Targeting Therapy Of Gastric Cancer By CD/TK Double Suicide Genes Driven By Survivin Promoter

Gastric cancer is one of the most common digestive system malignancies in clinical practice. There are more than10million new cancers worldwide every year-Gastric cancer has become the first cause of death of all malignant tumors in some areas of China. In recent years, with the deepening of the stomach cancer research, the pathogenesis and treatment of gastric cancer have made a lot of achievements. However, the diagnosis and treatment of gastric cancer still faces many difficulties, and the overall treatment effect is still not ideal. The clinical characteristics shows "three low and one high", namely the diagnosis is low (about10%), surgical excision rate low (＜70%),5years of survival rate low (about40%), radical surgery relapse and metastasis rate high (about50%).Therefore, to find a new stomach cancer prevention and control method, will have a positive and important practical significance to further improve the overall prevention level and cure of gastric cancer.Cancer gene therapy is usually transferring the exogenous genes into patients using expression vector and different methods, which produce the product to inhibit and kill cancer cells. Suicide gene therapy, also called gene-directed enzyme prodrug treatment, has become a new anti-tumor therapeutic approach after the traditional surgical resection, radiotherapy, chemical drug treatment.Suicide gene generally refers to the prodrug conversion gene or cytotoxic receptor gene from prokaryote or lower organisms (viruses and bacteria),which encode the specificity enzyme can convert the nontoxic prodrug (or cytotoxic factor) into toxic product (or cytotoxic factor) to achieve the purpose of killing cancer cells. At present, a variety of suicide genes have been found, including Escherichia coli cytosine deaminase (CD), herpes simplex virus-thymidine kinase (HSV-TK). Escherichia coli cytosine deaminase gene encodes the cytosine deaminase, which can convert the cytosine into uracil, change5-Fc into cells toxic metabolites5-Fu, to play cytotoxic effects through inhibiting RNA and DNA synthesis. HSV-TK gene encodes thymidine kinase, which can convert the nucleoside analogue (NA) into bisphosphate iodide, further turn into triphosphate compound to inhibit the DNA polymerases to play antitumor function.In cancer gene therapy, one of key issues is to improve the targeting expression of therapeutic gene. The targeted expression, is to make the gene function as in tumor cells as possible, and to reduce the influence to normal cells. Specifically expressed promoter sequence in tumor cells has been widely used, which can drive the downstream gene to express in tumor cells specifically, is an effective way to solve the problems of tumor targeted gene therapy. At present, the embryo carcinoma antigen (CEA) promoter, telomerase gene promoter, etc have been used as the regulated element for tumor target gene therapy, but the promoters have some disadvantages, such as application is limited, regulation effect is weak. Survivin is a member of apoptosis inhibiting protein family, it express highly in most tumors, but do not express or lower express in adult normal terminal differentiation tissue, has the obvious tumour specific. Therefore, survivn specific promoter can be used to drive the gene express specific in the tumor cells, to avoid the damage to normal cells.In the current study, we cloned the survivin promoter and constructed the CD/TK expression vector mediated by survivin promoter, then transfected into gastric cancers cells, established nude BALB/C mouse gastric cancers model, gave certain concentration of5-FC, GCV. We demonstrated that survivin-CD/TK systems have obvious target killing function to gastric cancer subcutaneous BALB/C rats.Our studies include the following four parts. Part I:Cloning of survivin promoter and express specific in the gastric cancer cellObjective:The aim of this study was to construct the recombinant EGFP expression vector driven by survivin promoter, and identify the promoter activity specific in gastric cancer cells.Methods:1. Human genome DNA was extracted from white blood cells. Primers were designed according to the sequence of survivin promoter. Survivin gene promoter was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.2. pMD-survivin vector was digested by double restriction endonuclease to release the survivin fragment, and ligated into the pEGFP-C1vector to generate the recombinant pEGFP-Su, and then identified by enzyme digestion.3. pEGFP-Su plasmid was transfected into gastric cancer SGC7901cells and normal gastric epithelial GES-1cell, and EGFP gene expression was detected by fluorescence microscope.Results:1. Human survivin promoter of910bp long was obtained via PCR method.2. We constructed the recombinant pEGFP-Su plasmid successfully.3. EGFP driven by survivin promoter can express specific in SGC7901gastric cancer cells, but not in the normal gastric epithelium GES-1cell.Part II:Construction and identification of the recombinant pEGS-CD/TK double suicide genes vectorObjective:To construct and identify the recombinant pEGS-CD/TK plasmid using molecular cloning technology Methods:1. E.coli BL21DNA was extracted. Primers were designed according to the sequence of CD gene. CD gene was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.2. pORF-HSVtk was extracted and used as PCR template. Primers were designed according to the sequence of TK gene. TK gene was obtained by PCR technology, and ligated into the pMD18-T vector, followed by digestion with restriction endonuclease and sequencing.3. pMD-CD vector was digested by double restriction endonuclease to release the CD gene fragment,and ligated into the pEGFP-Su vector to generate the recombinant pEGS-CD, then identified them by enzyme digestion.4. pMD-TK vector was digested by double restriction endonuclease to release the TK gene fragment, and ligated into the pEGS-CD vector to generate the recombinant pEGS-CD/TK, then identified them by enzyme digestion.Results:1. CD gene (1281bp) and TK gene (1161bp) were obtained through PCR method.2. We constructed the recombinant pEGS-CD plasmid successfully.3. We constructed the recombinant pEGS-CD/TK plasmid successfully.Part Ⅲ Killing effect of pEGS-CD/TK double suicide gene vector on the Gastric Cancer Cells in vitroObjective:To study the killing effect of CD/TK double suicide genes driven by survivin promoter on the gastric cancer cells in vitro.Methods:1. pEGS-CD/TK plasmid was transfected into SGC7901, GES-1cells using the liposome. 2. The prodrug5-FC and GCV were added into the SGC7901, GES-1cells, and then the cell survival rate was determined by MTT methods.3. The untransfected and transfected SGC7901were mixed, followed by adding the prodrug5-FC, GCV. The bystander effect was observed through MTT methods.4. CD, TK gene expression was detected by RT-PCR and Western blot.5. Cell apoptosis rate was detected by flow cytometry.Results:1. SGC7901cell survival rate decreased with the concentration5-FC, GCV increased, and the killing effect in vitro showed concentration dependent.The killing effect of drug combination on SGC7901cells was stronger than the drug used alone in vitro. However, the5-FC and GCV had little effect on GES-1cells survival rate.2. The transfected SGC7901cells can cause the untranfected cells apoptosis, showed the obvious bystander effect.3. CD, TK gene can express in the transfected in the SGC7901cell through RT-PCR and Western blot assay.4.The transfected SGC7901cells was treated by the prodrug for24h, SGC7901cell apoptosis rate of the treatment group was significantly higher than that of the control group (P<0.01).Part IV:Killing effect of pEGS-CD/TK double suicide gene on the nude mice model of gastric cancer cells in vivoObjective:To investigate the killing effect of CD/TK double suicide genes on the nude mice model of gastric cancer cells in vivo.Methods:1. The nude mice were subcutaneously inoculated by5×106gastric cancer cells, re inoculated at the third day to establish the gastric cancer nude mice model.2. After10days of SGC7901subcutaneous transplantation, the mice were divided into4groups (n=10). Group A:injected with recombinant pEGS-CD/TK and prodrug5-FC and GCV; Group B:only injected with recombinant pEGS-CD/TK; Group C:only injected prodrug5-FC and GCV; Group D:blank control, no any treatment. The size of the subcutaneous transplantation tumor was measured once every3days. The nude mice was killed and stained by HE method, the tumor pathological changes were observed.3. CD/TK gene expression was detected by RT-PCR method.Results:1.Ten days after tumor cells inoculation, subcutaneous tumor nodules appeared.The treatment began after the nude mice tumor achieved0.5cm.Compared with the only injected pEGS-CD/TK, prodrug GCV and5-FC and blank control group, the transplantable tumor size in the injected with pEGS-CD/TK and prodrug GCV and5-FC group was sigficantly reduced, and showed statistically significant (P＜0.05).2.Tumor cells number in the treatment group were visible scarce, had scant cytoplasm, nuclear was smaller and staining was deeper, the cell division was fewer, nuclear and cytoplasm condensed and necrotic area were scattered, fiber blood vessels were fewer, inflammatory cells infiltration was visible.3. CD, TK gene expression could be detected by RT-PCR in the treatment group.Conclusions1. Survivin promoter expresses specific in the gastric cancer cells.2. CD and TK genes have been obtained successfully by gene cloning technology.3. CD/TK double suicide genes plasmid driven by survivin promoter has been constructed.4. CD and TK double suicide genes have stronger killing effect on the SGC7901cells, and show the obvious bystander effect.SGC7901cell apoptosis rate of the treatment group is significantly higher than that of the control group (P＜0.01).5. CD/TK gene express in SGC7901cells successfully.6. Tumorigenic BALB/C nude mice have been successful established using SGC7901cells. 7. CD/TK double suicide gene driven by Survivin promoter can specifilly express in the gastric cancer cells.8. CD/TK double suicide gene driven by survivin promoter can inhibit the growth of gastric cancer in the presence of prodrug drug GCV and5-FC, has an inhibitory effect in vivo.