The Impacts Of Indoleamine 2,3-dioxygenase2 Expression On Murine Melanoma Growth And Its Application In Tumor Therapy

Part 1: The impacts of Indoleamine-2, 3-dioxygenase-2 expression on the biological behaviors of melanoma cells B16-BL6Objective: To study the impacts of indoleamine 2, 3 dioxygenase-2 on the biological behaviors of mouse melanoma cells.Methods: After silencing of IDO2 gene expression in melanoma cells B16-BL6 by using IDO2-si RNA, the expressions of IDO2 m RNA and protein levels were detected by Real-time quantitative PCR(RT-PCR) and Western blot, respectively. Following IDO2 gene silencing, the proliferation of B16-BL6 cells was measured by MTT assays, the cell cycle and apoptosis was analyzed by flow cytometry, the kynurenine generation was detected using Ehrlich’s method, the intracellular level of NAD+ was measured by NAD+/NADH kit and the intracellular expression of reactive oxygen species(ROS) was detected by H2 DCFDA staining.Results: The results of RT-PCR and Western blot showed that the expression of IDO2 was markedly decreased by more than 80% at both of the m RNA and protein levels after silencing of IDO2 gene in B16-BL6 cell. MTT assay results indicated that compared with the negative control group, silencing of IDO2 gene can significantly inhibit B16-BL6 cells proliferation. The Flow cytometry results demonstrated that following IDO2 gene silencing induced more apoptotic cells generation and increased cells arrest in G0/G1 phase whereas ratio of cells arrested in S phase and G2/M phase was notably reduced. The results of Ehrlich’s methods suggested that kynurenine generation was decreased after IDO2 gene silencing. Silencing of IDO2 gene, intracellular NAD + generation was decreased but the ROS level was increased. Adding the exogenous NAD+ can reduce the apoptosis which induced by IDO2 gene silencing.Conclusions: IDO2 has enzymatic activity in B16-BL6 cells and knockdown its expression can change the biological characteristics of the melanoma tumor cells including inhibit tumor cells proliferation, induce cells apoptosis and arrest cell cycles in G1 phase. After IDO2 gene silencing, the increase apoptosis in B16-BL6 cells is due to the decrease of NAD+ generation.Part 2: The impact of IDO2 expression in B16-BL6 cells on melanoma formation and growthObjective: To study the impact of IDO2 in B16-B6 cells on melanoma progression in the mouse model.Methods: We first constructed and validated the plasmid vector of IDO2-small hairpin RNA(IDO2-sh RNA) then transfected B16-BL6 cells with IDO2-sh RNA or scramble-sh RNA and established stable cell lines of B16-BL6/IDO2- and B16-BL6/IDO2 + by G418 selection. To confirm the established stable cell lines, GFP expression in the cells was detected by the flow cytometry and IDO2 m RNA expression was measured by RT-PCR. Next, to identify the impact of IDO2 expression on the melanoma formation and growth, the stable cell lines of B16-BL6/IDO2 + or B16-BL6/IDO2- were injected s.c. into the upper hind leg of the mouse and the tumor size was measured by caliper every other day. At the endpoint of experiments, tumor draining lymph nodes(LNs) were taken and flow cytometry was used to detect the percentage of Treg cells, populations of CD8+ /CD4+ T cells and T cells apoptosis.Results: We successfully constructed IDO2-sh RNA plasmid vector and established the B16-BL6/IDO2+and B16-BL6/IDO2- stable cell lines. The GFP expressions were more than 98% in both of the stable cell lines. In B16-BL6/IDO2-stable cell line, the IDO2 m RNA expression was kept in low level and didn’t increase by stimulating with IFN-g. Compare inoculation of B16-BL6/IDO2 + stable cell line, injected B16-BL6/IDO2- stable cell line into the mouse can delay the time point of tumor onset, decreased the tumor growth rate and reduced the tumor weight. Flow cytometry results showed that in tumor draining LNs, the percentage of Treg was decreased, CD8+T cell subset was increased and the apoptosis of T cell was decreased.Conclusion: The expression of IDO2 in B16-BL6 melanoma cells plays a promoting role in the growth of tumor and it can affect the immune response in the tumor draining lymph node.Part 3: IDO2 as a therapeutic target for anti-tumor therapyObjective: To study the role of IDO2 in anti-tumor therapy and its effect on the immune response when using IDO2 as therapeutic target.Methods: 1.IDO2-sh RNA or scrambled-sh RNA was injected by hydrodynamic injection via tail vein to treat melanoma; the tumor size was measured by Vernier caliper. The percentage of Treg cells, T cells apoptosis and co-stimulatory molecules expression of splenic dendritic cells(DCs) in spleen and draining lymph nodes from different treatment groups were analyzed by Flow cytometry. LDH assay was used to determine the Cytotoxic T lymphocytes(CTL) activity. Antigen-specific antitumor recall responses mediated by T cells were measured by 3H incorporation method. The level of TNF-a and IFN-g in serum was detected by ELISA. 2. After knocking down of IDO2 expression with IDO2-si RNA in dendritic cells in vitro, the expression of costimulatory molecules on the surface of DCs were measured by Flow cytometry. After labelled allogeneic T cells with Carboxyfluorescein succinimidyl ester(CFSE) and then cocultured with IDO2 silenced DCs, the CFSE fluorescence and Treg cells were measured by flow cytometry.Results: 1.The results demonstrated that in the IDO2-sh RNA treated group, the tumor formation time was delayed, tumor grew slowly, and excised tumor weight was significantly reduced. IDO2-sh RNA treatment also decreased the percentage of Treg and T cell apoptosis in spleen and draining LN and increased the expressions of co-stimulatory molecules of CD80 and CD86 on DCs, Furthermore, IDO2-sh RNA treatment improved the ability of T cell antigen memory capacity and enhance the CD8+T cell cytotoxic activity. The level of TNF-a,IFN-g upregulate. 2.Silencing of IDO2 gene in DCs increased the expression of surface costimulatory molecules of CD80 and CD86, enhanced the capacity of DCs to stimulate allogeneic T cells proliferation and decrease the induction of Treg cells.Conclusion: Silencing of IDO2 can effectively inhibit the growth of melanoma and improve the anti-tumor immune response in vivo.