华蟾素调节肠道菌群抑制胃癌细胞进展的机制研究

Study on the mechanism of cinobufotalin regulating intestinal microbiota and inhibiting the progression of gastric cancer cells

关键词:  华蟾素  胃癌细胞  原位移植瘤  肠道菌群

Key words:  cinobufotalin  gastric cancer cell  orthotopic transplantation tumor  intestinal microbiota

摘要:

目的:探究华蟾素在胃癌细胞以及胃癌移植瘤小鼠模型中,对胃癌细胞增殖、迁移、侵袭情况的影响,并探讨其调节小鼠肠道菌群发挥抗肿瘤作用的机制。方法:(1)体外实验:CCK8实验检测华蟾素对胃癌细胞的半数抑制浓度(IC50)；克隆形成实验检测华蟾素抑制胃癌细胞增殖的作用；划痕实验、Transwell实验检测华蟾素抑制胃癌细胞侵袭迁移能力的作用；细胞凋亡实验检测华蟾素促进胃癌细胞凋亡的作用；Western blot实验检测华蟾素对胃癌细胞EMT相关蛋白表达的影响。(2)体内实验:构建裸鼠异种原位移植瘤模型,随机分为3组,每组5只,分别为对照组、低剂量组(6 mg/mL)、高剂量组(12 mg/mL),给药干预4周,评估口服华蟾素对体内胃癌细胞增殖情况的影响。同时,收集小鼠粪便进行宏基因组学分析检测小鼠肠道细菌组成变化。结果:华蟾素在体外抑制胃癌细胞MKN1、HGC27的增殖、迁移、侵袭,同时显著促进胃癌细胞凋亡。华蟾素提高胃癌细胞E-cadherin的蛋白表达水平,降低N-cadherin、Vimentin的蛋白表达水平。华蟾素在体内抑制裸鼠移植瘤生长,并改变小鼠肠道菌群微生物组成。华蟾素干预后,肠道拟杆菌门丰度上升,厚壁菌门和变形菌门的丰度降低。结论:华蟾素可有效抑制体外及体内胃癌细胞增殖、迁移、侵袭情况,且华蟾素抗肿瘤作用与调节小鼠肠道菌群组成密切相关。

Abstract:

Objective: To investigate the efficiency and underlying mechanism of cinobufotalin in the treatment of gastric cancer cell proliferation, migration, invasion and mice with gastric cancer transplanted tumors, and to explore its effects on the intestinal flora of mice treated with cinobufotalin. Methods: (1) In vitro experiment: human gastric cancer cell line HGC27 and MKN1 were treated with different concentrations of cinobufotalin, cell viability and the half inhibitory concentration (IC50) of cinobufotalin was detected by cell counting kit 8 (CCK8); HGC27 and MKN1 cell proliferation was detected by clone formation experiment; HGC27 and MKN1 cell invasion and migration after treated by cinobufotalin was detected by scratch test and transwell assay; HGC27 and MKN1 cell apoptosis was detected by cell apoptosis assay; the expression of EMT related proteins in HGC27 and MKN1 was detected by western blot assay. (2) In vivo experiment: A nude mouse xenograft tumor model was constructed to evaluate the effect of cinobufotalin on the growth of gastric cancer in vivo. The model mice were randomly divided into 3 groups, with 5 mice in each group, namely the control group, low-dose group (6 mg/mL), and high-dose group (12 mg/mL). The intervention was given for 4 weeks to evaluate the effect of oral administration of cinobufotalin on the proliferation of gastric cancer cells in vivo. At the same time, mouse feces were collected for metagenomic analysis to detect changes in the composition of intestinal bacteria in mice. Results: Cinobufotalin inhibits the proliferation, migration, and invasion of gastric cancer cell line MKN1 and HGC27 in vitro as well as significantly promote apoptosis of gastric cancer cells. Cinobufotalin increases the protein expression level of E-cadherin in gastric cancer cells and reduces the protein expression level of N-cadherin and Vimentin. Cinobufotalin inhibits the growth of transplanted tumors in nude mice in vivo and alters the composition of gut microbiota in mice. After intervention with cinobufotalin, the abundance of Bacteroidetes in the intestine increased, while the abundance of Firmicutes and Proteobacteria decreased. Conclusion: Cinobufotalin can effectively inhibit the proliferation, migration, and invasion of gastric cancer cells in vitro and in vivo, and its anti-tumor effect is closely related to regulating the composition of mouse gut microbiota.