Induction effects of decitabine in combination with valproic acid sodium on apoptosis of myeloma cells and its underlying mechanism

Objective　 To investigate the effects of decitabine (DCA) combined with valproate sodium (VPA) on the apoptosis of multiple myeloma cell line RPMI 8226 and the demethylation of death-associated protein kinase (DAPK). Methods　 The RPMI 8226 cells were cultured and treated with DCA (1.5μmol/L, 3μmol/L), VPA (1μmol/L, 2μmol/L), VPA+DCA (DCA 3μmol/L+VPA 2μmol/L) and PBS, respectively, and those cells without treatment served as negative control. The survival status of cells was detected by MTT assay after culture for 24 and 72h. The apoptotic rate and cell cycle of RPMI 8226 cells were detected by flow cytometry (FCM), and the apoptosis of cell was observed under the transmission electron microscope (TEM). MSP method was used to detect the methylation of DAPK gene in the RPMI 8226 cells. The mRNA expression of DAPK was detected by RTPCR. Results　MTT assay showed that the cell activity decreased remarkably in VPA+DCA group after 72h compared with other groups, and the MTT value of VPA+DCA group was significantly different from those of PBS and negative control group (P<0.05). The cell activity in VPA and DCA group decreased remarkably, the MTT value of DCA 3μmol/L group was significantly different from those of PBS and negative control group (P<0.05). FCM analysis revealed that the apoptosis rate increased, the cells in G0/G1 phase increased and the cells in S phase decreased in VPA, DCA and VPA+DCA groups after 72h. The apoptosis rate was higher in DCA+VPA group than in DCA and VPA groups, and significantly higher than in PBS and negative control groups (P<0.05). The typical morphological changes of RPMI 8226 cells were observed under TEM in DCA+VPA group, such as chromatin condensation, pyknosis, margination and nuclear fragmentation. The demethylation of DAPK promoters was observed in all the VPA, DCA and VPA+DCA groups, with a highest degree in DCA+VPA group. The degree of DAPK demethylation was significantly different in DCA+VPA group from PBS and control group (P<0.05). The mRNA expression levels of DAPK were up-regulated in VPA, DCA and DCA+VPA groups. The relative mRNA ratio was higher in VPA+DCA group than in other groups, and significantly higher than in PBS and negative control group (P<0.05). Conclusion　DCA and VPA can induce the apoptosis of RPMI 8226 cells and the hypermethylation of DAPK promoter, which enables DAPK gene to be re-expressed. DOI: 10.11855/j.issn.0577-7402.2013.10.012