Antitumor Effects Of Interleukin-18Transected Rat Glioma Cells In Vitro And In Vivo

Objective: Retroviruses were used to transduce the mIL-18gene into ratglioma cells and the cell clones (9L/IL-18) which steadily expressing mIL-18gene were obtained; to observe the influence of the exogenous IL-18gene onthe biological characteristics of rat9L glioma cells in vitro and in vivo; toexplore the antitumor activity of IL-18and the related mechanisms to providbasis for clinical application.Methods:1The supernatant of PA317/1L-18and PA317/LXSN cells was collectedto incubate with9L cells and stable clones expressing IL-18gene (9L/IL-18)and the control cells of9L/LXSN were obtained by screening with G418.2Expression of IL-18gene in the9L/IL-18,9L/LXSN and9L cells wasdetected with RT-PCR; IL-18expression in9L/IL-18,9L/LXSN and9L cellswas analyzed with immunocytochemical assay; The level of IL-18secreted by9L/IL-18,9L/LXSN and9L cells and the IFN-γproduction by rat splenocytesinduced by IL-18were detected by ELISA.3MTT was used to analyze the growth of9L/IL-18,9L/LXSN and9Lcells in vitro.4Expression of MHCⅠ, MHCⅡ, CD80and CD86on the cell surface,the distribution of cell cycle and cell apoptosis in different cells were assessedby Flow cytometry.5The cell suspension of9L/IL-18,9L/LXSN and9L cells were inoculateinto the brain of F344rat to establish the animal models by the stereotactictechnique to investigate the role of different cells in tumorigenicity in vivo.6Flow cytometry was used to analyze the expression of MHCⅠ, MHCⅡ, CD80and CD86on the surface of tumor cells from rats implanted with9L/IL-18,9L/LXSN and9L cells and the percengage of CD4+, CD8+T cells in the peripheral blood of rats, calculated the ratio of CD4+/CD8+.7The level of TGF-βin the peripheral blood of rats was measured byELISA.8Cell apoptosis in tumor tissues was examined by TUNEL.9RT-PCR was used to analyze expression of IL-18, Bax, Bcl-2, Fgf2mRNA in tumor tissues, and the expression of Bax, Bcl-2protein wasinvestigated by Western Blot.10Angigenesis in tumor tissues was observed by HE staining.Results:19L/IL-18cells which express IL-18mRNA and produce IL-18proteinwere obtained through screening by G418; the level of IL-18in thesupernatant of9L/IL-18cells was112.00±4.30ng/L;In the presence of Con A,the culture supernatant of9L/IL-18cells can induce the production of IFN-γ(28.43±1.81pg/ml) by rat splenocytes which was higher than that of9L/LXSN cells (12.56±0.85pg/ml) and9L cells (10.43±0.57pg/ml)(p<0.05).2There was no apparent change of cell morphology among9L/IL-18,9L/LXSN and9L cells; the proliferation of9L/IL-18cells was slower than9L/LXSN and9L cells in vitro (p<0.05).3The expression of MHCⅠ, MHCⅡ, CD80and CD86on the surface of9L/IL-18,9L/LXSN and9L cells had no significant differences (p>0.05).The distribution of cell cycle in9L/IL-18cells revealed by flow cytometrywas: the G0/G1phase:59.70±2.02%, the G2/M phase:16.80±2.04%, the Sphase:23.70±3.12%compared with9L/LXSN (49.30±2.07%,19.10±1.94%,31.60±2.10%) and9L (35.70±2.33%,15.80±2.10%,48.50±2.63%), cellsin the G0/G1phase increased, in the S phase decreased (p<0.05).4The tumor-forming rates of9L/IL-18,9L/LXSN and9L cells were all100%, while the rat survival time implanted with9L/IL-18cells was22.75±1.11days, which was longer than9L/LXSN (15.00±0.91days)and9L (16.50±1.04days)(p <0.05).5Expressions of MHCⅠ, CD80and CD86in the tumor tissues from 9L/IL-18inoculated rats were (67.51±1.40%,12.51±1.57%,6.95±0.56%)which were higher than9L/LXSN (60.31±1.01%,6.77±0.86%,2.93±0.29%) and9L cells (61.08±0.46%,7.04±1.24%,3.06±0.28%) inoculatedrats respectively (p<0.05), while the expressiones of MHC Ⅱ were nosignificant difference (p>0.05).6The percengage of CD8+T cells in the peripheral blood of ratsinoculated with9L/IL-18was32.30±2.22%, which was higher than9L/LXSN (22.02±0.67%) and9L cells (22.34±0.76%) inoculated rats(p<0.05), the percengage of CD4+T cells and the ratio of CD4+/CD8+T weredecreased (p<0.05).7The level of TGF-βin the peripheral blood of rats inoculated with9L/IL-18cells (111.55±5.93ng/L) was weakend to that inoculated with9L/LXSN (127.89±4.01ng/L) and9L cells (133.47±3.68ng/L)(p<0.05).8Apoptosis ratio in9L/IL-18cells inoculated rats was higher than that of9L/LXSN and9L cells (22.20±1.02%,5.70±0.32%%and6.40±0.51%,respectively)(p<0.05).9Compared to9L/LXSN and9L cells inoculated rats, the expression ofIL-18and Bax were increased, while Bcl-2and Fgf2were decreased in thetumor tissues of rats inoculated with9L/IL-18cells.10The new blood vessels in the tumor tissues of9L/IL-18cells inoculatedrats were less than9L/LXSN and9L cells inoculated rats.Conclusion:1Morphology, the expression of MHCⅠ, MHCⅡ, CD80and CD86onthe surface of9L cells and the apoptosis ratio of9L cells were not affected bytransfected IL-18, but the distribution of cell cycle was changed: cells in theG0/G1phase increased, in the S phase decreased in vitro.2IL-18enhanced the tumor cells antigenicity and antigen presentation byup-regulate the expression of CD80, CD86and MHCⅠon the tumor cells,IL-18could decrease the level of TGF-βand enhance the ratio of CD8+T cellsin the peripheral blood of rats to improve the effect of immune system in vivo.3IL-18could induce antitumor activity through increasing cell apoptosis ratio and antiangiogenic activity by up-regulate the expression of Bax,down-regulate the expression of Bcl-2and Fgf2in vivo.