Biomedical subjects

Peter Hersey

Publications and source records attributed to Peter Hersey.

At least 19 recordsLinked to original sources

PubMed2006

Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group.

PURPOSE: Chemotherapy resistance in melanoma has been linked to antiapoptotic effects mediated by Bcl-2 protein. We evaluated whether targeting Bcl-2 using an antisense oligonucleotide (oblimersen sodium) could improve the efficacy of systemic chemotherapy in patients with advanced melanoma. PATIENTS AND METHODS: We randomly assigned chemotherapy-naïve patients with advanced melanoma to treatment with dacarbazine (1,000 mg/m2) alone or preceded by a 5-day continuous intravenous infusion of oblimersen sodium (7 mg/kg/d) every 3 weeks for up to eight cycles. Patients were stratified by Eastern Cooperative Oncology Group performance status, liver metastases, disease site, and serum lactate dehydrogenase (LDH). The primary efficacy end point was overall survival. RESULTS: Among 771 patients randomly assigned, the addition of oblimersen to dacarbazine yielded a trend toward improved survival at 24-month minimum follow-up (median, 9.0 v 7.8 months; P = .077) and significant increases in progression-free survival (median, 2.6 v 1.6 months; P < .001), overall response (13.5% v 7.5%; P = .007), complete response (2.8% v 0.8%), and durable response (7.3% v 3.6%; P = .03). A significant interaction between baseline serum LDH and treatment was observed; oblimersen significantly increased survival in patients whose baseline serum LDH was not elevated (median overall survival, 11.4 v 9.7 months; P = .02). Neutropenia and thrombocytopenia were increased in the oblimersen-dacarbazine group; however, there was no increase in serious infections or bleeding events. CONCLUSION: The addition of oblimersen to dacarbazine significantly improved multiple clinical outcomes in patients with advanced melanoma and increased overall survival in patients without an elevated baseline serum LDH.

Adolescent↗

PubMed2006

Progression in melanoma is associated with decreased expression of death receptors for tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in melanoma by interaction with death receptors TRAIL-R1 (DR4) or TRAIL-R2 (DR5) on melanoma cells or resists apoptosis by interaction with decoy receptors TRAIL-R3 (DcR1) or TRAIL-R4 (DcR2). Studies on cell lines suggest that there is a wide variation in TRAIL death receptor expression; however, their expression on excised human melanoma is not well documented. In view of this, we studied death receptor expression on melanomas using monoclonal antibodies specific for these receptors. Immunohistochemical staining for DR4, DR5, and DcR1/DcR2 was performed on formalin-fixed paraffin-embedded sections of 100 cases of primary melanoma, metastatic melanoma, and benign nevi. Percentage expressions of DR4 versus DR5 in benign nevi, primary melanoma, and melanoma metastases were 40% versus 90%, 69% versus 98%, and 55% versus 66%, respectively. There were significant differences in the mean percentage of DR5-positive cells between different groups of melanocytic lesions. Percent expression was higher in thin (< or =1.0 mm) compared with thick primary melanoma (88.9% versus 66.9%), and expression was less in subcutaneous metastases (49%) and lymph node metastases (30.6%) (P < .005). Expression was also higher in compound nevi (57%) than dysplastic nevi (49%). DcR1/DcR2 was found in 75% of benign nevi, 62% of primary melanomas, and 74% melanoma metastases. The results showed a wide variation in the expression of death receptors for TRAIL between and within primary and metastatic melanoma and a decreased expression on the thick primary melanoma and metastatic melanoma. This suggests that melanoma may not respond to treatment with TRAIL unless given with agents that increase the expression of TRAIL death receptors.

Adolescent↗

PubMed2006

Human melanoma cells selected for resistance to apoptosis by prolonged exposure to tumor necrosis factor-related apoptosis-inducing ligand are more vulnerable to necrotic cell death induced by cisplatin.

PURPOSE: Heterogeneous sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis may lead to outgrowth of TRAIL-resistant cells and limit successful treatment by TRAIL. The present study aims to better understand the biological characteristics of melanoma cells resistant to TRAIL-induced apoptosis. EXPERIMENTAL DESIGN: We generated TRAIL-resistant melanoma cells by prolonged exposure to TRAIL and characterized the cells in terms of their sensitivity to killing induced by a panel of cytotoxic agents using biological and biochemical methods. RESULTS: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by another death ligand FasL, the DNA-damaging agent cisplatin, the histone deacetylase inhibitor suberic bishydroxamate, and the antimicrotubule Vinca alkaloid, vincristine. The apoptotic signaling seemed to be inhibited upstream of mitochondrial apoptotic events and was associated with decreased expression of multiple apoptotic mediators, including pro-caspase-8, Fas-associated death domain, Bid, Bim, p53, and the products of its proapoptotic target genes. Despite being resistant to apoptosis, TRAIL-resistant melanoma cells were more vulnerable to cisplatin-induced nonapoptotic cell death. This was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase and p53 independence, and severe mitochondrial disruption, and was preceded by poly(ADP)ribose polymerase (PARP) activation and depletion of intracellular ATP, indicative of necrotic cell death. Inhibition of PARP activity partially converted the mode of cell death from necrosis to apoptosis. CONCLUSIONS: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by various apoptotic stimuli but are more sensitive to nonapoptotic cell death induced by cisplatin. Exploration of chemotherapy-induced nonapoptotic cell death may provide an alternative strategy in overcoming resistance of melanoma cells to TRAIL-induced apoptosis.

Antineoplastic Agents↗

PubMed2006

Bim plays a crucial role in synergistic induction of apoptosis by the histone deacetylase inhibitor SBHA and TRAIL in melanoma cells.

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor - induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X(L), Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim(EL). Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma.

Apoptosis↗

PubMed2006

Current strategies in overcoming resistance of cancer cells to apoptosis melanoma as a model.

Most anticancer agents mediate their effects through common pathways which induce apoptosis or in some cases necrosis of cancer cells. The apoptotic pathways are regulated by Bcl-2 family proteins, which include both pro- and anti-apoptotic members. Much is known about the interactions of these proteins involved in apoptosis and this information is being utilized in the development of new reagents that may be used to treat patients with cancers. The inhibitor of apoptosis family of proteins constitute a second group of proteins which inhibit the effector caspases. Reagents that inhibit their activity are also under development. Resistance of cancer cells to treatment can in many instances be attributed to activation of intracellular signal pathways involved in survival, such as the Ras-Raf-MEK-ERK1/2 or the P13K-Akt pathway. Again, much has been learned about the control of these pathways and their activation of resistance mechanisms. Inhibitors of such pathways are being evaluated in preclinical and clinical studies and are showing promise as a new class of anticancer agents. Much of the progress in future studies will likely depend on the ability to target these new treatments to particular subgroups of patients with tumor characteristics that make them responsive to the agents in question.

Antineoplastic Agents↗

PubMed2006

Involvement of BH3-only proapoptotic proteins in mitochondrial-dependent Phenoxodiol-induced apoptosis of human melanoma cells.

Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.

Apoptosis↗

PubMed2006

Apoptosis and melanoma: how new insights are effecting the development of new therapies for melanoma.

PURPOSE OF REVIEW: Melanoma has proven resistant to most available chemotherapy and immunotherapy. Despite a range of different biochemical targets, most agents kill cancer cells by induction of apoptosis. RECENT FINDINGS: Investigation of this process has provided insights into the resistance mechanisms in cancer cells and to development of a range of new agents that target apoptosis pathways. These include agents which inhibit antiapoptotic B cell lymphoma-2 family proteins and inhibitor of apoptosis proteins. In addition, a range of signal pathway inhibitors have become available that are able to inhibit signal pathways known to be associated with resistance to apoptosis. SUMMARY: Evaluation of most of these reagents are at a preclinical level but studies on some pathway inhibitors have passed from phase II into phase III studies. Similarly, evaluation of antisense reagents are at an advanced stage. These early trials show much promise and suggest this approach to development of new therapies will lead to much needed advances in treatment of this disease.

Apoptosis↗

PubMed2005

Selection for TRAIL resistance results in melanoma cells with high proliferative potential.

To better understand the outcome of the interaction between TNF-related apoptosis-inducing factor (TRAIL) and tumor cells, we studied TRAIL-resistant melanoma cells resulting from prolonged exposure to TRAIL and found that they had higher proliferative activity than the parental cells both in vitro and in vivo. This was associated with reduced p53 and p21 expression and increased activation of Erk1/2 and Akt. Accelerated proliferation was not due to TRAIL-mediated signaling but appeared to be the result of selection of previously existing, characteristically distinct cells. Moreover, responses of p53 to stimulation in the TRAIL-resistant cells appeared to be impaired.

Antineoplastic Agents↗

PubMed2005

Multi-centre Phase II trial of the polyamine synthesis inhibitor SAM486A (CGP48664) in patients with metastatic melanoma.

PURPOSE: To determine the activity and tolerability of SAM496A, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), in patients with metastatic melanoma who had not received prior chemotherapy. Selected patients were offered participation in two sub-studies examining early changes in tumor metabolism with FDG-PET and changes in tumor polyamine content. PATIENTS AND METHODS: Fifteen patients with measurable metastatic melanoma, normal cardiac function, and no known CNS metastases were eligible and received SAM486A by 1-hour IV infusion daily for 5 days every 3 weeks. Response was assessed by SWOG criteria. RESULTS: No patient had a confirmed partial response. Fatigue/lethargy, myalgia and neutropenia were the main toxicities but no febrile neutropenia or grade 4 non-hematological toxicity occurred. Five patients had PET scans pre-treatment and on days 8-12 of cycle 1. No patient had reduction of tumor metabolism. Serial biopsy in one patient showed alterations in polyamines consistent with SAMDC inhibition. CONCLUSIONS: Using the present dose and schedule of administration, SAM486A does not have significant therapeutic potential in patients with metastatic melanoma.

Adenosylmethionine Decarboxylase↗

PubMed2005

Treatment of metastatic melanoma using electroporation therapy with bleomycin (electrochemotherapy).

Electroporation therapy (EPT) is a novel treatment modality that uses brief, high-intensity, pulsed electrical currents to enhance the uptake of chemotherapeutic agents, vaccines and genes into cells. This technique is potentially useful for patients with secondary and, possibly, some primary tumours. Nineteen patients with metastatic melanoma were enrolled in a phase two, randomized, open-label study comparing intralesional bleomycin+EPT with intralesional bleomycin alone. Of 18 study lesions, 13 (72%) showed a complete response, one (5%) showed a partial response, three (18%) showed no change and one (5%) showed disease progression over a period of greater than 12 weeks. This represents a 78% objective response rate, which was significantly greater than the 32% response rate observed in the 19 patients with tumours treated with intralesional bleomycin alone (chi=7.94, 1 df, P=0.005). An additional 36 lesions, not enrolled in the study, were also treated with bleomycin+EPT. Of the total of 54 lesions treated with bleomycin+EPT, there was a 72% objective response rate. EPT treatment was well tolerated and was performed on an outpatient basis.

Adult↗

PubMed2005

Variable expression of protein kinase C epsilon in human melanoma cells regulates sensitivity to TRAIL-induced apoptosis.

Protein kinase C (PKC) activation is believed to protect against apoptosis induced by death receptors. We have found however that the effect of activation of PKC on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma differs between cell lines. Pretreatment with phorbol 12-myristate 13-acetate (PMA) led to inhibition of apoptosis in the majority of the melanoma cell lines, but those with relatively low PKC epsilon expression were sensitized to TRAIL-induced apoptosis. Introduction of PKC epsilon into PKC epsilon-low cell lines reversed sensitization of the cells to TRAIL-induced apoptosis by PMA. In contrast, a dominant-negative form of PKC epsilon caused an increase in sensitivity. The changes in sensitivity to TRAIL-induced apoptosis were reflected in similar changes in conformation of Bax and its relocation from the cytosol to mitochondria. Similarly, there were concordant increases or decreases in mitochondrial release of second mitochondria-derived activator of caspase/DIABLO, activation of caspase-3, and processing of its substrates. Activation of PKC seemed to mediate its effects upstream of mitochondria but downstream of caspase-8 and Bid in that pretreatment with PMA did not cause significant changes in the expression levels of TRAIL death receptors, alterations in the levels of caspase-8 activation, or cleavage of Bid. PKC activated the anti-apoptotic extracellular signal-regulated kinase 1/2 pathway, but inhibitors of this pathway only partially reversed the protective effect of PKC against TRAIL-induced apoptosis. These results provide further insights into the variable responses of melanoma to TRAIL-induced apoptosis and may help define responsive phenotypes to treatment of melanoma with TRAIL.

Adenoviridae↗

PubMed2004

Phase I/II study of immunotherapy with T-cell peptide epitopes in patients with stage IV melanoma.

Previous studies in small groups of patients suggested that immunization of melanoma patients with peptide epitopes recognized by T cells could induce regression of melanoma. This approach was tested in 36 patients with stage IV melanoma. The (MHC class I-restricted) peptides were from gp100, MART-1, tyrosinase, and MAGE-3. The gp100 and MART-1 peptides had been modified to increase their immunogenicity. In half the patients (groups 3 and 4) the peptides were given in the adjuvant Montanide-ISA-720, and half the patients in both groups were given GM-CSF s.c. for 4 days following each injection. Treatment was well tolerated except for two severe erythematous responses to Montanide-ISA-720 and marked inflammatory responses at sites of GM-CSF administration in three patients. There were no objective clinical responses but stabilization of disease for periods from 3 to 12 months were seen in seven patients. Five of these were patients given the peptides in Montanide-ISA-720. Delayed-type hypersensitivity (DTH) skin test responses were also seen mainly in the patients given the peptides in Montanide-ISA-720. GM-CSF did not increase DTH responses in patients in the latter group but may have increased DTH responses in those not given peptides in Montanide-ISA-720. Inflammatory responses around s.c. metastases or regional lymph nodes were observed in two patients. These results suggest that the peptides are more effective when given in the adjuvant Montanide-ISA-720. Nevertheless, results from this study, together with those from a number of comparable studies, indicate that peptide vaccines are currently of minimal benefit to patients and support the need for ongoing development of new strategies in treatment of this disease.

Adult↗

PubMed2004

The history and future of melanoma staging.

The evolution and progressive refinement of an internationally accepted melanoma staging system over the last 50 years has resulted in much greater accuracy and increased utility, but the staging process has become more complex and less intuitive. This raises the question of whether melanoma staging should continue to develop with ever-increasing levels of complexity, or whether attempts should be made to produce an alternative system that is simpler and more intuitive. The current, TNM-based American Joint Committee on Cancer (AJCC) staging system for melanoma incorporates only some of the prognostic factors of proven significance. However, the information that is now available about these and other, well-documented prognostic factors allows accurate prediction of an individual melanoma patient's prognosis using a computer-generated estimate. Thus an alternative staging strategy that could be considered in the future would be to use such an estimate to obtain a numerical score for each patient, based on all available information agreed to be of prognostic relevance. A stage grouping could then be assigned on the basis of that score, according to previously determined score ranges for each stage and substage. The advantages of such a system would be that it would allow more reliable comparison of treatment results within and between institutions, and would provide more equivalent stratification groups for patients entering clinical trials of new therapies and those entering adjuvant therapy trials. A further advantage would be that because there would be a direct link between staging and prognostic estimate, such a system would be more readily able to be understood in an intuitive fashion.

Antigens, Neoplasm↗

PubMed2004

Microarray expression profiling in melanoma reveals a BRAF mutation signature.

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Amino Acid Substitution↗

PubMed2004

Systemic therapy of malignant human melanoma tumors by a common cold-producing enterovirus, coxsackievirus a21.

PURPOSE: The incidence of malignant melanoma continues to increase worldwide; however, treatment of metastatic melanoma remains unsatisfactory, and there is an urgent need for development of effective targeted therapeutics. A potential biological target on the surface of malignant melanoma cells is the up-regulated expression of intercellular adhesion molecule (ICAM)-1 and decay-accelerating factor (DAF), relative to surrounding benign tissue. Coxsackievirus A21 (a common cold virus) targets and destroys susceptible cells via specific viral capsid interactions with surface-expressed virus receptors comprising ICAM-1 and DAF. EXPERIMENTAL DESIGN: The oncolytic capacity of a genetically unmodified wild-type common cold-producing human enterovirus (Coxsackievirus A21, CAV21) was assessed against in vitro cultures and in vivo xenografts of malignant human melanoma cells. RESULTS: In vitro studies established that human melanoma cells endogenously express elevated levels of ICAM-1/DAF and were highly susceptible to rapid viral oncolysis by CAV21 infection, whereas ICAM-1/DAF-expressing peripheral blood lymphocytes were refractile to infection. In vivo studies revealed that the tumor burden of nonobese diabetic severe combined immunodeficient mice bearing multiple s.c. melanoma xenografts was rapidly reduced by oncolysis mediated by a single administration of CAV21. The antitumor activity of CAV21 was characterized by highly efficient systemic spread of progeny CAV21, with oncolysis of tumors also occurring at sites distant to the primary site of viral administration. CONCLUSIONS: Overall, the findings presented herein demonstrate an important proof of principle using administration of replication-competent CAV21 as a potential biological oncolytic agent in the control of human metastatic melanoma.

Animals↗

PubMed2004

Rapid downregulation of antigen processing enzymes in ex vivo generated human monocyte derived dendritic cells occur endogenously in extended cultures.

Dendritic cells, the most potent antigen presenting cells, have been shown in murine models to induce immune responses against many antigens. Their role in the initiation of antitumour immunity has received enormous attention. Their ability to process and present antigen is dependent on their state of maturation. This study examines the activity of human monocyte-derived dendritic cells at two different time points and the corresponding changes in the proteolytic enzyme activity. Dendritic cells were produced from peripheral blood mononuclear cells of normal volunteers. Plastic adherent cells were cultured for 5 or 7 days with recombinant human (rh)GM-CSF and rhIL-4. Flow cytometry showed that day 5 dendritic cells (DC) were less mature than day 7 DC as indicated by the expression of CD1a, CD11c, CD14, CD80, CD83, CD86 and MHC-II. Proteolytic activity of the enzymes cathepsin C and cathepsin G and phagocytosis of particulate antigens also showed significant differences between d5 dendritic cells and d7 dendritic cells. Allogeneic costimulatory activity of d7 dendritic cells was also significantly increased. Induction of immunity requires active presentation of antigens by antigen processing cells on their MHC-I and/or MHC-II molecules. Study of peptide carriers and peptide precursor molecules showed a significant decrease in CLIP levels in the day 7 DC, suggesting their decreased ability to process antigens but no difference in their ability to load MHC-II molecules. These findings indicate that the length of time in culture, in the absence of exogenous maturation - inducing stimuli affects dendritic cell maturation. Intracellular enzymatic activities of dendritic cells also changed rapidly with small changes in phenotype.

Antigen Presentation↗

PubMed2004

Histological expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in human primary melanoma.

AIMS: Tumour necrosis factor-related apoptosis ligand (TRAIL) appears to selectively induce apoptosis in a wide range of cultured malignant cells, including melanoma. This study was designed to attempt to clarify the role of TRAIL in the biology of human melanoma. METHODS: Tissue sections cut from formalin-fixed, paraffin-embedded tissue blocks of 45 primary cutaneous melanomas were tested for expression of TRAIL using immunohistochemistry. The intensity, pattern of staining and percentage of positively stained tumour cells were evaluated in each melanoma. Breslow thickness, ulcerative state, dermal mitotic rate and the presence of tumour infiltrating lymphocytes were measured/determined in each case. Median follow up for the cohort of patients was 10 months (range 1-18). Survival analysis was conducted using the Kaplan-Meier method. The level of expression of TRAIL was compared with the various histological determinants using the two-tailed Fisher's exact test and the chi2-test. RESULTS: Overall and disease-free survival were 72 and 48%, respectively, and did not correlate with TRAIL expression. Among the pathological prognostic determinants, only mitotic rate showed a statistically significant correlation with TRAIL expression using the chi2-test (P=0.04). CONCLUSION: We conclude that TRAIL expression in melanoma defines a more aggressive/proliferative phenotype, either through selection of apoptotic resistant cells or by secondary induction of other factors enhancing proliferation of more malignant cells. Analysis of a larger group of patients with longer follow-up is required to determine whether TRAIL expression correlates with survival of patients.

Adult↗

PubMed2004

Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways.

Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In the present study, we have examined the apoptosis-inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine-induced apoptotic signaling pathway. We report that although staurosporine activated Bax and the mitochondrial caspase-dependent apoptotic pathway, it also induced apoptosis of melanoma by caspase-independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to staurosporine and was associated with release of cytochrome c and Smac/DIABLO from mitochondria and cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNase. This pathway was inhibitable by broad caspase inhibitors. A second apoptotic pathway that appeared to be involved in late apoptotic events was caspase independent in that inhibitors of caspases did not prevent the late onset of apoptosis. Overexpression of Bcl-2 inhibited the early onset of apoptosis but not the later, caspase-independent pathway. Apoptosis-inducing factor may be responsible for the late apoptotic execution in that its translocation from mitochondria into the nucleus coincided with the late onset of apoptosis and could not be inhibited by either a pan-caspase inhibitor or overexpression of Bcl-2. Our results indicate that staurosporine is able to bypass resistance of melanoma cells to mitochondrial caspase-dependent apoptotic pathways; hence, derivatives of staurosporine may warrant further evaluation either alone or with other apoptosis-inducing agents.

Apoptosis↗