Activation of PKC differentially regulates sensitivity of melanoma cells to TRAIL-induced apoptosis: Loss of PKCε contributes to apoptosis sensitization

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We have examined the potential crosstalk between PKC-mediated signal transduction and the TRAIL-induced apoptotic signaling pathway in melanoma cell lines. We report that activation of PKC can either sensitize melanoma cells to, or protect them from TRAIL-induced apoptosis in a cell line-dependent manner. This appeared to take place upstream of TRAIL-induced activation of Bax in that the conformational changes of Bax and its translocation from the cytosol to mitochondria were either promoted or suppressed by the PKC activator PMA, which was followed by an increase or a decrease in release of cytochrome C and Smac/DIABLO from mitochondria, activation of caspase-3 and cleavage of its substrates, ICAD and PARP. The PKC specific inhibitor, GF 109203X markedly reversed the effect of PMA on TRAIL-induced apoptosis. Overexpression of Bcl-2 inhibted TRAIL-induced apoptosis in the presence of either PMA or GF 109203X. Neither PMA nor GF 109203X caused significant changes in the expression levels of TRAIL-Rs on the cell surface. The protective effect of activation of PKC appeared to be associated with activation of down-stream kinases, Erk1/2 and Akt, as inhibition of either of them by specific inhibitors partially reversed resistance to TRAIL afforded by PMA. In contrast, inhibition of either P38 or JNK MAPKs had only minor effects on regulation of sensitivity to TRAIL by PMA. We examined the constitutive expression levels and phosphorylation status of PKC isozymes in the melanoma cell lines before and after treatment with TRAIL. PKCδ was expressed at relatively high levels in all the cell lines, and was constitutively phosphorylated in the majority of them. In contrast, PKCε expression was found to be lost in those cell lines that were sensitized to TRAIL-induced apoptosis by PMA. It was however expressed by all the cell lines that were protected from TRAIL by PMA. TRAIL-induced a rapid increase in the levels of phosphorylation of both PKCδ and PKCε. Notably, treatment with PMA induced rapid activation of Erk1/2 in all the lines, but activation of another MAPK, JNK, could only be observed in lines that had no PKCε expression. Re-introduction of PKCε into the PKCε-null cell lines resulted in a decrease in sensitivity to TRAIL in the presence of PMA. In contrast, a dominant negative form of PKCε caused an increase in sensitivity to TRAIL-induced apoptosis. These results demonstrate that activation of PKC differentially regulates sensitivity of melanoma to TRAIL-induced apoptosis, and that PKCε plays a critical role in resistance of melanoma to TRAIL.