Nimbolide induces mitochondrial-dependent apoptosis through activation of ERK1/2 mediated inhibition of clapsin expression in human hepatocellular carcinoma cells

[Speaker] Hui-Ling Chiou:1
[Co-author] Ching-Yi Lin:2, Yi-Hsien Hsieh:2,3
1:School of Medical Laboratory adn Biotechnology, Chung Shan Medical University, Taiwan, 2:Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan, 3:Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan

Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anti-tumor, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the antitumor effects and underlying mechanism of nimbolide on human hepatocellular carcinoma (HCC) cells remain unclear. Our results showed that nimbolide treatment had a cytotoxic effect on Huh-7 and PLC/PRF/5 cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment of HCC cells resulted in G2/M arrest and accompanied with a decrease expression of cyclin B1, cdc25c, and an increase in the phosphorylation of cdc25c and -H2AX and p27. Nimbolide also induced apoptosis in a dose-dependent manner as determined by Annexin V-FITC/ PI double-staining assay. The expression of caspase-7, -9, -3 and poly (ADP)-ribose polymerase (PARP) were increased in nimbolide-treated Huh-7 and PLC/PRF/5 cells. Additionally, nimbolide induced mitochondrial depolarization, which was accompanied by an increased level of Bax and decreased levels of Bcl-2 and Mcl-1. To identify the potential targets of nimbolide, human apoptosis antibody array analysis was performed, and the results indicated that the nimbolide treatment inhibited the protein and mRNA expression of claspin. Moreover, nimbolide induced activation of ERK1/2 phosphorylation, and treatment with a MEK inhibitor (PD98059) significantly reduced nimbolide-induced caspase-3/-9/-PARP activation, apoptosis and enhanced the claspin expression in Huh-7 cells. Using Huh-7 xenograft nude mice that orally intake of nimbolide dose-dependently inhibited tumor growth accompanied by decreased Ki-67 expression in tumor tissues. Taken together, our study demonstrated that Nimbolide may be a promising therapeutic strategy for the treatment of HCC.
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