Effect of diethylstilbestrol on Ca ²⁺ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca ²⁺ concentrations ([Ca ²⁺ ] _i ) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca ²⁺ ] _i in a concentration-dependent manner (EC ₅₀ =15 μM). DES-induced [Ca ²⁺ ] _i rise was reduced by 80 % by removal of extracellular Ca ²⁺ . DES-induced Mn ²⁺ -associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca ²⁺ influx. In Ca ²⁺ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca ²⁺ -ATPase, caused a monophasic [Ca ²⁺ ] _i rise, after which the increasing effect of DES on [Ca ²⁺ ] _i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca ²⁺ stores totally prevented thapsigargin from releasing more Ca ²⁺ , whereas ionomycin added afterward still released some Ca ²⁺ . These findings suggest that in human breast cancer cells, DES increases [Ca ²⁺ ] _i by stimulating extracellular Ca ²⁺ influx and also by causing intracellular Ca ²⁺ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 μM DES killed cells in a time-dependent manner.