| 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.
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