Therapy Experimental Study Of Recombinant Adenovirus-mediated Hsp70 Gene Expression Targeted On Pancreatic Cancer

Pancreatic cancer is the most common malignant tumor of the gastrointestinal tract. In spite of advances have been made in imageology, chemotherapy, peri-operation period and systemic therapy, significant progress in the treatment of pancreatic cancer remains elusive。Thus, the development of new therapeutic approaches for pancreatic cancer has become one of the major task for pancreatic cancer research. As the development of molecular biology and tumor immunology in recent yeas, immunogene therapy has become one of most effective methods for pancreatic cancer systemic therapy.Increasing evidence indicates that Hsp70 plays important roles in eliciting innate and adaptive immunity. Tumor-derived Hsp70 can induce a tumor-specific immune response。Hsp70 has the ability to chaperone a variety of tumor antigens. Hsp70 complex with antigenic peptides can gain access to the MHC class I and class II antigen-processing pathways in APC via receptor-mediated uptake leading to CD8+ and CD4+ T-cell responses. Hsp70 can induce the maturation of dendritic cells (DC) by upregulating the expression of costimulatory and antigen-presenting molecules such as B7-1, B7-2 and MHC II molecules. Hsp70 can also function as cytokines that can attract DC and T cells to tumors. Furthermore, Hsp70 has been shown to activate NK cells to specifically kill tumor cells that express Hsp70 at the cell surface。Tumor-derived Hsp70 provided effective tumor vaccines in animal models, and the ability of human tumor-derived Hsp70 to stimulate autologous tumor-specific T cells also has been shown in vitro.Adenoviral vector has a broad host range, high infection efficiency, and it can be insert a very large foreign gene. Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. They are one of the most attractive therapeutic vectors for several reasons. They have the ability to infect both dividing as well as non-dividing tumor cells. Furthermore, the techniques to produce high-titered preparations of adenoviral vectors are straightforward. In recent years, in order to improve adenovirus-mediated gene therapy efficiency, researchers have constructed tumor targeting adenovirus utilizing certain biological difference between tumor cells and normal cells. At present, a large number of tumor cell specific promoters have been identified and targeted for regulation of gene expression, such as MUC1 promoter targeting breast cancer, AFP promoter targeting liver cancer, PSA promoter targeting prostate cancer and EB virus promoter targeting NPC. Regulating the expression of foreign genes by tumor promoter can get the tumor specific targeting in cancer gene therapy. It can control the gene expression and reduce its toxic effects on normal cells. Studies have confirmed CEA does not express in normal pancreatic tissue, but it has a high frequency of positive expression rate in pancreatic carcinoma, indicating that CEA is closely related to pancreatic cancer. CEA is a relatively broad spectrum of the molecular target for pancreatic cancer gene therapy.Based on the research mentioned above, we clone the Hsp70 gene and CEA promoter firstly, and then construct a recombinant adenovirus expressing Hsp70 gene controlled by CEA promoter.1. Cloning of Hsp70 gene and CEA promoter and detection of the CEA promoter activityObjective To clone Hsp70 gene and CEA promoter, and to detect the activity of CEA promoterMethods The pancreatic cancer cell line Panc-1 was treated with heat shock and total cellular RAN was extracted. cDNA was synthesized by reverse transcriptase and Hsp70 gene was amplified by PCR. Human genome was extracted from peripheral blood of healthy volunteers and CEA promoter was amplified by PCR. Hsp70 gene and CEA promoter was subcloned into T vector, respectively, and for DNA sequencing. CEA promoter was inserted into the multiple cloning sites of plasmids pGL-6 using digestion method to construct plasmid pGL6-pCEA. CEA levels in the culture medium of pancreatic cancer cell lines Pac-1, SW1990, BxPC-3, Capa-Ⅱ, CF and DSL-6A/C1 were detected by ECLIA. pGL6-pCEA and pGL6 plasmid were transfected into the CEA-positive and CEA-negative cells by liposome respectively, and pRL-TK luciferase expression plasmid was co-transfected as the internal reference transfection efficiency. The activity of CEA promoter was detected by dual luciferase reporter gene system.Results PCR amplification products of Hsp70 gene was analyzed by agarose gel electrophoresis, and a 2000bp band could be seen obviously, consistent with the size of the target gene. The DNA sequencing results was consisted with the full length coding region of Hsp70 sequence in GenBank. PCR products of CEA promoter was analyzed by agarose gel electrophores, and a 380bp band could be seen obviously, consistent with the expected results. The DNA sequencing results was consisted with the reported CEA promoter sequence. CEA content analysis showed that CEA secretion in SW1990, Capa-ⅡPac-1 cell lines were negative, and were positive in BxPC-3, CF and DSL-6A/C1 cell lines. After a dual luciferase analysis, there was no significant difference of relative luciferase activity between pGL6-pCEA and the control pGL6 in CEA-negative pancreatic cancer cell line SW1990 and Capa-Ⅱ(P>0.05). However, compared with the control pGL6, pGL6-pCEA relative luciferase activity in CEA-positive pancreatic cancer cell lines CF, BxPC-3 and DSL-6A/C1 were higer and the difference was significant (P <0.01).Conclusion Hsp70 gene and CEA promoter are amplified successfully and the CEA promoter can drive the downstream gene expressed effectively in CEA-positive cancer cells.2. Construction and identification of recombinant adenovirus containing heat shock protein70 gene driven by CEA antigen promoterObjective To construct and identify a recombinant adenovirus containing Hsp70 gene driven by CEA promoter.Methods Hsp70 gene was inserted into plasmid PDC316 multiple cloning sites by digestion method, and the MCMV promoter of plasmid PDC316-Hsp70 was replaced by the CEA promoter to construct the shuttle plasmid PDC316-pCEA-Hsp70. The shuttle vector and the backbone plasmid pBHG lox△E1, 3Cre were recombined homologously in HEK293 cells to obtain the recombinant adenovirus Ad5-pCEA-Hsp70, and PCR method was used to identify the recombinant adenovirus. The recombinant adenovirus was purified by CsCl banding and titrated by TCID50 assay. After transfecting the recombinant adenovirus into human pancreatic cell lines SW1990,BxPC-3 and DSL-6A/C1 , the expression of mRNA and protein level of Hsp70 were determined by PCR and ELISA, respectivelyResults Restriction analysis showed that the shuttle PDC316-pCEA-Hsp70 plasmid was constructed successfully. PCR results showed that the same size fragment of Hsp70 could be amplified from the recombinant adenovirus as the positive control, indicating that the recombinant adenovirus Ad5-pCEA-Hsp70 was successfully constructed. Viral titer was determined by TCID50, and the recombinant adenovirus Ad5-pCEA-Hsp70 titer was 2.5×1010 PFU/ml, and the control virus Ad5-control titer was 2.2×1010 PFU/ml. After infected with Adenovirus Ad5-pCEA-Hsp70, the expression of the Hsp70 mRNA increased significantly in CEA-positive BxPC-3, DSL-6A/C1 cell lines. But there was no significant change of Hsp70mRNA in the CEA-negative cell line SW1990. Hsp70 protein content of Ad5-pCEA-Hsp70 group in BxPC-3 and DSL-6A/C1 cell lines were 398.0±76.9ng/ml and 210.7±39.1ng/ml, which were higher than the Ad5-control and PBS group(P <0.01). However, The Hsp70 protein content in SW1990 cell line did not change significantly between the three groups (P> 0.05).Conclusion We construct the recombinant adenovirus Ad5-pCEA-Hsp70 Successfully, which can be targeted expression of Hsp70 gene in the CEA-positive cells. Our study laid the foundation for further experiments.3. Experimental study of the therapeutic effect of recombinant adenovirus Ad5-pCEA-Hsp70 on pancreatic cancerObjective To observe the effect of recombinant adenovirus Ad5-pCEA-Hsp70 on proliferation and apoptosis of cultured pancreatic cancer cell, as well as the tumor growth in animal models of rat pancreatic cancer, and to analyze the underlying mechanism.Methods Pancreatic cancer cell lines were infected with recombinant adenovirus Ad5-pCEA-Hsp70, and cell proliferation was detected by MTT assay and cell apoptosis was analyzed by flow cytometry. Rat xenograft model of pancreatic cancer were established and were randomly divided into three groups, which were given Ad5-pCEA-Hsp70, Ad5-control and PBS treatment, respectively. Antitumor effect was evaluated by comparing tumor size of three groups at different time points. ELISA method was used to detect Hsp70 protein content and cytokines INF-γ, TNF-α, IL-6 levels in peripheral blood. HE staining was used in animal Xenografts to detected lymphocyte infiltration. CD4 + T and CD8 + T staining points were detected by Immunohistochemical and apoptosis of tumor cells were analyzed by TUNEL assay. Animal spleen mononuclear cells were isolated and the proportion of CD83+ cells was evaluated by flow cytometry. Cell-killing ability of Spleen lymphocytes were observed in vitro.Results When the MOI was 0.1 and 1, the recombinant adenovirus Ad5-pCEA-Hsp70 did not significantly affect the proliferation of pancreatic cancer cell lines (P> 0.5). When the MOI was10 and 100, compared with Ad5-control and PBS, Ad5-pCEA-Hsp70 inhibited the cell proliferation activity significantly in pancreatic cancer cell linesBxPC-3 and DSL-6A/C1 (P <0.5, P <0.01).When the MOI was 10, compared to Ad5-control and PBS, apoptosis of tumor cells did not obviously affected by Ad5-pCEA-Hsp70 in different pancreatic cancer cell lines (P>0.5).Animal experiments showed that 4, 6, 8 weeks after treatment, tumor volume in Ad5-CEA-Hsp70 grpup was 724.4±81.6mm3, 681.3±64.9 mm3, 648.0±65.9mm3, respectively. Compared with the Ad5-control group and PBS group, the difference was significant (P<0.01). The serum ALT, AST, BUN and Cr level had no statistically significant difference between different groups (P>0.05). ELISA results showed that, Hsp70 protein and cytokines INF-γ, TNF-α, IL-6 levels in peripheral blood in Ad5-pCEA-Hsp70 group were significantly higher than that of Ad5-control group and PBS group (P <0.01,).HE staining showed, compared to Ad5-control group and PBS group, Ad5-pCEA-Hsp70 group had more lymphocytic infiltration.TUNEL assay of apoptosis found that the apoptosis index in Ad5-pCEA-Hsp70 group was 12.6±3.2%, compared to the Ad5-control group (4.4±0.8%) and PBS group 3.6±0.8%) , there was a statistically significant difference (P<0.01) Immunohistochemistry showed that, CD4 and CD8 staining points of Ad5-pCEA-Hsp70 group was significantly higher than that of Ad5-control group and PBS group (P<0.01). By flow cytometry, the proportion of CD83 + cells in Ad5-pCEA-Hsp70 group was 10.8±1.3%, significantly higher than that of the Ad5-control group and PBS group (P<0.01). In the lymphocyte-mediated CTL experiment, when the effect target cell ratio was 1:1, there was no significant difference between groups (P>0.5), but with the increase of the effect target cell ratio, compared to Ad5-control group and PBS group, the cell killing ability of Ad5-pCEA-Hsp70 group was significantly increased (P<0.05, P<0.01).Conclusion Recombinant adenovirus Ad5-pCEA-Hsp70-mediated Hsp70 gene expression can inhibit the tumor growth definitely in rat animal model of pancreatic cancer. The mechanism is related to the promotion of DC maturation, induction of cytokines INF-γ, TNF-α, IL-6 secretion, and promoting CD4 + T and CD8 + T lymphocyte infiltration.From the above results, the following conclusions can be made:In our study, we cloned the Hsp70 gene and CEA promoter at first, and then constructed the recombinant adenovirus containing Hsp70 gene driven by CEA promoter. Animal experiments showed that the recombinant adenovirus Ad5-pCEA-Hsp70 -mediated Hsp70 gene expression inhibited the tumor growth definitely in rat animal model of pancreatic cancer. The mechanism was related to inducing anti-tumor immune response, and the body had no obvious toxicity. Our research provides theoretical and experimental basis for the further use of the recombinant targeted in treatment of pancreatic cancer patients.