p38MAPK信号转导通路在EGF诱导食管腺癌SEG-1细胞表达u-PA中的作用
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摘要
近年来,由Barrett食管(Barrett′s esophagus,BE)转化而来的食管腺癌的发病率明显升高,成为所有恶性肿瘤中增长速度最快的一种。食管腺癌是一种恶性度较高的消化道肿瘤,其侵袭力和转移率较高,许多患者在就诊时已处于肿瘤晚期,5年生存率很低。目前与食管腺癌侵袭转移有关的因子成为当前研究重点之一。
表皮生长因子(epidermal growth factor,EGF)通过其细胞表面的受体(epidermal growth factor receptor,EGFR)即表皮生长因子受体介导发挥多种生物学效应。有研究表明:EGF与受体结合后,通过激发多条信号传导通路,将有丝分裂信号传递到细胞核,引起细胞增殖,同时在细胞的侵袭、转移中起重要作用。另有研究发现EGF及其受体EGFR在食管腺癌组织中表达明显增高,并与肿瘤的发展和预后密切相关。提示EGF可能在刺激食管腺管癌细胞侵袭,促进肿瘤发展过程中发挥重要作用。
肿瘤侵袭是一个多阶段过程,包括细胞增殖、黏附、运动及各种酶的分泌。在这些环节中,细胞外基质和细胞基底膜的降解是肿瘤侵袭过程中的关键所在。研究证实,尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator, u-PA)作用系统被认为在细胞外基质和基底膜的降解、促进肿瘤侵袭转移过程中起核心作用。其他多种恶性肿瘤(包括肺癌、胰腺癌、胃癌、直结肠癌、前列腺癌等)研究中发现u-PA的激活与肿瘤侵袭力有关。现已证实体内多种因子如TGF、IGF、EGF、KGF、IL-1等均能调控u-PA表达,但迄今为止对其表达的分子调控机制研究较少。
EGF与其受体结合后诱导MAPK的磷酸化级联反应,是EGF诱导的重要反应。p38是MAPK家族中的一员,是一组细胞内信号转导分子,p38MAPK上游的MAPKK通过磷酸化p38MAPK的Thr-Gly-Tyr位点中的苏氨酸和酪氨酸使其活化,活化的p38MAPK进一步调控下游多种基因的表达,参与调控炎症、细胞生长、细胞分化,细胞周期及细胞死亡、侵袭表型等。近年来研究发现,p38MAPK信号转导通路参与胃癌、肺癌、淋巴瘤及乳腺癌等细胞u-PA的表达调控。目前尚未见关于p38MAPK信号转导通路与食管腺癌SEG-1细胞侵袭关系的相关报道。
为此,本研究从EGF着手,通过对p38MAPK信号转导通路的特异性阻断,观察阻断前后p38MAPK蛋白、u-PA表达变化,研究p38MAPK信号转导通路在EGF诱导SEG-1细胞表达u-PA中的作用,探讨食管腺癌细胞侵袭机制。
目的:探讨EGF通过p38MAPK信号转导通路诱导SEG-1细胞表达u-PA促进肿瘤侵袭的机制。
方法:采用体外细胞培养技术培养食管腺癌SEG-1细胞,以相同浓度的EGF (100ng/ml)按时间梯度刺激SEG-1细胞,应用Western blot法测定各时间点总p38MAPK蛋白、磷酸化p38MAPK蛋白、u-PA蛋白表达,应用RT-PCR方法检测各时间点u-PA mRNA表达。用p38MAPK特异抑制剂SB203580预处理细胞后,观察上述指标变化。
结果:①EGF对SEG-1细胞p38MAPK蛋白表达的影响:应用Western blot方法检测p38MAPK蛋白表达,结果显示大约在43KD位置出现p-p38MAPK特异性条带,在38KD位置出现t-p38MAPK特异性条带。EGF作用于SEG-1细胞后p38MAPK磷酸化程度有所升高,磷酸化程度随EGF作用时间的变化而变化:1h,6h,12h,24h,48h的p38MAPK磷酸化的百分比分别为11.2%,26.4%,37.6%,55.2%, 41.1%。可见24h的p38MAPK磷酸化水平最高,因此认为该时间点为p38MAPK最高大磷酸化时间点。②EGF对SEG-1细胞u-PA mRNA和蛋白表达的影响:应用RT-PCR方法检测u-PA mRNA表达,结果显示人食管腺癌细胞(SEG-1)中有u-PA基因表达,随着EGF作用时间的延长,u-PA mRNA的表达水平逐渐上升:EGF作用于SEG-1细胞1h后u-PA mRNA表达为:0.265±0.057,与对照组(0.259±0.064)相比,p>0.05,无统计学意义;EGF作用于SEG-1细胞6h后,u-PA mRNA表达为0.306±0.061,与对照组(0.259±0.064)相比,u-PA mRNA表达升高,有统计学意义(p<0.05);EGF作用于SEG-1细胞12h、24h、48h后u-PA mRNA分别表达为:0.642±0.049,0.894±0.045,0.725±0.051,与对照组(0.259±0.064)相比,u-PA mRNA表达明显升高,有统计学意义(p<0.01)。可见本实验中u-PA mRNA表达随EGF作用时间逐渐升高,于24h达高峰,后又逐渐下降。Westem blot方法检测u-PA蛋白表达,结果显示EGF刺激前SEG-1细胞u-PA蛋白表达量较低,刺激后6h开始上升,持续至24h达最高,后又逐渐下降,呈明显的时间依赖性。EGF作用于SEG-1细胞1h后u-PA蛋白表达为0.291±0.063,与对照组(0.288±0.036)比较,p>0.05,无统计学意义。作用6h后u-PA蛋白表达为0.458±0.069与对照组(0.288±0.036)比较,表达升高,p<0.05,有统计学意义。作用12h、24h、48h后,u-PA蛋白表达为0.705±0.056、0.0845±0.049、0.756±0.042,与对照组(0.288±0.036)比较,表达明显升高,p<0.01,有统计学意义。③p38MAPK特异抑制剂SB203580可明显抑制EGF诱导的p38MAPK激酶活力,对EGF诱导的SEG-1细胞内u-PA mRNA和蛋白的表达亦有明显的抑制作用。5μmol/L、10μmol/L、20μmol/L SB203580与EGF同时作用与细胞后p38MAPK磷酸化的百分比分别为36.2%,19.3%,7.1%,明显低于EGF组p38MAPK磷酸化的百分值51.4%。SB203580(5μmol/L)+EGF组、SB203580(10μmol/L)+EGF组u-PA mRNA表达分别为0.718±0.029,0.607±0.047,与EGF组0.853±0.034相比,p<0.05,表达下降,有统计学意义。EGF+ SB203580(20μmol/L)组表达量为0.313±0.034与EGF组0.853±0.034相比,p<0.01,表达明显下降,有统计学意义SB203580(5μmol/L)+EGF组、SB203580(10μmol/L)+EGF组u-PA蛋白表达分别为0.708±0.048,0.654±0.052,与EGF组0.805±0.042相比,p<0.05,表达下降,有统计学意义。SB203580(20μmol/L)+EGF组表达量为0.303±0.045与EGF组0.805±0.042相比,p<0.01,表达明显下降,有统计学意义。
结论:①EGF可引起p38MAPK蛋白磷酸化,在相同浓度EGF(100ng/ml)作用下,p38MAPK蛋白的磷酸化呈时间依赖性,最大磷酸化的作用时间点为24h。②相同浓度EGF(100ng/ml)可引起SEG-1细胞u-PA mRNA和蛋白表达增加,并呈明显的时间依赖性,其表达于24h达高峰。③SB203580可明显抑制SEG-1细胞p38MAPK蛋白的磷酸化;用其阻断p38MAPK信号转导通路后,EGF对u-PA的诱导作用受到明显抑制,并且具有剂量依赖性。
In recent years, esophageal adenocarcinoma progressed from Barrett’s esophagus has an obviously increased incidence rate, which is the highest among malignant tumors. Esophageal adenocarcinoma is a malignant digestive tract tumor with higher invasiveness and metastasis. Most esophageal adenocarcinoma are in late cancer stage when they are found. The five-year survival rate is low. Now, cancer invasion and metastasis related factors have become one focus of recent investigations.
Binding of epidermal growth factor (EGF) to the cell- surface domain of EGFR activates the receptor and its signaling pathways, which results in various biological effects. Recent works showed after EGF binding to its receptor, multiple signal transduction pathways would be activated, and mitogenic signals will be transmitted into cell nucleus to proliferate cell, while may play an important role in cell invasion and metastasis. Other works demonstrated the expression of EGF and its receptor EGFR was significantly increased in esophageal adenocarcinoma, with close relationship with the development and prognosis of esophageal adeno-carcinoma. More and more evidences indicate that EGF may participate in the esophageal adenocarcinoma cells invasion and accelerate the process of the tumor.
The invasion and metastasis of tumor is a very complicated process, including cell adhesion, cell migration, cell proliferation and the secretion of various enzymes. Among these links, the degradation of extra-cellular matrix and basement membrane is the key point. The overexpression of urokinase-type plasminogen activator (u-PA) is detected playing a key role in degradation of extra-cellular matrix and basement membrane and accelerating tumor invasion and metastasis. u-PA might be related to the invasion and metastasis of many malignant cancers including lung cancer, pancreatic carcinoma, gastric carcinoma, rectal carcinoma and prostate carcinoma. It has been confirmed many vivo factors can regulate the expression of u-PA,for example TGF, IGF, EGF, KGF, IL-1 and so on. However , less studies demonstrated the molecular regulation mechanisms for u-PA expression.
The mitogen-activated protein kinases (MAPKs) are serine/ threonine kinases, which generally exist in various cells. MAPKs have been shown to transduce extracellular signals into endocells and be involved in cell proliferation, differentiation and malignant transformation and play important roles in tumor generation and development. p38MAPK is one important member of mitogen-activated protein kinase (MAPK) family. The p38MAPK undergoes phosphorylation at both tyrosine and threonine sites and can be activated by a wide spectrum of stimuli, including inflammatory cytokines, growth factors and cellular stress. p38MAPK signaling pathway has been implicated in cell growth, apoptosis and tumor invasion and metastasis. Recent researches have indicated that p38MAPK signaling pathway participates in regulating u-PA expression of gastric, lung, leukoma and breast cancer cells. To our knowledge, it is the first report about the relation of p38MAPK and esophageal adenocarcinoma cells invasion and metastasis.
Therefore, we set about our research from EGF. By blocking p38MAPK signaling pathway, to study the effect of EGF on the expression of u-PA in esophageal adenocarcinoma cells and detect the role of p38MAPK signaling pathway in this process.
Objective: To study the effect of EGF on the expression of u-PA in esophageal adenocarcinoma cells and detect the role of p38MAPK signaling pathway in this process.
Methods: Esophageal adenocarcinoma cell line SEG-1 was treated with EGF (100ng/ml) based on time gradient, the protein expression of total p38MAPK, p38MAPK phosphory- lation and u-PA were determined by Western blot. The expression of u-PA mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR).The SEG-1 cells were pre-incubated with SB203580 (p38MAPK inhibitor) for two hours, the above mentioned were observed.
Results:①Western blot was used to detect the expression of p38MAPK protein. The result demonstrated phosphorylation of p38MAPK protein specific bands were present at about 43 KD and the total p38MAPK protein specific bands were present at about 38 KD. Phosphorylation of p38MAPK protein in EGF groups was increased than that in control group. The phosphorylation level of p38MAPK protein in SEG-1 cells changed as EGF action time prolonged. The protein expression of phosphorylation at 1, 6, 12, 24 and 48 h were 11.2%, 26.4%, 37.6%, 55.2%, 41.1%. Therefore, 24h is the time point of maximal phosphorylation.②u-PA mRNA expression variation in different groups detected by RT-PCR: u-PA mRNA was expressed in normal SEG-1 cells. The expression of u-PA mRNA changed as EGF action time prolonged. The expression at 1h there was no significance with the control group (0.265±0.057 VS 0.259±0.064, p>0.05); The expression at 6h were higher than that of the control group (0.306±0.061VS 0.259±0.064, p<0.05); The expression at 12, 24 and 48h were higher than that of the control group (0.642±0.049,0.894±0.045,0.725±0.051 VS 0.259±0.064, p<0.01). The u-PA mRNA levels peaked at 24h then decreased. The result of Western blot is in reasonable agreement with RT-PCR: There are lower- expression of u-PA protein in normal SEG-1 cells, the expression begin to increase as EGF action time prolonged and levels peaked at 24h. The expression at 1h was 0.291±0.063 VS 0.288±0.036, p>0.05; The expression at 6h were higher than that of the control group (0.458±0.069 VS 0.259±0.064, p<0.05) ; The expression at 12, 24 and 48h were higher than that of the control group (0.705±0.056, 0.845±0.049, 0.756±0.042 VS 0.259±0.064, p<0.01).③The p38MAPK inhibitor SB203580 could sufficiently suppress EGF-induced p38MAPK phosphorylation and significantly attenuate EGF-induced u-PA mRNA and protein expression in SEG-1 cells. Phosphorylation of p38MAPK protein in SB203580 groups was decreased .The protein expression of phosphorylation in SB203580 groups (5μmol/L, 10μmol/L, 20μmol/L) were lower than EGF group (36.2%,19.3%,7.1% VS 51.4%).u-PA mRNA expression variation in different SB203580 groups detected by RT-PCR: The expression of u-PA mRNA in 5μmol/L, 10μmol/L SB203580 groups were lower than that of the EGF group (0.718±0.029, 0.607±0.047 VS 0.853±0.034, p<0.05);The expre- ssion in 20μmol/L SB203580 group were significently lower than that of the EGF group(0.313±0.034 VS 0.853±0.034 p<0.01). Western blot was used to detect the u-PA protein: The expression of u-PA mRNA in 5μmol/L, 10μmol/L SB203580 groups were lower than that of the EGF group (0.708±0.048, 0.654±0.052 VS 0.805±0.042, p<0.05);The expression in 20μmol/L SB203580 group were significently lower than that of the EGF group (0.303±0.045 VS 0.805±0.042, p<0.01).
Conclusions:①EGF (100ngl/ml) could induce phosphorylation of p38MAPK in esophageal adenocarcinoma SEG-1 cells, The phosphorylation level is in a time-dependent manner.②EGF (100ngl/ml) could promote the expression of u-PA mRNA and u-PA protein in a time-dependent manner in esophageal adenocarcinoma SEG-1 cells.③The inhibitor SB203580 could sufficiently suppress EGF-induced p38MAPK phosphorylation and significantly attenuate EGF-induced u-PA mRNA and protein expressions in SEG-1 cells in a dose- dependent manner.
表皮生长因子(epidermal growth factor,EGF)通过其细胞表面的受体(epidermal growth factor receptor,EGFR)即表皮生长因子受体介导发挥多种生物学效应。有研究表明:EGF与受体结合后,通过激发多条信号传导通路,将有丝分裂信号传递到细胞核,引起细胞增殖,同时在细胞的侵袭、转移中起重要作用。另有研究发现EGF及其受体EGFR在食管腺癌组织中表达明显增高,并与肿瘤的发展和预后密切相关。提示EGF可能在刺激食管腺管癌细胞侵袭,促进肿瘤发展过程中发挥重要作用。
肿瘤侵袭是一个多阶段过程,包括细胞增殖、黏附、运动及各种酶的分泌。在这些环节中,细胞外基质和细胞基底膜的降解是肿瘤侵袭过程中的关键所在。研究证实,尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator, u-PA)作用系统被认为在细胞外基质和基底膜的降解、促进肿瘤侵袭转移过程中起核心作用。其他多种恶性肿瘤(包括肺癌、胰腺癌、胃癌、直结肠癌、前列腺癌等)研究中发现u-PA的激活与肿瘤侵袭力有关。现已证实体内多种因子如TGF、IGF、EGF、KGF、IL-1等均能调控u-PA表达,但迄今为止对其表达的分子调控机制研究较少。
EGF与其受体结合后诱导MAPK的磷酸化级联反应,是EGF诱导的重要反应。p38是MAPK家族中的一员,是一组细胞内信号转导分子,p38MAPK上游的MAPKK通过磷酸化p38MAPK的Thr-Gly-Tyr位点中的苏氨酸和酪氨酸使其活化,活化的p38MAPK进一步调控下游多种基因的表达,参与调控炎症、细胞生长、细胞分化,细胞周期及细胞死亡、侵袭表型等。近年来研究发现,p38MAPK信号转导通路参与胃癌、肺癌、淋巴瘤及乳腺癌等细胞u-PA的表达调控。目前尚未见关于p38MAPK信号转导通路与食管腺癌SEG-1细胞侵袭关系的相关报道。
为此,本研究从EGF着手,通过对p38MAPK信号转导通路的特异性阻断,观察阻断前后p38MAPK蛋白、u-PA表达变化,研究p38MAPK信号转导通路在EGF诱导SEG-1细胞表达u-PA中的作用,探讨食管腺癌细胞侵袭机制。
目的:探讨EGF通过p38MAPK信号转导通路诱导SEG-1细胞表达u-PA促进肿瘤侵袭的机制。
方法:采用体外细胞培养技术培养食管腺癌SEG-1细胞,以相同浓度的EGF (100ng/ml)按时间梯度刺激SEG-1细胞,应用Western blot法测定各时间点总p38MAPK蛋白、磷酸化p38MAPK蛋白、u-PA蛋白表达,应用RT-PCR方法检测各时间点u-PA mRNA表达。用p38MAPK特异抑制剂SB203580预处理细胞后,观察上述指标变化。
结果:①EGF对SEG-1细胞p38MAPK蛋白表达的影响:应用Western blot方法检测p38MAPK蛋白表达,结果显示大约在43KD位置出现p-p38MAPK特异性条带,在38KD位置出现t-p38MAPK特异性条带。EGF作用于SEG-1细胞后p38MAPK磷酸化程度有所升高,磷酸化程度随EGF作用时间的变化而变化:1h,6h,12h,24h,48h的p38MAPK磷酸化的百分比分别为11.2%,26.4%,37.6%,55.2%, 41.1%。可见24h的p38MAPK磷酸化水平最高,因此认为该时间点为p38MAPK最高大磷酸化时间点。②EGF对SEG-1细胞u-PA mRNA和蛋白表达的影响:应用RT-PCR方法检测u-PA mRNA表达,结果显示人食管腺癌细胞(SEG-1)中有u-PA基因表达,随着EGF作用时间的延长,u-PA mRNA的表达水平逐渐上升:EGF作用于SEG-1细胞1h后u-PA mRNA表达为:0.265±0.057,与对照组(0.259±0.064)相比,p>0.05,无统计学意义;EGF作用于SEG-1细胞6h后,u-PA mRNA表达为0.306±0.061,与对照组(0.259±0.064)相比,u-PA mRNA表达升高,有统计学意义(p<0.05);EGF作用于SEG-1细胞12h、24h、48h后u-PA mRNA分别表达为:0.642±0.049,0.894±0.045,0.725±0.051,与对照组(0.259±0.064)相比,u-PA mRNA表达明显升高,有统计学意义(p<0.01)。可见本实验中u-PA mRNA表达随EGF作用时间逐渐升高,于24h达高峰,后又逐渐下降。Westem blot方法检测u-PA蛋白表达,结果显示EGF刺激前SEG-1细胞u-PA蛋白表达量较低,刺激后6h开始上升,持续至24h达最高,后又逐渐下降,呈明显的时间依赖性。EGF作用于SEG-1细胞1h后u-PA蛋白表达为0.291±0.063,与对照组(0.288±0.036)比较,p>0.05,无统计学意义。作用6h后u-PA蛋白表达为0.458±0.069与对照组(0.288±0.036)比较,表达升高,p<0.05,有统计学意义。作用12h、24h、48h后,u-PA蛋白表达为0.705±0.056、0.0845±0.049、0.756±0.042,与对照组(0.288±0.036)比较,表达明显升高,p<0.01,有统计学意义。③p38MAPK特异抑制剂SB203580可明显抑制EGF诱导的p38MAPK激酶活力,对EGF诱导的SEG-1细胞内u-PA mRNA和蛋白的表达亦有明显的抑制作用。5μmol/L、10μmol/L、20μmol/L SB203580与EGF同时作用与细胞后p38MAPK磷酸化的百分比分别为36.2%,19.3%,7.1%,明显低于EGF组p38MAPK磷酸化的百分值51.4%。SB203580(5μmol/L)+EGF组、SB203580(10μmol/L)+EGF组u-PA mRNA表达分别为0.718±0.029,0.607±0.047,与EGF组0.853±0.034相比,p<0.05,表达下降,有统计学意义。EGF+ SB203580(20μmol/L)组表达量为0.313±0.034与EGF组0.853±0.034相比,p<0.01,表达明显下降,有统计学意义SB203580(5μmol/L)+EGF组、SB203580(10μmol/L)+EGF组u-PA蛋白表达分别为0.708±0.048,0.654±0.052,与EGF组0.805±0.042相比,p<0.05,表达下降,有统计学意义。SB203580(20μmol/L)+EGF组表达量为0.303±0.045与EGF组0.805±0.042相比,p<0.01,表达明显下降,有统计学意义。
结论:①EGF可引起p38MAPK蛋白磷酸化,在相同浓度EGF(100ng/ml)作用下,p38MAPK蛋白的磷酸化呈时间依赖性,最大磷酸化的作用时间点为24h。②相同浓度EGF(100ng/ml)可引起SEG-1细胞u-PA mRNA和蛋白表达增加,并呈明显的时间依赖性,其表达于24h达高峰。③SB203580可明显抑制SEG-1细胞p38MAPK蛋白的磷酸化;用其阻断p38MAPK信号转导通路后,EGF对u-PA的诱导作用受到明显抑制,并且具有剂量依赖性。
In recent years, esophageal adenocarcinoma progressed from Barrett’s esophagus has an obviously increased incidence rate, which is the highest among malignant tumors. Esophageal adenocarcinoma is a malignant digestive tract tumor with higher invasiveness and metastasis. Most esophageal adenocarcinoma are in late cancer stage when they are found. The five-year survival rate is low. Now, cancer invasion and metastasis related factors have become one focus of recent investigations.
Binding of epidermal growth factor (EGF) to the cell- surface domain of EGFR activates the receptor and its signaling pathways, which results in various biological effects. Recent works showed after EGF binding to its receptor, multiple signal transduction pathways would be activated, and mitogenic signals will be transmitted into cell nucleus to proliferate cell, while may play an important role in cell invasion and metastasis. Other works demonstrated the expression of EGF and its receptor EGFR was significantly increased in esophageal adenocarcinoma, with close relationship with the development and prognosis of esophageal adeno-carcinoma. More and more evidences indicate that EGF may participate in the esophageal adenocarcinoma cells invasion and accelerate the process of the tumor.
The invasion and metastasis of tumor is a very complicated process, including cell adhesion, cell migration, cell proliferation and the secretion of various enzymes. Among these links, the degradation of extra-cellular matrix and basement membrane is the key point. The overexpression of urokinase-type plasminogen activator (u-PA) is detected playing a key role in degradation of extra-cellular matrix and basement membrane and accelerating tumor invasion and metastasis. u-PA might be related to the invasion and metastasis of many malignant cancers including lung cancer, pancreatic carcinoma, gastric carcinoma, rectal carcinoma and prostate carcinoma. It has been confirmed many vivo factors can regulate the expression of u-PA,for example TGF, IGF, EGF, KGF, IL-1 and so on. However , less studies demonstrated the molecular regulation mechanisms for u-PA expression.
The mitogen-activated protein kinases (MAPKs) are serine/ threonine kinases, which generally exist in various cells. MAPKs have been shown to transduce extracellular signals into endocells and be involved in cell proliferation, differentiation and malignant transformation and play important roles in tumor generation and development. p38MAPK is one important member of mitogen-activated protein kinase (MAPK) family. The p38MAPK undergoes phosphorylation at both tyrosine and threonine sites and can be activated by a wide spectrum of stimuli, including inflammatory cytokines, growth factors and cellular stress. p38MAPK signaling pathway has been implicated in cell growth, apoptosis and tumor invasion and metastasis. Recent researches have indicated that p38MAPK signaling pathway participates in regulating u-PA expression of gastric, lung, leukoma and breast cancer cells. To our knowledge, it is the first report about the relation of p38MAPK and esophageal adenocarcinoma cells invasion and metastasis.
Therefore, we set about our research from EGF. By blocking p38MAPK signaling pathway, to study the effect of EGF on the expression of u-PA in esophageal adenocarcinoma cells and detect the role of p38MAPK signaling pathway in this process.
Objective: To study the effect of EGF on the expression of u-PA in esophageal adenocarcinoma cells and detect the role of p38MAPK signaling pathway in this process.
Methods: Esophageal adenocarcinoma cell line SEG-1 was treated with EGF (100ng/ml) based on time gradient, the protein expression of total p38MAPK, p38MAPK phosphory- lation and u-PA were determined by Western blot. The expression of u-PA mRNA was examined by reverse transcription-polymerase chain reaction (RT-PCR).The SEG-1 cells were pre-incubated with SB203580 (p38MAPK inhibitor) for two hours, the above mentioned were observed.
Results:①Western blot was used to detect the expression of p38MAPK protein. The result demonstrated phosphorylation of p38MAPK protein specific bands were present at about 43 KD and the total p38MAPK protein specific bands were present at about 38 KD. Phosphorylation of p38MAPK protein in EGF groups was increased than that in control group. The phosphorylation level of p38MAPK protein in SEG-1 cells changed as EGF action time prolonged. The protein expression of phosphorylation at 1, 6, 12, 24 and 48 h were 11.2%, 26.4%, 37.6%, 55.2%, 41.1%. Therefore, 24h is the time point of maximal phosphorylation.②u-PA mRNA expression variation in different groups detected by RT-PCR: u-PA mRNA was expressed in normal SEG-1 cells. The expression of u-PA mRNA changed as EGF action time prolonged. The expression at 1h there was no significance with the control group (0.265±0.057 VS 0.259±0.064, p>0.05); The expression at 6h were higher than that of the control group (0.306±0.061VS 0.259±0.064, p<0.05); The expression at 12, 24 and 48h were higher than that of the control group (0.642±0.049,0.894±0.045,0.725±0.051 VS 0.259±0.064, p<0.01). The u-PA mRNA levels peaked at 24h then decreased. The result of Western blot is in reasonable agreement with RT-PCR: There are lower- expression of u-PA protein in normal SEG-1 cells, the expression begin to increase as EGF action time prolonged and levels peaked at 24h. The expression at 1h was 0.291±0.063 VS 0.288±0.036, p>0.05; The expression at 6h were higher than that of the control group (0.458±0.069 VS 0.259±0.064, p<0.05) ; The expression at 12, 24 and 48h were higher than that of the control group (0.705±0.056, 0.845±0.049, 0.756±0.042 VS 0.259±0.064, p<0.01).③The p38MAPK inhibitor SB203580 could sufficiently suppress EGF-induced p38MAPK phosphorylation and significantly attenuate EGF-induced u-PA mRNA and protein expression in SEG-1 cells. Phosphorylation of p38MAPK protein in SB203580 groups was decreased .The protein expression of phosphorylation in SB203580 groups (5μmol/L, 10μmol/L, 20μmol/L) were lower than EGF group (36.2%,19.3%,7.1% VS 51.4%).u-PA mRNA expression variation in different SB203580 groups detected by RT-PCR: The expression of u-PA mRNA in 5μmol/L, 10μmol/L SB203580 groups were lower than that of the EGF group (0.718±0.029, 0.607±0.047 VS 0.853±0.034, p<0.05);The expre- ssion in 20μmol/L SB203580 group were significently lower than that of the EGF group(0.313±0.034 VS 0.853±0.034 p<0.01). Western blot was used to detect the u-PA protein: The expression of u-PA mRNA in 5μmol/L, 10μmol/L SB203580 groups were lower than that of the EGF group (0.708±0.048, 0.654±0.052 VS 0.805±0.042, p<0.05);The expression in 20μmol/L SB203580 group were significently lower than that of the EGF group (0.303±0.045 VS 0.805±0.042, p<0.01).
Conclusions:①EGF (100ngl/ml) could induce phosphorylation of p38MAPK in esophageal adenocarcinoma SEG-1 cells, The phosphorylation level is in a time-dependent manner.②EGF (100ngl/ml) could promote the expression of u-PA mRNA and u-PA protein in a time-dependent manner in esophageal adenocarcinoma SEG-1 cells.③The inhibitor SB203580 could sufficiently suppress EGF-induced p38MAPK phosphorylation and significantly attenuate EGF-induced u-PA mRNA and protein expressions in SEG-1 cells in a dose- dependent manner.
引文
1 Berger DH. Plasmin/plasminogen system in colorectal cancer. World J Surg, 2002,26:767-771
2 Han B, Nakamura M, Mori I, et al. Urokinase-type plasminogen activator system and breast cancer (Review). Oncol Rep, 2005,14:105-112
3 Widmann C, Gibson S, Jarpe MB. Mitogen-activated protein kinase: conservation of a three kinase module from yeast to human. Physiol Rev, 1999,79:143-180
4 Zhang H, Liu XF, Li YJ, et al. [Epidermal growth factor-mediated NF-kappaB activation promotes u-PA expression and invasiveness in pancreatic cancer cells]. Zhonghua Zhong Liu Za Zhi, 2007,29:909-912
5 Yoon JH, Gwak GY, Lee HS, et al. Enhanced epidermal growth factor receptor activation in human cholangiocarcinoma cells. J Hepatol, 2004,41:808-814
6 Mimeault M, Pommery N, Henichart JP. New advances on prostate carcinogenesis and therapies: involvement of EGF-EGFR transduction system. Growth Factors, 2003,21:1-14
7 Harper ME, Goddard L, Glynne-Jones E, et al. Multiple responses to EGF receptor activation and their abrogation by a specific EGF receptor tyrosine kinase inhibitor. Prostate, 2002,52:59-68
8 Tan X, Egami H, Nozawa F, et al. Analysis of the invasion-metastasis mechanism in pancreatic cancer: involvement of plasmin(ogen) cascade proteins in the invasion of pancreatic cancer cells. Int J Oncol, 2006,28:369-374
9 Pappot H, Pedersen AN, Brunner N, et al. The complex between urokinase (u-PA) and its type-1 inhibitor (PAI-1) in pulmonary adenocarcinoma: relation to prognosis. Lung Cancer, 2006,51:193-200
10 Okamoto T, Valacchi G, Gohil K, et al. S-nitrosothiols inhibit cytokine-mediated induction of matrix metalloproteinase-9 in airway epithelial cells. Am J Respir Cell Mol Biol, 2002,27:463-473
11 Schoppmeyer K, Fruhauf N, Oldhafer K, et al. Tumor cell dissemination in colon cancer does not predict extrahepatic recurrence in patients undergoing surgery for hepatic metastases. Oncol Rep, 2006,15:449-454
12 Behren A, Binder K, Vucelic G, et al. The p38 SAPK pathway is required for Ha-ras induced in vitro invasion of NIH3T3 cells. Exp Cell Res, 2005,303:321-330
13 Jo M, Thomas KS, Takimoto S, et al. Urokinase receptor primes cells to proliferate in response to epidermal growthfactor. Oncogene, 2007,26:2585-2594
14 Ke Z, Lin H, Fan Z, et al. MMP-2 mediates ethanol induced invasion of mammary epithelial cells over-expressing ErbB2. Int J Cancer, 2006,119:8-16
15 Yu J, Bian D, Mahanivong C, et al. p38 Mitogen- activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. J Biol Chem, 2004,279:50446-50454
16 Shin BA, Yoo HG, Kim HS, et al. p38MAPK pathway is involved in the urokinase plasminogen activator expression in human gastric SNU-638 cells. Oncol Rep, 2003,10:1467-1471
17 Saika S, Okada Y, Miyamoto T, et al. Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium. Invest Ophthalmol Vis Sci, 2004,45:100-109
18张梅,唐嘉微,李小玫. p38丝裂素活化蛋白激酶信号转录对IL- 1β诱导肾小管细胞转分化的影响.中华医学杂志, 2003,83:1161
19 Philippou A, Maridaki M, Koutsilieris M. The role of urokinase-type plasminogen activator (u-PA) and transforming growth factor beta 1 (TGFbeta1) in muscle regeneration. In Vivo, 2008,22:735-750
20 Estrella VC, Eder AM, Liu S, et al. Lysophosphatidic acid induction of urokinase plasminogen activator secretion requires activation of the p38MAPK pathway. Int J Oncol,2007,31:441-449
1 Schmassmann A, Gebbers JO. [Barrett's esophagus: diagnosis and therapy. Praxis (Bern 1994), 2005, 94: 861-868
2 Rabbani SA, Mazar AP. The role of the plasminogen activation system in angiogenesis and metastasis. Surg Oncol Clin N Am, 2001,10:393-415
3 Shin EY, Ma EK, Kim CK, et al. Src/ERK but not phospholipase D is involved in keratinocyte growth factor-stimulated secretion of matrix metalloprotease-9 and urokinase-type plasminogen activator in SNU-16 human stomach cancer cell. J Cancer Res Clin Oncol, 2002,128:596-602
4 Ploug M, Ellis V. Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett, 1994,349:163- 168
5 Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science,1996,273:1551-1555
6 Ploug M. Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor. Curr Pharm Des, 2003,9:1499-1528
7刘宇,张浩,郭仁宣.表皮生长因子介导的核转录因子-κB活化促进PC细胞尿激酶型纤溶酶原激活物表达及侵袭和转移.世界华人消化杂志, 2007,15:365-369
8 Xie X, Lu J, Kulbokas EJ, et al. Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature, 2005,434:338- 345
9 Milde-Langosch K, Roder H, Andritzky B, et al. The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas. Breast Cancer Res Treat, 2004,86:139-152
10 Kaneko T, Konno H, Baba M, et al. Urokinase-type plasminogen activator expression correlates with tumor angiogenesis and poor outcome in gastric cancer. Cancer Sci, 2003,94:43-49
11 Prager GW, Breuss JM, Steurer S, et al. Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-u-PA) activation on the surface of endothelial cells. Blood, 2004,103:955-962
12 Prager GW, Breuss JM, Steurer S, et al. Vascular endothelial growth factor receptor-2-induced initial endothelial cell migration depends on the presence of theurokinase receptor. Circ Res, 2004,94:1562-1570
13 Festuccia C, Angelucci A, Gravina G, et al. Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement. Exp Cell Res, 2002,280:1-11
14 Goscinski MA, Suo Z, Florenes VA, et al. FAP-alpha and u-PA show different expression patterns in premalignant and malignant esophageal lesions. Ultrastruct Pathol, 2008,32:89-96
15 Lagarde SM, ten Kate FJ, Richel DJ, et al. Molecular prognostic factors in adenocarcinoma of the esophagus and gastroesophageal junction. Ann Surg Oncol, 2007,14:977- 991
2 Han B, Nakamura M, Mori I, et al. Urokinase-type plasminogen activator system and breast cancer (Review). Oncol Rep, 2005,14:105-112
3 Widmann C, Gibson S, Jarpe MB. Mitogen-activated protein kinase: conservation of a three kinase module from yeast to human. Physiol Rev, 1999,79:143-180
4 Zhang H, Liu XF, Li YJ, et al. [Epidermal growth factor-mediated NF-kappaB activation promotes u-PA expression and invasiveness in pancreatic cancer cells]. Zhonghua Zhong Liu Za Zhi, 2007,29:909-912
5 Yoon JH, Gwak GY, Lee HS, et al. Enhanced epidermal growth factor receptor activation in human cholangiocarcinoma cells. J Hepatol, 2004,41:808-814
6 Mimeault M, Pommery N, Henichart JP. New advances on prostate carcinogenesis and therapies: involvement of EGF-EGFR transduction system. Growth Factors, 2003,21:1-14
7 Harper ME, Goddard L, Glynne-Jones E, et al. Multiple responses to EGF receptor activation and their abrogation by a specific EGF receptor tyrosine kinase inhibitor. Prostate, 2002,52:59-68
8 Tan X, Egami H, Nozawa F, et al. Analysis of the invasion-metastasis mechanism in pancreatic cancer: involvement of plasmin(ogen) cascade proteins in the invasion of pancreatic cancer cells. Int J Oncol, 2006,28:369-374
9 Pappot H, Pedersen AN, Brunner N, et al. The complex between urokinase (u-PA) and its type-1 inhibitor (PAI-1) in pulmonary adenocarcinoma: relation to prognosis. Lung Cancer, 2006,51:193-200
10 Okamoto T, Valacchi G, Gohil K, et al. S-nitrosothiols inhibit cytokine-mediated induction of matrix metalloproteinase-9 in airway epithelial cells. Am J Respir Cell Mol Biol, 2002,27:463-473
11 Schoppmeyer K, Fruhauf N, Oldhafer K, et al. Tumor cell dissemination in colon cancer does not predict extrahepatic recurrence in patients undergoing surgery for hepatic metastases. Oncol Rep, 2006,15:449-454
12 Behren A, Binder K, Vucelic G, et al. The p38 SAPK pathway is required for Ha-ras induced in vitro invasion of NIH3T3 cells. Exp Cell Res, 2005,303:321-330
13 Jo M, Thomas KS, Takimoto S, et al. Urokinase receptor primes cells to proliferate in response to epidermal growthfactor. Oncogene, 2007,26:2585-2594
14 Ke Z, Lin H, Fan Z, et al. MMP-2 mediates ethanol induced invasion of mammary epithelial cells over-expressing ErbB2. Int J Cancer, 2006,119:8-16
15 Yu J, Bian D, Mahanivong C, et al. p38 Mitogen- activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. J Biol Chem, 2004,279:50446-50454
16 Shin BA, Yoo HG, Kim HS, et al. p38MAPK pathway is involved in the urokinase plasminogen activator expression in human gastric SNU-638 cells. Oncol Rep, 2003,10:1467-1471
17 Saika S, Okada Y, Miyamoto T, et al. Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium. Invest Ophthalmol Vis Sci, 2004,45:100-109
18张梅,唐嘉微,李小玫. p38丝裂素活化蛋白激酶信号转录对IL- 1β诱导肾小管细胞转分化的影响.中华医学杂志, 2003,83:1161
19 Philippou A, Maridaki M, Koutsilieris M. The role of urokinase-type plasminogen activator (u-PA) and transforming growth factor beta 1 (TGFbeta1) in muscle regeneration. In Vivo, 2008,22:735-750
20 Estrella VC, Eder AM, Liu S, et al. Lysophosphatidic acid induction of urokinase plasminogen activator secretion requires activation of the p38MAPK pathway. Int J Oncol,2007,31:441-449
1 Schmassmann A, Gebbers JO. [Barrett's esophagus: diagnosis and therapy. Praxis (Bern 1994), 2005, 94: 861-868
2 Rabbani SA, Mazar AP. The role of the plasminogen activation system in angiogenesis and metastasis. Surg Oncol Clin N Am, 2001,10:393-415
3 Shin EY, Ma EK, Kim CK, et al. Src/ERK but not phospholipase D is involved in keratinocyte growth factor-stimulated secretion of matrix metalloprotease-9 and urokinase-type plasminogen activator in SNU-16 human stomach cancer cell. J Cancer Res Clin Oncol, 2002,128:596-602
4 Ploug M, Ellis V. Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett, 1994,349:163- 168
5 Wei Y, Lukashev M, Simon DI, et al. Regulation of integrin function by the urokinase receptor. Science,1996,273:1551-1555
6 Ploug M. Structure-function relationships in the interaction between the urokinase-type plasminogen activator and its receptor. Curr Pharm Des, 2003,9:1499-1528
7刘宇,张浩,郭仁宣.表皮生长因子介导的核转录因子-κB活化促进PC细胞尿激酶型纤溶酶原激活物表达及侵袭和转移.世界华人消化杂志, 2007,15:365-369
8 Xie X, Lu J, Kulbokas EJ, et al. Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature, 2005,434:338- 345
9 Milde-Langosch K, Roder H, Andritzky B, et al. The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas. Breast Cancer Res Treat, 2004,86:139-152
10 Kaneko T, Konno H, Baba M, et al. Urokinase-type plasminogen activator expression correlates with tumor angiogenesis and poor outcome in gastric cancer. Cancer Sci, 2003,94:43-49
11 Prager GW, Breuss JM, Steurer S, et al. Vascular endothelial growth factor (VEGF) induces rapid prourokinase (pro-u-PA) activation on the surface of endothelial cells. Blood, 2004,103:955-962
12 Prager GW, Breuss JM, Steurer S, et al. Vascular endothelial growth factor receptor-2-induced initial endothelial cell migration depends on the presence of theurokinase receptor. Circ Res, 2004,94:1562-1570
13 Festuccia C, Angelucci A, Gravina G, et al. Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement. Exp Cell Res, 2002,280:1-11
14 Goscinski MA, Suo Z, Florenes VA, et al. FAP-alpha and u-PA show different expression patterns in premalignant and malignant esophageal lesions. Ultrastruct Pathol, 2008,32:89-96
15 Lagarde SM, ten Kate FJ, Richel DJ, et al. Molecular prognostic factors in adenocarcinoma of the esophagus and gastroesophageal junction. Ann Surg Oncol, 2007,14:977- 991