雷公藤内酯醇衍生物对肝癌细胞增殖、凋亡的作用研究

Effects of Triptolide Derivatives on Hepatocellular Carcinoma Proliferation and Apoptosis

目的研究新型雷公藤内酯醇(Triptolide,TPL)衍生物对正常肝细胞的毒性以及对肝癌细胞的增殖、凋亡作用。方法通过对TPL进行化学修饰,获得2种新型衍生物:雷公藤内酯三醇(TP-3-OH)和丙烯酸雷公藤甲素酯(TPO)。通过体外培养人胚胎肝细胞LO2和肝癌细胞Hep G2、Hep3B、SMMC-7721,分为DMSO对照组,TPL/TP-3-OH/TPO低、中、高浓度组(25、50、100 nmol·L^(-1))。通过检测培养上清中乳酸脱氢酶(LDH)的活性来评价TP-3-OH、TPO对肝细胞的毒性;采用MTT实验检测细胞增殖情况;采用流式细胞术及Western Blot法检测细胞凋亡及凋亡通路相关蛋白表达情况;采用划痕实验检测细胞迁移能力。结果与TPL组比较,50、100 nmol·L^(-1)浓度条件下TP-3-OH、TPO组的LO2细胞培养上清中的LDH活性明显降低(P<0.05,P<0.01)。与DMSO对照组比较,TPO中、高浓度(50、100 nmol·L^(-1))组的LO2细胞增殖抑制率明显升高(P<0.05,P<0.01),TPO低、中、高浓度(25、50、100 nmol·L^(-1))组的Hep G2、Hep3B、SMMC-7721肝癌细胞增殖抑制率均明显升高(P<0.05,P<0.01);TPO高浓度(100 nmol·L^(-1))组的LO2、Hep G2和SMMC-7721细胞的早期、晚期细胞凋亡率均明显升高(P<0.05,P<0.01);TPO中、高浓度(50、100 nmol·L^(-1))组SMMC-7721、Hep G2细胞的cleaved-Caspase-3、cleaved-PARP蛋白表达显著上调(P<0.01);TPO中浓度(50 nmol·L^(-1))组SMMC-7721细胞的24 h迁移率明显降低(P<0.05)。结论经化学修饰获得的2种新型TPL衍生物对肝细胞的毒性降低,其中TPO具有抑制肝癌细胞增殖、促进肝癌细胞凋亡的作用。

Objective To investigate the effects of two triptolide derivatives on the cytotoxicity of normal hepatic cells and the proliferation and apoptosis of hepatocellular carcinoma cells.Methods Triptolide was modified by chemical method and transferred to two new monomers with similar structures,TP-3-OH and TPO.In vitro cultured normal hepatic cell(LO2),and three carcinoma cells(Hep G2,Hep3B,and SMMC-7721)were treated with DMSO or different concentrations of TPL,TP-3-OH or TPO(25,50,100 nmol·L^(-1)).Detection of LDH activity in the cell cultural supernatant was used to assess the cytotoxicity of these drugs to LO2 cells.MTT assay,flow cytometry and Western Blot were used to assess the effects of these drugs on proliferation and apoptosis of LO2 cells and carcinoma cells.Wound-healing assay was used to assess cell migration.Results Compared with TPL group,the LDH activities of LO2 cells cultural supernatant in TP-3-OH and TPO 50 and 100 nmol·L^(-1)groups were significantly reduced(P<0.05,P<0.01).Compared with DMSO group,the proliferation inhibition rates of LO2 cells in TPO 50 and 100 nmol·L^(-1)groups(P<0.05,P<0.01),the proliferation inhibition rates of carcinoma cells(Hep G2,Hep3B and SMMC-7721)in TPO 25,50 and 100 nmol·L^(-1)groups(P<0.05,P<0.01),the early and later apoptotic rates of carcinoma cells(LO2,Hep G2,and SMMC-7721)in TPO 100 nmol·L^(-1)groups(P<0.05,P<0.01),and the protein levels of cleaved-Caspase-3 and cleaved-PARP in TPO 50 and 100 nmol·L^(-1)groups(P<0.01),were significantly increased.The 24-hour migration rates of SMMC-7721 cells in TPO 50 nmol·L^(-1)group were significantly decreased(P<0.05).Conclusion We obtained two triptolide derivatives with attenuated hepatic cells toxicity,and TPO,one of them,inhibits proliferation and promotes apoptosis of hepatocellular carcinoma cells.