaluation of apoptosis the cells were stained with FITC-labelled annexin V and propidium iodide and the results were analysed by flow cytometry. 12H-benzo[alpha]phenothiazine [M627] induced apoptosis both in the parent cells and in the MDR cells. The apoptosis induction by [M627] was not affected significantly by post- or pre-treatment with resistance modifiers, while in the cells treated by (+/-)-verapamil before and after apoptosis induction with [M627], the apoptosis was somewhat higher. The resistance modifier compounds alone also induced apoptosis and it was slightly higher in the parent cells than its MDR1/A gene-transformed subline.
Abstract: Eleven analogues of nifedipine (NP) showed synergistic interactions with ampicillin (Ap) and erythromycin (Er) on Escherichia coli K12LE140/F'lac. The antibacterial effect of Ap was enhanced by most analogues but compound (G9) and (+/-)-verapamil (VP) were antagonistic. Two of the 11 compounds (G7, G8) were synergistic with Er and four were additive. With a sensitive clinical isolate of E. coli Gy-1/Ap(sens)Er(res), compound G1 antagonized the antibacterial effect of Ap and a synergistic effect was found in the combination of Er with G4, G5, G6 or G7. None of the drugs had any effect on a multidrug resistant (MDR) clinical isolate of E. coli Gy-2/Ap(res)Er(res).
Abstract: Chlorpromazine (CPZ) has in vitro antimicrobial activity against Staphylococcus aureus at concentrations that greatly exceed those achieved clinically. It is concentrated by tissues that are rich in macrophages and it is active against phagocytosed mycobacteria when the concentration in the medium is compatible with that achieved clinically. In this report we show that nontoxic concentrations of CPZ below clinical levels have killing activity against S. aureus phagocytosed by human monocyte-derived macrophages that have nominal killing activity against these bacteria. Little or no resistance to the antimicrobial activity of this compound is anticipated to result because of its large number of cellular targets. Therefore, CPZ may have a role in the management of intracellular staphylococcal infections that normally require the use of antibiotics whose potential toxicity exceeds that associated with short-term management with CPZ.
Abstract: A variety of local flaps have been described for chest wall and bronchopleural fistula reconstruction. When local options cannot be used because of previous surgery, trauma, radiation, or body habitus, free flaps become an acceptable option. The authors report a case of persistent bronchopleural-cutaneous fistula treated with a free latissimus dorsi musculocutaneous flap that obliterated the right chest cavity, closed the site of empyema drainage, and aided healing of a bronchopleural fistula. Surgical technique including anastomosis to the innominate vein is described.
Abstract: Fifteen 4-phenyl-3,5-dibenzoyl-1,4-dihydropyridines (BzDHPs) (1-15) substituted at the 4-phenyl ring were synthesized and compared to their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems. Among them, 2-CF3 (5) (IC50=8.7 microM), 2-Cl (11) (IC50=7.0 microM) and 3-Cl (12) (IC50=7.0 microM) derivatives showed the highest cytotoxic activity against human oral squamous carcinoma (HSC-2) cells. The activity of P-glycoprotein (Pgp) response for MDR in tumor cells was reduced by some of derivatives (3, 4, 8, 12), verapamil (VP) and nifedipine (NP). These data suggest that 3,5-dibenzoyl-4-(3-chlorophenyl)-1,4-dihydro-2,6-dimethylpyridine (12) can be recommended as a new drug candidate for MDR cancer treatment.
Abstract: A series of benzo[b]-1,8-naphthyridine derivatives branched with various side-chains and substituents were prepared with the aim of being investigated as multidrug resistance (MDR) modulators. The syntheses were achieved from 2-halonicotinic acid and suitable aryl-amines according to a three-step procedure. All the derivatives were tested in vitro on mouse T-Lymphoma cell line L5178 transfected by MDR1 gene and the chemosensitizing properties of the compounds were compared to those of verapamil and propranolol, as well as to several other tricyclic derivatives like phenothiazines and acridines. Most of the compounds tested reversed the MDR of tumour cells more effectively than the reference drugs did and they showed more potent chemosensitizing activity than phenothiazine and acridine derivatives have.
Abstract: Kiwi gold fruits were extracted successively with hexane, acetone, methanol and 70% methanol, and further fractionated by silica gel and ODS column chromatographies for the assays of various biological activities. Five fractions H1, H2 (hexane extract), Al, A2 (acetone extract) and M2 (methanol extract) showed selective cytotoxic activity against human oral tumor cell lines, which was more sensitive than human gingival fibroblasts. More hydrophilic fractions [70M3, 70M4, 70M5] of 70% methanol extract displayed higher anti-HIV activity, radical generation and O2- scavenging activity. The antibacterial activity of 70% methanol extracts [70M0, 70M1, 70M2, 70M3, 70M4] was generally lower than that of more lipophilic fractions (hexane, acetone, methanol extracts), although each fraction did not show any specific antimicrobial action. All fractions were inactive against Helicobacter pylori. These results demonstrate that gold kiwifruit extracts contain valuable, various bioactive materials, which can be separated with each other.
Abstract: Various bioactive substances in kiwifruit extracts were fractionated by organic solvent extractions, followed by silica gel and ODS chromatographies. Both cytotoxic activity and multi-drug resistance reversal activity were found in the less polar fractions. Cytotoxic activity was not always parallel the radical intensity. Antibacterial activity was distributed into various fractions and all fractions were inactive against Candida albicans and H. pylori. Only 70% methanol extracts showed anti-human immunodeficiency virus activity, and produced a broad ESR signal under alkaline conditions, in a fashion similar to lignin. These fractions also effectively scavenged O(2)(-) produced by the xanthine-xanthine oxidase reaction, suggesting a bimodal (pro-oxidant and antioxidant) action. These data suggest a medicinal efficacy of kiwifruit peel extracts.
Abstract: Solamargine, solasonine, ginsenosides and parishin-related compounds were investigated for their effects on mdr efflux pump of lymphoma cells, and their effects on T cell proliferative assays and cell mediated immune functions, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity of human peripheral mononuclear cells. Solamargine and solasonine were the only drugs which inhibited all of the tested immune functions; however, ginsenoside Rc and Rd enhanced T cell proliferative assays and marginally increased the NK cell activity. The majority of the compounds were not able to reverse the multidrug resistance of mouse lymphoma cells. However, ginsenosides Rc, Rd and parishin C were able to moderately reduce the activity of the efflux pump. Parishin, parishin C and crude extract significantly enhanced the ADCC reaction.
Abstract: Mycoviruses with double-stranded RNA (dsRNA) genomes are frequently encountered in Aspergillus isolates. A detailed study of such dsRNA elements in black Aspergillus isolates collected worldwide was carried out, and the data were analysed. The results indicate that about 10% of black Aspergilli are infected. However, the geographic distribution of infected isolates exhibits large variations; 3-13% of the isolates collected from different continents were found to carry dsRNA elements. Hybridization experiments indicated that electrophoretic banding patterns are not reliable tools for estimating the diversity of these mycovirus genomes. Among strains representing other Aspergillus sections, dsRNA segments indicative of mycovirus infection were observed for the first time in 4 species (A. leporis, A. petrakii, A. fumigatus and A. primulinus). The latter species is able to reproduce sexually. This is the second report on the detection of naturally-occurring dsRNAs in sexually reproducing Aspergillus species. The presence of virus-like particles in these and other Aspergilli was also examined by electron microscopy. Most infected Aspergillus isolates examined were found to carry virus-like particles in the size range 36-40 nm.
Abstract: Fifteen 3,5-dibenzoyl-1,4-dihydropyridines (BzDHP, GB1-GB15) (nifedipine (NP) analogs) were tested on three different E. coli strains. The compounds had relatively high MIC values on these strains. In combination with erythromycin (Er), compounds (G1,3,4,6,7,10,12) reduced MIC values of Er. When the BzDHPs were tested on E. coli Gy-1/Apsen.Erres strain isolated from a clinical specimen, the reduction of MIC values were similar to the previous strains, but not identical. In the polyresistant clinically isolated E. coli Gy-2/Apres.Erres strain, the MIC values of Er were slightly reduced in the presence of GB1-GB7. Compound GB12 was the most effective in enhancing the activity of Er, and was selected for plasmid elimination studies. However, GB12 itself had no antiplasmid effect and did not alter the promethazine induced plasmid elimination.
Abstract: Ten polycyclic derivatives related to ellipticine have been synthesised and tested for their intercalating, reverse transcriptase (RT) inhibitory and multidrug resistance efflux pump inhibitory properties. The intercalating activity and the RT inhibitory activity of the derivatives suggest that ellipticine analogues bind at an allosteric binding site on RT or that this inhibition could be controlled at the DNA level. The MDR efflux pump inhibitory activities of these derivatives, however, appears to be unrelated to the DNA binding ability.
Abstract: The antimicrobial effects of 30 trifluoromethyl ketones [1-30] were studied on various representative bacteria. Of the ketones, 4,4,4-trifluoro-1-phenyl-1,3-butanedione [10], 1,1,1-trifluoro-3-(4,5-dimethyloxazol-2-yl)-2-propanone [11] and 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone [18] were found to exhibit potent antibacterial activity against the Gram-positive Bacillus megaterium and Corynebacterium michiganese, but not against Gram-negative bacteria such as Pseudomonas aeruginosa and Serratia marcescens. Compounds 11 and 18 inhibited the Escherichia coli. Compound 18 was also effective against yeasts. The combination of promethazine with 18 was significantly synergistic against E. coli strains, especially the proton pump deficient mutant. The results suggest that membrane transporters are the target of trifluoromethyl ketones. The inhibition was more marked in the proton pump deficient E. coli mutant than in the wild type, which suggested that the antibacterial effect of trifluoromethyl ketones is partly prevented by the proton pump system.
Abstract: The combined antiviral effects of some benzo[a]phenothiazines and 9-[2-hydroxy(ethoxy)methyl]guanine (acycloguanosine, acyclovir, ACV) on the multiplication of herpes simplex virus type 2 (HSV-2) were studied using Vero cells. The antiviral effect of ACV on a wild strain of HSV-2 was enhanced in the presence of 5-oxo-5H-benzo[a]phenothiazine and 6-methyl-5-oxo-5H-benzo[a]phenothiazine in a yield reduction test. A mathematical formula was used to interpret the drug interaction and a synergistic effect was found with a combination of ACV and benzo[a]phenothiazines. The effect of simultaneous application of two benzo[a]phenothiazines on the multiplication of HSV-2 strain during serial passages was also investigated. The combinations of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine with ACV at a low concentration using serial passages of a plaque-purified ACV sensitive HSV-2 strain, reduced the infective virus population. A similar effect was also found on the activity of other benzo[a]phenothiazine derivatives. When the two most effective derivatives of 5-oxo-5H-benzo[a]phenothiazine or 6-methyl-5-oxo-5H-benzo[a]phenothiazine were simultaneously used with ACV against a wild type HSV-2 strain during consecutive passages, the infective virus titres were decreased, but their effect was only moderate. These results suggest that a combination of some benzo[a]phenothiazines with ACV might enhance their antiviral activity probably by reduction of the mutagenic rate in the virus populations.
Abstract: Immunosuppressive and carcinogenic Fusarium mycotoxins may appear in domestic food products. Therefore, the immunological effects of Fusarium mycotoxins were tested on human peripheral blood mononuclear cells from different blood donors. In the present study we investigated deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, zearalenone, alpha-zearalenol, beta-zearalenol and nivalenol for their effects on T and B cells in a proliferation assay, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity on human peripheral blood mononuclear cells. The concentrations applied in our experiments were similar to those which can be found in normal human peripheral blood system (0.2--1800 ng/ml). Among the eight mycotoxins tested, T-2 toxin, fusarenon X, nivalenol and deoxynivalenol exerted the highest immunosuppressing effect on human peripheral blood mononuclear cells in vitro. Mycotoxin-induced immunosupression was manifested as depressed T or B lymphocyte activity. Furthermore, by virtue of inhibition of NK cell activity, the protection against tumor development may also be attenuated.
Abstract: Russian green sweet pepper (Anastasia Green) was successively extracted with hexane, acetone, methanol and 70% methanol and the extracts were further separated into a total of twenty fractions by silica gel or ODS column chromatographies. The biological activities of these extracts and fractions were compared. The extracts and fractions showed higher cytotoxic activity against two human oral tumor cell lines than against normal human gingival fibroblasts, suggesting their tumor-specific action. Several fractions [H3, H4, A4] reversed the multidrug resistant gene (MDR1) against L5178 mouse T-cell lymphoma more effectively than (+/-) verapamil (positive control). All extracts and fractions showed no anti-human immunodeficiency virus (HIV) nor anti-Helicobacter pylori activity. These data suggest the medicinal importance of an Anastasia Green extract.
Abstract: A method to fluorometrically monitor efflux of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was developed. Genistein, daidzein, sophoraisoflavone A, and licoisoflavone A induced 50% inhibition (IC(50)) of BCPCF efflux at 15-70 microM. The IC(50) value of the most efficient isoflavone, licoisoflavone A (15-25 microM), was comparable to that of indomethacin (approximately 10 microM) and markedly lower than for probenecid (100-200 microM), both known MRP1 inhibitors. Our results indicate that the human erythrocyte is a useful cell model in screening potential MRP inhibitors, that BCPCF is a good substrate for MRP, and that some isoflavones at low concentrations inhibit MRP-mediated efflux.
Abstract: Allium victorialis L. (Liliaceae, "Hon-Gyoujya Nin-Niku" in Japanese) was successively extracted with hexane, acetone, methanol and 70% methanol and the extracts were further separated into a total of twenty-five fractions by silica gel and ODS column chromatographies. The biological activities of these four extracts and 25 column fractions were compared. The cytotoxic activity of all extracts and fractions against two oral tumor cell lines was significantly higher than that against normal human gingival fibroblasts, suggesting their tumor-specific action. Three methanol column fractions [M2, M3, M6] and a 70% methanol column fraction [70M6] most effectively reversed the multidrug resistance (MDR) against L5178 mouse T cell lymphoma. The electron spin resonance (ESR) spectroscopy showed that methanol column fractions and 70% methanol extracts produced the highest amount of radical(s) and most efficiently scavenging O2*-, generated by the hypoxanthine-xanthine reaction system, suggesting that the same substances in these fractions display both prooxidant and antioxidant properties. They showed no anti-human immunodeficiency virus (HIV) or anti-Helicobacterpylori activity. These data suggest the medicinal efficacy of Allium victorialis extract.
Abstract: The efflux pump of multidrug resistant mdr cells have different sensitivities to some stereoisomeric forms of CNS-active compounds. The ABC transporters of mdr cells were more sensitive to (-)butaclamol than to its stereoisomeric counterpart (8), which may function to alter the membrane structure. We suppose that the drug-accessible membrane structure possesses an important role in the induction (or prevention) of apoptosis. Therefore the apoptosis-inducing effect of three stereoisomeric pairs was studied on mouse lymphoma cells. In these experiments levo- and dextromepromazine had similiar effects. The cis- and trans-clopenthixol were less effective in apoptosis induction than the 12H-benzo(a)-phenothiazine used as a positive control. The effect of stereoisomeric pairs on induced apoptosis was studied when the cells were exposed to the stereoisomers for 60 minutes before subjection apoptosis induction by benzo(a)phenothiazine, a well-known apoptosis inducer. Then the pretreated cells were exposed to 12H-benzo(a)-phenothiazine for 60 minutes. The samples were washed and incubated for 24 hours. The cells were stained with annexin-V-FITC and propidium iodine and investigated by flow cytometry. The mdr cells with increased membrane integrity may result in the preferential killing of multidrug resistant cancer cells in the presence of some stereoisomers.
Abstract: Fractionated extracts of Feijoa peels were studied for cytotoxic activity, anti-human immunodeficiency virus (HIV) activity and antibacterial activity. Two most cytotoxic fractions A3 of acetone extract and M2 of methanol extract had potent inhibitory activity against Gram-positive and Gram-negative bacteria as well as fungi tested. Fraction A4 of acetone extract showed multidrug resistance (MDR)-reversal activity comparable with that of verapamil (positive control). These results indicate the therapeutic value of Feijoa peel extracts as potential antimicrobial and MDR-modulating agents.
Abstract: The effects of newly synthesized, reverse transcriptase inhibitors, 3-benzazepines, for their effects on natural killer (NK) cell and blast transformation in human peripheral blood mononuclear cells were investigated. The most effective reverse transcriptase inhibitors were KF1, KF2 and KF3, which primarily suppressed immunological functions. Besides the inhibition of T cell proliferation, the benzazepines also show inhibitory effect on NK cell functions, in particularly, against large granular lymphocytes and monocytes. The B lymphocytes and Fc mediated killer functions were less inhibited.
Abstract: Membrane functions in tumorous cells are different from those in healthy cells. The aim of the present study was to investigate the changes in pituitary cell membrane functions and hormone secretion after tumor induction in vivo and in vitro. Prolactinomas were induced in vivo in female Wistar rats with estrone acetate. Normal anterior pituitaries and prolactinomas of female Wistar rats were dissociated enzymatically and mechanically, then cultured on collagen-treated plastic dishes. Some normal anterior pituitary cultures were treated with benz(c)acridines as tumorigenic agents in vitro. Intracellular 3',5'-cyclic-adenosine monophosphate (cAMP) levels were determined by a competitive binding technique, membrane fluidity was assayed by fluorescence anisotropy, and ATP-ase activities were estimated via ATP loss. The results indicated decreased membrane fluidity in tumorous cell cultures. However, in vitro benz(c)acridine treatment exerted more pronounced effects than those observed after in vivo estrone treatment. The ATP-ase activities were highly increased in benz(c)acridine-treated cells and in estrogen-induced prolactinoma cells, more strongly so in the former ones. The intracellular cAMP levels were higher than normal in both of them. The results concerning the ACTH, alpha-MSH, PRL and GH levels of normal and tumorous cell cultures were published in our previous study. Our findings show that the tumorous transformation of pituitary cells can cause significant changes in functional membrane parameters and hormone secretion. Decreased membrane fluidity was accompanied by an increased exocytosis (hormone release) and adenylate cyclase activity in tumorous cells.
Abstract: The individual activity of antibiotics such as ampicillin, tetracycline, erythromycin and gentamicin in combination with compounds known to modify bacterial resistance to given antibiotics was studied using the checkerboard method. The combination of promethazine with either ampicillin, tetracycline or erythromycin or the combination of methylene blue and erythromycin produced significant synergistic activity against Escherichia coli. Verapamil, however, in combination with ampicillin reduced the activity of ampicillin against E. coli. Combinations of clomipramine with either tetracycline or erythromycin, promethazine and erythromycin or verapamil and ampicillin were synergistic against Staphylococcus epidermidis that was resistant to these antibiotics. The only synergy against Pseudomonas aeruginosa was shown by the combination of methylene blue and gentamicin.
Abstract: Previous studies have demonstrated the relationship between radical intensity and cytotoxic activity in water-soluble compounds. This relationship was investigated in lipophilic compounds. Several N-acylphenothiazines showed higher cytotoxic activity against human leukemic and squamous carcinoma cell lines than phenothiazine, the parent compound. Electron spin resonance (ESR) spectroscopy showed that these active compounds produced much lower amounts of radicals than phenothiazine. Several compounds failed to inhibit the cytopathic effects of human immunodeficiency virus (HIV) infection in MT-4 cells. It suggested that the radical-mediated-mechanisms has not involved in the induction of cytotoxic activity by lipophilic compounds, such as N-acylphenothiazines.
Abstract: The antiplasmid effects of promethazine on E. coli is the consequence of special complex formed with a covalently closed circular (ccc) form of plasmid DNA. The exact target in this macromolecule, however, was not clarified until recently. Caffeine and the chemically similar guanosine-5'- monophosphate (GMP) could compete with the antiplasmid effect of promethazine, showing that promethazine or other phenothiazines preferentially bind to xanthine type molecules. Among the xanthines, GMP was more effective at complex-forming than adenosine-5'-monophosphate (AMP). In addition, the Z-DNA was more susceptible than B-DNA. Therefore, one could suppose that guanine-cytosine (G-C) rich regions have higher affinity than adenine-thymine (A-T) rich region on phenothiazines. Because the G-C rich regions have a special role in the DNA stability via three hydrogen bonds, we suppose that these regions could have a key role in some biological effects such as antiplasmid and anticancer activity.
Abstract: Drug resistance to chemotherapy is rapidly emerging. Resistance to one drug carries over resistance to unrelated anticancer drugs leading to multidrug resistance (MDR). A major factor of MDR is P-glycoprotein (P-gp) mediated ABC transport found in many eukaryotic cells. P-gp acts as a drug eMux pump. The mdr1 gene involved in P-gp 170 protein production is localized in the human chromosome 7 band p2 1.0-21.1. Point mutations after cross-resistance patterns. A variety of stimuli increase the expression of the mdr1 gene: lowered extracellular pH, heat shock, arsenite, cytotoxic agents, anticancer drugs, transfection with oncogenes, HIV-I, and UV-irradiation. An alternative hypothesis to the efflux pump claims that P-gp modifies the intracellular environment to reduce accumulation of anticancer drugs in cancer cells by creating ionic or proton gradients. Chemosensitizers that block P-gp drug extrusion are generally lipid-soluble at physiological pH, possess a basic nitrogen atom and at least two co-planar rings. P-gp blocking does not depend on drug chirality. This opens the way of treating P-gp related MDR with chiral versions of drugs relatively harmless in terms of side-effects. We believe that resistance modifiers combined with cytostatics will chemotherapeutically be more effective for cancer patients.
Abstract: Eleven 4-phenyl-3,5-diacetyl-1,4-dihydropyridines (AcDHPs) [G1-11] substituted at the phenyl ring were synthesized and compared for their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems. Among them, compound [G7] showed the highest cytotoxic activity against human promyelocytic leukemia HL-60 and human squamous cell carcinoma HSC-2 cells. However, no compounds tested produced radicals at pH 7.4-12.5. The activity of P-glycoprotein (Pgp) responsible for MDR in tumor cells was reduced by compounds [G2, 3, 6, 5, 8, 1, 11], verapamil [VP] and nifedipine [NP]. However, compounds [G4, 7, 10] were hardly active while G9 did not show a MDR reversing effect at 2.0-20.0 micrograms/mL. These data show a relationship between chemical structures and MDR-reversing effect on tumor cells.
Abstract: In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions. Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells. Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL. Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells. The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells. Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression. However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL. The crocin had no antiviral effect [on HSV-2 infected vero cells] up to 25 micrograms/mL concentration. Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump. Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells. It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities.
Abstract: Organic pollution of water and soil has various harmful effects on biological systems (1). Chlorine substituted benzol compounds are one these xenobiotic substances, which are toxic to the environment (2). They can also accumulate in plant and animal tissues (3), which provides ample reason to study the effects of sublethal doses of chloro-benzols on various cell cultures. In this study the toxic effects of chloro-benzols were investigated on avian fibroblast and mammalian hepatocyte cultures. The fibroblast cultures were prepared from eggs preadapted to chloro-benzol during a fourteen-day-long incubation period. The Wistar rat hepatocyte monolayer cultures were exposed to a direct treatment of 1,2,4-tri-chloro-benzol (0.01 microgram/ml-1 microgram/ml) for 3 hours. Following the treatment with chloro-benzol, the viability of the cells was measured, together with lactic dehydrogenase activity, in both kinds of cultures. The effect of tri-chloro-benzol treatment on chicken eggs was not significant. The cells of chicken embryos were not damaged after the 1,2,4-tri-chloro-benzol treatment. The hepatocyte cultures showed the toxic effects of 1,2,4-tri-chloro-benzol after the direct and acute treatment. The cell viability decreased and the LDH activity increased significantly. These results show that the primary cell cultures are suitable for studying the effects of organochlorine compounds.
Abstract: The mutagenicity and carcinogenic properties of trichloroethylene (TCE) derivatives, and their correlation with its molecular properties were analyzed. The observed cancer incidence was compared to the predicted, calculated incidence. The predictions were based on the rodent bioassay results and were consistent with human data. The electrophilic data of molecules of the Ke system provided evidence for 205 rodent carcinogens, where Ke correlated with energy of the lowest unoccupied molecular orbital. The majority of carcinogenic compounds were found to be electron acceptors with decreased lowest unoccupied molecular orbital (LUMO) energy, indicating the particular DNA-reactivity leading to mutations and abnormal cell division. Based on the mutagenic activity in Ames test, the affinity of target organs for mutagens and non mutagens were compared in 351 rodent carcinogens. Nearly 80% of carcinogens (mutagenic and non mutagenic ones) were positive in the mouse and rat, in at least one of the most frequent target organs, i.e. liver, lung, mammary gland, stomach, kidney, hematopoietic system, urinary bladder and vascular system. Several predictive methods have been developed over the last 5 years based on structure-activity relationship studies known as US National Toxicology Program. One of these programs, called "PROGOL" is widely used for the prediction of carcinogenesis for a wide variety of compounds, e.g., nitro aromatics and suramin analogs. This program provides a simple model for predictive carcinogenesis, despite of the fact that the very first steps of carcinogenesis are not fully understood yet.
Abstract: Among twelve phenothiazine-related compounds, the cytotoxic activity of six "half-mustard type" phenothiazines [7-12] was significantly higher than that of six phthalimido compounds [1-6]. 1-(2-Chloroethyl)-3-(2-chloro-10H-phenothiazin-10-yl)propylurea [9], 1-(2-chloroethyl)-3-(2-chloro-10H-phenothiazin-10-yl)butylurea+ ++ [10] and 1-(2-chloroethyl)-3-(2-trifluoromethyl-10H-phenothiazin-10-yl)b utylurea [12] showed the highest cytotoxic activity, in parallel with high delta mu (difference between two dipole moments, mu g and mu e). There was also positive relationship between cytotoxic activity and molecular orbital energy such as pi-LUMO, pi-HOMO, and lone pair orbitals originated from O, N1, and N3 atoms. The present study demonstrated that cytotoxic activity of "half-mustard type" phenothiazines can be predicted by their dipole moments and molecular orbital energies.
Abstract: Among 54 cancer cell lines, colon-cancer cells were the most sensitive to six "half-mustard type" phenothiazines [7-12], followed by leukemia, melanoma, prostate-, CNS-, breast-, lung-, renal and ovarian cancer cells. The distribution of electrostatic potential (ESP) of "half-mustard type" phenothiazines [7-12] suggests that the ESP surface of urea site is important for the interaction between "half-mustard type" phenothiazines [7-12] and target cancer cell structures (or DNA base sequence). Actually, the urea site of "half-mustard type" phenothiazines displayed extensive variability in the energy of lone pair orbital and net atomic charges of N1, 0 and N3 atoms.
Abstract: This paper present a short overview on the new perspectives in drug-design against drug resistance phenomena focusing on the infectious antibiotic resistance of bacteria. The role of plasmids in resistance of bacteria, the mode of transmissions were analyzed in details. The elimination of plasmids--the genetic basis of bacterial resistance located extrachromosomally--was studied systematically with several heterocyclic compounds. Correlations were analyzed between the anti-plasmids++ effects and chemical structure of tricyclic compounds in quantitative structure activity relationship (QSAR) studies. A hypothesis was presented to exploit the analogous mechanisms of multidrug resistance in bacterial and cancer cells by inhibiting the action of drug exporter proteins in the presence of compounds synthetized on the basis of computer aided drug design.
Abstract: The antiproliferative activity of six "half-mustard type" phenothiazines against a total of 54 tumor cell lines: 4 leukemia, 9 non-small-cell lung, 7 colon-, 5 CNS-, 8 melanoma, 6 ovarian-, 8 renal-, 1 prostate and 6 breast cancer was determined by NCI-Information Intensive-Approach. The C-2 position of phenothiazines were substituted with H, Cl and CF3 groups. The half-mustard and ring system was linked either by a propylene or a butylene bridge. Colon-cancer cell showed the highest sensitivity against "half-mustard type" phenothiazines, followed by leukemia, melanoma, prostate-, CNS-, breast-, lung-, renal and ovarian cancer cells. These data suggest the "cancer-type-specific" antitumor action of "half-mustard type" phenothiazines.
Abstract: For studying the mechanisms of biological activity on 3-benzazepines, antimicrobial effect, F'lac plasmid elimination activity (a plasmid curing effect on F'lac plasmid) and antibody-dependent cellular cytotoxicity (ADCC) test were performed. A weak antiplasmid effect was found at sub-inhibitory concentrations. A combination of [KF4] with verapamil [2] did not alter the ineffectivity, however, [KF4] could inhibit the antiplasmid effect of promethazine, as compared to the control (promethazine alone) plasmid curing effect. A competition between promethazine and [KF4] might exist in plasmid elimination effect. ADCC activity of human leukocytes was enhanced by KF1, KF2, KF3, DA and NE at 1.0 microgram/mL concentrations. The majority of 3-benzazepines [KS02, KM57, KN50, KE04, KI10, KP80] was ineffective for plasmid curing, however, inhibited the ADCC reaction, but they did not show a real dose-dependent effect.
Abstract: We have previously reported on the diverse biological activities of benzo[a]phenothiazines, such as the induction of antitumor and antimutagenic activity in vivo, and differentiation and apoptosis in vitro. The relationship of radical generation and pi-spin density or dipole moment was investigated, using quantum-chemical calculation with UHF/PM3. These data suggest that the origin of radical generation by active benzo[a]phenothiazines, which affect such biological activities might be N-atom at position 12.
Abstract: The cancer inducing effect of trichloroethylene (TCE) was studied by various methods. DNA complexing activity and apoptosis inhibition were found to be the key elements of the carcinogenicity of TCE and its metabolites. The ability of TCE to interact with DNA was low, but its incorporation into the RNA and DNA of the brain, testis, pancreas, kidney, liver, lung and spleen, cannot be excluded. Exposure to TCE and its metabolites provides a selective growth advantage to spontaneously occurring mutations in some K- and H-ras oncogenes (as non specific results of secondary DNA or RNA damage). The amount of DNA-TCE adducts was higher in mouse hepatocytes than in rat hepatocytes. These differences may explain the species difference in carcinogenicity of TCE, which was dose dependent (due to metabolism) in mice but independent in rats. The blood level kinetics of TCE confirmed the faster metabolic rate in mice, including peroxisome proliferation and induction in hepatocytes. Dichloroacetic- and trichloroacetic acid were found to be hepatic carcinogens in mice, and the specificity depends on peroxisome proliferation induction. Possibly, TCE and related compounds down regulated apoptosis in mouse liver, and the reduced ability to remove initiated cells by apoptosis could be responsible for liver cancer induction by TCE.
Abstract: delta 9-Tetrahydrocannabinol, cannabidiol, cannabidiolic acid, tetrahydrocannabidiolic acid, cannabispirol, acetylcannabispirol, cannabispirone, and cannabispirenone in a low concentration did not affect the adhesion of Escherichia coli on cultured HEp-2 cells. Cannabinoids at 10(-6) M increased the chemiluminescence of human polymorphonuclear leukocytes, while the cannabispiro compounds failed to enhance the oxidative burst of leukocytes. In lymphocyte and granulocyte function tests (E- and EA-rosette formation, blast transformation of T-lymphocytes in the presence of phytohaemagglutinin and concanavalin-A, ADCC and phagocytosis) all compounds displayed immunosuppressive effect at 1.5 X 10(-5) M. Tetrahydrocannabidiolic acid exerted the weakest immunosuppression on human leukocyte functions.
Abstract: Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.
Abstract: A 12-year-old girl was admitted to the hospital for evaluation of an abdominal mass. A preoperative computed tomography scan showed a large tumor in the pelvis. The serum alpha-fetoprotein level was 2,170,000 ng/ml. A 3000-g left ovarian neoplasm was resected. It was encapsulated and showed focal microcystic degeneration, necrosis, and hemorrhage. Microscopically, it was composed of gland-like spaces containing mucin-positive material and surrounded by scant fibrovascular tissue. The epithelial cells were low columnar with immature oval, basophilic nuclei. Immunoperoxidase staining for alpha-fetoprotein and alpha1-antitrypsin were positive. Enzyme histochemistry was negative for alkaline phosphatase and positive for alpha-naphthyl acetate esterase. Electron microscopy, including freeze-fracture analysis, showed desmosomes and tight junctions. No gap junctions were identified. Actin filaments, glycogen, and microvilli were abundant. This is the first case of an ovarian endodermal sinus tumor with exclusive enteric differentiation.
Abstract: Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.
Abstract: Total body electrical conductivity (TOBEC) provides a rapid and safe noninvasive technique for the assessment of total body water in animals and man. An instrument employing this principle has been shown to measure body water in healthy Sprague-Dawley rats. With the exception of adult obesity in humans, alterations in body fluid compartments that could theoretically affect the utility of conductivity measurements have not been studied. We, therefore, applied the total body electrical conductivity measurement in rats following perturbations of body fluid/electrolyte spaces including obesity, furosemide diuresis, severe burn, and low protein diet. Our findings confirm that total body water can be accurately measured by TOBEC in conditions of abnormal body fluid distribution. However, when the ratio of intracellular to extracellular fluid is significantly reduced, such as the severe burn or low protein intake, TOBEC does not reflect the intracellular (potassium) space but does predict total water and extracellular (sodium) space.
Abstract: Recent research on nutrition needs following thermal injury suggests that carbohydrate and caloric excess must be avoided while an attempt is made to meet relatively large protein requirements. The nutrition regimen in this investigation for adult and pediatric burned patients provides calories at twice the predicted basal metabolic rate, protein at 2.5 gm/kg/day, and carbohydrate at 5 mg/kg/minute. Lipids are provided to meet the caloric deficit after protein and carbohydrate administration. Even though such nutrients are readily provided parenterally, the high carbohydrate content of standard tube feedings makes it difficult to provide adequate protein without excess of carbohydrate and calories. As a result, a modular tube feeding, containing appropriate macro- and micronutrients and tailored to the individual patient, has been used in our burn units. The adequacy of the diet was assessed by the effect on nitrogen balance, weight change, selected serum indexes of visceral metabolism, and compliance with planned dietary goals. Patients remained in positive nitrogen balance more than 80% of the time while the modular diet was used in conjunction with other modes of therapy (parenteral and/or oral). More than 90% of the time, positive balance was achieved when the modular diet was used as the sole source of nutrition. Compliance with the caloric goal and physicians' diet orders for the modular diet exceeded, on average, the 80% level, which has previously been the established lower limit. The results in this study suggest that the modular diet is an appropriate method of nutrition support.
Abstract: A closed-circuit metabolic system has been designed and tested for multiple applications. Air pressure within a closed chamber is regulated electronically while allowing for respiratory gas exchange. Compared with a previously reported standard indirect calorimetry system, the new device had by virtue of longer duration of measurement improved precision (coefficient of variation 3% vs. 14%) during studies of O2 consumption both at room temperature and at 5 degrees C. In addition, a more physiological atmospheric environment is maintained. This system has also been utilized for simultaneously labeling groups of up to 20 weanling rats with 18O2 over a 2-day period and for exposure of rats to a hyperoxic (84% O2), normobaric environment for 4-day periods. Potential applications include maintenance of pressure (hypobaric through hyperbaric) and O2 (hypoxic through hyperoxic) controlled environments, exposure to toxic gases, study of diurnal variations in metabolic rate, measurement of metabolic expenditure with activity, and adaptation to other species including humans.
Abstract: Weight change, defined as the difference between the preadmission and discharge weights, has often been used as an indicator of the adequacy of nutrition support. Weight change has been found to be uninterpretable in burned patients because excision and grafting contribute to weight loss but do not reflect systemic physiology. Also, normal growth during hospitalization contributes to weight gain but is not included in traditional methods of assessing weight change of children. We examined weight change corrected for escharectomies and growth in 42 adult and pediatric burned patients. Thirty-five of the patients had weight loss within 10% of their premorbid weight. Moreover, weight loss was correlated with three observable patient characteristics: age, percent initial burn size, and preburn weight. It was concluded that the dietary regimen of providing twice the predicted basal metabolic rate in terms of calories, based on ideal body weight, was adequate to prevent significant weight loss in most patients but that further refinement is necessary to improve individual reliability. Furthermore, significant weight losses may not affect morbidity/mortality as previously thought.
Abstract: Gonadotropin releasing hormone (GnRH) content of the two halves of the median eminence of the rat hypothalamus was determined by radioimmunoassay three weeks after three different unilateral knife cuts around the preoptic area. A unilateral cut in front or above the area caused a more than 25% decrease in the GnRH content of the two halves of the median eminence. A cut lateral to the preoptic region had only a slight effect similar to that observed after sham operations. The data suggest that probably more than 50% of the rat median eminence GnRH derives from outside the preoptic-suprachiasmatic region. The GnRH fibres projecting to the median eminence but arising from outside the preoptic region, probably mainly from GnRH perikarya in the limbs of the diagonal band of Broca and septum, enter this area partly from rostral and partly from above, but not from lateral direction. partly from rostral and partly from above, but not from lateral direction. Several of these fibres probably cross before terminating in the median eminence.
Abstract: Some cannabispiro compounds and tetrahydrocannabidiolic acid were tested for antibacterial plasmid curing activity and inhibition of plasmid transfer. MIC values of the compound were above 1500 micrograms/ml. Cannabispirol and tetrahydrocannabidiolic acid eliminated the F'lac plasmid from Escherichia coli, but acetylcannabispirol, cannabispirone and cannabispirenone were ineffective as curing agents. Each compound, except acetyl-cannabispirol, selectively killed plasmid carrying bacteria. The compounds inhibited R144 plasmid transfer from E. coli into E. coli cells via inhibition of mating pair formation, zygotic killing and inhibition of transconjugal DNA synthesis in a lesser extent. All of the cannabispiro compounds and tetrahydrocannabidiolic acid inhibited the transformation with pBR322 plasmid DNA when the bacteria were pretreated with the compounds, via inhibition of the DNA penetration or decreasing the synthesis of plasmid DNA during bacterial growth. Although each of the compounds, except acetyl-cannabispirol, had a weak antibacterial effect which was more definite on plasmid carrying bacteria than plasmidless ones, and inhibited intercellular plasmid transfer and transforming activity of plasmid DNA, only two of them were able to cure F'lac plasmid showing that plasmid elimination is a complex process which strictly depends on the stereochemical configuration of curing agents.
Abstract: In 364 patients with myasthenia gravis the morphology of thymus glands, thymomas and muscle samples was studied and compared to different clinical, therapeutic and prognostic parameters of the disease. The role of active thymus as the most outstanding finding, was considered for the result of thymectomy and for the prognosis to be positive: The more active the thymus the better the outcome of thymectomy and the final and lasting prognosis of the disease.
Abstract: Group motility was recorded continuously in male rats during the inhalation of benzene, toluene, ethylbenzene, o-, m- and p-xylene vapours. The solvents were applied in at least six concentrations, up to those inducing anaesthesia. Minimum narcotic concentrations (ppm) were: 5940 (benzene), 3590 (toluene), 2180 (ethyl-benzene), 2180 (0-xylene), 2100 (m-xylene), and 1940 (p-xylene). The results indicate that prenarcotic concentrations of these structurally related aromatic hydrocarbons and also the xylene isomers elicit qualitatively and quantitatively different acute behavioral effects. Except o-xylene which caused depression only the agents produced bell-shaped concentration-action curves characteristic of the biphasic effect, i.e., activation at lower and depression at higher concentrations. The curves differed in form and magnitude depending on the stimulatory potency and on the range of effective concentrations. Based on arbitrary assessment of central excitation, the five aromatics may be ranked as follows: benzene and toluene (striking activation), p-xylene (marked activation), ethylbenzene (moderate activation), m-xylene (slight activation). At the same time, high degree of motor incoordination, and in the case of benzene and p-xylene, also marked tremor could be seen.
Abstract: Various supra- and retrochiasmatic cuts injuring different parts of the septo-preoptico-infundibular GnRH pathway were made in adult female rats, and their effects on ovulation, median eminence (ME) GnRH content and on plasma LH and FSH levels were studied. Extended retrochiasmatic frontal cut just behind the optic chiasm, or a frontal cut in front of the suprachiasmatic nucleus presumably interrupting the whole GnRH pathway blocked ovulation, led to persistent estrus with polyfollicular ovaries, and reduced the ME GnRH content to 10 and 32%, respectively, expressed in percentage of unoperated control value. Severance of the GnRH pathway on one side or partial interruption of the pathway on the two sides in the retrochiasmatic area did not interfere with ovulation, and the ME GnRH content was 50% or more of the control value. Disconnection of the GnRH fibers coming from the septum resulted in a more than 30% decrease in the GnRH content of the ME, but did not block ovulation. Two separate symmetrical lateral cuts in the suprachiasmatic area leaving intact the presently known GnRH pathway reduced the ME GnRH content to 40% of the intact value, but did not interfere with ovulation. Plasma LH and FSH levels of the animals with different knife cuts were considerably dissociated and there were no apparent correlations between brain interventions and the concentrations of these two hormones in the blood. Our previous findings together with the present data suggest that: half of the GnRH pathway, medial or lateral bundles of the pathway on the two sides are sufficient for ovulation and cyclic gonadotrophic function; persistent estrus develops if the ME GnRH content is below 40% of the control value, and about 60% of the ME GnRH originates outside the preoptic, supra- and retrochiasmatic region. Half of this 60% may come from the septum and the vertical part of the diagonal band of Broca, the other half from the region in front of the preoptic area. The remaining 40% presumably arises from the preoptic (7-10%), supra- (15-20%) and retrochiasmatic region (8-10%).
Abstract: In the course of thymectomy of patients with myasthenia gravis, surprising data concerning the recovery or considerable improvement of other immune disorders have been observed. Among these disorders rheumatoid arthritis figured in six instances, immune thrombocytopenia in two cases, polymyositis and psoriasis in one case each. Thymectomy as a powerful immunosuppressive procedure may have a role in the therapeutic management of some immune disorders other than myasthenia gravis.
Abstract: The purpose of this study was to obtain a quantitative assessment of the behavioral retardation caused by prenatal ethanol exposure in mice and to test the hypothesis that gamma-linolenic acid (GLA) supplementation would prevent such effects. Pregnant B6D2F1 mice were fed liquid diets containing 25% ethanol-derived calories from days 7-17 of gestation. The experimental groups were given GLA via subcutaneous injection; the control was administered vehicle only. All groups were pair fed to this ethanol control, including a second control group which received sucrose substituted isocalorically for ethanol. Additional control groups included one fed lab chow ad libitum and two further ethanol groups, one treated with coconut oil, the other with arachidonic acid (AA). Behavioral development of the pups was measured on day 32 postconception and open field behavior was measured on day 50. Body and brain weight were also measured. The results indicated that reproductive outcome, as measured by animals which produced live pups, was worse in the GLA- and AA-treated groups. Ethanol produced significant behavioral retardation of the order of 1.7 days. Body and brain weight were lower in ethanol-treated pups. Covariance analysis indicated that the effect on brain weight was independent of the effect on body weight. Open field scores suggested that ethanol-treated males were more active then sucrose controls. The data did not support the hypothesis that GLA would prevent the deleterious effects of prenatal ethanol exposure; in no instance was a GLA-treated group different from the ethanol control.
Abstract: Although many of the current nutrition assessment techniques are inappropriate for burned patients, we have found nitrogen balance to be a helpful measure of the effectiveness of nutrition therapy if it is used in conjunction with other measures of nutritional status. Many clinicians suggest that a 24-hour urinary urea nitrogen (UUN) measurement, in addition to a correction term of 2 to 4 gm nitrogen, will accurately represent all nitrogen losses (urinary non-urea nitrogen, fecal, integumental). However, since no burned patients were used in the development of that method, its use may not be appropriate for burned patients. As an alternative to total urinary nitrogen (TUN), which is costly and time consuming, we have developed a simple way to predict TUN from UUN, percent initial total body surface area burn, and age for pediatric and adult burned patients. We propose that nitrogen balance may then be determined from the predicted TUN by an equation showing that nitrogen balance equals daily nitrogen intake minus predicted TUN plus fecal and normal nitrogen loss.
Abstract: Dermal and respiratory exposure to pirimiphosmethyl, dimethoate and permethrin were determined for applicators and operators in greenhouse tomato spraying operations. Dermal exposure is several times higher than the degree of respiratory exposure. Dermal exposure in terms of different parts of the body shows significant differences. For applicators the exposure of hands, arms and legs is the greatest, and the operators are the most exposed on their hands and to a small extent on legs. This fact should be taken into account when providing the workers with suitable protective clothing. The carefully selected technology of spraying also has great significance in decreasing the degree of exposure. Because of the chronic toxicity of dimethoate, all possible methods should be taken to reduce exposure.
Abstract: Lactate dehydrogenase (LDH) isoenzyme pattern and the percent of H-subunit content were determined in the thymus of 62 patients (55 with hyperplasia, 7 with tumours) after thymectomy. An increase in LDH1 relative activity indicates that in the thymus of patients with myasthenia gravis the ratio of mature differentiated thymocytes was higher than in the thymus of control subjects. LDH isoenzyme profiles of thymus tumours were similar to those described in other neoplasms, except that thymomas with apparent predominance of epithelial cells and with minimal lymphocytic reaction exhibited a marked elevation only in LDH2 relative activity, presumably associated with the specific (secretory) function of epithelial cells. The elevation of H-subunit content, a parameter characteristic of both thymic components (lymphoid and epithelial), correlated closely with a poor clinical condition in patients several years after surgery.
Abstract: The effects of rutin-N-mustard, amantadine-N-mustard, chlorpromazine and human interferon types alpha, beta and gamma (IFN-alpha, -beta and -gamma) were studied on the DNA, RNA and protein synthesis of K-562 cells. Monocyte-mediated cytotoxicity and immune spleen cell activity were examined in the presence of the same compounds (except for IFN-beta). The natural killer (NK) cell activity was tested in the presence of the two chlorpromazine compounds and the two N-mustard derivatives. Only 7,8-dioxochlorpromazine exerted an inhibitory effect on DNA synthesis. The protein synthesis of the cells was inhibited in the presence of IFN-alpha, -beta and -gamma. 7,8-Dioxochlorpromazine exerted some inhibition on both NK and immune spleen cell activity, while monocyte-mediated cytolysis was not altered. IFN-alpha, -beta and -gamma activated the cytolytic activity of monocytes and the NK activity in control experiments. Chlorpromazine, rutin-N-mustard and amantadine-N-mustard were ineffective in both tests in vitro. Rutin-N-mustard, 7,8-dioxochlorpromazine and the interferons may be assumed to have quite different antiproliferative mechanisms of actions.
Abstract: Pentoxifylline decreases the cerebral edema resulting from cortical freezing lesions in cats, produces mitochondrial hypertrophy with preservation of structure in gerbils, and increases survival rate in gerbils rendered ischemic by temporary bilateral carotid occlusion. Since all these findings may be related to energy metabolism, the effect of the drug on postischemic cerebral phosphocreatine and ATP concentrations and on cytochrome oxidase activity has been studied. Brain slices prepared from animals subjected to bilateral carotid occlusion for 30 min and treated with pentoxifylline at release of occlusion, then allowed 3 h for recovery, exhibited higher concentrations of ATP and higher levels of cytochrome oxidase activity than did those of the untreated animals.
Abstract: Alkali burns of the cornea were made bilaterally in 16 albino rabbits. The eyes were then treated four times a day in a masked fashion with a solution either of fibronectin (250 micrograms/ml) plus gentamicin (1.5 mg/ml) or of gentamicin (1.5 mg/ml) plus hydroxypropyl methylcellulose (Tears Naturale). The eyes were examined daily in a masked manner for evidence of a corneal epithelial defect. On days 6-14, the fibronectin-treated eyes had more healed corneal defects (p less than 0.05, McNemar's test for matched pairs) than the control eyes. Fibronectin aided the healing of corneal alkali burns by decreasing the peeling back of the healing epithelium and by allowing re-epithelialization if peeling back had occurred. These properties of fibronectin helped to preserve a stable, intact corneal surface.
Abstract: The prominent role of plasma fibronectin (pFN) in the host defense system as an opsonin for gelatin (collagen)-coated colloids has been established. In the present study we investigated the interaction of pFN and membrane isolates from cells devoid of collagen, as well as several tissues. In a liver slice assay system it was shown that subcellular membrane fractions from lung macrophages, peritoneal exudate cells, spleen, tests, and liver were able to competitively inhibit the pFN-mediated uptake of 125I-gelatin coated latex beads (gLtx) at low concentrations. Endocytosis of 125I-labeled membrane isolates by macrophage monolayers was also promoted by addition of pFN. In an attempt to characterize the membrane component(s) interacting with pFN, it was found that mild extraction procedure with 1 M KBr could release a significant amount of this inhibitory activity. Further studies demonstrated that the agent(s) responsible for inhibition of gLtx uptake was heat sensitive, not altered by trypsin treatment, and did not contain actin, a protein known to interact with pFN. This work indicates that pFN interacts specifically with an as yet unknown membrane component(s) and that such interaction will promote clearance of cellular debris by macrophages. This suggests that pFN may be an important opsonin for the reticuloendothelial system in clearance of collagenous and noncollagenous cellular debris once they are exposed to interact with it.
