Resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells.
Resveratrol is one of the most extensively studied cancer chemopreventive agents; however, its mechanisms of action are not completely understood. Here, we observed that resveratrol induces S phase arrest via Tyr15 phosphorylation of Cdc2 in human ovarian carcinoma Ovcar-3 cells. Overexpression of Cdc2AF, a mutant resistant to Thr14 and Tyr15 phosphorylation, ablated resveratrol-induced S phase arrest. Further upstream, we observed that resveratrol causes phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinases Chk1 and Chk2, which in turn were activated via ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia-Rad3-related) kinase in response to DNA damage, as resveratrol also increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM/ATR in response to DNA damage. The involvement of these molecules in resveratrol-induced S phase was also supported by the studies showing that addition of ATM/ATR inhibitor caffeine reverses resveratrol-caused activation of ATM/ATR-Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X, and S phase arrest. In additional studies assessing whether observed effects of resveratrol are specific to Ovcar-3 cells, we observed that it also induces S phase arrest and H2A.X (Ser139) phosphorylation in other ovarian cancer cell lines PA-1 and SKOV-3, albeit at different levels; whereas, resveratrol showed only marginal S phase arrest in normal human foreskin fibroblasts with undetectable level of phospho-H2A.X (Ser139). These findings for the first time identify that resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for DNA damage and S phase arrest selectively in ovarian cancer cells, and provide a rationale for the potential efficacy of ATM/ATR agonists in the prevention and intervention of cancer.
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Cancer Center, Denver, CO 80262, USA.
Alpna
A
Tyagi
Rana P
RP
Singh
Chapla
C
Agarwal
Sunitha
S
Siriwardana
Robert A
RA
Sclafani
Rajesh
R
Agarwal
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001260:Ataxia Telangiectasia / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077185:Resveratrol; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors; D016196:S Phase / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*; D013267:Stilbenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D020687:cdc25 Phosphatases / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D013267:Stilbenes; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D012264:Ribonucleotide Reductases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D020687:cdc25 Phosphatases; D000077185:Resveratrol