Abstract: Multiple myeloma (MM) is an incurable hematological disorder characterized by dysregulated proliferation of terminally differentiated plasma cells. Aberrant histone acetylation has been observed in the development of numerous malignancies. Histone deacetylase inhibitors such as valproic acid (VPA) are promising drugs for cancer therapy since they have been reported to have antiproliferative effects and to induce differentiation in carcinoma and leukemic cells. Considering the advantage of being already in clinical use for epilepsy treatment, valproic acid might be a promising therapeutic candidate drug in the management of multiple myeloma. In this study, we show that the short fatty acid VPA has a time and dose-dependent cytotoxic effect on the MM cell lines OPM2, RPMI and U266. The influence of VPA on cell cycle and apoptosis have been evaluated by flow cytometry. Our results show that the three cell lines are blocked in G0/G1 phase. The observed sensitivity to VPA can be partially explained by late apoptosis. Since caspase 3 is activated in all tested cell lines after VPA treatment, a caspase-dependent pathway seems to be involved but not activated by the classic apoptotic pathways. We have also studied another mechanism of cell death, the senescence-like phenotype, but did not find any evidence for its implication. Thus, treatment with VPA may imply other alternative cell death mechanisms.
Abstract: The aspartic protease cathepsin D (cath-D) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, cath-D is translocated to the cytosol. Because cath-D is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically active relative to the cysteine lysosomal enzymes such as cath-B and -L, it is therefore open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of wild-type cath-D and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of cath-D catalytic function. We demonstrate that wild-type or mutated catalytically inactive cath-D strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wild-type and mutated inactive cath-D are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and -3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly therefore, the stimulatory effect of cath-D on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic cath-D stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).
Abstract: BACKGROUND: Intracoronary infusion of autologous bone marrow cells (CTX) has been shown to improve myocardial function in postinfarct patients and in patients with chronic ischemic cardiomyopathy. Whether CTX affects exercise-induced changes in cardiac deformation and mitral regurgitation (MR) in patients with end-stage heart failure has not been studied. METHODS AND RESULTS: In this small pilot study, 11 patients with chronic ischemic cardiomyopathy, ejection fraction (EF) <25%, no inducible ischemia and heart failure class New York Heart Association (NYHA) III underwent CTX. Symptom-limited bicycle exercise echocardiography was performed pre- and 4 months post-CTX and maximum systolic strain (msyepsilon), peak systolic strain rate (psysr), and effective regurgitant orifice of MR (ERO) were determined. There were no complications related to the procedure. The overall clinical benefit of CTX was limited with a trend towards improvement (NYHA 3.0 +/- 0.1 pre- and 2.7 +/- 0.2 post-CTX, P = .06). The EF did not improve after CTX. The wall motion score index did not change at rest but decreased significantly during exercise (1.48 +/- 0.16 versus 1.44 +/- 0.17, P = .01). In patients with non-viable areas, msyepsilon, psysr, and ERO were not affected by CTX. However, in patients with viable areas, msyepsilon and psysr appeared to increase during exercise and ERO appeared to decrease from 19 +/- 5 to 16 +/- 5 mm(2). This effect was not apparent at rest and more pronounced with inferior viability. CONCLUSION: CTX may improve cardiac deformation and MR during exercise in patients with severe chronic heart failure when viable areas are targeted.
Abstract: The ability to induce apoptosis in tumor cells is critical to elicit a positive response to cytotoxic chemo-therapy. In this study, we investigated the effect of the topoisomerase I inhibitors camptothecin and SN-38, known to cause an unusual form of DNA damage, on apoptotic pathways using the leukemic cell line HL-60 and its vincristine-resistant variant HL-60 VCR. Both camptothecin and SN-38 induced high levels of apoptosis in sensitive cells when compared to the multidrug-resistant ones. Interestingly, a higher BCL-2/BAX ratio was observed in HL-60 VCR at the basal state and during treatments. Moreover, these cells which did not exhibit Bcr-abl translocation or bcrp efflux pump, overexpressed topoisomerase I protein. The data provide evidence that BCL-2 protein could protect HL-60 VCR from mitochondrial membrane depolarization and block ROS production in these cells. Finally, our results suggest that dysregulation of proteins associated with DNA replication and apoptotic process could contribute to the multidrug-resistance phenotype.
Abstract: For several years it has been possible to routinely detect numerous mutations in the human genome by different methods. The most common technique is a standard PCR, but real time fluorescence PCR is increasingly being used. The purpose of this paper is to compare these two different techniques from the point of view of reliability, time consumption, and cost. More than 600 DNA samples of prevalence studies and from cancer patients were used to determine mutations in the genes of coagulation factor V Leiden, prothrombin, and methylenetetrahydrofolate reductase using standard PCR. A subset of 132 samples from the same pool was also tested by LightCycler PCR for the same coagulation gene mutations. Originally LightCycler techniques were applied for quantitative PCR by real time fluorescence measuring. Adding a melting curve analysis allows mutation detection. The results were perfectly concordant. The cost for the reagents is nearly the same for both methods but the time consumption for standard PCR is much higher than for the LightCycler method, resulting in higher laboratory personnel costs.
Abstract: Functional tumor imaging using Fluorodeoxyglucose positron emission tomography (FDG-PET) is a new method in clinical oncology. The 18 FDG, is a glucose analog that accumulates in cells in proportion to the rate of glucose metabolism, and increased carbohydrate metabolism has been recognized as a feature of malignant cells versus normal cells. In addition, it permits the detection of metastases or synchronous tumours not discovered by anatomic imaging. Although detection of the primary site of disease is usually accomplished well with conventional techniques, the performance of FDG-PET may be useful to determine tumours that are not clinically evident. The authors describe a case of early detection of synchronous thyroid carcinoma by FDG-PET in a young patient opereted on for a malignant melanoma on his arm.
Abstract: Cathepsin-D is an independent marker of poor prognosis in human breast cancer. We previously showed that human wild-type cathepsin-D, as well as its mutated form devoid of proteolytic activity stably transfected in 3Y1-Ad12 cancer cells, stimulated tumor growth. To investigate the mechanisms by which human cathepsin-D and its catalytically-inactive counterpart promoted tumor growth in vivo, we quantified the expression of proliferating cell nuclear antigen, the number of blood vessels and of apoptotic cells in 3Y1-Ad12 tumor xenografts. We first verified that both human wild-type and mutated cathepsin-D were expressed at a high level in cathepsin-D xenografts, whereas no human cathepsin-D was detected in control xenografts. Our immunohistochemical studies then revealed that both wild-type cathepsin-D and catalytically-inactive cathepsin-D, increased proliferating cell nuclear antigen expression and tumor angiogenesis. Interestingly, wild-type cathepsin-D significantly inhibited tumor apoptosis, whereas catalytically-inactive cathepsin-D did not. We therefore propose that human cathepsin-D stimulates tumor growth by acting-directly or indirectly-as a mitogenic factor on both cancer and endothelial cells independently of its catalytic activity. Our overall results provide the first mechanistic evidences on the essential role of cathepsin-D at multiple tumor progression steps, affecting cell proliferation, angiogenesis and apoptosis.
Abstract: Advances continue in erythropoietin biology, and additional data reviewed here have recently become available on complex feedback mechanisms describing the interrelations of hypoxia and its effects on anemia and tumor behavior (eg, apoptosis, angiogenesis). In addition to biology, other clinically relevant data in oncology are included and an attempt is made to identify patients who are most likely to benefit from treatment. The latter aspects will better define the profile of the target patient, probably prevent overtreatment, and improve cost-benefit ratios. Interesting data on radiotherapy results improved by increasing tissue hemoglobin have been published but will need further confirmation.
Abstract: Docetaxel (Taxotere), a member of the taxoid family of chemotherapy drugs is currently being tested in clinical trials simultaneously with other apoptosis inducing drugs like doxorubicin. We show, in vitro, in MCF-7 breast cancer cells that when it is used at doses as low as 5nM, 24 hours before either doxorubicin or etoposide, docetaxel is capable of inducing a significant increase in cell death compared to the reverse sequence or simultaneous treatment. We further show that this increase in cell death is due to an increase in apoptosis, and that this sensitization coincides with a docetaxel induced G2-M arrest and phosphorylation of the bcl-2 oncoprotein. We speculate that this phosphorylation of the apoptosis blocker bcl-2 might be responsible for the sensitization, and we suggest a clinical study comparing a 24 hour docetaxel pretreatment to the current simultaneous schedules.
Abstract: Some epidemiological studies have suggested that aspirin could be a chemopreventive agent against breast cancer. We tested the effects of the aspirin metabolite salicylate (SA) on four (Hs578T, MCF-7, MDA-MB-231, and T-47D) breast cancer cell (BCC) lines in vitro. Two features were studied: the proliferation of BCC and their production of the osteolytic cytokines interleukins-6 (IL-6) and -11 (IL-11) since BCC frequently metastasize to bone and induce tumor-induced osteolysis. SA, from 0.5 to 5 mM, caused BCC growth inhibition by up to 70% (IC50 range 2.54 to 4.28 mM). At high concentrations, the drug induced apoptosis only (MDA-MB-231), or both apoptosis and primary necrosis (MCF-7). SA, as well as indomethacin (INDO), reduced the synthesis of IL-6 and -11, at both the protein and mRNA levels, in the two cell lines producing these cytokines (MDA-MB-231 and Hs578T). This latter effect seemed to be mediated by PGE2 since SA and INDO reduced PGE2 levels in MDA-MB-231 and Hs578T cells, PGE2 was not detected in MCF-7 and T-47D cells and exogenous PGE2 increased IL-6 and -11 expression by MDA-MB-231 cells. Collectively, our results suggest that SA could reduce the growth of breast tumors and inhibit to some extent the ability of BCC to induce osteoclast recruitment and osteolysis. These data indicate the need for further epidemiological and experimental studies.
Abstract: Due to the frequent diagnosis at a late inoperable stage and the bad prognosis of metastatic disease, lung cancer has become the first cause of cancer mortality. Early detection is thus the only way to influence mortality as there is no good treatment available for advanced disease. In the eighties, large screening studies using standard chest X Ray and sputum cytology have not been able to show a significant reduction in global lung cancer mortality. However these studies are now largely criticized for their methodological flaws. Recently, a new technique using auto-fluorescence fibroscopy has been developed, which is able to detect dysplastic and in situ neoplastic lesions which are invisible to standard fibroscopic techniques. This technique holds great promise in the detection of early lesions. In addition, molecular biology techniques are being developed which aim at detecting early invasive lesions at a stage where surgical treatment is still curative. The addition of these two techniques will probably in the future increase the efficiency of lung cancer screening. Therefore we think that new large scale screening studies are needed.
Abstract: Retinoids inhibit the growth and reverse aberrant differentiation of squamous cell carcinoma (SCC) cells in vitro. To investigate the potential mechanisms of antitumor activity of retinoids in vivo, we used the cervical SCC cell line ME-180 as a s.c. tumor xenograft in athymic nude mice. After s.c. injection, tumor cells were allowed to form visible tumors and antitumor activity of all-trans-retinoic acid (tRA) was studied. tRA was administered daily for a 1-week or a 2-week period at 60 mg/kg/day. Tumor specimens were then analyzed using immunohistochemical staining for the number of blood vessels and apoptotic cells and for proliferating cell nuclear antigen expression. Furthermore, we studied the effect of the tRA treatment on the expression of a binding protein for fibroblast growth factors (BP; Gen-Bank accession no. M60047) that is a candidate angiogenesis modulator in SCC (F. Czubayko et al., J. Biol. Chem., 269: 28243-28248, 1994). We found that in vivo tRA treatment reduces BP expression in SCC xenografts, inhibits their angiogenesis, induces apoptosis of the tumor cells, and leads to a decrease of the tumor growth rate. We speculate that the tRA down-regulation of BP is responsible for the reduction of angiogenesis.
Abstract: PURPOSE: Benign prostatic hyperplasia (BPH) is related to advancing age and the presence of androgens and occurs in virtually all older men. BPH causes morbidity, most often by urinary obstruction, in a substantial fraction of men over sixty. Both finasteride and androgen ablation induce partial diminution in BPH that occurs over weeks to months. This is in contrast to the often rapid involution seen in both normal prostatic epithelium and prostatic carcinoma in response to androgen withdrawal. This study was performed to analyze the response of prostatic cells, and in particular BPH, to acute androgen ablation. MATERIALS AND METHODS: We subjected a cohort of 26 men to androgen ablation with goserelin, a gonadotrophin releasing hormone agonist, for 3-4 weeks prior to radical prostatectomy for prostate cancer. Preablation biopsy specimens and prostatectomy specimens were immunohistochemically stained for apoptotic cells and for expression of apoptosis regulatory proteins Bcl-2, Bax, Bcl-x, and Bak. RESULTS: Normal prostatic epithelial cells and prostate cancer responded to hormone deprivation by undergoing apoptosis, but in 19/26 specimens prostatic hyperplasia had a total absence of apoptosis. In all 26 specimens, benign prostatic hyperplasia demonstrated increased expression of the Bcl-2 protein, but no change in the expression of Bax, Bcl-x, and Bak. In contrast, adjacent normal and malignant prostatic epithelium showed positive staining for apoptosis and did not alter Bcl-2 expression in response to androgen ablation. CONCLUSIONS: BPH demonstrated increased staining for Bcl-2 after androgen deprivation that may render hyperplastic epithelium relatively resistant to apoptosis induced acutely by androgen withdrawal.
Abstract: The growth and metastatic spread of cancer is directly related to tumor angiogenesis, and the driving factors need to be understood to exploit this process therapeutically. However, tumor cells and their normal stroma express a multitude of candidate angiogenic factors, and very few specific inhibitors have been generated to assess which of these gene products are only innocent bystanders and which contribute significantly to tumor angiogenesis and metastasis. Here we investigated whether the expression in tumors of a secreted fibroblast growth factor (FGF)-binding protein (FGF-BP) that mobilizes and activates locally stored FGFs (ref. 11) can serve as an angiogenic switch molecule. Developmental expression of the retinoid-regulated FGF-BP gene is prominent in the skin and intestine during the perinatal phase and is down-modulated in the adult. The gene is, however, upregulated in carcinogen-induced skin tumors, in squamous cell carcinoma (SCC) and in some colon cancer cell lines and tumor samples. To assess the significance of FGF-BP expression in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their endogenous FGF-BP by targeting with specific ribozymes. We found that the reduction of FGF-BP reduced the release of biologically active basic FGF (bFGF) from cells in culture. Furthermore, the growth and angiogenesis of xenograft tumors in mice was decreased in parallel with the reduction of FGF-BP. This suggests that human tumors can utilize FGF-BP as an angiogenic switch molecule.
Abstract: Epirubicin is one of the less cardiotoxic alternatives to doxorubicin. We were interested in studying the cardiotoxic effect of the total cumulative dose, and weekly schedules of low compared to high dose intensity. Fifty-seven patients were treated with different epirubicin-containing regimens. We confirm the classical notion that total cumulative doses of less than 600 mg/m2 do not induce significant cardiotoxicity, whereas doses above 600 mg/m2 are associated with a trend towards cardiotoxicity. Patients receiving a high weekly dose intensity (> 40 mg/m2), however, did have a significantly lower incidence of cardiotoxicity than those receiving a low dose intensity per week (< 40 mg/m2) (22.8% versus 50%; P < 0.05). We identified the association of a dose intensity of more than 40 mg m-2/ week-1 and a cumulative dose of 400-899 mg/m2 or a dose intensity of less that 40 mg m-2/week-1 and a cumulative dose of less than 400 mg/m2 to have the lowest incidence rate of cardiotoxicity. We conclude from this study that epirubicin in weekly schedules of high dose intensity is not more cardiotoxic than in weekly schedules of low dose intensity.
Abstract: Clinical and experimental evidence suggests that spreading of malignant cells from a localized tumor (metastasis) is directly related to the number of microvessels in the primary tumor. This tumor angiogenesis is thought to be mediated by tumor-cell-derived growth factors. However, most tumor cells express a multitude of candidate angiogenesis factors and it is difficult to decipher which of these are rate-limiting factors in vivo. Herein we use ribozyme targeting of pleiotrophin (PTN) in metastatic human melanoma cells to assess the significance of this secreted growth factor for angiogenesis and metastasis. As a model we used human melanoma cells (1205LU) that express high levels of PTN and metastasize from subcutaneous tumors to the lungs of experimental animals. In these melanoma cells, we reduced PTN mRNA and growth factor activity by transfection with PTN-targeted ribozymes and generated cell lines expressing different levels of PTN. We found that the reduction of PTN does not affect growth of the melanoma cells in vitro. In nude mice, however, tumor growth and angiogenesis were decreased in parallel with the reduced PTN levels and apoptosis in the tumors was increased. Concomitantly, the metastatic spread of the tumors from the subcutaneous site to the lungs was prevented. These studies support a direct link between tumor angiogenesis and metastasis through a secreted growth factor and identify PTN as a candidate factor that may be rate-limiting for human melanoma metastasis.
Abstract: We have previously shown that TGF alpha and c-Myc interact in a strong, synergistic fashion to induce mammary gland tumors in double transgenic mice. Here we show this interaction can be explained, at least in part, by a cooperative growth stimulus by the two proteins, and by TGF alpha-mediated inhibition of c-Myc-induced apoptosis. We initially compared rapidly progressing mammary tumors from double transgenic mice to long latency tumors from single transgenic mice and observed a striking difference in the occurrence of apoptosis among the three groups. Tumors exhibiting apoptosis were derived exclusively from mice that expressed the c-myc transgene in the absence of the TGF alpha transgene, indicating that TGF alpha might protect c-Myc-overexpressing cells from programmed cell death. Cell lines were derived from single and double transgenic mammary tumors to examine further the mechanism underlying the cooperative interaction between the two gene products. In accordance with our in vivo data, apoptosis was only detected when the c-myc transgene was expressed without the TGF alpha transgene. Furthermore, exogenous addition of TGF alpha inhibited apoptosis in cells overexpressing c-Myc alone. In addition, tumor-derived cells that overexpressed both TGF alpha and c-Myc exhibited faster growth rates in vitro and in vivo and were less sensitive to the inhibitory effects of TGF beta in vitro compared to cell lines expressing only one of the transgenes. Based on our findings we propose that TGF alpha acts both as a proliferative and a survival factor for c-Myc-expressing tumor cells. Our results indicate that TGF alpha and c-Myc cooperate in tumorigenesis via a dual mechanism: TGF alpha can inhibit c-Myc-induced apoptosis and both proteins provide a growth stimulus.
Abstract: We describe an in vitro model for prostate cancer treatment that suggests a potential benefit for combined androgen ablation and cytotoxic chemotherapy. Androgen treatment of the LNCaP hormone-dependent human prostate cancer cell line induces increased expression of the BCL-2 protein. Increased levels of this protein are known to mediate inhibition of apoptosis. LNCaP cells, however, did not undergo apoptosis in response to androgen withdrawal. Etoposide exerts its cytotoxicity on LNCaP and other cells by inducing apoptosis. In vitro etoposide cytotoxicity was diminished 83% in the presence of either 10(-8) M dihydrotestosterone or 10(-9) M R1881 in LNCaP cells. The interaction between androgen and etoposide was mediated through the BCL-2 protein, since bcl-2 antisense oligonucleotides blocked the protective effect of androgens on etoposide cytotoxicity.
