Nrf2信号蛋白与卵巢癌顺铂耐药相关性的初步研究
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摘要
肿瘤耐药是影响肿瘤治疗效果的主要问题。肿瘤细胞产生耐药的机制及如何逆转耐药依旧是亟待解决的重要问题。已知肿瘤耐药机制包括肿瘤细胞内药物泵出增多、体内非特异解毒过程加强、DNA损伤修复能力增强、细胞凋亡抑制等。最近有研究发现,以转录因子Nrf2为主体的Nrf2-Keapl-ARE信号通路可能串连多种耐药机制,从而解释多种肿瘤耐药的原因,因此本课题利用卵巢癌A2780cp、A2780、SKOV3、Hey细胞株,研究Nrf2蛋白在卵巢癌顺铂耐药及敏感细胞株中的表达情况,观察Nrf2siRNA干扰细胞株后Nrf2表达及耐药情况的变化,希望为卵巢癌顺铂耐药的综合治疗提供理论基础。本实验主要分为以下两部分：
一.多种卵巢癌细胞株对顺铂的耐药性及Nrf2蛋白在其中的表达
目的：了解顺铂对卵巢癌细胞株A2780cp、A2780、SKOV3、Hey四种细胞增殖的抑制作用及Nrf2在A2780cp、A2780、SKOV3、Hey细胞株中的表达情况。方法：卵巢癌A2780cp、A2780、SKOV3、Hey细胞株复苏后培养,将四种细胞株分为两组：组为A2780cp及A2780,另一组为SKOV3及Hey。应用SRB方法检测顺铂对A2780cp、A2780、SKOV3、Hey四种细胞株增殖的抑制作用,其中顺铂浓度取1μg／ml,2μg／ml,4μg／ml,8μg／ml。观察72hr。应用western-blot方法检测四种细胞株中Nrf2蛋白表达情况。应用SPSS16.0软件对数据进行统计分析,所有数据采用单因素方差分析。结果：顺铂对卵巢癌细胞株A2780cp、A2780、SKOV3、Hey均有增殖抑制作用,其抑制作用与浓度呈正相关。对每组中：A2780cp和A2780,SKOV3和Hey细胞抑制率行统计学分析,各浓度间抑制率比较p<0.05。说明A2780cp和SKOV3为顺铂相对耐药细胞株,A2780和Hey为顺铂相对敏感细胞株。Nrf2在卵巢癌细胞株A2780、Hey中呈相对低表达,在卵巢癌细胞株A2780cp、SKOV3中呈相对高表达。每组中两细胞间表达差异比较p<0.05。
二.Nrf2 siRNA转染后卵巢癌细胞株中Nrf2蛋白表达变化及细胞株对顺铂敏感性的
变化
目的：了解经Nrf2 siRNA转染后卵巢癌细胞株对顺铂敏感性的变化情况。方法：以6孔板培养卵巢癌细胞株A2780cp、A2780,将Nrf2 siRNA(终浓度为30 nM)加入其中,作用6-8h；采用western-blot方法检测细胞中Nrf2蛋白表达情况。以96孔板培养卵巢癌细胞株A2780cp、A2780,将Nrf2 siRNA(终浓度为30 nM)加入其中,作用6-8h；应用SRB检测顺铂对A2780cp、A2780cp_kd、A2780、A2780_kd细胞株增殖的抑制作用,摸索实验条件后调整顺铂浓度,顺铂浓度取A2780cp及A2780cp_kd：4μg／ml,8μg／ml,16μg／ml,32μg／ml：A2780及A2780_kd：1μg／ml,2μg／ml,4μg／ml,8gg／ml,观察72hr。采用流式细胞仪检测卵巢癌细胞株A2780cp及A2780cp_kd经顺铂作用72hr后凋亡情况。应用SPSS16.0软件对数据进行统计分析,所有数据采用单因素方差分析。结果：经Nrf2 siRNA转染后,Nrf2在卵巢癌细胞株A2780cp中表达量前后比较p<0.05；而在卵巢癌细胞株A2780中表达量前后比较p>0.05。顺铂对转染后的卵巢癌A2780cp细胞株增殖抑制作用明显增强,与卵巢癌A2780细胞株相比无明显差别。各浓度之间抑制率比较p<0.05。经顺铂作用72hr后,卵巢癌细胞株A2780cp_kd存活率明显低于A2780cp细胞株,抑制率比较p<0.05。
结论：本实验结果提示,顺铂对卵巢癌细胞株A2780cp、SKOV3的生长抑制作用明显低于对卵巢癌细胞株A2780、Hey增殖的抑制作用。Nrf2在卵巢癌细胞株A2780cp、SKOV3中呈高表达,在卵巢癌细胞株A2780、Hey中呈低表达。Nrf2 siRNA可抑制Nrf2蛋白在卵巢癌细胞中的表达,Nrf2蛋白表达被干扰后,卵巢癌A2780cp细胞株的顺铂耐药性被逆转,其作用结果与对顺铂敏感的卵巢癌A2780细胞株相似。由此可见Nrf2蛋白与卵巢癌细胞顺铂耐药有一定相关性。
Chemoresistance is the main obstacle which restricts the effect of chemotherapy on cancer cells. Why cancer cells produce chemoresistance and how to solve this problem is an urgent concern for oncologists. The major factor or dominant mechanism underling chemoresistance is still unknown, though some mechanisms have been reported, such as the increase of drug effluence, the strengthening of non-specific detoxification process, the enhanced DNA damage repair and apoptosis inhibition. Recent study has shown that the Nrf2-Keapl-ARE signaling pathway may be the key chain linking a variety of mechanisms for chemoresistance, and cancer cells may regain chemosensitivity through genetic engineering on this pathway. We observed the different Nrf2 expressions in A2780cp, A2780, SKOV3 and Hey cell lines as well as the change of their chemosensitivity through Nrf2 siRNA transfection. The result may provide a theoretical basis for the treatment of ovarian cancer.
1.The cisplatin chemoresistance of several ovarian cancer cells and the Nrf2 protein expression in these cells
Objective:To detect the Nrf2 protein expression in A2780cp、A2780、SKOV3 and Hey cell and explore the effects of cisplatin on A2780cp, A2780, SKOV3 and Hey cell lines.
Methods:Ovarain cancer cell line A2780cp, A2780, SKOV3 and Hey were resuscitated, incubated and divided into 2 groups:one included A2780cp and A2780 cell lines, another included SKOV3 and Hey cell lines.SRB was used to investigate cisplatin effects on their growth. The concentration of cisplatin was 1μg/ml,2μg/ml,4μg/ml,8μg/ml.The samples were observed for 72hr respectively. Western-blot was used to investigate Nrf2 protein expression in these cells. SPSS 16.0 software was used to analysis the data by single factor variance method. Results:The effect of the cisplatin inhibiting to this four cell lines proliferation is related with the concentration. There are difference in inhibition between the two cell lines in each group(p<0.05).So the A2780cp and SKOV3 cell lines are realatively chemoresistance to cisplatin while A2780 and Hey cell lines are relatively sensitive. The Nrf2 protein expression is quite different in each two cell lines (p<0.05). The Nrf2 protein is highly expressed in A2780cp and SKOV3 cells and extremely low expressed in A2780 and Hey cells.
2. The effect to the ovarian cancer cells of siRNA transfection in the Nrf2 protein expression and the cisplatin sensitivity
Objective:To observe the change of the Nrf2 protein expression in A2780cp and A2780 cell lines after Nrf2 siRNA transfection.Explore the effects of cisplatin on A2780cp and A2780 cell lines both before and after trasfection. Methods:Ovarain cancer cell line A2780cp and A2780 were incubated and be trasfected before western-blot was used to investigate Nrf2 protein expression in the treated cells. SRB was used to investigate the difference in cisplatin effects. The concentration of cisplatin was:A2780cp and A2780cp_kd:4μg/ml,8μg/ml,16μg/ml,32μg/ml.A2780 and A2780_kd:1μg/ml,2μg/ml, 4μg/ml,8μg/ml. FCA was used to detect the A2780cp and A2780cp_kd cells apoptosis rate. SPSS 16.0 software was used to analysis the data by single factor variance method. Results: The Nrf2 protein is low expressed in A2780cp_kd and A2780 cell lines. The chemoresistence of the A2780cp cells will be reversed through the Nrf2 siRNA transfection. The apoptosis rate of A2780cp_kd cell line was higher than the A2780cp cell group(p<0.05).
Conclusion:The results of this study point out:The inhibitory effect of cisplatin to the growth of A2780cp and SKOV3 cell lines is not as significant as to the A2780 and Hey cell lines. The Nrf2 protein showed a high expression in ovarian cancer A2780cp and SKOV3 cell lines and a low expression in ovarian cancer A2780 and Hey cell lines.The Nrf2 protein can be interferenced through Nrf2 siRNA transfection. Thus, the chemoresistence of the A2780cp cells would be reversed.The summery is:the Nrf2 protein has a certain correlation with platinum-resistance in ovarian cancer cells.
一.多种卵巢癌细胞株对顺铂的耐药性及Nrf2蛋白在其中的表达
目的：了解顺铂对卵巢癌细胞株A2780cp、A2780、SKOV3、Hey四种细胞增殖的抑制作用及Nrf2在A2780cp、A2780、SKOV3、Hey细胞株中的表达情况。方法：卵巢癌A2780cp、A2780、SKOV3、Hey细胞株复苏后培养,将四种细胞株分为两组：组为A2780cp及A2780,另一组为SKOV3及Hey。应用SRB方法检测顺铂对A2780cp、A2780、SKOV3、Hey四种细胞株增殖的抑制作用,其中顺铂浓度取1μg／ml,2μg／ml,4μg／ml,8μg／ml。观察72hr。应用western-blot方法检测四种细胞株中Nrf2蛋白表达情况。应用SPSS16.0软件对数据进行统计分析,所有数据采用单因素方差分析。结果：顺铂对卵巢癌细胞株A2780cp、A2780、SKOV3、Hey均有增殖抑制作用,其抑制作用与浓度呈正相关。对每组中：A2780cp和A2780,SKOV3和Hey细胞抑制率行统计学分析,各浓度间抑制率比较p<0.05。说明A2780cp和SKOV3为顺铂相对耐药细胞株,A2780和Hey为顺铂相对敏感细胞株。Nrf2在卵巢癌细胞株A2780、Hey中呈相对低表达,在卵巢癌细胞株A2780cp、SKOV3中呈相对高表达。每组中两细胞间表达差异比较p<0.05。
二.Nrf2 siRNA转染后卵巢癌细胞株中Nrf2蛋白表达变化及细胞株对顺铂敏感性的
变化
目的：了解经Nrf2 siRNA转染后卵巢癌细胞株对顺铂敏感性的变化情况。方法：以6孔板培养卵巢癌细胞株A2780cp、A2780,将Nrf2 siRNA(终浓度为30 nM)加入其中,作用6-8h；采用western-blot方法检测细胞中Nrf2蛋白表达情况。以96孔板培养卵巢癌细胞株A2780cp、A2780,将Nrf2 siRNA(终浓度为30 nM)加入其中,作用6-8h；应用SRB检测顺铂对A2780cp、A2780cp_kd、A2780、A2780_kd细胞株增殖的抑制作用,摸索实验条件后调整顺铂浓度,顺铂浓度取A2780cp及A2780cp_kd：4μg／ml,8μg／ml,16μg／ml,32μg／ml：A2780及A2780_kd：1μg／ml,2μg／ml,4μg／ml,8gg／ml,观察72hr。采用流式细胞仪检测卵巢癌细胞株A2780cp及A2780cp_kd经顺铂作用72hr后凋亡情况。应用SPSS16.0软件对数据进行统计分析,所有数据采用单因素方差分析。结果：经Nrf2 siRNA转染后,Nrf2在卵巢癌细胞株A2780cp中表达量前后比较p<0.05；而在卵巢癌细胞株A2780中表达量前后比较p>0.05。顺铂对转染后的卵巢癌A2780cp细胞株增殖抑制作用明显增强,与卵巢癌A2780细胞株相比无明显差别。各浓度之间抑制率比较p<0.05。经顺铂作用72hr后,卵巢癌细胞株A2780cp_kd存活率明显低于A2780cp细胞株,抑制率比较p<0.05。
结论：本实验结果提示,顺铂对卵巢癌细胞株A2780cp、SKOV3的生长抑制作用明显低于对卵巢癌细胞株A2780、Hey增殖的抑制作用。Nrf2在卵巢癌细胞株A2780cp、SKOV3中呈高表达,在卵巢癌细胞株A2780、Hey中呈低表达。Nrf2 siRNA可抑制Nrf2蛋白在卵巢癌细胞中的表达,Nrf2蛋白表达被干扰后,卵巢癌A2780cp细胞株的顺铂耐药性被逆转,其作用结果与对顺铂敏感的卵巢癌A2780细胞株相似。由此可见Nrf2蛋白与卵巢癌细胞顺铂耐药有一定相关性。
Chemoresistance is the main obstacle which restricts the effect of chemotherapy on cancer cells. Why cancer cells produce chemoresistance and how to solve this problem is an urgent concern for oncologists. The major factor or dominant mechanism underling chemoresistance is still unknown, though some mechanisms have been reported, such as the increase of drug effluence, the strengthening of non-specific detoxification process, the enhanced DNA damage repair and apoptosis inhibition. Recent study has shown that the Nrf2-Keapl-ARE signaling pathway may be the key chain linking a variety of mechanisms for chemoresistance, and cancer cells may regain chemosensitivity through genetic engineering on this pathway. We observed the different Nrf2 expressions in A2780cp, A2780, SKOV3 and Hey cell lines as well as the change of their chemosensitivity through Nrf2 siRNA transfection. The result may provide a theoretical basis for the treatment of ovarian cancer.
1.The cisplatin chemoresistance of several ovarian cancer cells and the Nrf2 protein expression in these cells
Objective:To detect the Nrf2 protein expression in A2780cp、A2780、SKOV3 and Hey cell and explore the effects of cisplatin on A2780cp, A2780, SKOV3 and Hey cell lines.
Methods:Ovarain cancer cell line A2780cp, A2780, SKOV3 and Hey were resuscitated, incubated and divided into 2 groups:one included A2780cp and A2780 cell lines, another included SKOV3 and Hey cell lines.SRB was used to investigate cisplatin effects on their growth. The concentration of cisplatin was 1μg/ml,2μg/ml,4μg/ml,8μg/ml.The samples were observed for 72hr respectively. Western-blot was used to investigate Nrf2 protein expression in these cells. SPSS 16.0 software was used to analysis the data by single factor variance method. Results:The effect of the cisplatin inhibiting to this four cell lines proliferation is related with the concentration. There are difference in inhibition between the two cell lines in each group(p<0.05).So the A2780cp and SKOV3 cell lines are realatively chemoresistance to cisplatin while A2780 and Hey cell lines are relatively sensitive. The Nrf2 protein expression is quite different in each two cell lines (p<0.05). The Nrf2 protein is highly expressed in A2780cp and SKOV3 cells and extremely low expressed in A2780 and Hey cells.
2. The effect to the ovarian cancer cells of siRNA transfection in the Nrf2 protein expression and the cisplatin sensitivity
Objective:To observe the change of the Nrf2 protein expression in A2780cp and A2780 cell lines after Nrf2 siRNA transfection.Explore the effects of cisplatin on A2780cp and A2780 cell lines both before and after trasfection. Methods:Ovarain cancer cell line A2780cp and A2780 were incubated and be trasfected before western-blot was used to investigate Nrf2 protein expression in the treated cells. SRB was used to investigate the difference in cisplatin effects. The concentration of cisplatin was:A2780cp and A2780cp_kd:4μg/ml,8μg/ml,16μg/ml,32μg/ml.A2780 and A2780_kd:1μg/ml,2μg/ml, 4μg/ml,8μg/ml. FCA was used to detect the A2780cp and A2780cp_kd cells apoptosis rate. SPSS 16.0 software was used to analysis the data by single factor variance method. Results: The Nrf2 protein is low expressed in A2780cp_kd and A2780 cell lines. The chemoresistence of the A2780cp cells will be reversed through the Nrf2 siRNA transfection. The apoptosis rate of A2780cp_kd cell line was higher than the A2780cp cell group(p<0.05).
Conclusion:The results of this study point out:The inhibitory effect of cisplatin to the growth of A2780cp and SKOV3 cell lines is not as significant as to the A2780 and Hey cell lines. The Nrf2 protein showed a high expression in ovarian cancer A2780cp and SKOV3 cell lines and a low expression in ovarian cancer A2780 and Hey cell lines.The Nrf2 protein can be interferenced through Nrf2 siRNA transfection. Thus, the chemoresistence of the A2780cp cells would be reversed.The summery is:the Nrf2 protein has a certain correlation with platinum-resistance in ovarian cancer cells.
引文
[1]Chobanian N,Dietrich CS 3rd. Ovarian cancer. Surg Clin North Am.2008, 88(2):285-299.
[2]Ries LAG,Melbert D, Krapcho M et al.(eds).SEER Cancer Statistics Review, 1975-2005,National Cancer Institute. Bethesda,MD.Available at: http://seer.cancer.gov/csr/1975_2005/. Based on November 2007 SEER data submission, posted to the SEER web site 2008.
[3]Anzenbacher P,Anzenbacherova E. Cytochromes P450 and metabolism of xenobiotics[J].Cell Mol Life Sci,2001,58(5/6):737-747.
[4]Wilkinson JT,Clapper ML.Detoxication enzymes and chemoprevention[J].Proc Soc Exp Biol Med,1997,216(3):192-200.
[5]Itoh K,Ishii T,Wakabayashi N,et al.Regulatory mechanisms of cellar response to oxidative stress [J].FreeRadic Res,1999,31(4):319-324.
[6]Dhakshinamoorthy S, Long DJ 2nd, Jaiswal AK. Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens[J].Curr Top Cell Regul, 2000,36:201-216.
[7]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.
[8]Kim HR,Kim S,Kim EJ,et al. Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin[J].Lung Cancer,2008,60:47-56.
[9]Kim SK,Yang JW,Kim MR,et al.Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells[J].Free Radic Biol Med,2008,45:537-546.
[10]Rushmore TH,Morton MR,Pickett CB.The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity [J].J Biol Chem,1991,266(18):11632-11639.
[11]Kobayashi A,Ohta T,Yamamoto M.Unique function of the Nrf22keap1 pathway in the inducible expression of antioxidant and detoxifying enzynmes[J].Methods Enzymol,2004,378:273-286.
[12]Dinkova-Kostova AT,HoltzclawWD,Cole RN,et al. Direct evidence that sulfhydryl groups of Keapl are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proc Natl Acad Sci USA,2002,99 (18):11908-11913.
[13]Zhang DD. Mechanistic studies of the Nrf2-Keapl signaling pathway[J].Drug Metab Rev,2006,38:769-789.
[14]Sun Z,Zhang S,Chan JY,et al.Keapl controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2[J].Mol Cell Biol,2007,27(18):6334-6349.
[15]Lau A,Villeneuve NF,Sun Z,et al.Dual roles of Nrf2 in cancer[J].Pharmacol Res, 2008,58(5/6):262-270.
[16]Itoh K,Wakabayashi N,Katoh Y,et al. Keapl represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain[J].Genes Dev,1999,13(1):76-86.
[17]Kang MI,Kobayashi A,Wakabayashi N,et al. Scaffolding of Keapl to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes[J].Proc Natl Acad Sci USA,2004,101(7):2046-2051.
[18]Kensler TW,Wakabayashi N,Biswal S.Cell survival responses to environmental stresses via the Keapl-Nrf2-ARE pathway [J].Annu Rev Pharmacol Toxicol,2007, 47(2):89-116.
[19]Yates MS,Kensler TW. Chemopreventive promise of targeting the Nrf2 pathway[J]. Drug News Perspect,2007,20(2):109-117.
[20]Satoh T,Okamoto SI,Cui J,et al.Activation of the Keapl/Nrf2 pathway for neuroprotection by electrophilic phase II inducers [J].Proc Natl Acad Sci U S A, 2006,103(13):768-773.
[21]Itoh K,Chiba T,Takahashi S et al.An Nrf2/small Maf heterodimer mediates the induction of phase Ⅱ detoxifying enzyme genes through antioxidant response elements.Biochem Biophys Res Commun.1997,236(2):313-322.
[22]Kweon MH,Adhami VM,Lee JS,et al.Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate[J].J Biol Chem,2006,281:33761-33772.
[23]Singh A,Boldin-Adamsky S,Thimmulappa RK,et al. RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy[J].Cancer Res, 2008,68(19):7975-7984.
[24]Homma S,IshiiY,MorishimaY,et al.Nrf2 Enhances Cell Proliferation and Resistance to Anticancer Drugs in Human Lung Cancer[J].Clin Cancer Res,2009,15(10): 3423-3432.
[25]Song NY,Kim DH,Kim EH,et al.15-Deoxy-Δ12,14-prostaglandin J2 Induces Upregulation of Multidrug Resistance-Associated Protein 1 via Nrf2 Activation in Human Breast Cancer Cell[J].Ann NY Acad Sci,2009,1171:210-216.
[26]Akhdar H,Loyer P,Rauch C,et al.Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells[J].Europ J Cancer,2009,45(12): 2219-2227.
[27]Carette J E,Overmeer R M,Schagen FHE,et al.Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a traget gene in cancer cells. Cancer Res,2004,64:2663-2667.
[1]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.
[2]Kim HR,Kim S,Kim EJ,et al.Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin[J].Lung Cancer,2008,60(1):47-56.
[3]Kim SK,Yang JW,Kim MR,et al.Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells[J].Free Radic Biol Med,2008,45(4):537-546.
[4]Kobayashi A,Ohta T,Yamamoto M. Unique function of the Nrf22keapl pathway in the inducible expression of antioxidant and detoxifying enzynmes[J]. Methods Enzymol,2004,378:273-286.
[5]Zhang DD.Mechanistic studies of the Nrf2-Keapl signaling pathway[J].Drug Metab Rev,2006,38(4):769-789.
[6]Sun Z,Zhang S,Chan JY,et al.Keapl 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]Lau A,Villeneuve NF,Sun Z,et al.Dual roles of Nrf2 in cancer[J].Pharmacol Res,2008,58(5/6):262-270.
[8]Dinkova-Kostova AT,HoltzclawWD,Cole RN,et al.Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proc Natl Acad Sci U S A,2002,99(18):11908-11913.
[9]Kang MI,Kobayashi A,Wakabayashi N,et al.Scaffolding of Keapl 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.
[10]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(2):89-116.
[11]Yates MS,Kensler TW. Chemopreventive promise of targeting the Nrf2 pathway[J].Drug News Perspect,2007,20(2):109-117.
[12]Satoh T,Okamoto SI,Cui J,et al.Activation of the Keapl/Nrf2 pathway for neuroprotection by electrophilic phase II inducers [J].Proc Natl Acad Sci U S A,2006,103(13):768-773.
[13]Hong F,Freeman ML,Liebler DC. Identification of sensor cysteines in human Keapl modified by the cancer chemopreventive agent sulforaphane [J]. Chem Res Toxicol,2005,18(12):1917-1926.
[14]Li W,Jain MR,Chen C,et al.Nrf2 Possesses a redoxinsensitive nuclear export signal overlapping with the leucine zipper motif [J].J Biol Chem, 2005,280(31):28430-28438.
[15]Kweon MH,Adhami VM,Lee JS,et al.Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate[J].J Biol Chem,2006, 281(44):33761-33772.
[16]Singh A,Boldin-Adamsky S,Thimmulappa RK,et al.RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy[J].Cancer Res,2008,68(19):7975-7984.
[17]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.
[18]Song NY,Kim DH,Kim EH, et al.15-Deoxy-Δ12,14-prostaglandin J2 induces upregulation of multidrug resistance-associated protein 1 via Nrf2 activation in human breast cancer cell[J].Ann NY Acad Sci,2009,1171: 210-216.
[19]Akhdar H,Loyer P,Rauch C,et al.Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells[J].Europ J Cancer,2009,45(12):2219-2227.
[2]Ries LAG,Melbert D, Krapcho M et al.(eds).SEER Cancer Statistics Review, 1975-2005,National Cancer Institute. Bethesda,MD.Available at: http://seer.cancer.gov/csr/1975_2005/. Based on November 2007 SEER data submission, posted to the SEER web site 2008.
[3]Anzenbacher P,Anzenbacherova E. Cytochromes P450 and metabolism of xenobiotics[J].Cell Mol Life Sci,2001,58(5/6):737-747.
[4]Wilkinson JT,Clapper ML.Detoxication enzymes and chemoprevention[J].Proc Soc Exp Biol Med,1997,216(3):192-200.
[5]Itoh K,Ishii T,Wakabayashi N,et al.Regulatory mechanisms of cellar response to oxidative stress [J].FreeRadic Res,1999,31(4):319-324.
[6]Dhakshinamoorthy S, Long DJ 2nd, Jaiswal AK. Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens[J].Curr Top Cell Regul, 2000,36:201-216.
[7]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.
[8]Kim HR,Kim S,Kim EJ,et al. Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin[J].Lung Cancer,2008,60:47-56.
[9]Kim SK,Yang JW,Kim MR,et al.Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells[J].Free Radic Biol Med,2008,45:537-546.
[10]Rushmore TH,Morton MR,Pickett CB.The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity [J].J Biol Chem,1991,266(18):11632-11639.
[11]Kobayashi A,Ohta T,Yamamoto M.Unique function of the Nrf22keap1 pathway in the inducible expression of antioxidant and detoxifying enzynmes[J].Methods Enzymol,2004,378:273-286.
[12]Dinkova-Kostova AT,HoltzclawWD,Cole RN,et al. Direct evidence that sulfhydryl groups of Keapl are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proc Natl Acad Sci USA,2002,99 (18):11908-11913.
[13]Zhang DD. Mechanistic studies of the Nrf2-Keapl signaling pathway[J].Drug Metab Rev,2006,38:769-789.
[14]Sun Z,Zhang S,Chan JY,et al.Keapl controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2[J].Mol Cell Biol,2007,27(18):6334-6349.
[15]Lau A,Villeneuve NF,Sun Z,et al.Dual roles of Nrf2 in cancer[J].Pharmacol Res, 2008,58(5/6):262-270.
[16]Itoh K,Wakabayashi N,Katoh Y,et al. Keapl represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain[J].Genes Dev,1999,13(1):76-86.
[17]Kang MI,Kobayashi A,Wakabayashi N,et al. Scaffolding of Keapl to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes[J].Proc Natl Acad Sci USA,2004,101(7):2046-2051.
[18]Kensler TW,Wakabayashi N,Biswal S.Cell survival responses to environmental stresses via the Keapl-Nrf2-ARE pathway [J].Annu Rev Pharmacol Toxicol,2007, 47(2):89-116.
[19]Yates MS,Kensler TW. Chemopreventive promise of targeting the Nrf2 pathway[J]. Drug News Perspect,2007,20(2):109-117.
[20]Satoh T,Okamoto SI,Cui J,et al.Activation of the Keapl/Nrf2 pathway for neuroprotection by electrophilic phase II inducers [J].Proc Natl Acad Sci U S A, 2006,103(13):768-773.
[21]Itoh K,Chiba T,Takahashi S et al.An Nrf2/small Maf heterodimer mediates the induction of phase Ⅱ detoxifying enzyme genes through antioxidant response elements.Biochem Biophys Res Commun.1997,236(2):313-322.
[22]Kweon MH,Adhami VM,Lee JS,et al.Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate[J].J Biol Chem,2006,281:33761-33772.
[23]Singh A,Boldin-Adamsky S,Thimmulappa RK,et al. RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy[J].Cancer Res, 2008,68(19):7975-7984.
[24]Homma S,IshiiY,MorishimaY,et al.Nrf2 Enhances Cell Proliferation and Resistance to Anticancer Drugs in Human Lung Cancer[J].Clin Cancer Res,2009,15(10): 3423-3432.
[25]Song NY,Kim DH,Kim EH,et al.15-Deoxy-Δ12,14-prostaglandin J2 Induces Upregulation of Multidrug Resistance-Associated Protein 1 via Nrf2 Activation in Human Breast Cancer Cell[J].Ann NY Acad Sci,2009,1171:210-216.
[26]Akhdar H,Loyer P,Rauch C,et al.Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells[J].Europ J Cancer,2009,45(12): 2219-2227.
[27]Carette J E,Overmeer R M,Schagen FHE,et al.Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a traget gene in cancer cells. Cancer Res,2004,64:2663-2667.
[1]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.
[2]Kim HR,Kim S,Kim EJ,et al.Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin[J].Lung Cancer,2008,60(1):47-56.
[3]Kim SK,Yang JW,Kim MR,et al.Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells[J].Free Radic Biol Med,2008,45(4):537-546.
[4]Kobayashi A,Ohta T,Yamamoto M. Unique function of the Nrf22keapl pathway in the inducible expression of antioxidant and detoxifying enzynmes[J]. Methods Enzymol,2004,378:273-286.
[5]Zhang DD.Mechanistic studies of the Nrf2-Keapl signaling pathway[J].Drug Metab Rev,2006,38(4):769-789.
[6]Sun Z,Zhang S,Chan JY,et al.Keapl 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]Lau A,Villeneuve NF,Sun Z,et al.Dual roles of Nrf2 in cancer[J].Pharmacol Res,2008,58(5/6):262-270.
[8]Dinkova-Kostova AT,HoltzclawWD,Cole RN,et al.Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants[J].Proc Natl Acad Sci U S A,2002,99(18):11908-11913.
[9]Kang MI,Kobayashi A,Wakabayashi N,et al.Scaffolding of Keapl 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.
[10]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(2):89-116.
[11]Yates MS,Kensler TW. Chemopreventive promise of targeting the Nrf2 pathway[J].Drug News Perspect,2007,20(2):109-117.
[12]Satoh T,Okamoto SI,Cui J,et al.Activation of the Keapl/Nrf2 pathway for neuroprotection by electrophilic phase II inducers [J].Proc Natl Acad Sci U S A,2006,103(13):768-773.
[13]Hong F,Freeman ML,Liebler DC. Identification of sensor cysteines in human Keapl modified by the cancer chemopreventive agent sulforaphane [J]. Chem Res Toxicol,2005,18(12):1917-1926.
[14]Li W,Jain MR,Chen C,et al.Nrf2 Possesses a redoxinsensitive nuclear export signal overlapping with the leucine zipper motif [J].J Biol Chem, 2005,280(31):28430-28438.
[15]Kweon MH,Adhami VM,Lee JS,et al.Constitutive overexpression of Nrf2-dependent heme oxygenase-1 in A549 cells contributes to resistance to apoptosis induced by epigallocatechin 3-gallate[J].J Biol Chem,2006, 281(44):33761-33772.
[16]Singh A,Boldin-Adamsky S,Thimmulappa RK,et al.RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy[J].Cancer Res,2008,68(19):7975-7984.
[17]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.
[18]Song NY,Kim DH,Kim EH, et al.15-Deoxy-Δ12,14-prostaglandin J2 induces upregulation of multidrug resistance-associated protein 1 via Nrf2 activation in human breast cancer cell[J].Ann NY Acad Sci,2009,1171: 210-216.
[19]Akhdar H,Loyer P,Rauch C,et al.Involvement of Nrf2 activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells[J].Europ J Cancer,2009,45(12):2219-2227.