基于FOXO3a调控下的糖酵解途径探讨康艾注射液改善A549/DDP细胞顺铂耐药性研究

Study on Improvement of Cisplatin Resistance of A549/DDP Cells by Kang’ai Injection(康艾注射液) Based on FOXO3a Regulated Glycolysis Pathway

目的基于肿瘤细胞糖酵解途径探讨康艾注射液改善非小细胞肺癌顺铂耐药的分子机制。方法应用CCK-8法测定人肺腺癌顺铂耐药A549/DDP细胞对顺铂的耐药指数,并观察康艾注射液联合顺铂对细胞生长的影响;利用Kaplan-Meier Plotter数据库分析叉头转录因子O3a(FOXO3a)基因表达水平与肺腺癌患者预后的相关性;康艾注射液联合顺铂干预A549/DDP细胞,分别利用荧光定量PCR方法检测转录因子FOXO3a基因表达水平,化学显色法检测肿瘤细胞葡萄糖消耗及乳酸生成情况,Western blot方法检测FOXO3a、葡萄糖转运蛋白及糖酵解相关酶的表达水平。结果A549及A549/DDP细胞的IC_(50)值分别为8.89μg/mL、18.97μg/mL,A549/DDP细胞的耐药指数为2.13;康艾注射液可显著抑制A549/DDP细胞生存活力,联合顺铂后可明显改善A549/DDP细胞对顺铂的耐药性;FOXO3a基因表达水平与肺腺癌患者预后成正相关;康艾注射液联合顺铂则可明显上调A549/DDP细胞FOXO3a基因及蛋白表达水平,抑制肿瘤细胞葡萄糖消耗及乳酸生成,抑制葡萄糖转运蛋白1(GLUT1)、己糖激酶1(HK1)、己糖激酶2(HK2)及血小板型磷酸果糖激酶(PFKP)表达水平。结论康艾注射液联合顺铂可通过上调FOXO3a表达,抑制肿瘤细胞糖酵解,改善A549/DDP细胞顺铂耐药性。

Objective To investigate the molecular mechanism of Kang’ai Injection(康艾注射液)in the reversal of cisplatin resistance in non-small cell lung cancer cells based on the pathway of glycolysis of tumor cells.Methods CCK-8 method was used to determine the cisplatin resistance index of human lung adenocarcinoma A549/DDP cells,and to observe the effect of Kang’ai Injection combined with cisplatin on cancer growth.Kaplan-Meier Plotter database was used to analyze the correlation between FOXO3a gene expression and prognosis of patients with lung adenocarcinoma.The gene expression level of FOXO3a was detected by fluorescence quantitative PCR,the glucose consumption and lactate production of tumor cells were determined by chemical colorimetric assay,and the protein expression levels of FOXO3a,glucose transporter and glycoly-related enzymes were analysed by Western blot when combined Kang’ai Injection with cisplatin in A549/DDP cells.Results The IC_(50) values of A549 and A549/DDP cells were 8.89 g/mL and 18.97 g/mL,respectively,and the drug resistance index of A549/DDP cells was 2.13.Kang’ai Injection could significantly inhibit the viability of A549/DDP cells,and combination with cisplatin could significantly improve the cisplatin resistance of A549/DDP cells.FOXO3a gene expression was positively correlated with prognosis in patients with lung adenocarcinoma.Co-treatment Kang’ai Injection with cisplatin could significantly up-regulate the gene and protein expression levels of FOXO3a in A549/DDP cells,inhibit glucose consumption and lactic acid production in tumor cells,down-regulate the protein expression levels of glucose transporter 1(GLUT1),hexokinase 1(HK1),hexokinase 2(HK2)and platelet-type phosphofructokinase(PFKP).Conclusion Combination of Kang’ai Injection and cisplatin could inhibit the glycolysis pathway and improve the cisplatin resistance of A549/DDP cells by up-regulating the expression of FOXO3a.