Studies Of IL-24 Gene Targeting To Kill Ovarian Cancer Cells And Its Influence On Chemtherapy Drug Sensitivity In Vitro

Ovarian cancer has a third morbidity in the female reproductive system malignancies, and the mortality rate is the first. Due to lacking of effective preventive measures and screening methods to Ovarian cancer, which invades hidingly,75%-80% of patients present with advanced stage disease while diagnosed. Although the majority of patients who received initial surgery and chemotherapy could be eased, the recurrence rate is still high, which makes 5-year survival rate is always at around 30%. Therefore, finding new therapy strategies of ovarian cancer is urgent for the truth of lacking of an effective treatment.Recent years, several gene therapy strategies have been used in the treatment of ovarian cancer, such as molecular chemotherapy, mutation compensation, immune enhancement, oncolytic virus, RNAi and multi-gene therapy, etc, some of which have been applied in clinical trials, but the efficacy and safety of gene therapy has always been the limites of clinical applications in a wide range. In order to improve the safety of gene therapy, people began gene targeted therapy, the aim gene is highly transfered into tumor cells by vector system, and only kill the tumor cells without affecting the normal tissue. Meanwhile, in order to improve the efficacy of gene therapy, people are constantly looking for effective tumor-killing genes, as well as which is combined with chemotherapeutics to enhance the effectiveness of gene therapy.These years, some ovarian tissue specific promoters have been reported one after another, such as cytochrome P450 aromatase (P450c19/CYP19) promoter, inhibin a promoter and Mullerian inhibiting substance typeⅡreceptor (MISⅡR) promoter, etc. On the following study, researchers found that these promoters also express in non-ovarian tissues, and their non-highly specific to ovarian tissue limits the application in researches and clinical trials. Ovarian-specific promoter-1(OSP-1) is a retro virus-like elements, which expresses specifically in the mouse ovary tissue, acts highly in majority ovarian cancer cells and normal ovarian cells but none in non-ovarian cells. Some vivo and vitro studies confirmed that the promoter can start tumor-killing genes expressing specifically in ovarian cancer cells. Therefore, specific expression of tumor therapy gene in ovarian cancer tissue can be achieved by OSP-1 promoter, which can minimize side effects to other organizations and enhance the safety of gene therapy for ovarian cancer.The ideal therapy gene should inhibit the growth of tumor cells selectively as well as result in cell apoptosis, while it has anti-angiogenic effect and strong bystander effect, which means transduced cells have cytotoxicity on non-transduced cells, so that expanding the anti-tumor effect and inhibiting metastasis and diffusion of tumor. Combinating with other treatments (radiotherapy or chemotherapy), the gene can create synergy, which can improve the efficacy of gene transfer in many ways, so that change the low effect of tumor gene therapy. Interleukin-24 (IL-24) is a multi-functional tumor suppressor gene, which has these advantages mentioned above. It has been hailed as the tumor's "magic bullet", based on the good preliminary experiments of IL-24 gene therapy for Cancer, In this study, IL-24 gene therapy is to be applied for ovarian cancer At present, the physiological function of IL-24 is not clear, what will happen to human system after receiving high dose IL-24 therapy is unknown. How to consolidate and strengthen the anti-tumor capacity of IL-24 and minimize the side effects to improve security at the same time is the key to widely clinical application of IL-24. Therefore, our study plans to adopt gene-targeting therapy in the application of IL-24 gene therapy for ovarian cancer, so that IL-24 gene only expresses in the ovarian cancer cells, concentrating on tumor tissue to avoid the potential side effects to human system. Meanwhile, we want to detect whether IL-24 gene can enhance the sensitivity of ovarian cancer cells to chemotherapeutic drugs or not, such as paclitaxel and cisplatin, as well as explore the mechanism, so that we can try to provide experimental basis for IL-24 gene therapy for ovarian cancer safely and efficiently in clinical. This study was as follows:1. Construct eukaryotic expression vector targeting ovarian cancer cellMethods:Compose OSP-1 promoter through gene synthesis method, insert the promoter fragment into the eukaryotic expression vector named pcDNA3.0 by genetic recombination method, which replaces the cytomegalovirus promoter and enhancer sequences in the vector, so that pcDNA3.0-OSP-1 drived by OSP-1 was constructed. On this basis, clone the report gene namedβ-galactosidase gene (β-gal) into the vector, ensure it is in the downstream of OSP-1 promoter to construct pcDNA3.0-OSP-1--β-gal vector, of which report gene is drived by OSP-1. Then we transfer pcDNA3.0-OSP-1--β-gal vector into ovarian cancer cell line SKOV3, human hepatoma cell line HepG2 and human fibroblast cell line BJ cells by Liposomes, and obeserve the expression of (3-gal in the three groups of cells byβ-galactosidase staining cells in situ afer 72h.Result:In these three groups of cells, only SKOV3 cells expressβ-galactosidase, while the other two groups not.Conclusion:The pcDNA3.0-OSP-1--β-gal vector can drive foreign gene expressing specifically in ovarian cancer cells.2. Specific expression of IL-24 gene in ovarian cancer cellsMethod:Human IL-24 cDNA fragments was amplified from peripheral blood mononuclear cells by RT-PCR, clone the products of RT-PCR into T-vector, then identified the results by DNA sequencing. We clone the IL-24 gene into pcDNA3.0 and pcDNA3.0-OSP-1 vector by genetic recombination method respectively, construct pcDNA3.0-IL-24 and pcDNA3.0-OSP-1-IL-24 expression vectors. These two expression vectors are transferred into ovarian cancer cell line SKOV3, human hepatoma cell line HepG2 and human fibroblast cell line BJ cells by Liposomes, after the stable selection of G418, we detect the expressing level of IL-24 gene mRNA in cells by RT-PCR and the protein product of IL-24 gene in cell supernatant by ELISA.Result:In the three groups of cells transfected with pcDNA3.0-IL-24 expression vector, the expressing levels of IL-24 gene mRNA in cells and the protein product of IL-24 gene in cell supernatant are highly more than nontransfected cells, as the same as the SKOV3 cells transfected with pcDNA3.0-OSP-1-IL-24 expression vectors, but the other two groups had no differences between transfected cells and not.Conclusion:The pcDNA3.0-OSP-1-IL-24 expression vectors constructed in this study can achieve specific expression in ovarian cancer cells.3. Inhibition of IL-24 gene targeting of ovarian cancer cellsMethod:Plate the transfected cells in 24-well culture plates with the dose of 1×104 cells per well, count the number of cells in 8 days by cell counting method. Then describe the growth curve to identify the influence of IL-24 gene on cells, and detect the cell cycle distribution by flow cytometry. Dilute the supernatant collected from the SKOV3 cells to different concentrations, which were stably transfected with pcDNA3.0-OSP-1-IL-24 plasmids and cultured for 6 days,then deal SKOV3 cells with the supernatant。After 24 hours,detect the cells activity by MTT, as well as 48 hours and 72 hours later.Result:In the three groups of cells transfected with pcDNA3.0-IL-24 expression vector, there is no difference between transfected cells and nontransfected cells in the growth curve of BJ cells. While, the growth of SKOV3 and HepG2 cells were significantly inhibited since the 5th day, compared with nontransfected cells, there are distinct differences. In the three groups transfected with pcDNA3.0-OSP-1-IL-24 expression vector, only the growth of SKOV3 cells was significantly inhibited since the 6th day compared to nontransfected cells, and the other two groups had no differences. The result of flow cytometry showed the G1 phase cells of SKOV3 increased mostly and the S phase cells of SKOV3 decreased mostly in the two groups transfected with pcDNA3.0-IL-24 and pcDN A3.0-OSP-1-IL-24 expression vectors comparing to nontransfected cells. The result of MTT showed that the supernatant of the SKOV3 cells stably transferred with pcDNA3.0-OSP-1-IL-24 plasmids could inhibit the growth of SKOV3 cells.The inhibition capability depended on the concentration of the supernatant.Conclusion:The expression production of pcDNA3.0-OSP-1-IL-24 can specifically inhibits the proliferation of ovarian cancer cells, and increases the G1 phase cells. The IL-24 product expressed by pcDNA3.0-OSP-1-IL-24 plasmids can effectively inhibit the growth of non-transfected SKOV3 cells, which play the role of anti-tumot bystander effect.4. Influence of IL-24 gene on the sensitivity of ovarian cancer cells to chemotherapeutic drugsMethod:We use paclitaxel and cisplatin respectively to deal with SKOV3 cells, SKOV3 cells stably transfected with pcDNA3.0-IL-24 and pcDNA3.0-OSP-1-IL-24 respectively, then detect the cells activity by MTT after 72 hours and calulate the cell inhibition rate by chemotherapeutics according to the detected result, as well as the IC50% value of cells inhibiton. Detected the expression of ERCC1and TUBB 3 gene by fluorescence quantitative PCR.Result:The sensitivity of SKOV3 cells transfected with IL-24 gene to paclitaxel and cisplatin were increased evidently comparing to nontransfected cells, and their IC50% values were reduced by 6 times and 10 times respectively, Comparing to nontransfected cells, the expression of ERCC1 and TUBB3 gene mRNA of transfecred SKOV3 cells reduced by 10 times and 4 times respectively.Conclusion:Transfecting IL-24 gene can enhance the senstivity of SKOV3 cells to paclitaxel and cisplatin, and the machanism maybe down-regulating the expression of TUBB3 gene and ERCCl gene.Above all, we successfully constructed eukaryotic expression vector pcDNA3.0-OSP-1-IL-24 targeting ovarian cancer cells, and achieved the specific expression and anti-tumor function of IL-24 gene in ovarian cancer in vitro, and confirmed that transfecting IL-24 gene to ovarian cancer cells can effectively increase their sensitivity of paclitaxel and cisplatin. This study provid strong experimental evidences in viro for improving the safety and efficacy of ovarian cancer gene therapy and applying IL-24 gene trerapy combinating with chemotherapy in Clinical treatment of ovarian cancer.