Delayed micromolar elevation in intracellular calcium precedes induction of apoptosis in thapsigargin-treated breast cancer cells

Thapsigargin (TG), a highly specific inhibitor of the sacroplasmic reticulum and endoplasmic reticulum Ca²⁺-ATPase pump, can induce apoptosis in a variety of epithelial and lymphoid cell types. In prostate cancer cell lines, TG induces an initial 5- to 10-fold elevation of intracellular calcium ([Ca²⁺](i)) within a few minutes of exposure. With prolonged exposure times (i.e., 12-36 h) a second elevation of [Ca²⁺](i) to >10 μM is observed. In this study, the human breast carcinoma cell lines MCF-7 and MDA MB 468 cells were used to determine the temporal relationship between TG-induced elevation of [Ca²⁺](i) and activation of programmed cell death. Using a microinjection method that allows for long-term analysis of [Ca²⁺](i) changes, we found that after TG exposure, calcium measurements in these cells demonstrated an initial rise (>4-fold) in [Ca²⁺](i) that occurred within minutes and returned to baseline within a few hours. With prolonged TG exposure, the cells underwent a second elevation (>5 μM) of [Ca²⁺](i) occurring stochastically between 12 and 36 h after initial exposure to TG. Both of the cell lines were growth-inhibited by 100 nM TG after only 1 h of exposure, but clonogenic ability in the MCF-7 cells was significantly reduced only after 48 h of exposure. The induction of apoptosis by TG was demonstrated by morphological changes typical for programmed cell death and DNA fragmentation (both high molecular weight and oligonucleosomal-sized fragments were detected) after 48 h of treatment. TG induction of apoptosis in these breast cancer cells occurred subsequent to the secondary rise in [Ca²⁺](i), which confirmed that this secondary rise in [Ca²⁺](i) is not prostate cancer-specific. The secondary rise in [Ca²⁺](i) to micromolar levels may directly activate the endonucleases responsible for DNA fragmentation that occurs as part of the apoptotic process. These studies indicate that TG is an active agent in vitro against breast cancer cells. Inactive prodrug analogues of TG are currently being developed that can be activated by tissue-specific proteases, and further pursuit of this strategy as a potential treatment for breast cancer is warranted.