Celastrol Synergistically Enhances Temozolomide Cytotoxicity in Melanoma Cells

Efforts to improve melanoma response rates to temozolomide (TMZ) have thus far been unsuccessful. We screened a library of 2,000 marketed drugs and natural products to identify agents with the potential to sensitize melanoma cells to the effects of TMZ. Celastrol (CEL), a natural compound found in the Thunder of God vine, was identified based on its ability to enhance cell death in TMZ-resistant melanoma cells. A cell proliferation assay was used to compare the growth-inhibitory effects of TMZ alone versus TMZ/CEL combination treatment. Cytotoxic synergy was assessed using combination-index methods. The expression of nuclear factor-kappa B (NF-kappa B), I kappa B, mitogen-activated protein kinase, and ubiquitinated proteins were examined using Western blotting, and the localization of NF-kappa B in CEL-treated melanoma cells was evaluated using immunofluorescence microscopy. The CEL/TMZ combination synergistically inhibited cell proliferation in melanoma cells. CEL treatment increased the levels of ubiquitinated proteins, reduced the levels of tumor necrosis factor-alpha-induced I kappa B phosphorylation, and blocked NF-kappa B translocation to the nucleus. Inhibition of NF-kappa B with small interfering RNA mimicked the ability of CEL to sensitize melanoma cells to TMZ, suggesting that inhibition of NF-kappa B may play a role in TMZ/CEL-induced cytotoxicity. The TMZ/CEL combination induced the phosphorylation of c-Jun NH2-terminal kinase, implicating the mitogen-activated protein kinase pathway in the treatment effects. Our data suggest that CEL may be effective in sensitizing resistant melanoma cells to the effects of TMZ. (Mol Cancer Res 2009;7(12):1946-53)