Synergistic anti-tumor activity of andrographolide and gemcitabine in intrahepatic cholangiocarcinoma through inhibition of RRM2 and the JAK/STAT3 pathway.

PURPOSE: Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy frequently characterized by intrinsic resistance to standard gemcitabine (Gem) chemotherapy. This study aimed to investigate whether andrographolide (Andro) could sensitize intrinsically gemcitabine-resistant ICC cells to gemcitabine and to explore the underlying molecular mechanisms.

METHODS: Naturally Gem-resistant ICC cell lines (HUCCT-1 and QBC-939) were treated with Andro, Gem, or their combination. Synergistic effects were quantified using the Chou-Talalay method. Anti-tumor efficacy was assessed via CCK-8, colony formation, EdU incorporation, wound healing, and Transwell invasion assays. ROS accumulation, apoptosis, mitochondrial membrane potential, and cell-cycle distribution were assessed by fluorescence microscopy and flow cytometry, as appropriate. Molecular targets were identified using molecular docking, RT-qPCR, and Western blotting.

RESULTS: Andro and Gem exhibited strong synergistic anti-tumor activity (Combination Index < 1.0) in both cell lines. The combination significantly suppressed proliferation, motility, and epithelial-mesenchymal transition. Mechanistically, the synergy was driven by a surge in intracellular ROS, triggering mitochondrial apoptosis and G1/S phase arrest. Furthermore, Ribonucleotide-diphosphate reductase subunit M2 was identified as a candidate molecular target associated with the effects of andrographolide, leading to its downregulation and the subsequent inactivation of the JAK/STAT3 signaling pathway.

CONCLUSION: Andrographolide overcomes intrinsic Gem resistance in ICC, potentially through modulation of the RRM2/JAK/STAT3 axis. This combination therapy represents a promising strategy for treating chemoresistant intrahepatic cholangiocarcinoma.