[The role and mechanism of 2-deoxyglucose in reversing osimertinib-acquired resistance of non-small cell lung cancer cell line].

Objective: To explore the role and mechanism of 2-deoxyglucose (2-dg) in reversing osimertinib- acquired resistance of non-small cell lung cancer(NSCLC)cell line. Methods: The NSCLC line H1975 (purchased from the American Type Culture Collection) was conducted by induction method in vitro to construct the osimertinib-resistance NSCLC cell line H1975-OR. The osimertinib-resistance of H1975-OR cell line was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony-formation assay, Ki67 incorporation assay and the expression of apoptosis-related protein. The glycolysis level was assayed by the lactic acid production measured in the culture medium supernatant of H1975 and H1975-OR. The expression of glycolysis key enzymes (HK2, GLUT1, P-PKM2) and apoptosis-related protein (BIM, Bcl-2) were detected by Western blot. The cells were divided into control group, 2-deoxyglucose (4 mmol/L) monotherapy group, osimertinib (3 μmol/L) monotherapy group and 2-deoxyglucose (4 mmol/L)+ osimertinib (3 μmol/L) combination therapy group, then the apoptosis rate of cells was measured by flow cytometry to evaluate the pro-apoptotic ability of drugs. Date were analyzed by Independent-Samples t-test using SPSS 16.0 statistical software. Results: The glycolysis level of osimertinib-sensitive cell line H1975 was lower than that of osimertinib-resistance cell line H1975-OR [the yield of lactic acid, respectively, was (21.0±0.9) and (26.5±2.8) mmol·L(-1)·10(4)cells(-1), P<0.05]. The osimertinib- acquired resistance of H1975-OR could be reversed by 4 mmol/L 2-deoxyglucose(the IC(50) value of osimertinib in H1975-OR cell line decreased from (7.0±1.9) μmol/L to (1.4±0.1) μmol/L, which was close to the IC(50) value of osimertinib in H1975 cell line (1.0±0.2) μmol/L. The apoptosis rate of H1975-OR was significantly higher in 2-deoxyglucose + osimertinib combination therapy group (26.7±2.4)%, compared to control group (5.1±0.7)%, 2-deoxyglucose monotherapy group (6.1±2.5)% and osimertinib monotherapy group (11.4±2.7)%(all P<0.05). The expression of pro-apoptotic protein BIM in H1975-OR was significantly higher in 2-deoxyglucose+ osimertinib combination therapy group (177.8±28.1)% and the expression of anti-apoptotic protein Bcl-2 in H1975-OR was significantly lower in 2-deoxyglucose+ osimertinib combination therapy group (24.6±5.2)%, compared to control group (100±0)%, all P<0.05. Conclusion: 2-deoxyglucose can reverse the acquired resistance of NSCLC cell line to osimertinib, which may be related to the inhibition of cell glycolysis and the induction of apoptosis.目的： 探究2-脱氧葡萄糖（2-DG）逆转非小细胞肺癌细胞对奥希替尼继发性耐药的作用及机制。 方法： 将非小细胞肺癌（NSCLC）细胞株H1975（购自美国国家细胞库）通过体外诱导法处理后建立奥希替尼继发性耐药肺癌细胞株H1975-OR，用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐（MTT）法、克隆形成实验、Ki67蛋白荧光染色、凋亡通路相关蛋白表达水平检测，评估细胞株H1975-OR对奥希替尼的耐药性；通过测定细胞株H1975和H1975-OR培养基上清液中乳酸含量确定细胞糖酵解水平；Western blot法检测糖酵解关键酶（己糖激酶2、葡萄糖转运子1、磷酸化丙酮酸激酶M2亚型）及凋亡相关蛋白（B淋巴细胞瘤-2、Bcl-2蛋白相互作用的细胞死亡调解子）的表达；分为空白对照、2-脱氧葡萄糖（4 mmol/L）单药处理、奥希替尼（3 μmol/L）单药处理、2-脱氧葡萄糖（4 mmol/L）+奥希替尼（3 μmol/L）联合处理组，用流式细胞分析仪检测药物处理后的细胞凋亡率，评估药物的促凋亡能力。采用SPSS 16.0统计软件，用独立t检验对结果进行统计分析。 结果： 奥希替尼敏感细胞株H1975比奥希替尼继发性耐药细胞株H1975-OR的糖酵解水平低[乳酸产生量分别为（21.0±0.9）和（26.5±2.8）mmol/（L×10(4)cells），P<0.05]；细胞株H1975-OR经4 mmol/L的2-脱氧葡萄糖处理后可逆转其对奥希替尼的继发性耐药，对奥希替尼的IC(50)值由（7.0±1.9）μmol/L降至（1.4±0.1）μmol/L，接近敏感细胞株H1975对奥希替尼的IC(50)值（1.0±0.2）μmol/L；细胞株H1975-OR经2-脱氧葡萄糖+奥希替尼联合处理后细胞凋亡率为（26.7±2.4）%，显著高于对照组的（5.1±0.7）%、2-脱氧葡萄糖单药处理组的（6.1±2.5）%和奥希替尼单药处理组的（11.4±2.7）%（均P<0.05）；细胞株H1975-OR经2-脱氧葡萄糖+奥希替尼联合处理后，BIM蛋白表达为（177.8±28.1）%，显著高于对照组的（100.0±0）%（P<0.05）；Bcl-2蛋白表达为（24.6±5.2）%，显著低于对照组的（100±0）%（P<0.05）。 结论： 2-脱氧葡萄糖可以逆转NSCLC细胞对奥希替尼继发性耐药，其机制可能与抑制细胞糖酵解进而诱导凋亡增加有关。.