Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer Curtis Balch, 1,4 Pearlly Yan, 2 Teresa Craft, 1 Suzanne Young, 3 David G. Skalnik, 3,5,6 Tim H-M. Huang, 2 and Kenneth P. Nephew 1,4,6 1 Medical Sciences Program, Indiana University, Bloomington, Indiana; 2 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio; Departments of 3 Biochemistry and Molecular Biology and 4 Cellular and Integrative Physiology, Indiana University School of Medicine; 5 Department of Hematology/Oncology, Herman B Wells Center for Pediatric Research; and 6 Indiana University Cancer Center, Indianapolis, Indiana Abstract Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the develop- ment and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemo- sensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(B-D-ribofurano- syl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2V -deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5- aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebular- ine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1 . RASSF1A gene reexpres- sion was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU , although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as deter- mined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug- resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC 50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regi- mens, for the therapy of drug-resistant ovarian cancer. [Mol Cancer Ther 2005;4(10):1505 – 14] Introduction The acquisition of drug resistance is a severe impediment to the successful therapy of ovarian cancer. Although most patients initially respond to a taxane/platinum regimen, relapse occurs in 80% of cases following a median period of 15 months (1). Although chemoresistance has a number of causative factors, one frequent determinant is the positive selection of clones harboring genotypes that allow survival from the intended drug insult (2, 3). Such genotypes can be inherent to the tumor or acquired during treatment but generally involve up-regulation of antiapoptotic oncogenes and/or down-regulation of proapoptotic tumor suppres- sors (3). Tumor suppressors are often rendered nonfunc- tional by deletion, mutation, or translocation; however, in a growing number of cases, loss of function is associated with nongenetic (i.e., epigenetic) modifications (4), primarily DNA methylation. Aberrations of DNA methylation are now accepted as a common instigator in the development and progression of many cancers (4), including ovarian (5–7). In general, neoplasia is correlated with overall genomic hypomethyla- tion, whereas isolated, normally unmethylated CG-rich regions, known as ‘‘CpG islands’’ (often associated with transcriptionally active genes), frequently become hyper- methylated (8). Tumor suppressor genes such as ras- associated domain family 1A (RASSF1A), p16 , and human MutL homologue-1 (hMLH1 ) are frequently silenced by CpG island methylation in numerous tumor types (9); the subsequent interruption of proapoptotic pathways is thought to contribute to increased proliferation and/or drug resistance (2). In particular, loss of mismatch repair gene expression, including that of hMLH1 , is believed to result in inadequate response to platinum agents, resulting in enhanced resistance to those therapies (10). As both hMLH1 and RASSF1A are believed to promote apoptotic responses (9, 11), their silencing likely contributes significantly to the progression of drug-resistant ovarian cancer. To reverse methylation-induced silencing of tumor sup- pressors, a number of inhibitors are currently under investigation in clinical and preclinical trials (12). In several Received 6/30/05; revised 7/29/05; accepted 8/17/05. Grant support: National Cancer Institute grant CA085289 (K.P. Nephew) and US4CA11300 (T.H-M. Huang), National Science Foundation grant MCB0344870 (D.G. Skalnik), and American Heart Association grant 0315222Z (S. Young). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Kenneth P. Nephew, Medical Sciences Program, Indiana University, 302 Jordan Hall, 1001 East Third Street, Bloomington, IN 47405. Phone: 812-855-9445; Fax: 812-855-4436. E-mail: knephew@indiana.edu Copyright C 2005 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-05-0216 1505 Mol Cancer Ther 2005;4(10). October 2005 on June 13, 2020. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from