Interactions of vitamin D analogue CB1093, TNFα and ceramide on breast cancer cell apoptosis

Abstract

Mechanisms by which vitamin D analogues promote apoptosis in tumour cells are unclear. In this study we have examined possible interactions between the synthetic vitamin D analogue CB1093 and two other known mediators of apoptosis, TNFα and ceramide, in MCF-7, T47D and Hs578T breast cancer cells. These studies indicated that cytosolic phospholipase A 2 (cPLA 2) is involved in CB1093 as well as TNFα-mediated cell death. CB1093 promoted both TNFα and ceramide-induced c-PLA 2 activation, which was inversely related to loss of cell viability in MCF-7 and Hs578T cells. TNFα alone (5–20 ng/ml) failed to induce cytotoxicity and activation of cPLA 2 in T47D cells. However, pretreatment of these cells with CB1093 potentiated C 2-ceramide-induced cPLA 2 activation and cell death. Treatment with CB1093 alone induced loss of cell viability and DNA fragmentation in all three cell lines by 5 days and these effects were accompanied by activation of cPLA 2. Furthermore, co-treatment with the cPLA 2 inhibitor AACOCF 3 led to partial protection against loss of cell viability induced by CB1093 in Hs578T and T47D cells as well as MCF-7 cells. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFα but not C 2-ceramide and CB1093-mediated release of arachidonic acid and cell death in MCF-7 cells. These results indicate that CB1093 potentiates responsiveness of breast cancer cells to TNFα and suggest that ceramide and/or cPLA 2 might be involved as downstream effectors in vitamin D-mediated caspase-independent cell death.