Chemoresistant Melanoma Cells Mebendazole Induces Apoptosis via Bcl-2 Inactivation in

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 Mmol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC50 of 0.32 Mmol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent.(Mol Cancer Res 2008;6(8):1308–15) Introduction The lack of response of metastatic melanoma to a wide range of antineoplastic treatments suggests the presence of complex drug resistance mechanisms. Although most cancers selectively acquire drug resistance following drug treatment, drug resistance in melanoma is thought to be largely due to intrinsic survival factors (1). In this regard, several lines of evidence suggest that defects in apoptotic signaling cascades may represent a critical event in melanoma progression (2). Consistent with this notion are reports of modified expression of proapoptotic and antiapoptotic factors such as Bcl-2, Bcl-XL, and Mcl-1 in melanoma (3, 4). In this study, we screened a library of 2,000 small molecules to identify new antimelanoma agents. The major advantage of this library is its collection of well-characterized drugs, many of which are already approved for human use in the United States, allowing a reduction in the time and effort required to advance promising candidates in a clinical setting. Our study describes the therapeutic utility and mechanism of action of mebendazole, one of the compounds identified from this screen as an antimelanoma agent. Antitumor activity of mebendazole has been previously described in lung and adrenocortical carcinomas (5, 6). Furthermore, mebendazole has been shown to induce depolymerization of tubulin in a variety of cancer models (6, 7). However, to date, the effect of this well-tolerated drug in melanoma cells has not been examined. The data presented in this report show that mebendazole selectively inhibits growth and induces apoptosis in melanoma cells at clinically achievable doses that are largely nontoxic to melanocytes. Our data provide evidence that the antineoplastic effects of mebendazole in human melanoma cells result from differential Bcl-2–mediated cellular responses to mebendazoleinduced tubulin disruption. Results Library Screening Identifies Mebendazole as an Inhibitor of Melanoma Cell Proliferation To discover new antineoplastic agents useful in the treatment of melanoma, we did a screen of 2,000 small molecules in chemoresistant melanoma cell lines. M-14 and SK-Mel-19 melanoma cell lines display resistance to a variety of traditional chemotherapies, including temozolomide, cisplatin, and arsenic trioxide (8-10). Additionally, these cell lines were chosen based on their differential p53 status—M-14 expresses mutant Received 11/14/07; revised 4/4/08; accepted 5/5/08. Grant support: Jacklyn and Miguel Bezos (S.J. Orlow). 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: Seth J. Orlow, New York University School of Medicine, 560 First Avenue, New York, NY 10016. Phone: 212-263-5245; Fax: 212-2638752. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-2159 5 Unpublished results. Mol Cancer Res 2008;6(8). August 2008 1308 American Association for Cancer Research Copyright © 2008 on October 13, 2011 mcr.aacrjournals.org Downloaded from Published OnlineFirst July 30, 2008; DOI:10.1158/1541-7786.MCR-07-2159