Antitumor Effects Of Murine Interleukin-23 Mediated By Retrovirus And Related Mechanism

Breast carcinoma is one of the serious diseases threatening female's health in developed country and some big cities of our country. Development of tumors is related to a defect of a host immunosurveillance system and an escape mechanism of tumors from host immune responses. At present, operation, chemothreapy and radiotherapy are the main methods to treat tumors with some limitations. Immunotherapy of tumors which can not only enhance the immune function of host, kill tumor cells in specific way, but also has less side-effects on normal tissues and more and more people have pay attention to immunotherapy. Cytokines possess pleiotropic functions and mediate systemic and local biological actions, which relate to cell growth and differentiation, immune function and inflammation. There are some effects by administration of some cytokines directly injected in treatment of tumors. Because of the short half-life, large and lasting usage and strong side-effects of cytokines, they are limited in clinical application. Therefore, it is necessary to looking for more effective cytokines and new administration approaches. Cytokine gene therapy is one of the therapeutic strategies of tumors, in which cytokine genes are transduced into some vectors such as tumor cells, dendritic cells and fibroblasts. The vectors carried cytokine genes in host can last production of cytokines that can induce immune responses to tumors and overcome the drawbacks of short half-life, large and lasting usage and strong side effects of cytokines by direct administration. Besides, tumor cells and dendritic cells loaded tumor antigens as cytokine gene therapeutic vectors can provide the antigens to induce specific antitumor responses.Interleukin-23 (IL-23) is a cytokine reported by Oppmann et al in 2000, which can enhance proliferation of memory T cells and production of interferon-γ(IFN-γ) and IL-12 from activated T cells and dendritic cells. IL-23 has also been considered essential to development and control of autoimmune diseases, chronic inflammatory response and some infections. Several reports have shown that expression of IL-23 in murine tumor cells can induce antitumor response by enhancing CTL activity and production of IFN-γ. So, IL-23 can be a candidate of cytokine gene therapy of tumors. The aim of this study was to research the antitumor activity of IL-23 and relate mechanisms and provided basis for clinical application.The research was devided into four parts:Part 1 Expression of murine IL-23 gene in murine mammary carcinoma cellsObjective: To obtain the murine mammary carcinoma cell lines steadily expressing murine IL-23.Methods: Retrovirus was generated by transfecting retroviral vector (LXSN/IL-23) intoψ2 and then PA317 packaging cells. Retroviruses were used to transduce the mIL-23 gene into murine mammary carcinoma cells and stable clones expressing mIL-23 gene (IL-23/MA-891) were obtained by screening with G418. Expression of mIL-23 gene was detected with RT-PCR. The level of mIL-23 secreted by IL-23/MA-891 cells and IFN-γproduction by murine splenocytes induced with mIL-23 were determined by ELISA. Growth of IL-23/MA-891cells in vitro was assessed by MTT colorimetry. Flow cytometry was used to analyze the expression of MHCⅠ,MHCⅡ,CD80,CD86 and Fas on the surface of cells.Results: 1 Construction of IL-23 gene in MA-891 cells: IL-23 gene was transferred into two packing cells (ecotropicψ2 and amphotropic PA317) in sequence by a retrovirus vector (LXSN), and the positive cellular clones were screened by G418. The retroviruses were used to transduce the mIL-23 gene into MA-891 cells. IL-23/MA-891 cells which express mIL-23mRNA and produce mIL-2(3136.5±2.8)ng/L were obtained. This result suggests that mIL-23 gene has been integrated into the genome of IL-23/MA-891 cells.2 Biological character of IL-23/MA-891 cells: In the presence of Con A, the culture supernatant of IL-23/MA-891 cells can induce the production of IFN-γ(28.5±2.7) ng/L by murine splenocytes which was higher than that of LXSN/MA-891 cells and MA-891 cells supernatants(5.1±0.3,4.7±0.6)ng/L can induce respectively. The growth of IL-23/MA-891 cells was similar to that of LXSN/MA-891 and MA-891 cells in vitro. The expression level of MHCⅠ, MHCⅡ, CD80 and CD86 molecules on surface of IL-23/MA-891, LXSN/MA-891 and MA-891 cells were showing no significant difference (P>0.05).Conclusion: IL-23/MA-891 cell secreting mIL-23 was obtained. Transfection of mIL-23 gene did not affect the growth of MA-891 cells in vitro. The culture supernatant of IL-23/MA-891 cells can induce murine splenocytes to produce higher level IFN-γ.Part 2 Antitumor activity and effects of IL-23 on immune function in the murine carring tumor modelsObjective: To study antitumor activity and effects of IL-23 secreted from IL-23/MA-891 cells on immune function of the murine which carried the grafted tumors.Methods: IL-23/MA-891, LXSN/MA-891 and the parental MA-891 cells were respectively inoculated to TA-2 mice in the subcutaneous tissue. The mouse carring tumor models were used to observe the tumorigenic activity of the IL-23/MA-891 cells. Three mice of every group were killed on the thirtieth day after inoculation, the spleens and tumors were obtained from mice. The other mice of every group were used to observe survival and tumor size. LDH releaese, [3H]-TdR incorporation were used to determine CTL activity, proliferation of splenocytes respectively. IFN-γ, TNF-α, IL-12 and IL-4 producted by splenocytes were detected with ELISA. The expression of the MHCⅠ, MHCⅡ, CD80 and CD86 molecules on the surface of tumor cells and the positive cells to expression of CD11c, CD4 and CD8 in splenocytes from three kinds of mice were detected with flow cytometry. The infiltration of CD4+ and CD8~+ T cells in tumor tissues from three kinds of mice were detected by immunohistochemistry.Results: 1 Antitumor activity of IL-23 in vivo: The tumor-forming rates of IL-23/MA-891, LXSN/MA-891 and MA-891 cells were all 100%, while the tumors inoculated with IL-23/MA-891 cells developed obviously slower than inoculated with other two cells, and the mice survival time was more longer than 120 days. The tumor growth speed of the mice inoculated with LXSN/MA-891and MA-891 cells was similar, their mean survival times were 51±3 days and 50±3 days respectively.2 The splenocytes from the mice inoculated with IL-23/MA-891 cells can secret IFN-γ,TNF-αand IL-12 that were higher than with other two cells(P<0.01), but the secretion of IL-4 has no difference in the three kinds of mice(P>0.05).3 In the splenocytes from the mice inoculated with IL-23/MA-891 cells, the ratio of CD4~+, CD8~+ T cells and CD11c positive cells was also obviously higher than those in the splenocytes from the mice inoculated with LXSN/MA-891 and MA-891 cells(P<0.01).4 Compared with the tumor tissues from LXSN/MA-891 and MA-891inoculated mice, the infiltration amounts of CD4~+ and CD8~+ T cells in the tumor tissues from IL-23/MA-891 inoculated mice was significantly increased(P<0.01).5 The expression level of MHCⅠ, MHCⅡ, CD80 and CD86 molecules in the tumor tissues from IL-23/MA-891 inoculated mice were also higher than those from other two kinds inoculated mice(P<0.01).Conclusion: The antitumor effect of IL-23 secreted from IL-23/MA-891 cells was obviously in vivo, which enhanced the cellular immune function by increasing the infiltration amounts of CD4~+ and CD8~+ T cells in the tumor tissues and promoting secretion of IFN-γ,TNF-αand IL-12 cytokines. IL-23 can also increase the amounts of DC of murine splenocytes, antigenicity of tumor tissues and co-stimulation of angtigen presentation. So IL-23 can prevent tumor escaping from immune system and play an important role in inhibiting the growth of tumor.Part 3 The effects of IL-23 on the apoptosis of the murine mammary cancer cells and discussion of the mechanism. Objective: To study the effects of IL-23 on the apoptosis of the inoculated tumor cells and research the possible mechanism.Methods: The three kinds of TA2 mouse with graft tumor was killed on day 30 after s.c. injection with tumor cells. The inoulated tumors were harvested respectively. The three kinds of tumors were used to observe the cell apoptosis which was detected by TUNEL, flow cytometry and electron microscope. The expression of Fas and Suvivin on three kinds of tumor cells were assessed with RT-PCR, Western-blot and immunohistochemistry methods.Results: 1 The result of flow cytometry instructed that the apoptotic rates of three kinds of tumor cells were all very low in vitro. There had no difference among three groups. Nevertheless, on day 30 after s.c. injection with tumor cells, the apoptotic rates of IL-23/MA-891 cells (37.3±1.2%) was increased significantly compared to LXSN/MA- 891(18.7±0.5%) and MA-891 cells(18.4±1.3%) and the difference had statistical significance (p<0.01).2 The results of TUNEL and electron microscope were also instructed the obvious apoptosis in the experimental group. At the same time, the apoptotic rates detected by TUNEL of IL-23/MA-891 cells was 39.0±1.0%, which was increased significantly compared to MA-891 (11.6±1.2%) and LXSN/ MA-891 cells (11.1±0.9%) (p<0.01).3 The effect of IL-23 on the expression of Fas: By RT-PCR, Western-blot and immunohistochemistry, the mRNA and protein expression of Fas in the IL-23/MA-891 tumor cells on day 30 after s.c. injection were also increased markedly compared to MA-891 and LXSN/ MA-891 cells (p<0.01).4 The effect of IL-23 on the expression of Suvivin: By RT-PCR, Western-blot and immunohistochemistry, the mRNA and protein expression of Suvivin in the IL-23/MA-891 tumor cells on day 30 after s.c. injection were also decreased markedly compared to MA-891 and LXSN/ MA-891 cells (p<0.01).Conclusion: There were no direct effects of IL-23 on the apoptosis in tumor cells in vitro. But in vivo, IL-23 could induce the apoptosis of the inoculated tumor cells. In our experiment , the expression of Fas was up-regulated in IL-23 transfected groupes, but the expression of Survivin was down-regulated in IL-23 transfected groupes. Anyway, IL-23 inhibit proliferation of tumor cell and increase in apoptosis. Increase in apoptoais was related partly to induction of expression of Fas and reduction of Suvivin of tumor cells.Part 4 The effection of angiogenesis on IL-23 gene transfected murine mammary cancersObjective: To examine whether the retrovirus-mediated mIL-23 gene transfer into mammary carcinoma cells (IL-23/MA-891) could inhibit angiogenesis in mice.Methods: Three kinds of inoculated tumors were harvested on day 30 after s.c injection respectively. The expression of mRNA and protein of VEGF were detected by RT-PCR and immunohistochemistry. The microvessel density (stained with CD34) in the grafted tumor tissues were detected by immunohistochemistry, observed under the optical micro- scope and the value of MVD was calculated.Results: In the IL-23 gene transfected tumor group, the expression of mRNA and protein of VEGF was lower than LXSN/MA-891 group and MA-891 group. Meanwhile, the expression of the microvessel density of IL-23/MA-891 tumor cell group (18.23±6.92)was decreased compared with LXSN/MA-891(36.13±10.40)and MA-891(38.16±12.30) and the difference has statistical significance(P<0.01).Conclusion: IL-23 secreted from IL-23/MA-891 cells reduced the expression of VEGF and MVD, inhibited the angiogenesis in tumors, which induced the regression of the IL-23 gene transfected tumors.