左金丸醇提物调控糖代谢改善结肠癌细胞西妥昔单抗抵抗的作用机制

Mechanism of Zuo Jin Wan alcohol extract reversing cetuximab resistance via regulating glucose metabolism incolorectal cancer cells

目的探讨左金丸醇提物通过能量代谢糖酵解对人结肠癌细胞西妥昔单抗(cetuximab,CET)抵抗的调节机制。方法将人结肠癌SW620细胞分为对照组、CET组和左金丸醇提物联合CET组,CET组加入含有100μL不同浓度(0.5、1、2、4和8 mg/mL)的CET的培养液,左金丸醇提物联合CET组加入含有100μL不同浓度(0.5、1、2、4和8 mg/mL)的CET以及50μg/mL左金丸醇提物的培养液,对照组不作处理,每组设4个复孔。培养24、48和72 h后采用Cell Counting Kit-8(CCK-8)法检测细胞增殖。应用Western blot法检测各组表皮生长因子受体(epidermal growth factor receptor,EGFR)、磷酸化EGFR(phosphorylated EGFR,p-EGFR)、蛋白激酶B(protein kinase B,Akt)、p-Akt、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)和p-mTOR等EGFR通路上下游蛋白的表达情况。采用相关试剂盒分别检测细胞葡萄糖摄取、乳酸产生量和ATP含量。Seahorse能量代谢检测仪检测细胞的基础耗氧率(oxygen comsumpition rate,OCR)、细胞外酸化率(extracellular acidification rate,ECAR)和代谢表型变化。结果左金丸醇提物对SW620细胞的增殖作用呈浓度及时间依赖性,72 h的IC50为62.59μg/mL,本研究采用低于该值的无毒剂量50μg/mL。与CET组比较,左金丸醇提物联合CET组在24、48和72 h时对细胞增殖的抑制作用均增强,且呈时间依赖性,差异均具有统计学意义(均P<0.01);并伴随着EGFR、Akt和mTOR等蛋白磷酸化表达的下调(均P<0.01)。与对照组比较,CET组和左金丸醇提物联合CET组的葡萄糖摄取量、乳酸产生量、ATP含量及ECAR均降低(均P<0.05)。与CET组比较,左金丸醇提物联合CET组的葡萄糖摄取量、乳酸产生量、ATP含量及ECAR也均下降(均P<0.05)。结论左金丸醇提物联合CET抑制人结肠癌SW620细胞增殖具有协同作用。左金丸醇提物可能通过调控人结肠癌细胞糖酵解以及降低p-EGFR、p-Akt和p-mTOR等蛋白表达来逆转CET耐药。

Objective To investigate the mechanism of the reversal effects of Zuo Jin Wan alcohol extract on cetuximab(CET)resistance through glycolysis in colon cancer cells.Methods Human colon cancer SW620 cells were divided into the control group,CET group and Zuo Jin Wan alcohol extract combined CET group.The CET group was cultured with 100μL medium containing different concentrations of CET(0.5,1,2,4 and 8 mg/mL),the Zuo Jin Wan alcohol extract combined CET group was cultured with 100μL medium containing different concentrations of CET(0.5,1,2,4 and 8 mg/mL)and 50μg/mL Zuo Jin Wan alcohol extract,while no treatment was given to the control group.Four wells were set in each group.After 24,48 and 72 h of culture,cell proliferation was detected by the cell counting kit-8(CCK-8)method,respectively.The expressions of epidermal growth factor receptor(EGFR),phosphorylated EGFR(p-EGFR),protein kinase B(AKT),p-AKT,mammalian target of rapamycin(mTOR),and p-mTOR were detected by Western blot.Glucose uptake,lactate production,and ATP content of cells were detected by the corresponding kits.The changes of oxygen consumption rate(OCR),extracellular acidification rate(ECAR)and metabolic phenotype were detected by Seahorse energy metabolism detector.Results The effect of Zuo Jin Wan alcohol extract on the proliferation of SW620 cells was in a dose-dependent and time-dependent manner,with an IC50 in 72 h of 62.59μg/mL.Therefore,50μg/mL Zuo Jin Wan alcohol extract was used as the non-toxic dose for SW620 cells in this study.Compared with the CET group,the Zuo Jin Wan alcohol extract combined CET group showed increased inhibitory effect on cell proliferation at 24,48 and 72 h in a time-dependent manner,with statistically significant differences(all P<0.01).The levels of p-EGFR,p-Akt,and p-mTOR were all significantly decreased after treatment with Zuo Jin Wan alcohol extract combined with CET(all P<0.01).Compared with the control group,glucose uptake,lactate production,ATP content,and ECAR of the CET group and Zuo Jin Wan alcohol extract combined CET group were all decreased(all P<0.05).Compared with the CET group,the glucose uptake,lactic production,ATP content and ECAR of the Zuo Jin Wan alcohol extract combined CET group were also decreased(all P<0.05).Conclusions Zuo Jin Wan alcohol extract combined with CET has a synergistic effect on the proliferation of human colon cancer SW620 cells.Zuo Jin Wan alcohol extract can reverse CET resistance by regulating glucose metabolism and inhibiting the phosphorylation of EGFR,Akt and mTOR in human colon cancer cel ls.