北豆根总碱对Hela细胞抑制作用及其机制研究
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摘要
目的:研究北豆根总碱对宫颈癌细胞Hela凋亡、细胞周期及侵袭性的影响,探讨北豆根总碱抗肿瘤作用机制,为抗肿瘤新药的研究和开发提供科学依据。
方法:1采用四甲基偶氮唑蓝法(MTT)检测不同浓度北豆根总碱(3.125、6.25、12.5、25、50μg/ml)处理不同时间(24、48和72小时)对Hela细胞增殖反应的抑制作用。
2采用流式细胞技术(flow cytometry,FCM)检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用48小时,对Hela细胞凋亡及周期变化的影响。
3瑞氏-姬姆萨染色后显微镜观察北豆根总碱(0、40μg/ml)作用24小时后Hela细胞形态学变化。
4采用Transwell小室法检测北豆根总碱(0、40μg/ml)作用24小时后对Hela细胞侵袭性的影响。
5采用免疫印迹(Western blot)方法检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用24小时后,Hela细胞中磷酸化ERK(p-ERK)、ERK、C-myc、CyclinD1的表达变化。
6采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测北豆根总碱(0、4、16、40μg/ml)作用24小时,Hela细胞抗凋亡基因bcl-2、促凋亡基因bax、基质金属蛋白酶-9(MMP-9)的表达变化。
7应用激光共聚焦显微镜(laser scanning microscope,LSM)观察北豆根总碱(0、40μg/ml)作用24小时后,Hela细胞内P-ERK、ERK、Bcl-2、Bax蛋白的表达变化。
结果:1北豆根总碱对Hela细胞增殖有较强的抑制作用,随着北豆根总碱浓度及作用时间的增加,对细胞的抑制率升高。其中50μg/ml北豆根总碱作用72小时对细胞抑制率最高(85.37%);与对照组相比,不同浓度北豆根总碱对细胞增殖的抑制率均有显著性差异(p<0.05)。
2北豆根总碱能明显诱导Hela细胞发生凋亡和细胞周期阻滞。随着北豆根总碱作用浓度的增大,细胞的凋亡率升高,G0/G1期细胞明显增多,S期细胞明显减少,对照组与试验组之间比较有显著性差异(p<0.05)。
3北豆根总碱作用24小时后,Hela细胞形态呈现凋亡前期改变。
4 Transwell小室侵袭实验结果显示,经北豆根总碱(0,40μg/ml)作用24小时后,穿膜细胞数较对照组显著减少,存在显著差异(p<0.05)。
5 Western blot分析结果显示,经4、16、40μg/ml北豆根总碱处理24小时后,Hela细胞中磷酸化ERK、C-myc、CyclinD1表达水平与对照组细胞比较均有所下降,ERK表达无显著性变化。
6半定量RT-PCR检测结果显示,经不同浓度北豆根总碱处理后,Hela细胞抗凋亡基因bcl-2、基质金属蛋白酶-9 MMP-9 mRNA表达水平逐渐降低,促凋亡基因bax mRNA表达水平逐渐增高,与对照组比较有显著性差异(p<0.05),该现象随着浓度北豆根总碱浓度的升高而更加明显,呈明显的浓度依赖性。
7 LSM荧光图像分析结果显示,北豆根总碱(40μg/ml)作用24小时后,Hela细胞内抗Bax蛋白抗染色的荧光强度较对照组明显增加,抗P-ERK、Bal-2抗体荧光强度较对照组明显降低,ERK抗体荧光强度较对照组无明显变化。
结论:1北豆根总碱在一定浓度范围内,能抑制宫颈癌细胞Hela的增殖,具有明显的量效关系。
2北豆根总碱可通过降低Hela细胞中磷酸化ERK(P-ERK)水平,抑制Hela细胞ERK1/2信号转导通路。
3北豆根总碱通过抑制Hela细胞ERK1/2信号转导通路,减弱bcl-2,MMP-9 mRNA表达,增强bax mRNA表达,减弱Hela细胞中C-myc、Cyclin D1蛋白的表达,进而促进Hela细胞凋亡,抑制Hela细胞侵袭力,使细胞阻滞于G0/G1期。
Objective: To study the effects of Total Alkaloid of Menispermum (TAM) on apoptosis, cell cycle and invasive ablity to the Hela cell. To investigate the antitumor mechanisms of TAM. To produce the scientific evidence for developing and manufacturing new antitumor drugs.
Methods: 1 The inhibitory effect on cell growth of Hela was measured by MTT assay in treated or untreated groups (3.125, 6.25, 12.5, 25, 50μg/ml TAM and control) for three different treatment times (24h, 48h and 72h).
2 Apoptosis and cell cycle were measured by FCM in four experimental groups (0, 4, 16, 40μg/ml TAM) for 48h.
3 Adopting Wright and Giemse's staining to observe the morphology of Hela cells which treated with 40μg/ml TAM.
4 Using invasion experiment to detect the Hela cells’invasive abilities which treated with 40μg/ml TAM.
5 The protein expressional levels of P-ERK, ERK, C-myc and Cyclin D1 in Hela cells untreated or treated with 4, 16, 40μg/ml TAM for 24h were measured by Western blot.
6 Expression of anti-apoptotic gene bcl-2, apoptotic gene bax and MMP-9 in Hela cells of four experimental groups (0, 4, 16, 40μg/ml TAM for 24h), were observed by revers transcription PCR (RT-PCR).
7 The protein expression of P-ERK, ERK, Bcl-2 and Bax in Hela cells treated with 40μg/ml TAM for 24h observed by laser scanning microscopes (LSM).
Results: 1 TAM could significantly inhibit the growth of Hela cells in vitro (p<0.05). The growth inhibition was in dose-dependent. The greatest inhibit rate was 85.37% observed in the treatment group of Hela cells by 50μg/ml TAM for 72 hours.
2 The results of FCM showed that TAM can induce apoptosis and cell cycle arrest of Hela cells. After treated by TAM for 48h, the apoptosis rate of Hela cells increased markely with a dose-dependent manner (p<0.05). Meanwhile, after treated with TAM, the cell cycles of Hela cells were changed, the cells in G_0/G_1 phase were increased and cells in S phase were decreased.
3 Wright and Giemsa's staining revealed that the Hela cells treated by TAM had had proapoptotic morphology.
4 The results of invasion experiment showed that the invasive ability of Hela cells treated with TAM had decreased remarkable (p<0.05), compared with the contral group.
5 The results of western blotting revealed that TAM had down- regulated the level of phopholated ERK, C-myc and CyclinD1 expression in Hela cells in treated group with 4, 16, 40μg/ml TAM for 24 hours, compared with the control group (untreated) (p<0.05),but the level of ERK expression didn’t change significantly.
6 TAM inhibited the mRNA expression of the bcl-2 and MMP- 9, increased the mRNA expression of apoptotic gene bax in Hela cells treated by 4, 16, 40μg/ml periplocin for 24 hours compared with untreated cells (p<0.05).
7 LSM images show that after treated with 40μg/ml TAM, the fluorescence intensity of P-ERK, Bcl-2 were significantly weaker, and fluorescence intensity of Bax was more increased than that in control group. but fluorescence intensity of ERK was no significant change.
Conclusion: 1 TAM could inhibit significantly proliferation of the human cervix cancer Hela cells in vitro in dose-dependent and time-dependent manner.
2 In the Hela cells, TAM could repress the ERK1/2 signaling pathway through downregulating the expression of P-ERK.
3 TAM could inhibit significantly the expression of bcl-2 ,MMP-9 mRNA, increase the expression of bax mRNA, affect the expression of C-myc and CyclinD1, accordingly, the treatment of TAM induced apoptosis , arrested the cell cycle in G_0/G_1 phase and debilitated invasion ability of Hela cells in vitro.
方法:1采用四甲基偶氮唑蓝法(MTT)检测不同浓度北豆根总碱(3.125、6.25、12.5、25、50μg/ml)处理不同时间(24、48和72小时)对Hela细胞增殖反应的抑制作用。
2采用流式细胞技术(flow cytometry,FCM)检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用48小时,对Hela细胞凋亡及周期变化的影响。
3瑞氏-姬姆萨染色后显微镜观察北豆根总碱(0、40μg/ml)作用24小时后Hela细胞形态学变化。
4采用Transwell小室法检测北豆根总碱(0、40μg/ml)作用24小时后对Hela细胞侵袭性的影响。
5采用免疫印迹(Western blot)方法检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用24小时后,Hela细胞中磷酸化ERK(p-ERK)、ERK、C-myc、CyclinD1的表达变化。
6采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测北豆根总碱(0、4、16、40μg/ml)作用24小时,Hela细胞抗凋亡基因bcl-2、促凋亡基因bax、基质金属蛋白酶-9(MMP-9)的表达变化。
7应用激光共聚焦显微镜(laser scanning microscope,LSM)观察北豆根总碱(0、40μg/ml)作用24小时后,Hela细胞内P-ERK、ERK、Bcl-2、Bax蛋白的表达变化。
结果:1北豆根总碱对Hela细胞增殖有较强的抑制作用,随着北豆根总碱浓度及作用时间的增加,对细胞的抑制率升高。其中50μg/ml北豆根总碱作用72小时对细胞抑制率最高(85.37%);与对照组相比,不同浓度北豆根总碱对细胞增殖的抑制率均有显著性差异(p<0.05)。
2北豆根总碱能明显诱导Hela细胞发生凋亡和细胞周期阻滞。随着北豆根总碱作用浓度的增大,细胞的凋亡率升高,G0/G1期细胞明显增多,S期细胞明显减少,对照组与试验组之间比较有显著性差异(p<0.05)。
3北豆根总碱作用24小时后,Hela细胞形态呈现凋亡前期改变。
4 Transwell小室侵袭实验结果显示,经北豆根总碱(0,40μg/ml)作用24小时后,穿膜细胞数较对照组显著减少,存在显著差异(p<0.05)。
5 Western blot分析结果显示,经4、16、40μg/ml北豆根总碱处理24小时后,Hela细胞中磷酸化ERK、C-myc、CyclinD1表达水平与对照组细胞比较均有所下降,ERK表达无显著性变化。
6半定量RT-PCR检测结果显示,经不同浓度北豆根总碱处理后,Hela细胞抗凋亡基因bcl-2、基质金属蛋白酶-9 MMP-9 mRNA表达水平逐渐降低,促凋亡基因bax mRNA表达水平逐渐增高,与对照组比较有显著性差异(p<0.05),该现象随着浓度北豆根总碱浓度的升高而更加明显,呈明显的浓度依赖性。
7 LSM荧光图像分析结果显示,北豆根总碱(40μg/ml)作用24小时后,Hela细胞内抗Bax蛋白抗染色的荧光强度较对照组明显增加,抗P-ERK、Bal-2抗体荧光强度较对照组明显降低,ERK抗体荧光强度较对照组无明显变化。
结论:1北豆根总碱在一定浓度范围内,能抑制宫颈癌细胞Hela的增殖,具有明显的量效关系。
2北豆根总碱可通过降低Hela细胞中磷酸化ERK(P-ERK)水平,抑制Hela细胞ERK1/2信号转导通路。
3北豆根总碱通过抑制Hela细胞ERK1/2信号转导通路,减弱bcl-2,MMP-9 mRNA表达,增强bax mRNA表达,减弱Hela细胞中C-myc、Cyclin D1蛋白的表达,进而促进Hela细胞凋亡,抑制Hela细胞侵袭力,使细胞阻滞于G0/G1期。
Objective: To study the effects of Total Alkaloid of Menispermum (TAM) on apoptosis, cell cycle and invasive ablity to the Hela cell. To investigate the antitumor mechanisms of TAM. To produce the scientific evidence for developing and manufacturing new antitumor drugs.
Methods: 1 The inhibitory effect on cell growth of Hela was measured by MTT assay in treated or untreated groups (3.125, 6.25, 12.5, 25, 50μg/ml TAM and control) for three different treatment times (24h, 48h and 72h).
2 Apoptosis and cell cycle were measured by FCM in four experimental groups (0, 4, 16, 40μg/ml TAM) for 48h.
3 Adopting Wright and Giemse's staining to observe the morphology of Hela cells which treated with 40μg/ml TAM.
4 Using invasion experiment to detect the Hela cells’invasive abilities which treated with 40μg/ml TAM.
5 The protein expressional levels of P-ERK, ERK, C-myc and Cyclin D1 in Hela cells untreated or treated with 4, 16, 40μg/ml TAM for 24h were measured by Western blot.
6 Expression of anti-apoptotic gene bcl-2, apoptotic gene bax and MMP-9 in Hela cells of four experimental groups (0, 4, 16, 40μg/ml TAM for 24h), were observed by revers transcription PCR (RT-PCR).
7 The protein expression of P-ERK, ERK, Bcl-2 and Bax in Hela cells treated with 40μg/ml TAM for 24h observed by laser scanning microscopes (LSM).
Results: 1 TAM could significantly inhibit the growth of Hela cells in vitro (p<0.05). The growth inhibition was in dose-dependent. The greatest inhibit rate was 85.37% observed in the treatment group of Hela cells by 50μg/ml TAM for 72 hours.
2 The results of FCM showed that TAM can induce apoptosis and cell cycle arrest of Hela cells. After treated by TAM for 48h, the apoptosis rate of Hela cells increased markely with a dose-dependent manner (p<0.05). Meanwhile, after treated with TAM, the cell cycles of Hela cells were changed, the cells in G_0/G_1 phase were increased and cells in S phase were decreased.
3 Wright and Giemsa's staining revealed that the Hela cells treated by TAM had had proapoptotic morphology.
4 The results of invasion experiment showed that the invasive ability of Hela cells treated with TAM had decreased remarkable (p<0.05), compared with the contral group.
5 The results of western blotting revealed that TAM had down- regulated the level of phopholated ERK, C-myc and CyclinD1 expression in Hela cells in treated group with 4, 16, 40μg/ml TAM for 24 hours, compared with the control group (untreated) (p<0.05),but the level of ERK expression didn’t change significantly.
6 TAM inhibited the mRNA expression of the bcl-2 and MMP- 9, increased the mRNA expression of apoptotic gene bax in Hela cells treated by 4, 16, 40μg/ml periplocin for 24 hours compared with untreated cells (p<0.05).
7 LSM images show that after treated with 40μg/ml TAM, the fluorescence intensity of P-ERK, Bcl-2 were significantly weaker, and fluorescence intensity of Bax was more increased than that in control group. but fluorescence intensity of ERK was no significant change.
Conclusion: 1 TAM could inhibit significantly proliferation of the human cervix cancer Hela cells in vitro in dose-dependent and time-dependent manner.
2 In the Hela cells, TAM could repress the ERK1/2 signaling pathway through downregulating the expression of P-ERK.
3 TAM could inhibit significantly the expression of bcl-2 ,MMP-9 mRNA, increase the expression of bax mRNA, affect the expression of C-myc and CyclinD1, accordingly, the treatment of TAM induced apoptosis , arrested the cell cycle in G_0/G_1 phase and debilitated invasion ability of Hela cells in vitro.
引文
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6 McCubrey JA, Steelman LS, Chappell WH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta, 2007, 1773(8):1263-1284.
7齐洁敏,马卫军,白连宏.宫颈鳞癌PTEN和P-ERK的表达及其临床意义.中国妇幼保健,2007,22:1213-1214.
8殷飞,赵军艳,姚树坤.苦参碱对SMMC-7721细胞MAPK、JAK-STAT信号通路的影响.肿瘤防治研究,2008, 35(2):84-87.
9 So EY, Oh J, Jang JY, et al. Ras/Erk pathway positivelyregulates Jak1/STAT6 activity and IL-4 gene expression in Jurkat T cells. Molecular Immunology, 2007,44: 3416–3426.
10腊蕾,饶进军,吴曙光. Wnt2β链接素通道阻断在肿瘤治疗中的意义.国外医学肿瘤学分册,2003,30(6):433-435.
11 Park KS, Jeon SH, Kim SE, et al. APC inhibits ERK pathway activation and cellular proliferation induced by RAS. Journal of Cell Science,2006,119 (5):819-827.
12 Soung HJ, Yoon JY, Park YN, et al. Axin Inhibits Ex- tracellular Signal-regulated Kinase Pathway by Ras Degradation viaβ-Catenin. JBiol Chem, 2007,282(9): 14482–14492.
13 Ding Q, Xia W, liu JC. et al. Erk associates with and primes GSK-3 beta for its inactivation resulting in upregulation of beta-catenin. Mol Cell, 2005, 19(2):159-170.
14王兵,周士福,蔡凤林,等. PTEN,P-AKT,P-ERK蛋白在胃癌组织中的表达及意义.现代肿瘤医学,2008, 16(9): 1568- 1570.
15周树伟,陈静. PTEN和P-ERK蛋白在皮肤鳞状细胞癌中的表达和意义.中国美容医学,2008,17(10):1483-1485.
16王树森,管忠震,向燕群,等.鼻咽癌组织中EGFR和p-ERK蛋白表达的检测及意义.中华肿瘤杂志,2006, 28(1):28-31.
17乔玉环,侯艺芳,郭瑞霞,等.人子宫内膜癌组织中磷酸化细胞外信号调节激酶的检测.,郑州大学学报(医学版),2008, 43(2):273-275.
18关剑,李柏林,宋敏,等.乳腺浸润性导管癌组织学分级与BAD、p-BAD及p-ERK蛋白的表达.,肿瘤,2006, 26(3): 267-270.
19 Margherita B, Marco C, Donatella S, et al. Activation of the ERK/MAP Kinase Pathway in Cervical Intra- epithelial Neoplasia Is Related to Grade of the Lesion but Not to High-Risk Human Papillomavirus, Virus Clearance, or Prognosis in Cervical Cancer. Am J Clin Pathol, 2004, 122:902-911.
20 Lee HJ, Kim DI, Kang G H, et al. Phosphorylation of ERK1/2 and Prognosis of Clear Cell Renal Cell Carcinoma. Urology, 2009, 73(2) :394-399.
21柏素云,李冠武,张立民,等. RAS/MAPK通路在食管癌中的活化改变及其意义.国际检验医学杂志,2006, 27(9): 771-773.
22 Yamamoto H, Toyooka S, Mitsudomi T. Impact of EGFR mutation analysis in non-small cell lung cancer. Lung Cancer, 2009,63 (3): 315-321.
23 Kotoula V, Sozopoulos E, Litsiou H, et al. Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas. Endocr Relat Cancer. 2009 . [Epub ahead of print].
24 Leo SH, Lee JW, Soung YH, et al. Colorectal tumors frequently express phosphorylated mitogen-activated protein kinase [J]. APMIS, 2004,112(4-5):233-238.
25 Liang B, Wang S, Zhu XG, et al. Increased expression of mitogenactivated protein kinase and its upstream regulatingsignal in human gastric cancer. World J Gastroenterol., 2005, 11(5):623-628.
26 Ning L, Kunnimalaiyaan M, Chen H, et al. Regulation of cell–cell contact molecules and the metastatic phenotype of medullary thyroid carcinoma by the Raf-1/MEK/ERK pathway [J]. Surgery, 2008,144:920-925.
27 Jukka W, Veli-Matti K. Regulation of matrix metallopro- teinase expression in tumor invasion[J]. FASEB J, 1999 , 13 : 781-792.
28 Simon C, Hicks MJ, Nemechek AJ, et al. PD098059 , an inhibitor of ERK1activation,attenuates the in vivo inva- siveness of head and neck squamous cell carcinoma [J]. Br J Cancer, 1999 , 80 ( 9) :1412-1419.
29 Bancroft CC, Chen Z, Dong G, et al. Coexpression of proangiogenic factors IL28 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF-kappaB signal pathways[J]. Clin Cancer Res, 2001, 7 ( 2) : 435-442.
30缪泽鸿,丁健.抗肿瘤多药耐药的研究进展.癌症Chinese Journal of Cancer, 2003,22(8):886-892.
31 Kazuhiro K, Sho Y, Satomi T. Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein. Mol Cancer Ther, 2007, 6(7):2092-2102.
32 Jiang K, Coppola D, Crespo NC, et al. The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis[J]. Mol Cell Biol, 2000, 20(1): 139-148.
33 End DW, SmetsG, Todd AV, et al. Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro[J]. Cancer Res, 2001, 61(1): 131-137.
34 Sebolt-Leopold JS, Herrera R. Targeting the mitogenac- tivated protein kinase cascade to treat cancer[J]. NatRev Cancer, 2004, 4(12): 937-947.
35 Bergo MO, Ambroziak P, Gregory C, et al. Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation[J]. MolCellBiol, 2002, 22(1): 171-181.
36 Hagemann C, Rapp UR. Isotype-specific functions of Raf kinases.Exp. Cell Res, 1999, 253: 34-46.
37 Mason CS, Springer CJ, Cooper RG, et al. Serine and tyrosine phosphorylations cooperate in Raf-1,but not B-Raf activation. Embo J, 1999, 18: 2137–2148.
38 Li N, Batt D, Warmuth M. B-Raf kinase inhibitors for cancer Treatment [J], Curr Opin Invest Drugs, 2007, 8: 452–456.
39 Garnett MJ, Marais R. Guilty as charged: B-RAF is a human Oncogene [J]. Cancer Cell, 2004, 6: 313–319.
40 Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF[J]. Cell, 2004, 116: 855–867.
41 Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis [J]. Cancer Res, 2004, 64: 7099–7109.
42 Lyons JF, Wilhelm S, Hibner B, et al. Discovery of a novel Rafkinase inhibitor [J]. Endocr Relat Cancer, 2001, 8(3): 219-225.
43 Escudier B, Eisen T, Stadler WM, et al. Sorafenib in ad- vanced clear-cell renal-cell carcinoma [J]. Med, 2007, 356: 125–134.
44 Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo -controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma [J]. Clin. Oncol, 2006, 24: 2505–2512.
45 Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in ad- vanced hepatocellular carcinoma [J]. Med, 2008, 359: 378–390.
46 Stuart D, Aardalen K, Venetsanakos E, et al. RAF265 is a potent Raf kinase inhibitor with selective anti-proliferative activity in vitro and in vivo[J]. Cancer Res, 2008, 68:4876.
47 Venetsanakos E, Stuart D, Tan N, et al. CHIR-265, a novel inhibitor that targets B-Raf and VEGFR, shows efficacy in a broad range of preclinical models[J].Cancer Res,2006, 47:4854.
48 Stuart D, Aardalen K, Lorenzana E,et al. Characterization of a novel Raf kinase inhibitor that causes target dependenttumor regression in human melanoma xenografts expressing mutant B-Raf[J]. Cancer Res, 2006, 47: 4856.
49 Wang JY, Wilcoxen KM, Nomoto K, et al. Recent advances of MEK inhibitors and their clinical progress[J]. Chem, 2007, 7: 1364–1378.
50罗威,曹亚.靶向ERK信号转导通路抗肿瘤的研究进展.国际病理与临床杂志,2006,26(3):200-203.
51 Sebolt-Leopold JS, Dudley DT, Herrera R, er al. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo[J]. Med,1999, 5: 810–816.
52 Lorusso PM, Adjei AA, Varterasian M, et al. Phase I and pharmacodynamic study of the oral MEK inhibitor CI-1040 in patients with advanced malignancies[J]. Oncol, 2005,23: 5281–5293.
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54 Tan W, DePrimo S, Krishnamurthi S. Pharmacokinetic (PK) and pharmacodynamic (PD) results of a phase I study of PD-0325901, a second generation oral MEK inhibitor, in patients with advanced cancer[J]. Cancer Ther, 2007, 6: B109.
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1 Gray P, Fred R. Tara Beers Gibson. Mitogen Activated Protein (MAP) Kinase Pathways:Regulationand Phy- siological Functions. Endocrine Reviews, 2001,22(2): 153–183.
2 Liebmann C. Regulation of MAP kinase activity by peptide receptor signalling pathway:Paradigms of multiplicity. Cellular Signalling, 2001.13: 777– 785.
3袁向飞,陆敏. Ras/MAPK与PI3K/Akt信号转导通路及其相互作用.国际检验医学杂志,2006,27(3):251-263.
4 Carmen BA, Oliver R, Juan F, et al. PTEN more than the AKT pathway. Carcinogenesis, 2007,28 (7) :1379–1386.
5 Sheng H, Shao J, Dubois RN. Akt/PKB activity is required for Ha-Ras-mediated transformation of intestinal epithelial cells. Biol Chem, 2001, 276(17):14498-14504.
6 McCubrey JA, Steelman LS, Chappell WH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta, 2007, 1773(8):1263-1284.
7齐洁敏,马卫军,白连宏.宫颈鳞癌PTEN和P-ERK的表达及其临床意义.中国妇幼保健,2007,22:1213-1214.
8殷飞,赵军艳,姚树坤.苦参碱对SMMC-7721细胞MAPK、JAK-STAT信号通路的影响.肿瘤防治研究,2008, 35(2):84-87.
9 So EY, Oh J, Jang JY, et al. Ras/Erk pathway positivelyregulates Jak1/STAT6 activity and IL-4 gene expression in Jurkat T cells. Molecular Immunology, 2007,44: 3416–3426.
10腊蕾,饶进军,吴曙光. Wnt2β链接素通道阻断在肿瘤治疗中的意义.国外医学肿瘤学分册,2003,30(6):433-435.
11 Park KS, Jeon SH, Kim SE, et al. APC inhibits ERK pathway activation and cellular proliferation induced by RAS. Journal of Cell Science,2006,119 (5):819-827.
12 Soung HJ, Yoon JY, Park YN, et al. Axin Inhibits Ex- tracellular Signal-regulated Kinase Pathway by Ras Degradation viaβ-Catenin. JBiol Chem, 2007,282(9): 14482–14492.
13 Ding Q, Xia W, liu JC. et al. Erk associates with and primes GSK-3 beta for its inactivation resulting in upregulation of beta-catenin. Mol Cell, 2005, 19(2):159-170.
14王兵,周士福,蔡凤林,等. PTEN,P-AKT,P-ERK蛋白在胃癌组织中的表达及意义.现代肿瘤医学,2008, 16(9): 1568- 1570.
15周树伟,陈静. PTEN和P-ERK蛋白在皮肤鳞状细胞癌中的表达和意义.中国美容医学,2008,17(10):1483-1485.
16王树森,管忠震,向燕群,等.鼻咽癌组织中EGFR和p-ERK蛋白表达的检测及意义.中华肿瘤杂志,2006, 28(1):28-31.
17乔玉环,侯艺芳,郭瑞霞,等.人子宫内膜癌组织中磷酸化细胞外信号调节激酶的检测.,郑州大学学报(医学版),2008, 43(2):273-275.
18关剑,李柏林,宋敏,等.乳腺浸润性导管癌组织学分级与BAD、p-BAD及p-ERK蛋白的表达.,肿瘤,2006, 26(3): 267-270.
19 Margherita B, Marco C, Donatella S, et al. Activation of the ERK/MAP Kinase Pathway in Cervical Intra- epithelial Neoplasia Is Related to Grade of the Lesion but Not to High-Risk Human Papillomavirus, Virus Clearance, or Prognosis in Cervical Cancer. Am J Clin Pathol, 2004, 122:902-911.
20 Lee HJ, Kim DI, Kang G H, et al. Phosphorylation of ERK1/2 and Prognosis of Clear Cell Renal Cell Carcinoma. Urology, 2009, 73(2) :394-399.
21柏素云,李冠武,张立民,等. RAS/MAPK通路在食管癌中的活化改变及其意义.国际检验医学杂志,2006, 27(9): 771-773.
22 Yamamoto H, Toyooka S, Mitsudomi T. Impact of EGFR mutation analysis in non-small cell lung cancer. Lung Cancer, 2009,63 (3): 315-321.
23 Kotoula V, Sozopoulos E, Litsiou H, et al. Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas. Endocr Relat Cancer. 2009 . [Epub ahead of print].
24 Leo SH, Lee JW, Soung YH, et al. Colorectal tumors frequently express phosphorylated mitogen-activated protein kinase [J]. APMIS, 2004,112(4-5):233-238.
25 Liang B, Wang S, Zhu XG, et al. Increased expression of mitogenactivated protein kinase and its upstream regulatingsignal in human gastric cancer. World J Gastroenterol., 2005, 11(5):623-628.
26 Ning L, Kunnimalaiyaan M, Chen H, et al. Regulation of cell–cell contact molecules and the metastatic phenotype of medullary thyroid carcinoma by the Raf-1/MEK/ERK pathway [J]. Surgery, 2008,144:920-925.
27 Jukka W, Veli-Matti K. Regulation of matrix metallopro- teinase expression in tumor invasion[J]. FASEB J, 1999 , 13 : 781-792.
28 Simon C, Hicks MJ, Nemechek AJ, et al. PD098059 , an inhibitor of ERK1activation,attenuates the in vivo inva- siveness of head and neck squamous cell carcinoma [J]. Br J Cancer, 1999 , 80 ( 9) :1412-1419.
29 Bancroft CC, Chen Z, Dong G, et al. Coexpression of proangiogenic factors IL28 and VEGF by human head and neck squamous cell carcinoma involves coactivation by MEK-MAPK and IKK-NF-kappaB signal pathways[J]. Clin Cancer Res, 2001, 7 ( 2) : 435-442.
30缪泽鸿,丁健.抗肿瘤多药耐药的研究进展.癌症Chinese Journal of Cancer, 2003,22(8):886-892.
31 Kazuhiro K, Sho Y, Satomi T. Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein. Mol Cancer Ther, 2007, 6(7):2092-2102.
32 Jiang K, Coppola D, Crespo NC, et al. The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis[J]. Mol Cell Biol, 2000, 20(1): 139-148.
33 End DW, SmetsG, Todd AV, et al. Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro[J]. Cancer Res, 2001, 61(1): 131-137.
34 Sebolt-Leopold JS, Herrera R. Targeting the mitogenac- tivated protein kinase cascade to treat cancer[J]. NatRev Cancer, 2004, 4(12): 937-947.
35 Bergo MO, Ambroziak P, Gregory C, et al. Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation[J]. MolCellBiol, 2002, 22(1): 171-181.
36 Hagemann C, Rapp UR. Isotype-specific functions of Raf kinases.Exp. Cell Res, 1999, 253: 34-46.
37 Mason CS, Springer CJ, Cooper RG, et al. Serine and tyrosine phosphorylations cooperate in Raf-1,but not B-Raf activation. Embo J, 1999, 18: 2137–2148.
38 Li N, Batt D, Warmuth M. B-Raf kinase inhibitors for cancer Treatment [J], Curr Opin Invest Drugs, 2007, 8: 452–456.
39 Garnett MJ, Marais R. Guilty as charged: B-RAF is a human Oncogene [J]. Cancer Cell, 2004, 6: 313–319.
40 Wan PT, Garnett MJ, Roe SM, et al. Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF[J]. Cell, 2004, 116: 855–867.
41 Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis [J]. Cancer Res, 2004, 64: 7099–7109.
42 Lyons JF, Wilhelm S, Hibner B, et al. Discovery of a novel Rafkinase inhibitor [J]. Endocr Relat Cancer, 2001, 8(3): 219-225.
43 Escudier B, Eisen T, Stadler WM, et al. Sorafenib in ad- vanced clear-cell renal-cell carcinoma [J]. Med, 2007, 356: 125–134.
44 Ratain MJ, Eisen T, Stadler WM, et al. Phase II placebo -controlled randomized discontinuation trial of sorafenib in patients with metastatic renal cell carcinoma [J]. Clin. Oncol, 2006, 24: 2505–2512.
45 Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in ad- vanced hepatocellular carcinoma [J]. Med, 2008, 359: 378–390.
46 Stuart D, Aardalen K, Venetsanakos E, et al. RAF265 is a potent Raf kinase inhibitor with selective anti-proliferative activity in vitro and in vivo[J]. Cancer Res, 2008, 68:4876.
47 Venetsanakos E, Stuart D, Tan N, et al. CHIR-265, a novel inhibitor that targets B-Raf and VEGFR, shows efficacy in a broad range of preclinical models[J].Cancer Res,2006, 47:4854.
48 Stuart D, Aardalen K, Lorenzana E,et al. Characterization of a novel Raf kinase inhibitor that causes target dependenttumor regression in human melanoma xenografts expressing mutant B-Raf[J]. Cancer Res, 2006, 47: 4856.
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50罗威,曹亚.靶向ERK信号转导通路抗肿瘤的研究进展.国际病理与临床杂志,2006,26(3):200-203.
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