摘要
为了探讨通关藤苷G(tenacissoside G,TG)对人结直肠癌细胞增殖的影响及其分子机制,本研究采用CCK-8和平板克隆检测通关藤苷G对结直肠癌RKO和LoVo细胞增殖水平的影响,并利用流式细胞仪观察细胞周期变化、彗星实验检查DNA损伤情况、免疫荧光检测γ-H2AX表达水平以及Western blot检测细胞周期与DNA损伤相关蛋白表达。实验结果显示,TG作用于细胞48 h后,IC_(50)分别为91.71(RKO)和88.34(LoVo)μM,可显著抑制结直肠癌细胞增殖;TG作用于细胞48 h后,细胞在G0/G1期比例显著上升(P<0.01),周期相关蛋白CDK2、CDK4、CDK6、Cyclin D1、Cyclin E表达水平显著下降(P<0.01),DNA损伤明显,γ-H2AX、cleaved PARP、cleaved Caspase 3、p-ATM、p-CHK2、p-p53蛋白表达水平均显著上调(P<0.01),同时,p-ATM/ATM、p-CHK2/CHK2和p-p53/p53均显著升高(P<0.01)。说明TG通过诱导DNA损伤抑制结直肠癌细胞增殖,其作用机制可能为激活ATM-CHK2-p53信号通路介导的细胞凋亡和周期阻滞。
To explore the effects and molecular mechanisms of tenacissoside G(TG)on the proliferation of colorectal cancer,we determined the proliferation inhibitory effects of TG on colorectal cancer(CRC)cell lines RKO and LoVo by CCK-8 assay and colony formation.After 48 h treatment of TG,flow cytometry was used to analyze the cell cycle distribution of CRC cell lines;the comet assay and immunofluorescence were adopted to detect the DNA damage degree and the expression level ofγ-H2 AX;and the expression level of cell cycle and apoptosis related proteins was detected by Western blot.The results showed that TG significantly inhibited the proliferation and colony formation of RKO and LoVo in a dose and time dependent manner.After 48 h treatment of TG,the IC_(50) values of TG on RKO and LoVo were 91.71 and 88.34μM respectively;the proportion of cells of G0/G1 phase significantly increased(P<0.01)and marked DNA damage was detected with an obvious increase in the expression ofγ-H2 AX.The expression of cell cycle related proteins including CDK2,CDK4,CDK6,Cylin D1,and Cyclin E was remarkably downregulated(P<0.01),however,the expression of cleaved Caspase3,cleaved PARP,p-ATM,p-CHK1,and p-p53 was remarkably upregulated(P<0.01).Meanwhile,p-ATM/ATM,p-CHK2/CHK2 and p-p53/p53 were significantly increased(P<0.01).In conclusion,TG exerted proliferation inhibition through DNA damage,which further activated cell cycle arrest and apoptosis mediated by ATM-CHK2-p53 signaling pathway.
作者
王恺纯
徐勤芬
刘微
胡道德
WANG Kai-chun;XU Qin-fen;LIU Wei;HU Dao-de(Department of Clinical pharmacology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China;Department of Clinical pharmacology,Shanghai General Hospital,Nanjing Medical University,Nanjing 211166,China)
出处
《天然产物研究与开发》
CAS
CSCD
2021年第4期554-562,共9页
Natural Product Research and Development
基金
上海市转化医学协同创新中心项目(TM201816)
上海市科委项目(16401901400)。
关键词
通关藤苷G
结直肠癌
DNA损伤
周期阻滞
凋亡
tenacissoside G
colcorectal cancer
DNA damage
cell cycle arrest
apoptosis
