CK2抑制剂CX4945对肺癌A549/DDP细胞顺铂耐药性的逆转作用
|
摘要
目的 :探讨蛋白激酶CK2(casein kinase 2,曾称酪蛋白激酶Ⅱ)抑制剂CX4945对顺铂(cisplatin,DDP)耐药性肺癌A549/DDP细胞生长活性的影响及其可能的作用机制。方法 :CCK-8法检测肺癌A549细胞及其耐药性A549/DDP细胞的DDP半数抑制浓度(half maximal inhibitory concentration,IC50),以及抑制剂CX4945对A549/DDP细胞耐药性的影响。蛋白质印迹法检测A549及A549/DDP细胞在DDP干预前后Wnt信号通路相关蛋白(CK2α、β-catenin和cyclin D1)、耐药相关蛋白[多药耐药相关蛋白1(multidrug resistance-associated protein 1,MRP1)和肺耐药相关蛋白(lung resistance-related protein,LRP)]和凋亡相关蛋白[剪切型半胱天冬酶-3(cleaved caspase-3,c-caspase-3)]的表达变化。将A549/DDP细胞分为未处理对照组、CX4945组、DDP组及CX4945+DDP组,各组药物处理后,采用蛋白质印迹法和FCM法分别检测Wnt信号通路、耐药及凋亡相关蛋白的表达以及细胞凋亡情况。结果:DDP对A549/DDP细胞的IC50值是A549细胞的4.59倍,并在CX4945预处理后A549/DDP细胞耐药性明显降低(P <0.001)。与A549细胞相比,A549/DDP细胞中CK2α、β-catenin、cyclin D1、MRP1和LRP蛋白的表达水平明显较高,并在DDP处理后进一步升高(P值均<0.001);然而A549细胞中,DDP处理后CK2α、β-catenin和cyclin D1蛋白表达水平明显降低(P值均<0.01),而MRP1和LRP蛋白表达则无明显变化(P值均>0.05)。与未处理对照组和DDP组相比,CX4945组和CX4945+DDP组的A549/DDP细胞中β-catenin、cyclin D1、MRP1和LRP蛋白表达水平均明显降低(P值均<0.01)。此外,CX4945+DDP组的A549/DDP细胞凋亡率和c-caspase-3表达水平均较未处理对照组和DDP组明显升高(P值均<0.001)。结论 :CK2抑制剂CX4945可通过抑制Wnt通路,降低耐药相关蛋白的表达,从而逆转肺癌A549/DDP细胞的顺铂耐药性。
Objective: To investigate the effect of CK2(casein kinase 2) inhibitor CX4945 on the cisplatin(DDP)-resistance of lung cancer A549/DDP cells and the underlying molecular mechanism.Methods: The CCK-8 assay was used to detect the half maximal inhibitory concentration(IC50) of DDP in lung cancer A549 and A549/DDP cells, and to compare the DDP-resistance of two cell lines. The effect of CX4945 on DDP-resistance of A549/DDP cells was tested by CCK-8 method. Western blotting was used to detect the expressions of Wnt signaling pathway-related proteins(CK2α, β-catenin and cyclin D1), drug resistance-related proteins [multidrug resistance-associated protein 1(MRP1) and lung resistance-related protein(LRP)] and apoptosis-related protein [cleaved caspase-3(c-caspase-3)] in A549 and A549/DDP cells treated with DDP or not. The A549/DDP cells were treated with no drug(as the control group), CX4945, DDP and their combination(as CX4945+DDP group), then the expressions of Wnt signaling pathway-, drug resistance-and apoptosis-related proteins were detected by Western blotting, and the apoptosis of A549/DDP cells was detected by FCM method.Results: The IC50 value of DDP in A549/DDP cells was 4.59 times higher than that in A549 cells, and the DDP-resistance of A549/DDP cells was decreased by CX4945 pretreatment(P < 0.001). The expression levels of CK2α, β-catenin, cyclin D1, MRP1 and LRP proteins were significantly increased in A549/DDP cells as compared with A549 cells(all P < 0.001), and the levels of these proteins in A549/DDP cells were further increased after DDP treatment(all P < 0.001). In A549 cells after treatment with DDP, the expression levels of CK2α, β-catenin and cyclin D1 proteins were reduced(all P < 0.01), but the levels of MRP1 and LRP proteins were not significantly changed(both P > 0.05). Compared with the control group and DDP group, the expression levels of β-catenin, cyclin D1, MRP1 and LRP proteins in A549/DDP cells of CX4945 group and CX4945+DDP group were significantly declined(all P < 0.01). In addition, the apoptosis rate of A549/DDP cells and the expression level of c-caspase-3 in CX4945+DDP group were significantly higher than those in the control group and DDP group(all P < 0.001).Conclusion: CK2 inhibitor CX4945 can reverse the DDP resistance of lung cancer A549/DDP cells through blocking Wnt signal pathway and decreasing the expressions of drug resistance-relatied proteins.
Objective: To investigate the effect of CK2(casein kinase 2) inhibitor CX4945 on the cisplatin(DDP)-resistance of lung cancer A549/DDP cells and the underlying molecular mechanism.Methods: The CCK-8 assay was used to detect the half maximal inhibitory concentration(IC50) of DDP in lung cancer A549 and A549/DDP cells, and to compare the DDP-resistance of two cell lines. The effect of CX4945 on DDP-resistance of A549/DDP cells was tested by CCK-8 method. Western blotting was used to detect the expressions of Wnt signaling pathway-related proteins(CK2α, β-catenin and cyclin D1), drug resistance-related proteins [multidrug resistance-associated protein 1(MRP1) and lung resistance-related protein(LRP)] and apoptosis-related protein [cleaved caspase-3(c-caspase-3)] in A549 and A549/DDP cells treated with DDP or not. The A549/DDP cells were treated with no drug(as the control group), CX4945, DDP and their combination(as CX4945+DDP group), then the expressions of Wnt signaling pathway-, drug resistance-and apoptosis-related proteins were detected by Western blotting, and the apoptosis of A549/DDP cells was detected by FCM method.Results: The IC50 value of DDP in A549/DDP cells was 4.59 times higher than that in A549 cells, and the DDP-resistance of A549/DDP cells was decreased by CX4945 pretreatment(P < 0.001). The expression levels of CK2α, β-catenin, cyclin D1, MRP1 and LRP proteins were significantly increased in A549/DDP cells as compared with A549 cells(all P < 0.001), and the levels of these proteins in A549/DDP cells were further increased after DDP treatment(all P < 0.001). In A549 cells after treatment with DDP, the expression levels of CK2α, β-catenin and cyclin D1 proteins were reduced(all P < 0.01), but the levels of MRP1 and LRP proteins were not significantly changed(both P > 0.05). Compared with the control group and DDP group, the expression levels of β-catenin, cyclin D1, MRP1 and LRP proteins in A549/DDP cells of CX4945 group and CX4945+DDP group were significantly declined(all P < 0.01). In addition, the apoptosis rate of A549/DDP cells and the expression level of c-caspase-3 in CX4945+DDP group were significantly higher than those in the control group and DDP group(all P < 0.001).Conclusion: CK2 inhibitor CX4945 can reverse the DDP resistance of lung cancer A549/DDP cells through blocking Wnt signal pathway and decreasing the expressions of drug resistance-relatied proteins.
引文
[1] TORRE LA, SIEGEL RL, JEMAL A. Lung cancer statistics[J]. Adv Exp Med Biol,2016, 893:1-19.
[2] HELLMANN MD, LI BT, CHAFT JE, et al.Chemotherapy remains an essential element of personalized care for persons with lung cancers[J]. Ann Oncol, 2016, 27(10):1829-1 835.
[3]张雨,陆红,徐刚. PI3K/AKT通路在非小细胞肺癌顺铂耐药中的作用[J].中国肺癌杂志,2014, 1 5(8):635-642.
[4] GAO Y, LIU Z, ZHANG X, et al.Inhibition of cytoplasmic GSK-3beta increases cisplatin resistance through activation of Wnt/β-catenin signalingin A549/DDP cells[J]. Cancer Lett,2013, 336(1):231-239.
[5] LITCHFIELD DW. Protein kinase CK2:structure, regulation and role in cellular decisions of life and death[J].Biochem J, 2003, 369(Pt 1):1-15.
[6] SELDIN DC, LANDESMAN-BOLLAG E,FARAGO M, et al. CK2 as a positive regulator of Wnt signalling and tumourigenesis[J]. Mol Cell Biochem,2005, 274(1/2):63-67.
[7] KIM J, KIM SH. Druggability of the CK2inhibitor CX-4945 as an anticancer drug and beyond[J]. Arch Pharm Res,2012, 35(8):1293-1296.
[8] KU MJ, PARK JW, RYU BJ, et al. CK2inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells[J].Bioorg Med Chem Lett, 2013,23(20):5609-5613.
[9] KIM J, HWAN KIM S. CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells[J]. PLoS One, 201 3, 8(9):e74342.
[10] SO KS, RHO JK, CHOI YJ, et al. AKT/mTOR down-regulation by CX-4945,a CK2 inhibitor, promotes apoptosis in chemorefractory non-small cell lung cancer cells[J]. Anticancer Res, 201 5,35(3):1 537-1 542.
[11] GOTTESMAN MM. Mechanisms of cancer drug resistance[J]. Annu Rev Med, 2002, 53:61 5-627.
[12] WEI H, LU W, LI M, et al. Concomitance of P-gp/LRP expression with EGFR mutations in exons 19 and 21 in nonsmall cell Llung cancers[J]. Yonsei Med J,2016, 57(1):50-57.
[13] ZHU Z. Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways[J]. Oncol Lett, 2018,15(6):884-8846.
[14]徐立群,薛兴阳,邬晓东,等.参慈胶囊联合顺铂对A549/DDP肺癌组织Wnt/β-catenin信号通路DKK-3、β-catenin、Cyclin-D1 mRNA表达的影响[J].中药材,2016, 39(1 0):2363-2365.
[15] HUANG H, LIU J, MENG Q, et al.Multidrug resistance protein and topoisomerase 2 alpha expression in non-small cell lung cancer are related with brain metastasis postoperatively[J]. IntJ Clin Exp Pathol,201 5, 8(9):11 537-11542.
[16] KELLAND L. The resurgence of platinum-based cancer chemotherapy[J]. Nat Rev Cancer,2007, 7(8):573-584.
[17] XIA Y, H E Z, LI U B, et al.Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/beta-caten in signaling pathway[J]. Mol Med Rep, 2015, 12(3):4530-4537.
[18] ZHANG Q, ZHANG B, SUN L, et al.MicroRNA-130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/betacatenin pathway[J]. Cell BiochemFunct, 2018, 36(4):194-202.
[19]阮杰,刘新光.非小细胞肺癌组织中蛋白激酶CK2的表达及其意义[J].肿瘤,2009, 29(12):1090-1094.
[20] MACDONALD BT, TAMAI K, HE X. Wnt/beta-catenin signaling:components,mechanisms, and diseases[J]. Dev Cell,2009, 17(1):9-26.
[21] ANGERS S, MOON RT. Proximal events in Wnt signal transduction[J]. Nat Rev Mol Cell Biol, 2009, 10(7):468-677.
[22] JAVID J, MIR R, MIRZA M, et al. CC genotype of anti-apoptotic gene BCL-2(-938 C/A)is an independent prognostic marker of unfavorable clinical outcome in patients with nonsmall-cell lung cancer[J]. Clin Transl Oncol, 2015, 17(4):289-295.
[23] NICHOLSON DW, AU A, THORNBERRY NA,et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis[J]. Nature,1995, 376(6535):37-43.
[2] HELLMANN MD, LI BT, CHAFT JE, et al.Chemotherapy remains an essential element of personalized care for persons with lung cancers[J]. Ann Oncol, 2016, 27(10):1829-1 835.
[3]张雨,陆红,徐刚. PI3K/AKT通路在非小细胞肺癌顺铂耐药中的作用[J].中国肺癌杂志,2014, 1 5(8):635-642.
[4] GAO Y, LIU Z, ZHANG X, et al.Inhibition of cytoplasmic GSK-3beta increases cisplatin resistance through activation of Wnt/β-catenin signalingin A549/DDP cells[J]. Cancer Lett,2013, 336(1):231-239.
[5] LITCHFIELD DW. Protein kinase CK2:structure, regulation and role in cellular decisions of life and death[J].Biochem J, 2003, 369(Pt 1):1-15.
[6] SELDIN DC, LANDESMAN-BOLLAG E,FARAGO M, et al. CK2 as a positive regulator of Wnt signalling and tumourigenesis[J]. Mol Cell Biochem,2005, 274(1/2):63-67.
[7] KIM J, KIM SH. Druggability of the CK2inhibitor CX-4945 as an anticancer drug and beyond[J]. Arch Pharm Res,2012, 35(8):1293-1296.
[8] KU MJ, PARK JW, RYU BJ, et al. CK2inhibitor CX4945 induces sequential inactivation of proteins in the signaling pathways related with cell migration and suppresses metastasis of A549 human lung cancer cells[J].Bioorg Med Chem Lett, 2013,23(20):5609-5613.
[9] KIM J, HWAN KIM S. CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells[J]. PLoS One, 201 3, 8(9):e74342.
[10] SO KS, RHO JK, CHOI YJ, et al. AKT/mTOR down-regulation by CX-4945,a CK2 inhibitor, promotes apoptosis in chemorefractory non-small cell lung cancer cells[J]. Anticancer Res, 201 5,35(3):1 537-1 542.
[11] GOTTESMAN MM. Mechanisms of cancer drug resistance[J]. Annu Rev Med, 2002, 53:61 5-627.
[12] WEI H, LU W, LI M, et al. Concomitance of P-gp/LRP expression with EGFR mutations in exons 19 and 21 in nonsmall cell Llung cancers[J]. Yonsei Med J,2016, 57(1):50-57.
[13] ZHU Z. Miltirone-induced apoptosis in cisplatin-resistant lung cancer cells through upregulation of p53 signaling pathways[J]. Oncol Lett, 2018,15(6):884-8846.
[14]徐立群,薛兴阳,邬晓东,等.参慈胶囊联合顺铂对A549/DDP肺癌组织Wnt/β-catenin信号通路DKK-3、β-catenin、Cyclin-D1 mRNA表达的影响[J].中药材,2016, 39(1 0):2363-2365.
[15] HUANG H, LIU J, MENG Q, et al.Multidrug resistance protein and topoisomerase 2 alpha expression in non-small cell lung cancer are related with brain metastasis postoperatively[J]. IntJ Clin Exp Pathol,201 5, 8(9):11 537-11542.
[16] KELLAND L. The resurgence of platinum-based cancer chemotherapy[J]. Nat Rev Cancer,2007, 7(8):573-584.
[17] XIA Y, H E Z, LI U B, et al.Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/beta-caten in signaling pathway[J]. Mol Med Rep, 2015, 12(3):4530-4537.
[18] ZHANG Q, ZHANG B, SUN L, et al.MicroRNA-130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/betacatenin pathway[J]. Cell BiochemFunct, 2018, 36(4):194-202.
[19]阮杰,刘新光.非小细胞肺癌组织中蛋白激酶CK2的表达及其意义[J].肿瘤,2009, 29(12):1090-1094.
[20] MACDONALD BT, TAMAI K, HE X. Wnt/beta-catenin signaling:components,mechanisms, and diseases[J]. Dev Cell,2009, 17(1):9-26.
[21] ANGERS S, MOON RT. Proximal events in Wnt signal transduction[J]. Nat Rev Mol Cell Biol, 2009, 10(7):468-677.
[22] JAVID J, MIR R, MIRZA M, et al. CC genotype of anti-apoptotic gene BCL-2(-938 C/A)is an independent prognostic marker of unfavorable clinical outcome in patients with nonsmall-cell lung cancer[J]. Clin Transl Oncol, 2015, 17(4):289-295.
[23] NICHOLSON DW, AU A, THORNBERRY NA,et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis[J]. Nature,1995, 376(6535):37-43.