ATP7A对结肠癌细胞耐奥沙利铂的机制初探
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摘要
目的探讨铜转运蛋白ATP7A对人结肠癌耐药细胞HCT116/L-OHP耐奥沙利铂的作用机制。方法选用RNA沉默(siRNA)ATP7A稳转结肠癌耐药细胞siATP7A-HCT116/L-OHP,同时设立空转染组negATP7A-HCT116/L-OHP,以化疗药奥沙利铂(oxaliplatin,L-OHP)作为用药组,分别采用四甲基偶氮唑盐比色法(MTT)、Annexin V-FITC/PI双染法及蛋白免疫印迹法检测ATP7A蛋白沉默前后对HCT116/L-OHP细胞抑制率、凋亡率及ATP7A蛋白表达的影响。结果 (1)随着L-OHP浓度的增加对细胞抑制率增强,IC50(μg/mL)由85.81±2.76降至41.30±3.31,逆转耐药2.07倍。(2)negATP7A-HCT116/L-OHP和siATP7A-HCT116/L-OHP细胞空白组48 h凋亡率分别为(1.4±0.1)%和(4.47±0.15)%,L-OHP组细胞凋亡率分别为(4.97±0.06)%和(25.87±0.21)%。与negATP7A-HCT116/L-OHP细胞相比,同一组别siATP7A-HCT116/L-OHP细胞凋亡率均显著升高(P <0.001)。(3)与negATP7A-HCT116/L-OHP细胞L-OHP组相比,siATP7A-HCT116/L-OHP细胞L-OHP组内除CTR1蛋白上调外,ATP7A、ATP7B、ATOX1蛋白均呈不同程度的下调(P <0.001)。结论 ATP7A降低结肠癌耐药细胞HCT116/L-OHP对L-OHP的敏感性及细胞凋亡,沉默后能上调铜转运相关蛋白CTR1的表达、下调ATP7B、ATOX1表达,可能是人结肠癌细胞耐奥沙利铂的重要机制之一。
Objective To investigate the mechanism of copper transporter ATP7A on oxaliplatin resistance in human colon cancer cell line HCT116/L-OHP.Methods RNAi(siRNA)ATP7A was used to stably transfect the colon cancer cell line siATP7A-HCT116/L-OHP.At the same time,empty-transfected group negATP7A-HCT116/L-OHP was established.Chemotherapy drug oxaliplatin(L-OHP)was used as the medical group.The inhibitory rates,apoptosis rates and the expression of ATP7A protein in HCT116/L-OHP cells before and after the RNA silence of it were detected by MTT assay,Annexin V-FITC/PI double staining and Western blot(WB)respectively.ResultsThe inhibitory rates(%)of L-OHP at different concentrations were-8.51±58.52,25.37±6.19,32.72±2.92,53.81±6.96,72.23±3.32,93.37±2.82(μg/mL),which decreased from 85.81±2.76 to41.30±3.31,reversing the resistance by 2.07 times.In addition,the apoptotic rates of blank groups in negATP7A-HCT116/L-OHP and siATP7A-HCT116/L-OHP were(1.4±0.1)%and(4.47±0.15)%respectively after 48h,while the ones of L-OHP groups in them were(4.97±0.06)%and(25.87±0.21)%.Compared with the apoptosis rates in negATP7A-HCT116/L-OHP cells,the ones in the same group of siATP7A-HCT116/L-OHP increased significantly(P<0.001).Moreover,the expressions of ATP7A,ATP7B and ATOX1 protein were down-regulated differently except the up-regulation of CTR1 protein in siATP7A-HCT116/L-OHP cells compared with negATP7A-HCT116/L-OHP group(P<0.001).Conclusion ATP7A can reduce the sensitivity and apoptosis of L-OHP induced by HCT116/L-OHP in colon cancer cells.After silencing,ATP7A can up-regulate the expression of CTR1and down-regulate the expression of ATP7B and ATOX1,which may be one of the important mechanisms of oxaliplatin resistance in human colon cancer cells.
Objective To investigate the mechanism of copper transporter ATP7A on oxaliplatin resistance in human colon cancer cell line HCT116/L-OHP.Methods RNAi(siRNA)ATP7A was used to stably transfect the colon cancer cell line siATP7A-HCT116/L-OHP.At the same time,empty-transfected group negATP7A-HCT116/L-OHP was established.Chemotherapy drug oxaliplatin(L-OHP)was used as the medical group.The inhibitory rates,apoptosis rates and the expression of ATP7A protein in HCT116/L-OHP cells before and after the RNA silence of it were detected by MTT assay,Annexin V-FITC/PI double staining and Western blot(WB)respectively.ResultsThe inhibitory rates(%)of L-OHP at different concentrations were-8.51±58.52,25.37±6.19,32.72±2.92,53.81±6.96,72.23±3.32,93.37±2.82(μg/mL),which decreased from 85.81±2.76 to41.30±3.31,reversing the resistance by 2.07 times.In addition,the apoptotic rates of blank groups in negATP7A-HCT116/L-OHP and siATP7A-HCT116/L-OHP were(1.4±0.1)%and(4.47±0.15)%respectively after 48h,while the ones of L-OHP groups in them were(4.97±0.06)%and(25.87±0.21)%.Compared with the apoptosis rates in negATP7A-HCT116/L-OHP cells,the ones in the same group of siATP7A-HCT116/L-OHP increased significantly(P<0.001).Moreover,the expressions of ATP7A,ATP7B and ATOX1 protein were down-regulated differently except the up-regulation of CTR1 protein in siATP7A-HCT116/L-OHP cells compared with negATP7A-HCT116/L-OHP group(P<0.001).Conclusion ATP7A can reduce the sensitivity and apoptosis of L-OHP induced by HCT116/L-OHP in colon cancer cells.After silencing,ATP7A can up-regulate the expression of CTR1and down-regulate the expression of ATP7B and ATOX1,which may be one of the important mechanisms of oxaliplatin resistance in human colon cancer cells.
引文
[1]潘刚,殷佩浩,范跃祖.靶向治疗大肠癌的纳米药物及其载体进展[J].国际外科学杂志,2016,43(2):124-128.
[2]SOUTHON A,BURKE R,CAMAKARIS J.What can flies tell us about copper homeostasis?[J].Metallomics,2013,5(10),1346-1356.
[3]KIM H W,CHAN Q,AFTON S E,et al.Macrophage ATP7A is localized in the trans-golgi apparatus,controls intracellular copper levels,and mediates macrophage responses to dermal wounds[J].Inflammation,2012,35(1):167-175.
[4]TADINI-BUONINSEGNI,BARTOLOMMEI,ROSA MONCEL-LI,et al.Anticancer drugs by human copper ATPases ATP7Aand ATP7B[J].Angew Chem Int Ed Engl,2014,53(5):1297-1301.
[5]GOURDON P,SITSEL O,LYKKEGAAR DKARLSEN J,et al.Structural models of the human copper P-type ATPases ATP7Aand ATP7B[J].Biol Chem,2012,393(4):205-216.
[6]CHEN H H,KUO M T.Overcoming platinum drug resistance with copper-lowering agents[J].Anticancer Res,2013,33(10):4157-4161.
[7]KILARI D,GUANCIAL E,KIM E S.Role of copper transporters in platinum resistance[J].World J Clin Oncol,2016,7(1):106-113.
[8]ABADA P B,LARSON C A,MANOREK G,et al.SEC61βcontrols sensitivity toplatinum-containing chemotherapeutic agents through modulation of the copper-transporting ATPase ATP7A[J].Mol Pharmacol,2012,82(3):510-520.
[9]TANG H,LIU Y J,LIU M,et a1.Establishment and gene analysis of an oxaliplatin-resistant colon cancer cell line THC8307/L-OHP[J].Anticancer Drugs,2007,18(6):633-639.
[10]MATIN L P,HAMILTON T C,SCHILDR R J.Platinum resistance:the role of DNA repair pathways[J].Clin Cancer Res,2008,14(5):1291-1295.
[11]SAMIMI G,SAFAEI R,KATANO K,et al.Increased expression of the copper efflux transporter ATP7A mediates resistance to cisplatin,carboplation,and oxaliplatin in ovarian cancer cells[J].Clin Cancer Res,2004,10(14):4661-4669.
[12]石铸,闫振文.铜伴侣蛋白研究进展[J].国外医学遗传学分册,2002,25(4):204-206.
[13]CARA A R,EDWARD M.Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant cells[J].Cancer Chemotherapy and Pharmacology,2009,64(1):133-142.
[14]SHARON La F,JULIAN M.Trafficking of the copper-ATPases,ATP7A and ATP7B:Role in copper homeostasis[J].Biochemistry and Biophysics,2007,463(2):149-167.
[2]SOUTHON A,BURKE R,CAMAKARIS J.What can flies tell us about copper homeostasis?[J].Metallomics,2013,5(10),1346-1356.
[3]KIM H W,CHAN Q,AFTON S E,et al.Macrophage ATP7A is localized in the trans-golgi apparatus,controls intracellular copper levels,and mediates macrophage responses to dermal wounds[J].Inflammation,2012,35(1):167-175.
[4]TADINI-BUONINSEGNI,BARTOLOMMEI,ROSA MONCEL-LI,et al.Anticancer drugs by human copper ATPases ATP7Aand ATP7B[J].Angew Chem Int Ed Engl,2014,53(5):1297-1301.
[5]GOURDON P,SITSEL O,LYKKEGAAR DKARLSEN J,et al.Structural models of the human copper P-type ATPases ATP7Aand ATP7B[J].Biol Chem,2012,393(4):205-216.
[6]CHEN H H,KUO M T.Overcoming platinum drug resistance with copper-lowering agents[J].Anticancer Res,2013,33(10):4157-4161.
[7]KILARI D,GUANCIAL E,KIM E S.Role of copper transporters in platinum resistance[J].World J Clin Oncol,2016,7(1):106-113.
[8]ABADA P B,LARSON C A,MANOREK G,et al.SEC61βcontrols sensitivity toplatinum-containing chemotherapeutic agents through modulation of the copper-transporting ATPase ATP7A[J].Mol Pharmacol,2012,82(3):510-520.
[9]TANG H,LIU Y J,LIU M,et a1.Establishment and gene analysis of an oxaliplatin-resistant colon cancer cell line THC8307/L-OHP[J].Anticancer Drugs,2007,18(6):633-639.
[10]MATIN L P,HAMILTON T C,SCHILDR R J.Platinum resistance:the role of DNA repair pathways[J].Clin Cancer Res,2008,14(5):1291-1295.
[11]SAMIMI G,SAFAEI R,KATANO K,et al.Increased expression of the copper efflux transporter ATP7A mediates resistance to cisplatin,carboplation,and oxaliplatin in ovarian cancer cells[J].Clin Cancer Res,2004,10(14):4661-4669.
[12]石铸,闫振文.铜伴侣蛋白研究进展[J].国外医学遗传学分册,2002,25(4):204-206.
[13]CARA A R,EDWARD M.Role of human copper transporter Ctr1 in the transport of platinum-based antitumor agents in cisplatin-sensitive and cisplatin-resistant cells[J].Cancer Chemotherapy and Pharmacology,2009,64(1):133-142.
[14]SHARON La F,JULIAN M.Trafficking of the copper-ATPases,ATP7A and ATP7B:Role in copper homeostasis[J].Biochemistry and Biophysics,2007,463(2):149-167.