摘要
目的:研究应用JAK酶抑制剂AG490对乳腺癌细胞MDA-MB-231 STAT3和ERK磷酸化的影响,探讨JAK/STAT3和MAPK/ERK两条信号转导通路的交互作用以及在乳腺癌细胞侵袭转移中的调控意义。
方法:以人乳腺癌细胞株MDA-MB-231为研究对象;以JAK酶抑制剂AG490处理乳腺癌细胞MDA-MB-231;Western blot检测细胞中P-STAT3、P-ERK蛋白表达水平变化;RT-PCR检测细胞中STAT3、ERK1、ERK2 mRNA表达变化;MTT法检测AG490对MDA-MB-231细胞的生长抑制作用;Western blot检测细胞中MMP-9表达及明胶酶谱法检测细胞分泌MMP-2、MMP-9的变化;MTT法检测AG490对MDA-MB-231细胞黏附人工基底膜(Matrigel)的能力变化;Transwell小室进行人工重组基底膜侵袭和迁移实验。
结果:应用JAK酶抑制剂AG490 40μmol/L处理人乳腺癌细胞MDA-MB-231 24h后P-STAT3、P-ERK蛋白表达均减少,分别为未处理组的37.6%、48.9%;STAT3、ERK1、ERK2 mRNA表达减少,分别为未处理组的41.8%、44.8%、56.2%,表达差异有统计学意义(P<0.01)。AG490对乳腺癌细胞MDA-MB-231有生长抑制作用,呈时效-量效依赖关系。40μmol/L AG490处理MDA-MB-231细胞48h后其黏附基底膜能力和侵袭迁移能力均降低(P<0.05)。40μmol/L AG490处理MDA-MB-231细胞24h后MMP-9的表达减少,是未处理组的44.9%;MMP-2、MMP-9的分泌亦减少,分泌量分别是未处理组的30.6%、62.2%,差异有统计学意义(P<0.01)。
结论:通过JAK酶抑制可改变转录因子STAT3和激酶ERK磷酸化水平,进而可以交互影响其基因转录的表达,JAK/STAT3和MAPK/ERK两条信号转导通路之间存在交互作用;JAK酶抑制对两条信号转导通路的激活有阻断作用而可以抑制乳腺癌细胞的侵袭转移。
Objective: To explore the effect of Janus kinase inhibitor AG490 on phosphorylation of STAT3 and ERK of the human breast cancer cell MDA-MB-231 and elucidate the cross-talking between JAK/STAT3 and MAPK/ERK signal transduction pathways and roles of two pathways on invasion and metastasis of human breast cancer cells.
Methods: MDA-MB-231 cell line was used as the research model, and they were treated with Janus kinase inhibitor AG490. The expression of P-STAT3 and P-ERK proteins were detected by Western-blot.The expression of STAT3, ERK1, ERK2 mRNA were measured by RT-PCR. Proliferation of MDA-MB-231 cell was measured with MTT assay. The expression of MMP-9 protein was detected by Western-blot and the secretion of MMP-2 and MMP-9 were tested by gelatin zymography. Adhesion of MDA-MB-231 cells to matrigel was measured with MTT assay. Invasion and migration of MDA-MB-231 cells was investigated with transwell chamber.
Results: In MDA-MB-231 cells the expression level of P-STAT3, P-ERK proteins were reduced obviously to 37.6%, 48.9% respectively and the expression level of STAT3, ERK1 and ERK2 mRNA were decreased to 41.8%, 44.8% and 56.2% respectively after treated with 40μmol/L AG490 for 24 hours compared with untreated group,(P<0.01). The proliferation was inhibited on a time and dose-dependent manner after treated with AG490.The adhesion, invasion and migration of human breast cancer cells were also depressed after treated with 40μmol/L AG490 for 48 hours compared with that of untreated group, (P<0.05).The expression of MMP-9 was reduced to 44.9% and the secretion of MMP-2 , MMP-9 were diminished to 30.6%, 62.2% respectively after treated with 40μmol/L AG490 for 24 hours compared with untreated group, (P<0.01).
Conclusion: Our study demonstrates transcription factor STAT3 and kinase ERK could affect gene expression each other likely by phosphorylation interactions.Cross-talking occurs between JAK/STAT3 and MAPK/ERK signal transduction pathways.The inhibitory effects of JAK kinase on MMPs expression and invasion of breast cancer cells was associated with the down-regulation of these two pathways.
引文
[1] Nakagawa T, Murakami A, Torii M,et al. E-cadherin increases squamous cell carcinoma antigen expression through phosphatidylinositol-3 kinase-Akt pathway in squamous cell carcinoma cell lines[J].Oncol Rep.2007,18(1):175-179.
[2] Liu KD,Gaffen SL,Goldsmith MA.JAK/STAT signaling by cytokine receptors. Curr Opin Immunol.1998,10(3):271-278.
[3] Zhang YH, Wei W, Xu H,et al.Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways[J]. Chin Med J (Engl). 2007, 120(9):743-748.
[4] Huang C,Qiu ZJ, Cao J,et al. Inhibitory effect of AG490 on invasion and metastasis of human pancreatic cancer cells in vitro[J]. Zhonghua Zhong Liu Za Zhi. 2006,28(12):890-893.
[5] Shankar S, Ganapathy S, Hingorani SR,et al.EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer[J]. Front Biosci. 2008,13: 440-452.
[6] Shukla S, Maclennan GT, Hartman DJ,et al. Activation of PI3K-Akt signaling pathway promotes prostate cancer cell invasion[J]. Int J Cancer. 2007, 121(7): 1424-1432.
[7] Inamoto T, Azuma H, Sakamoto T,et al.Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases[J]. Cancer Invest. 2007,25(7):574-583.
[8] Itoh M, Murata T, Suzuki T, et al.Requirement of STAT3 activation for maximal collagenase-1 (MMP-1) induction by epidermal growth factor and malignant characteristics in T24 bladder cancer cells[J].Oncogene.2006,25(8):1195-1204.
[9] Jing N,Tweardy D J.Targeting Stat3 in cancer therapy[J].Anticancer Drugs.2005, 16(6):601-607.
[10] Saxena NK, Sharma D, Ding X,et al.Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells[J]. Cancer Research. 2007, 67(6): 2497-2507.
[11] Neilson LM,Zhu J,Xie J,et al.Coactivation of janus tyrosine kinase (Jak)1 positively modulates prolactin-Jak2 signaling in breast cancer: recruitment of ERK and signal transducer and activator of transcription (Stat)3 and enhancement of Akt and Stat5a/b pathways[J]. Mol Endocrinol. 2007,21(9):2218-2232.
[12] So EY,Oh J,Jang JY,et al.Ras/Erk pathway positively regulates Jak1/STAT6 activity and IL-4 gene expression in Jurkat T cells[J]. Mol Immunol.2007,44(13): 3416-3426.
[13] Galdiero M, Vitiello M, D'Isanto M,et al. STAT1 and STAT3 phosphorylation by porins are independent of JAKs but are dependent on MAPK pathway and plays a role in U937 cells production of interleukin-6[J]. Cytokine.2006,36(5-6):218-228.
[14] Rahaman SO,Harbor PC,Chernova O,et a1.Inhibition of constitutively active STAT3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells[J].Oncogene.2002,21(55):8404-8414.
[15] Calo V,Migiliavacca M,Bazan V,et a1.STAT proteins:from normal control of cellular events to tumorigenesis[J].J Cell Physiol.2003,197(2):157-168.
[16] Byun H J, Hong I K, Kim E,et al.A splice variant of CD99 increases motility and MMP-9 expression of human breast cancer cells through the AKT-, ERK-, and JNK-dependent AP-1 activation signaling pathways[J].J Biol Chem.2006,281(46): 34833-34847.
[17] Dien J, Amin HM, Chiu N,et al.Signal transducers and activators of transcription-3 up-regulates tissue inhibitor of metalloproteinase-1 expression and decreases invasiveness of breast cancer[J]. Am J Pathol.2006,169(2):633-642.
[18] Kunigal S, Lakka SS, Gondi CS,et al.RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth[J]. Int J Cancer. 2007,121(10):2307-2316.
[19] Liotta LA,steeg PS,Stetler-Steveson WG,et a1.Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation[J].Cell,1991, 151(5):5054-5062.
[20] Aaronson DS,Horvath CM.A road map for those who don't know JAK-STAT [J]. Science.2002,296(5573):1653-1655.
[21] Rahaman SO,Harbor PC,Chernova O,et a1.Inhibition of constitutively active STAT3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells[J].Oncogene.2002,21(55):8404-8414.
[22] Calo V,Migiliavacca M,Bazan V,et a1.STAT proteins:from normal control of cellular events to tumorigenesis[J].J Cell Physiol.2003,197(2):157-168.
[23] Bromber JF,Wrzeszczymska MH,Devgan G,et a1.Stat3 as an oncogene[J].Cell. 1999,98(3):295-303.
[24] Yi Z,James T,Christin C,et a1.Activation of STAT3 in v-Src transformed fibroblasts requires cooperation of Jakl kinase activity[J].Biol Chem.2000,275(32):24935.
[25] Haura EB,Turkson J,Jove R.Mechanisms of disease:Insights into the emerging role of signal transducers and activators of transcription in cancer[J].Nat Clin Pratt Oncol.2005,2(6):3l5-324.
[26] Hsieh FC,Cheng G,Lin J.Evaluation of potential Stat3-regulated genes in human breast cancer[J].Biochem Biophys Res Commun.2005,335(2):292-299.
[27] Widmann C,Gibson S, Jarpe MB,et al.Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human[J]. Physiol Rev. 1999,79(1):143-180.
[28] Reddy KB,Nabha SM,Atanaskova N. Role of MAP kinase in tumor progression and invasion[J]. Cancer Metastasis Rev.2003,22(4): 395-403.
[29] Dong C,Davis RJ,Flavell RA.Signalling by the JNK group of MAP Kinases-c-jun N-terminal kinase[J].J Clin Immunol.2001,21(4):253-257.
[30] Obata T, Brown GE, Yaffer MB.MAP kinase pathways activated by stress:the p38MAPK pathways[J].Crit Care Med.2000,28(4):67-77.
[31] Arredondo J, Chernyavsky, AI, Jolkovsky DL,et al.Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of α7 nicotinic receptor in oral keratinocytes[J]. FASEB J. 2006,20: 2093-2101.
[32] Song H, Jin X, Lin J. Stat3 upregulates MEK5 expression in human breast cancer cells[J].Oncogene.2004,23(50):8301-8309.
[33] Bai Jing,Liu Xian-Sheng,Xu Yong-Jian.et al.Extracellular signal-regulated Kinase activation in airway smooth muscle cell proliferation in chronic asthmatic rats[J]. Acta Physiologica Sinica.2007,59(3):311-318.
[34] Wang LH,Robert AK,Rebecca AE,et a1.JAK3,STAT, and MAPK signaling pathways as novel molecular targets for the tyrphostin AG-490 regulation of IL-2-mediated T cell response[J].J Immunol.1999,l62(7):3897-3904.
[35] Lai SY,Childs EE, Xi S,et al. Erythropoietin-mediated activation of JAK-STATsignaling contributes to cellular invasion in head and neck squamous cell carcinoma[J].Oncogene.2005,24(27):4442–4449.
[36] Opdam FJ,Kamp M,de Bruijn R,et al. Jak kinase activity is required for lymphoma invasion and metastasis[J]. Oncogene.2004,23(39):6647–6653.
[37] Huang C,Cao J,Huang KJ.et al.Inhibition of STAT3 activity with AG490 decreases the invasion of human pancreatic cancer cells in vitro[J]. Cancer Sci. 2006,97(12):1417-1423.
[1] Kobel M, Pohl G, Schmitt WD,et al.Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor [J].Am J Patho.2005,167(3):879-885.
[2] Liotta LA,steeg PS,Stetler-Steveson WG.et a1.Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation[J].Cell.1991, 64(2):327-336.
[3] Reddy KB,Nabha SM,Atanaskova N. Role of MAP kinase in tumor progression and invasion[J]. Cancer Metastasis Rev.2003,22(4): 395-403.
[4] Raviv Z,Kalie E,Segar R.MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells,while MEKK2 translocates from the cytosol to the nucleus upon stimulation[J].J Cell Sci.2004,117(Pt 9):1773-1784.
[5] Dong C,Davis RJ,Flavell RA.Signalling by the JNK group of MAP kinases.c-jun N-terminal kinase[J].J Clin Immunol.2001,21(4):253-257.
[6] Obata T,Brown GE,Yaffer MB.MAP kinase pathways activated by stress:the p38MAPK pathways[J].Crit Care Med.2000,28(4 Suppl):N67-77.
[7] 田芳,缪泽鸿,章雄文等.整合蛋白信号转导研究进展[J].生命科学.2005,17(3): 240-245.
[8] Giancotti FG,Ruoslahti E.Integrin signaling[J].Science.1999,285(5430): 1028-1032.
[1] Kobel M, Pohl G, Schmitt WD,et al.Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor [J].Am J Patho.2005,167(3):879-885.
[2] Liotta LA,steeg PS,Stetler-Steveson WG.et a1.Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation[J].Cell.1991, 64(2):327-336.
[3] Reddy KB,Nabha SM,Atanaskova N. Role of MAP kinase in tumor progression and invasion[J]. Cancer Metastasis Rev.2003,22(4): 395-403.
[4] Raviv Z,Kalie E,Segar R.MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells,while MEKK2 translocates from the cytosol to the nucleus upon stimulation[J].J Cell Sci.2004,117(Pt 9):1773-1784.
[5] Dong C,Davis RJ,Flavell RA.Signalling by the JNK group of MAP kinases.c-jun N-terminal kinase[J].J Clin Immunol.2001,21(4):253-257.
[6] Obata T,Brown GE,Yaffer MB.MAP kinase pathways activated by stress:the p38MAPK pathways[J].Crit Care Med.2000,28(4 Suppl):N67-77.
[7] 田芳,缪泽鸿,章雄文等.整合蛋白信号转导研究进展[J].生命科学.2005,17(3): 240-245.
[8] Giancotti FG,Ruoslahti E.Integrin signaling[J].Science.1999,285(5430): 1028-1032.