Caspase-independent induction of apoptosis in human melanoma cells by the proapoptotic Bcl-2-related protein Nbk Bik

The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk / Bik) has been described to inhibit Bcl-2 and Bcl-x L , thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk / Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk / Bik only weakly at the mRNA and protein level. Conditional expression of Nbk / Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk / Bik in vivo , stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk / Bik expression. By investigating the mechanism of Nbk / Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk / Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk / Bik.