冬凌草甲素抑制胃癌细胞增殖及增强西妥昔单抗化疗敏感性的机制研究

Mechanism of oridonin in inhibiting proliferation of gastric cancer cells and enhancing chemosensitivity of cetuximab

目的探讨冬凌草甲素抑制胃癌细胞增殖及增强西妥昔单抗化疗敏感性的机制。方法采用不同浓度冬凌草甲素或相同浓度冬凌草甲素在不同时间处理SGC7901胃癌细胞株,通过MTT法检测其增殖水平。冬凌草甲素、西妥昔单抗、冬凌草甲素联合西妥昔单抗与SGC7901胃癌细胞株共培养后,采用流式细胞技术检测细胞凋亡率和细胞周期的变化,Western blot检测细胞中表皮生长因子受体(EGFR)、蛋白激酶B(AKT)、细胞外调节蛋白激酶(ERK)、信号传导及转录激活蛋白3(STAT3)磷酸化表达水平,Real-time PCR检测Bcl-xL、Cyclin D1的转录水平。结果冬凌草甲素对SGC7901胃癌细胞株的增殖作用呈浓度及时间依赖性,浓度越高则增殖水平越低,相同浓度冬凌草甲素对细胞的增殖抑制作用随时间延长而增强,差异有统计学意义(P<0.05)。流式细胞技术检测细胞凋亡结果显示,SGC7901胃癌细胞株无药物干预时凋亡率为(2.90±0.90)%;西妥昔单抗作用细胞后,细胞凋亡率为(9.30±2.20)%;冬凌草甲素作用后细胞凋亡率为(18.20±4.20)%;冬凌草甲素联合西妥昔单抗处理组细胞凋亡率高达(57.10±8.70)%,显著高于单药组细胞凋亡率(P<0.05)。冬凌草甲素联合西妥昔单抗处理SGC7901胃癌细胞后,磷酸化表皮生长因子受体(pEGFR)、磷酸化蛋白激酶B(pAKT)、磷酸化细胞外调节蛋白激酶(pERK)、磷酸化信号传导及转录激活蛋白3(pSTAT3)表达水平减少,与冬凌草甲素组、西妥昔单抗组比较有显著差异(P<0.05),Bcl-xL、Cyclin D1 mRNA转录减少,差异有统计学意义(P<0.05)。结论冬凌草甲素通过抑制EGFR/STAT3通路来抑制胃癌细胞增殖及增强西妥昔单抗化疗敏感性。

Objective To explore the mechanism of oridonin in inhibiting proliferation of gastric cancer cells and enhancing chemosensitivity of cetuximab.Methods The SGC7901 gastric cancer cell lines were treated with different concentrations of oridonin or the same concentration of oridonin at different time points,and the proliferation levels were detected by MTT.After oridonin,cetuximab,oridonin combined with cetuximab were co-cultured with SGC7901 gastric cancer cell lines,the flow cytometry was used to detect the apoptosis rate and cell cycle.Western blot was used to detect the phosphorylation of epidermal growth factor receptor(EGFR),protein kinase B(Akt),extracellular regulatory protein kinase(ERK),signal transduction and activator of transcription 3(STAT3)in cells,and real-time PCR was used to detect the transcriptional level of Bcl-xL and Cyclin D1.Results Oridonin had a time and concentration dependent effect on the proliferation of SGC7901 gastric cancer cell lines.The higher the concentration was,the lower the proliferation level was.The inhibitory effects of oridonin at the same concentration on cell proliferation increased with time,and there was significant difference(P<0.05).The results of apoptosis detected by flow cytometry showed that the mortality of SGC7901 gastric cancer cell line was(2.90±0.90)%without drug intervention,and cetuximab was(9.30±2.20)%,the apoptosis rate of oridonin was(18.20±4.20)%,and the apoptosis rate of oridonin combined with cetuximab was(57.10±8.70)%,which were significantly higher than that of other groups(P<0.05).The expression levels of pEGFR,pAKT,pERK and pSTAT3 decreased in SGC7901 gastric cancer cells treated with oridonin and cetuximab,Bcl-xL and Cyclin D1 mRNA transcription decreased,and there were significant differences when compared with oridonin group and cetuximab group(P<0.05).Conclusion Oridonin inhibits proliferation of gastric cancer cells and enhances chemosensitivity of cetuximab by inhibiting EGFR/STAT3 pathway.