丙戊酸钠对多发性骨髓瘤细胞株KM3细胞增殖及其VEGF受体表达的影响

Effect of a novel histone deacetylase inhibitor valproic acid on cell proliferation and apoptosis in multiple myeloma cells KM3 in vitro

目的:研究丙戊酸钠(VPA)对多发性骨髓瘤细胞株KM3细胞增殖和凋亡的影响,探讨其抗多发性骨髓瘤细胞的分子生物学机制。方法:采用MTT法检测细胞增殖,流式细胞仪检测凋亡率,RT-PCR检测KM3细胞VEGFR mRNA的表达;采用免疫细胞化学法观察KM3细胞VEGFR、ac-H4蛋白的表达。结果:VPA可明显抑制KM3细胞增殖,且具有时间剂量依赖性(P<0.05);不同浓度VPA处理48 h可以明显诱导细胞凋亡,具有剂量依赖性(P<0.05),VPA(0.5、1.0、2.0、4.0 mmol/L)诱导总凋亡率分别为(11.77±4.64)%、(22.13±1.20)%、(23.95±2.57)%和(42.72±4.61)%;RT-PCR结果显示,KM3细胞仅表达VEGFR-1(flt-1),且VPA能在mRNA水平抑制VEGFR-1的表达;免疫细胞化学结果显示,VPA(4 mmol/L)作用48 h后,KM3细胞中ac-H4吸光度值明显增加而VEGFR-1的吸光度明显降低(P<0.05)。结论:VPA通过增加组蛋白乙酰化程度,下调骨髓瘤细胞表面VEGFR的表达,对KM3细胞的增殖起抑制作用。

Objective：To define the effects of VPA on multiple myeloma cell line KM3 cells in vitro and the underlying mechanism of VPA in the treatment of MM were investigated. Method： KM3 cells were cultured in RP- MI1640 medium and treated with VPA in different concentrations. Cell proliferation was measured by 3-[4,5-dim- ethyl-thiazol-2-yl]-2, 5-dipbenyl tetrazolium bromide （MTT） assay. AnnexinV and PI staining were used to detect the apoptosis rates, the mRNA level of VEGFR was determined by RT-PCR, and immunocytochemistry was used to detect the protein level of acetylated-histone H4 （ac-histone H4） and VEGFR. Result：The proliferation rate of KM3 cells in VPA-treated group decreased in a time-dose dependent manner （ P 〈0.05）. And the AnnexinV and PI staining showed VPA could induce apoptosis of KM3 cells. After treatment with VPA （0.5, 1.0, 2.0 and 4.0 mmol/L） for48 h, the apoptosis rates of KM3 ceils were （11. 77±4. 64）%, （22. 13±1. 20）%, （23. 95±2.57） % and （42. 72±4.61）% respectively. The RT-PCR results demonstrated the KM3 cells only expressed VEGFR-1 and VEGFR expression decreased in VPA （0.5, 1.0, 2.0 and 4.0 mmol/L for 48 h） treated groups compared with control （ P 〈 0. 05）. Results of immunocytochemistry showed the protein of ac-histone H4 increased significantly in VPA （4 mmol/L for 48 h） treated group whereas the VEGFR-1/fh-1 expression decreased （ P 〈0.05）. Conclusion： VPA, as an histone deacetylase inhibitor, can increase the expression of ae-histone H4 and inhibit the expression of VEGFR-1 in KM3 cells and it plays an important role in regulating proliferation and apoptosis of multiple myeloma cell line KM3 cells in vitro.