| Background: Intrahepatic cholangiocarcinoma(ICC)is a malignant tumor that originates from the epithelial cells of the bile ducts within the liver parenchyma.It is characterized by high malignancy,insidious symptoms,and poor prognosis.Surgery and chemotherapy are the main treatment options for ICC,but due to limited efficacy and drug resistance of current chemotherapy drugs,it is crucial to explore safe,universal,and effective anti-cholangiocarcinoma drugs.Cinobufotalin has strong antitumor effects,but its impact on ICC and the specific mechanisms of action remain unclear.Objective: This study aims to investigate the effects of cinobufotalin on the biological characteristics(proliferation and apoptosis)of ICC cells by in vitro intervention with cinobufotalin.Furthermore,the study will explore the molecular mechanisms underlying its actions based on proteomics and phosphoproteomics analysis,aiming to provide theoretical support for clinical treatment.Methods:(1)Cinobufotalin with different final concentrations(0,0.1,0.25,0.5,1.0,and 2.0 μM)were used to treat RBE and HCCC-9810 cells for 24,48,and 72 hours,and cell viability was measured using the CCK-8 assay.(2)The colony formation assay was used to investigate the effect of cinobufotalin on the clonogenic ability of RBE and HCCC-9810 cells.(3)Flow cytometry was used to detect the effect of cinobufotalin on the apoptosis of RBE and HCCC-9810 cells.(4)Proteomic and phosphoproteomic techniques were used to identify changes in protein and phosphorylation site expression levels in RBE cells treated with cinobufotalin.(5)Data processing and visualization analysis were conducted using R language.(6)Bioinformatics analysis such as GO,KEGG,protein interaction,and kinase substrate regulatory network analysis were performed using databases and online websites such as DAVID,Metascape,SIGNOR,and Phospho Site Plus.(7)Western blot was used to detect changes in the expression of relevant proteins in enriched pathways.Results:(1)The CCK-8 assay showed that cinobufotalin significantly inhibited the activity of intrahepatic cholangiocarcinoma cells,with IC50 values of 0.342 μM and 0.421 μM for RBE and HCCC-9810 cells,respectively.The colony formation assay demonstrated that cinobufotalin significantly inhibited the clonogenicity of intrahepatic cholangiocarcinoma cells.Flow cytometry analysis revealed that cinobufotalin significantly promoted apoptosis of intrahepatic cholangiocarcinoma cells.(2)A total of 6,740 proteins were identified by proteomics,with 200 proteins significantly upregulated(FC(CB/Ctr)>1.25,p<0.05)and 418 proteins significantly downregulated(FC(CB/Ctr)<0.8,p<0.05)after cinobufotalin intervention.KEGG pathway analysis showed that the upregulated proteins were mainly enriched in the p53 signaling pathway,amino acid biosynthesis,and cell apoptosis,while the downregulated proteins were mainly enriched in steroid biosynthesis,terpenoid biosynthesis,and ubiquitin-mediated proteolysis.(3)A total of 16,445 phosphorylation sites were identified by phosphoproteomics,with 321 sites significantly upregulated(FC(CB/Ctr)>1.25,p<0.05)and 1,541 sites significantly downregulated(FC(CB/Ctr)<0.8,p<0.05)after cinobufotalin intervention.Enrichment analysis of biological processes showed that the upregulated phosphorylation sites were mainly enriched in the response of cells to DNA damage stimulus,and kinase substrate enrichment analysis indicated significant activation of ATM kinase.(4)Western blot analysis further confirmed that the expression levels of apoptosis-and DNA damage-related proteins,including Cleaved caspase-3,γH2AX,p-ATM-S1981,p-CHK2-T68,and p-p53-S15,were significantly upregulated after cinobufotalin intervention,while the expression of the apoptotic regulatory protein Bcl-2 was downregulated.Conclusion: Cinobufotalin can inhibit the proliferation of intrahepatic cholangiocarcinoma cells and promote cell apoptosis by activating the ATM/CHK2/p53 signaling pathway through DNA damage.Therefore,cinobufotalin has the potential to become a promising anti-cholangiocarcinoma drug. |
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