Molecular Echanism Of TrkB In The Carcinogenesis Of Prostate Cancer And Mechanism Of Sur-D71A In The Induction Of Apoptosis Of Prostate Cancer Cells

The incidence and mortality of adenocarcinoma of the prostate continue to rise and will continue to pose a major public health problem.Prostate cancer is the second most common cause of cancer deaths on men.Despite progress in diagnosis and local therapy,fundmental questions remain with regard to the etiology, prevention,and treatment of prostate cancer.Our knowledge of prostate adenocarcinoma remains significantly less than of most other neoplasm.Finding a more sensitivity marker or oncogenic potential gene will provide us a useful tool to diagnose and cure prostate carcinoma. In our study, we focus our research on the prostate cancer gene therapeutics and the biological function of a novel identified tumor-associated molecule in the program of prostate cancer. The first part of our study is about the molecular mechanism of TrkB, a receptor-like tyrosine kinase, in the program of prostate carcinogenesis. And the second part is about the further mechanistic explanation of how dominant negative form of Survivin—Sur-D71A does induce apoptosis in prostate cancer cells.TrkB, together with TrkA and TrkC are neurotrophin receptors regulating the proliferation and differentiation of neuronal cells. Upon binding to its ligand brain-derived neurotrophic factor (BDNF), TrkB will dimerise, auto- phosphorylate and initiate multiple signal transduction pathways that are involved in neurite growth, morphological plasticity and the synthesis of proteins for differentiated function of neurons and synapses. Recent reports showed that TrkB may also have important fuhnction outside of the central neural system. Though the investigations are limited and the underlying mechanism remains unclear, yet it strongly indicates that TrkB may be involved in the tumorigenesis. In the study of prostate cancer, the relationship between TrkB and carcinogenesis has not been established. In this study, we investigated the expression pattern of TrkB in prostate cancer tissues with different pathological degrees. We performed RNAi technology to knockdown TrkB expression in prostate cancer cell lines and detected the malignant phenotype of the cells. To further analysis the cellular signal pathway mediated by TrkB,we used immunostaining to determine the changes of MAPK and PI3K/Akt signal pathwayhs.Objective:To determine the expression of TrkB in the prostate cancer tissues and to estimate its clinical significance. To investigate the biological function of TrkB in the malignant behavior of prostate cancer cell lines. To prove if TrkB can be a proming target in the prostate cancer gene therapeutics. To detect the change of cellular signal pathway mediated by TrkB in prostate cancer cells. Method:Immunohistochemistry was performed to detect TrkB expression in malignant tumor of prostate and the tissue of benign neoplasia was used as the control. Vector-based siRNA was constructed and the stable transfectants were established. Cell counting assay was used to detect the proliferation rate of prostate cancer cells with a low level of TrkB expresion. And soft agar assay was performed to investigate the transformation capacity of prostate cancer cells with a low level of TrkB expresion. Wester blot was used to detect the change of cellular signal pathway involved in TrkB-mediated tumor growth.Result:TrkB expressed with a much higher level in prostate cancer tissues with comparison to that in the benign neoplasia. And its expression trend was correlated with the malignant degree of the carcinoma. TrkB was mainly expressed on the cellular membrane and the cytoplasma, however, in some samples, TrkB was obviously localized in the nucleus of cancer cells. Its clinical significance was not clear since there is no sufficient tumor tissues to be detected. We successfully constructed the plasmid expression siRNA targeting TrkB. It can effectively downregulate TrkB expression in cancer cells. The proliferation rate and transformation capacity of prostate cancer cells decreased with the downregulation of TrkB by RNAi technology. Downregulation of TrkB decreased the phosphorylation level of Akt kinase while had slight effect on MAPK signal pathway.Conclusion:Receptor-like tyrosine kinase TrkB involved in the program of prostate carcinogenesis. It accelerated the proliferation rate of prostate cancer cells and played an important role in the process of transformation. TrkB-mediated cell survival, proliferation and transformation were mediated by PI3K/Akt signal pathway. And TrkB targeting therapeutics may be a promising strategy for cancer therapy of prostate cancer.Survivn is over expressed in many types of cancers. It plays important roles in cell division and antiapoptosis. Survivin-targeting therapeutics is a proming strategy for the cancer therapy. Our previous study demonstrated that adenovirus-mediated a novel dominant negative form of Survivin, Sur-D71A,increased the apoptosis rate of prostate cancer cells. But its molecular mechanism has not been elucidated. In our study, we investigated the expression pattern of Bcl-2 family members and detected its capacity to form homodimer or heterodimer with itself or wild-type Survivin protein.Objective: To investigated the expression pattern of Bcl-2 family members in prostate cancer cells after infected with the adenovirus expressing Sur-D71A or GFP control. To detect the capacity of Sur-D71A to form homodimer or heterodimer with itself or wild-type Survivin protein. Method: To package, amplify and purify the adenovirus expressing Sur-D71A. Western blot was used to detect the expression of Sur-D71A in prostate cancer cells after infection with adenovirus expressing Sur-D71A. We used western blot to detect the expression of Bcl-2 family members after transduction of Ad-Sur-D71A. We fused Flag-tag or Myc-tag to the terminal of Survivin or Sur-D71A. and also inserted the open reading frame of survivin or sur-D71A into the prokaryotic expression vector pGEX-4T-1.We used coimmunoprecipitation and GST-pull down assay to detect the capacity of Sur-D71A to form homodimer or heterodimer with itself or wild-type Survivin protein.Result: Adenovirus mediated expression of Sur-D71A induced apoptosis of prostate cancer cells by robustly changing the expression patterns of mitochandria-associated Bcl-2 family members. Antiapoptotic molecule Bcl-2 and Bcl-xl were downregulated and proapoptotic molecules Bax was upregulated in prostate cancer cells transfected with Ad-Sur-D71A in comparison with Ad-GFP-infected control or intact cells. Coimmunoprecipitation and GST-pull down assay showed that Sur-D71A had the similar binding affinity to form homodimer with Sur-D71A or heterodimer with wild-type Survivin.Conclusion: Adenovirus mediated expression of Sur-D71A induced obvious apoptosis of prostate cancer cells by disrupting the balance of antiapoptosis maintained by the homodimer of wild-type Survivin in a Sur-D71A dependent competitive binding manner to form heterodimer of wild-type Survivin and Sur-D71A, instead of the functional homodimer of wild type Survivin. And on the other hand, downregulation of Bcl-2 and Bcl-xl and upregulation of Bax also involved in the apoptosis of prostate cancer cells induced by Sur-D71A. This study provided a molecular mechanistic explaination for the Sur-D71A induced apoptosis in cancer cells.