The Pharmacological Mechanism Of A Novel Anthraquinone BH3Mimetic Compound6That Efficiently Induces Apoptosis In Melanoma Cells

The Bcl-2family plays a key role in regulating apoptosis in response to various stimuli. Bcl-2like inhibitor mimic the structure and action of BH3-only proteins and interact with anti-apoptotic Bcl-2proteins at their BH3-binding groove and then suppress their anti-apoptotic function, which could provide a promising therapeutic strategy for malignant melanoma.Mitogen-activated protein kinases (MAPKs) pathway is constitutively activated in virtually all melanomas, which may directly or indirectly result in the extraordinary resistance to existing therapies and a very poor prognosis. For example, melanoma cells are relatively resistant to Bcl-2like inhibitors, which was not associated with MAPK pathway. Therefore, study on the molecular mechanism that Bcl-2inhibitors induce endogenous apoptosis in melanoma cells could contribute to provide a much improved understanding the resistance of melanoma cells and opportunities for new therapeutic approaches to this disease.We carried out a study to comparatively investigate the apoptosis-inducing effect of gossypol and S1in melanoma cell lines. To comfirm our hypothesis, we tested the interference of these BH3mimetics on Bcl-2family protein heterodimers. The results revealed that phosphorylation of Mcl-1at T163facilitate its interaction with pro-apoptotic protein to antagonize BH3mimetics in melanoma cells. Based on the newly detected molecular target in melanoma cells, we screened and identified a series of high-efficiency and low side effects anthraquinone compounds. Unlike the known BH3mimetics, these compounds were designed and evaluated to mimic two faces of Bim α-helix. They could induce mitochondrial apoptosis via targeting whole anti-apoptotic proteins. Compound6could efficiently induce apoptosis in melanoma cellsNext we performed co-immunoprecipitation (co-IP) experiments in order to evaluate the influence of Compound6on the heterodimers of anti-apoptotic proteins and pro-apoptotic proteins and activation of Bak and Bax proteins, cytochrome c release, PARP cleavage and caspase-3/9activation. These data suggested that the activation of mitochondria mediated apoptosis by compound6was induced by antagonizing pMcl-1to release their pro-apoptotic partners Bim or Bak.Result of treatment with increasing concentrations of ERK1/2inhibitor PD98059or JNK inhibitor SP610025for24in melanoma cell indicated that ERK1/2and JNK mediated phosphorylation of Mcl-1in melanoma cells. In addition, the result that treatment with S1at a proper concentration in combination with PD98059and SP610025each inhibitor alone significantly sensitized melanoma to S1, they progressively increased S1cytotoxicity in combination, suggesting that Mcl-1phosphorylation was mediated by ERK1/2and JNK combination led to the resistant of melanoma to BH3mimetics.We then examined whether the proteasome inhibitor MG132or wide-spectrum general caspase inhibitor zVADfmk could prevent Mcl-1from degradation during apoptosis mediated by Compound6. Western blotting analysis showed MG132could not affect the decrease of Mcl-1, while zVADfmk prevented Mcl-1from compound6induced-degradation, suggesting that Mcl-1degradation in the case of compound6treatment was caspase-dependent.We examined Noxa protein and mRNA levels upon compound6treatment by western blotting and quantitative PCR. These results indicated that up-regulation of Noxa following compound6treatment was at transcriptional level. Finally, we investigate the apoptosis-inducing effect of Compound6in melanoma cell following Noxa shRNA treatment. The result show that compound6killed melanoma cells independent of Noxa.For the first time, we illustrated it was pMcl-1that antagonize the known BH3mimetics and pMcl-1was a therapeutic target for melanoma. We also provided evidence that both MEK/ERK and JNK contributed to the Mcl-1phosphorylation and thus enhanced its anti-apoptotic function. In the meanwhile, Compound6is the first report of a pMcl-1inhibitor. Taken together, these results contribute to have an improve understanding of regulatory mechanism of pMcl-1and provide theoretical foundations for solutions in the future treatment of melanoma.