Effect of Diethylstilbestrol (DES) on Ca^2+ Homeostasis and Viability in OC2 Human Oral Cancer Cells

Abstract

The effect of diethylstilbestrol (DES), a synthetic estrogen, on cytosolic free Ca^2+ concentrations ([Ca^2+]i) in OC2 human oral cancer cells is unclear. This study explored whether DES elevated basal [Ca^2+]i levels in suspended cells by using fura-2 as a Ca^2+-sensitive fluorescent dye. DES at concentrations between 10-150 μM increased [Ca^2+]i in a dose-dependent manner. The Ca^2+signal was reduced partly by removing extracellular Ca^2+. DES-induced Ca^2+influx was inhibited by nifedipine, econazole, SK&F96365, phorbol 12-myristate 13 acetate and GF109203X. In Ca^2+-free medium, 150 μM DES pretreatment abolished the [Ca^2+]i rise induced by the endoplasmic reticulum Ca^2+pump inhibitors thapsigargin/ 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin/BHQ partly reduced DES-induced [Ca^2+]i rise. Inhibition of inositol 1, 4, 5-trisphosphate formation with U73122 did not alter DESinduced [Ca^2+]i rise. At concentrations between 10 and 50 μM DES killed cells in a dose-dependent manner. The cytotoxic effect of DES was not reversed by prechelating cytosolic Ca^2+with an acetoxy-methyl ester of 1,2-bis(2- aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA/AM). Annexin V/propidium iodide staining data suggest that DES induced apoptosis in a dosedependent manner. DES did not increase levels of reactive oxygen species (ROS). Together, in OC2 human oral cancer cells, DES induced a [Ca^2+]i rise by inducing phospholipase C-independent Ca^2+release from the endoplasmic reticulum and Ca^2+entry via protein kinase C-sensitive store-operated Ca^2+channels. DES induced cell death that involved apoptosis via non-mitochondrial pathways.