Gossypol Suppresses In Vitro Head and Neck Squamous Cell Carcinoma Growth through Its Inhibition of DNA Methyltransferase 1 and Epigenetic Activation of Tumor Suppressor Genes
C-Y Fan, C Xie, H Zhang, YC Lin, JY Suen. University of Arkansas for Medical Sciences, Little Rock, AR; John L. McClellan Memorial Veterans Hospital, Little Rock, AR; The Ohio State University, Columbus, OH
Background: Most forms of human malignancy display frequent epigenetic alterations in association with promoter hypermethylation. A key enzyme involved in the initiation and maintenance of DNA hypermethylated state is DNA methyltransferase (DNMT) 1 and this gene is overexpressed in many forms of human malignancies. Gossypol, a male antifertility agent and a natural polyphenolic compound present in cottonseeds, has been shown to possess antiproliferative and pro-apoptotic effects both in vivo and in vitro in a variety of human tumors through its potent inhibition of Bcl2 family of antiapoptotic proteins, protein kinase C, and Cyclin D1. Here, we reported that gossypol is a novel small molecule inhibitor of DNA methyltransferase 1 and can suppress in vitro head and neck cancer growth through its epigenetic effects.
Design: In vitro methylation assay was used to evaluate the inhibition of DNMT1 by gossypol. DNA pyrosequencing and methylation-specific PCR were used to analyze changes in DNA methylation. Real-time RT-PCR and western blot analysis were used to characterize expression of tumor suppressor genes. For in vitro tumor suppression, 6 HNSCC cell lines (UMSCC1, SQ-20B, T-167, T409, TU167, and MDA1986) and a normal oral keratinocyte cell line (HOK-16B) were treated with gossypol followed by determination of cell viability.
Results: Gossypol showed dose-dependent inhibition of DNMT1 in in vitro cell-free methylation assay. As a result, gossypol significantly reduces DNA methylation in the promoter region of MGMT and E-cadherin genes, resulting in reactivation of these 2 tumor suppressor genes. Gossypol also showed dose-dependent inhibition of 5 out of 6 (80%) HNSCC cell lines (UMSCC1, SQ-20B, T-167, T409, and TU167) at biologically achievable doses (less than 10 M) with an IC50 ranging from 2.8 M to 6.2 M for these 5 cell lines.
Conclusions: Gossypol is a novel and potent small molecule inhibitor of DNMT1 with effective DNA demethylating property. In HNSCC, gossypol can suppress in vitro tumor growth in part through epigenetic activation of tumor suppressor genes. Therefore, gossypol.
Tuesday, March 10, 2009 1:00 PM
Poster Session IV # 199, Tuesday Afternoon