The Study Of TrKB On The Mechanism Of Angiogenesis In Prostate Cancer

Objective:Prostate carcinoma(PCa)is a malignant tumor derived from prostate glandular epithelium.Current research suggests that the formation of new blood vessels is closely related to the growth,invasion,metastasis,and prognosis of malignant tumors.Vascular endothelial growth factor(VEGF)promotes tumor growth by accelerating angiogenesis at the tumor site.In addition,tyrosine kinase B(TrKB)plays an indispensable role in tumor angiogenesis,growth,invasion,metastasis,and prognosis.K252 a can block TrKB-BDNF pathway and observe proliferation,migration,cell cycle,cell apoptosis and VEGF expression in prostate cancer PC3 cells cultured in vitro.Recent studies have found that VEGF promotes angiogenesis by inducing angiogenesis and promoting the division of vascular endothelial cells,and angiogenesis and microvessels are closely related to tumor invasion and metastasis,and suggest that microvessel density(MVD)is related to tumor metastasis and prognosis..The results of in vitro cell experiments show that the intervention of TrKB expression can affect the expression of VEGF in prostate cancer PC3 cells.In this part of the experiment,we established a nude mouse xenograft tumor model,and then treated with K252 a to observe the changes of VEGF expression and MVD in animals to explore the regulation of TrKB.The possible mechanism of neovasculature in prostate cancer is a new theoretical basis for anti-angiogenic gene therapy in prostate cancer.Methods:The expression of VEGF and TrKB proteins in 235 human prostate cancer tissues and 250 benign prostatic hyperplasia tissues was measured by immunohistochemistry.Patients were grouped according to their age,clinical classification,primary tumor size,lymph node metastasis,and distant metastases.Bivariate correlation Pearson test was used to analyze the relationship between VEGF and TrKB expression and clinicopathological features.Prostate cancer PC3 cells were treated with different concentrations of K252 a to block the TrKB-BDNF pathway.The effects of different concentrations of K252 a on the growth of prostate cancer PC3 cells were compared by MTT assay,and the cell survival rate was calculated and the K252 a optimal intervention concentration was selected;The healing test was performed to observe the changes in the migration ability of prostate cancer PC3 cells after blocking the TrKB-BDNF pathway;flow cytometry was used to analyze the changes and apoptosis of prostate cancer PC3 cell cycle after blocking the TrKB-BDNF pathway;RT-PCR,Western-blot was used to detect the expression of VEGF and TrKB in prostate cancer PC3 cells after blocking TrKB-BDNF pathway.The BALB/C-nu/nu nude mice aged 4-6,male,and weighing 16-20 g were selected and injected into the right quasi region of the nude mice to generate prostate cancer PC3 cells.The nude mice bearing human prostate cancer cells were established subcutaneously.Transplantation tumor model.The experimental animals were randomly divided into 2 groups,group A: K252 a intervention group,10;group B: PBS group,10;group 6,nude mice,tumors in group A,6,8,10,12 and 14 days after inoculation The K252 a drug was injected at a dose of 450 nmol/L and 200 ?l/time.The two groups of models were observed for 18 days.The tumor volume was measured once every two days after the first injection of the drug and the tumor growth curve was plotted.The nude mice were sacrificed on the 18 th day after inoculation,the tumor specimens were detached,the tumor volume was measured,and the overall characteristics of the specimens were observed.Specimens taken out of some tissues were fixed in 10% formalin solution,paraffin-embedded and serially sectioned for routine HE staining and immunohistochemical staining for VEGF,TrKB,and CD31.Results:1.The expression of TrKB protein was located in the cancer cell plasma,and the expression of VEGF protein was localized in cancer cell plasma and vascular endothelial cells.186 patients presented TrKB positive expression(the positive expression rate was 79.15%)and 192 patients presented VEGF positive expression in 235 cases of prostatic cancer(the positive expression rate was 81.70%).21 patients presented TrKB positive expression(the positive expression rate was 8.40%)and 26 patients presented VEGF positive expression in 250 cases of benign prostatic hyperplasia(The proportion of positive expression occupies 10.40%.The positive expression of TrKB and VEGF in human cancer tissues has increased significantly than that in cancer tissues of benign prostatic hyperplasia(p<0.05).There was significant correlation between VEGF and TrKB expression in cancer tissues(correlation coefficient R=0.602.p<0.05).2.TrKB protein expression was significantly correlated with PCa clinical staging(p=0.004),the Gleason grade(p=0.004)and the presence of lymph node metastasis(p=0.002),regardless of diameter of tumor,age and distant metastasis(p>0.05).The expression of VEGF protein was significantly correlated with the clinical stages of PCa(p=0.003),the Gleason grade of the tumor(p=0.005)and the presence of lymph node metastasis(p=0.001),regardless of diameter of tumor,age,and distant metastasis(p>0.05).3.MTT assay: The proliferation of PC3 cells in prostate cancer was significantly inhibited by K252 a,which showed that cell number,density and cell survival rate decreased.The inhibition of PC3 cell growth in prostate cancer was positively correlated with K252 a intervention time and concentration.After the intervention of K252 a for 48 hours,the proliferation rate of PC3 cells in prostate cancer was the most obvious.The IC50 of K252 a was 450 nmol/L.4.Scratch healing experiment: After the TrKB-BDNF pathway was blocked by K252 a,the scar healing time was delayed.Compared with the control group,the migration distance of PC3 cells was decreased in 12 hours,but the statistical difference was not significant(p=0.0.99).But the migration distance of PC3 cells in 24 h hours of prostate cancer was significantly reduced(p=0.026).It was shown that inhibition of TrKB expression could inhibit the mobility of PC3 cells in prostate cancer.5.Flow cytometry: After K252 a intervention,the PC3 prostate cancer cells in G0 / G1 phase cells was significantly increased(69.66+3.56 vs 51.22+6.21,p=0.026),significantly less cells in S phase(16.25+1.26 vs32.21+2.31,p=0.033)compared with control group,the statistical difference was significant(p < 0.05).The apoptosis rate of K252 a intervention group higher than the control group(25.31+4.11vs4.23+0.99,p=0.016).6.Western blot:Compared with the control group,the expression level of TrKB protein in K252 a intervention group was down-regulated(0.312�0.019 vs 0.659+0.023),and the expression level of VEGF protein was down-regulated(0.323�0.042 vs 0.689�0.012),and the difference was statistically significant(p<0.05).7.RT-PCR:Compared with the control group,the expression of TrkB mRNA in K252 a intervention group was down-regulated(0.413+0.065 vs 0.682�0.025).The expression of VEGF mRNA was down-regulated(0.412+0.015 vs 0.659�0.050),and the difference was statistically significant(p<0.05).8.The morphology and HE staining of tumor specimens: On the 4th day after inoculation,the experimental animals became tumorigenic.The tumor has a relatively complete envelope,with a clearer boundary with the surrounding tissue.In the K252 a intervention group,the subcutaneous tumors were small in size,irregularly spherical,lobulated,grayish white in section,hard in texture,and white in section.The volume of the tumor in the control group was significantly larger than that of the intervention group,with uneven surface,nodule-like nodule,and grayish-white cut surface with slight grayish red;9.Changes in tumor volume of experimental animals: Since the beginning of administration,tumor growth was slow in the K252 a intervention group,tumor volume was(361.26�49.6)mm3 at the 18 th day,and(1132�201.26)mm3 in the control group:10.The comparison of CD31-labeled MVD with VEGF and TrKB immunohistochemical staining and correlation analysis: The positive vascular counts in the intervention group and the control group were 25.65�5.36 and 49.36�4.36,respectively.The analysis of variance showed that there was a significant difference between the groups(p<0.05).Compared with the control group,the expression of VEGF in the intervention group was significantly decreased with a statistically significant difference(3.32�0.54 vs6.56�1.02,p=0.007).TrKB expression in the intervention group also decreased significantly,with a statistical difference(3.93� 0.36 vs 6.53�1.03,p=0.035),VEGF expression in each group was correlated with MVD(control group: r=0.726,p=0.008;K252a group: r=0.956,p=0.02),TrKB expression and MVD Positive correlation(control group: r=0.801,p=0.031,K252 a group: r=0.865,p=0.03).Conclusions:1.The increased expression of VEGF and TrKB in prostate cancer tissues is related to the occurrence and development of prostate cancer and angiogenesis.2.Blocking of the TrKB-BDNF pathway can reduce the growth and migration of prostate cancer PC3 cells.At the same time,it can inhibit the growth and migration of prostate cancer PC3 by promoting apoptosis,changing cell cycle and reducing VEGF expression.3.TrKB affects prostate cancer angiogenesis by regulating VEGF expression.4.Inhibition of TrKB expression can inhibit the growth of prostate cancer xenografts in animals and reduce the formation of neovascularization in cancer tissues by reducing the expression of VEGF;5.TrKB may become a new target for anti-prostate cancer neovascular treatments.