Apoptosis induced by doxorubicin in neurotumor cells is divorced from drug effects on ceramide accumulation and may involve cell cycle-dependent caspase activation

Doxorubicin (0.5 μg/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident ~ 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B₁ (25 μM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G₂/M phase block in both cell lines, but whereas the G₁ phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G₁ and G₂/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G₂/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G₁ cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G₁ cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.