Expression Of TrKB And VEGF And Its’ Regulation Mechanisms Of The Invasiveness And Metastasis In Nasopharyngeal Carcinoma In Vivo And Vitro

ObjectiveNaropharyngeal carcinoma (NPC) is one of the commonest tumors in the head and neck region, locoregional recurrence, cervical lymph nodes metastases and distant metastases are the factors that significantly affect the prognosis in NPC patients. Rencent study showed that the development and metastasis of malignants is vascular dependence. Angiogenesis is extremely relate with the development, metastases and prognosis of tumors. The purpose of this study was to investigate the significance of TrKB, VEGF expression in naropharyngeal carcinomaMethods:The level of TrKB, VEGF expression in55tumor tissues and30normal nasopharyngeal tissues were determined by immunohisto-chemistry. Patients were classified according to age, gender, T stage, lymph node metastases and distant metastases. Staining grades were obtained for each group, and the correlation were analysed within groups stratified with respect to clinicopathological parameters by pearson correlation test. Research on the relationship between them and clinicopathology parameters in NPC respectively Result1. Both of VEGF and TrKB expression located in cytoplasm. In55cases of NPC, positive expression of VEGF and TrKB was47and45cases respectively. The positive rate of TrKB and VEGF in NPC tissues was82.1%and85.7%respectively. However, in30cases of normal nasopharyngeal tissues, the cases of TrKB and VEGF positive expression was3,5respectively. the positive expression rate of VEGF and TrKB in NPC were significantly higher than the normal nasopharyngeal tissues (P<0.05), A correlation analysis indicates a significant correlation between TrKB and VEGF expression in NPC(related coefficient R=0.586, P<0.01);2. The differences of VEGF expression were significantly correlated with different T stage (P=0.009), clinic stage (P=0.002), lymph node metastases (P=001). The differences of VEGF expression between the different age, gender and distant metastases group were not statistically significant; the differences of TrKB expression were significantly correlated with different T stage (P=0.00), clinic stage (P=0.00) and lymph node metastases groups (P=0.006). The differences of TrKB expression between the different age, gender, and distant metastases groups were not statistically significant.Conclusion:1. The expression of VEGF and TrKB is increased in the tumor tissues, which indicated that both of them may have been involved in the occurrence and development of NPC.2. The expression of TrKB is probably related to angiogenesis of NPC PurposeTo investigate the impact on the proliferation, cell cycle, cell movement and VEGF expression in human NPC cell line CNE-1after suppression of TrKB expression through K252a-the inhibitor TrKB receptor tyrosine kinase.MethodHuman NPC cell line CNE-1was treated with different concentrations of K252a, Then, cell proliferation in different groups was measured by MTT assay. We would select the optimal concentration of intervention of K252a. After treated with optimal concentration of intervention of K252a, wound healing assay were used to detect cell motility, cell cycle and apoptosis were analysed by Flow Cytometry, Changes in expression of VEGF and TrKB expression were examined by western-blot and real-time PCR.Result1. MTT assay:K252a inhibited proliferation of cell line CNE-1, which induced the decreases in cell number, density and survival rate. The impaction of K252a which inhibited proliferation on cell line CNE-1 had positive correlation with the intervention time and Intervention concentration of K252a.2. Wound healing assay:K252a delayed the wound healing, and partly inhibited cell motility and movement. The12h distance of cell migration in the two experimental group had no significantly different than the control group (P=0.18).The24h distance of cell migration in the two experimental group were significantly different than the control group (P=0.01);3. Flow Cytometry:Cell line CNE-1treated with K252a exhibited the increases in phase G1and decreases in phase S (P<0.05), and cell apoptosis rate was significantly increased than the control group (P<0.05).4. Western blot:K252a led to significantly decreased TrKB protein expression (K252a group:Relative protein quantity0.313±0.03vs control groups:Relative protein quantity0.63±0.01), K252a led to significantly decreased VEGF protein expression(K252a group:Relative protein quantity0.38±0.04vs control groups:Relative protein quantity0.6883±0.02) which had significant difference with control group (P﹤0.05).5. In the presence of K252a treatment, there is significant changes in expression of VEGF mRNA (K252a group:0.43±0.02vs control groups:0.69±0.05) and TrkB mRNA in CNE-1cells (K252a group:0.44±0.06vs control groups:0.67±0.037)Conclusion:Inhibiting TrKB expression led to the inhibition of tumor growth, migration and invasion in vitro which were caused by the increase of cell apoptosis rate and down regulation of VEGF expression. These data indicated that TrKB played a role in angiogenesis of NPC via regulating VEGF expression ObjectiveTumor growth is dependent on the angiogenic factors which induces angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specefic mitogen and an angiogenesis inducer in vivo. TrKB and VEGF play an important role in angiogenesis of NPC. Previous studies have showed K252a have ability to inhibit expression of TrKB and VEGF protein and gene. The aim of present study is to establish the model of nasopharyngeal subcutaneous xenograft in nude mice and to discuss the impact on the development of NPC by the use of K252a interfering to block the production of TrKB and VEGF in human nasopharyngeal cancer cell line CNE-1in vivo. Thus, it can provide information for the anti-angiogenesis gene therapyMethodThe nasopharygeal axenografts were established by subcutaneous injection of human CNE-1nasopharyngeal cancer cells into14nude mice (Balb/c) which weighed16-20grams, and were4-6weeks old. Every mouse was injected to1×10cells. These mice were divided randomly into2groups. A:K252a interfere group,7mice. B:PBS group,7mice. The mice were treated with K252a and PBS respectively intra-tumor at "6","8","10","12","14" day after the implantation of CNE-1cells. The drug was injected at200u1400nmol/L each time. The next indexes were detected. The volume of tumor was measured every two days after the first injection and the growth curve was drawn. All mice were sacrificed at "21t" day after injection. The tumor samples were collected and weighted. These index were detected such as sample observation and weight, HE staining, VEGF, TrKB and CD31immunocytochemical analysis.Results1. Observation of tumor sample and HE staining:tumors could be seen at4th day after the implantation of CNE-1cells. NPC Xenografts in K252a group were small, elliptical, HE staining showed a few number of tumor cells, big cell interspace, few blood capillaries, whereas, tumors in the control group were big. HE staining showed large amount of cells, small cell interspace, little stroma, plenty of blood capillaries.2. The development of tumor growth:the tumors of K252a group grew slowly till to the end of drug injection. At21th day the average volume of K252a group was (328±326.7) mm3, whereas the volume of control group was (1044±818) mm3. Comparison between group showed significant differences (P<0.05).3. The numers of microvessel density (MVD) labeled by CD31immunocytochemical analysis among K252a group and control group of were22.385±4.718and49.785±11.12, Comparison between groups showed significant differences (P<0.05).4. Expression of TrKB, VEGF analysis:Comparison of VEGF expression between K252a group and control group showed significant difference (K252a group:7.57±2.82, control group:9.86±2.79P=0.007), Comparison of TrKB expression between K252a group and control group showed significant difference (K252a group:5.86±2.79, control group:7.43±2.64, P=0.005).5. Correlation analysis of MVD and VEGF:the Pearson correlation coefficient of K252a group was r=0.96,P=0.01. the Pearson correlation coefficient of control group was r=0.88, P=0.009. They were positively correlated.6. Correlation analysis of MVD and TrKB:the Pearson correlation coefficient was control group:r=0.79, P=0.04, K252a group:r=0.85, P=0.01). They were positively correlated.7. TrKB and VEGF were uncorrelated.Conclusions1. Inhibiting the expression of TrKB led to the inhibition of tumor growth and metastasis in nasopharygeal axenografts model.2. Immunocytochemical analysis shows K252a may inhabit the expression of VEGF, TrKB and interfere with new vessels’angiogenesis.3. VEGF, TrKB has a close relationship with MVD in NPC.