Effect And Mechanism Of Carboxyamidotriazole Combined With Nifuroxazide In Treating Colon Cancer

BackgroundColon cancer is one of the most common gastrointestinal malignant tumors in the world,which seriously threatens people's lives.Therefore,it is urgent for researchers to solve the problem of finding effective,low-adverse reactions and definite therapeutic drugs and the combination therapy.Carboxyamidotriazole?CAI?is a non-cytotoxic anti-tumor drug primarily studied at the National Cancer Center of the United States,which has been shown to have anti-tumor properties and can inhibit angiogenesis.However,CAI showed mild antitumor effect in phase?clinical trials and increased the expression of Indoleamine 2,3-dioxygenase-1?IDO1?,inhibiting the immune system's ability to recognize and kill tumor cells.In order to enhance its therapeutic effect and overcome the disadvantage of CAI-induced immunosuppression,we tried to find a drug that can enhance its anti-tumor effect.Nifuroxazide?NFX?is an oral nitrofuran antibiotic,which is mainly used to prevent and treat bacterial dysentery and enteritis.In recent years,some studies have found that NFX is an effective inhibitor of signal transduction and activator of transcription 3?STAT3?and has potential anti-tumor effect.Scholars have reported its effect on the proliferation and metastasis of tumor cells in liver cancer,breast cancer,multiple myeloma and melanoma.Since the activation of STAT3 was accompanied by the up-regulation of IDO1 expression,we conclud that the combination of the two drugs might enhance the anti-tumor efficacy from the perspective of immunity.The purpose of this study was to evaluate the synergistic antitumor effect of the two drugs in combination and explore the possible mechanism,and to further explore the effect of CAI on CD8⁺T cells activated in vitro.MethodsThe proliferation inhibition of CAI and/or NFX in two colon cancer cell lines was detected by CCK8 method,and the combined drug index was calculated.Caspase 3 activity assay kit and flow cytometry were used to detect apoptosis.The intracellular reactive oxygen species was stained by DCFH-DA and was quantitatively analyzed.The effect of drugs on cell migration was detected by cell scratch test.The expression of MMP-2 and MMP-9 was detected by RT-qPCR.EstablishIng a model of tumor-bearing mice and evaluate the effect of CAI and NFX on tumor inhibition in vivo.H&E staining was used to detect tumor necrosis.After stripping tumor tissues from tumor-bearing mice,fluorescence quantitative PCR and western blot were used to detect the differences in mRNA and protein expression levels of IDO1.Western blot were used to detect differences in the expression of STAT3,pSTAT3proteins.The expression differences of c-c chemokine?c-c motif?receptor 5?CCR5?and c-c chemokine?c-c motif?ligand 5?chemokine?c-c motif?ligand 5,CCL5?were detected by flow cytometry.Spleen lymphocytes of tumor-bearing mice were isolated and the proportions of CD4⁺and CD8⁺T cells were detected by flow cytometry.Mice CD8⁺T cells were isolated by immunomagnetic beads and divided into control group,CAI group,ZK756326(Ca²⁺activator)group and CAI+ZK756326 group.Flow cytometry was used to detect the level of intracellular free calcium ions in CD8⁺T cells;Enzyme-linked immunosorbent assay was used to detect the expression of intracellular calcineurin?CaN?;Immunofluorescence staining was used to detect NFAT2 nuclear transportation;Chromatin immunoprecipitation-qPCR was used to detect the expression of PD-1 regulated by NFAT2;Isolating mice spleen cytotoxic T lymphocytes?CTLs?and detecting the expression of PD-1 in CD8⁺T cells by flow cytometry.Results1.In colon cancer cell lines C26 and HCT116,both CAI and NFX showed inhibitory effects,and the inhibitory effects were time-dependent and dose-dependent?P<0.01?.2.CAI and NFX had a synergistic effect when used at the concentration of 5-10?M,CI<1.3.The combination of CAI and NFX significantly increased the level of apoptosis and the activity of caspase 3?P<0.01?.4.The combination of CAI and NFX can significantly increase the intracellular ROS level?P<0.0001?5.The combination of CAI and NFX can inhibit cell migration and reduce the expression levels of MMP2 and MMP9?P<0.05?.6.The combination of CAI and NFX can inhibit tumor volume growth,reduce tumor weight and prolong the survival of tumor bearing mice?P<0.05?.7.The combination of CAI and NFX can induce the mice tumor tissue necrosis.8.Protein expressions of IDO1 and p-stat3 were decreased in the drug combination group compared with the CAI monotherapy group?P<0.001?.9.CAI combined with NFX increased the proportion of CD4+and CD8+T cells in spleen lymphocytes of tumor-bearing mice?P<0.01?.10.The combination of CAI and NFX reduced the expression of CCL5 and CCR5 in tumor cells of tumor-bearing mice?P<0.01?.11.Intracellular Ca²⁺concentration in CD8⁺T cells was significantly decreased in the CAI group?P<0.001?,while intracellular Ca²⁺concentration was increased in the CAI+ZK756326 group?P<0.01?;CAI significantly decreased the content of calcineurin phosphatase in CD8⁺T cells?P<0.001?.12.CAI significantly inhibited NFAT2 nuclear transportation?P<0.001?.13.CAI significantly reduced the PD-1 transcription process depend on NFAT2?P<0.001?.14.CAI significantly reduced the proportion of PD-1⁺CD8⁺T cells in mice spleen CTLs cells ?P<0.001?.ConclusionCAI and NFX in the experiments in vivo and in vitro showed good synergistic tumor suppression effect on colon cancer,its may be NFX inhibits the STAT3/IDO1 signal transduction pathway and the inhibits IDO1 expression caused by CAI,elevated CD⁴+and CD8⁺T cell infiltration in immune organs,inhibited CCL5/CCR5 signal axis,so as to activate the immune system for tumor destruction.In addition,in artificially activated T cells in vitro,CAI can inhibit NFAT2 nuclear transport by inhibiting the level of calcium ions and the expression of calcine phosphatase in CD8⁺T cells,thereby reducing the expression of pd-1 in CD8⁺T cells and further producing immunotherapeutic intervention.It is suggested that CAI can be used in combination with drugs to further enhance its clinical anti-tumor effect.