The Actions And Its Mechanism Of CXCR4in Invasion And Metastasis Of Esophageal Squamous Carcinoma

Objective:Esophageal cancer is one of10kind of most common malignant tumor in the world and more than300000new patients suffer from it every year, which has become one of the fastest rising incidence rates of malignant tumors as well as a serious disease,China has the highest incidence rates of esophageal cancer in the world,211thousand people with esophageal cancer died in2008which were more than half of the world, and among them87%of the pathological types were esophageal squamous carcinoma. It shows that the occurrence&development of esophageal cancer are not a single molecular event, but are a complex biological processes with multi-step, multi-stage, multi-gene involved and long-time duration, and It has mostly passed through a procedure of epithelial hyperplasia, epithelial neoplasia, invasive carcinoma,as well as related to many factors, such as oncogenes&antioncogenes, cell signal transduction pathway changing, cell proliferation regulation disorders,et al. Meanwhile, there are evidences that environmental exposure, tumor site, tumor size, degree of tumor invasion, lymph node metastasis and other factors have certain relationship between the occurrence&development of esophageal cancer, which even be important to the early diagnosis, prognosis, treatment and prevention from esophageal cancer, as well as provide a scientific theoretical basis and a new way for the diagnosis&therapy of esophageal cancer. After all,esophageal cancer has the highly aggressive and transfer tendency biological characteristics as well as poor overall treatment effects, which long-term survival rate has not been improved significantly, postoperative recurrence and metastasis are the main causes of treatment failure. How to control the recurrence&metastasis of the tumor has become the key to heal esophageal carcinoma, So studying the mechanism of recurrence&metastasis, looking for new methods and new targets in treatment have become the focus of medical workers.By IHC and rank correlation analysis, the expression of CXCR4in tissues of patients were associated significantly with malignant metastasis and TNM stage, which implied that high expressions of CXCR4are related to malignant, invasion and lymphatic metastasis, CXCR4has an important impact on tumor in the occurrence&development. Furthermore,using cultured tumor cell line EC9706and by the assays of cell adhesion and transwell chamber, the activities of CXCR4in invasion&metastasis of esophageal squamous carcinoma were investigated, and the action mechanisms were determined by examinating EGFR mRNA&protein through RT-PCR&westemblotting, which aim to provide theoretical basis for that CXCR4may become a new method and target of tumor treatment.Methods:1. IHC was performed to measure CXCR4protein expressions in tissues of both174patients with esophageal squamous cell carcinoma and50cases of normal tissues adjacent to carcinoma, whose clinical pathological factors including gender, age, tumor size, smoking habit, drinking habit, metastasis of lymph nodes, tumor differentiation, TNM stage, etal were recorded and with which rank correlation analysis was used to determine correlativity of the expression of CXCR4protein in tissues, meanwhileχ2test (chi-square test) was performed to evaluate the difference of the positive rate of CXCR4protein expressions in each group.2. EC9706cells in logarithmic phase were adjusted to the concentration of1×106/mL after trypsinization, then were inoculated in the culture plates and incubated for12h, then were treated with chemokine CXCL12of the final concentration of10μg/mL,5μg/mL,as well as, the negative control group were treated with blank medium. Tumor cells were collected after24h, and the activities of invasion&migration were evaluated by the assays of cell adhesion and transwell chamber. 3. EC9706cells in logarithmic phase was adjusted to the cell concentration of1×106/mL after trypsinization, then were inoculated in the culture plate and incubated for12h, then were treated with chemokine CXCL12of the final concentration of10μg/mL,5μg/mL,as well as, the negative control group were treated with blank medium. Tumor cells were collected after24h, and RT-PCR&westenblotting were performed to determine the expressions of EGFR mRNA&protein.Results:1. By IHC, different degree of brown yellow or brown staining appeared in174patients’specimens while nonstaining or a few with light brown in negative control group and normal control group. The positive expression ratio of CXCR4protein in174cases with esophageal squamous cell carcinoma and50cases of normal tissue adjacent to carcinoma were67.8%(118/174),32.5%(16/50) respectively. CXCR4protein had higher expressions in esophageal squamous carcinoma tissues and lower expressions in normal tissue adjacent to carcinoma, the differences were statistically significant (χ2=10.16, P<0.01).By rank correlation analysis, relationship between expressions of CXCR4protein in esophageal squamous cell carcinoma tissues and clinicopathologic factors were as follows:the expressions of CXCR4protein were correlated with lymphatic metastasis and TNM stage (r>0, P<0.05), the higher TNM stage, the stronger CXCR4protein expressions,which implied that expressions of CXCR4protein are closely related to the malignant degree, invasion and metastasis of esophageal squamous cell carcinomas. There were not relevant relations between the expressions of CXCR4protein in esophageal squamous carcinoma and the other clinicopathologic factors.2. The result of cell adhesion assays showed as follows:Treated with chemotatic factor CXCL12of different concentrations, the activities of adhesion were different. The mean values of optical density were0.12±0.021,0.25±0.011and0.38±0.006respectively, after treated with blank medium and CXCL12of5μg/mL,10μg/mL concentration, there were significant differences among them (paired comparison, P <0.01). The results showed that the tumor cell activities of adhesion is correlated with CXCL12, and there are dose dependent relationship between them, the higher concentration, the stronger activities. Treated with blank medium and CXCL12of5μg/mL,10μg/mL concentration, The mean number of tumor cells penetrated through the Matrigel coated polycarbonate filter were5.3±1.8,18.9±2.1and41.2±4.6respectively, there were significant differences among them(paired comparison, P<0.01). The results showed that the tumor cell activities of invasion and migration is correlated with CXCL12, and there are dose dependent relationship between them, the higher concentration, the stronger activities.Treated with chemotatic factor CXCL12of different concentrations, the activities of adhesion, invasion and migration of EC9706cells rised up, which were higher than negative group as well as there were dose dependent relationship and significant differences among them (paired comparison,P<0.01) and the higher concentration, the stronger activities.The results showed that CXCL12is correlated with the tumor cell activities of adhesion, invasion and metastasis closely, while combined with and upgrade its receptor CXCR4, it results in the invasion and metastasis of carcinomas cells. So high expressions of CXCR4are related to tumor cell invasion and metastasis.3. Treated with chemotatic factor CXCL12of different concentrations, the expressions of EGFR mRNA were different. After standardization by β-actin, The ratios were0.103±0.044、0.851±0.167and1.114±0.732respectively, after treated with blank medium and CXCL12of5p.g/mL,10μg/mL concentration, there were significant differences among them(paired comparison, P<0.01). The results showed that the expressions of EGFR mRNA in tumor cells is correlated with CXCL12, and there are dose dependent relationship between them, the higher concentration, the stronger expressions.Treated with chemotatic factor CXCL12of different concentrations, the expressions of EGFR protein were different. After standardization by β-actin, The ratios were0.065±0.033,0.443±0.101and0.907±0.226respectively after treated with blank medium and CXCL12of5μg/mL,10μg/mL concentration, there were significant differences among them(paired comparison, P<0.01). The results showed that the expressions of EGFR protein in tumor cells is correlated with CXCL12, and there are dose dependent relationship between them, the higher concentration, the stronger expressions.By RT-PCR and Western blotting, treated with chemotatic factor CXCL12of different concentrations, the expressions of EGFR mRNA&protein of EC9706cells increased, which were higher than negative group as well as there were dose dependent relationship and significant differences among them (paired comparison, P<0.01), the higher concentration, the stronger expressions. While combined with and upgrade its receptor CXCR4, it results in that the expressions of EGFR mRNA&protein of tumor cells increase, a series of signal pathway are activated, cell abnormal proliferation and apoptosis disorder appear, which lead to tumor in the occurrence and development.Conclusion:1.The expressions of CXCR4protein were high in esophageal squamous carcinoma tissues and were correlated positive with lymphatic metastasis and TNM stage. CXCR4can promote the occurrence, gowth and development of tumors as well as play an important role in early diagnosis, prognosis, treatment selection of esophageal squamous carcinoma.2. High expressions of CXCR4are related to tumor invasion and metastasis, between which there are dose dependent relationships, the higher expression of CXCR4, the stronger tumor cells activities.3. CXCR4maybe take part in invasion and metastasis of esophageal squamous cell carcinoma via regulating the expressions of EGFR mRNA&protein, which is one of the mechanism getting involved in invasion and metastasis.