南蛇藤多萜通过抑制Chk1介导胃癌细胞DNA损伤并诱导其凋亡

目的从DNA损伤应答角度探讨南蛇藤多萜(the total terpenoids of Celastrus orbiculatus Thunb.,TTC)对胃癌细胞的抑制作用。方法CCK-8实验考察不同浓度(0、20、40、80、160、320 mg·L^-1)的南蛇藤多萜对胃癌细胞的毒性影响;克隆形成率实验检测TTC对胃癌细胞的克隆形成的影响;彗星实验分析南蛇藤多萜对胃癌细胞DNA的影响;Western blot实验检测DNA损伤相关蛋白γH2AX、Poly-PAR、p-Chk1以及cleaved-PARP1的影响。结果TTC在24 h,48 h,72 h对AGS和MKN-45细胞的IC 50分别是421.1、99.68、58.57 mg·L^-1和308.2、124.1、68.21 mg·L^-1;克隆形成实验表明5、10 mg·L^-1的TTC对AGS和MKN-45细胞的克隆形成抑制率分别为29.67%、61.46%和42.73%、64.39%;TTC能够加重胃癌细胞DNA损伤而同等剂量对正常胃粘膜上皮细胞GES-1细胞DNA几乎无损伤;TTC能够增加DNA单双链断裂Poly-PAR、γH2AX蛋白的表达;抑制Chk1的磷酸化水平表达,并增加cleaved-PARP1的蛋白表达。结论TTC能够抑制DNA损伤应答激酶Chk1的激活,从而诱导胃癌细胞DNA单双链断裂加剧最终诱导细胞凋亡。

The total terpenoids of Celastrus aggravate DNA damage and induce apoptosis of gastric cancer cells by inhibiting Chk1

Aim To explore the inhibitory effect of the total terpenoids of Celastrus orbiculatus Thunb(TTC)on gastric cancer cells from the perspective of DNA damage response.Methods CCK-8 experiment was conducted to investigate the toxic effects of different concentrations(0,20,40,80,160,320 mg·L^-1)of TTC on gastric cancer cells;the clone formation rate experiment was used to detect the clones of TTC on gastric cancer cellformation.Comet assay was used to analyze the effects of celosia polyterpene on the DNA of gastric cancer cells,and Western blot was employed to detect the effects of DNA damage-related proteinsγH2AX,Poly-PAR,p-Chk1 and cleaved-PARP1.Results The IC 50 of TTC on AGS and MKN-45 cells at 24 h,48 h and 72 h was 421.1,99.68,58.57 mg·L^-1 and 308.2,124.1,68.21 mg·L^-1,respectively;the clone formation experiment showed that 5 mg·L^-1,10 mg·L^-1 TTC clone formation inhibition rate on AGS and MKN-45 cells were 29.67%,61.46%and 42.73%,64.39%,respectively.TTC could aggravate the DNA damage of gastric cancer cells,while TTC at the same dose almost had no DNA damage to normal gastric mucosal epithelial cells(GES-1 cells).TTC could increase the expression of DNA single-and double-strand break Poly-PAR andγH2AX protein,inhibit the phosphorylation level of Chk1 and increase the protein expression of cleaved-PARP1.Conclusions TTC can inhibit the activation of DNA damage response kinase Chk1,thereby inducing DNA single-and double-strand breaks in gastric cancer cells to aggravate and ultimately induce cell apoptosis.