Calcium as a mediator of 1,25-dihydroxyvitamin D 3-induced apoptosis

Abstract

Cellular calcium has been implicated in induction of apoptosis. We have shown that 1,25(OH) 2D 3-induced apoptosis is associated with a sustained increase in concentration of intracellular Ca 2+ ([Ca 2+] i) resulting from depletion of the endoplasmic reticulum (ER) Ca 2+ stores and activation of the voltage-insensitive Ca 2+ entry pathway [1,25-Dihydroxyvitamin D 3, intracellular Ca 2+ 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) 2D 3-induced apoptosis in breast cancer cells and compare effects of the hormone on Ca 2+ and apoptosis in cancer and normal human mammary epithelial cells. The treatment of MCF-7 breast cancer cells with 1,25(OH) 2D 3 induced a sustained increase in [Ca 2+] i and activated the Ca 2+-dependent proapoptotic proteases, μ-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the calpain substrate. The selective inhibition of Ca 2+ binding sites of μ-calpain decreased apoptotic indices in the 1,25(OH) 2D 3-treated cells. 1,25(OH) 2D 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) 2D 3 triggered a transient Ca 2+ response, which was not accompanied by the calpain and caspase activation. HMEC, but not MCF-7 cells expressed the Ca 2+ binding protein calbindin-D 28k and buffered Ca 2+ increases induced by a Ca 2+ ionophore ionomycin. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH) 2D 3: increase in [Ca 2+] i →μ-calpain activation → caspase-12 activation → apoptosis. Our findings also imply that differences of Ca 2+ regulatory mechanisms in breast cancer versus normal mammary epithelial cells underlay resistance of normal cells and susceptibility of cancer cells to 1,25(OH) 2D 3-induced Ca 2+-mediated apoptosis.