Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells.
BACKGROUND
Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro.


METHODS
We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively.


RESULTS
Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin.


CONCLUSIONS
Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.
Department of Surgery, Mater Misericordiae University Hospital, Conway Institute of Biomolecular and Biomedical Research, Dublin Molecular Medicine Centre, University College Dublin, Dublin, Ireland.
Colm
C
Morrissey
Amanda
A
O'Neill
Barbara
B
Spengler
Volker
V
Christoffel
John M
JM
Fitzpatrick
R William G
RW
Watson
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D047310:Apigenin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004347:Drug Interactions; D004847:Epithelial Cells / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D048789:Phytoestrogens / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D011480:Protease Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012838:Silymarin / Q000037:antagonists & inhibitors / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D009842:Oligopeptides; D048789:Phytoestrogens; D011480:Protease Inhibitors; D017382:Reactive Oxygen Species; D012838:Silymarin; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D047310:Apigenin; D020169:Caspases