ABCG2在非小细胞肺癌吉非替尼耐药中的作用研究
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摘要
目的研究ATP结合膜转运蛋白超家族G成员2(ABCG2)在非小细胞肺癌吉非替尼耐药中的作用,对吉非替尼耐药的非小细胞肺癌治疗提供依据。方法培养人肺腺癌吉非替尼敏感细胞株A549及吉非替尼耐药的人肺腺癌细胞系A549/GR,A549/GR细胞的耐药性检测应用MTT法。用蛋白质印迹法(Western Blot)检测被吉非替尼作用的A549/GR细胞及A549细胞中的ABCG2、p-AKT蛋白的表达情况。分别加入磷醇酰肌醇3激酶/丝苏氨酸蛋白激酶(PI3K/AKT)通路激活剂胰岛素样生长因子-1(IGF-1)及抑制剂LY294002,分析作用于上述两种细胞的p-AKT、ABCG2蛋白表达情况。应用流式细胞仪测定A549及A549/GR细胞株中的ABCG2外排情况。收集2015年4月～2017年4月我院收治的非小细胞肺癌患者及健康人血清,分为非小细胞肺癌非耐药组(20例)、非小细胞肺癌吉非替尼耐药组(20例)和健康者组(20例)。应用ELISA法检测三组的ABCG2表达。结果随着剂量的增加,吉非替尼对A549细胞及A549/GR细胞的抑制作用均增强(P<0.05)。A549/GR的耐药指数为13.4。Western Blot检测耐药相关蛋白,结果显示,耐药细胞A549/GR中的ABCG2和p-AKT均高于亲代细胞A549。IGF-1作用的A549细胞,p-AKT、ABCG2蛋白表达均提高(P<0.05)。LY294002作用A549/GR细胞,p-AKT、ABCG2均下调(P<0.05)。IGF-1作用A549细胞后,较未加入激活剂的A549细胞,其外排作用增强(P<0.05)。LY294002作用A549/GR细胞后,较未加入激活剂的A549/GR细胞,其外排作用减少(P<0.05)。健康人的ABCG2表达明显低于非小细胞肺癌非耐药组患者(P<0.05),非小细胞肺癌吉非替尼耐药组患者的ABCG2表达均高于非小细胞肺癌非耐药组患者及健康人(P<0.05)。结论在非小细胞肺癌吉非替尼耐药中ABCG2起到了重要的作用,逆转ABCG2介导的NSCLC靶向耐药主要通过调节PI3K/AKT信号通路。
Objective To investigate the effect of ATP-binding cassette superfamily G member 2(ABCG2) in gefitinib resistance in non-small cell lung cancer(NSCLC), and to provide a basis for gefitinib-resistance of NSCLC. Methods The human lung adenocarcinoma gefitinib-sensitive cell line A549 and gefitinib-resistant human lung adenocarcinoma cell line A549/GR were cultured. A549/GR cells were tested for drug resistance by MTT assay. Western blot was used to detect the expression of ABCG2 and p-AKT proteins in A549/GR and A549 cells. Phosphatidylinositol 3 kinase/serine-threonine kinase(PI3 K/AKT) pathway activator insulin-like growth factor-1(IGF-1) and LY294002 acted as inhibitor were added, respectively. The expression of p-AKT and ABCG2 proteins on the above two kinds of cells were analyzed. The efflux of ABCG2 in A549 and A549/GR cell lines was determined by flow cytometry. The serum of nonsmall cell lung cancer(NSCLC) patients and healthy people treated in our hospital from April 2015 to April 2017 were collected, they were divided into NSCLC non-drug resistant group(20 cases), NSCLC Gefitinib resistance group(20 cases) and healthy group(20 cases). The expression of ABCG2 in the three groups were detected by ELISA method.Results With the increase of dose, the inhibitory effect of gefitinib on A549 cells and A549/GR cells were enhanced(P<0.05). The resistance index of A549/GR was 13.4. Western Blot detected for drug-resistance proteins showed that the levels of ABCG2 and p-AKT in drug-resistant A549/GR cell were both higher than those in the parental cell A549. The expression of p-AKT and ABCG2 proteins were increased in A549 cells treated with IGF-1(P <0.05), while treated by LY294002, the levels of p-AKT and ABCG2 were both down-regulated in A549/GR cells(P<0.05). After IGF-1 acting on A549 cells, the efflux was enhanced in A549 cells compared with that without activation(P<0.05). After A549/GR cells being treated with LY294002, the efflux effect was decreased in A549/GR cells compared with that without activator(P<0.05). The expression of ABCG2 in healthy participants was obviously lower than that in patients of NSCLC non-drug resistant group(P<0.05). The expression of ABCG2 in patients of NSCLC Gefitinib resistance group was higher than that in NSCLC non-drug resistant group and healthy participants(P<0.05). Conclusion ABCG2 plays a key role in gefitinib-resistant NSCLC. Reversal of ABCG2-mediated NSCLC-targeted drug resistance is mainly through regulation of the PI3 K/AKT signaling pathway.
Objective To investigate the effect of ATP-binding cassette superfamily G member 2(ABCG2) in gefitinib resistance in non-small cell lung cancer(NSCLC), and to provide a basis for gefitinib-resistance of NSCLC. Methods The human lung adenocarcinoma gefitinib-sensitive cell line A549 and gefitinib-resistant human lung adenocarcinoma cell line A549/GR were cultured. A549/GR cells were tested for drug resistance by MTT assay. Western blot was used to detect the expression of ABCG2 and p-AKT proteins in A549/GR and A549 cells. Phosphatidylinositol 3 kinase/serine-threonine kinase(PI3 K/AKT) pathway activator insulin-like growth factor-1(IGF-1) and LY294002 acted as inhibitor were added, respectively. The expression of p-AKT and ABCG2 proteins on the above two kinds of cells were analyzed. The efflux of ABCG2 in A549 and A549/GR cell lines was determined by flow cytometry. The serum of nonsmall cell lung cancer(NSCLC) patients and healthy people treated in our hospital from April 2015 to April 2017 were collected, they were divided into NSCLC non-drug resistant group(20 cases), NSCLC Gefitinib resistance group(20 cases) and healthy group(20 cases). The expression of ABCG2 in the three groups were detected by ELISA method.Results With the increase of dose, the inhibitory effect of gefitinib on A549 cells and A549/GR cells were enhanced(P<0.05). The resistance index of A549/GR was 13.4. Western Blot detected for drug-resistance proteins showed that the levels of ABCG2 and p-AKT in drug-resistant A549/GR cell were both higher than those in the parental cell A549. The expression of p-AKT and ABCG2 proteins were increased in A549 cells treated with IGF-1(P <0.05), while treated by LY294002, the levels of p-AKT and ABCG2 were both down-regulated in A549/GR cells(P<0.05). After IGF-1 acting on A549 cells, the efflux was enhanced in A549 cells compared with that without activation(P<0.05). After A549/GR cells being treated with LY294002, the efflux effect was decreased in A549/GR cells compared with that without activator(P<0.05). The expression of ABCG2 in healthy participants was obviously lower than that in patients of NSCLC non-drug resistant group(P<0.05). The expression of ABCG2 in patients of NSCLC Gefitinib resistance group was higher than that in NSCLC non-drug resistant group and healthy participants(P<0.05). Conclusion ABCG2 plays a key role in gefitinib-resistant NSCLC. Reversal of ABCG2-mediated NSCLC-targeted drug resistance is mainly through regulation of the PI3 K/AKT signaling pathway.
引文
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[3]钟锐,邬麟,江美林,等.EGFR-TKIs获得性耐药的机制及其治疗的研究进展[J].肿瘤药学,2017,7(6):641-647.
[4]刘红柳,杨家梅.培美曲塞联合吉非替尼治疗EGFR-TKI耐药后晚期非小细胞肺癌临床观察[J].中国癌症杂志,2017,27(2):135-139.
[5]Guo J,Jin D,Wu Y,et al.The miR 495-UBE2C-ABCG2/ERCC1 axis reverses cisplatin resistance by downregulating drug resistance genes in cisplatin-resistant non-small cell lung cancer cells[J].Ebio Med,2018,35(9):204-221.
[6]Zhang GN,Zhang YK,Wang YJ,et al.Epidermal growth factor receptor(EGFR)inhibitor PD153035 reverses ABCG2-mediated multidrug resistance in non-small cell lung cancer:In vitro and in vivo[J].Cancer Lett,2018,28(6):19-29.
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[8]Tang L,Zhang C,He H,et al.Associations between ABCG2gene polymorphisms and gefitinib toxicity in non-small cell lung cancer:a meta-analysis[J].Onco Targets Ther,2018,11(1):665-675.
[9]Biedler JL,Riehm H.Cellular resistance to actinomycin D in Chinese hamster cells in vitro:cross-resistance,radioautographic,and cytogenetic studies[J].Cancer Res,1970,30(4):1174-1184.
[10]Wu Q,Yang Z,Nie Y,et al.Muti-drug resistance in cancer chemotherapeutics:mechanisms and lab approaches[J].Cancer Lett,2014,347(2):159-166.
[11]Xie T,Mo L,Li L,et al.Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer[J].Oncol Lett,2018,15(4):4900-4906.
[12]严晓丽,杜梦楠,郑纪宁.ABC转运蛋白相关micro RNA与肿瘤多药耐药的研究进展[J].广东医学,2017,38(23):3685-3688.
[13]王诗琦,刘克辛.外排型转运体与肿瘤多药耐药[J].药物评价研究,2018,41(6):952-957.
[14]Hegedus C,Truta-Feles K,Antalffy G,et al.Interaction of the EGFR inhibitors gefitinib,vandetanib,pelitinib and neratinib with the ABCG2 multidrug transporter:implications for the emergence and reversal of cancer drug resistance[J].Biochem Pharmacol,2012,84(3):260-267.
[15]Maricla G,Giorgio PP,Claudia F,et al.Effect of ABCG2/BCRP expression on efflux and uptake of gefitinib in NSCLC cell lines[J].PLoS One,2015,10(11):e0141795.
[16]Ho MM,Ng AV,Lam S,et al.Side population in human lung cancer cell lines and tumors is enriched with stemlike cancer cells[J].Cancer Res,2007,67(10):4827-4833.
[17]Chen YJ,Huang WC,Huang MC,et al.Elecated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells[J].PLoS One,2011,6(6):214-228.
[18]李静,朱冰.PI3K/AKT信号通路与EGFR-TKIs治疗NSCLC耐药的相关性研究进展[J].重庆医学,2018,47(17):2340-2343.
[19]胡丽娜,彭兴春,郭显智,等.抑制Notch和PI3K/Akt信号通路对食管腺癌细胞增殖、侵袭及迁移的影响[J].肿瘤防治研究,2016,43(8):653-658.
[20]杨金成,陈福宝,周丽娟.LY294002对人肾透明细胞腺癌细PI3K/Akt/mTOR信号通路的影响[J].宁夏医学杂志,2018,40(10):896-898.
[21]Hu J,Li J,Yue X,et al.Expression of the cancer stem cell markers ABCG2 and OCT-4 in right-sided colon cancer predicts recurrence and poor outcomes[J].Oncotarget,2017,8(17):28 463-28 470.
[22]赖红锦,林锋,陈楠,等.肺癌干细胞作为靶点的肺癌治疗策略研究进展[J].中国肺癌杂志,2018,21(1):57-62.
[23]王大星,谢彤.手术前后非小细胞肺癌血清ABCG2水平的影响[J].中国癌症防治杂志,2011,3(1):56-58.
[2]Siegel RL,Miller KD,Jemal A.Cancer statistics,2016[J].CA Cancer J Clin,2016,66(1):7-30.
[3]钟锐,邬麟,江美林,等.EGFR-TKIs获得性耐药的机制及其治疗的研究进展[J].肿瘤药学,2017,7(6):641-647.
[4]刘红柳,杨家梅.培美曲塞联合吉非替尼治疗EGFR-TKI耐药后晚期非小细胞肺癌临床观察[J].中国癌症杂志,2017,27(2):135-139.
[5]Guo J,Jin D,Wu Y,et al.The miR 495-UBE2C-ABCG2/ERCC1 axis reverses cisplatin resistance by downregulating drug resistance genes in cisplatin-resistant non-small cell lung cancer cells[J].Ebio Med,2018,35(9):204-221.
[6]Zhang GN,Zhang YK,Wang YJ,et al.Epidermal growth factor receptor(EGFR)inhibitor PD153035 reverses ABCG2-mediated multidrug resistance in non-small cell lung cancer:In vitro and in vivo[J].Cancer Lett,2018,28(6):19-29.
[7]张靖,马虎,陈正堂,等.ABCG2在非小细胞肺癌厄洛替尼耐药中的作用[J].第三军医大学学报,2014,36(10):987-991.
[8]Tang L,Zhang C,He H,et al.Associations between ABCG2gene polymorphisms and gefitinib toxicity in non-small cell lung cancer:a meta-analysis[J].Onco Targets Ther,2018,11(1):665-675.
[9]Biedler JL,Riehm H.Cellular resistance to actinomycin D in Chinese hamster cells in vitro:cross-resistance,radioautographic,and cytogenetic studies[J].Cancer Res,1970,30(4):1174-1184.
[10]Wu Q,Yang Z,Nie Y,et al.Muti-drug resistance in cancer chemotherapeutics:mechanisms and lab approaches[J].Cancer Lett,2014,347(2):159-166.
[11]Xie T,Mo L,Li L,et al.Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer[J].Oncol Lett,2018,15(4):4900-4906.
[12]严晓丽,杜梦楠,郑纪宁.ABC转运蛋白相关micro RNA与肿瘤多药耐药的研究进展[J].广东医学,2017,38(23):3685-3688.
[13]王诗琦,刘克辛.外排型转运体与肿瘤多药耐药[J].药物评价研究,2018,41(6):952-957.
[14]Hegedus C,Truta-Feles K,Antalffy G,et al.Interaction of the EGFR inhibitors gefitinib,vandetanib,pelitinib and neratinib with the ABCG2 multidrug transporter:implications for the emergence and reversal of cancer drug resistance[J].Biochem Pharmacol,2012,84(3):260-267.
[15]Maricla G,Giorgio PP,Claudia F,et al.Effect of ABCG2/BCRP expression on efflux and uptake of gefitinib in NSCLC cell lines[J].PLoS One,2015,10(11):e0141795.
[16]Ho MM,Ng AV,Lam S,et al.Side population in human lung cancer cell lines and tumors is enriched with stemlike cancer cells[J].Cancer Res,2007,67(10):4827-4833.
[17]Chen YJ,Huang WC,Huang MC,et al.Elecated BCRP/ABCG2 expression confers acquired resistance to gefitinib in wild-type EGFR-expressing cells[J].PLoS One,2011,6(6):214-228.
[18]李静,朱冰.PI3K/AKT信号通路与EGFR-TKIs治疗NSCLC耐药的相关性研究进展[J].重庆医学,2018,47(17):2340-2343.
[19]胡丽娜,彭兴春,郭显智,等.抑制Notch和PI3K/Akt信号通路对食管腺癌细胞增殖、侵袭及迁移的影响[J].肿瘤防治研究,2016,43(8):653-658.
[20]杨金成,陈福宝,周丽娟.LY294002对人肾透明细胞腺癌细PI3K/Akt/mTOR信号通路的影响[J].宁夏医学杂志,2018,40(10):896-898.
[21]Hu J,Li J,Yue X,et al.Expression of the cancer stem cell markers ABCG2 and OCT-4 in right-sided colon cancer predicts recurrence and poor outcomes[J].Oncotarget,2017,8(17):28 463-28 470.
[22]赖红锦,林锋,陈楠,等.肺癌干细胞作为靶点的肺癌治疗策略研究进展[J].中国肺癌杂志,2018,21(1):57-62.
[23]王大星,谢彤.手术前后非小细胞肺癌血清ABCG2水平的影响[J].中国癌症防治杂志,2011,3(1):56-58.
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