帕瑞昔布对肝癌细胞凋亡及增殖的影响

Effect of parecoxib on apoptosis and proliferation of hepatocellular cancer cells

目的评价帕瑞昔布对肝癌细胞系HepG2和QGY-7703增殖及凋亡的影响。方法取对数生长期HepG2和QGY-7703细胞,消化后制成单细胞悬液接种于6孔板,将细胞分为对照组和10、20、30、40、50 mg·L-1帕瑞昔布处理组。对照组细胞用正常培养基培养,帕瑞昔布处理组细胞分别用含有10、20、30、40、50 mg·L-1帕瑞昔布的培养基培养24 h。采用Annexin V标记法检测各组细胞凋亡情况,5-乙炔基-2′脱氧尿嘧啶核苷嵌入法检测各组细胞增殖效率,流式细胞仪检测对照组和20 mg·L-1帕瑞昔布处理组细胞周期分布,免疫印迹法检测各组细胞中c-myc、cyclin D1、mcl-1蛋白表达。结果不同浓度帕瑞昔布处理组HepG2细胞凋亡率均显著高于对照组(P<0.05),不同浓度帕瑞昔布处理组HepG2细胞凋亡率随帕瑞昔布浓度的增加而升高(P<0.05)。10 mg·L-1帕瑞昔布处理组QGY-7703细胞凋亡率与对照组比较差异无统计学意义(P>0.05),20、30、40、50 mg·L-1帕瑞昔布处理组QGY-7703细胞凋亡率均显著高于10 mg·L-1帕瑞昔布处理组和对照组(P<0.05);20、30、40、50 mg·L-1帕瑞昔布处理组QGY-7703细胞凋亡率比较差异均无统计学意义(P>0.05)。与对照组比较,不同浓度帕瑞昔布处理组HepG2细胞增殖率均降低,且随帕瑞昔布浓度的增加呈下降趋势,QGY-7703细胞增殖率无明显变化。20 mg·L-1帕瑞昔布处理组HepG细胞处于S期的比例显著低于对照组(P<0.05);20 mg·L-1帕瑞昔布处理组和对照组QGY-7703细胞处于S期的比例比较差异无统计学意义(P>0.05)。不同浓度帕瑞昔布处理组HepG2细胞中c-myc、cyclin D1和mcl-1蛋白表达随帕瑞昔布浓度的升高呈下降趋势。各组QGY-7703细胞中c-myc、cyclin D1、mcl-1蛋白表达水平无明显变化。结论帕瑞昔布可以通过不同机制调控肝癌细胞系HepG2和QGY-7703的存活,其可通过下调c-myc、cyclin D1和mcl-1蛋白表达促进HepG2细胞凋亡并抑制增殖,可通过下调mcl-1蛋白表达水平促进QGY-7703细胞凋亡。2种细胞中只有HepG2呈现出药物剂量依赖性,且HepG2细胞较QGY-7703细胞对帕瑞昔布更为敏感。

Objective To evaluate the effect of parecoxib on proliferation and apoptosis of hepatocellular cancer cells HepG2 and QGY-7703.Methods HepG2 and QGY-7703 cells in logarithmic growth phase were taken and digested into single cell suspension and then were inoculated into 6-well plate.Both the HepG2 and QGY-7703 cells were divided into control group and 10,20,30,40,50 mg·L-1 parecoxib treatment group.The cells in control group were cultured in normal medium and the cells in parecoxib treatment group were cultured in medium with 10,20,30,40,50 mg·L-1 parecoxib for 24 h respectively.The cell apoptosis was detected by annexin V labeling method,the proliferation efficiency was detected by 5-ethynyl-29-deoxyuridine embedding method,the cell cycle distribution of the two kinds of cells in the control group and 20 mg·L-1 parecoxib treatment group was detected by flow cytometry and the expression of c-myc,cyclin D1,mcl-1 protein was detected by Western blot.Results The apoptosis rate of HepG2 cells in different concentrations of parecoxib treatment groups was higher than that in the control group(P<0.05),and the apoptosis rate of HepG2 cells increased with the increase of the concentration of parecoxib(P<0.05).There was no significant difference in the apoptosis rate of QGY-7703 cells between 10 mg·L-1 parecoxib treatment group and the control group(P>0.05);the apoptosis rate of QGY-7703 cells in the 20,30,40,50 mg·L-1 parecoxib treatment group was higher than that in the 10 mg·L-1 parecoxib treatment group and the control group(P<0.05);there was no significant difference in the apoptosis rate of QGY-7703 cells among the 20,30,40 and 50 mg·L-1 parecoxib treatment group(P>0.05).Compared with the control group,the proliferation rate of HepG2 cells in different concentrations of parecoxib group decreased and the proliferation rate decreased with the increase of the concentration of parecoxib;while the proliferation rate of QGY-7703 cells did not change significantly.The percentage of HepG cells in S phase in the 20 mg·L-1 parecoxib treatment group was lower than that in the control group(P<0.05);there was no significant difference in the percentage of QGY-7703 cells in S phase between the 20 mg·L-1 parecoxib treatment group and the control group(P>0.05).The expressions of c-myc,cyclin D1,mcl-1 protein in HepG2 cells in different concentration of parecoxib treatment groups decreased with increase of parecoxib.The expressions of c-myc,cyclin D1,mcl-1 protein in QGY-7703 cells had no significant change in each group.Conclusion Parecoxib can regulate the survival of HepG2 and QGY-7703 cells through different mechanisms.Parecoxib can promote apoptosis and inhibit proliferation of HepG2 cells by down regulating the expression of c-myc,cyclin D1 and mcl-1 protein;and parecoxib can promote the apoptosis of QGY-7703 cells by down regulating the expression of mcl-1 protein.Only HepG2 cells show a dose-dependent manner,and the HepG2 cells were more sensitive to parecoxib than QGY-7703 cells.