The Role of Calcium, pH, and Cell Proliferation in the Programmed (Apoptotic) Death of Androgen-independent Prostatic Cancer Cells Induced by Thapsigargin

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-dependent ATPase family of intracellular Ca2+-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene γ-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca2+-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca2+-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Ca,) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Ca, is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian Mr 28,000 calbindin D Ca2+-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (>1 h) elevation in Ca1 and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., >1 h) elevation in Ca,.