Aspirin (Acetylsalicylic Acid) Exerts Antineoplastic Effects on Bile Duct Carcinoma Cells Through Modulation of COX-2/ EGFR, AMPK, and IGF-1R Signaling Pathways.

Background/Aims: Bile duct carcinoma (BDC) is a highly aggressive malignancy. While epidemiological evidence suggests that acetylsalicylic acid (ASA [aspirin]) reduces BDC risk, the underlying molecular mechanisms have not been fully elucidated. This investigation explored the antineoplastic mechanisms of ASA in BDC cells. Materials and Methods: The human BDC cell line SNU-245 was used in all experiments. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, whereas apoptosis and caspase-3 levels were evaluated using enzyme-linked immunosorbent assay. Protein expression was analyzed using Western blotting and immunofluorescence. Functional pathway interactions were investigated using siRNA-mediated gene silencing. Results: ASA reduced cell viability and increased apoptosis markers, accompanied by increased Bax and p53 expression and decreased Bcl-2 levels. ASA treatment reduced cyclooxygenase-2 (COX-2) expression and decreased epidermal growth factor receptor (EGFR)levels. COX-2 knockdown markedly attenuated deoxycholic acid (DCA)-induced EGFR phosphorylation, whereas EGFR silencing partially reduced DCA-induced COX-2 expression. These results suggest reciprocal signaling interactions, with COX-2 exerting a relatively stronger upstream influence. ASA increased phosphorylation of AMP-activated protein kinase at threonine 172 (AMPKᵀʰʳ¹⁷²) and reduced insulin like growth factor-1 receptor (IGF-1R)/insulin receptor substrate-1 (IRS-1) signaling and decreased mammalian target of rapamycin (mTOR) phosphorylation. ASA also attenuated epidermal growth factor (EGF)-induced changes in epithelial-mesenchymal transition- related markers, including preservation of E-cadherin and reduction of N-cadherin expression. Conclusion: In this in vitro model, ASA exposure was associated with coordinated modulation of multiple cell-survival-related signaling pathways in BDC cells, including COX-2/EGFR signaling, AMPK activation, and IGF-1R-mediated mTOR regulation. These findings provide mechanistic insight into the potential antineoplastic effects of ASA and support further translational studies in BDC.