[Phenoxodiol Induces G1 Arrest by Specific Loss in CDK2 Activity by p53-Independent Induction of p21WAF1/CIP1]
>>Cancer Research 65, 3364-3373, April 15, 2005
Experimental Therapeutics, Molecular Targets, and Chemical Biology
Phenoxodiol, a Novel Isoflavone, Induces G1 Arrest by Specific Loss in Cyclin-Dependent Kinase 2 Activity by p53-Independent Induction of p21WAF1/CIP1
Martin F. Aguero, Maria M. Facchinetti, Zhanna Sheleg and Adrian M. Senderowicz
Molecular Therapeutics Unit, Oral & Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland
Requests for reprints: Adrian M. Senderowicz, Molecular Therapeutics Unit, Oral & Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Building 30, Room 212, Bethesda, MD 20892-4330. Phone: 301-594-5270; Fax: 301-402-0823; E-mail: sendero@helix.nih.gov.
Phenoxodiol, an isoflavone derivative of genistein with unknown mechanism of action, is currently being evaluated in early human cancer clinical trials. To determine the mechanism of antiproliferative effects of phenoxodiol, we examined its effects in a battery of human cell lines. Although we observed caspase-dependent apoptosis in HN12 cells as early as 24 hours after exposure, clonogenic death occurred only after 48-hour exposure despite caspase blockade by the general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD)-fmk. Moreover, clear evidence of cell death as determined by nuclear morphology and plasmatic membrane damage occur despite ZVAD, suggesting that another mechanism besides caspase-dependent apoptosis is required for clonogenic death induced by phenoxodiol. In search for other potential antiproliferative effects, we assessed the effects of phenoxodiol in the cell cycle progression of human carcinoma cell lines. A significant G1-S arrest was observed by 12 hours of exposure in HN12 cell lines at concentrations 5 µg/mL. Cell cycle arrest occurred several hours (12 hours) before induction of apoptosis. Analysis of in vitro purified cyclin-dependent kinase (cdk) activity showed that phenoxodiol did not inhibit cdk activity. In contrast, cellular cdk2 activity obtained from HN12 cell lines exposed to phenoxodiol for 12 hours decreased by 60%, whereas cdk6 activity remained unaltered, suggesting that the loss of cdk2 activity was specific. Loss in cdk2 activity was preceded by the accumulation of the endogenous cdk inhibitor p21WAF1. To assess the role of p21WAF1 induction by phenoxodiol, we used HCT116 isogenic cell lines and showed that phenoxodiol induced G1 arrest together with p21WAF1 expression in wild-type clones. In contrast, p21–/– variants failed to show G1 arrest. Finally, induction of p21 by phenoxodiol is p53 independent, as phenoxodiol induced p21 in HCT116 lacking p53. These data therefore indicate that phenoxodiol promotes G1-S arrest by the specific loss in cdk2 activity due to p53-independent p21WAF1 induction. This novel feature of phenoxodiol may have clinical implications, as the majority of human malignancies have aberrations in cell cycle progression regulation. <<
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