Study On Antitumor Activity Of IL-27 Gene Transfected Human Pancreatic Cancer Cells

Objective: Pancreatic cancer is the most serious tumor in digestive system, which is hardly to detect at early stage. However, it often has lymphoid nudes metastasis at advanced stage. In recent years, tumor biotherapy is becoming a new method and more and more people have pay attention to it. Cytokine gene therapy is one of the biotherapeutic strategies for tumors, in which cytokine genes are transduced into tumor cells or other immune effector cells, which can secret cytokines, inducing tumor cell apoptosis and strengthening the immune functions to accelerate tumor regression. Interleukin-27 (IL-27) is a novel IL-6/IL-12 family member [1], reported by Pflanz in 2002. IL-27 plays a wide role of immunological regulation in various kinds of cells of natural and adaptive immune system. Besides the important contribution to the Th1 reaction, IL-27 has various biological activities [2], such as anti-infection, anti-tumor and inducing inflammatory reaction. But, for IL-27, some mecha- nisms biological activily is still not clear. In this study, we transfected IL-27 gene into human pancreatic cancer cell line (Aspc1), set up the animal model to investigate its antitumor mechanisms.Methods: 1 Establishment of IL-27 gene transfected cell line Retrovirus vector LXSN was used to transfect IL-27 into Aspc1 cell selected by G418. Meanwhile transfcted LXSN and Aspc1 cells as control groups.2 Observation morphologic change of Aspc1/IL-27,Aspc1/ LXSN and Aspc1 cells by light microscope, IL-27 gene expression was detected by RT-PCR and the secretion of IL-27 protein was measured by ELISA.3 The production of IFN-γand IL-4 induced by IL-27 were detected by ELISA from human peripheral blood mononuclear cells (hPBMCs).4 Cell proliferation analysis MTT was used to detect the proliferation of Aspc1/IL-27, Aspc1/LXSN and Aspc1cells in vitro and draw the growth curve.5 Expression of molecular on cell surface Flow cytometry was used to analyze the expression of CD80, CD86 and MHCI, MHCII molecular on the surface of Aspc1/IL-27, Aspc1/LXSN and Aspc1 cells.6 Animal experiment in vivo Aspc1/IL-27, Aspc1/LXSN and Aspc1 cells were subcutaneously inoculated into nude mice respectively and the tumor volumn and the survival time were observed. RT-PCR was used to detect the expression of IL-27 in tumor tissues. The IFN-γsecretion in spleen cells was measured by ELISA.7 Cell apoptosis analysis Cell apoptosis in vitro and in vivo were analyzed by flow cytometry and the ultramicrostru- cture of apoptosis was observed by electron microscope.8 Analysis of expression of Fas and Survivin RT-PCR was used to investigate gene expression of Fas and Survivin in the transplanted tumor tissues of Aspc1/IL-27,Aspc1/LXSN and Aspc1 cells, the protein expression was detected by Western-blot.Results: 1 Aspc1/IL-27 and Aspc1/LXSN cells were obtained after G418 selection. Positive expression of IL-27p28 and IL-27EBI3 gene in Aspc1/IL-27 cell were showed by RT-PCR. ELISA result indicated that the supernature of Aspc1/ IL-27 cell could produce higher level of IL-27 compard to Aspc1/LXSN and Aspc1 cells (P<0.01), suggested that IL-27 gene transfected into pancreatic cancer cell lines in gene and protein level.2 The supernature of Aspc1/IL-27 cell could induce hPBMCs to produce higher level IFN-γthan that of Aspc1/LXSN and Aspc1 cells (P<0.01). However, for IL-4 production, there was no significance difference in the three groups (P>0.05).3 The growth of Aspc1/IL-27 cells was similar to that of Aspc1 cells and Aspc1/LXSN cells in vitro (P>0.05).4 There was no difference of expression of CD80, CD86 and MHCI, MHCII molecular in Aspc1/IL-27, Aspc1/LXSN and Aspc1 cells (P>0.05).5 The growth of tumor in nude mice inoculted with Aspc1/IL-27 cells was significantly retarded (P<0.05) and the survival time was longer compared to that of Aspc1/LXSN and Aspc1 groups (P<0.01). IFN-γproduction from spleen cells of Aspc1/IL-27 inoculted mice was higher than that of two control groups (P<0.01).6 There was no difference of cell apoptosis rate in Aspc1/IL-27, Aspc1/LXSN and Aspc1 cells in vitro (P>0.05). But, after the inoculation nude mice, cell apoptosis rate of Aspc1/IL-27 group was higher than that of Aspc1/LXSN and Aspc1 groups (P<0.01). Electron microscopc also showed the morphology of apoptosis.7 Compared to Aspc1/LXSN and Aspc1 groups, the expression of Fas was increased (P<0.01) and Survivin is decreased (P<0.01) in the tumor tissue of nude mice inculated Aspc1/IL-27 cells detected in gene and protein level.Conclusions: Successfully establish IL-27 transfacted pancreatic cancer cell line (Aspc1/IL-27) with the retrovirus vector. The gene transfaction of IL-27 had no effect on cell growth in vitro; IL-27 could induce the secretion of IFN-γby hPBMCs. In nude mice, IL-27 could decrease the expression of Survivin and increase the expression of Fas in tumor tissue to induce cell apoptosis, which was one of the antitumor mechanisms of IL-27.