| Prostate cancer is most frequently diagnosed in male in western countries. Recently, It has been becoming a major disease which affects the living quality and anticipated life span of Chinese elder men because of some changes of their daily habits, such as increase consumption of animal fat, obesity and physical inactivity. Current standard therapeutic approaches including radical prostatectomy, irradiation, chemotherapy and hormonal therapy, shows effective in patients with organ-confined prostate cancer. However, a significant proportion of patients, who initially present with localized disease, relapse ultimately.The application of these therapeutic approaches are often limited by their significant side effects and effectless on metastasized and hormone-refractory disease. Thus, it is urgent to develop a novel therapeutic strategy to cure prostate cancer efficiently.Gene therapy which can target key points in any phase during prostate cancer genesis and development has become the hottest research in prostate cancer therapy. Adenovectors are the most popular vectors in gene therapy of prostate cancer. Some adenoviral therapeutic regimens have been approved in clinical trials. Oncolytic adenovirus, also termed conditionally-replicating adenoviruses (CRADs), can specifically replicate in tumor cells leading to their death finally. Another advantage of CRADs is its high efficiency for therapeutic gene delivery and expression. Therefore, the therapeutic effect of CRADs armed with effective genes is more effective than that of single CRADs or replication defective adenoviruses carrying therapeutic genes.The ultimate effector cells that mediate the rejection of solid tumors in preclinical animal models are the cytolytic T lymphocytes. And the activation of professional antigen-presenting cells (APC) is essential to induce effective T cell immunity. CD40 is highly expressed on dendritic cells(DC).CD40 and CD40 Ligand(CD40L) is a pair of important co-stimulatory molecules of specific immune system.And it plays an important roles in regulation of T lymphocyte activation and effector cytokines production. Latest researches show that fusion protein of CD40L and tumor antigen can activate immune response through binding of CD40L and CD40 on DC.Oncolytic therapy and immunotherapy are combined in this study. We developed a prostate cancer specific CRADs carrying PSA-IZ-CD40L gene(fusion protein gene of prostate cancer antigen and CD40L,PL)and GM-CSF gene, named Ad-psh-PL-PPT. Followed by evaluating its oncolytic and immuno activation effects both in vitro and in vivo.Firstly, according to our oncolytic adenovirus preparation system, we designed and constructed a 712bp length prostate specific promoter PPTp,which composed of prostate specific antigen enhancer(PSAe), prostate specific membrane antigen enhancer(PSMAe) and TARPp in proper order. Moreover we evaluated PPTp on plasmid and adenovirus level. Results show that the report genes, controlled by PPTp, could be expressed effectively and specifically in various prostate cancer cells.Secondly, recombinant adenovirus Ad-psh-PL-PPT was prepared using oncolytic adenovirus preparation system, which established by our laboratory. (1) pshuttle-cmv-PL was obtained by insert PL gene into pshuttle-cmv.(2)Recombinant plasmid TE-SV-PPT-IR-55K,carrying genes of PPTp,E1A,E1Bp,E1B55K and GM-CSF(PPT-E1A),was constructed by replace TP gene of TE-SV-IR-TP-55K with PPTp. (3)PPT-E1A was obtained by MfeI cutting, and then fragment was inserted into pshuttle-cmv in forward direction. Recombinant plasmid pshuttle-cmv-PL-PPT-E1A was obtained by screening. (4) Ad-psh-PL-PPT was prepared by Adeasy system..(5) The expression of fusion protein PL and GM-CSF was confirmed by Western-blot and ELISA post infected byAd-psh-PL-PPT. (4) Ad-psh-PL-PPT was purified by density gradient centrifugation of cesium chloride. The purity was 1.2338 ,and the infectious units(IU) was 3.2×1010 IU per milliliter(IU/ml).In order to evaluate the function of Ad-psh-cmv-PL-PPT objectively, we prepared control adenovirus, Ad-psh-cmv-PL.Thirdly, the specific replicate ability, oncolytic efficacy and immune activation of this recombinant adenovirus were detected in vitro. (1)Cytopathic effect(CPE) was observed 96h post infected by 10 IU/cell Ad-psh-PL-PPT in DU145.And CPE was detected again in DU145 after infected by supernatant of freeze thawing. (2)The cell viability of various cells was measured by MTT assay and crystal violet staining 6d post infect. Results showed that Ad-psh-PL-PPT could kill the PC cells effectively and specifically. When infect with more than 10IU/cell Ad-psh-PL-PPT could kill PC cells more effectively than Ad-GFP. And at the IU/cell of 250, 60% of PC3,70% of DU145,80% of PC3M,and 90% of LNCaP were killed, while it could not kill other cells, such as 7721,HepG2 and ECV304. (3) 24h,48h and 72h post infect by 25IU/cell Ad-psh-PL-PPT, We detected the replicate ability of Ad-psh-PL-PPT.. The results showed that Ad-psh-PL-PPT could replicate selectively in prostate cancer cell lines, such as PC3,PC3M and DU145. And the titers increased with the time extending. (4)We collected the cell supernatant of PC cells infected by Ad-psh-PL-PPT. Mononuclear cells isolated from peripheral blood(PB)was stimulated by supernatant, then the proliferation ability of mononuclear cells was identified. The outcome showed that supernatants from PC3M and DU145 could stimulate the proliferation of mononuclear cells more effectively (P<0.01,comparing with the supernatants from Ad-GFP infection) (5) Mononuclear cells co-cultured with PC cells infected by Ad-psh-PL-PPT could kill PC cells stronger.Finally, we evaluated the oncolytic effects and immune response in vivo. In order to evaluate the anti-tumor effect, we established the transplantation tumor model of prostate cancer by injected PC3 cells subcutaneouly in Babl/c nude mice. After tumor formed, 1×109 IU/50μl Ad-psh-PL-PPT was administered intratumorally. The result shows that adenovirus titers in tumors of Ad-psh-PL-PPT therapy was much higher than that of Ad-GFP therapy. We observed the basic states of mice and growth curve of tumors. The results showed that Ad-psh-PL-PPT could delay the growth of tumor.In order to evaluate the immune activation ability, we replace the CD40L gene of PL with mouse CD40L(mCD40L) generating a recombinant plasmid pUDK-PmL-IR-GM, carrying PmL (fusion protein of PSA and mCD40L) gene and GM-CSF gene.(1)We confirm the expression of PmL and GM-CSF by Western-blot and ELISA in 293 cells transfected by pUDK-PmL-IR-GM. (2)pUDK-PmL-IR-GM was administered into muscle of normal Babl/c mice every 10 days for four times. We isolated T lymphocyte from spleen of mice 10 days after the latest administration, then measured the subset of T lymphocyte. The results showed that CD4+T was increased after immuned by pUDK-PmL-IR-GM, and the ratio of CD4+T and CD8+T was elevated obviously comparing control group(P<0.05).(3)MTT results of T lymphocytes showed that PSA could stimulate the proliferation of T lymphocytes from PUDK-PmL-IR-GM group(P <0.05,comparing negative control).And the results also showed that PSA could stimulate the proliferation of T lymphocytes more effectively in PUDK-PmL-IR-GM group than in other groups(P <0.01).The results showed that pUDK-PmL-IR-GM could provoke the immune activation of T lymphocytes to kill LNCaP(P <0.05.in E/T ration of 5:1,comparing with pUDK group).In conclusion, we developed a CARDs armed with PL fusion gene and GM-CSF gene and evaluated its therapeutic effects on prostate cancer. In vitro, it could specifically replicate in prostate cancer cells leading to cell death.; it also could stimulate the proliferation and enhance the toxicol effect of mononuclearcells in human peripheral blood. In vivo, it could encourage the immune activation of T lymphocytes in normal mice and induce effective antitumor response in nude mice PC xenograft tumor model.
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