The cytotoxic effects of Graptopetalum paraguayense (N.E.Br.) E. Walther extract on human melanoma cells

Abstract

IntroductionThis study was conducted to investigate the mechanisms of apoptosis inducing by 50% ethanolic G. paraguayense (N.E.Br.) E. Walther extract (GE50) in human melanoma cells. MethodsThe polyphenols in GE50 were analyzed by HPLC. After GE50 treatment, cell viability, cell cycle distribution, DNA damage, Ca2+, mitochondrial membrane potential (MMP), caspase-3 activity, and cellular redox status were determined. Additionally, the expression of cell cycle-regulated and apoptosis-related proteins in GE50-treated cells was determined by Western blotting. The translocation of GADD153, Endo G, AIF, and cytochrome c proteins was examined by immunostaining. ResultsHPLC analysis showed that flavone, quercetin, and daidzein were the major constituents in GE50. GE50 significantly prohibits cell proliferation and arrests the cell cycle in the G2/M phase. Results of DAPI staining and DNA gel electrophoresis showed that GE50-treatment can cause chromatin condensation and DNA fragmentation in the cells. GE50 stimulated Ca2+ release, decreased MMP, but inhibited ROS production. GE50 promoted caspase-3 activity in cells. The protein levels of cyclin A, cyclin B, CDC2, CDC25c, and Bcl-2 were decreased; and that of CHK1, CHK2, Weel, p21, p53, GRP78, GADD153, caspase-7, caspase-9, Bax, and AIF were increased in GE50-treated cells. The proteins of GADD153, Endo G, AIF, and cytochrome c were released in the nucleus or cytosol. The GE50-treatment can significantly decrease the glutathione content and antioxidant enzyme activity but significantly increase the MDA levels in the cells. ConclusionsGE50 may have potential cytotoxic effect in human melanoma through G2/M phase arrest, ER stress, and intrinsic apoptotic pathways.