Tamoxifen-induced increases in cytoplasmic free Ca2+ levels in human breast cancer cells.

Abstract

Tamoxifen has been shown to increase cytoplasmic free Ca2+ levels [Ca2+]i in renal tubular cells and bladder cancer cells, and to after Ca2+ signaling in MCF-7 breast cancer cells. The present study examined the effect of tamoxifen on [Ca2+], in ZR-75-1 human breast cancer cells using fura-2 as an indicator. Tamoxifen increased [Ca2+]i at a concentration above 2 microM with an EC50 of 5 microM. Removing extracellular Ca2+ reduced the response by 48+/-2%. In Ca2+-free medium, after tamoxifen-induced [Ca2+]i increased had returned to baseline, adding 3 mM Ca2+ increased [Ca2+]i in a concentration-dependent manner. Further, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor); and conversely, pretreatment with thapsigargin prevented tamoxifen from releasing more Ca2+. Tamoxifen (10 microM)-induced Ca2+ release was not changed by inhibiting phospholipase C activity with 2 microM U73122. Trypan blue exclusion assay revealed that tamoxifen (1-10 microM) did not alter viability after 1 min of incubation, but killed 10% of cells after 3-10 min of incubation. Together, this study shows that tamoxifen (>2 microM) induced a significant, immediate increase in [Ca2+]i in ZR-75-1 breast cancer cells. Tamoxifen acted by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from extracellular medium. Tamoxifen may be of mild cytotoxicity after acute exposure.