VPA和As2O3对Molt-4细胞的协同作用及可能机制

本研究探讨丙戊酸钠（sodium valproate，VPA）单药或与亚砷酸（arsenie trioxide，As2O，3）联用对人急性T细胞白血病细胞株Molt-4增殖活力的抑制、去甲基化作用及其可能机制。用MTT法检测药物对细胞增殖的抑制作用，利用金氏公式观察两药联用体外抗癌的协同作用。采用半巢式甲基化特异PCR（hnMS—PCR）检测p15INK4B基因甲基化，RT—PCR方法检测p15基因、DNA甲基转移酶DNMT-1、DNMT3A、DNMT3B的表达。结果表明：VPA和As2O3均可明显抑制细胞增殖，二者联用在体外有相加作用（Q值均大于0．85），在5mmol／LVPA与10μmol／LAs2O3联用时抑制率达（70．31±2．54）％。Molt-4细胞p15基因因高甲基化而失表达，经VPA和As2O3联合作用后p15基因甲基化程度明显下降，p15基因表达增强，两药联用时DNMT-1、DNMT-3A、DNMT3B mRNA的表达都有下降。结论：VPA可能通过抑制DNA甲基转移酶DNMT-1和（或）DNMT3B使p15INK4B基因去甲基化，使p15基因表达上调，恢复其活性。VPA和As2O3均可明显抑制Mol-4细胞增殖，两药合用有协同相加作用，可减少砷剂的副作用。

Synergistic Effects of VPA and As2O3 on Molt-4 Cells in Vitro and Its Possible Mechanisms

This study was purposed to investigate the synergistic effects of sodium valproate （VPA） and As2O3 on the proliferation of Molt-4 cells in vitro and its possible mechanisms. Cell viability and growth curve were assessed by the MTT assay. The synergistic activity in combination of 2 drugs was determined by the Q format. The expression levels of p15, DNA methyltransferase 1 （ DNMT-1 ）, DNMT3A and DNMT 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR. The results indicated that the VPA and As2O3 both inhibited proliferation of Molt-4 cells. The combination of two drugs showed an additive effect （ values of Q were ≥0.85 ）. The inhibitory rate in combination of 5 mmol/L of VPA with 10 μmol/L of As2O3 was （70.31 ±2.54） %. The p15 gene in Molt-4 cell line failed to express due to its hypermethylation. The level of p15 gene mRNA expression increased significantly after exposure to VPA in combination with As2O3 for 48h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner, whereas DNMT3A had no significant differences from the control. The level of expression of DNMT3B seemed to decrease at 10 mmol/L concentration. There were significant differences between the combination of the two drugs and the control group. The gray value of methylated bands decreased after the treatment of VPA alone and in combination with As2O3 in a dose-dependent manner. It is concluded that VPA induces demethylation of p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities, which upregulates the p15 gene, recovers its activity. The combination of VPA with As2O3 has the synergetic additive effect on the inhibition of cell viabilitiy, so that VPA can reduce the side effect of As2 O3 on liver function, which would be verified in the clinical practice.