神经生长因子β对椎管内神经鞘瘤细胞增殖的影响
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摘要
背景:研究已经证实,在人的良恶性肿瘤中NGF/TrkA信号通路促进肿瘤细胞增殖、分化的效应与PI3K/AKT信号通路密切相关,然而,关于NGF/TrkA信号通路在椎管内神经鞘瘤发病机制的研究甚少。目的:观察神经生长因子β对椎管内神经鞘瘤细胞增殖的影响及初步研究NGF/TrkA信号通路在椎管内神经鞘瘤中的发病机制。方法:收集肿瘤标本并消化培养获得高纯度肿瘤细胞作为实验细胞;分别给予细胞不同浓度的神经生长因子β(15,30,60,120,240μg/L)、K252a(100,200,300,400,500,600 nmol/L)、LY294002(10,20,30,40,50,60μmol/L)、神经生长因子β(120μg/L)+K252a(TrkA受体抑制剂,400 nmol/L)、神经生长因子β(120μg/L)+LY294002(PI3K抑制剂,50μmol/L)作用一定时间;通过MTT法评估细胞增殖情况,Western blot检测TrkA、AKT、p-AKT(Ther308)、p-GSK-3β蛋白质的表达情况,RT-PCR检测TrkA、AKT蛋白质对应的mR NA表达情况。结果与结论:①与对照组比较,神经生长因子β组细胞吸光值(A值)随神经生长因子β的质量浓度增加而升高(P <0.05),于120μg/L时升高幅度最大(P <0.001),K252a、LY294002组细胞A值不断下降(P <0.05),分别于浓度400nmol/L、50μmol/L时下降幅度最大(P<0.001);②神经生长因子β组中TrkA、p-AKT(Ther308)、p-GSK-3β蛋白质的表达上调(P <0.05),TrkA mRNA的表达也上调(P <0.05);③神经生长因子β(120μg/L)+K252a(400 nmol/L)组细胞A值下降(P <0.001),TrkA、p-AKT(Ther308)、p-GSK-3β蛋白质的表达下调(P <0.05),TrkA mR NA的表达亦下调(P <0.05);④神经生长因子β(120μg/L)+LY294002(50μmol/L)组细胞A值下降(P <0.01),p-AKT(Ther308)、p-GSK-3β蛋白质的表达下调(P <0.05);⑤各组中AKT蛋白质及其对应的mR NA的表达无明显变化(P>0.05);⑥结果表明,神经生长因子β能够促进椎管内神经鞘瘤细胞增殖,其增殖可能与NGF/TrkA信号通路中TrkA、p-AKT(Ther308)及p-GSK-3β等蛋白质的表达水平有关。
BACKGROUND: Existing evidence has shown that that the effect of NGF/TrkA signaling pathway on proliferation and differentiation of tumor cells is closely related to PI3 K/AKT signaling pathway in human benign and malignant tumors. However, there is little information on the NGF/TrkA signaling pathway in pathogenesis of intraspinal schwannomas. OBJECTIVE: To investigate the effect of nerve growth factor-beta on the proliferation of interspinal schwannoma cells and to explore on the pathogenesis of NGF/TrkA signaling pathway in interspinal schwannoma. METHODS: Tumor samples were collected and digested to obtain high purity tumor cells as experimental cells. Then the cells were given different concentrations of nerve growth factor-beta(15, 30, 60, 120 and 240 μg/L), K252 a(100, 200, 300, 400, 500 and 600 nmol/L), LY294002(10, 20, 30, 40, 50 and 60 μmol/L), nerve growth factor-beta(120 μg/L) plus K252 a(TrkA inhibitor, 400 nmol/L), and nerve growth factor-beta(120 μg/L) plus LY294002(P13 K inhibitor, 50 μmol/L), respectively, for a certain time. The cell proliferation was detected by MTT assay. TrkA, AKT, p-AKT(Ther308), p-GSK-3 beta protein expression was detected by western blot assay. TrkA and AKT mRNA expression was detected by RT-PCR. RESULTS AND CONCLUSION:(1) Compared with the control group, the absorbance value of cells in the nerve growth factor-beta groups was increased in a concentration-dependent manner(P < 0.05), and increased obviously at the concentration of 120 μg/L(P < 0.001). The absorbance value of cells in the K252 a and LY294002 groups was decreased continuously(P < 0.05), and decreased obviously at the concentration of 400 nmol/L and 50 μmol/L, respectively(P< 0.001).(2) The expression levels of TrkA, p-AKT(Ther308), and p-GSK-3 beta protein were upregulated in the nerve growth factor-beta group(P < 0.05), and the expression level of TrkA mRNA was upregulated(P < 0.05).(3) In the nerve growth factor-beta(120 μg/L) plus K252 a(400 nmol/L) group, the absorbance value of cells decreased(P < 0.001). The expression levels of TrkA, p-AKT(Ther308), and p-GSK-3 beta protein downregulated(P < 0.05), and the expression level of TrkA mRNA downregulated(P < 0.05).(4) In the nerve growth factor-beta(120 μg/L) plus LY294002(50 μmol/L) group, the absorbance value of cells decreased(P < 0.01), and the expression levels of p-AKT(Ther308), and p-GSK-3 beta protein downregulated(P < 0.05).(5)There was no significant change in AKT protein and mRNA in each group(P > 0.05).(6) These results suggest that nerve growth factor-beta can promote interspinal schwannoma cell proliferation, which may be related to the expression of TrkA, p-AKT(Ther308) and p-GSK-3 beta protein in NGF/TrkA signaling pathway.
BACKGROUND: Existing evidence has shown that that the effect of NGF/TrkA signaling pathway on proliferation and differentiation of tumor cells is closely related to PI3 K/AKT signaling pathway in human benign and malignant tumors. However, there is little information on the NGF/TrkA signaling pathway in pathogenesis of intraspinal schwannomas. OBJECTIVE: To investigate the effect of nerve growth factor-beta on the proliferation of interspinal schwannoma cells and to explore on the pathogenesis of NGF/TrkA signaling pathway in interspinal schwannoma. METHODS: Tumor samples were collected and digested to obtain high purity tumor cells as experimental cells. Then the cells were given different concentrations of nerve growth factor-beta(15, 30, 60, 120 and 240 μg/L), K252 a(100, 200, 300, 400, 500 and 600 nmol/L), LY294002(10, 20, 30, 40, 50 and 60 μmol/L), nerve growth factor-beta(120 μg/L) plus K252 a(TrkA inhibitor, 400 nmol/L), and nerve growth factor-beta(120 μg/L) plus LY294002(P13 K inhibitor, 50 μmol/L), respectively, for a certain time. The cell proliferation was detected by MTT assay. TrkA, AKT, p-AKT(Ther308), p-GSK-3 beta protein expression was detected by western blot assay. TrkA and AKT mRNA expression was detected by RT-PCR. RESULTS AND CONCLUSION:(1) Compared with the control group, the absorbance value of cells in the nerve growth factor-beta groups was increased in a concentration-dependent manner(P < 0.05), and increased obviously at the concentration of 120 μg/L(P < 0.001). The absorbance value of cells in the K252 a and LY294002 groups was decreased continuously(P < 0.05), and decreased obviously at the concentration of 400 nmol/L and 50 μmol/L, respectively(P< 0.001).(2) The expression levels of TrkA, p-AKT(Ther308), and p-GSK-3 beta protein were upregulated in the nerve growth factor-beta group(P < 0.05), and the expression level of TrkA mRNA was upregulated(P < 0.05).(3) In the nerve growth factor-beta(120 μg/L) plus K252 a(400 nmol/L) group, the absorbance value of cells decreased(P < 0.001). The expression levels of TrkA, p-AKT(Ther308), and p-GSK-3 beta protein downregulated(P < 0.05), and the expression level of TrkA mRNA downregulated(P < 0.05).(4) In the nerve growth factor-beta(120 μg/L) plus LY294002(50 μmol/L) group, the absorbance value of cells decreased(P < 0.01), and the expression levels of p-AKT(Ther308), and p-GSK-3 beta protein downregulated(P < 0.05).(5)There was no significant change in AKT protein and mRNA in each group(P > 0.05).(6) These results suggest that nerve growth factor-beta can promote interspinal schwannoma cell proliferation, which may be related to the expression of TrkA, p-AKT(Ther308) and p-GSK-3 beta protein in NGF/TrkA signaling pathway.
引文
[1]Sakai Y,Hirose T,Tomono A,et al.Angiosarcoma arising in schwannoma of cerebellopontine angle and later associating with meningioma in a patient with neurofibromatosis type 2.Brain Tumor Pathol.2014;31(4):293-298.
[2]Verma A,Banerjee K,Verma A,et al.Maxillary neurilemmomaRarest of the rare tumour:Report of 2 cases.Int J Surg Case Rep.2013;4(11):1044-1047.
[3]李嘉瑶,戚基萍.459例神经鞘瘤多发部位临床分析[J].中国初级卫生保健,2014,28(01):120-121.
[4]Suchomel P,Jurak L,Antinheimo J,et al.Does sagittal position of the CTDR-related centre of rotation influence functional outcome?Prospective 2-year follow-up analysis.Eur Spine J.2014;23(5):1124-1134.
[5]王献印.颈椎管内肿瘤的外科治疗:濮阳市首届学术年会,中国河南濮阳,2006[C].
[6]黄建华,张明贵,高伟,等.椎管内肿瘤13例诊治分析[J].临床骨科杂志,2000,3(4):271-272.
[7]刘树义,陈实,张开创,等.椎管内神经鞘瘤NF2基因的突变分析[J].中华医学遗传学杂志,2017,34(5):637-641.
[8]Sudhakar D,Nelson L,Herrera L.Chest wall malignant peripheral nerve sheath tumor in a 21-year old female.Asian Cardiovasc Thorac Ann.2015;23(2):227-228.
[9]Jacoby LB,MacCollin M,Louis DN,et al.Exon scanning for mutation of the NF2 gene in schwannomas.Hum Mol Genet.1994;3(3):413-419.
[10]Hondermarck H.Neurotrophins and their receptors in breast cancer.Cytokine Growth Factor Rev.2012;23(6):357-365.
[11]Ruggeri P,Farina AR,Di Ianni N,et al.The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria,within the context of a tumour stem cell-like phenotype.PLoS One.2014;9(4):e94568.
[12]Yu X,Liu Z,Hou R,et al.Nerve growth factor and its receptors on onset and diagnosis of ovarian cancer.Oncol Lett.2017;14(3):2864-2868.
[13]Heinen TE,Dos SR,Da RA,et al.Trk inhibition reduces cell proliferation and potentiates the effects of chemotherapeutic agents in Ewing sarcoma.Oncotarget.2016;7(23):34860-34880.
[14]梁家宏,郑周红,尹朝晖.神经生长因子及受体在结直肠肿瘤中的表达及临床意义[J].当代医学,2013,19(28):1-3.
[15]Zhang KC,Chen S,Lin J,et al.Primary Culture of Human Interspinal Schwannoma.J Biomat Tissue Eng.2016;6(4):263-269.
[16]Levi-Montalcini R.The nerve growth factor:thirty-five years later.Biosci Rep.1987;7(9):681-699.
[17]Aloe L,Chaldakov GN.The multiple life of nerve growth factor:tribute to rita levi-montalcini(1909-2012).Balkan Med J.2013;30(1):4-7.
[18]Yajima I,Kumasaka MY,Thang ND,et al.RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy.Dermatol Res Pract.2012;2012:354191.
[19]Testa JR,Bellacosa A.AKT plays a central role in tumorigenesis.Proc Natl Acad Sci U S A.2001;98(20):10983-10985.
[20]Descamps S,Pawlowski V,Revillion F,et al.Expression of nerve growth factor receptors and their prognostic value in human breast cancer.Cancer Res.2001;61(11):4337-4340.
[21]Rende M,Rambotti MG,Stabile AM,et al.Novel localization of low affinity NGF receptor(p75)in the stroma of prostate cancer and possible implication in neoplastic invasion:an immunohistochemical and ultracytochemical study.Prostate.2010;70(5):555-561.
[22]Vivanco I,Sawyers CL.The phosphatidylinositol 3-Kinase AKTpathway in human cancer.Nat Rev Cancer.2002;2(7):489-501.
[23]Stegeman H,Span PN,Kaanders JH,et al.Improving chemoradiation efficacy by PI3-K/AKT inhibition.Cancer Treat Rev.2014;40(10):1182-1191.
[24]Li B,Cai S,Zhao Y,et al.Nerve growth factor modulates the tumor cells migration in ovarian cancer through the WNT/beta-catenin pathway.Oncotarget.2016;7(49):81026-81048.
[25]高春英,刘金钰,李慧.神经生长因子及其受体TrkA在卵巢癌中的作用[J].中国妇幼保健,2015,30(8):1310-1312.
[26]Meco D,Di Francesco AM,Melotti L,et al.Ectopic nerve growth factor prevents proliferation in glioma cells by senescence induction.J Cell Physiol.2018 Nov 11.
[27]张靖,马虎,韩静,等.ABCG2在非小细胞肺癌厄洛替尼耐药中的作用[J].第三军医大学学报,2014,36(10):987-991.
[28]吴海峰,李猛,高裕,等.PI3K抑制剂LY294002对人膀胱尿路上皮癌细胞增殖与凋亡的影响[J].宁夏医学杂志,2016,38(1):4-6.
[29]黄轶群,马旭东.LY294002对套细胞淋巴瘤增殖、凋亡及Notch1信号通路的影响[J].华中科技大学学报(医学版),2015,44(2):156-159.
[30]耿英华,武文娟,于北凯,等.LY294002靶向抑制PI3K/Akt信号通路干预K562细胞增殖的研究[J].安徽医科大学学报,2014,49(3):295-299.
[31]石雪萍,李静,冉建华,等.人参皂苷Rh2调控PI3K/AKT/GSK-3β信号通路诱导人结肠癌细胞凋亡[J].中国药理学通报,2017,33(1):114-119.
[32]陈普建,熊二梦,张严,等.DAPT与K252a联用体外抑制人脑胶质瘤U251MG细胞的生长[J].江苏医药,2014,40(15):1746-1749.
[2]Verma A,Banerjee K,Verma A,et al.Maxillary neurilemmomaRarest of the rare tumour:Report of 2 cases.Int J Surg Case Rep.2013;4(11):1044-1047.
[3]李嘉瑶,戚基萍.459例神经鞘瘤多发部位临床分析[J].中国初级卫生保健,2014,28(01):120-121.
[4]Suchomel P,Jurak L,Antinheimo J,et al.Does sagittal position of the CTDR-related centre of rotation influence functional outcome?Prospective 2-year follow-up analysis.Eur Spine J.2014;23(5):1124-1134.
[5]王献印.颈椎管内肿瘤的外科治疗:濮阳市首届学术年会,中国河南濮阳,2006[C].
[6]黄建华,张明贵,高伟,等.椎管内肿瘤13例诊治分析[J].临床骨科杂志,2000,3(4):271-272.
[7]刘树义,陈实,张开创,等.椎管内神经鞘瘤NF2基因的突变分析[J].中华医学遗传学杂志,2017,34(5):637-641.
[8]Sudhakar D,Nelson L,Herrera L.Chest wall malignant peripheral nerve sheath tumor in a 21-year old female.Asian Cardiovasc Thorac Ann.2015;23(2):227-228.
[9]Jacoby LB,MacCollin M,Louis DN,et al.Exon scanning for mutation of the NF2 gene in schwannomas.Hum Mol Genet.1994;3(3):413-419.
[10]Hondermarck H.Neurotrophins and their receptors in breast cancer.Cytokine Growth Factor Rev.2012;23(6):357-365.
[11]Ruggeri P,Farina AR,Di Ianni N,et al.The TrkAIII oncoprotein inhibits mitochondrial free radical ROS-induced death of SH-SY5Y neuroblastoma cells by augmenting SOD2 expression and activity at the mitochondria,within the context of a tumour stem cell-like phenotype.PLoS One.2014;9(4):e94568.
[12]Yu X,Liu Z,Hou R,et al.Nerve growth factor and its receptors on onset and diagnosis of ovarian cancer.Oncol Lett.2017;14(3):2864-2868.
[13]Heinen TE,Dos SR,Da RA,et al.Trk inhibition reduces cell proliferation and potentiates the effects of chemotherapeutic agents in Ewing sarcoma.Oncotarget.2016;7(23):34860-34880.
[14]梁家宏,郑周红,尹朝晖.神经生长因子及受体在结直肠肿瘤中的表达及临床意义[J].当代医学,2013,19(28):1-3.
[15]Zhang KC,Chen S,Lin J,et al.Primary Culture of Human Interspinal Schwannoma.J Biomat Tissue Eng.2016;6(4):263-269.
[16]Levi-Montalcini R.The nerve growth factor:thirty-five years later.Biosci Rep.1987;7(9):681-699.
[17]Aloe L,Chaldakov GN.The multiple life of nerve growth factor:tribute to rita levi-montalcini(1909-2012).Balkan Med J.2013;30(1):4-7.
[18]Yajima I,Kumasaka MY,Thang ND,et al.RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy.Dermatol Res Pract.2012;2012:354191.
[19]Testa JR,Bellacosa A.AKT plays a central role in tumorigenesis.Proc Natl Acad Sci U S A.2001;98(20):10983-10985.
[20]Descamps S,Pawlowski V,Revillion F,et al.Expression of nerve growth factor receptors and their prognostic value in human breast cancer.Cancer Res.2001;61(11):4337-4340.
[21]Rende M,Rambotti MG,Stabile AM,et al.Novel localization of low affinity NGF receptor(p75)in the stroma of prostate cancer and possible implication in neoplastic invasion:an immunohistochemical and ultracytochemical study.Prostate.2010;70(5):555-561.
[22]Vivanco I,Sawyers CL.The phosphatidylinositol 3-Kinase AKTpathway in human cancer.Nat Rev Cancer.2002;2(7):489-501.
[23]Stegeman H,Span PN,Kaanders JH,et al.Improving chemoradiation efficacy by PI3-K/AKT inhibition.Cancer Treat Rev.2014;40(10):1182-1191.
[24]Li B,Cai S,Zhao Y,et al.Nerve growth factor modulates the tumor cells migration in ovarian cancer through the WNT/beta-catenin pathway.Oncotarget.2016;7(49):81026-81048.
[25]高春英,刘金钰,李慧.神经生长因子及其受体TrkA在卵巢癌中的作用[J].中国妇幼保健,2015,30(8):1310-1312.
[26]Meco D,Di Francesco AM,Melotti L,et al.Ectopic nerve growth factor prevents proliferation in glioma cells by senescence induction.J Cell Physiol.2018 Nov 11.
[27]张靖,马虎,韩静,等.ABCG2在非小细胞肺癌厄洛替尼耐药中的作用[J].第三军医大学学报,2014,36(10):987-991.
[28]吴海峰,李猛,高裕,等.PI3K抑制剂LY294002对人膀胱尿路上皮癌细胞增殖与凋亡的影响[J].宁夏医学杂志,2016,38(1):4-6.
[29]黄轶群,马旭东.LY294002对套细胞淋巴瘤增殖、凋亡及Notch1信号通路的影响[J].华中科技大学学报(医学版),2015,44(2):156-159.
[30]耿英华,武文娟,于北凯,等.LY294002靶向抑制PI3K/Akt信号通路干预K562细胞增殖的研究[J].安徽医科大学学报,2014,49(3):295-299.
[31]石雪萍,李静,冉建华,等.人参皂苷Rh2调控PI3K/AKT/GSK-3β信号通路诱导人结肠癌细胞凋亡[J].中国药理学通报,2017,33(1):114-119.
[32]陈普建,熊二梦,张严,等.DAPT与K252a联用体外抑制人脑胶质瘤U251MG细胞的生长[J].江苏医药,2014,40(15):1746-1749.
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