| Research background and purpose:Colon cancer is one of the common and highly malignant gastrointestinal tumors with high harm to humans,With the improvement of people’s dietary level,the risk of developing colon cancer is increasing.At present,the treatment method for colon cancer is still mainly radical surgery,supplemented by chemotherapy.Postoperative adjuvant chemotherapy can reduce the risk of colon cancer metastasis and recurrence,but with the increasing resistance of chemotherapy drugs,old drugs are no longer able to meet current treatment needs.Therefore,we need to seek new chemotherapy drugs to improve patient prognosis.Among the current various pathogenic factors,chronic inflammation is more closely related to colon cancer,and most colon cancer patients have inflammatory infiltration in their tumor tissue.Pyroptosis is a new type of programmed cell death since the discovery of apoptosis and autophagy.It is characterized by cell swelling,large bubbles emerging from the cell membrane,accompanied by the release of inflammatory factors,and finally inducing cascade inflammatory reaction.Previous experiments found that DS6051 b could cause caspase-3/GSDME mediated pyroptosis in colon cancer cells.However,the specific mechanism is not yet clear,and whether DS6051 b has a significant inhibitory effect on the growth and replication of colon cancer cells still needs further verification.Therefore,this paper elucidates the specific molecular mechanisms by which DS6051 b inhibits colon cancer cells and induces their pyroptosis through research methods such as cell experiments and gene intervention.Materials and Methods:1.Screening and analysis of drug DS6051 b with killing effect on colon cancer cellsFrom 39 small molecular compounds in the pyroptosis compound library,we used CCK-8 for preliminary screening and twice screening.In order to analyze the screening results,we performed fusion gene detection on colon cancer cells.After treating colon cancer cells with DS6051 b,the damage effects of different concentrations of DS6051 b on colon cancer cells HCT116 and Lo Vo were evaluated through CCK-8.Crystal violet staining was used to detect cell proliferation.2.DS6051 b induces pyroptosis in colon cancer cellsAfter treating colon cancer cells with DS6051 b at a concentration of 5u M for 24 hours,the morphological changes of the cells were observed through microscopy and scanning electron microscopy,respectively.Detect the proportion of annexin V-PE and7-AAD double positive cells in colon cancer cells through flow cytometry experiments;Evaluate the damage effect of DS6051 b on colon cancer cells through TUNEL assay and LDH cytotoxic release assay.3.DS6051 b mediates pyroptosis of colon cancer cells through SRCThrough databases and literature,we screened the targets of DS6051 b and related targets of colon cancer.After intersection,they were used as PPI protein interaction networks to predict the most likely target SRC of DS6051 b on colon cancer.Western Blot was used to detect the expression of SRC and then the phosphorylation of AKT/m TOR.We overexpressed SRC through viral plasmid transfection,and after 24 hours of DS6051 b treatment,we used WB again to detect the expression of SRC and AKT/m TOR phosphorylation.CCK-8 evaluated the damage effect of DS6051 b and SRC overexpression on HCT116 and Lo Vo colon cancer cells,LDH test evaluated the damage effect of DS6051 b and SRC overexpression on colon cancer cells,and then flow cytometry was used to detect the double positive ratio of Annexin V-PE and 7-AAD in cells after DS6051 b and SRC overexpression;Western blot was used to detect the changes of pyroptosis related proteins Caspase-3,PARP,GSDME-N,etc.Result:1.The best small molecule compound DS6051 b for inhibiting the survival of colon cancer cells has been screenedWe conducted initial screening on 39 small molecules and identified 5 small molecule compounds with inhibition rates greater than 70%.Then,we conducted secondary screening on these 5 small molecule compounds and identified the drug DS6051 b with inhibition rates greater than 50% for both colon cancer cells.According to literature reports,DS6051 b has therapeutic effects on tumors with ROS1/NTRK gene rearrangements,and our colon cancer cells do not have ROS1/NTRK gene rearrangements.HCT116 and Lo Vo cells were treated with different concentrations of DS6051 b at 1.25,2.5,5,10,and 20 u M for 24 hours,respectively.The cell activity significantly decreased with the increase of DS6051 b concentration.2.DS6051 b induces caspase-3/GSDME mediated pyroptosis in colon cancer cellsAfter 24 hours of treatment with DS6051 b on colon cancer cells,we observed under a microscope that compared with the control group,the number of cells in the DS6051 b group decreased and morphological changes such as blistering occurred.We observed under a scanning electron microscope that compared with the control group,the DS6051 b group showed changes such as pores and blistering.Flow cytometry also showed an increase in the number of annexin V-PE and 7-AAD double positive cells.We found through WB detection that DS6051 b treatment upregulated the expression of Cleared Caspase-3,GSDME-N,and PARP-CL,and increased LDH toxicity release in both cell lines.In order to verify whether DS6051 b can mediate pyroptosis through Caspase-3/GSDME after treatment,we pretreated HCT116 and Lo Vo cells with Z-VAD for 1h.After DS6051 b treated HCT116 and Lo Vo colon cancer cells for 24 h respectively,we observed with a microscope that pyroptosis was significantly reversed,and the expression of Cleaved Caspase-3,GSDME-N,PARP-CL was reversed.CCK8 showed that compared with the DS6051 b treated group,the inhibitor treated group of HCT116 cells showed a significant increase in cell activity(p<0.01),while the inhibitor treated group of Lo Vo cells showed a significant increase in cell activity(p<0.05);The LDH toxicity release experiment showed that in HCT116 cells,the LDH toxicity release was reduced in the inhibitor treated group compared to the DS6051 b treated group(p<0.001),and the LDH toxicity release in the inhibitor treated group in Lo Vo cells was also relatively significantly reduced(p<0.05).3.DS6051 b mediates colon cancer pyroptosis through SRC/AKT/m TOR signaling pathway,thus leading to colon cancer cell damage.By obtaining 100 potential targets of DS6051 b from the Swiss Target Prediction website(Table S1),202 effective kinase targets of DS6051 b were obtained from the literature(Table S2),and 28 potential targets of DS6051 b were obtained by intersection.A total of 2388 known colon cancer related genes were collected from the Dis Ge NET,Genecard,OMIM,and uniport databases(Table S3),After intersecting the potential target of DS6051 b with colon cancer related targets,10 candidate targets were obtained.After conducting PPI network construction analysis on the 10 candidate targets,SRC was determined as the most likely target for DS6051 b to act on colon cancer.After treating HCT116 and Lo Vo cells with DS6051 b,we found that the expression of SRC was significantly inhibited,and AKT and m TOR phosphorylation were also significantly reduced.After overexpressing SRC through viral plasmid transfection,the phosphorylation inhibition of SRC,AKT,and m TOR was reversed,and the expression of upregulated pyroptosis related factors such as Cleared Caspase-3,GSDME-N,and PARPCL was also reversed.The proportion of annexin V-PE and 7-AAD double positive was also increased.Compared with the DS6051 b treated group,the SRC overexpression group also significantly reduced the toxic release of LDH(HCT116 cells: p<0.01;Lo Vo cells: p<0.05).Conclusion:1.DS6051 b inhibits the proliferation of colon cancer cells,and its inhibition rate is positively correlated with the concentration of DS6051 b.2.DS6051 b induces swelling,blistering,and pore formation in colon cancer cells,releases LDH,and activates Caspase-3 to lyse GSDME,leading to pyroptosis of colon cancer cells;3.DS6051 b activates Caspase-3/GSDME mediated pyroptosis by inhibiting SRC/AKT/m TOR signaling pathway. |
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