Potentiation by vitamin D analogs of TNFα and ceramide-induced apoptosis in MCF-7 cells is associated with activation of cytosolic phospholipase A2

Abstract

Synthetic analogs of Vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFα and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFα as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFα or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFα-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFα or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFα and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.