己糖激酶2在NSCLC厄洛替尼耐药中的作用及机制

Role and mechanism of hexokinase 2 in erlotinib resistance in non-small cell lung cancer

目的观察糖酵解关键酶己糖激酶2(hexokinase 2,HK2)在人非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达,探讨HK2在NSCLC厄洛替尼耐药中的作用及机制。方法在线数据库分析肺癌组织中HK2的表达与预后的关系以及厄洛替尼对NSCLC HK2基因表达的影响;q-PCR体外验证HK2在正常肺上皮细胞、肺癌细胞中的表达差异;q-PCR、Western blot检测厄洛替尼敏感细胞(PC9)、耐药细胞(PC9/ER)中HK2的基础表达以及厄洛替尼干预后HK2的表达情况;进而使用HK2抑制剂3-溴丙酮酸处理PC9/ER,采用CCK-8法检测PC9/ER耐药性、流式细胞术分析PC9/ER的增殖能力。结果HK2与肺癌患者预后不良相关,HK2高表达组患者的总体生存期(overall survival,OS)较低表达组明显缩短(P<0.05);细胞实验证实HK2在PC9/ER中相对高表达,且厄洛替尼可降低PC9细胞中HK2的表达,而对PC9/ER细胞HK2的表达无显著影响;3-溴丙酮酸可有效抑制PC9/ER中HK2的表达,CCK-8法结果表明,PC9/ER 3-溴丙酮酸处理组厄洛替尼IC_(50)值较PC9/ER对照组显著降低[(0.005±0.001)vs(1.967±0.436)μmol/L,P=0.016],提示3-溴丙酮酸处理后能让耐药的PC9/ER细胞恢复对厄洛替尼的敏感性;3-溴丙酮酸处理组增殖指数为(14.1±0.8)%,较对照组(30.4±2.3)%显著减少(P=0.008),提示3-溴丙酮酸能明显抑制耐药细胞增殖。结论HK2是NSCLC患者的不良预后指标,可诱导NSCLC厄洛替尼耐药。可能和其促进糖酵解的机制有关,抑制其表达有望成为逆转NSCLC厄洛替尼耐药的有效策略。

Objective To observe the expression of hexokinase 2(HK2),a key enzyme of glycolysis,in human non-small cell lung cancer(NSCLC)tissues,and to investigate the role and mechanism of HK2 in the resistance of NSCLC to erlotinib.Methods The relationship between the expression of HK2 and prognosis in lung cancer and the effect of erlotinib on NSCLC HK2 gene exlpression were analyzed through online databases(Kaplan-Meier Plotter,GEPIA and GEO DataSets).q-PCR was used to verify the expression differences of HK2 in normal lung epithelial cells and lung cancer cells in vitro.The basic expression of HK2 in erlotinib-sensitive cells(PC9)and erlotinib-resistant cells(PC9/ER)and the expression of HK2 after erlotinib intervention were detected by q-PCR and Western blotting.After 3-bromopyruvate,a HK2 inhibitor,was used to treat PC9/ER cells,the erlotinib resistance and cell proliferation in PC9/ER cells were detected by CCK-8 assay and flow cytometry.Results HK2 was associated with poor prognosis in NSCLC patients,and overall survival(OS)in the group with high expression of HK2 was significantly shortened when compared with the group with low expression(P<0.05).Cell experiments confirmed that HK2 was relatively highly expressed in PC9/ER cells,and erlotinib treatment could reduce HK2 expression in PC9 cells,but had no significant effect on PC9/ER cells.3-bromopyruvate successfully inhibited the expression of HK2 in PC9/ER cells.CCK-8 assay showed that IC50 value of erlotinib was significantly lower in the PC9/ER 3-bromopyruvate treated group than the PC9/ER control group(0.005±0.001 vs 1.967±0.436μmol/L,P=0.016),suggesting that the inhibitor treatment can restore the sensitivity of PC9/ER cells to erlotinib.In addition,the treatment also resulted in significantly decreased proliferation index in the 3-bromopyruvate treatment group than the control group[(14.1±0.8)%vs(30.4±2.3)%,P=0.008),indicating that the inhibitor could significantly inhibit the proliferation of drug-resistant cells.Conclusion HK2 is an indicator for poor prognosis in NSCLC patients.It can induce erlotinib resistance to NSCLC,which may be associated with its promotion in glycolysis,and inhibiting its expression may be an effective strategy to reverse erlotinib resistance in NSCLC.