Calcium as a mediator of 1,25-dihydroxyvitamin D3-induced apoptosis

Cellular calcium has been implicated in induction of apoptosis. We have shown that 1,25(OH)(2)D-3-induced apoptosis is associated with a sustained increase in concentration of intracellular Ca2+ ([Ca2+](i)) resulting from depletion of the endoplasmic reticulum (ER) Ca2+ stores and activation of the voltage-insensitive Ca2+ entry pathway [1,25-Dihydroxyvitamin D-3, intracellular Ca2+ and apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid Hormone, University of California, Riverside, 1997, pp. 473-474; Vitamin D and intracellular calcium, in: P. Quinn, V. Kagan (Eds.), Subcellular Biochemistry: Fat-Soluble Vitamins, Plenum Press, New York, 1998, pp. 271-297; 1,25-Dihydroxyvitamin D-3 and calcium signaling, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 715-718; 1,25-Dihydroxyvitamin D-3 triggers calcium-mediated apoptosis in breast cancer cells, in: A.W. Norman, R. Bouillon, M. Thomasset (Eds.), Vitamin D Endocrine System: Structural, Biological, Genetic and Clinical Aspects, University of California, Riverside, 2000, pp. 399-402; Endocrine 9 (1998) 321]. This study was undertaken to investigate mechanism of 1,25(OH)(2)D-3-induced apoptosis in breast cancer cells and compare effects of the hormone on Ca2+ and apoptosis in cancer and normal human mammary epithelial cells. The treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D-3 induced a sustained increase in [Ca2+](i) and activated the Ca2+-dependent proapoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain substrate. The selective inhibition of Ca2+ binding sites of I-L-calpain decreased apoptotic indices in the 1,25(OH)(2)D-3-treated cells. 1,25(OH)(2)D-3 did not induce apoptosis in normal human mammary epithelial cells (HMECs), as evaluated by DNA fragmentation (TUNEL), loss of the plasma membrane asymmetry (Annexin V assay) and morphological criteria. In these cells, 1,25(OH)(2)D-3 triggered a transient Ca2+ response, which was not accompanied by the calpain and caspase activation. HMEC, but not MCF-7 cells expressed the Ca2+ binding protein calbindin-D-28k and buffered Ca2+ increases induced by a Ca2+ ionophore ionomycin. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D-3: increase in [Ca2+](i) --> mu-calpain activation --> caspase-12 activation --> apoptosis. Our findings also imply that differences of Ca2+ regulatory mechanisms in breast cancer versus normal mammary epithelial cells underlay resistance of normal cells and susceptibility of cancer cells to 1,25(OH)(2)D-3-induced Ca2+-mediated apoptosis. (C) 2004 Elsevier Ltd. All rights reserved.