| Objective: Leukemia is one of the common cancers in hematological system. At present, the minority of leukemia patients could receive complete remission after treatment with chemotherapeutics and bone marrow transplantation. But majority of patients have treatment failure due to drug resistance and relapse. The immunotherapy is an important approach for further prolonging remission duration and eradicating residual leukemia cells.Interleukin-23(IL-23) is a heterodimeric cytokine reported by Oppmann et al in 2000, which consists of a novel P19 chain and IL-12p40 chain. IL-23 can enhance proliferation of memory T cells, as well as induce production of interferon-γ(IFN-γ) and IL-12 from activated T cells and dendritic cells. Additionally, recent studies demonstrated that IL-23 has the functions of anti-tumor and anti-metastatic, and it may be involved in the development of autoimmune diseases and inflammatory diseases. IL-23 can be a safe candidate of cytokine gene therapy for tumors, because of it is limited effect on inducing IFN-γ, production in T cells. IL-2 is an important cytokine of interleukine family, It can enhance proliferation of T cells and cytotoxic T lymphocyte (CTL), and also improve cytotoxicity of natural killer (NK) cell. The evidences confirmed that IL-2 has obvious anti-tumor effect in vivo, but the effect depends on continuous infusion of exogenous high-dose IL-2 and associated with serious side effects. To reduce the toxic side effects, many researchers reported that the anti-cancer effect of peripheral blood mononuclear cell (PBMC) was induced by combating low-dose IL-2 and other cytokines. The aim of this study was to explore the killer effect of PBMC on K562 cells induced by IL-23 alone or combined with IL-2 We want to provide a new treatment for leukemia, and improve the clinical outcome of hematological malignancies. Methods: We separated human peripheral blood mononuclear cells by Ficoll density gradient centrifugation.In vitro incubation of PBMC with IL-23 and IL-2 at the concentration of 50ng/mL,100IU/mL respectively and leukemia cell lines K562 for 72h,detecting the cytotoxicity of PBMC with CCK-8,measuring the production of IFN-γin the culture supematants with ELISA,detecting perforin, granzymes B with RQ-PCR.Results:1. IL-23 alone or combined with IL-2 stimulated the human PBMC anti-leukemia cell line K562 activity: The rate of killing K562 cells of PBMC after treated with IL-23(50ng/mL) for 24h, 48h, 72h were: (15.12±0.58)%, (18.71±1.07)%, (24.36±1.59)%; The rate of killing K562 cells of PBMC after treated with IL-2(100IU/mL) for 24h, 48h, 72h were: (15.64±0.60) %, (19.75±0.96) %, (25.34±1.71)%; The rate of killing K562 cells of PBMC after treated with IL-23(50ng/mL) and IL-2(100IU/mL) for 24h, 48h, 72h were: (22.58±0.67)%, (27.84±1.31)%, (34.4±1.24)%. Statistical analysis showed that the rate of killing K562 cells of PBMC in experiment groups after treated for 24h, 48h, 72h were significantly different (P<0.05), at the same time, there was a significant difference among intervention groups (P<0.05). IL-23 can promote the rate of killing K562 cells of PBMC, IL-23 combined with IL-2 can further enhance the rate, and at 72h, the cytotoxicity was the highest.2. The production of IFN-γin the supermatants of human PBMC culture was induced by IL-23 alone or combined with IL-2: The level of IFN-γin supermatants of human PBMC culture after treated with IL-23(50ng/mL), IL-2(100IU/mL), IL-23(50ng/mL)+IL-2(100IU/mL) for 72h were: 156.08±12.40, 191.68±16.92, 615.83±21.39, while the control was 44.39±5.90. Statistical analysis showed that the IFN-γlevels in the supermatants of human PBMC culture in the presence of cytokines significantly increased, and the IFN-γlevels in IL-23(50ng/mL) + IL-2(100IU/mL) group were significantly higher than that in others (P<0.05). Although the level of IFN-γin IL2 (100IU/mL) group seem to be slightly higher than that in IL-23(50ng/mL) group, there was no statistic difference (p>0.05). 3. The expression of perforin in human PBMC was induced by IL-23 alone or combined with IL-2: RQ-PCR showed, the RQ value of perforin on human PBMC after treated with IL-23 (50ng/mL), IL-2(100IU/mL), IL-23(50ng/mL) + IL-2(100IU/mL) for 72h were: 1.39±0.23, 1.54±0.16, 2.32±0.22, while the control was 0.51±0.12. Statistical analysis showed that the expressions of perforin mRNA of expanded human PBMC cells in groups cultured with cytokines was significantly higher than that in group without (p<0.05) , and the expressions of perforin mRNA in IL-23(50ng/mL) + IL-2(100IU/mL) group were significantly higher than that in others (P<0.05). Although the expressions of perforin mRNA in IL-2(100IU/mL) group seem to be slightly higher than that in IL-23(50ng/mL) group, there was no statistic difference (p>0.05).4. The expression of granzymes B in human PBMC was induced by IL-23 alone or combined with IL-2: RQ-PCR showed, the RQ value of granzymes B on human PBMC after treated with IL-23(50ng/mL), IL-2 (100IU/mL), IL-23(50ng/mL) + IL-2(100IU/mL) for 72h were: 1.57±0.33, 1.74±0.11, 2.298±0.21, while the control was 0.38±0.12. Statistical analysis showed that the expressions of granzymes B mRNA of expanded human PBMC cells in groups cultured with cytokines was significantly higher than that in group without (p<0.05), and the expressions of granzymes B mRNA in IL-23(50ng/mL) + IL-2(100IU/mL) group were significantly higher than that in others (P<0.05). Although the expressions of granzymes B mRNA in IL-2(100IU/mL) group seem to be slightly higher than that in IL-23 (50ng/mL) group, there was no statistic difference (p>0.05).Conclusion:1. IL-23 improves the killer effect of PBMC on K562 cells. The significant effect of combing IL-2 with IL-23 on inducing PBMC is in a time dependent manner.2. IL-23 enhances the expression of IFN-γ, perforin, granzyme B in PBMC. The combined application of IL-23 and IL -2 has a synergistic effect. In this study, we demonstrate that IL-23 possesses anti-leukemic activity and unravels its mechanisms.
|
PDF Full Text Request