| Gene therapy is a promising molecular alternative in the treatment of gastric cancer, including the replacement of defective tumor suppressor genes, the inactivation of oncogenes, the introduction of suicide genes, genetic immunotherapy, antiangiogenic gene therapy, and virotherapy. Replication-deficient adenovirus (Ad vector) is one of the most effective gene transfer systems. The adenoviral vectors offer many advantages for gene delivery. Specifically, adenoviral vectors possess efficient nuclear entry mechanisms and low pathogenicity for humans; they infect most cell types regardless of their growth state; they are easy to propagate to high titers; and they can accommodate large DNA inserts and do not integrate into the host cell genome. Therefore, adenovirus vector is capable of mediating gene transfer with high efficiency and has good prospects in gene therapy.Several factors affect the treatment of gastric carcinoma, one of which is that these patients have a lower immune response. In order to improve host immune function and inhibit tumor growth, many kinds of immunotherapy have been tried, but the conventional immunotherapy against gastric carcinoma shows little effects. Interleukin-12(IL-12) has generated much interest due to its central role in the immune system and its potential anti-tumor effects. Among all the cytokines tested, IL-12 seems to possess the strongest anti-tumor ability. IL-12 plays a key role in activating cellular immunity by augmenting the cytotoxic activity of T-cells and NK cells, by inducing the production of IFN-γand other cytokines, and by promoting the differentiation of uncommitted T cells (Th0) to Th1 cells which subsequently initiate the cell-mediated immunity pathway. An antiangiogenic effect of IL-12 has also been reported. In fact, systemic administration of IL-12 has been proved highly effective in inducing tumor regression and reducing metastases in diverse murine models of solid tumors. However, severe hematologic, hepatotoxic and muscular side effects limit the systemic administration of recombinant IL-12 as an anti-tumor agent.With the progress of molecular biology and immunotherapy, cytokines gene therapy could provide an alternative method. The gene IL-12 can be inserted into the genome of the adenovirus vector and can be highly expressed in the tumor cells by the viruses. The new scheme that can exploit the virtues of virus therapy and gene therapy is named gene-virus therapeutics. Nevertheless, constitutive or inappropriate expression of the IL-12 gene with traditional expression system may interfere with the effect of the gene therapy,and may even lead to lethal side effect, so it is important to build regulations governing the gene expression level and expression time in gene therapy and acquisition of the gene function. Inducible expression systems can be used to regulate the expression level of genes of interest in appropriate time according to the aim of the experiments and gene therapy. Up till now, several inducible expression systems have been established and become the potent tools for gene therapy and studying the function of genes of interest. One of the most promising regulatory systems is the mifepristone/RU486-regulated system, which has much lower basal transcriptional activity and high inducibility.Gastric cancer is one of the most prevalent malignancies and remains an important cause of death worldwide. The incidence of primary malignant gastric carcinoma ranks the fourth and the mortality ranks the second in the world. The Situation is worse in China. The latest statistics shows that every year in China 400,000 people suffer from gastric cancer and about 300,000 people die of it. The gastric cancer is the third common cancer and the leading cause of cancer mortality in China. The morbility and mortality of gastric cancer are twice as high in china as in the world. The prognosis for patients with primary malignant gastric carcinoma remains poor, in spite of various different forms of treatment. There is still no effective treatment for patients with advanced gastric carcinoma. Surgery, radiotherapy, and chemotherapy or combined therapeutic procedures rarely succeed in curing the disease. It is urgent to seek new strategies for this disease.In our studies, the RU486-regulated system was introduced into adenovirus genomic DNA and a regulatory adenovirus vector carrying interleukin-12 was constructed. The new regulatory vector can be used to regulate the expression level of genes of interest in appropriate time by the inducer RU486. This regulatory expression recombination adenovirus vector can provide a platform for the gene therapy of gastric caner. 1.The preparation and construction of the regulatable adenovirus vector carrying mouse Interleukin-12 gene.(1)Ad-RUmIL-12 adenovirus vectors were constructed through recombination technique. The PRS plasmid vector encoding a GLP65 chimeric regulator and GAL4 hybrid promoter was modified with molecular biological methods. The BGHpolyA fragment was amplified by PCR technique and introduced several enzyme cutting sites. A hCMV promoter replaced the TTR promoter to control the GLP65 regulator, and mIL-12 gene was inserted into downstream of the GAL4 promoter. To minimize any potential interference, the two transcriptional elements were spaced with a 1.2kb chicken beta-globin insulator. The double cassettes were cloned into the plasmid pDC313 to construct shuttle plasmids PDC-RUmIL-12. The recombination vector was identified by different restriction endonuclease reactions, sequencing analysis and PCR assay. (2) Recombinant adenoviruses were generated by Admax system. The shuttle plasmids PDC-RUmIL-12 were cotransfected HEK 293 cells with plasmid pBHGlox(delta)E1,3Cre(circularized adenovirus genome) to achieve recombinant adenoviral vectors. The cytopathic effects and PCR using primers specific for mIL-12 were used to identify the recombinant adenoviral vectors. The correct recombinant adenovirus was named Ad-RUmIL-12.Then Ad-RUmIL-12 were transfected into the 293 cell and amplified, and purified by cesium chloride density purification, titrated by TCID50 method and then stored at -80℃for usage. Finally the titers of Ad-RUmIL-12 were the 4.62×1010 pfu/ml.(3) In order to identify the effect of the RU486-regulated system, recombinant adenoviruses Ad-RULUC and Ad-RURED which contain the reported gene Luciferase and DsRed were generated in the same way.2 . Effects of the regulatable adenovirus vector carrying mouse Interleukin-12 gene in vitro.(1) Transient Cotransfection and Luciferase Assays: MFC cells were plated in complete medium to achieve 70–80% confluence. Transient transfections of the PDC-RULUC using Polyfect Transfection Reagent(Qiagen) followed manufacturer's recommendations. pGL3-control and pGL3-basic were used as positive and negative control respectively. All groups were cotransfected with pGL-TK Renilla luciferase reporter vector (Promega) to normalize for transfection efficiency differences. Cells were incubated Forty-eight hours in different concentration of RU486 after transfection and harvested for luciferase assays by using the Dual-Luciferase Reporter Assay System (Promega). In the presence of RU486 a 40-fold maximum activation of the luciferase reporter gene was observed in cultured cells, whereas in the absence of RU486, no significant activation was observed. There was a positive correlation between the luciferase activation and RU486 concentration in a definite range. (2) Expression of Ad-RURED in MFC Cells:MFC Cells were infected with Ad-RURED at a MOI of 100 pfu/cell. Different dosage of RU486 was added in each hole after infection. As a control, RU486 was not added in the two holes.DsRed expression was followed by observing the cells under a fluorescence microscope. Infected cells were also subjected to flow cytometry analysis by use of the FCM apparatus. The data demonstrated that the positive cell was very few without inducer RU486,and proportion of the positive cell increased in parallel with RU486 concentration under fluorescence microscopy and FCM.(3) mIL-12 was expressed in gastric cancer MFC cells transfected with recombinant adenovirus:After generating the adenoviral particles, we examined the ability of the viral vector to infect MFC cells and regulate expression of mIL-12 in cell culture. The infection was carried out for 2h at MOI=100, then the medium was changed, and mifepristone was added as appropriate. mIL-12 was measured in the medium after 48 h by a ELISA. Adenoviral vectors regulated the expression of mIL-12 in a RU486- dependent manner and highly express in the presence of RU486 up to 1×10-6(mol/L) of medium, whereas Ad-RU-mIL12 seemed to express mIL-12 at a very low level in the absence of the ligand. The level of the expression of the mIL-12 was in a dose-dependent manner with respect to RU486 concentration. Besides, if mIL-12 was measured again 72 h after changing fresh medium without RU486, the expression deceased significantly.3.Treatment of gastric cancer in vivo by the regulatable adenovirus vector with Interleukin-12 gene.(1)615 mice (4–6 weeks old) with normal immune response were used for in vivo studies. Tumor xenografts were established by subcutaneous injection of 5×106 MFC cells into the right flank of mice. After a week, when the diameter of tumors reached about 6-7mm, mice were randomly divided into four groups (PBS,Ad-RUmIL-12,Ad-RUmIL-12+RU486 and Ad-mIL-12, n=12). To assess the ability of the adenoviral constructs to effect regulatory expression of mIL-12 in vivo, we infected 12 mice with 1.5×1010 infectious viral particles by tumor injection. The adenoviral particles were injected in tumors alternate day for 5 times. RU468 was administrated intraperitoneally at 0.05 mg/kg body weight. Tumor size was determined by measuring two diameters perpendicular to each other with a caliper every 3 days, and tumor volumes were estimated by using the equation V=1/2×length×width2. The survival time was also calculated.(2)measure of the tumor apoptosis :Two of the mice were killed by cervical dislocation after treatment in seven days, others were still observed. Intratumoral administration of a recombinant adenovirus encoding IL-12 to animals combination with RU486 caused obviously tumor regression in most animals and increased longterm survival rate. Ad-RUmIL-12 group showed little anticancer effect on gastric cancer model compared with the Ad-RUmIL-12+RU486 group. The tumors were made to monoplast suspension and measured with FCM after labeling with Annexin V-FITC. The percentage of cells in each phase was calculated with ModFit software. Flow cytometry (FCM) demonstrated that obvious apoptosis occurred in Ad-RUmIL-12+ RU486 and Ad-mIL-12 group, and few apoptosis cells were founded in the other two groups.(3)measure of serum mIL-12 : The transgene expression in Ad-RUmIL-12 group remained as low as in PBS group. There was no signification different between these them. At day 7 from viral and RU486 infection the concentration of serum mIL-12 reached high level in Ad-RUmIL-12+ RU486 and Ad-mIL-12 group, whereas at day 14, the levels sharply decreased in Ad-RUmIL-12+ RU486 group, but the levels remained high in Ad-mIL-12 group. These results indicate that the expression of Ad-RUmIL-12 vector can be regulated by administration of RU486 in vivo. Conclusion : Above all, the regulatory recombination adenovirus vectors containing a mifepristone (RU486) inducible system was successfully constructed for controlled expression of mouse IL-12.When the vectors was induced by the RU486, the IL-12 gene can be highly expressed in the gastric carcinoma cells. Adenovirus vectors can be well regulated expression of IL-12 protein, which effectively induce anticancer immune responses. In conclusion, regulatory recombination adenovirus vectors provide us an effective and safe tool for gene therapy of gastric cancer in the future.
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