硫酸右旋糖酐对卵巢癌细胞A2780增殖和迁移及侵袭机制研究

Inhibitory effect of dextran sulfate on proliferation,migration and invasion of ovarian cancer cells

目的观察硫酸右旋糖酐(DS)对人卵巢癌细胞A2780生物学行为的影响及可能的分子机制。方法体外培养人浆液性卵巢癌细胞A2780,将其分为空白对照组、不同浓度(质量分数分别为0.1%、0.3%、0.6%和1%)DS处理组及1%DS+AKT激动剂组。通过细胞增殖实验、划痕实验、Transwell迁移和侵袭实验检测A2780细胞的增殖、迁移和侵袭的影响;蛋白质印迹法检测AKT和p-Akt的表达。两独立样本比较采用t检验,双因素数据间比较采用两因素方差分析。结果细胞实验表明,DS对A2780细胞的增殖具有抑制作用,并呈浓度和时间依赖性,差异有统计学意义(F_(处理)=81.66;F_(时间)=321.2;F_(处理×时间)=15.84,均P<0.0001)。划痕实验显示,DS呈剂量依赖性地抑制A2780细胞迁移,0、0.3%和1%DS处理A2780细胞不同时间后,差异有统计学意义(F_(处理)=40.96,P<0.0001;F_(时间)=73.32,P<0.0001;F_(处理×时间)=5.075,P=0.0017)。Transwell实验结果表明,空白对照组、0.3%DS处理组和1%DS处理组迁移细胞数分别为(514.13±19.56)、(251.59±15.95)和(101.67±10.07)个/HP,两处理组分别与空白对照组相比差异均有统计学意义,t值分别为32.47和18.02,均P<0.0001;0.3%DS处理组和1%DS处理组相比差异有统计学意义,t=13.77,P<0.001。空白对照组、0.3%DS处理组和1%DS处理组侵袭细胞数分别为(553.27±18.79)、(231.67±17.75)和(91.54±7.13)个/HP,两处理组分别与空白对照组相比差异有统计学意义,t值分别为21.55和39.79,均P<0.0001;0.3%DS处理组和1%DS处理组相比差异有统计学意义,t=12.69,P<0.001。蛋白质印迹结果显示,0.3%和1%DS处理A2780细胞24 h后,AKT表达量与对照组相比没有改变;p-AKT蛋白相对表达量均低于对照组,差异均有统计学意义,t值分别为8.143和26.19,均P<0.005;0.3%DS处理组与1%DS处理组相比差异有统计学意义,t=11.67,P<0.001。加入AKT激动剂后,DS对卵巢癌细胞的抑制作用消失。结论DS能够抑制A2780细胞的增殖、迁移和侵袭能力,其机制可能与人卵巢癌细胞A2780 p-AKT蛋白表达量降低与抑制PI3K/AKT信号通路有关。

Objective To analyze the effect of dextran sulfate(DS)on the biological behavior of human ovarian cancer cell line A2780 and its molecular mechanism were observed in vitro.Methods Human serous ovarian cancer cells(A2780)were cultured in vitro and divided into blank control group,DS treatment group(concentration of 0.1%,0.3%,0.6%and 1%)and 1%DS+AKT agonist group.The effects of proliferation,migration and invasion of A2780 cells were detected by cell proliferation experiment,scratch test,Transwell migration and invasion test;and the expression of AKT and p-Akt were detected by western blotting.t-test was used to compare the two independent samples,and two-way ANOVA was used to compare the two-factor data.Results CCK8 results showed that DS inhibited the proliferation of A2780 cells in a concentration-and time-dependent manner,and the difference was statistically significant(F_(treatment)=81.66,F_(time)=321.2,F_(treatment×time)=15.84,all P<0.0001).Scratch test showed that DS inhibited the migration of A2780 cells in a dose-dependent manner,and there was significant difference between 0,0.3%and 1%DS treatment of A2780 cells at different times(F_(treatment)=40.96,P<0.0001;F_(time)=73.32,P<0.0001;F_(treatment×time)=5.075,P=0.0017).The results of Transwell experiment showed that the number of migrating cells in the blank control group,0.3%DS treatment group and 1%DS treatment group was(514.13±19.56),(251.59±15.95)and(101.67±10.07)/HP,there were significant differences between the two treatment groups and the blank control group(t=32.47,18.02,P<0.0001),and between the 0.3%DS treatment group and the 1%DS treatment group(t=13.77,P<0.001).The numbers of invasive cells in blank control group,0.3%DS treatment group and 1%DS treatment group were(553.27±18.79),(231.67±17.75),(91.54±7.13)/HP,there were significant differences between the two treatment groups and the blank control group(t=21.55,39.79,P<0.0001),and between the 0.3%DS treatment group and the 1%DS treatment group(t=12.69,P<0.001).Western blotting showed that after A2780 cells were treated with 0.3%and 1%DS for 24 h,the expression of AKT did not change compared with the control group,but the relative expression of p-AKT protein was lower than that of the control group(t=8.143,26.19,P<0.005).There was significant difference between 0.3%DS group and 1%DS group,t=11.67,P<0.001.After the addition of AKT agonist,the inhibitory effect of DS on ovarian cancer cells disappeared.Conclusion DS can inhibit the proliferation,migration and invasion of A2780 cells,which may be related to the decreased expression of p-AKT protein and inhibition of PI3 K/AKT signal pathway in human ovarian cancer cells(A2780).