Synergy between enzastaurin doxorubicin in inducing melanoma apoptosis

Melanoma is resistant to most standard chemotherapeutics. We analysed the combined effect of doxorubicin and enzastaurin on cell death of four melanoma cell lines, namely G361, SK-MEL3, A375 and SAN. Enzastaurin IC50 was calculated by measure of growth inhibition with MTS assay and corresponded to 2 μM; the half maximal cytotoxicity of doxorubicin was obtained at 3 μM dose. Evaluation of combination index showed synergism (CI > 1) or additive effect (CI = 1) with all melanoma cell lines, with enzastaurin doses ≥0.6 μM and doxorubicin doses ≥1 μM. Combination of the two drugs resulted in increase in caspase 3 and 8 activation, in comparison with activation by single agents. Caspase 8 activation was impaired by TNFR-1 blocking. Our results show doxorubicin-stimulated production of TNFα, whereas enzastaurin-stimulated TNFR-1 expression on plasma membrane. The effect on TNFR-1 appeared to be mediated by PKCζ inhibition. Taken together, our findings suggest that enzastaurin increases doxorubicin-induced apoptosis of melanoma by a mechanism involving, at least in part, activation of the TNF-α signal.