Carrimycin modulates malignant behavior of intrahepatic cholangiocarcinoma cells through inhibition of the AXL/c-Met/c-Myc signaling axis

WANG Tongshu; ZHOU Min; LIU Huimin; PIAO Hongxin

doi:10.3969/j.issn.1006-5725.2025.15.003

Abstract：Objective The effects of carrimycin(CAM)on the biological functions of intrahepatic cholan-giocarcinoma HuCCT1 cells were examined through in vitro experiments,and a preliminary investigation was conducted into its mechanism of action.Methods The intrahepatic cholangiocarcinoma cell line HuCCT1 was selected for the study.The effect of CAM on cell viability was assessed using the CCK-8 assay,and the IC50 concen-tration was determined accordingly.The impact of CAM on cell migration was evaluated through a scratch wound healing assay.In addition,the effect of CAM on clonogenic ability was examined using a colony formation assay.Cell invasion capacity was assessed using a Transwell invasion assay.Flow cytometry was employed to analyze the effect of CAM on cell cycle progression.Furthermore,Western blotting was conducted to evaluate the expression levels of key proteins associated with epithelial-mesenchymal transition and the cell cycle.Finally,the influence of CAM on the AXL/c-Myc/c-Met signaling axis was also investigated.Results Compared with the control group,CAM significantly inhibited the proliferation of HuCCT1 cells in a concentration-dependent manner(P<0.05).Plate cloning assays demonstrated that CAM markedly suppressed the colony-forming ability of HuCCT1 cells(P<0.05).Scratch wound healing assays confirmed that CAM treatment significantly reduced the migration speed and narrowed the migration area of HuCCT1 cells(P<0.05).Flow cytometry analysis revealed that CAM treatment led to a significant increase in the proportion of cells in the G0/G1 phase and a decrease in the S phase(P<0.05).Western blot analysis further confirmed that the expression levels of key regulatory proteins CCND1 and CDK4,which are involved in the G1/S phase transition,were down-regulated,while the expression of p21 was up-regulated(P<0.05).Transwell invasion assays indicated that CAM inhibited the invasive capacity of HuCCT1 cells.Consis-tently,Western blot results showed that E-Cadherin expression was increased(P<0.05),whereas the expression levels of N-Cadherin and Vimentin were decreased(P<0.05).Moreover,Western blot analysis verified that the expression of AXL,c-Met,and c-Myc was up-regulated in HuCCT1 cells treated with AXL recombinant protein(P<0.05).However,co-treatment with CAM and AXL recombinant protein significantly attenuated the expression of these proteins(P<0.05).Conclusions CAM inhibits the proliferation,migration,and invasion of intrahe-patic cholangiocarcinoma HuCCT1 cells,thereby demonstrating antitumor effects,which may be associated with the AXL/c-Met/c-Myc signaling pathway.