Mechanistic Study Of Dioscin In Regulating Chemotherapy Sensitivity Of Colon Cancer Via ABCA1

Objective(s):Colon cancer has the third-highest incidence rate globally.The standard clinical approach for patients with advanced colon cancer involves surgical removal of primary or metastatic tumors,followed by combination chemotherapy using5-fluorouracil/capecitabine and oxaliplatin.Nonetheless,the long-term effectiveness of chemotherapy can be hindered by the development of resistance and drug-related toxicity.Chemotherapy resistance is the main cause of high mortality in patients with refractory colon cancer,with a 5-year survival rate of only 12%.Actively exploring molecular mechanisms and intervention methods to overcome chemotherapy resistance is an essential strategy to improve the effectiveness of chemotherapy.During pre-experiments,we found that dioscin(DIO),a small molecule compound from Chinese medicine,significantly inhibited various colon cancer cell lines.This study aims to investigate the chemosensitivity of DIO in colon cancer,with the objective of providing valuable laboratory data that can be used to develop sensitizers for traditional cancer chemotherapy regimens.Methods:1.Chemosensitivity of dioscin on colon cancer cellsThe HCT116 human colon cancer cell line and its lymph node metastatic derivative SW620 were revived and cultured until reaching the logarithmic phase.Cells were treated with varying concentrations of Dioscin(DIO),5-Fluorouracil(5-Fu),and Oxaliplatin(L-OHP)for 24-48 hours to calculate the half inhibitory concentration(IC50)using the MTT assay.According to IC50,cells were then co-treated with 5-Fu and L-OHP(6.4 μM,12.8 μM)in combination with DIO(0.4-3.6μM)for 24 hours,and the combined drug index was calculated using the MTT method.Cells were grouped and treated with 5-Fu and L-OHP(6.4 μM)individually or in combination with DIO(0.8 μM)for 24 h.The proliferation,apoptosis,cycle,invasion,and migration ability of the cells were analyzed,and the related phenotypes and Notch pathway proteins were detected using Western blotting(WB).A nude mouse model of transplanted tumor was constructed,and the mice were treated with L-OHP alone or in combination with DIO to observe the drug’s growth inhibition on the transplanted tumor.The growth curve was plotted,and blood samples were collected from the peripheral veins of the eye to test for liver function and evaluate the drug’s hepatotoxicity.2.Study on chemosensitivity targets of dioscin in colon cancerThe main experimental cell used was HCT116,and the groups were divided into HCT116-0,HCT116-DIO 0.8μM,HCT116-L-OHP 6.4μM,and HCT116-DIO 0.8μM+ L-OHP 6.4μM treated cells for 24 hours,followed by RNA sequencing(RNA-seq)analysis.DIO and colon cancer disease targets were identified in the database,and differentially expressed genes in the database were intersected with the candidate gene to obtained candidate intersection genes.Functional enrichment analysis was performed on candidate intersection genes,and protein protein interaction(PPI)network was constructed.The colon cancer dataset was further mined based on the TCGA database,and differentially expressed genes between tumor and non-tumor groups were intersected with candidate intersection genes,which were named potential active targets.The clinical correlation and chemosensitivity of potential active targets were analyzed.Potential active targets transcription factors(TF)were predicted,and the potential active targets-TF regulatory network was constructed,followed by a molecular docking experiment.Finally,cell validation was performed using real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting(WB).3.Study on the mechanism of dioscin regulating chemotherapy sensitization of colon cancer by ABCA1First,multiple colon cancer cells were tested for ABCA1 mRNA expression using RT-qPCR,and HCT116 and SW620 were selected for further experiments.Cells were divided into control,DIO 0.8μM,DIO 0.8μM + si-NC,and DIO 0.8μM +si-ABCA1 groups.WB was used to evaluate the expression of apoptosis,cycle,and pathway-related proteins,and MTT was used to measure cell proliferation.Results confirmed that DIO inhibited tumor growth by modulating the Notch pathway through ABCA1.Secondly,a SW620-5Fu-resistant cell line was generated and identified to further explore the chemosensitivity-enhancing effect of DIO in colon cancer.Drug-resistant strains were divided into groups and WB was used to detect the expression levels of Notch1,Hes1,and drug-resistant proteins ABCC1 and P-gp.The sensitization mechanism of DIO on colon cancer through ABCA1 was further elucidated.Lastly,the tumor tissues of nude mice transplanted with tumors and drugs were subjected to immunohistochemistry(IHC)to analyze the correlation between Ki-67,Notch1,and ABCA1.Results:1.Sensitization of colon cancer cells by DIO in cooperation with 5-Fu and L-OHPThe inhibitory effect of DIO,5-Fu,and L-OHP drugs on HCT116 and SW620 cell lines exhibited a dose and time-dependent relationship.A comparison of the IC50 values showed that the IC50 concentration of DIO is the smallest,and L-OHP and5-Fu could easily induce resistance in HCT116 and SW620 cell lines.The combined effect of chemotherapy drugs and DIO on cells for 24 hours showed a significantly higher cell inhibition rate in the dual-drug group compared to the single-drug group.The combined effect of the two drugs depended on the concentration of DIO,and the combined drug index CI <1.Cell phenotype verification shows showed that the dual drug group canould significantly inhibit cell proliferation compared to the single drug group,and play a role againstin resisting cell invasion and migration by inducing cell apoptosis and GO/G1 cycle arrest.It is widely thought that the Notch signaling pathway is a key regulatory drug resistance pathway positively correlated with colon cancer pathogenesis.Notch1 and Hes1 protein expression decreased gradually when the two cells were treated with DIO in a dose-dependent manner.The experiments involving HCT116 cells in both single and dual-drug groups demonstrated that combining the two drugs could reverse the aberrant activation of Notch1 induced by L-OHP and substantially decrease Hes1 protein expression.The results confirmed that DIO could promote chemosensitivity through the Notch pathway.In vivo experiments results in mice showed that daily intragastric administration of DIO daily intragastric administration canexhibited better performance in inhibiting the growth of transplanted tumors more than L-OHP regular administration,and the combination of two drugs significantly inhibited the growth of tumorumor growth.Moreover,DIO did not exhibit any additional burden on liver function and displayed low toxicity,indicating its potential for use in clinical settings.2.Prediction of active targets of DIO on colon cancer cellsAfter database retrieval and transcriptome sequencing data intersection analysis,we identified 31 genes that were common targets of DIO in colon cancer(candidate intersection genes).GO enrichment analysis of candidate intersection genes genes showed that most were related to cell cycle and apoptosis regulation.KEGG enrichment analysis was significantly correlated with the p53 signal pathway,platinum drug resistance,cell cycle,cell apoptosis,and other metabolic and signal transduction pathways.A protein-protein interaction network was constructed for candidate intersection genes and analyzed with the TCGA-COAD dataset,resulting in the identification of five potential active targets for DIO in colon cancer.The upregulated genes included cell death receptor(FAS),cyclin-dependent kinase inhibitor 1A(CDKN1A/P21),ATP binding cassette transporter A1(ABCA1),and peroxisome proliferators-activated receptors α(PPAR α/ PPARA).On the other hand,cyclin-dependent kinase 1(CDK1)was found to be downregulated.Further clinical data analysis shows that the expression of FAS,CDKN1 A,ABCA1,and PPARA in cancer tissue was lower than that in paracancerous tissue,while CDK1 expression was higher than that in paracancerous tissue.Chemotherapy drug sensitivity analysis showed a significant correlation between the potential active targets and many drugs.Finally,the cell experiment confirmed that DIO could increase the chemosensitivity of colon cancer through the potential active targets;the observed trend in gene expression aligned with earlier predictions,and the effectiveness of the treatment was concentration-dependent.3.DIO regulates the Notch signaling pathway through ABCA1 to enhance the chemosensitivity of colon cancer cellsAfter analyzing and comparing multiple colon cancer cell lines and normal colon epithelial cells by RT-qPCR,we found that ABCA1 expression was downregulated in colon cancer cells.ABCA1 knockdown reversed the proliferation inhibition,apoptosis induction,and cycle arrest of DIO on HCT116 and SW620 and reversed the inhibition of DIO on the Notch pathway.The SW620-5Fu drug-resistant cell line was next constructed.Upon treatment with 5Fu,the cell morphology of SW620 changed from a "pebble" to a "spindle" shape,and there was an increase in the expression of drug-resistant proteins such as ABCC1 and P-gp.However,when ABCA1 was transfected into a drug-resistant cell line and treated with DIO at a concentration of0.8 μM,the inhibition of the Notch pathway and drug resistance proteins were reversed.These results suggest that DIO may inhibit the proliferation of HCT116 and SW620 cells via ABCA1,while also promoting sensitization to traditional chemotherapy drugs,which is likely regulated by the Notch signaling pathway.Immunohistochemical results of transplanted tumors in nude mice showed that Ki-67 expression was comparable after L-OHP treatment,suggesting that the drug yielded a weak inhibitory effect on tumor proliferation.Compared with the control group,Ki-67 and Notch1 expression in the DIO synergetic L-OHP group gradually decreased,and ABCA1 expression gradually increased.The in vivo experiment results were consistent with those of cell experiments.Conclusion(s):1.Dioscin inhibits the proliferation of colon cancer cells by inducing cell apoptosis,cell cycle arrest,and inhibiting cell invasion and migration while enhancing the sensitivity of oxaliplatin and 5-fluorouracil to chemotherapy of colon cancer cells.2.Dioscin exerts its anticancer effect by inhibiting the Notch signal pathway.3.Transcriptome analysis combined with high-throughput pharmacoinformatics analysis revealed several genes including FAS,CDKN1 A,ABCA1,PPARA,and CDK1 as potential active targets of dioscin in colon cancer.4.Dioscin regulates the Notch pathway through ABCA1 to inhibit the proliferation of colon cancer cells and enhance their chemosensitivity.