Regulation of demethylation and re-expression of RASSF1A gene in hepatocellular carcinoma cell lines treated with NCTD in vitro

Abstract

BACKGROUND: Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD), the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear. PURPOSE: The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells. MATERIALS AND METHODS: Human HepG2 cell lines were treated with NCTD at different concentrations (2.50, 5.00, 10.00, 20.00, 40.00 μg/mL) for 24 hours. Cell proliferation was evaluated by measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The methylation levels of RASSF1A (Ras-association domain family 1 A) in HepG2 cells were detected by methylation-specific PCR (MSP). The mRNA levels of RASSF1A in HepG2 cells were detected by real-time fluorescent quantitative PCR (RT-PCR). The levels of RASSF1A protein expression of HepG2 cells were detected by Western blotting assay. RESULTS: The inhibition of cell proliferation was observed when treated with NCTD at concentrations (2.5 μg/mL), and as concentration increased, the proliferation of HepG2 cells was markedly inhibited by NCTD in dose-dependent manners. The levels of methylation of RASSF1A decreased at the increasing concentration of 10, 20 and 40 μg/mL. The levels of RASSF1A mRNA and protein were decreased when treated with NCTD at the concentrations of 10, 20 and 40 μg/mL, which were also in a dose-dependent manner. CONCLUSION: NCTD can reverse the methylation state of RASSF1A gene and induce its re-expression, which will provide the theoretical basis for the clinical practice.