Inhibitory effect of caffeine combined with ionizing radiation on proliferation of hepatocellular carcinoma stem cells silenced by Chk-1 and its apoptosis-induced effect / 吉林大学学报(医学版)

ABSTRACT

Objective:To treat the hepatocellular carcinoma stem cells silenced by Chk-1 with caffeine combined with 4 Gy X-ray irradiaition,and to explore their synergistic killing effects on the hepatocellular carcinoma stem cells by detecting the cell proliferation,cell cycle and apoptosis.Methods:The lentivirus silencing Chk-1 was transfected into the 293T cells;after the HepG2 cells were infected by the lentivirus,the Chk-1 protein expression was detected by Western blotting to confirm the silencing efficency,and non-target control was established,and HepG2-Chk-1 and HepG2-control were named.The cells highly expressed CD133 were cultured by suspension culture method,S-HepG2-Chk-1 and S-HepG2-control were named respectively.After the cells were treated by caffeine,they were irradiated by 4 Gy X-ray;the proliferation activity was measured by MTT,and the cell cycle distribution and the apoptotic rate were measured by PI single staining and AnnexinⅤ-FITC double staining using FCM,respectively.For proliferation,cell cycle and apoptosis assays,there were control,caffeine,4 Gy and caffeine+4 Gy groups.Results:The Western blotting results showed that after the HepG2 cells were infected by lentivirus,the Chk-1 protein expression was significantly decreased,but it was not obvious in non-target control group,it demonstrated that the cell models HepG2-Chk-1 and HepG2-control were obtained successfully.After the HepG2-Chk-1 and HepG2-control cells were cultivated by suspension,the CD133 protein expression were increased,it demonstrated that there were high proportion of CD133+ cells,they were hepatocellular carcinoma stem cells.Compared with control group,the proliferation activities in caffeine group and 4 Gy group were significantly decreased (P＜0.05 or P＜0.01),the percentages of cells at G2/M phage and the apoptotic rates were significantly increased (P＜0.05 or P＜0.01),and the percentage of cells at S phage in caffeine group was significantly increased (P＜0.05);the percentage of cells at G0/G1 phage in 4 Gy group was increased (P＜0.05 or P＜0.01),the effect was more stronger in caffeine+4 Gy group.Compared with S-HepG2-control,the proliferation activities of S-HepG2-Chk-1 ceils in caffeine group and 4 Gy group were decreased,the apoptotic rates were increased (P＜0.05 or P＜0.01),and the percentage of cells at G1/M phage was significantly decreased (P＜0.05 or P＜0.01).CollusionThe hepatoccellular carcinoma stem cells silenced by Chk-1 with positive CD133 are obtained successfully;Caffeine combined with X-ray irradiation can inhibit the cell proliferation and induce the apoptosis,and enhance the G2/M arrest,and both of them have synergistic effects.