Effect of celastrol on temozolomide cytotoxicity in melanoma cells and inhibition of NF-kB signaling

Abstract

9076 Background: Temozolomide (TMZ) exhibits clinical activity in the treatment of melanoma and glioblastoma, but response rates are low. Identification of agents that improve TMZ's efficacy and overcome resistance is of great interest. In this study, we have identified celastrol as a natural product that significantly enhances TMZ-induced cytotoxicity by testing a library of drugs and natural products for cytotoxic activity against glioma and melanoma cell lines and have examined its mechanism of action in melanoma cells. Methods: A preliminary screening of a library of 2000 drugs and natural products was performed and a short list of drugs was identified as able to enhance TMZ-induced cell killing in TMZ-resistant cancer cell lines. The effects of these compounds were further confirmed in five melanoma cell lines. A cell proliferation assay was used to compare growth inhibitory effects of single agent TMZ versus combination treatments. Synergy in inhibiting cell proliferation was assessed using combination-index methods. The expression of NF-kB, IkB, MAPK, and PARP were examined using Western blot analysis. The effect of treatments on the cell cycle was examined by flow cytometry. The localization of NF-kB in melanoma cells was evaluated through immunofluorescence microscopy. Results: Combining celastrol and TMZ synergistically inhibited cell proliferation, enhanced cell cycle arrest, and increased apoptosis in a series of melanoma cell lines, compared to treatment with TMZ alone. We further found that celastrol inhibited proteasome activity, TNF-α induced IkB phosphorylation and NF-kB translocation to the nucleus. Inhibition of NF-kB with siRNA mimicked the ability of celastrol to sensitize melanoma cells to TMZ-induced cell killing, suggesting inhibition of NF-kB was indeed involved in TMZ/celastrol-induced cytotoxicity. Furthermore, combination treatment induced phosphorylation of JNK. Conclusions: Our data suggest that combined use of TMZ with celastrol, a natural product derived from a vine extract that has been used orally in Chinese medicine for over a thousand years, may enhance the chemotherapeutic efficacy of TMZ in melanoma. No significant financial relationships to disclose.