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Carcinogenesis Advance Access originally published online on February 12, 2009
Carcinogenesis 2009 30(4):662-670; doi:10.1093/carcin/bgp042
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

BTG3 tumor suppressor gene promoter demethylation, histone modification and cell cycle arrest by genistein in renal cancer

Shahana Majid, Altaf A. Dar1, Ardalan E. Ahmad, Hiroshi Hirata, Kazumori Kawakami, Varahram Shahryari, Sharanjot Saini, Yuichiro Tanaka, Angela V. Dahiya, Gaurav Khatri and Rajvir Dahiya*

Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, CA 94121, USA
1 Department of Dermatology, University of California, San Francisco, San Francisco, CA 94121, USA

* To whom correspondence should be addressed. Urology Research Center (112F), Veterans Affairs Medical Center and University of California at San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA. Tel: +1415 750 6964; Fax: +1 415 7506639; Email: rdahiya{at}urology.ucsf.edu

BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2'-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and RNA polymerase II at the BTG3 promoter indicative of active histone modifications. Enzymatic assays showed genistein and 5Aza-C decreased DNA Methyltransferase, methyl-CpG-binding domain 2 activity and increased HAT activity. Cell cycle and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide cell proliferation assays showed that genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. This is the first report to show that BTG3 is epigenetically silenced in RCC and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein had similar effects to that of 5Aza-C, which is a potent demethylating agent with high toxicity and instability. Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of RCC cells, making it a promising candidate for epigenetic therapy in renal carcinoma.

Abbreviations: 5Aza-C, 5-aza-2'-deoxycytidine; DNMTase, DNA Methyltransferase; HAT, histone acetylase; HDAC, histone deacetylase; MBD, methyl-CpG-binding domain; mRNA, messenger RNA; PCR, polymerase chain reaction; RCC, renal cell carcinoma; TSG, tumor suppressor gene

Received November 20, 2008; revised February 5, 2009; accepted February 5, 2009.


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