乳腺癌细胞NGF信号通路抑制性多肽适配体筛选
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摘要
目的研究乳腺癌MCF-7细胞中神经生长因子(nerve growth factor,NGF)信号通路,筛选抑制乳腺癌细胞增殖的多肽适配体,从而探索治疗乳腺癌的潜在靶点。
方法运用免疫荧光与蛋白质印迹(western blotting)检测NGF及其高亲和力受体TrkA(酪氨酸激酶受体A)在MCF-7细胞中的表达;运用细胞计数方法(cellcounting kit-8,CCK-8)检测不同浓度的NGF阻断剂Ro 08-2750和酪氨酸酶阻断剂K252a对细胞增殖和存活的影响;运用western blotting检测不同浓度Ro 08-2750处理后的细胞表达活性促分裂素原活化蛋白激酶(active-mitogen-activated proteinkinases,active-MAPK)与p53蛋白变化情况;运用流式细胞仪检测不同浓度Ro08-2750处理后的细胞凋亡情况以及细胞周期分布的变化;运用多肽适配体技术将5种特异性结合Ras蛋白不同位点的多肽适配体转染MCF-7细胞,并运用CCK-8法检测MCF-7细胞转染后的增殖情况,运用western blotting检测表达active-MAPK蛋白与p53蛋白的变化,以及运用流式细胞仪检测细胞凋亡情况。
结果实验结果显示MCF-7细胞同时表达NGF及其受体TrkA蛋白;NGF阻断剂Ro 08-2750能够明显地抑制细胞增殖与存活,并具有剂量效应;经不同浓度Ro08-2750处理细胞后p-ERK 1/2表达量上调,p53表达下调,细胞出现凋亡峰,并出现S期细胞含量上升,62期细胞含量下降:转染的5种多肽适配体中,pPER39R能明显抑制细胞的增殖和存活,p53的表达上调,并出现显著的凋亡峰,然而pPER39R对正常背根神经节细胞DRG(dorsal root ganglion)细胞没有任何抑制作用。
结论乳腺癌MCF-7细胞中存在NGF信号通路,当使用特异性的NGF阻断剂Ro 08-2750处理,MCF-7细胞的增殖和存活受到明显抑制,出现S期停滞与细胞凋亡;通过对结合Ras的的多肽适配体的筛选,pPER39R可以明显抑制MCF-7细胞的增殖与存活且引起细胞的凋亡,这个结果可能与引起p53上调有关。并且pPER39R对存在正常NGF信号通路的DRG细胞没有任何抑制效应。提示pPER 39R具有突变Ras的抑制特异性,具有进一步深入研究的较大价值。
Objective:To investigate the nerve growth factor signaling pathway in human breast cancer MCF-7 cells,to select the breast cancer cell proliferation inhibitory peptide aptamers and to find the potential therapeutic target for breadst cancer.
Methods:Immunofluorescence and western blotting method to detect NGF and its receptor TrkA protein in MCF-7 cells;The use of cell counting kit-8(CCK-8) to detect the effect of different concentrations of NGF antagonist Ro 08-2750 and K252a on cell proliferation and survival;Western blotting to detect the changes of active-MAPK and p53 protein in MCF-7 cells being treated with different concentrations of Ro 08-2750;the use of flow cytometry to detect the apotosis as well as changes in cell cycle distribution of the cells when being treated with different concentrations of Ro 08-2750 for 24h;the transfection technology was used to study the action of 5 specific Ras binding peptide aptamers on MCF-7 cells,and to detect the proliferation,survival of MCF-7 cells 24h after transfection,as well as active-MAPK and p53 proteins expression and apotosis of MCF-7 cells.
Results:Immunofluorescence results showed MCF-7 cells express NGF and its receptor TrkA protein;cell counting kit(CCK-8) showed that Ro 08-2750 can inhibit cell proliferation and survival with the dosage,Western blot showed that the expression of active-MAPK increase while the expression of p53 decreased with the dosage;Flow cytometry results suggest that Ro 08-2750 treated cells appeared apoptotic peak,the content of S-phase cells increased,and G2 phase cells decreased;Fluorescence microscopy showed that transfection efficiency was above 70%in MCF-7 cells,Cell counting kit(CCK-8) showed that cell proliferation and survival after being transfected with pPER39R decreased significantly;Peptide aptamer pPER39R can induce the expression of p53 and significant cell apoptosis;pPER39R have no effect on normal DRG cells.
Conclusion:Nerve growth factor signaling pathway exists in human breast cancer cells MCF-7.NGF-specific antagonist Ro 08-2750 can inhibit the growth and survival of breast cancer cell in vitro,and induce apoptosis which might be associated with reduced expression of p53 and increased expression of p-ERK 1/2 protein.Aptamer pPER39R can significantly inhibit the proliferation and survival of MCF-7 cells.Aptamer pPER39R can induce apoptosis of MCF-7 cells.This result may be caused by the increase of the p53.And pPER 39R had no inhibitory effect on normal NGF signaling pathway of DRG cells.It is suggested that pPER 39R has mutation-specific inhibition of Ras and has a larger value in further study.
方法运用免疫荧光与蛋白质印迹(western blotting)检测NGF及其高亲和力受体TrkA(酪氨酸激酶受体A)在MCF-7细胞中的表达;运用细胞计数方法(cellcounting kit-8,CCK-8)检测不同浓度的NGF阻断剂Ro 08-2750和酪氨酸酶阻断剂K252a对细胞增殖和存活的影响;运用western blotting检测不同浓度Ro 08-2750处理后的细胞表达活性促分裂素原活化蛋白激酶(active-mitogen-activated proteinkinases,active-MAPK)与p53蛋白变化情况;运用流式细胞仪检测不同浓度Ro08-2750处理后的细胞凋亡情况以及细胞周期分布的变化;运用多肽适配体技术将5种特异性结合Ras蛋白不同位点的多肽适配体转染MCF-7细胞,并运用CCK-8法检测MCF-7细胞转染后的增殖情况,运用western blotting检测表达active-MAPK蛋白与p53蛋白的变化,以及运用流式细胞仪检测细胞凋亡情况。
结果实验结果显示MCF-7细胞同时表达NGF及其受体TrkA蛋白;NGF阻断剂Ro 08-2750能够明显地抑制细胞增殖与存活,并具有剂量效应;经不同浓度Ro08-2750处理细胞后p-ERK 1/2表达量上调,p53表达下调,细胞出现凋亡峰,并出现S期细胞含量上升,62期细胞含量下降:转染的5种多肽适配体中,pPER39R能明显抑制细胞的增殖和存活,p53的表达上调,并出现显著的凋亡峰,然而pPER39R对正常背根神经节细胞DRG(dorsal root ganglion)细胞没有任何抑制作用。
结论乳腺癌MCF-7细胞中存在NGF信号通路,当使用特异性的NGF阻断剂Ro 08-2750处理,MCF-7细胞的增殖和存活受到明显抑制,出现S期停滞与细胞凋亡;通过对结合Ras的的多肽适配体的筛选,pPER39R可以明显抑制MCF-7细胞的增殖与存活且引起细胞的凋亡,这个结果可能与引起p53上调有关。并且pPER39R对存在正常NGF信号通路的DRG细胞没有任何抑制效应。提示pPER 39R具有突变Ras的抑制特异性,具有进一步深入研究的较大价值。
Objective:To investigate the nerve growth factor signaling pathway in human breast cancer MCF-7 cells,to select the breast cancer cell proliferation inhibitory peptide aptamers and to find the potential therapeutic target for breadst cancer.
Methods:Immunofluorescence and western blotting method to detect NGF and its receptor TrkA protein in MCF-7 cells;The use of cell counting kit-8(CCK-8) to detect the effect of different concentrations of NGF antagonist Ro 08-2750 and K252a on cell proliferation and survival;Western blotting to detect the changes of active-MAPK and p53 protein in MCF-7 cells being treated with different concentrations of Ro 08-2750;the use of flow cytometry to detect the apotosis as well as changes in cell cycle distribution of the cells when being treated with different concentrations of Ro 08-2750 for 24h;the transfection technology was used to study the action of 5 specific Ras binding peptide aptamers on MCF-7 cells,and to detect the proliferation,survival of MCF-7 cells 24h after transfection,as well as active-MAPK and p53 proteins expression and apotosis of MCF-7 cells.
Results:Immunofluorescence results showed MCF-7 cells express NGF and its receptor TrkA protein;cell counting kit(CCK-8) showed that Ro 08-2750 can inhibit cell proliferation and survival with the dosage,Western blot showed that the expression of active-MAPK increase while the expression of p53 decreased with the dosage;Flow cytometry results suggest that Ro 08-2750 treated cells appeared apoptotic peak,the content of S-phase cells increased,and G2 phase cells decreased;Fluorescence microscopy showed that transfection efficiency was above 70%in MCF-7 cells,Cell counting kit(CCK-8) showed that cell proliferation and survival after being transfected with pPER39R decreased significantly;Peptide aptamer pPER39R can induce the expression of p53 and significant cell apoptosis;pPER39R have no effect on normal DRG cells.
Conclusion:Nerve growth factor signaling pathway exists in human breast cancer cells MCF-7.NGF-specific antagonist Ro 08-2750 can inhibit the growth and survival of breast cancer cell in vitro,and induce apoptosis which might be associated with reduced expression of p53 and increased expression of p-ERK 1/2 protein.Aptamer pPER39R can significantly inhibit the proliferation and survival of MCF-7 cells.Aptamer pPER39R can induce apoptosis of MCF-7 cells.This result may be caused by the increase of the p53.And pPER 39R had no inhibitory effect on normal NGF signaling pathway of DRG cells.It is suggested that pPER 39R has mutation-specific inhibition of Ras and has a larger value in further study.
引文
郝晶,高英茂,刘凯,史桂芝,等.NGF的研究进展[J].解剖科学进展,1999,5(3):203-207
厉元红,吴凯南,刘胜春.血管生成对乳腺癌的预后价值与P53表达的关系[J].中国普通外科杂志,2003,12(5):341-343
李东亮,张朝.基础神经生物学[M].北京:人民军医出版社,2006:332-336
李书芹,季艳霞,等.沙立度胺抑制乳腺癌细胞增殖的作用机制[J].肿瘤防治研究,2008,35(8):560-562
卿素珠,徐永平,张涌.山羊胎儿脑室脉络丛发育中NGF及其受体TrKA的表达[J].畜牧兽医学报,2005,36(3):250-254
阮芳铭,高文元,路长林.神经生长因子家族在外周听觉神经系统中的作用研究进展[J].海军医学杂志,1999,20(1):84-86
孙剑瑞,杨波,杜英,等.神经生长因子对神经干细胞增殖的影响[J].郑州大学学报,2006,41(2):241-244
汪进富,安景华.神经生长因子及其临床应用[J].航空军医,1995,23(1):50-52
王深明,王劲松.乳腺癌基因治疗现状,中华医学会第十五次全国外科学学术会议论文汇编:605-607
吴玉臣,王艳玲,等.神经生长因子的研究进展[J].甘肃畜牧兽医,2004(3):33-37
熊亮,林观平,周克元,等.基因治疗乳腺癌的研究进展[J].广东医学院学报,2006,24(3):314-316
严开平.生长因子及其受体介导的RAS信号系统研究进展[J].国外医学生理、病理科学与临床分册,1995,15(4):221-223
赵丹丹,朱中元,张春发.乳腺癌靶向治疗药物研究进展[J].中国热带医学,2007,7(4):620-622
周雪瑞,黄选东,等.乳腺与乳腺癌[J].生物学通报,2004,39(7):26-27
张万福,帅晋豪,罗开元.神经生长因子及受体对乳腺癌作用机制的研究进展[J].中国现代普通外科进展,2009,2(12):48-50
Anton,E.S.,Weskamp,G.,Reichardt,L.F.,and Matthew,W.Barbacid M:Neurotrophic factors and their receptors.Curr Opin Cell Biol.1995,7:148-155.
Bucker ED.Implantation of tumors in the hind limb field of the embryonic chick and the developmental response of the lumbosacral nervors system.Anat Rec, 1948,102:369-390
Davidson B, Reich R, Lazarocivi P, et al.Altered expression and activation of the nerve growth factor receptors TrkA and p75 provide the first evidence of tumor progression to effusion in breast carcinoma.Breast cancer Res Treat, 2004, 83(2): 119-282
Deseamps S,Lebonrhis X,DeleheddeMetal .Nerve growth factor is Mitogenic for cancerous but not normal human breast epithelial cells.Biol Chem, 1998,273(27): 16659-16662
Deseamps S,PawlowskiV, Renllion Fetal. Expression of nerve growth factor receptors and their pregnosticvalue in human breast cancer . CancerRes, 2001,61(11):4337-4340
Dolle L, Adriaenssens E, El Yazidi-Belkoura I, et al.Nerve growth factor receptors and signaling in breast cancer.Curr Cancer Drug Targets, 2004,4(6):463-470
Dolle L, Yazidi-Belkoura IE, Adriaenssens E, et al. Never Growth Factor overexpression and autocrine loop in breast cancer cells.Oneogene, 2003, 36:5592-5601
Di Marco, E., Mathor, M., Bondanza, S., Cutuli, N., Marchisio, P. C.,Cancedda, R., and De Luca, M.,1993
Ehrhard, P. B., Ganter, U., Stalder, A., Bauer, J., and Otten, U., 1993
Ethier SP. J. Natl. Cancer Inst, 1995, 87:964-973.
Eric Adriaenssens etal.Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer.Cancer Res 2008; 68(2):346-51.
Friedman, W. J., and Greene, L. A. Exp. Cell Res. 1999,253:131-142
Koh S,Higgins GA.Differential regulation of the low-affinity NGFR during postnatal development the rat brain. J Comp Neurol, 1991, 31 (3):494
Kaplan DR,Martin-Zanca D,Parada LF.Tyrosine phosphorylation and tyrosine kinase activity of the Trk protooncogene product induced by NGF.Nature,1991,350:158-160
Laurent Dolle et al. Nerve growth factor overexpression and autocrine loop in breast cancer cells.Nature, 2003(22), 5592-5601
Lee A, Christenson LK, Patton PE, Burry KA, Stouffer RL. Vascular endothelial growth factor production by human luteinized granulosa cells in vitro.Hum Reprod, 1997,12(27):56-61
Lehner R,Enomoto T,McGregor JA,et al.Correlation of survivin mRNA detection with histologic diagnosisi in normal endometrium and endometrial carcinoma.Acta Obstet Gynecol Scand ,2002,81(2):162-167
Levi-Montacini R. The nerve growth factor 35 years later.Science, 1987,237:1154
Levi-Montacini R. Hamburger V.Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetiv nerous system of the chick emcryo.J Exp Zool, 1951,116:321-362
Lewin, G. R., and Barde, Y. A. Ann. Rev. Neurosci. 1996,19: 289-317
Lillien, L. E, and Claude, P. Nature, 1985, 317:632-634
Montero S, Guzman C, Vargas C, et al .Prognostic value of cytosolic P53 protein in breast cancer.Tumor Biol, 2001, 22(5):337
Mulcahy LS et al,Nature,1985,313:241-24
Neet KE, Campenot RB.Receptor binding, internalization, and retrograde transport of neurotrophic factors.Cell Mol Life Sci, 2001, 58(8):1021-1035
Norberg T,Klaar S,Karf G,et al.Increased P53 mutation frequency during tumor progpression,results from a breast cancer cohort.Cancer Ros, 2001,61 (22):8317-8321
Proc.Natl. Acad. Sci. U. S. A. 91:2795-2799
Proc.Natl. Acad. Sci. U. S. A. 90:5423-5427
Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res, 2001, May 1,29(9): e45
Otten, U., Ehrhard, P., and Peck, R. Proc. Natl. Acad. Sci. U. S. A. 1989,86:10059-10063.
O. Niederhauser et al.,NGF Ligand Alters NGF Signaling Via p75NTR and TrkA,Journal of Neuroscience Research 61,2000:263-272
Simpson P, Mcgrath A, Savion S. Myocyte hypertrophy inneonatal rat heart cultures and its regulation by serum andcatecholamines.Circ Res, 1982, 51:787-801
Simon Descampuse et al.Nerve Growth Factor Stimulates Proliferation and Survival of Human Breast Cancer Cells through Two Distinct Signaling Pathways.The journal of bioloicalchemistry, 2001 , 276 (21): 17864-17870.
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Tapley,P.,Larnballe,F.,and Barbacid,M.Oncogene 7:371-381
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Zhu Z,Kleeff J,Kayed H,et al.Nerve growth factor and enhancement of proliferation,invasion,and tumor igenicitv of pancreatic cancer cells.Mol Carcinog,2002,35(3):138-147
厉元红,吴凯南,刘胜春.血管生成对乳腺癌的预后价值与P53表达的关系[J].中国普通外科杂志,2003,12(5):341-343
李东亮,张朝.基础神经生物学[M].北京:人民军医出版社,2006:332-336
李书芹,季艳霞,等.沙立度胺抑制乳腺癌细胞增殖的作用机制[J].肿瘤防治研究,2008,35(8):560-562
卿素珠,徐永平,张涌.山羊胎儿脑室脉络丛发育中NGF及其受体TrKA的表达[J].畜牧兽医学报,2005,36(3):250-254
阮芳铭,高文元,路长林.神经生长因子家族在外周听觉神经系统中的作用研究进展[J].海军医学杂志,1999,20(1):84-86
孙剑瑞,杨波,杜英,等.神经生长因子对神经干细胞增殖的影响[J].郑州大学学报,2006,41(2):241-244
汪进富,安景华.神经生长因子及其临床应用[J].航空军医,1995,23(1):50-52
王深明,王劲松.乳腺癌基因治疗现状,中华医学会第十五次全国外科学学术会议论文汇编:605-607
吴玉臣,王艳玲,等.神经生长因子的研究进展[J].甘肃畜牧兽医,2004(3):33-37
熊亮,林观平,周克元,等.基因治疗乳腺癌的研究进展[J].广东医学院学报,2006,24(3):314-316
严开平.生长因子及其受体介导的RAS信号系统研究进展[J].国外医学生理、病理科学与临床分册,1995,15(4):221-223
赵丹丹,朱中元,张春发.乳腺癌靶向治疗药物研究进展[J].中国热带医学,2007,7(4):620-622
周雪瑞,黄选东,等.乳腺与乳腺癌[J].生物学通报,2004,39(7):26-27
张万福,帅晋豪,罗开元.神经生长因子及受体对乳腺癌作用机制的研究进展[J].中国现代普通外科进展,2009,2(12):48-50
Anton,E.S.,Weskamp,G.,Reichardt,L.F.,and Matthew,W.Barbacid M:Neurotrophic factors and their receptors.Curr Opin Cell Biol.1995,7:148-155.
Bucker ED.Implantation of tumors in the hind limb field of the embryonic chick and the developmental response of the lumbosacral nervors system.Anat Rec, 1948,102:369-390
Davidson B, Reich R, Lazarocivi P, et al.Altered expression and activation of the nerve growth factor receptors TrkA and p75 provide the first evidence of tumor progression to effusion in breast carcinoma.Breast cancer Res Treat, 2004, 83(2): 119-282
Deseamps S,Lebonrhis X,DeleheddeMetal .Nerve growth factor is Mitogenic for cancerous but not normal human breast epithelial cells.Biol Chem, 1998,273(27): 16659-16662
Deseamps S,PawlowskiV, Renllion Fetal. Expression of nerve growth factor receptors and their pregnosticvalue in human breast cancer . CancerRes, 2001,61(11):4337-4340
Dolle L, Adriaenssens E, El Yazidi-Belkoura I, et al.Nerve growth factor receptors and signaling in breast cancer.Curr Cancer Drug Targets, 2004,4(6):463-470
Dolle L, Yazidi-Belkoura IE, Adriaenssens E, et al. Never Growth Factor overexpression and autocrine loop in breast cancer cells.Oneogene, 2003, 36:5592-5601
Di Marco, E., Mathor, M., Bondanza, S., Cutuli, N., Marchisio, P. C.,Cancedda, R., and De Luca, M.,1993
Ehrhard, P. B., Ganter, U., Stalder, A., Bauer, J., and Otten, U., 1993
Ethier SP. J. Natl. Cancer Inst, 1995, 87:964-973.
Eric Adriaenssens etal.Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer.Cancer Res 2008; 68(2):346-51.
Friedman, W. J., and Greene, L. A. Exp. Cell Res. 1999,253:131-142
Koh S,Higgins GA.Differential regulation of the low-affinity NGFR during postnatal development the rat brain. J Comp Neurol, 1991, 31 (3):494
Kaplan DR,Martin-Zanca D,Parada LF.Tyrosine phosphorylation and tyrosine kinase activity of the Trk protooncogene product induced by NGF.Nature,1991,350:158-160
Laurent Dolle et al. Nerve growth factor overexpression and autocrine loop in breast cancer cells.Nature, 2003(22), 5592-5601
Lee A, Christenson LK, Patton PE, Burry KA, Stouffer RL. Vascular endothelial growth factor production by human luteinized granulosa cells in vitro.Hum Reprod, 1997,12(27):56-61
Lehner R,Enomoto T,McGregor JA,et al.Correlation of survivin mRNA detection with histologic diagnosisi in normal endometrium and endometrial carcinoma.Acta Obstet Gynecol Scand ,2002,81(2):162-167
Levi-Montacini R. The nerve growth factor 35 years later.Science, 1987,237:1154
Levi-Montacini R. Hamburger V.Selective growth stimulating effects of mouse sarcoma on the sensory and sympathetiv nerous system of the chick emcryo.J Exp Zool, 1951,116:321-362
Lewin, G. R., and Barde, Y. A. Ann. Rev. Neurosci. 1996,19: 289-317
Lillien, L. E, and Claude, P. Nature, 1985, 317:632-634
Montero S, Guzman C, Vargas C, et al .Prognostic value of cytosolic P53 protein in breast cancer.Tumor Biol, 2001, 22(5):337
Mulcahy LS et al,Nature,1985,313:241-24
Neet KE, Campenot RB.Receptor binding, internalization, and retrograde transport of neurotrophic factors.Cell Mol Life Sci, 2001, 58(8):1021-1035
Norberg T,Klaar S,Karf G,et al.Increased P53 mutation frequency during tumor progpression,results from a breast cancer cohort.Cancer Ros, 2001,61 (22):8317-8321
Proc.Natl. Acad. Sci. U. S. A. 91:2795-2799
Proc.Natl. Acad. Sci. U. S. A. 90:5423-5427
Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res, 2001, May 1,29(9): e45
Otten, U., Ehrhard, P., and Peck, R. Proc. Natl. Acad. Sci. U. S. A. 1989,86:10059-10063.
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