Thapsigargin induces a calmodulin/calcineurin-dependent apoptotic cascade responsible for the death of prostatic cancer cells

BACKGROUND. New agents are required for the treatment of androgen-independe nt prostate cancer. Due to the low rate of proliferation of these malignant cells, agents which can activate the apoptotic death of these cells withou t requiring the cells being in the proliferative cell cycle are critically required. Thapsigargin (TG), via its ability to perturb intracellular free calcium [Ca2+](i), is such a cell proliferation-independent cytotoxic agent . The present study focuses on more completely describing the biochemical c ascade during the apoptotic death of androgen-independent prostate cancer c ells induced by TG and on the mechanistic requirements for this death. METHODS. A variety of cell and molecular biology techniques (e.g., time-lap se video, fluorescence image analysis, Northern and Western blotting) were used to examine the temporal relationship between changes in [Ca2+](i) GADD 153 transcription, translocation of the NFATc transcription factor to the nucleus, translocation of BAD from the cytosol to the mitochondria, caspase 9 activation, DNA fragmentation, and the loss of clonogenic survival induc ed by TG treatment of both human TSU-prl and rat AT3.1 prostate cancer cell s in vitro. Additional studies using both microinjection of inhibitors of c almodulin and DNA transfections to induce expression of Ca2+ binding protei ns, e.g., calbindin, were performed to evaluate the causal relationship bet ween [Ca2+](i) elevation, calmodulin/calcineurin activation, and apoptosis of prostate cancer cells. RESULTS. Using simultaneous fluorescence ratiometric and phase contrast ima ge analysis in individual cells followed longitudinally for several days, i t was documented that TG induced early (1-12 hr) moderate (i.e., <500 nM) e levation in [Ca2+](i). During this early rise in [Ca2+](i), genes like GADD 153 are induced at the transcriptional level. This early rise is followed by a return of [Ca2+](i) to baseline (i.e., similar to 50 nM) before the in duction of a delayed (i.e., >12 hr) secondary rise (similar to 10 mu M) in [Ca2+](i). During the secondary rise in [Ca2+](i) Ca2+ binds to calcineurin and calmodulin, allowing these proteins to form a complex which activates calcineurin's latent phosphatase activity. Once activated, calcineurin deph osphorylates NFATc and BAD, allowing translocation of these proteins to the nucleus and mitochondria, respectively. BAD translocation induces the rele ase of cytochrome C from the mitochondria into the cytoplasm, which results in activation of caspase 9 and DNA fragmentation. If the TG-induced rise i n [Ca2+](i) is blocked by overexpressing calbindin, or if calmodulin functi on is inhibited, these apoptotic events are prevented. CONCLUSIONS. TG induces the apoptotic death of prostate cancer cells via th e activation of a reversible signaling phase induced by a transient nanomol ar rise in [Ca2+], which involves new gene transcription and translation. T his reversible signaling phase is followed by an irreversible commitment to undergo the execution phase which is induced by a secondary micromolar ris e in [Ca2+](i). This secondary [Ca2+](i) rise irreversibly commits the cell to a calmodulin/calcineurin-dependent cascade, which results in DNA and ce llular fragmentation into apoptotic bodies. (C) 2000 Wiley-Liss, Inc.