Abstract: There are mainly three types of propolis whose major anticancer ingredients are entirely different: (1) CAPE (caffeic acid phenethyl ester)-based propolis in Europe, Far East and New Zealand, (2) artepillin C (ARC)-based Brazilian green propolis and (3) Brazilian red propolis. It was shown previously that NF (neurofibromatosis)-associated tumors require the kinase PAK1 for their growth, and CAPE-based propolis extracts such as Bio 30 suppress completely the growth of NF tumors in vivo by blocking PAK1 signaling. Also it was demonstrated that ARC suppresses angiogenesis, suggesting the possibility that ARC also blocks oncogenic PAK1 signaling. Here it is shown for the first time that both ARC and green propolis extract (GPE) indeed block the PAK1 signaling selectively, without affecting another kinase known as AKT. Furthermore, it was confirmed that ARC as well as GPE suppress almost completely the growth of human NF tumor xenografts in mice, as does Bio 30. These results suggest that both CAPE-based and ARC-based propolis extracts are natural anti-PAK1 remedies and could be among the first effective NF therapeutics available on the market. Since more than 70% of human cancers such as breast and prostate cancers require the kinase PAK1 for their growth, it is quite possible that GPE could be potentially useful for the treatment of these cancers, as is Bio 30.
Abstract: Angiotensin I-converting enzyme (ACE) inhibitory and hypotensive effects of 7 peptide fractions (Frs) of royal jelly protein hydrolysate (RJPH) were studied in comparison with those of RJPH alone. Fr 4 and Fr 5 were the highest in ACE inhibitory activity and yield, respectively. Molecular weights (MWs) of RJPH and Fr 1-Fr 7 were distributed from 100 to 5,000 and those of Fr 1-Fr 7 increased in order from Fr 1 to Fr 7. RJPH, Fr 3 and Fr 4 at doses of 10, 30 and 100mg/kg i.v. and Fr 5 and Fr 6 at doses of 30 and 100mg/kg i.v. caused transiently significant hypotensive effects in spontaneously hypertensive rats (SHR). Fr 3, Fr 4, Fr 5 and Fr 6 at a dose of 1,000mg/kg also caused significant hypotensive effects 3h, 4-5h, 7-8h and 8h after oral administration in SHR, respectively. RJPH caused a long-lasting hypotensive effect in proportion to the magnitude of the MWs of RJPH fractions. The hypotensive pattern of RJPH was similar to the combined pattern of Fr 3-Fr 6. From these results, it can be concluded that the long-lasting hypotensive effect of oral administration of RJPH is dependent on the MWs of its ACE inhibitory peptides and the time required to digest them.
Abstract: We investigated whether artepillin C, a major component of Brazilian propolis, acts as a neurotrophic-like factor in rat PC12m3 cells, in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of PC12m3 cells were treated with artepillin C at a concentration of 20 microM, the frequency of neurite outgrowth induced by artepillin C was approximately 7-fold greater than that induced by NGF alone. Artepillin C induced-neurite outgrowth of PC12m3 cells was inhibited by the ERK inhibitor U0126 and by the p38 MAPK inhibitor SB203580. Although artepillin C-induced p38 MAPK activity was detected in PC12m3 cells, phosphorylation of ERK induced by artepillin C was not observed. On the other hand, artepillin C caused rapid activation of ERK and the time course of the activation was similar to that induced by NGF treatment in PC12 parental cells. However, NGF-induced neurite outgrowth was inhibited by artepillin C treatment. Interestingly, inhibition of ERK by U0126 completely prevented artepillin C-induced p38 MAPK phosphorylation of PC12m3 cells. These findings suggest that artepillin C-induced activation of p38 MAPK through the ERK signaling pathway is responsible for the neurite outgrowth of PC12m3 cells.
Abstract: The aim of this study was to evaluate the effects of bee products such as honey, royal jelly and propolis on 5-fluorouracil-induced experimental oral mucositis in hamsters. Oral mucositis was induced in hamsters through a combination of 5-fluorouracil and mild abrasions that were made on the cheek pouch. Honey, royal jelly and propolis were thereafter topically administered to the oral mucosa, and then the healing process was examined by measuring the size of the mucositis. Honey (1%, 10% and 100%) and propolis (0.3%, 1% and 3%) ointments did not reduce the size of the mucositis in comparison to the vaseline-treated control group. However, the royal jelly (3%, 10% and 30%) ointments significantly improved the recovery from 5-fluorouracil-induced damage in a dose-dependent manner. These results suggest the possibility that the topical application of royal jelly has a healing effect on severe oral mucositis induced by chemotherapy.
Abstract: BACKGROUND AND OBJECTIVE: We studied the effects of honeybee products on the in vitro formation of calcium phosphate precipitates. MATERIAL AND METHODS: Screening tests of the in vitro formation of calcium phosphate precipitates using 20 types of honey and four types of propolis were carried out using the pH drop method. RESULTS: The inhibitory effect on the rate of amorphous calcium phosphate transformation to hydroxyapatite and on the induction time varied greatly among the 20 types of honey and four types of propolis. We classified them according to their effects on decreasing the rate of amorphous calcium phosphate transformation to hydroxyapatite and/or increasing the induction time. Two of the 20 honeys showed little or no inhibition, either on the rate of amorphous calcium phosphate transformation to hydroxyapatite or on the induction time. Six of the honeys reduced the rate of amorphous calcium phosphate transformation to hydroxyapatite by 12-35% and with a 2.5- to 4.4-fold increase in the induction time. The remaining 12 honeys showed even greater activity. Because four of these 12 honeys had an inhibitory effect on the rate of amorphous calcium phosphate formation, they were excluded as candidates for anticalculus agents. Furthermore, three of the four types of propolis showed an inhibitory effect that was the same as or greater than 1-hydroxyethylidene- 1,1-bisphosphonate. CONCLUSION: These results suggest that eight honeys and three types of propolis may have potential as anticalculus agents in toothpastes and mouthwashes.
Abstract: Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17beta-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17beta-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5-2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17beta-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.
Abstract: The effect of the combination of beta-cryptoxanthin and zinc sulfate (zinc) on bone components in the femoral-diaphyseal and -metaphyseal tissues of young rats in vitro was investigated. Bone tissues were cultured for 48 h in a serum-free Dulbecco's modified Eagle's medium containing either vehicle, beta-cryptoxanthin (10(-9)-10(-7) M) or zinc sulfate (10(-6)-10(-4) M). The presence of beta-cryptoxanthin (10(-9) M) or zinc (10(-6) M) did not have a significant effect on calcium content in the femoral-diaphyseal or -metaphyseal tissues. However, culture which combined beta-cryptoxanthin (10(-9) M) and zinc (10(-6) M) caused a significant increase in calcium content in the femoral-diaphyseal and -metaphyseal tissues. Such an effect was not observed by the combination of beta-cryptoxanthin (10(-9) M) plus genistein (10(-6) M) or menaquinone-7 (10(-6) M), or zinc (10(-6) M) plus genistein (10(-6) M) or menaquinone-7 (10(-6) M). Also, the combination of beta-cryptoxanthin (10(-9) M) plus zinc (10(-6) M) caused a remarkable increase in alkaline phosphatase activity and deoxyribonucleic acid (DNA) in the femoral-diaphyseal and -metaphyseal tissues, while their application alone did not have an effect on the enzyme activity or DNA content in the femoral tissues. The effect of the combination of beta-cryptoxanthin (10(-9) M) plus zinc (10(-6) M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DBR), an inhibitor of transcriptional activity. This study demonstrates that the combination of beta-cryptoxanthin and zinc at a lower concentration has a synergistic effect on bone components in vitro.
Abstract: We studied pharmacologic profiles of KRH-594, dipotassium (Z)-2-[[5-ethyl-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl-1,3,4 -thiadiazolin-2-ylidene]aminocarbonyl]-1-cyclopentenecarb oxylate, a novel angiotensin II (AII)-receptor antagonist. KRH-594 potently displaced specific binding of [125I]-AII from AT1 receptor with a Ki of 0.39 nM in rat liver membranes, but not from AT2 receptor in bovine cerebellar membranes (Ki > 10 microM). KRH-594 exhibited no affinity for 21 other receptors and two enzymes [50% inhibitory concentration (IC50) > 10 microM], demonstrating its high specificity toward AT1 receptors. In isolated rabbit aorta, KRH-594 caused nonparallel shifts to the right of the dose-response curve to AII and decreased the maximal response with a pK(B) of 10.4. We evaluated the in vivo efficacy and the duration of action in freely moving rats under nonfasting conditions. In normotensive rats, orally administered KRH-594 inhibited AII-induced pressor responses with a 50% inhibitory dose (ID50) of 0.39 mg/kg. In spontaneously hypertensive rats (SHRs), both KRH-594 (1 mg/kg p.o.) and losartan (10 mg/kg p.o.) exerted similar blood pressure-reducing effects, and their effects were still significant at 24 h after drug administration. We concluded that KRH-594 is a specific and efficacious AT1 antagonist that may find its use in the treatment of human hypertension.
Abstract: This report describes the in vitro pharmacological properties of dipotassium (Z)-2-[[5-ethyl-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl-1, 3,4-thiadiazolin-2-ylidene]aminocarbonyl]-1-cyclopentenec arboxylate, called KRH-594, a novel angiotensin II (AII) type 1 (AT1) receptor antagonist. We exposed rabbit aortic rings to KRH-594 (0.1 nM) for increasing contact times and observed an increasing degree of insurmountable suppression of AII-induced contractions. KRH-594 (0.01, 0.1 and 1.0 nM) caused a concentration-related, insurmountable suppression of the AII concentration-response curve. Repeated washing of rabbit aortic rings preincubated with KRH-594 (0.1, 1.0 and 10 nM) slowly reversed the insurmountable suppression. The marked suppression of AII-induced contractions by KRH-594 (0.1 nM) was restored by co-incubation with losartan (100 nM). KRH-594 (10 microM) had no effect on bradykinin-, acetylcholine-, or histamine-induced contractions of guinea pig ileum, demonstrating its high specificity for AT1 receptors. These results demonstrate that KRH-594 is a potent, specific and insurmountable AT1 receptor antagonist. KRH-594 activity in rabbit aorta appears to be that of a slowly reversible (pseudo-irreversible) antagonist.
Abstract: We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.
Abstract: The mechanism of the protection by human epidermal growth factor (hEGF) against the gastric mucosal lesions induced by acidified ethanol was studied in rats. At different times following the subcutaneous administration of hEGF (30 micrograms/kg), intragastric acidified ethanol (EtOH: 0.125 M HC1 = 50:50 v/v%) was administered to induce an experimental gastric mucosal lesion. Mean length of the lesion in the gastric mucosa was used as a lesion index. Extravasation of intravenously injected Evans blue into the gastric wall and gastric contents was used as an indicator of vascular permeability. Pretreatment with hEGF decreased both the gastric mucosal lesions and the increase of vascular permeability caused by acidified ethanol with similar time profiles relative to pretreatment with hEGF. Maximal protective actions of hEGF occurred about 10 to 30 min after the observed peak plasma concentration of hEGF. Indomethacin and N-ethylmaleimide, but not iodoacetamide, blocked the protective action of hEGF, indicating that endogenous prostaglandins and/or sulfhydryls may participate in the protective action of hEGF. The content of endogenous nonprotein sulfhydryls in the gastric mucosa decreased markedly after acidified ethanol. However, pretreated hEGF did not restore the sulfhydryl contents. Thus, it seemed that endogenous prostaglandins, but not sulfhydryls, are the probable mediators for protection against gastric mucosal injury caused by acidified ethanol.
Abstract: We previously demonstrated that the antitumor efficacy of various antitumor agents such as 5-fluorouracil and cisplatin against experimental solid tumors was enhanced by pre- or simultaneous administration of human epidermal growth factor (hEGF). In the present study, the combined therapy by hEGF and mitomycin C (MMC) as an antitumor agent was studied in A431 solid tumor-bearing mice to determine the dosage schedule of hEGF. When MMC alone was injected intraperitoneally (2 mg/kg) every 7th day to the tumor-bearing mice, tumor weights increased to 2138 +/- 285 mg from 282 +/- 41 mg during 22 d. Tumor weight in every day treatment of hEGF alone for 21 d increased to the same extent in the treatment by MMC alone. On the other hand, the increase of the solid tumor weight in the every day treatment and in the every 7th day treatment of hEGF, in combination with the every 7th day administration of MMC, were as follows; from 282 +/- 41 mg to 1522 +/- 357 mg (71.2 +/- 16.7% of MMC alone) and from 280 +/- 44 mg to 1245 +/- 150 mg (58.2 +/- 7.0% of MMC alone), respectively, demonstrating a greater antitumor potency of MMC in the combination with the every 7th day treatment of hEGF. Both combined therapies did not affect the toxicity of MMC as evaluated by decrease in nontumorous body weight. Single subcutaneous administration of hEGF to A431 tumor-bearing mice caused the decrease of the binding capacity of hEGF to A431 tumor cells by 80% 24 h after the administration.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Severe toxic side effects of antiproliferative agents limit their clinical usefulness as antitumor drugs. Recently we observed that the antitumor efficacy of various antitumor agents (5-fluorouracil, tegafur, adriamycin, mitomycin C, cyclophosphamide, and cisplatin) against experimental solid tumors was enhanced by prior or simultaneous administration of human epidermal growth factor (EGF). However, coadministration of EGF did not enhance the toxicity of antitumor agents as measured by LD50 and body weight loss. The above selective potentiation of efficacy of the antitumor agents by human EGF can be characterized as follows. In a dose-dependent manner, human EGF enhanced the efficacy of an antitumor agent (5-FU) treatment against human epidermoid carcinoma A431 transplanted sc in athymic nude mice [ED50 = 2.9 (0.2-49.7, 95% confidence interval) microgram/kg, sc]. Various degrees of enhancement were also observed against other experimental tumors transplanted sc. The degrees of enhancement were directly proportional to the numbers of human EGF binding sites present on tumor cell plasma membrane (threshold of binding site density = 1.5 X 10(3) sites/cell) using 5-FU or cisplatin as an antitumor agent, thus suggesting that the binding of EGF to the receptors on tumor cells is an essential process in enhancing the susceptibility of tumor cells to antitumor agents. Normal cells including intestinal epithelial and bone marrow cells are endowed with fewer EGF binding sites (less than 10(3) sites/cell). This may explain partially the absence of EGF-enhanced cytotoxicity by antitumor agents toward normal cells.
Abstract: A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.
Abstract: Free fatty acids (FFA) are known to uncouple oxidative phosphorylation in mitochondria. However, their mechanism of action has not been elucidated as yet. In this study we have investigated in detail the patterns of uncoupling by the FFA oleate and palmitate in rat liver mitochondria and submitochondrial particles. The patterns of uncoupling by FFA were compared to uncoupling induced by the ionophores valinomycin (in the presence of K+) and gramicidin (in the presence of Na+) and the proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP). The most striking difference in the pattern of uncoupling relates to the effect on the proton electrochemical potential gradient, delta mu H. Uncoupling by ionophores, particularly valinomycin, is associated with and most likely caused by a major reduction of delta mu H. In contrast, uncoupling by FFA is not associated with a significant reduction of delta mu H, indicating another mechanism of uncoupling. We suggest the use of the term decouplers for uncoupling agents such as FFA and general anesthetics that do not collapse the delta mu H [Rottenberg, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3313-3317]. The protonophore CCCP and to some extent the ionophore gramicidin indicate a mixed mode of uncoupling since their effect on delta mu H is moderate when compared to that of valinomycin. Another distinguishing feature of uncouplers that collapse the delta mu H is their ability to stimulate ADP-stimulated respiration (state 3) further. Decouplers such as FFA and general anesthetics do not stimulate state 3 respiration.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The interaction of the cationic spin probe 4-(N,N-dimethyl-N-dodecyl)-ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (Cat12) with intact mitochondria and submitochondrial particles was investigated as a function of salt concentration, pH and energization by ATP. In the presence of 1 mM Fe(CN)-36, which inhibits the probe reduction by the mitochondria, the probe signal is stable and shows both bound and free forms. The partition of the probe into mitochondrial membranes is decreased by various salts depending on the cation valency, indicating that the membrane is negatively charged (-10 to -15 mV at pH 7.0). The surface potential increases with pH from -3 mV at pH 5.0 to -18 mV at pH 8.0. Energization of intact mitochondria by ATP reduces the magnitude of both bound and free signals by more than 50%; the signal of the bound form slowly disappears on further incubation. The ATP effect is inhibited and also reversed by either oligomycin or CCCP. Similar effects of ATP were observed in mitoplasts but not in submitochondrial particles. In submitochondrial particles ATP has no effect on the probe signal or binding. These results suggest that the formation of membrane potential in mitochondria induces uptake and internal binding of the probe which results in broadening of the EPR signal of the internally bound probe. It is concluded that Cat12 is not a suitable probe for measurement of surface potential in energized mitochondria.
Abstract: The binding and phosphorescence of Tb3+ in rat liver mitochondria and submitochondrial particles were investigated. Mitochondria were treated briefly with N-ethyl-maleimide (NEM) to prevent phosphate leak and Tb3+ chelation. Up to 30 nmol of Tb3+/mg of protein binds to mitochondrial membranes with high apparent affinity (Kd congruent to 6 microM). Generation of a membrane potential had no significant effect on the apparent affinity or capacity of Tb3+ binding in NEM-treated mitochondria. Mitochondrial bound Tb3+ phosphorescence can be induced selectively by excitation of aromatic amino acid residues. The decay of mitochondrial bound Tb3+ phosphorescence is biphasic. The phosphorescence of the slow phase (t1/2 = 0.45-0.70 ms) is quenched by monovalent salts, indicating a negative surface potential at low salt medium of -5.4 +/- 2.8 mV [10 mM 3-(N-morpholino)-propanesulfonic acid, pH 7.2, 5 microM Tb3+]. In submitochondrial particles, a surface potential of -6.5 +/- 2.7 mV was estimated under the same conditions. Energization did not affect the surface potential significantly in submitochondrial particles and only slightly in mitochondria. Analysis of the phosphorescence of mitochondrial bound Tb3+ reveals two binding sites with high (Kd = 1.5 microM) and low affinity (Kd = 29 microM). The high-affinity site is tentatively identified as the Ca2+ carrier. A fraction of the carrier-bound Tb3+ phosphorescence decays rapidly, presumably as a result of energy transfer to cytochromes in the membrane core. These intramembrane sites appear to move to the surface on the generation of a membrane potential. We conclude that the salt effect on the phosphorescence of the slow phase may serve as a reliable measure of delocalized surface potential in mitochondria and submitochondrial particles. Tb3+ binding to the high-affinity site may be useful as a probe for the mitochondrial Ca2+ translocator.
Abstract: 1. The dependences of the reduction of ferricytochrome c-555 in the reaction center-cytochrome c complex on the redox potential and pH were investigated using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), ferrocyanide, and reduced 2,5-dimethyl-p-quinone as electron donors. 2. In the reduction of cytochrome c-555 by TMPD, the unprotonated form was the exclusive electron donor to the cytochrome with a second-order rate constant of 1.0 X 10(5) M-1.s-1. 3. Ferrocyanide reduced cytochrome c-555 slowly with a rate constant of 7.8 X 10(3) M-1.s-1 at infinite salt concentration. The value of -5.2 X 10(-4) elementary charge/A2 was estimated as the surface charge density in the vicinity of cytochrome c-555 by analyzing the salt effect on the cytochrome reduction using the Gouy-Chapman theory. 4. The characteristics of the dependences of the reduction of cytochrome c-555 by reduced 2,5-dimethyl-p-quinone on the redox potential and pH were well explained by the redox potential and pH dependences of the formation of the semiquinone. In the neutral-to-alkaline pH range the anionic semiquinone was the main electron-donating species with a second-order rate constant of 6.0 X 10(7) m-1.s-1.
