摘要
目的观察不同浓度华蟾素联合吉非替尼对人非小细胞肺腺癌细胞株A549增殖、凋亡的影响,并探讨其作用机制。方法将A549细胞分为吉非替尼组、华蟾素组、联合药物组和对照组,吉非替尼组分别加入1、5、10、20、40μmol/L的吉非替尼,记为A1、A2、A3、A4、A5组;华蟾素组分别加入0.005、0.01、0.05、0.1、0.5 mg/mL的华蟾素,记为B1、B2、B3、B4、B5组;联合药物组分别加入1μmol/L吉非替尼+0.005 mg/mL华蟾素、5μmol/L吉非替尼+0.01 mg/mL华蟾素、10μmol/L吉非替尼+0.05 mg/mL华蟾素、20μmol/L吉非替尼+0.01 mg/mL华蟾素、40μmol/L吉非替尼+0.5 mg/mL华蟾素,记为C1、C2、C3、C4、C5组;对照组加入等量DMSO。培养24、48、72 h时,采用MTT法观察各组细胞增殖能力(以OD值表示细胞增殖能力),采用流式细胞术测算A5、B5、C5和对照组细胞凋亡率,采用Western Blotting法检测A5、B5、C5和对照组细胞c-met蛋白。结果A1、A2、A3、A4、A5组细胞培养24 h时的OD值分别为0.410±0.030、0.371±0.037、0.322±0.010、0.284±0.025、0.196±0.020,B1、B2、B3、B4、B5组分别为0.463±0.021、0.454±0.017、0.472±0.013、0.460±0.009、0.452±0.028,C1、C2、C3、C4、C5组分别为0.412±0.016、0.359±0.023、0.306±0.035、0.249±0.020、0.174±0.024,对照组为0.458±0.025;其中,吉非替尼组、联合药物组与对照组相比,P均<0.05;吉非替尼组、联合药物组内各浓度组间相比,P均<0.05;C2、C3、C4、C5组与相同浓度的A2、A3、A4、A5组和B2、B3、B4、B5组相比,P均<0.05。A1、A2、A3、A4、A5组细胞培养48 h时的OD值分别为0.541±0.010、0.453±0.016、0.396±0.011、0.344±0.014、0.261±0.011,B1、B2、B3、B4、B5组分别为0.688±0.035、0.687±0.018、0.677±0.018、0.641±0.036、0.556±0.019,C1、C2、C3、C4、C5组分别为0.526±0.033、0.431±0.019、0.376±0.027、0.296±0.022、0.209±0.027,对照组为0.684±0.019;其中,吉非替尼组、B3组、B4组、B5组、联合药物组与对照组相比,P均<0.05;吉非替尼组、华蟾素组、联合药物组内各浓度组间相比,P均<0.05;联合药物组与相同浓度的吉非替尼组、华蟾素组相比,P均<0.05。A1、A2、A3、A4、A5组细胞培养72 h时的OD值分别为0.755±0.021、0.590±0.024、0.499±0.023、0.353±0.022、0.267±0.005,B1、B2、B3、B4、B5组分别为1.088±0.176、0.990±0.085、0.849±0.038、0.758±0.050、0.704±0.041,C1、C2、C3、C4、C5组分别为0.714±0.033、0.481±0.036、0.383±0.026、0.310±0.006、0.232±0.011,对照组为1.089±0.190;吉非替尼组、华蟾素组、联合药物组与对照组相比,P均<0.05;吉非替尼组、华蟾素组、联合药物组内各浓度组间相比,P均<0.05;联合药物组与相同浓度的吉非替尼组、华蟾素组相比,P均<0.05。各组细胞培养24、48、72 h时,相同浓度组间相比,P均<0.05。A5、B5、C5、对照组细胞培养48 h时的凋亡率分别为14.37%±0.48%、9.76%±0.37%、35.01%±0.62%、4.13%±0.65%,组间相比,P均<0.01。A5、B5、C5、对照组细胞中c-met蛋白的相对表达量分别为0.477±0.034、0.629±0.007、0.332±0.010、0.738±0.023,组间相比,P均<0.001。结论与单独应用吉非替尼或华蟾素相比,华蟾素联合吉非替尼可抑制人非小细胞肺腺癌细胞株A549等增殖,促进其凋亡;华蟾素联合吉非替尼可通过下调c-met蛋白表达来发挥抗肿瘤作用。
Objective To observe the effects of different concentrations of cinobufacini combined with gefitinib on the proliferation and apoptosis of non-small-cell lung adenocarcinoma A549 cells and to discuss its mechanism.Methods A549 cells were divided into the gefitinib group,the cinobufacini group,the drug combination group and the control group.In the gefitinib group,1,5,10,20 and 40 μmol/L gefitinib were added,and marked as the group A1,group A2,group A3,group A4 and group A5;in the cinobufacini group,0.005,0.01,0.05,0.1 and 0.5 mg/mL cinobufacini were add ed separately,and marked as group B1,group B2,group B3,group B4 and group B5;in the drug combination group,1 μmol/L gefitinib+0.005mg/mL cinobufacini,5 μmol/L gefitinib+0.01 mg/mL cinobufacini,10 μmol/L gefitinib+0.05 mg/mL cinobufacini,20 μmol/L gefitinib+0.01 mg/mL cinobufacini,40 μmol/L gefitinib+0.5mg/mL cinobufaci-ni were added separately,and marked as the group C1,group C2,group C3,group C4 and group C5;in the control group,equivalent DMSO was added.At 24,48,and 72 h after culture,the cell proliferative ability(expressed by the OD value)was observed by MTT,the apoptosis rates of group A5,group B5,group C5 and the control group were calculated by flow cytometry,and the cell c-met protein of group A5,group B5,group C5 and the control group was detected by Western Blotting.Results At 24 h,the OD values of group A1,group A2,group A3,group A4,and group A5 were 0.410±0.030,0.371±0.037,0.322±0.010,0.284±0.025,and 0.196±0.020;the OD values of group B1,group B2,group B3,group B4,and group B5 were 0.463±0.021,0.454±0.017,0.472±0.013,0.460±0.009,0.452±0.028;the OD values of group C1,group C2,group C3,group C4,and group C5 were 0.412±0.016,0.359±0.023,0.306±0.0350.249±0.0200.174±0.024;the OD value of the control group was 0.458±0.025;significant differ ence was found between the control group and the gefitinib group and the drug combination group between the gefitinib group and the drug combination group and between the group C2 group C3 group C4 and group C5 and the group A2 group A3 group A4 and group A5 and the group B2 group B3 group B4 and group B5(all P<0.05).At 48 h the OD values of group A1 group A2 group A3 group A4 and group A5 were 0.541±0.0100.453±0.0160.396±0.011,0.344±0.014,and 0.261±0.011;the OD values of group B1,group B2,group B3,group B4,and group B5 were 0.688±0.0350.687±0.0180.677±0.0180.641±0.036 and 0.556±0.019;the OD values of group C1 group C2 group C3 group C4 and group C5 were 0.526±0.0330.431±0.0190.376±0.0270.296±0.022 and 0.209±0.027;the OD value of the control group was 0.684±0.019;significant difference was found between the control group and the gefitinib group B3 group B4 group B5 group and the drug combination group between the gefitinib group cinobufacini group and the drug combination groups and between the drug combination group and the gefitinib group and the cinobufacini group with different concentrations(all P<0.05).At 72 h the OD values of group A1 group A2 group A3 group A4 and group A5 were 0.755±0.0210.590±0.0240.499±0.0230.353±0.022 and 0.267±0.005;the OD values of group B1,group B2,group B3,group B4,and group B5 were 1.088±0.176,0.990±0.0850.849±0.0380.758±0.050 and 0.704±0.041;the OD values of group C1 group C2 group C3 group C4 and group C5 were 0.714±0.0330.481±0.0360.383±0.0260.310±0.0060.232±0.011;and the OD value of the control group was 1.089±0.190;significant difference was found between the control group all the gefitinib groups cinobufacini group and the drug combination group between the gefitinib group cinobufacini group and the drug combi nation group and between the drug combination group and the gefitinib group and the cinobufacini group with different con centrations(all P<0.05).At 48 h the apoptosis rates of the group A5 group B5 group C5 and the control group were 14.37%±0.48%,9.76%±0.37%,35.01%±0.62%,and 4.13%±0.65%;the relative expression of c-met protein in the group A5 group B5 group C5 and the control group were 0.477±0.0340.629±0.0070.332±0.010 and 0.738±0.023,with statistically significant difference(all P<0.001).Conclusions Compared with the using of ge-fitinib or cinobufacini alone cinobufacini combined with gefitinib can inhibit the proliferation of A549 cells and promote its apoptosis.Cinobufacini combined with gefitinib can exert anti-tumor effect by down-regulating c-met protein expression.
作者
马荣辉
董春岚
韩有溪
曹国磊
罗琴
MA Ronghui;DONG Chunlan;HAN Youxi;CAO Guolei;LUO Qin(The Third Affiliated Tumor Hospital of Xinjiang Medical University,Urumchi 830000,China)
出处
《山东医药》
CAS
2019年第36期27-31,共5页
Shandong Medical Journal
基金
新疆维吾尔自治区自然科学基金资助项目(2017D01C411)
关键词
华蟾素
吉非替尼
肺癌
非小细胞肺癌
非小细胞肺腺癌
C-MET蛋白
细胞实验
cinobufacini
gefitinib
lung carcinoma
non-small-cell lung cancer
non-small-cell lung adenocarcino ma
c-met protein
cell experiment
