Pharmacodynamic mechanism of Myristicae Semen's active ingredient dehydrodiisoeugenol regulating MDM2/p53 signaling pathway against colorectal cancer
Objective To investigate the effect and mechanism of dehydrodiisoeugenol(DDIE)in inhibiting colorectal cancer.Methods DDIE targets were predicted through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and the SwissTargetPrediction database.Disease targets for colorectal cancer were gathered from the GeneCards and Online Mendelian Inheritance in Man(OMIM)databases.The intersection of drug and disease targets was analyzed using the String database to assess protein-protein interactions(PPI).The Cytoscape 3.9.1 software plug-in was employed to evaluate node topology parameters and identify core targets.The Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analyses were conducted through Metascape database.The enriched core targets and key pathways were validated through in vivo and in vitro experiments.The impact of DDIE on the viability of mouse colorectal cancer cells(CT26),human colorectal cancer cells(HCT116),and human colorectal epithelial cells(NCM460)was assessed using a cell counting kit(CCK-8).The migration of HCT116 cells was evaluated through a scratch assay,while the apoptosis of CT26 and HCT116 cells was analyzed via Hoechst staining and flow cytometry.Additionally,Western blot was performed to measure the expression levels of pro-apoptotic proteins[B lymphoblastoma-2-associated X protein(Bax)and Caspase-3]in CT26 and HCT116 cells.A subcutaneous transplantation tumor model of CT26 colon cancer was established using 24 SPF-grade male Balb/c mice,which were divided into 4 groups:a model group,an irinotecan(CPT11)group(60 mg/kg,intraperitoneal injection),a low-dose DDIE group(25 mg/kg,gavage),and a high-dose DDIE group(50 mg/kg,gavage).Body mass and tumor size were recorded.The histopathological changes of tumor tissues were monitored using hematoxylin-eosin(HE)staining.Western blot was used to detect the effect of DDIE on the expression of tumor protein p53(p53)and murine double minute2(MDM2)in tumor tissues.Results The network pharmacological analysis results indicated that the anti-colon cancer effect of DDIE were associated with the p53 signaling pathway.CCK-8 results revealed that DDIE could inhibit the viability of CT26 and HCT116 colon cancer cells,with IC50 values of 53.56 μmol/L and 63.36 μmol/L,respectively,had little effect on the viability of normal epithelial cells NCM460 with IC50>100 μmol/L.Scratch assay results demonstrated that 10 μmol/L and 20 μmol/L DDIE significantly inhibited the migration of HCT116 cells in a dose-dependent manner(P<0.001).Hoechst staining assays and flow cytometry results revealed that 50 μmol/L DDIE significantly increased the apoptosis rates of CT26 and HCT116 cells(P<0.05,P<0.001).Western blot results analysis indicated that DDIE significantly enhanced the expression levels of pro-apoptotic proteins Bax and Caspase-3 in HCT116 cells(P<0.05,P<0.01,P<0.001),as well as Bax expression level in CT26 cells(P<0.05,P<0.01).Animal experiment result demonstrated that DDIE significantly reduced tumor mass and tumor volume of mice(P<0.05,P<0.001).Western blot results indicated that DDIE significantly decreased the protein expression levels of MDM2 in CT26 and HCT116 cells(P<0.05,P<0.01),while increased p53 expression(P<0.01,P<0.001);DDIE significantly decreased MDM2 expression level and increased p53 expression level in tumor tissues(P<0.05,P<0.01).Conclusion DDIE has an anti-colorectal cancer effect,and its mechanism is related to up-regulation of p53 protein expression,inhibition of tumor cell proliferation and migration,and promotion of tumor cell apoptosis.
colorectal cancerdehydrodiisoeugenolMyristicae Semenanti-tumournetwork pharmacologymouse modeltraditional Chinese medicine research
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