摘要
目的:基于有氧糖酵解探讨四君子汤合沙参麦冬汤加减化裁方[益气养阴解毒方(YQYYJD)]提高表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)耐药肺腺癌细胞顺铂敏感性的作用机制.方法:细胞增殖与活性检测(CCK-8)法检测YQYYJD(0、2、3、4、5、6、7、8 g·L-1)和顺铂(0、3、6、9、12、15、18、21、24、27 mg·L-1)干预 24 h对 PC9/GR细胞增殖活力的影响,分别计算其对PC9/GR细胞的半数抑制浓度(IC50)作为后续实验浓度;将PC9/GR细胞分为空白组(完全培养基)、YQYYJD组(5 g·L-1)、顺铂组(12 mg·L-1)、联合组(YQYYJD 5 g·L-1+顺铂12 mg·L-1),按分组干预24 h后,采用CCK-8法检测细胞存活率;克隆形成实验检测细胞增殖能力;划痕实验、Transwell实验检测细胞迁移能力;比色法检测细胞葡萄糖的消耗量、乳酸及三磷酸腺苷(ATP)的生成量;蛋白免疫印迹法(Western blot)检测糖酵解相关蛋自己糖激酶2(HK2)、P型磷酸果糖激酶(PFKP)、M2型丙酮酸激酶(PKM2)、乳酸脱氢酶A(LDHA)、葡萄糖转运体1(GLUT1)、单羧酸转运蛋白4(MCT4)表达水平.结果:YQYYJD与顺铂均呈浓度依赖性抑制PC9/GR细胞的活力,YQYYJD和顺铂的IC5.值分别为5.15 g·L-1和12.91 mg·L-1.细胞增殖方面,与空白组比较,YQYYJD组、顺铂组、联合组的细胞存活率、克隆形成数目均显著下降(P＜0.01);与YQYYJD组、顺铂组比较,联合组的细胞存活率、克隆形成数目均显著下降(P＜0.01).细胞迁移方面,与空白组比较,YQYYJD组、顺铂组、联合组的细胞迁移率、穿过Transwell小室膜的细胞数均显著下降(P＜0.01);与YQYYJD组、顺铂组比较,联合组的细胞迁移率、穿过Transwell小室膜的细胞数均显著下降(P＜0.01).糖酵解方面,与空白组比较,YQYYJD组、顺铂组、联合组细胞的葡萄糖消耗量、乳酸及ATP生成量均显著下降(P＜0.01);与YQYYJD组、顺铂组比较,联合组葡萄糖消耗量、乳酸及ATP生成量均明显下降(P＜0.05).与空白组比较,YQYYJD组、顺铂组、联合组细胞中HK2、PFKP、PKM2、LDHA蛋白表达水平均明显下降(P＜0.01);与YQYYJD组、顺铂组比较,联合组上述蛋白表达水平显著下降(P＜0.01).结论:YQYYJD能协同顺铂抑制PC9/GR细胞的增殖、迁移,提高细胞对顺铂的敏感性,其机制可能与下调糖酵解相关限速酶HK2、PFKP、PKM2、LDHA的表达,抑制糖酵解水平有关.
Abstract
Objective:To investigate the mechanism of modified Si Junzitang and Shashen Maidong Tang[Yiqi Yangyin Jiedu prescription(YQYYJD)]in enhancing the sensitivity of cisplatin in epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)-resistant lung adenocarcinoma cells based on aerobic glycolysis.Methods:The effects of different concentrations of YQYYJD(0,2,3,4,5,6,7,8 g·L-1)and cisplatin(0,3,6,9,12,15,18,21,24,27 mg·L-1)on the proliferation and activity of PC9/GR cells were detected by the cell counting kit-8(CCK-8)assay after 24 hours of intervention.The half-maximal inhibitory concentration(IC50)for PC9/GR cells was calculated to determine the concentrations used in subsequent experiments.PC9/GR cells were divided into blank group(complete medium),YQYYJD group(5 g·L-1),cisplatin group(12 mg·L-1),and combined group(YQYYJD 5 g·L-1+cisplatin 12 mg·L-1).After 24 hours of intervention,cell viability was measured using CCK-8 assay.Cell proliferation was assessed by colony formation assay,and cell migration was evaluated by scratch and Transwell assays.Glucose consumption,lactate production,and adenosine triphosphate(ATP)levels were measured by colorimetric assays.The expression levels of glycolysis-related proteins,including hexokinase 2(HK2),phosphofructokinase P(PFKP),pyruvate kinase M2(PKM2),lactate dehydrogenase A(LDHA),glucose transporter 1(GLUT1),and monocarboxylate transporter 4(MCT4),were determined by Western blot.Results:Both YQYYJD and cisplatin inhibited the viability of PC9/GR cells in a concentration-dependent manner.The IC50 of PC9/GR cells for YQYYJD and cisplatin were 5.15 g·L-1 and 12.91 mg·L-1,respectively.In terms of cell proliferation,compared with the blank group,the cell survival rate and the number of colonies formed in the YQYYJD group,cisplatin group,and combined group were significantly decreased(P＜0.01).Compared with the YQYYJD and cisplatin groups,the combined group showed a further significant reduction in cell survival rate and colony formation(P＜0.01).In terms of cell migration,compared with the blank group,the cell migration rate and the number of cells passing through the Transwell membrane in the YQYYJD group,cisplatin group,and combined group were significantly decreased(P＜0.01).Compared with the YQYYJD and cisplatin groups,the combined group exhibited a further significant reduction in cell migration rate and the number of cells passing through the Transwell membrane(P＜0.01).In terms of glycolysis,compared with the blank group,glucose consumption,lactate production,and ATP levels in the YQYYJD group,cisplatin group,and combined group were significantly decreased(P＜0.01).Compared with the YQYYJD and cisplatin groups,the combined group showed a further significant reduction in glucose consumption,lactate production,and ATP levels(P＜0.05).Compared with the blank group,the protein expression levels of HK2,PFKP,PKM2,and LDHA in the YQYYJD,cisplatin,and combined groups were significantly decreased(P＜0.01).The combined group showed a further significant reduction in the expression levels of these proteins compared with the YQYYJD and cisplatin groups(P＜0.01).No significant differences were observed in the protein expression levels of GLUT1 and MCT4 among the groups.Conclusion:YQYYJD can synergistically inhibit the proliferation and migration of PC9/GR cells and enhance their sensitivity to cisplatin.The mechanism may be related to the downregulation of the expression of glycolysis-related rate-limiting enzymes,including HK2,PFKP,PKM2,and LDHA,thereby inhibiting glycolysis.
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维普
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