Synergistic Anti-Tumor Activity of Andrographolide and Gemcitabine in Intrahepatic Cholangiocarcinoma Through Inhibition of RRM2 and the JAK/STAT3 Pathway

Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy with limited treatment options, often exhibiting intrinsic resistance to gemcitabine (Gem), the standard first-line chemotherapy. This study investigates the synergistic anti-tumor effects of andrographolide (Andro), a natural diterpenoid from Andrographis paniculata, combined with Gem in naturally Gem-resistant ICC cell lines (HUCCT-1 and QBC-939). Using Chou-Talalay analysis, we demonstrated strong synergy (combination index <1.0) in inhibiting cell viability. The combination potently suppressed proliferation (via colony formation and EdU assays), migration/invasion (wound healing and Transwell assays), and epithelial-mesenchymal transition (downregulation of N-Cadherin and Vimentin). Mechanistically, it induced reactive oxygen species-dependent apoptosis (Annexin V/PI and JC-1 assays) and G1/S cell cycle arrest. Furthermore, bioinformatics and RT-qPCR identified ribonucleotide reductase M2 (RRM2) as a key Andro target, confirmed by molecular docking. Andro-Gem synergistically downregulated RRM2, leading to inactivation of the JAK/STAT3 pathway (reduced p-JAK1 and p-STAT3). Gene set enrichment analysis linked RRM2 to JAK/STAT3 activation. These findings suggest Andro overcomes Gem resistance by targeting the RRM2-JAK/STAT3 axis, proposing Andro-Gem as a promising therapeutic strategy for ICC, potentially eradicating cancer stem cell-like populations.