低毒剂量丹参酮ⅡA联合顺铂通过下调糖酵解途径抑制A549/DTPs的形成

Low-toxic dose of tanshinone ⅡA combined with cisplatin inhibits the formation of A549/DTPs by down-regulating glycolysis pathway

目的 探讨低毒剂量丹参酮ⅡA联合顺铂对药物耐受人肺腺癌A549持留细胞(A549/DTPs)的影响及与糖酵解途径的关系。方法 (1)用4μg/ml(IC50)顺铂处理A549细胞10 d,构建顺铂诱导的A549/DTPs模型;采用CCK-8法检测A549、A549/DTPs对顺铂的耐受情况。(2)采用CCK-8法检测丹参酮ⅡA、顺铂以及两药联用对A549/DTPs的细胞抑制率,筛选低毒剂量丹参酮ⅡA、顺铂以及两药联用对A549/DTPs作用的终浓度,并通过Isobologram分析两药联用的协同作用。(3)将A549/DTPs分为4组:对照组、顺铂组、低毒剂量丹参酮ⅡA组、低毒联合组(低毒剂量丹参酮ⅡA联合顺铂组)。采用CCK-8法检测细胞抑制率;ATP含量检测试剂盒检测ATP含量;qRT-PCR和Western blot法检测葡萄糖转运蛋白(GLUT4)、己糖激酶(HK2)、果糖磷酸激酶(PFKFP3)和丙酮酸激酶(PKM2)蛋白和mRNA的表达情况。结果 (1)CCK-8结果显示:当顺铂浓度≥0.5μg/ml时,与A549细胞比较,A549/DTPs的细胞抑制率降低(P<0.05)。(2)CCK-8结果显示:与单用顺铂比较,浓度≥0.1μg/ml顺铂联合低毒剂量丹参酮ⅡA后细胞抑制率升高(P<0.05)。故选择0.1μg/ml顺铂联合20μg/ml(IC10)丹参酮ⅡA进行后续实验研究,Isobologram分析结果表明低毒剂量丹参酮ⅡA联合顺铂对A549/DTPs有协同作用。(3)与对照组相比,低毒剂量丹参酮ⅡA组和顺铂组ATP含量、GLUT4、HK2、PFKFP3和PKM2的mRNA和蛋白水平差异均无统计学意义(P>0.05);与对照组相比,低毒联合组ATP含量降低,GLUT4、HK2、PFKFP3和PKM2的mRNA和蛋白水平均下调(P<0.05);与低毒联合组相比,低毒剂量丹参酮ⅡA组和顺铂组细胞抑制率降低,ATP含量升高,GLUT4、HK2、PFKFP3和PKM2的mRNA和蛋白水平均上调(均P<0.05)。结论 低毒剂量丹参酮ⅡA联合顺铂抑制A549/DTPs形成,即抑制顺铂诱导的DTPs形成,可能与糖酵解途径有关。

Objective To investigate the effect of low-dose tanshinone ⅡA combined with cisplatin on drug-tolerant persister A549 cells model(A549/DTPs) and its relationship with glycolysis pathway. Methods(1)A549 cells were treated with 4 μg/ml(IC50) cisplatin for 10 d, the cisplatin-induced A549/DTPs model was constructed, and then CCK-8 method was used to detect the tolerance of A549 and A549/DTPs to cisplatin.(2)The cell inhibition rate of drugs to A549/DTPs was detected after treatment with tanshinone ⅡA, cisplatin and their combination by CCK-8 method to screen the final drug concentration, and the synergistic effect of two drugs was analyzed by Isobologram.(3)The A549/DTPs were divided into four groups: control group, cisplatin group, low-dose tanshinone ⅡA group, and low-toxicity combination group(low-dose tanshinone ⅡA combined with cisplatin group). The cell inhibition rate was detected by CCK-8 method, ATP content was detected by ATP content detection kit, and the protein and mRNA expression levels of glucose transporter(GLUT4), hexokinase(HK2), phosphofructose kinase(PFKFP3), and pyruvate kinase(PKM2) were detected by qRT-PCR and Western blot. Results(1)When the concentration of cisplatin was ≥0.5 μg/ml, the cell inhibition rate of A549/DTPs was lower than that of A549 cells by CCK-8(P<0.05).(2)When the concentration of cisplatin was ≥ 0.1 μg/ml, the cell inhibition rate after treated with low-dose tanshinone ⅡA combined with cisplatin was higher than that of cisplatin alone by CCK-8(P<0.05). Therefore, 0.1 μg/ml cisplatin combined with 20 μg/ml(IC10) tanshinone ⅡA was selected for the following experiments. Results of Isobologram analysis showed that low-dose tanshinone ⅡA combined with cisplatin had a synergistic effect on A549/DTPs.(3)Compared with control group, ATP content, the mRNA and protein levels of GLUT4, HK2, PFKFP3 and PKM2 showed no significant difference in low-dose tanshinone ⅡA group and cisplatin group(P>0.05). Compared with control group, ATP content, the mRNA and protein levels of GLUT4, HK2, PFKFP3 and PKM2 were decreased in low-toxicity combination group(P<0.05). Compared with low-toxicity combination group, the cell inhibition rate was decreased, ATP content and the mRNA and protein levels of GLUT4, HK2, PFKFP3, and PKM2 were increased in low-dose tanshinone ⅡA group and cisplatin group(P<0.05). Conclusion Low-toxic dose of tanshinone ⅡA combined with cisplatin inhibits the formation of A549/DTPs induced by cisplatin, which may be related to the glycolysis pathway.