苯己异硫氰酸酯通过抑制组蛋白去乙酰化和诱导去甲基化来抑制MDS细胞生长

Phenylhexyl Isothiocyanate Can Inhibit MDS Cell Growth as Histone Deacetylase Inhibitor and Hypomethylating Agent

目的为了探索治疗骨髓增生异常综合征(mylodysplasia syndrome,MDS)新的一类靶向药物,笔者研究了苯己异硫氰酸酯(phenylhexyl isothiocyanate,PHI),1种合成的萝卜硫素(sulforphate,SFN)衍生物对MDS细胞作用。方法 MDS细胞株SKM-1经过不同浓度PHI处理3个平行孔后使用台盼蓝拒染法测定细胞活力,流式细胞术分析细胞周期相,荧光染料JC-1检测线粒体膜电位改变;甲基化特异性PCR检测去甲基化改变,Western blot法蛋白印迹分析p^(15)基因表达和组蛋白乙酰化程度,以及ELISA法测定细胞培养上清中血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。结果经过5μmol/L PHI处理后MDS细胞增殖受到抑制和产生凋亡,在诱导浓度低至10μmol/L细胞增殖减少了50%,和细胞周期分析显示MDS细胞停滞在细胞G_1期,PHI诱导p^(15)基因去甲基化呈现浓度依赖性,进一步证明也以浓度依赖方式PHI诱导组蛋白H3高乙酰化。同时也表明,PHI抑制MDS细胞中VEGF产生,以及通过破坏线粒体膜电位诱导细胞凋亡。结论 PHI可以诱导p^(15)基因去甲基化和组蛋白H3高乙酰化,具有对MDS p^(15)基因去甲基化和组蛋白高乙酰化的双重表观遗传效应,从而启动了靶向途径抑制MDS细胞增殖。

Objective A new class of chemotherapeutic agents is being developed for myelodysplasia syndrome（MDS）. In this study we examined whether phenylhexyl isothiocyanate（PHI）,one kind of synthetic sulforphate（SFN） derivatives,has its effects on MDS cells. Methods SKM-1 of MDS cell line were exposed to PHI at various concentrations and cell viability was determined from at least triplicate cultures by trypan blue exclusion method. Analysis of cell cycle phases was performed using a BD FACScan Flow Cytometry. The effects of PHI treatment on mitochondrial membrane potential was measured using a potential sensitive dye JC-1. Both p15 hypomethylation and histone H3 hyperacetylation in PHI treated SKM-1 cell line by using methylation specific PCR （MS-PCR） and Western blot analyses. The contents of Vascular Endothelial Growth Factor （VEGF）in the culture supernatants were determined with ELISA kit. Results PHI inhibited the proliferation of the MDS cells and induced apoptosis in a concentration as low as 5μmol/L. Cell proliferation was reduced to 50% of control with PHI concentration of 10μmol/L. Cell cycle analysis revealed that PHI caused G1-phase arrest of SKM-1 cells. PHI induced p15 hypomethylation in a concentration-dependent manner. PHI was further shown to induce histone H3 hyperacetylation in a concentration-dependent manner. It was also demonstrated that PHI inhibited VEGF production in the SKM-1 cells, and It was found that PHI induced apoptosis through disruption of mitochondrial membrane potential. Conclusion It shows that PHI can induce both p15 hypomethylation and histone H3 hyperacetylation in SKM-1 cell line. PHI has dual epigenetic effects on p15 hypomethylation and histone hyperacetylation in MDS cells and targets several critical processes of MDS proliferation.