不同浓度维替泊芬对鼻咽癌细胞株CNE1增殖、侵袭、凋亡的影响及其机制

Effects of different concentrations of veteporfin on proliferation, invasion, and apoptosis of nasopharyngeal carcinoma cells

目的观察不同浓度维替泊芬对鼻咽癌细胞株CNE1增殖、侵袭、凋亡、细胞周期的影响,并探讨及其机制。方法取对数生长期CNE1细胞分为实验组和对照组,实验组分别加入1、2、4μmol/L的维替泊芬,记为A、B、C组,对照组加入等量培养基。采用CCK-8法测算各组细胞存活率(以细胞存活率表示细胞增殖能力),采用细胞克隆形成实验测算细胞克隆形成个数(以细胞克隆形成个数表示细胞克隆形成能力),采用Transwell小室侵袭实验测算侵袭细胞数(以侵袭细胞数表示细胞侵袭能力),采用流式细胞术测算各组细胞凋亡率及各细胞周期细胞百分比,采用实时荧光定量PCR法检测B组及对照组细胞中YAP1、TEAD、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、B细胞瘤-2(Bcl-2)、细胞周期素D1(CyclinD1)、髓细胞组织增生蛋白(MYC)、Axl mRNA,采用Western Blotting法检测B组及对照组细胞中YAP1、TEAD、caspase-3、Bcl-2、CyclinD1、MYC、Axl蛋白。结果A、B、C及对照组细胞培养24 h时细胞存活率分别为85.27%±0.63%、74.18%±1.01%、57.67%±0.92%、100.00%±0.42%,组间相比,P均<0.05;A、B、C及对照组细胞培养48 h时细胞存活率分别为70.35%±0.61%、59.33%±1.27%、41.39%±0.46%、100.00%±0.85%,组间相比,P均<0.05;A、B、C组细胞在培养48 h时的细胞存活率,与24 h时相比,P均<0.05。A、B、C及对照组细胞克隆形成个数分别为183.67±12.92、118.67±10.50、47.33±5.31、253.67±8.18个,组间相比,P均<0.05。A、B、C及对照组细胞侵袭细胞数分别为44.67±2.87、28.67±4.11、16.67±3.68、56.67±5.31个,组间相比,P均<0.05。A、B、C及对照组细胞凋亡率分别为14.52%±1.03%、26.56%±2.10%、38.64%±1.42%、5.31%±0.77%,组间相比,P均<0.05。A组G2/M、S、G0/G1期细胞分别为15.46%±1.74%、42.37%±2.69%、42.17%±1.73%,B组G2/M、S、G0/G1期细胞分别为8.39%±0.77%、34.48%±0.57%、57.13%±0.39%,C组G2/M、S、G0/G1期细胞分别为3.82%±1.89%、26.16%±0.81%、70.02%±1.42%,对照组G2/M、S、G0/G1期细胞分别为21.52%±0.75%、53.14%±1.31%、25.34%±0.95%,各细胞周期比例组间相比,P均<0.05。B组细胞中YAP1、TEAD、caspase-3、Bcl-2、CyclinD1、MYC、Axl mRNA的相对表达量分别为0.37±0.12、0.56±0.17、0.35±0.25、0.43±0.29、0.48±0.27、0.39±0.35、0.35±0.71,对照组细胞中YAP1、TEAD、caspase-3、Bcl-2、CyclinD1、MYC、Axl mRNA的相对表达量分别为1.00±0.33、1.00±0.67、1.00±0.14、1.00±0.28、1.00±0.81、1.00±0.33、1.00±0.11,两组相比,P均<0.05。B组细胞中YAP1、TEAD、caspase-3、Bcl-2、CyclinD1、MYC、Axl蛋白的相对表达量分别为0.51±0.45、0.44±0.31、0.42±0.23、0.61±0.09、0.39±0.41、0.52±0.38、0.48±0.21,对照组细胞中YAP1、TEAD、caspase-3、Bcl-2、CyclinD1、MYC、Axl蛋白的相对表达量分别为1.00±0.56、1.00±0.82、1.00±0.73、1.00±0.32、1.00±0.34、1.00±0.14、1.00±0.29,两组相比,P均<0.05。结论1、2、4μmol/L的维替泊芬均可抑制CNE1细胞的增殖、侵袭能力,诱导细胞凋亡,阻滞生长周期,呈浓度依赖性,其作用机制可能与抑制YAP1及其下游因子的表达有关。

Objective To explore the effects of different concentrations of veteporfin on the proliferation,invasion,apoptosis,and cell cycle of nasopharyngeal carcinoma cell line CNE1 and their mechanism.Methods CNE1 cells were divided into the experimental group and control group.Cells in the experimental group were treated with 0,1,2 and 4 μmol/L veteporfin and were taken as the groups A,B and C,respectively.Cells in control group were added with equal a mount of drug-free medium.CCK8 was used to measure the cell survival rate of each group(the capability of cell prolifera tion is indicated by its cell survival rate).Colony forming assay was applied to evaluate the number of cell colony formation(the capability of cell colony formation is indicated by its cell colony numbers).The number of cells going through Tran swell chamber was detected and calculated by invasion tests(the capability of cell invasion is indicated by its invasive cell numbers).Flow cytometry was applied to examine the apoptotic cell proportion and cell cycle of each group.Quantitative real-time PCR was applied to detect the mRNA expression of YAP1,TEAD,Caspase-3,Bcl-2,CyclinD1,MYC and Axl in the group B and control group.Western blotting was used to evaluate the protein expression of YAP1,TEAD,Caspase-3,Bcl-2,CyclinD1,MYC and Axl in the group B and control group.Results At 24 h,the cell survival rates of the control group,group A,group B and group C were 85.27%±0.63%,74.18%±1.01%,57.67%±0.92%,and 100.00%±0.42%,respectively,and significant difference was found between groups(all P<0.05).At 48 h,the cell survival rates of the control group,group A,group B and group C were 70.35%±0.61%,59.33%±1.27%,41.39%±0.46%,and 100.00%±0.85%,respectively,with significant difference(all P<0.05).The cell survival rates of groups A,B and C at 48 h was lower than that at 24 h(all P<0.05).The cell colony numbers of the control group,group A,group B,and group C were 183.67±12.92,118.67±10.50,47.33±5.31,and 253.67±8.18,respectively;sig nificant difference was found between groups(all P<0.05).The invasive cell numbers of the control group,group A,group B and group C were 44.67±2.87,28.67±4.11,16.67±3.68,and 56.67±5.31,respectively;significant difference was found between groups(all P<0.05).The apoptosis rates of the control group,group A,group B and group C were 14.52%±1.03%,26.56%±2.10%,38.64%±1.42%,and 5.31%±0.77%,respectively,with significant difference was found between groups(all P<0.05).In the group A,the proportion of cells in the G2/M,S and G0/G1 phases were 15.46%±1.74%42.37%±2.69%and 42.17%±1.73%respectively.In the group B the proportion of cells in the G2/M,S and G0/G1 phases were 8.39%±0.77%,34.48%±0.57%,and 57.13%±0.39%,respec tively.In the group C,the proportion of cells in the G2/M,S and G0/G1 phases were 3.82%±1.89%,26.16%±0.81%,and 70.02%±1.42%,respectively.In the control group,the proportion of cells in the G2/M,S and G0/G1 phase were 21.52%±0.75%,53.14%±1.31%,and 25.34%±0.95%,respectively,with significant difference be tween groups(all P<0.05).In the group B the mRNA expression of YAP1 TEAD Caspase-3 Bcl-2 CyclinD1 MYC and Axl was 0.37±0.12,0.56±0.17,0.35±0.25,0.43±0.29,0.48±0.27,0.39±0.35,and 0.35±0.71,re spectively.In the control group the mRNA expression of YAP1 TEAD Caspase-3 Bcl-2 CyclinD1 MYC and Axl was 1.00±0.33,1.00±0.67,1.00±0.14,1.00±0.28,1.00±0.81,1.00±0.33,and 1.00±0.11,respectively;sig nificant difference was found between these two groups(all P<0.05).In the group B the protein expression of YAP1 TEAD Caspase-3 Bcl-2 CyclinD1 MYC and Axl was 0.51±0.450.44±0.310.42±0.230.61±0.090.39±0.410.52±0.38 and 0.48±0.21 respectively.In the control group the protein expression of YAP1 TEAD Caspase-3 Bcl-2 CyclinD1 MYC and Axl was 1.00±0.561.00±0.821.00±0.731.00±0.321.00±0.341.00±0.14,and 1.00±0.29,respectively;significant difference was found between these two groups(all P<0.05).Conclusions Veteporfin(1,2 and 4 μmol/L)can suppress the proliferation and invasion of CNE1 cells,induce the ap optosis and block the cell cycle with a concentration dependent manner.The mechanism may be related to the inhibition of YAP1 and its downstream factors.