| Colon cancer is a sort of common malignant tumors of the digestive tract with high morbidity and mortality.There are always no obvious symptoms in the early-stage of colon cancer,and the patients are diagnosed when the cancer has reached advanced stage.For the treatment of patients with advanced colon cancer,the combination of radiotherapy and chemotherapy is used.But the combination treatment may have toxic side effects,and the results are often unsatisfactory.So it is vital to find new drugs with low toxicity and to identify their targets of action.Coumarins can be widely extracted in rutaceae,umbelliferae,leguminosae,asteraceae and orchidaceae.They have biological activities such as antitumor effect,vasodilation,abirritation,antioxidation,anti-inflammation and neuroprotection.Coumarins are also commonly used in drug design due to their easily derivatized molecular structures.Our group designed the coumarin,which exhibiting a significant agonistic activity on GPR35(EC50=0.8 n M),but its applications and the mechanism of actions have not been investigated in depth.It has been reported that GPR35 is related to numerous disease,pain,tumours and hypertension,for example.The natural agonist of GPR35,kynurenine uric acid,can inhibit the proliferation of HT-29 cells(IC50=0.9m M),which is a kind of colon cancer cells.Therefore,we speculate that the coumarin may have an inhibitory effect on colon cancer.ObjectiveTo investigate the protein targets,and the inhibitory effect of the coumarin on colon cancer and its mechanism of action are analyzed by bioinformaticsMethods1.The GPR35 agonistic activity of the photoaffinity probes was evaluated by the EPIC BT system.2.“light” and “heavy” HT-29 cells were labeled with coumarin compound probes and control probes respectively,then the proteins that bind with probes were detected by mass spectrometry.We regard the proteins with SILAC ratio greater than 2 as the target proteins.3.The TBtools software were used to analyze physicochemical properties of the target proteins,and the post-translational modification of the target proteins were analyzed by the results directly.4.Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of target proteins were carried out to preliminarily investigate the targets and the mechanisms of the action of the coumarin.5.By using the GEO online database,the dataset with accession number GSE74604 was downloaded and selected for finding differentially expressed genes,and the differential genes were considered as the colon cancer-related genes as well as the targets for colon cancer therapy.6.By comparing the dataset of the target proteins and the dataset of colon cancer-related genes,the overlap between the two datasets was obtained,and the potential targets which exert antitumor effects of coumarin were inferred.7.By performing GO enrichment analysis and KEGG enrichment analysis on the potential targets,the mechanisms of coumarin how to exert antitumor effects were predicted.Results1.The photoaffinity probe has GPR35 agonistic activity,EC50=14.38 n M.2.A total of 1537 target proteins were identified.Among the 1537 drug target proteins,655 proteins only bind to drug probes,and 882 proteins can bind to both drug and control probes.By analyzing the post-translational modifications of the target proteins,it was found that 290 proteins had post-translational modifications.3.It is found that the mass of the target protein are mostly within 100 kd;the lengths are mostly within 1000 amino acids;the isoelectric points of the target proteins are mostly less than 7,which are acidic proteins;and GRAVY values are mostly less than 0,which are hydrophilic proteins.4.The target proteins were analyzed by GO analysis and found that the target proteins are widely distributed in cells,which are mainly related to a variety of binding modes,and participate in a variety of cellular biological processes.5.KEGG analysis of the drug target proteins found that they were mainly related to metabolism.6.By utilizing the GEO online database,the GSE74604 dataset was selected and downloaded for cancer-related gene analysis.A total of 1661 differential genes were identified,including 671 up-regulated genes and 990 down-regulated genes.They are the therapeutic targets for colon cancer.7.By comparing the datasets of cancer-related genes and drug target proteins,171 potential targets were identified,which are the therapeutic targets of coumarin for colon cancer.8.Through GO analysis and KEGG analysis of the therapeutic targets,it was found that they were associated with the formation of multiple complexes and the activity of various proteins.The coumarin has an anti-colon cancer property by regulating metabolism as well as various biosynthesis.Conclusion1.The photoaffinity probes have GPR35 agonistic activity.2.1537 target proteins of the coumarin were identified by quantitative chemical proteomics approach.3.By comparing the datasets of cancer-related genes and drug target proteins,171 potential targets were identified.The coumarin has the potential to be used in colon cancer treatment,and the mechanism of the action of anti-colon cancer are related to the regulation of metabolism and various biosynthesis... |
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