斑蝥素对肝癌HepG2细胞增殖、凋亡的影响及其可能机制  

Effect of Cantharidin on Growth,Survival and Mechanism in HepG2 Cells

作　　者：封艳艳 马齐襄 隋彤彤[1] 孙笑[1] 邵好珍 金琳[1] 胡晓炜[1] 邓恺文 范潇婷 罗广彬[1] 马志涛[1] FENG Yan-yan MA Qi-xiang SUI Tong-tong SUN Xiao SHAO Hao-zhen JIN Lin HU Xiao-wei DENG Kai-wen FAN Xiao-ting LUO Guang-bin MA Zhi-tao(School of Life Science, Beijing University of Chinese Medicine, Beijing 100029, China)

机构地区：[1]北京中医药大学生命科学院,北京100029

出　　处：《中国实验方剂学杂志》2017年第15期112-117,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：北京中医药大学自主选题项目--特聘教授启动基金项目(1040053310002/002)

摘　　要：目的:探讨不同浓度斑蝥素(cantharidin,CTD)对肝癌细胞HepG2细胞增殖、凋亡以及其可能机制。方法:应用长时间细胞观察及功能分析系统(IncuCyte^(TM) ZOOM)以及克隆形成实验,分析药物处理后肝癌细胞HepG2细胞增殖的影响。应用流式细胞术检测CTD对肝癌细胞HepG2凋亡的影响。采用蛋白免疫印迹法(Western blot)检测技术,检测CTD处理后,HepG2细胞pro半胱氨酸蛋白酶-3(Caspase-3)及聚二磷酸腺苷核糖多聚酶(PARP)蛋白表达的变化以及细胞外信号调节激酶1/2(ERK1/2),蛋白激酶B(Akt)磷酸化水平的变化。采用长时间细胞观察及功能分析系统检测ERK1/2抑制剂(PD98059),磷脂酰肌醇3激酶(PI3K)抑制剂(Wortmannin)与CTD共处理对HepG2细胞抑制作用的变化。结果:CTD在2.5μmol·L^(-1)时就能够明显抑制HepG2细胞的生长,但不出现明显凋亡。CTD 5μmol·L^(-1)时HepG2的生长几乎完全受到抑制,流式细胞术结果显示,HepG2细胞凋亡率达40.4%。同时Western blot结果显示CTD 5μmol·L^(-1)组细胞pro Caspase-3蛋白表达明显降低,Cleaved PARP蛋白表达明显上升,说明CTD 5μmol·L^(-1)组会出现细胞凋亡,与流式结果相符。CTD能够促进ERK磷酸化,但不能促进Akt磷酸化。结论:CTD 2.5μmol·L^(-1)具有抑制HepG2细胞生长的作用,5μmol·L^(-1)时能够促进HepG2细胞凋亡,提示CTD既具有抑制细胞生长作用,也具有一定的细胞毒作用。CTD能够促进ERK磷酸化,但不能促进Akt磷酸化。Objective：To investigate the effect of cantharidin on proliferation,apoptosis and MAPK signaling in hepatocellular carcinoma HepG2 cells.Method：IncuCyteTM ZOOM-based proliferation assay and clonogenic assay were used to assess the inhibitory effect of cantharidin（CTD） on HepG2 cells.Flow cytometry was used to define the effect of CTD on apoptosis.Western blot was used to monitor the changes in Caspase-3,poly adeno-sine diphosphate ribose polymerase（PARP）,extraeellular-signalregulatedki-nase（ERK1/2） and proteinkinase B（Akt） phosphorylation after CTD treatment.Finally,ERK1/2 inhibitor（PD98059）,and Akt inhibitor（Wortmannin） were used to assess the potential effect of ERK1/2 and Akt on the inhibitory and/or proapoptotic effect of CTD.Result：CTD at 2.5 μmol·L-1significantly inhibited the growth of HepG2,but did not significantly affect cell survival.However,CTD at 5 μmol·L-1resulted in not only most a complete inhibitory effect on growth,but also a significant apoptosis rate（40.4%）.Western blot result showed that 5 μmol·L-1of CTD decreased pro Caspase-3 expression and increased cleaved PARP expression in HepG2.Besides,CTD promoted the phosphorylation of ERK,but not Akt.Conclusion：CTD exhibited a potent growth inhibitory（cytostatic） effect at2.5 μmol·L-1,but could show a strong pro-apoptotic（cytotoxic） effect at 5 μmol·L-1for HepG2 cells.Therefore,the study reveals a potent cytostatic effect of CTD and a certain cytotoxic potency,suggesting that the cytostatic effect is an important anticancer mechanism.

关 键 词：斑蝥素 HEPG2 增殖 凋亡 生长抑制 细胞毒作用 

分 类 号：R285.5[医药卫生—中药学]