| Background:Advances in harnessing the immune system for cancer treatment have been spectacular in recent years.The discovery of immune checkpoints has provided new ideas for the treatment of tumors.PD-1 on the T cells and PD-L1 on the tumor cells are a pair of important immune checkpoints.Blocking the binding of PD-1 and PD-L1 has become a hotspot in the field of tumor immunotherapy.Compared with antibodies targeting PD-1/PD-L1 axis,small-molecule checkpoint inhibitors which have natural advantages in pharmacokinetic properties,tissue and tumor permeability,and production and price,are urgently needed.There is no small molecule tumor immunotherapy drug targeting PD-1/PD-L1 in the market so far.Berberine(BBR)was screened from a series of Traditional Chinese Medicine(TCM)chemical monomers which could significantly down-regulate PD-L1 expression in lung cancer cells.Although previous studies have shown that BBR has pharmacological activities such as anti-inflammatory,antibacterial,and hypolipidemic effects,no research has been reported on its antitumor effect by activating the immune system.Therefore,we investigated the role of BBR in killing tumor cells by regulating the tumor microenvironment and its mechanism.Methods:We start our chemical screen by testing the ability of a panel of 55 Traditional Chinese Medicine(TCM)chemical monomers to alter PD-L1 protein expression in H460 cells,a human non-small cell lung cancer cell line.The levels of PD-L1 protein were determined by western blot.The effect of BBR on the proliferation and apoptosis of tumor cells were determined by MTT,flow cytometry assay,and western blot.Changes of membrane PD-L1 were determined by flow cytometry assay and PD-1/PD-L1 interaction experiment.The cytotoxicity of activated human T cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment.The antitumor effect of BBR in vivo was examined by C57BL/6 mice and BALB/C nude mice Lewis xenograft tumor models.The activity of tumor-infiltrating T cell were detected by multicolor flow cytometry assay,including the number of CD8+T cell,the ratio of CD8+ to CD4+T cell and the level of Interferon-γ(IFN-γ)and Granzyme B(GzmB).The activity of MDSCs and Tregs were also measured by flow cytometric assay.The levels of PD-L1,Ki67,Cleaved caspase 3 were determined by immunohistochemistry(IHC)assay.Viscera including liver,spleen,lung and kidney were taken out for H&E staining.The serum biochemical indices were measured by serum biochemical analyzer.Changes of the mRNA level of PD-L1 were measured by qRT-PCR assay.Biotin-conjugated BBR probe(BBR-biotin)was synthesized and the interaction between BBR and CSN5 was determined by surface plasmon resonance(SPR)analysis and BBR-biotin pull-down assay.Changes of the activity of the CSN5 were measured by deubiquitinating activity assay.The binding site of BBR was analyzed by Discovery studio 4.0 sofeware and confirmed by site-directed mutagenesis and pull down assay.Results:Western blot results showed that BBR can significantly downregulate the expression of constitutive and inducible PD-L1 in NSCLC cells.In addition,BBR has no significant effect on the expression of other immune checkpoints such as CD47 and IDO1.Flow cytometry assay revealed that BBR dramatically attenuated the cell surface PD-L1 in NSCLC cells.In addition,BBR had little toxicity on cells and did not induce cell apoptosis within a concentration of 20 μM.PD-1/PD-L1 binding experiment results showed that BBR can reduce the binding of tumor cells to recombinant PD-1 protein.Cell impedance and crystal violet staining showed that BBR could enhance the killing activity of co-cultured T cells toward tumor cells.Animal experiments revealed that BBR treatment displayed significant suppression in the growth of Lewis tumor xenografts in C57BL/6 mice with an inhibition rate of 46.8%and 75.8%at 4 mg/kg and 8 mg/kg,respectively,and tumor-infiltrating T cell was activated.The number of killer T cells CD8+ T cell and the ratio of CD8+ to helper T cells CD4+T cell in BBR-treated mice were increased.In addition,BBR treatment boosted the level of IFN-y and GzmB.At the same time,BBR can inhibit the activity of MDSCs and Tregs cells.These findings demonstrated BBR switches the immune microenvironment from immunosuppressive to immunoactivation.IHC assay showed that the levels of PD-L1 and Ki67(a marker of proliferation)were decreased,while the level of cleaved caspase 3 was increased in BBR-treated tumor xenografts mice.BBR has no systemic toxicity on mice.IHC assay,and serum biochemical indices showed that BBR had no significant toxicity to major organs,and BBR had no obvious inhibitory effect on nude mice.Mechanism studies found that BBR had no significant effect on the transcription level of PD-L1,but promoted ubiquitination and degradation of PD-L1 by acting on the ubiquitin-proteasome pathway.Streptavidin Magnetic Beads pull down experiment found that BBR can directly interacte with CSN5.BBR was demonstrated to directly bind to CSN5 protein with an affinity(KD)value of 16.25 μM via SPR method.Site-directed Mutagenesis and Co-Immunoprecipitation experiments found that BBR selectively bound to glutamate 76 of CSN5 and subsequently inactivated CSN5,which led to degradation of PD-L1 and activation of tumor-infiltrating T cells.Conclusion:Our study reveals a new mechanism that BBR exerts its anti-tumor activity by regulating T cell activity in the tumor microenvironment.BBR specific binds to CSN5 and inhibit its deubiquitination activity,which led to ubiquitination and subsequent degradation of PD-L1.At the same time,BBR can activate cytotoxic T cell(CD8+)in tumor microenvironment.This study provides an important material basis and scientific basis for developing BBR into a new small molecule drug for tumor immunotherapy... |
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