Antimicrotubule agent-induced activation of ERK1/2 protects human melanoma cells from apoptosis mediated by c-Jun N-terminal kinase

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The antimicrotubule agents vincristine and docetaxel are commonly used in the treatment of metastatic melanoma. The clinical response to the agents remains, however, often disappointing. To better understand the mechanism(s) underlying the resistance of melanoma to the antimicrotubule agents, we examined vincristine- and docetaxel-induced cytotoxic pathways and their potential crosstalk with survival signalling pathways. Both vincristine and docetaxel predominantly killed melanoma cells by induction of caspase-dependent, mitochondria-mediated apoptosis. This was evidenced by caspase activation, externalization of phosphatidylserine, cleavage of caspase-3 substrates PARP, ICAD, and XIAP, and increased sub-G1 DNA content. Over-expression of Bcl-2 or treatment with a general caspase inhibitor markedly inhibited apoptosis induced by either vincristine or docetaxel. The BH3-only protein of the Bcl-2 family, Bim, was rapidly down-regulated, suggesting that it plays a limited role in induction of apoptosis by these drugs. In support of this, silencing of Bim by siRNA did not effect apoptosis induced by the compounds. Two other BH-3 only proteins, Noxa and PUMA, were transiently up-regulated during the early stages of treatment. This did not, however, appear to be associated with sensitivity of melanoma cells to apoptosis induced by the drugs. Treatment with either vincristine or docetaxel induced phosphorylation of Bcl-2, which was more pronounced in cell lines sensitive to apoptosis. Notably, capsase-2 appears to be the apical caspase in the caspase-cascade induced by docetaxel but not vincristine. Both docetaxel and vincristine induced rapid activation of the MAPKs, JNK and ERK1/2, but not p38. Inhibition of JNK inhibited apoptosis, whereas inhibition of ERK1/2 sensitized melanoma to apoptosis induced by the drugs. Furthermore, inhibition of JNK markedly inhibited docetaxel-induced caspase-2 activation and down-stream apoptotic events. Inhibition of ERK1/2 blocked Bcl-2 phosphorylation induced by either docetaxel or vincristine. These results demonstrate that the antimicrotubule agents vincristine and docetaxel induce apoptosis of melanoma by activation of JNK, and that the agent-mediated activation of ERK1/2 plays a role in protection of melanoma cells from apoptosis. While JNK-mediated caspase-2 appears to be the initiating factor in docetaxel-induced apoptosis, the factor(s) that is responsible for JNK-mediated activation of the caspase-cascade in the case of vincristine remains to be elucidated. Phosphorylation of Bcl-2 may also play a role in regulation of sensitivity of melanoma cells to the antimicrotubule agents. Taken together, exploration of a combination of ERK1/2 specific inhibitors and antimicrotubule agents may provide a more effective therapeutic regimen in treatment of melanoma.