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 4cells）， 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。
ObjectiveTo explore the role and mechanism of 2-deoxyglucose (2-dg) in reversing osimertinib- acquired resistance of non-small cell lung cancer(NSCLC)cell line.
MethodsThe 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.
ResultsThe 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 4cells -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.
Conclusion2-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.
郝帅,卢从华,林采余,等. 2-脱氧葡萄糖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[J]. 中华结核和呼吸杂志,2019,42(3)：198-205.
DOI:10.3760/cma.j.issn.1001-0939.2019.03.010版权归中华医学会所有。
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