Effect of the anti-breast cancer drug tamoxifen on Ca²⁺ movement in human osteosarcoma cells

The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca²⁺]_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 Ca²⁺ signaling in hmnan osteoblast-like MG63 cells. Cytosolic free Ca²⁺ levels were recorded by using the Ca²⁺-sensitive dye fura-2. Tamoxifen induced a sustained [Ca²⁺]_i increase at concentrations above 1 μM with an EC₅₀ of 8 μM. Removal of extracellular Ca²⁺ reduced the response by 40%, suggesting that tamoxifen induced both Ca²⁺ influx and store Ca²⁺ release. Tamoxifen-induced Ca²⁺ influx was confirmed as tamoxifen caused Mn²⁺ influx-induced quench of fura-2 fluorescence. In Ca²⁺-free medium, pretreatment with 10 μM tamoxifen abolished the [Ca²⁺]_i increase induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), and by 2 μM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m-chlorophenylhydrazone only reduced 64% of tamoxifen-induced [Ca²⁺]_i increases. Addition of 2 μM U73122 to inhibit phospholipase C activity abolished the [Ca²⁺]_i increase induced by 1 μM histamine, a phospholipase C-dependent Ca²⁺ mobilizer, without affecting 10 μM tamoxifen-induced Ca²⁺ release. The [Ca²⁺]_i increase induced by 10 μM tamoxifen was not altered by 10 μM of nifedipine, verapamil and diltiazem. Together, the data show that tamoxifen induced a lasting increase in [Ca²⁺]_i in human osteoblast-like cells by causing Ca²⁺ influx and releasing Ca²⁺ from multiple stores in a phospholipase C-independent manner.