DHA联合5-FU通过Rho/ROCK调控胃癌SGC7901细胞增殖、凋亡的体外实验

The Effect of DHA Combined with 5-FU on the Proliferation and Apoptosis in Gastric SGC7901 Cells by Rho/ROCK

该文研究了二十二碳六烯酸(doeosahexaenoic acid,DHA)联合5-氟尿嘧啶(5-fluorouracil,5-FU)对胃癌SGC7901细胞增殖、凋亡及其Rho家族基因表达的影响。实验分为对照组、DHA组、5-FU组和DHA联合5-FU组,用CCK-8法分别检测药物作用24、48、72 h后对人胃癌细胞增殖的抑制作用,流式细胞术检测细胞凋亡情况,Real-time PCR和Western blot分别检测细胞RhoA、RhoC和ROCK1的mRNA水平和蛋白质水平。结果显示,5-FU单独作用时,随着作用时间延长和剂量加大,对胃癌细胞的增殖抑制作用增强,DHA单独作用时,低浓度抑制作用不明显,较高浓度时有显著抑制作用,40μg/mL DHA与4μg/mL 5-FU联合时有明显的增效作用。与对照组相比,40μg/mL DHA对细胞凋亡作用不明显,60μg/mL DHA主要引起细胞晚期凋亡,16μg/mL 5-FU主要引起细胞早期凋亡,两者联合时对细胞晚期凋亡有显著的增强作用。与对照组相比,DHA组及5-FU组RhoAmRNA水平下降,5-FU及联合组RhoC mRNA水平升高。与对照组相比,DHA组RhoA、RhoC蛋白质水平下降,5-FU组RhoA、ROCK1蛋白质水平下降,而联合组RhoA蛋白质水平下降显著。综上所述,DHA联合5-FU可增强对胃癌SGC7901细胞增殖的抑制作用,两者作用于细胞凋亡的不同时期且联合用药对晚期凋亡有增强作用。DHA与5-FU联合应用对细胞增殖和凋亡作用机制可能通过抑制RhoA蛋白表达起作用。

The objective of the present study is to explore the synergistic effect of docosahexaenoic acid（DHA） and 5-fluorouracil（5-FU） on the human gastric cancer cell line SGC7901 and its underlying mechanism. The experiments were divided into four groups： control group, DHA group, 5-FU group and combination treatment group. The inhibition of gastric cancer cell proliferation was determined by CCK-8 assay after stimulated 24, 48 and 72 h. Flow cytometric analysis was used to assess cell apoptosis. Real-time PCR and Western blot were used to detect the mRNA and protein levels of RhoA, RhoC and ROCK1 genes, respectively. The results showed 5-FU could increasingly suppress the proliferation of SGC7901 cells with higher doses and longer response time. However, the inhibitory effect, which is not significant with lower doses, appeared significantly in higher doses treated with DHA alone. The inhibitory effect increased when combining 40 μg/mL DHA and 16 μg/mL 5-FU. Compared with the control group, the pro-apoptotic effect with 40 μg/mL DHA was not obvious, 60 μg/mL DHA mainly increased the late apoptosis cells and 16 μg/mL 5-FU induced early apoptosis effect respectively. Co-treatment with DHA and 5-FU enhanced the post-apoptotic effects. The levels of RhoA mRNA decreased in DHA group and 5-FU group while the levels of RhoC mRNA increased in 5-FU group and combination treatment group compared with control group. Meanwhile, the levels of RhoA and RhoC proteins declined with DHA treatment and 5-FU decreased the levels of RhoA and ROCK1 proteins. Combination treatment group resulted in a significantly decrease of RhoA protein level. These results showed that DHA and 5-FU synergetic inhibited the proliferation of gastric SGC7901 cells and affected the cell apoptosis in different periods. Mechanism of synergistically affecting cell proliferation and apoptosis might work through reducing the level of RhoA protein in SGC7901 cells.