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-independent prostate cancer. Due to the low rate of proliferation of these malignant cells, agents which can activate the apoptotic death of these cells without requiring the cells being in the proliferative cell cycle are critically required. Thapsigargin (TG), via its ability to perturb intracellular free calcium [Ca(2+)](i), is such a cell proliferation-independent cytotoxic agent. The present study focuses on more completely describing the biochemical cascade during the apoptotic death of androgen-independent prostate cancer cells induced by TG and on the mechanistic requirements for this death.


METHODS
A variety of cell and molecular biology techniques (e.g., time-lapse video, fluorescence image analysis, Northern and Western blotting) were used to examine the temporal relationship between changes in [Ca(2+)](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 induced by TG treatment of both human TSU-prl and rat AT3.1 prostate cancer cells in vitro. Additional studies using both microinjection of inhibitors of calmodulin and DNA transfections to induce expression of Ca(2+) binding proteins, e.g., calbindin, were performed to evaluate the causal relationship between [Ca(2+)](i) elevation, calmodulin/calcineurin activation, and apoptosis of prostate cancer cells.


RESULTS
Using simultaneous fluorescence ratiometric and phase contrast image analysis in individual cells followed longitudinally for several days, it was documented that TG induced early (1-12 hr) moderate (i.e., <500 nM) elevation in [Ca(2+)](i). During this early rise in [Ca(2+)](i), genes like GADD 153 are induced at the transcriptional level. This early rise is followed by a return of [Ca(2+)](i) to baseline (i.e., approximately 50 nM) before the induction of a delayed (i.e., >12 hr) secondary rise ( approximately 10 microM) in [Ca(2+)](i). During the secondary rise in [Ca(2+)](i), Ca(2+) binds to calcineurin and calmodulin, allowing these proteins to form a complex which activates calcineurin's latent phosphatase activity. Once activated, calcineurin dephosphorylates NFATc and BAD, allowing translocation of these proteins to the nucleus and mitochondria, respectively. BAD translocation induces the release of cytochrome C from the mitochondria into the cytoplasm, which results in activation of caspase 9 and DNA fragmentation. If the TG-induced rise in [Ca(2+)](i) is blocked by overexpressing calbindin, or if calmodulin function is inhibited, these apoptotic events are prevented.


CONCLUSIONS
TG induces the apoptotic death of prostate cancer cells via the activation of a reversible signaling phase induced by a transient nanomolar rise in [Ca(2+)](i), which involves new gene transcription and translation. This reversible signaling phase is followed by an irreversible commitment to undergo the execution phase which is induced by a secondary micromolar rise in [Ca(2+)](i). This secondary [Ca(2+)](i) rise irreversibly commits the cell to a calmodulin/calcineurin-dependent cascade, which results in DNA and cellular fragmentation into apoptotic bodies.
Tombal
B
B
Johns Hopkins Oncology Center, Baltimore, Maryland 21231, USA.
Weeraratna
A T
AT
Denmeade
S R
SR
Isaacs
J T
JT
D000818:Animals; D017209:Apoptosis; D019703:Calcineurin / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D006801:Humans; D008297:Male; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011471:Prostatic Neoplasms / Q000473:pathology*; D051381:Rats; D015398:Signal Transduction; D019284:Thapsigargin / Q000627:therapeutic use*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051025:bcl-Associated Death Protein
C494819:BAD protein, human; C494821:Bad protein, rat; D002147:Calmodulin; D002352:Carrier Proteins; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; D019284:Thapsigargin; D019703:Calcineurin; D002118:Calcium