六神丸和雄黄诱导子宫内膜癌细胞凋亡和DNA损伤的机制

Mechanism of Liushenwan and Realgar in Inducing Apoptosis and DNA Damage in Human Endometrial Cancer JEC Cells

目的:比较经典名药六神丸和雄黄(As4S4)对子宫内膜癌细胞JEC的抗肿瘤作用及其机制。方法:原子吸收法测定六神丸与As4S4中砷(As)的溶解度;噻唑蓝(MTT)比色法比较六神丸与As4S4对细胞增殖的抑制作用,细胞迁移实验比较六神丸与As4S4对细胞迁移的抑制作用;磷脂酰丝氨酸蛋白抗体/碘化丙啶(Annexin V-FITC/PI)双染结合流式细胞术比较六神丸与As4S4对细胞凋亡的影响,蛋白免疫印迹法(Western blot)比较六神丸与As4S4对凋亡和DNA损伤相关蛋白的影响。结果:原子吸收法测定结果显示六神丸的As溶出量为17.4%,As4S4的As溶出量仅为1.6%。但在相同As含量的情况下,与As4S4组比较,六神丸3,10 mg·L^-1组的细胞存活率明显降低(P<0.05),六神丸0.25,0.5,1 mg·L-1组早期凋亡率明显增高(P<0.05),六神丸1 mg·L^-1组细胞迁移率明显下降(P<0.05),六神丸1 mg·L^-1组剪切的半胱氨酸天冬氨酸蛋白酶-3(cleaved Caspase-3),剪切的半胱氨酸天冬氨酸蛋白酶-7(cleaved Caspase-7),剪切的聚ADP核糖聚合酶(cleaved PARP)以及磷酸化的组蛋白(p-H2AX),磷酸化的检测点激酶2(p-CHK2),人共济失调毛细血管扩张症突变激酶(ATM)蛋白表达水平升高(P<0.05),人共济失调毛细血管扩张症Rad3相关激酶(ATR)蛋白表达水平降低(P<0.05),而Caspase-3和Caspase-7蛋白表达水平无明显改变。结论:在等As剂量下,六神丸与As4S4均能抑制JEC细胞的增殖和迁移,诱导细胞凋亡和DNA损伤,但六神丸的效果优于As4S4,提示中药复方六神丸中还有其他成分协同As共同参与抗肿瘤作用。

Objective:Compare the anti-tumor effect and mechanism of Liushenwan and realgar(As4S4)on human endometrial cancer cells JEC.Method:The release of Asin Liushenwan and As4S4 was measured by atomic absorption spectrometry.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used for cell proliferation,and cell migration was measured by Transwell assay.Flow cytometry and Western Blot were used to determine apoptosis and DNA damage.Result:The dissolution of As in Liushenwan was 17.4%,and that of As4S4 was only 1.6%according to atomic absorption assay.With the same content of As,compared with the As4S4 group,the cell viability in the 3,10 mg·L^-1 Liushenwan groups was decreased(P<0.05),the early apoptosis rate was significantly increased in 0.25,0.5,1 mg·L^-1 Liushenwan groups(P<0.05),the rate of cell migration was decreased in 1 mg·L^-1 Liushenwan group(P<0.05),the expressions of cleaved cysteinyl aspartate-specific proteinase-3(cleaved Caspase-3),cleaved cysteinyl aspartate-specific proteinase-7(cleaved Caspase-7),cleaved poly ADP-ribose polymerase(cleaved PARP),phosphorylated histone(p-H2AX),phosphorylation of checkpoint kinase 2(p-CHK2)and ataxiatelangiectasia mutated(ATM)were increased in 1 mg·L^-1 Liushenwan group(P<0.05),while the expression of phosphorylation of ataxia-telangiectasia mutated rad3-related(ATR)was decreased in 1 mg·L^-1 Liushenwan group(P<0.05),with no significant changes in the expressions of cysteinyl aspartate-specific proteinase-3(Caspase-3)and cysteinyl aspartate-specific proteinase-7(Caspase-7).Conclusion:With the same content of As,both Liushenwan and As4S4 could inhibit JEC cell proliferation and migration,and induce cell apoptosis and DNA damage.Liushenwan has a stronger effect than As4S4.It is suggested that there are other components in Liushenwan with an anti-tumor effect in cooperation with As.