Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells

The active form of vitamin D-3 (1,25(OH)(2)D-3) induces an increase in the intracellular free calcium ([Ca2+](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D-3 or its chemotherapeutic analog, EB 1089, releases Ca2+ from the endoplasmic reticulum. The increase in [Ca2+](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D-28k, in MCF-7 cells not only attenuated the elevation in [Ca2+](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D-3 or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.