Abstract:The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca 2π ] i in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to interact with oestrogens leading to modulation of bone metabolism, the present study was aimed at exploring whether tamoxifen could alter Ca 2π signaling in human osteoblastlike MG63 cells. Cytosolic free Ca 2π levels were recorded by using the Ca 2π -sensitive dye fura-2. Tamoxifen induced a sustained [Ca 2π ] i increase at concentrations above 1 mM with an EC 50 of 8 mM. Removal of extracellular Ca 2π reduced the response by 40%, suggesting that tamoxifen induced both Ca 2π influx and store Ca 2π release. Tamoxifen-induced Ca 2π influx was confirmed as tamoxifen caused Mn 2π influx-induced quench of fura-2 fluorescence. In Ca 2π -free medium, pretreatment with 10 mM tamoxifen abolished the [Ca 2π ] i increase induced by 1 mM thapsigargin (an endoplasmic reticulum Ca 2π pump inhibitor), and by 2 mM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m-chlorophenylhydrazone only reduced 64% of tamoxifeninduced [Ca 2π ] i increases. Addition of 2 mM U73122 to inhibit phospholipase C activity abolished the [Ca 2π

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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.

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