In Vitro and In Vivo Studies of Pharmacological Effects of Zerumbone on Human Malignant Melanoma

Abstract

This study aims to dissect the molecular mechanisms by which sesquiterpene zerumbone (ZER) exerts its inhibitory effects on human melanoma cells, and examine the potential therapeutic effect of ZER on melanoma development and metastasis in mouse xenograft model. Methods: The inhibitory effect of ZER at physiological concentration on WM1552C cell was examined by MTT (methyl thiazolyl tetrazolium) assay. Subcutaneous inoculation and tail vein injection of melanoma cells in murine model were used for in vivo study of potential therapeutic effect of ZER. The roles of apoptosis, autophagy, and oxidative stress in the ZER-induced cell death of WM1552C cells were assessed using the according inhibitors. Autophagy was confirmed by MDC staining, and detecting autophagic marker LC3B. The expression of apoptosis, autophagy, and cancer- related genes were measured by real-time RT-PCR and Western Blot at mRNA and protein level. The expression profile of microRNA in ZER-treated WM1552C cells was analyzed by miRNA PCR array. Results: ZER inhibited the proliferation of WM1552C in long term at physiological concentrations and induced apoptosis, autophagy, and oxidative stress in cells, which lead to cell death. Meanwhile, in vivo study showed that ZER significantly reduced the tumor mass and lung metastasis (p < 0.05) in C57 BL/6 mice. In line with this finding, ZER was found to induce apoptosis and autophagy in melanoma cell lines. In addition, ZER inhibited the activation of Akt and MAPK and abolished NF-κB activation in does- dependant manners. In WM1552C cells treated with ZER, 6 human miRNAs were up-regulated and 13 miRNAs were down-regulated as detected with miRNA PCR array analysis. Conclusion: ZER may exert potent anti-melanoma effects by inducing apoptosis, autophagy, and oxidative stress, and regulating cancer survival pathway and expression of miRNA in vitro. The in vivo study suggests that zerumbone can be a potential chemotherapeutic candidate for deterring growth and metastasis of melanoma.