Hao S, Lu C H, Lin C C, Chen H Y, Li L, Wang Y B, Feng M X, He Y
Department of Respiratory Disease, Daping Hospital, Army Military Medical University, Chongqing 400042, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2019 Mar 12;42(3):198-205. doi: 10.3760/cma.j.issn.1001-0939.2019.03.010.
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. The NSCLC line H1975 (purchased from the American Type Culture Collection) was conducted by induction method 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 -test using SPSS 16.0 statistical software. 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), 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 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 0.05. 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.
探讨2-脱氧葡萄糖（2-dg）在逆转非小细胞肺癌（NSCLC）细胞系对奥希替尼获得性耐药中的作用及机制。采用诱导法对NSCLC细胞系H1975（购自美国模式培养物集存库）进行处理，构建奥希替尼耐药的NSCLC细胞系H1975-OR。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法、集落形成试验、Ki67掺入试验及凋亡相关蛋白表达检测H1975-OR细胞系对奥希替尼的耐药性。通过检测H1975和H1975-OR培养基上清液中乳酸生成量来测定糖酵解水平。采用蛋白质免疫印迹法检测糖酵解关键酶（HK2、GLUT1、P-PKM2）及凋亡相关蛋白（BIM、Bcl-2）的表达。将细胞分为对照组、2-脱氧葡萄糖（4 mmol/L）单药治疗组、奥希替尼（3 μmol/L）单药治疗组和2-脱氧葡萄糖（4 mmol/L）+奥希替尼（3 μmol/L）联合治疗组，然后通过流式细胞术检测细胞凋亡率，以评估药物的促凋亡能力。使用SPSS 16.0统计软件通过独立样本t检验分析数据。奥希替尼敏感细胞系H1975的糖酵解水平低于奥希替尼耐药细胞系H1975-OR[乳酸产量分别为(21.0±0.9)和(26.5±2.8) mmol·L(-1)·10(4)细胞(-1)，P<0.05]。4 mmol/L的2-脱氧葡萄糖可逆转H1975-OR对奥希替尼的获得性耐药（奥希替尼在H1975-OR细胞系中的IC(50)值从(7.0±1.9) μmol/L降至(1.4±0.1) μmol/L，接近奥希替尼在H1975细胞系中的IC(50)值(1.0±0.2) μmol/L）。与对照组（5.1±0.7）%、2-脱氧葡萄糖单药治疗组（6.1±2.5）%和奥希替尼单药治疗组（11.4±2.7）%相比，2-脱氧葡萄糖+奥希替尼联合治疗组中H1975-OR的凋亡率显著更高（(26.7±2.4)%，均P<0.05）。与对照组（100±0）%相比，2-脱氧葡萄糖+奥希替尼联合治疗组中H1975-OR促凋亡蛋白BIM的表达显著更高（(177.8±28.1)%），抗凋亡蛋白Bcl-2的表达显著更低（(24.6±5.2)%），均P<0.05。2-脱氧葡萄糖可逆转NSCLC细胞系对奥希替尼的获得性耐药，这可能与抑制细胞糖酵解和诱导凋亡有关。
