Study On Combination Gene Therapy For Gastric Cancer

Backgroud and Objective:Gastric cancer (GC) is one of the most common malignancies, the incidence rate has been increasing year by year. Most patients with GC are diagnosed at an advanced stage because of non-specific clinical symptoms. Despite a variety of traditional treatments, the prognosis for patients with advanced GC remains poor, and the recurrence rate is still high. New therapeutic approaches with more efficacy are urgently needed. With the development in molecular biology, gene therapy has been researched extensively. However, clinical trials have revealed that single-gene therapy has limited effects. The main reason might be that multiple-gene mutations are involved in the occurrence and development of tumors. These genes could co-ordinate with each other, and promote tumor progression. Thus, combination gene therapy using two or more related therapeutic genes might enhance anti-tumor effect. Suicide gene therapy has received much attention due to its direct killing mechanism and "by-stander effect". Two of the best characterized suicide gene therapy systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminase/5-fluorocytosine (CD/5-FC). Each suicide gene/prodrug system has is own features, for example, HSV-TK/GCV has a stronger killing effect, while CD/5-FC shows a more powerful "by-stander effect". In addition, cell type dependency might exist with one suicide gene therapy system. Therefore, combination of HSV-TK/GCV and CD/5-FC is expected to have synergistic effects. Angiogenesis plays an essential role in tumor growth, infiltration and metastasis, and inhibition of angiogenesis is a new target for cancer therapy. Vascular endothelial growth factor (VEGF) is a most important pro-angiogenic factor, and is overexpressed in the majority of human cancers. VEGF expression level has a close relationship with prognosis and recurrence rate of malignant cancers. RNA interference (RNAi) technology could inhibit gene expression at mRNA level efficiently and specificly. RNAi could downregulate VEGF expression, resulting in a decreased micro vessel density (MVD) and tumor growth delay in several cancer cell lines, such as nasopharyngeal caner cells, colon cancer cells, and gastric cancer cells. To explore the feasibility of combination gene therapy for gastric cancer using suicide gene and anti-angiogenic genem therapy, a combination gene vector, co-expressing VEGF-shRNA and fusion suicide gene yCDglyTK was constructed in this research, and its inhibiting effect on gastric cancer in vivo and in vitro was also studied.Methods:(1) RNAi targeting VEGF was applied to construct an interfering plasmid pGenesil-shVEGF. Then, the VEGF-shRNA expression cassette (including the U6 promoter) was amplified by PCR and subcloned into pcDNA3.1(-)CV-yCDglyTK to build a novel combination gene plasmid pcDNA3.1(-)shVEGF-yCDglyTK. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. (2) A blank plasmid pcDNA3.1(-)null, pGenesil-shVEGF, pcDNA3.1(-)CV-yCDglyTK and pcDNA3.1(-)shVEGF-yCDglyTK were transfected into SGC7901 cells using calcium phosphate nanoparticles (CPNPs), and stable transfected SGC7901 cell lines were established by G418 selection. Expressions of yCDglyTK and VEGF were detected by RT-PCR, Western-blot and immunofluorescence assays. (3) The killing efficacy and "by-stander effecf"of shVEGF-yCDglyTK/5-FC were checked using MTT assays. Plate colony forming assays were carried out to study the impact of combination gene/prodrug on SGC7901 colony forming rate. Levels of 5-FC and 5-FU in cell supernatant were determined by high-performance liquid chromatography (HPLC). Cell apoptosis were detected by Hoechst 33258 staining and flow cytometry. (4) Nude mice bearing gastric cancer were established by transplantation of SGC7901 cells. There were five groups:group A was the blank control and received no treatment; group B received intratumoral injection of CPNPs-pcDNA3.1(-)null complex and intraperitoneal injection of 5-FC; group C received intratumoral injection of CPNPs-pGenesil-shVEGF complex; group D received intratumoral injection of CPNPs-pcDNA3.1(-) CV-yCDglyTK complex and intraperitoneal injection of 5-FC; and group E received intratumoral injection of CPNPs-pcDNA3.1(-)shVEGF-yCDglyTK complex and intraperitoneal injection of 5-FC. The tumor growth was observed. When the test was terminated, tumor tissue was subjected to hematoxylin and eosin staining for histological examination. Immunohistochemistry was performed to study the expression of yCDglyTK and VEGF, and MVD was detected also.Results:(1) Both of the interfering plasmid pGenesil-shVEGF and combination gene plasmid pcDNA3.1(-)shVEGF-yCDglyTK were successfully constructed. (2) Four stable transfectants were established. They were named SGC/null, SGC/shVEGF, SGC/CDTK and SGC/shVEGF-CDTK. In SGC/shVEGF-CDTK cells, yCDglyTK gene expression could be detected, and VEGF gene expression was significantly decreased compared with wild-type SGC7901; (3) In the presence of 5-FC, cell survival rate of SGC/shVEGF, SGC/CDTK and SGC/shVEGF-CDTK was reduced, and the proportion of apoptotic cells increased. SGC/shVEGF-CDTK were the most sensitive to 5-FC (P< 0.01); Conversion efficiency of 5-FC to 5-FU was 92.00% in SGC7901/ shVEGF-CDTK cell supernatant; When SGC/shVEGF-CDTK accounted for 40%, cell relative viability was only (19.00±2.44)%, exhibiting a powerful "by-stander effect". (4) in vivo experiments:Compared with group A and group B, tumor growth in group C, group D and group E was inhibited. CPNPs/shVEGF-CDTK+5-FC system was the most efficient in suppressing tumor growth compared with CPNPs/shVEGF therapy or CPNPs/CDTK+5-FC threatment. At the end of observation, the growth inhibition ratio in group E was 72.91%. There were obvious cell necrosis in tumor tissue of group E. Immunohistochemistry revealed yCDglyTK expression, decreased VEGF and MVD. Conclusion:(1) The combination gene plasmid pcDNA3.1(-) shVEGF-yCDglyTK was successfully constructed, and in this novel vector, both VEGF-shRNA and yCDglyTK could expressed normally. (2) shVEGF-yCDglyTK/5-FC treatment system could kill SGC7901 cells effectively and exhibit powerful "by-stander effect"; Inducing cell apoptosis might be an important mechanism. (3) Intratumoral injection of CPNPs-pcDNA3.1(-)shVEGF-yCDglyTK, in combination with intraperi-toneal injection of 5-FC could inhibit tumor growth effectively, and reduce neovascularization. Our research suggested a promising strategy for clinical gastric cancer therapy.