Study On The Antitumor Effects Of IL-17 Gene Transfected Mouse Breast Cancer Cells

Objective: In the past few years, the biotherapy is becoming a new method for tumor and a lot of people have paid attention to it. The 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, induce tumor cells apoptosis and strengthen the immune functions to accelerate tumor regression. In this study, we transfected interleukin-17 gene into mouse breast cancer cells(4T1), set up the animal model to investigate its antitumor mechanisms.Methods: 1 By plasmid vector, IL-17 gene was transfected into mouse breast cancer cells (4T1) and stable clones expressing IL-17(4T1/IL-17) were obtained by selecting with G418. Meanwhile transfected empty plasmid vector(pcDNA3.1) and 4T1 cells were as control groups.2 The morphologic changes of 4T1/IL-17, 4T1/ pcDNA3.1 and 4T1 cells were observed by light microscope, IL-17 gene expression was detected by RT-PCR and the secretion of IL-17 protein was measured by LSM and Western-blot .3 The proliferation of cells in vitro was detected by MTS and then draw the growth curve. Flow cytometry was used to analyze the expression of MHCI, MHCII and LFA-1 molecular on the surface of 4T1/IL-17,4T1/pcDNA3.1 and 4T1 cells.4 The production of IL-6 from mouse macrophage RAW264.7 induced by IL-17 was detected by ELISA.5 4T1/IL-17, 4T1/pcDNA3.1 and 4T1 cells were subcutaneously inoculated into mice respectively and the tumor volumn and the survival time were observed. Cell apoptosis in vitro and in vivo were analyzed by flow cytometry. The expression of the MHCI, MHCII and LFA-1 molecules on the surface of tumor cells were also detected by flow cytometry. LDH releaese, MTS were used to determine CTL activity, proliferation of the splenocytes respectively. The production of IFN-γand IL-12 from splenocytes were detected by ELISA.Results: 1 4T1/IL-17 cell stable expressing IL-17 were successfully set up. IL-17 gene expression in 4T1/IL-17 cell was detected by RT-PCR.The protein level was showed by LSM and Western-blot, which suggested that IL-17 gene transfected into breast cancer cell lines and could express in gene and protein level.2 The growth of 4T1/IL-17 cell was similar to 4T1 cell and 4T1/pcDNA3.1 cell in vitro (P>0.05). There were no differences of expression of MHCI, MHCII and LFA-1 molecular in 4T1/IL-17, 4T1/pcDNA3.1, 4T1 cells (P>0.05).3 The supernatant of 4T1/IL-17 cell could induce RAW264.7 cell to produce higher level of IL-6 than 4T1/pcDNA3.1 and 4T1 cells (P<0.01).4 The growth of tumor in mice inoculted with 4T1/IL-17 cells was significantly retarded (P<0.05), and the survival time was longer compared to 4T1/pcDNA3.1 and 4T1 groups (P<0.05).5 There was no difference of cell apoptosis rate in 4T1/IL-17, 4T1/pcDNA3.1, 4T1 cells in vitro (P>0.05). But, in the mice of inoculation, cell apoptosis rate of 4T1/IL-17 group was higher than those of 4T1/pcDNA3.1, 4T1 groups (P<0.05). The expression of MHCI, MHCII and LFA-1 molecules in the tumor tissues from 4T1/IL-17 inoculated mice were also higher than two other groups (P<0.01).6 The CTL activity and proliferation of the splenocytes from 4T1/IL-17 inoculated mice was higher than those of 4T1/pcDNA3.1, 4T1 groups (P<0.01). The splenocytes from the mice inoculated with 4T1/IL-17 cells can secret higher IFN-γand IL-12 than two other groups (P<0.01). Conclusions: Successfully constructed the IL-17 transfected breast cancer cell line (4T1/IL-17) with the pcDNA3.1 vector. The gene transfection of IL-17 had no effect on cell growth and apoptosis in vitro; But in the mice inoculated with 4T1/IL-17, IL-17 could enhance the immune function and induce the antitumor activity in mice.