Study On The Effects Of Adenovirus-mediated Human FEZ1 Gene On The Growth Of Prostate Cancer Cell Line DU145

Background Prostate cancer is the most frequent malignant tumor in western counties.Its incidence rate situated the first or second place in maleness tumors in the United States and Western Europe.The mortality rate situate the first place in maleness malignant tumors in the United States and Western Europe. At present, the incidence of prostate cancer in our country is much lower than western countries, but in recent years, it's incidence ascended significantly and situated the third place in urinary tract tumors. More than half patients with prostate cancer were already partly progressive and metastatic prostate cancer and missed opportunity of radical treatment; they can only be applied endocrine hormone treatment. Moreover 70%-80% of prostate cancer patients were to gradually transformed into a state of tolerance or lack of response to hormone, namely, the so-called androgen-independent prostate carcinoma (AIPC). At present, the efficacy of any therapy to AIPC efficacy is very limited. It is extremely urgent to find new treatments.Gene therapy is a new method of treatment that created by combining modern medicine and molecular biology. The gene therapy has been growing rapidly in this 20 year since the first success of clinical gene therapy for a four-year-old girl with immunodeficiency disorder (SCID) causing by adenosine deaminize (ADA) deficiency in 1990. Adenovirus vector for gene therapy is the most commonly used carrier. Ishii H(1999), found a candidate fragment F37 which was found in normal cell lines and not found in cancer cell lines by cloning, sequencing methods to analysis of tumor and cell line cDNA in the study of primary esophageal cancer. Ishii H named F37/Esophageal cancer-related gene-coding leucine-zipper motif (F37/esophageal cancer related gene encoding leucine-zipper gene sequences, known FEZ1/LZTS1). At present FEZ1 gene is considered a candidate tumor suppressor gene, FEZ1 gene may inhibit the tumor cell growth by regulating the microtubule and mitotic.The FEZ1 gene mRNA and protein expression of the FEZ1 were found missing or decreased significantly in the lung, stomach, esophagus, bladder cancer, breast cancer cells lines.Application of gene therapy to breast cancer cell line MCF7, AT6.2 (mouse prostate cancer cell line) and LNCaP (androgen-dependent prostate cancer cell line) SW780 cells (bladder cancer line), all the cell growth and proliferation had a noticeable inhibition.First We Use RT-PCR and Western blot to detect FEZ1 gene expression in prostate cell lines. The next we use PCR amplify FEZ1 gene fragments, use CloneEZ technology and LR in vitro recombinant technology clone FEZ1 gene fragment into adenovirus vector. Then we use PCR and sequencing Methods identify recombinant adenovirus vector pAd-FEZ1. At last we use MTT method, Hoechst staining, flow cytometer analyze, to analyze the proliferation, apoptosis of the DU145 cell lines.We wish that we can give a new idea for androgen-independent prostate carcinoma.Objective:To study the FEZ1 gene expression in different prostate cell lines. Methods:RT-PCR and Western blot were used to examine the expression of FEZ1 gene in normal prostate epithelial cells (RWPE-1), androgen-independent prostate cancer cell line DU-145, prostate cancer cell line ALAV. Results:All the cells have the FEZ1 gene expression, and the RWPE-1 FEZ1 gene expression was the maximum, two kinds of tumor cell lines expression have no significant difference. Conclusion: All the cells have the FEZ1 gene expression; the RWPE-1 expression was the highest. This work provides the base for the following study.Objective:To amplify human FEZ1 gene, construct and identify the FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145.Methods:(1) We used PCR to amplify FEZ1 gene fragment., used cloneEZ technology and LR in vitro recombination technology clone FEZ1 gene fragment into adenovirus vector. Constructed recombinant adenoviral vector pAd-FEZ1 well, we identified FEZ1 gene recombinant adenovirus vector by the enzyme digestion and sequencing. (2) Choosed the best multiplicity of infection (multiplicity ofinfection, MOI) of rAd-FEZ1 infected DU145, constructed the recombinant adenovirus rAd-FEZ1 DU145 group, adenovirus vector DU145 group, and blank control DU145 group. To study FEZ1 gene expression with RT-PCR, Western-Blot technique and immunofluorescence technique to confirm construct recombinant adenovirus rAd-FEZ1 DU145 group successfully. Results: The FEZ1 gene was amplified successfully and the FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145 were constructed and identified successfully. Conclusion:The FEZ1 gene recombinant adenovirus vector and FEZ1 gene stably expressing prostate cancer cell line DU145 were constructed and identified successfully. This work provided the base for the following study. Objective:To observe the biology effect of the FEZ1 gene recombinant adenovirus to prostate cancer cell line DU145. Methods: Measured the cell growth curves by MTT method. Hoechst staining cells in morphology, flow cytometry analysis method explored cell proliferation and apoptosis. Results:(1) MTT method results:the growth rate of the recombinant adenovirus rAd-FEZ1 infection DU145 cell line group was lower than the adenovirus vector control DU145 group and blank control DU145 group. The growth rate of the adenovirus control group and blank control group, had no significant difference (p> 0.05), There was a significant difference (p<0.05). between the growth rate of the two groups of cells and recombinant adenovirus rAd-FEZ1 group, (2) Hoechst staining showed dense nucleus wound stain or a dense stain Chunky, abnormal nuclei increased significantly, a marked apoptosis after being infected for 48 hours (3) Annexinâ…¤/PI flow cytometry analysis showed the apoptosis rate of recombinant adenovirus rAd-FEZ1 infection DU145 group was higher than the blank control group and the control adenovirus group. The apoptosis rate of the adenovirus control group and blank control group, had no significant difference (p> 0.05) There was a significant difference (p<0.05). between the apoptosis rate of the two groups of cells and recombinant adenovirus rAd-FEZ1 group Conclusion:FEZ1 gene can FEZ1 protein induce apoptosis and inhaibit proliferation androgen indepent prostate cancer DU145 cell lines. FEZ1 gene shows great potential for the gene therapy of androgen independent carcinoma of prostate.