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

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 μmol/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 μmol/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)

[1]  B. Wüthrich,et al.  Treatment of human echinococcosis with mebendazole. Preliminary observations in 28 patients. , 1982, Hepato-gastroenterology.

[2]  S. Abuzar,et al.  The benzimidazole anthelmintics--chemistry and biological activity. , 1983, Progress in drug research. Fortschritte der Arzneimittelforschung. Progres des recherches pharmaceutiques.

[3]  The mode of action of some benzimidazole drugs on Trichinella spiralis. , 1987, Parasitology.

[4]  D. Bennett,et al.  A line of non‐tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth , 1987, International journal of cancer.

[5]  E. Lacey.  The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. , 1988, International journal for parasitology.

[6]  E. Lacey.  Mode of action of benzimidazoles. , 1990, Parasitology today.

[7]  C. Garbe,et al.  P53 mutation and c-fos overexpression are associated with detection of the antigen VLA-2 in human melanoma cell lines. , 1994, Pigment cell research.

[8]  H. Kerl,et al.  bcl‐2 Protein Expression in Cutaneous Malignant Melanoma and Benign Melanocytic Nevi , 1995, The American Journal of dermatopathology.

[9]  C. Croce,et al.  Inactivation of Bcl-2 by phosphorylation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  E. Lacey,et al.  Inhibition of [3H]mebendazole binding to tubulin by structurally diverse microtubule inhibitors which interact at the colchicine binding site. , 1995, Biochemistry and molecular biology international.

[11]  W. B. Derry,et al.  Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. , 1996, Cancer research.

[12]  T. Fojo,et al.  Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. , 1997, Cancer research.

[13]  W. May,et al.  Bcl-2 Phosphorylation Required for Anti-apoptosis Function* , 1997, The Journal of Biological Chemistry.

[14]  T. Kurosaki,et al.  Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. , 1998, Cancer detection and prevention.

[15]  C. Croce,et al.  Serine-70 is one of the critical sites for drug-induced Bcl2 phosphorylation in cancer cells. , 1998, Cancer research.

[16]  H. Pehamberger,et al.  Expression of Bcl-2 family members in human melanocytes, in melanoma metastases and in melanoma cell lines , 1998, Melanoma research.

[17]  P Wadsworth,et al.  Taxol suppresses dynamics of individual microtubules in living human tumor cells. , 1999, Molecular biology of the cell.

[18]  S. Korsmeyer.  BCL-2 gene family and the regulation of programmed cell death. , 1995, Cancer research.

[19]  E. Nogales.  Structural insights into microtubule function. , 2000, Annual review of biochemistry.

[20]  W. Kratzer,et al.  Benzimidazoles in the treatment of alveolar echinococcosis: a comparative study and review of the literature. , 2000, The Journal of antimicrobial chemotherapy.

[21]  S. Orlow,et al.  Inverse correlation between pink-eyed dilution protein expression and induction of melanogenesis by bafilomycin A1. , 2001, Pigment cell research.

[22]  D. Morris,et al.  Pilot Study of Albendazole in Patients with Advanced Malignancy , 2001, Oncology.

[23]  D. Schadendorf,et al.  Drug‐resistance in human melanoma , 2001, International journal of cancer.

[24]  D. Morris,et al.  In vitro and in vivo suppression of growth of hepatocellular carcinoma cells by albendazole. , 2001, Cancer letters.

[25]  E. Nogales.  Structural insight into microtubule function. , 2001, Annual review of biophysics and biomolecular structure.

[26]  L. Yépez-Mulia,et al.  Synthesis, antiprotozoal and anticancer activity of substituted 2-trifluoromethyl- and 2-pentafluoroethylbenzimidazoles. , 2002, European journal of medicinal chemistry.

[27]  Jack A Roth,et al.  Mebendazole elicits a potent antitumor effect on human cancer cell lines both in vitro and in vivo. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[28]  E. McClay,et al.  Systemic chemotherapy for the treatment of metastatic melanoma. , 2002, Seminars in oncology.

[29]  Jack A Roth,et al.  The anthelmintic drug mebendazole induces mitotic arrest and apoptosis by depolymerizing tubulin in non-small cell lung cancer cells. , 2002, Molecular cancer therapeutics.

[30]  John A Hadfield,et al.  Tubulin and microtubules as targets for anticancer drugs. , 2003, Progress in cell cycle research.

[31]  H. Pehamberger,et al.  Small interfering RNA targeting bcl-2 sensitizes malignant melanoma. , 2003, Oligonucleotides.

[32]  J. Jiricny,et al.  The Effect of O 6-Alkylguanine-DNA Alkyltransferase and Mismatch Repair Activities on the Sensitivity of Human Melanoma Cells to Temozolomide, 1,3-bis(2-Chloroethyl)1-nitrosourea, and Cisplatin , 2003, Journal of Pharmacology and Experimental Therapeutics.

[33]  S. Lowe,et al.  Apoptosis and melanoma chemoresistance , 2003, Oncogene.

[34]  M. Iqbal,et al.  Immunomodulatory effect of various anti-parasitics: a review , 2005, Parasitology.

[35]  S. Lowe,et al.  Differential regulation of noxa in normal melanocytes and melanoma cells by proteasome inhibition: therapeutic implications. , 2005, Cancer research.

[36]  D. Boykin,et al.  Synthesis and potent antibacterial activity against MRSA of some novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidines. , 2005, European journal of medicinal chemistry.

[37]  H. Pehamberger,et al.  Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the Oblimersen Melanoma Study Group. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[38]  G. Tchernev,et al.  Downregulation of cell cycle modulators p21, p27, p53, Rb and proapoptotic Bcl‐2‐related proteins Bax and Bak in cutaneous melanoma is associated with worse patient prognosis: preliminary findings , 2007, Journal of cutaneous pathology.

[39]  R. Scolyer,et al.  Mcl-1, Bcl-XL and Stat3 expression are associated with progression of melanoma whereas Bcl-2, AP-2 and MITF levels decrease during progression of melanoma , 2007, Modern Pathology.

[40]  E. Livingston,et al.  Screening and detection of apoptosis. , 2007, The Journal of surgical research.

[41]  Giovanni Mazzoni,et al.  Mebendazole inhibits growth of human adrenocortical carcinoma cell lines implanted in nude mice , 2008, Cancer Chemotherapy and Pharmacology.

[42]  N. Doudican,et al.  Inhibition of mitochondrial protein translation sensitizes melanoma cells to arsenic trioxide cytotoxicity via a reactive oxygen species dependent mechanism , 2008, Cancer Chemotherapy and Pharmacology.