The Study On Epigenetic Alteration In Multiple Myeloma Regulated By Triptolide

Objective:Multiple myeloma is a kind of malignant plasma disease that originated from B lymphocyte and secrete great amount of monoclonal immunoglobulin. It is still one of the refractory diseases at present. Numerous studies show that there is an intensive relationship between the disequilibrium of epigenetics and the occurance of multiple myeloma. Here we investigated the effects of triptolide on cell proliferation, cell cycle arrest, apoptosis, histone H3 and H4 acetylation, histone H3K9 and H3K27 trimethylation, and expression of the corresponding histone deacetylase and histone methyltransferases in vitro, trying to explore the anti-myeloma mechanism.Methods:The effect of triptolide on the growth of RPMI8226 was studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay and compared with PBMC. Effects of triptolide on the cell cycle distribution of RPMI8226 cells were studied by propidium iodide method. Apoptosis was detected by TUNEL and Annexin V-FITC/PI double-labeled cytometry and Caspase-3 and Caspase-9 activities were measured by spectrophotometry. The protein expression of acetyl-histone H3 and H4, histone H3K9me3, H3K27me3 and H3K36me3, HDAC8, SUV39H1 and EZH2 were determined by Western blot. The mRNA expression of hdac8 was assessed by RT-PCR, while the mRNA expression of suv39hl and ezh2 were measured by Real-time RT-PCR. Confocal microscopy was used to evaluate the subcellular disposition and expression of HDAC8, SUV39H1 and EZH2.Results:Triptolide inhibited the proliferation of RPMI8226 dose-and time-dependently, IC50for24h,48hand72h was 105.37±0.19,76.89±0.02 and39.17±0.13 nmol/L, respectively. Triptolide caused G0/G1 arrest and induced apoptosis in a time-and dose-dependent manner. Triptolide remarkably increased the acetylation of histone H3 and H4 by decreasing the expression of HDAC8 while declined histone H3K9me3 and H3K27me3 via inhibiting the expression of SUV39H1 and EZH2 respectively.Conclusion:Triptolide can inhibit cell proliferation, cause G0/G1 arrest and induce caspase dependent-apoptosis of RPMI8226 cells significantly. Triptolide remarkably increased the acetylation of histone H3 and H4 by decreasing the expression of HDAC8 while declined histone H3K9me3 and H3K27me3 via inhibiting the expression of SUV39H1 and EZH2 respectively, which is possibly the anti-myeloma mechanism of triptolide.Objective:To investigated the effect of triptolide on cell proliferation, cell cycle arrest, apoptosis, histone H3K4, H3K27 and H3K36 trimethylation, and the expression of corresponding histone methyltransferases in vitro, trying to explore the anti-myeloma mechanism.Methods:The effect of triptolide on the growth of U266 was studied by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium(MTT) assay and compared with PBMC. Effects of triptolide on the cell cycle distribution of U266 cells were studied by propidium iodide method. Apoptosis was detected by Annexin V-FITC/PI double-labeled cytometry and Caspase-3 and Caspase-9 activities were measured by spectrophotometry. The protein expression of histone H3K4me3, H3K27me3 and H3K36me3, SMYD3, EZH2 and NSD1 were determined by Western blot. The mRNA expression of smyd3, ezh2 and nsdl were assessed by RT-PCR. Confocal microscopy was used to evaluate the subcellular disposition and expression of SMYD3, EZH2 and NSD1.Results:Triptolide inhibited the proliferation of U266 cells significantly with the IC50 of 157.19±0.38,57.19±0.38 and 41.20±0.13 nmol/L for 24 h,48 h and 72 h respectively. Triptolide caused G2/M cell cycle arrest and caspase-dependent apoptosis. In U266 cells triptolide reduced histone H3K4me3, H3K27me3 and H3K36me3 along with the decline of the mRNA and protein expression of SMYD3,EZH2 and NSD1.Conclusion:Triptolide presented potent effects on growth inhibition, G2/M cell cycle arrest and induction of caspase-dependent apoptosis in U266 cells in vitro. The study provides the first evidence that triptolide induces epigenetic alterations by regulating histone lysine methylation, offering a novel view of anti-myeloma mechanism of triptolide.Objective:To investigate the effects of triptolide on the apoptosis in human multiple myeloma cell line U266 in vitro, and the regulation of histone H3K9 monomethylation (H3K9me1) via RIZ1 by triptolide.Methods:The effect of triptolide on cell apoptosis was detected through Hoechst 33258 staining and the mRNA expresson of caspase-3 was measured by RT-PCR. Western blot, flow cytometry and RT-PCR were applied to assess the expression of RIZ1, while the location and expression of H3K9mel regulated by RIZ1 were detected by confocal microscope and Western blot.Results:It was shown that triptolide could induce typical apoptotic morphological changes and concentration-dependent apoptosis that was caspase-3 dependent. Compared with PBMC from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, while the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. The protein expression of histone H3K9me1 regulated via RIZ1 was detected significantly elevated by triptolide. Conclusion:Triptolide exerted caspase-dependent apoptosis-inducing potency in U266 cells. The expression of RIZ1 in U266 cells was relatively low while it appeared remarkably upregulatd when treated with triptolide.