The Anti-tumor Effect Of Romidepsin And Its Synergistic Effect With Anti-PD-1 Antibody

Background: Romidepsin,a histone deacetylase inhibitor,is also an epigenetic regulation drug that has been used for the treatment of skin T cell lymphoma.Experiments had shown that HDACi had anti-tumor effects on solid tumors such as meningioma.A large number of studies confirmed that HDACi could be used as an immunosuppressive agent for organ transplantation to maintain the survival of transplanted organs.Studies also suggested that HDACi could upregulate PD-L1 molecule in tumor cells.In cancer cells,the PD-L1 on the surface of the tumor cells could combine with the PD-1 molecules on the T cells to help the tumor cells escape from the attack of the T cells and promote the immune escape.Anti-PD-1 antibody could combine with PD-1 of T cells,restoring the function of T cells.Colon cancer is common in the digestive cancers.The purpose of this study is to explore whether romidepsin has anti-tumor effects in colon cancer,how romidepsin influences the immune system,and whether the combined use of anti-PD-1 antibody could relieve the effects of romidepsin.Methods: 1.WST,western blotting and flow cytometry were used to investigate the effects of romidepsin on the proliferation,cell cycle and apoptosis of CT28 and MC38 cells.2.Flow cytometry was used to detect the expression of MHC-I molecules on CT28 and MC38 cells.3.Mice subcutaneous tumor model of colon cancer was used to detect the effect of romidepsin on colon cancer.4.RT-PCR,Western blotting and flow cytometry were used to detect the effect of romidepsin on the expression of PD-L1 in CT28/MC38 cells.5.Western blotting and IP were used to detect the regulation effects in acetylation level and acetylation sites of histone H3/H4 by romidepsin.6.si-RNA and sh-RNA were used to knock down the expression of BRD4 in CT26/MC38,and RT-PCR,western blotting and flow cytometry were used to detect the PD-L1 expression level.7.Mice colon cancer subcutaneous tumor model and colon cancer in situ models were established.The mice were randomly divided into four groups: 1)the physiological saline treatment group;2)the injection of romidepsin(10mg/kg);3)the injection of anti-PD-1 antibody(300?g/mice);4)the romidepsin(10mg/kg)+anti-PD-1 antibody(300?g/mice).After three cycles of administration,the mice were killed.We measured the size and quality of the tumors.The percentage of Treg in peripheral blood,spleen,bone marrow and tumor infiltrating lymphocytes of mice and the percentage of CD4+IFN-?+T/CD4+IL4+T cells,CD8+ IFN-?+T cells in peripheral blood and tumor infiltrating were measured by flow cytometry.Results: 1.Romidepsin inhibited the proliferation and cell cycle of CT28 and MC38 cells,and increased cell apoptosis through inhibiting the AKT / MAPK/NFkappa B signaling pathways.2.Romidepsin upregulated the MHC-I molecule on the surface of colon cancer cells.3.In mice colon cancer xenograft models,romidepsin inhibited colon cancer growth.4.Romidepsin increased the expression of PD-L1 in CT26 and MC38 cells.5.Romidepsin regulate the expression level of PD-L1 on cell surface by increasing the acetylation level of histone H3,H4,and regulating transcription factor BRD4.6.In subcutaneous transplantation tumor and colon cancer in situ mice,romidepsin increased the percentage of FOXP3+Treg in peripheral blood,spleen,bone marrow and tumor local infiltrating lymphocytes,reduced the percentage of CD4+IFN-?+T cells and decreased the ratio of Th1/Th2 in peripheral blood and tumor infiltrating lymphocytes.After treatment with romidepsin,the percentage of CD8+IFN-?+T cells decreased in peripheral blood and tumor infiltrating lymphocytes.After combining with anti-PD-1 antibody,the percentage of FOXP3+Treg decreased,the percentage of CD4+IFN-?+T cells CD8+IFN-?+ T cells increased,the ratio of Th1/T h2 increased.Conclusion: 1.Romidepsin has anti-tumor effect in colon cancer,through inducing apoptosis,inhibiting cell proliferation and arresting cell cycle at G0/ G1 phase.2.Romidepsin upregulated MHC-1 molecules on the surface of CT26 and MC38 cells.3.Romidepsin upregulated the acetylation level of H3 and H4,and promoted the binding of RD4 to the PD-L1 promoter region,and increased the expression level of PD-L1.4.In subcutaneous transplantation tumor and colon cancer in situ mice,romidepsin increased the FOXP3+Treg,inhibited the activation of CD4+T and CD8+T cells,and promoted the transformation of Th cells to Th2 type.After combining with anti-PD-1 antibody,the percentage of FOXP3+Treg decreased and the activation level of CD4 + T / CD8 + T cells increased,the transformation of Th cells to Th1 type increased.That combination of anti-PD-1 antibody will enhance the antitumor effect of romidepsin.