Triptolide Induces Apoptosis Of Multiple Myeloma Through Epigenetic Mechanism

Part Ⅰ. Triptolide induces cell apoptosis by targeting H3K4me3and downstream effector proteins in multiple myelomaKM3cellsObjective: H3K4me3is proposed to promote gene expression, and accumulating ofH3K4me3at transcriptional start site of oncogenes is involved in carcinogenesis.Interestingly, our previous study showed that triptolide induced multiple myeloma(MM) U266and KM3cells apoptosis accompanied by a global reduction oftrimethyl-lysine4on histone H3(H3K4me3). In this article, we try to reveal thecorrelation between reduction of H3K4me3and MM cells apoptosis induced bytriptolide, meanwhile, provide theory support for cllnical application of triptolide.Methods: The MM KM3cell line was cultured with triptolide. Apoptosis wasevaluated by Annexin-V-FITC/PI-labeled flow cytometry. Combined ChIP-on-chipand realtime-PCR to detect the occupancy pattern of H3K4me3on KM3cell genepromoters. Western blotting and H3K4me3-targeted siRNAs were assayed to identifythe effect of H3K4me3on gene promoters of KM3cells.Results: Triptolide strongly inhibited cell proliferation and induced apoptosis ofKM3cells. Western blotting revealed that triptolide inhibited the protein expressionof H3K4me3dramaticlly. Combined analyses with ChIP-on-chip and western blottingshowed that highly accumulation of H3K4me3on gene promoters of c-Myc andVEGFA was associated with the up-regulation of both genes in MM. Treatment withtriptolide and siRNA-ASH2L reduced expression of c-Myc and VEGFA in KM3 cells.Conclusions: These results demonstrates that triptolide could drive MM cellsapoptosis through blocking H3K4me3accumulating at the VEGFA and c-Myc genepromoters. This could partly explain the antitumor mechanism of MM cells inducedby triptolide. Part Ⅱ. Triptolide induces cell-cycle arrest and apoptosis andalters the expression of the histone demethylasesLSD1and JMJD2B in human multiple myeloma cellsObjective: We aimed to elucidate the association between the anti-tumor effects oftriptolide on multiple myeloma (MM) and triptolide-induced changes in histonemethylation and the expression of the histone demethylases LSD1and JMJD2B.Methods: The human multiple myeloma cell line RPMI8226was cultured withtriptolide. Apoptosis was evaluated using Annexin-V-FITC/PI-labeled flow cytometry,Hoechst33258staining, and transmission electron microscopy. Flow cytometry wasused to detect the cell cycle distribution of the apoptotic cells. The presence of theLSD1, JMJD2B, H3K4me2, H3K9me2, and H3K36me2proteins was verified bywestern blot analysis. Semiquantitative real-time PCR was also performed for LSD1and JMJD2B.Results: We found that triptolide repressed the proliferation of the MM cell lineRPMI8226in a dose-and time-dependent manner. Moreover, triptolide inducedapoptosis and G0/G1cell cycle arrest in vitro. Target analysis revealed that triptolideinhibited the expression of dimethylated H3K4, dimethylated H3K9and dimethylatedH3K36by regulating the expression of the histone demethylases LSD1and JMJD2B.Conclusion: These results suggest that abnormal histone methylation and LSD1andJMJD2B expression play key roles in the genesis of MM. Triptolide strongly inhibitsthe growth of MM cells via regulation of LSD1and JMJD2B.