Experimental Study Of Human Colon Cancer Cells Transfected With Interleukin-18 Gene And Its Antitumor Effects

It has long been assumed that the immune system may play an important role in controlling tumor growth and development. However, the immune interaction between tumor and host appears to be much more complex and difficult than the original immune surveillance theories suggested. A number of very rigorous conditions must be met for effective anti-tumor immunity to be induced that is capable of destroying tumor cells.The ability of various cytokines to hamper tumor growth or to induce antitumor immune response has resulted in their studies as antitumor agents in gene therapy approaches. In this study, we will concentrate on the antitumor immune responses using modification of SW480 cells by cytokine IL-18 gene.IL-18, formerly called interferon- λ (IFN- λ ) inducing factor(IGIF), is a recently discovered cytokine that plays an important role in the Thl response, primarily by its ability to induce IFN-λ production in T cells and natural killer (NK) cells. IL-18 is produced by Kupffer cells, activated macrophages, keratinocytes, intestinal epithelial cells. Numerous investigations have noted the importance of IL-18 as a Th1 cytokine, especially in cooperation with IL-12, in antitumor immunity. At the tumor site, locally decreased IFN production after decreased or abolished IL-18 production was correlated with an unfavorable outcome for patients with colon carcinoma. IL-18 stimulates NKcells, T cells, B cells and cells of the monocyte lineage to express IFN- y at high level. Furthermore ,IL-18 plays an important role in T cells proliferation, CTL activation and enhancement of NK cell activity primarily through Fas-FasL mechanism. Systemic administration of rIL-18 is associated with significant in vivo antitumor effects that seem to be primarily mediated by enhanced NK cells activity. Recent studies including one using IL-18-deficient mice, support the notion that IL-18 plays an important role in the development of cellar immunity (Thl response) following antigen presentation. These informations suggest that IL-18 has an important role in local antitumor immune responses.Many studies have demonstrated that tumor cells transduced with cytokine genes showed increased immunogenicity and decreased tumorigenicity, and that tumor vaccine prepared with tumor cells transfected with cytokine genes can induce potent antitumor cellular immunity. IL-18 exhibits antitumor activity in various tumor models. IL-18 induced the sequential activation of NK cells and CTL to protect syngeneic mice from transplantation with Meth A scarcoma cells. Protective immunity was induced by the immunization with colon carcinoma cells genetically modified to express IL-18. Colon 26 cells transduced with the IL-18 gene (colon26/IL-18) could not form subcutaneous tumors in immunocompetent mice, and the mice became resistant to rechallenge with wild-type colon 26 cells.Part one: Experimental study of construction of recombinant plasmid containing secreted human IL-18Aim: To construct the eukaryotic expression plasmid of human IL-18 cDNA possessed by a proper structure and biological activities.Methods: The human IL-18 cDNA was obtained by RT-PCR amplification from human normal heaptic tissue, and the pGEM-T-Easy-hIL-18 plasmid was constructed. The pGEM-T-Easy-hIL-18 plasmid and the eukaryotic expression plasmid pcDNA3.1 containing a IgG kappa signal peptide gene were enzymolysed by ECOR I and BamH I. The two enzymolysed fragments were connected with T4 Ligation enzyme and the hIL-18 eukaryotic expression plasmid pcDNA3.1-hIL-18 was constructed successfully.Results: IL-18 mRNA was extracted with Trizol from human normal heaptic tissue. Theresults of RT-PCR and Sequencing showed that the fragment of IL-18 were inconformity with the sequence from Gene bank.Conclusion: The eukaryotic expression plasmid of human IL-18 Cdna was constructedsuccessfully.Part two: A study of interleukin-18 transduced into human colon adenocarcinoma SW480 cells.Aim: To study the effect of interleukin-18 gene transfer into colon carcinoma cell lineSW480 cells.Methods: SW480 cells were...