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

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, TNF alpha and ceramide, Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFa as assessed by cell viability assay, DNA fragmentation and videomicroscopy, No significant chan ges in the levels of TNF alpha or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C-2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A(2) (cPLA(2)) has been implicated in TNF alpha-mediated apoptosis, As assessed by [H-3]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM)showed enhanced cPLA(2) activation in response to TNF alpha or ceramide. CB1093 treatment alone led to cPLA(2) activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF(3), These results suggest that TNF alpha and vitamin D analogs share a common pathway leading to apoptosis involving cPLA(2) activation and/or ceramide generation.