Enhancing drug sensitivity in melanoma cells through pro-apoptotic pathways that subvert chemoresistance mechanisms

The general ineffectiveness of current chemotherapeutic agents for malignant melanoma warrants the investigation into biological approaches that increase sensitivity to the cytotoxic effects of anticancer drugs. The majority of anticancer agents induce cancer cell death by means of apoptosis. An escape by cancer cells from apoptosis seems to be closely associated with the development of anticancer drug resistance; therefore, determining what mechanisms melanoma cells utilize for survival is our objective. Using three distinct strategies, we demonstrate the importance of apoptotic pathways for melanoma chemoresistance after drug treatment. In the first approach, we found that the mutational status of p53 can dictate several key phenotypes of a cell population. A dominant negative p53 stimulated faster growth in melanoma cells compared to the parental cell line and after treatment with the DNA-damaging drug, camptothecin, cell survival was significantly greater in the mutant /?53-bearing cell line attributable to a general decrease in apoptosis. We established that chemosensitivity of melanoma cells with normal p53 can occur through selective suppression of anti-apoptotic proteins like Bcl-2 and of drug transporters like P-glycoprotein while dysfunction of p53 inhibits the suppression leading to upregulation of these proteins and increasing chemoresistance. We further evaluated the relationship between p53 and P-glycoprotein in vivo using a mouse model to confirm our in vitro results that dysfunction of p53 causes increased expression of P-glycoprotein. Since dysfunction of p53 contributes to chemoresistance, overcoming the p53 block through the overexpression of other pro-apoptotic genes was explored and validated in a second strategy. Our previous data has suggested that the mitochondria-mediated apoptosis pathway plays a critical role in druginduced apoptosis for melanoma. Using ectopic overexpression of upstream pro-apoptotic genes such as Bax and the newly identified PUMA and Noxa that are involved in pore formation within the outer membrane of mitochondria, we could reduce the threshold of melanoma resistance to drug treatment. By several distinct methods, we determined that an increase in apoptosis was the mechanism for increased sensitivity to drug treatment. A further analysis of genes acting downstream of the mitochondria, such as Apaf-1 and caspase-3, that play a more terminal role in executing apoptogenic signals from the mitochondria also proved useful for the sensitization of melanoma cells to anticancer agents. We observed a p53-dependent activation of some candidate genes as they were more effective in a cell line with wild-type p53, function than a mutant p53 cell line. In the third approach, we focussed on the antitumour properties of the powerful antioxidant, curcumin. Our endeavour to find alternative compounds that may improve today's therapy regimes for melanoma yielded useful biochemical data for this compound. Contrary to other reports in some tumour cell lines demonstrating a p53-dependence for curcumin action, we showed that curcumin-treated melanoma cells undergo apoptosis independently of the p53 pathway. Our results also suggest that curcumin-induced apoptosis is not modulated through the mitochondria but rather the Fas death receptor-mediated pathway is responsible for curcumin activity on melanoma cells since inhibition of Fas activation could increase cell survival after curcumin treatment. In this thesis, we have employed specific approaches targeting particular molecular abnormalities for the induction of apoptosis by upregulating apoptotic signals. Although based on different targets and delivery methods, our strategies have the common goal of eliminating melanoma cells by restoration of the apoptotic function. Our hope is that the mechanistic understanding of cell death will have profound impacts upon the practice of medical oncology and outlook for many patients.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat