Keap1-Nrf2信号通路参与子宫内膜癌细胞增殖、转移、耐药机制的研究
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摘要
目的:研究Kelch样环氧氯丙烷相关蛋白1(Keap1)-核转录因子E2相关因子(Nrf2)信号通路对子宫内膜癌细胞生长、转移及耐药的影响。方法:选择代表I型子宫内膜癌细胞的KLE细胞株和代表II型子宫内膜癌的ARK2细胞株,采用real-time PCR和Western blot法测定两细胞株Keap1和Nrf2基因m RNA、蛋白表达,MTT法检测两细胞株对顺铂耐药性。对Keap1表达更低、耐药性更强的ARK2细胞株的Keap1基因过表达。Real-time PCR、Western blot、平板克隆实验、MTT法、Transwell迁徙、侵袭实验等方法研究ARK2细胞中Keap1过表达对Nrf2及下游靶基因表达、细胞生长、侵袭、耐药的影响。结果:在ARK2和KLE细胞中Nrf2基因m RNA表达无明显差异,ARK2细胞中Keap1基因表达无论是m RNA还是蛋白水平在的表达均要低于KLE细胞株,而Nrf2蛋白水平的表达在ARK2细胞株中则要明显高于KLE细胞株。Keap1表达较低的ARK2细胞较Keap1高表达的KLE细胞耐药性更高。ARK2细胞株中Keap1过表达能够明显下调Nrf2蛋白及下游基因ABCC2、γ-GCS表达;MTT实验和平板克隆实验表明Keap1过表达能够降低ARK2细胞增殖能力;Keap1过表达能够降低ARK2细胞迁徙和侵袭能力;Keap1过表达能够显著提高ARK2细胞对顺铂的敏感性。结论:Keap-Nrf2信号通路在子宫内膜浆液性癌细胞株ARK2中活化程度显著高于子宫内膜样癌细胞株KLE,Keap1对Nrf2蛋白表达调控是基于转录后的,且低表达的Keap1及高表达的Nrf2蛋白和肿瘤细胞的高耐药性有关。在子宫内膜浆液性癌细胞株ARK2中上调Keap1基因表达能够有效的下调Nrf2蛋白及其下游基因的表达,并降低肿瘤细胞的生长、迁徙、侵袭能力,且能提高肿瘤细胞对顺铂的敏感性。
Objective: To investigate the effect of Keap1-Nrf2 pathway on the cell proliferation, metastasis, and drug resistance of endometrial cancer cells. Methods: The m RNA and protein levels of Keap1, Nrf2 as well as its downstream genes were assayed in KLE and ARK2 cell lines(which represent type I and II endometrial cancer), using real-time PCR and Western blot. MTT assay to detect the sensitivities of these two cell lines to cisplatin with different concentrations. Keap1 was overexpressed in ARK2 cell line with a lower Keap1 expression and higher drug resistance. Real-time PCR, Western blot, MTT, colony formation assay and transwell assay were performed to evaluate the effect of overexpression of keap1 on the expression of Nrf2 and its downstream gene, cell proliferation,invasion, migration and drug resistance of ARK2 cells. Results: There was no significant difference in m RNA expression of Nrf2 between ARK2 and KLE cells, but the expression of Keap1 gene in ARK2 cells was lower than that of KLE cell line both m RNA and protein expression. The expression of Nrf2 protein in the ARK2 cell line was significantly higher than that in the KLE cell line. Keap1 expression was lower in ARK2 cells than KLE cells; overexpression of Keap1 in ARK2 cell lines could significantly down-regulate expression of Nrf2 protein and of its downstream genes ABCC2 and γ-GCS; MTT experiments and colony formation assay showed overexpression of Keap1 could decrease the cell proliferation, migration and invasion of ARK2 cells, and enhanced its sensitivity to cisplatin. Conclusions: Activation of Keap-Nrf2 signaling pathway in endometrial serous carcinoma cell ARK2 was considerably higher than that in endometrioid adenocarcinoma cell KLE. The low expression of Keap1 and high expression of Nrf2 protein was associated with drug resistance of ARK2 cells. Overexpression of Keap1 gene expression could effectively down-regulate the expression of Nrf2 protein and its downstream genes in ARK2 cells, reduce the growth, migration and invasion of tumor cells, and enhance the sensitivity of tumor cells to chemotherapeutic drugs.
Objective: To investigate the effect of Keap1-Nrf2 pathway on the cell proliferation, metastasis, and drug resistance of endometrial cancer cells. Methods: The m RNA and protein levels of Keap1, Nrf2 as well as its downstream genes were assayed in KLE and ARK2 cell lines(which represent type I and II endometrial cancer), using real-time PCR and Western blot. MTT assay to detect the sensitivities of these two cell lines to cisplatin with different concentrations. Keap1 was overexpressed in ARK2 cell line with a lower Keap1 expression and higher drug resistance. Real-time PCR, Western blot, MTT, colony formation assay and transwell assay were performed to evaluate the effect of overexpression of keap1 on the expression of Nrf2 and its downstream gene, cell proliferation,invasion, migration and drug resistance of ARK2 cells. Results: There was no significant difference in m RNA expression of Nrf2 between ARK2 and KLE cells, but the expression of Keap1 gene in ARK2 cells was lower than that of KLE cell line both m RNA and protein expression. The expression of Nrf2 protein in the ARK2 cell line was significantly higher than that in the KLE cell line. Keap1 expression was lower in ARK2 cells than KLE cells; overexpression of Keap1 in ARK2 cell lines could significantly down-regulate expression of Nrf2 protein and of its downstream genes ABCC2 and γ-GCS; MTT experiments and colony formation assay showed overexpression of Keap1 could decrease the cell proliferation, migration and invasion of ARK2 cells, and enhanced its sensitivity to cisplatin. Conclusions: Activation of Keap-Nrf2 signaling pathway in endometrial serous carcinoma cell ARK2 was considerably higher than that in endometrioid adenocarcinoma cell KLE. The low expression of Keap1 and high expression of Nrf2 protein was associated with drug resistance of ARK2 cells. Overexpression of Keap1 gene expression could effectively down-regulate the expression of Nrf2 protein and its downstream genes in ARK2 cells, reduce the growth, migration and invasion of tumor cells, and enhance the sensitivity of tumor cells to chemotherapeutic drugs.
引文
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[15]Wang XJ,Sun Z,Villeneuve NF,et al.Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs,the dark side of Nrf2[J].Carcinogenesis,2008,29(6):1235-1243
[16]Kensler TW,Wakabayashi N,Biswal S.Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway[J].Annu Rev Pharmacol Toxicol,2007,47:89-116
[17]Jeong WS,Jun M,Kong AN.Nrf2:a potential molecular target for cancer chemoprevention by natural compounds[J].Antioxid Redox Signal,2006,8(1-2):99-106
[18]Ohta T,Iijima K,Miyamoto M,et al.Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth[J].Cancer Res,2008,68(5):1303-1309
[19]Kim YR,Oh JE,Kim MS,et al.Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin[J].J Pathol,2010,220(4):446-451
[20]Pi J,Diwan BA,Sun Y,et al.Arsenic-induced malignant transformation of human keratinocytes:involvement of Nrf2[J].Free Radic Biol Med,2008,45(5):651-658
[21]Homma S,Ishii Y,Morishima Y,et al.Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer[J].Clin Cancer Res,2009,15(10):3423-3432
[22]Xia M,Yu H,Gu S,et al.p62/SQSTM1 is involved in cisplatin resistance in human ovarian cancer cells via the Keap1-Nrf2-AREsystem[J].Int J Oncol,2014,45(6):2341-2348
[23]Chen N,Yi X,Abushahin N,et al.Nrf2 expression in endometrial serous carcinomas and its precancers[J].Int J Clin Exp Pathol,2010,4(1):85-96
[24]Jain AK,Bloom DA,Jaiswal AK.Nuclear import and export signals in control of Nrf2[J].J Biol Chem,2017,292(5):2052
[25]Shen H,Yang Y,Xia S,et al.Blockage of Nrf2 suppresses the migration and invasion of esophageal squamous cell carcinoma cells in hypoxic microenvironment[J].Dis Esophagus,2014,27(7):685-692
[2]Bokhman JV.Two pathogenetic types of endometrial carcinoma[J].Gynecol Oncol,1983,15(1):10-17
[3]Alvarez T,Miller E,Duska L,et al.Molecular profile of grade 3endometrioid endometrial carcinoma:is it a type I or type IIendometrial carcinoma?[J].Am J Surg Pathol,2012,36(5):753-761
[4]Moore KN,Fader AN.Uterine papillary serous carcinoma[J].Clin Obstet Gynecol,2011,54(2):278-291
[5]Sykiotis GP,Habeos IG,Samuelson AV,et al.The role of the antioxidant and longevity-promoting Nrf2 pathway in metabolic regulation[J].Curr Opin Clin Nutr Metab Care,2011,14(1):41-48
[6]Sun Z,Zhang S,Chan JY,et al.Keap1 controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2[J].Mol Cell Biol,2007,27(18):6334-6349
[7]Kang MI,Kobayashi A,Wakabayashi N,et al.Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes[J].Proc Natl Acad Sci U S A,2004,101(7):2046-2051
[8]Mc Mahon M,Lamont DJ,Beattie KA,et al.Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide,zinc,and alkenals[J].Proc Natl Acad Sci U S A,2010,107(44):18838-18843
[9]Padmanabhan B,Tong KI,Ohta T,et al.Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer[J].Mol Cell,2006,21(5):689-700
[10]Singh A,Misra V,Thimmulappa RK,et al.Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer[J].PLo SMed,2006,3(10):e420
[11]Giudice A,Montella M.Activation of the Nrf2-ARE signaling pathway:a promising strategy in cancer prevention[J].Bioessays,2006,28(2):169-181
[12]Kwak MK,Kensler TW.Targeting NRF2 signaling for cancer chemoprevention[J].Toxicol Appl Pharmacol,2010,244(1):66-76
[13]Zhang L,Wang N,Zhou S,et al.Propofol induces proliferation and invasion of gallbladder cancer cells through activation of Nrf2[J].JExp Clin Cancer Res,2012,31:66
[14]Cho JM,Manandhar S,Lee HR,et al.Role of the Nrf2-antioxidant system in cytotoxicity mediated by anticancer cisplatin:implication to cancer cell resistance[J].Cancer Lett,2008,260(1-2):96-108
[15]Wang XJ,Sun Z,Villeneuve NF,et al.Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs,the dark side of Nrf2[J].Carcinogenesis,2008,29(6):1235-1243
[16]Kensler TW,Wakabayashi N,Biswal S.Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway[J].Annu Rev Pharmacol Toxicol,2007,47:89-116
[17]Jeong WS,Jun M,Kong AN.Nrf2:a potential molecular target for cancer chemoprevention by natural compounds[J].Antioxid Redox Signal,2006,8(1-2):99-106
[18]Ohta T,Iijima K,Miyamoto M,et al.Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth[J].Cancer Res,2008,68(5):1303-1309
[19]Kim YR,Oh JE,Kim MS,et al.Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin[J].J Pathol,2010,220(4):446-451
[20]Pi J,Diwan BA,Sun Y,et al.Arsenic-induced malignant transformation of human keratinocytes:involvement of Nrf2[J].Free Radic Biol Med,2008,45(5):651-658
[21]Homma S,Ishii Y,Morishima Y,et al.Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer[J].Clin Cancer Res,2009,15(10):3423-3432
[22]Xia M,Yu H,Gu S,et al.p62/SQSTM1 is involved in cisplatin resistance in human ovarian cancer cells via the Keap1-Nrf2-AREsystem[J].Int J Oncol,2014,45(6):2341-2348
[23]Chen N,Yi X,Abushahin N,et al.Nrf2 expression in endometrial serous carcinomas and its precancers[J].Int J Clin Exp Pathol,2010,4(1):85-96
[24]Jain AK,Bloom DA,Jaiswal AK.Nuclear import and export signals in control of Nrf2[J].J Biol Chem,2017,292(5):2052
[25]Shen H,Yang Y,Xia S,et al.Blockage of Nrf2 suppresses the migration and invasion of esophageal squamous cell carcinoma cells in hypoxic microenvironment[J].Dis Esophagus,2014,27(7):685-692