为了评估同时抑制 FGFR 和 PI3K/mTOR 信号通路对胆管癌 (CCA) 细胞致癌特征（包括增殖、自噬和细胞死亡）的影响。对 KKU-213A、KKU-100 和 KKU-213C 细胞进行处理单独或联合使用 infigratinib 或 PKI-402。使用磺基罗丹明 B (SRB) 测定和吖啶橙/溴化乙锭 (AO/EB) 染色评估细胞活力和细胞死亡。通过流式细胞术分析细胞周期进展和细胞凋亡。进行蛋白质印迹以评估参与细胞周期调节和自噬的蛋白质的表达。此外，采用 AO 染色来评估自噬诱导。与单一抑制剂治疗相比，infigratinib 和 PKI-402 的组合在两种 CCA 细胞系中显示出对细胞活力的显着协同抑制。这种抗增殖作用与细胞周期停滞在 G2-M 期以及 CCA 细胞中细胞周期蛋白 A 和细胞周期蛋白 B1 表达的减少有关。此外，联合治疗比单一抑制剂治疗更大程度地诱导凋亡细胞死亡。 Infigratinib 增强了 PKI-402 对自噬的诱导，吖啶橙染色的自噬空泡、LC3B-II 水平显着增加以及 p-mTOR 和 p-mTOR 水平的抑制证明了这一点。值得注意的是，氯喹抑制自噬流可防止联合治疗诱导的细胞死亡。这项研究表明，同时抑制 CCA 致癌过程中关键的 FGFR/PI3K/mTOR 通路可增强对 CCA 细胞的抑制。目前的研究结果表明在 CCA 治疗中使用联合治疗方式具有潜在的临床意义。© 2024 作者。

To assess the impact of concurrent inhibition of the FGFR and PI3K/mTOR signaling pathways on oncogenic characteristics in cholangiocarcinoma (CCA) cells, including proliferation, autophagy, and cell death.KKU-213A, KKU-100, and KKU-213C cells were treated with either infigratinib or PKI-402 alone or in combination. Cell viability and cell death were evaluated using the sulforhodamine B (SRB) assay and acridine orange/ethidium bromide (AO/EB) staining. Cell cycle progression and apoptotic cell death were analyzed by flow cytometry. Western blotting was performed to assess the expression of proteins involved in cell cycle regulation and autophagy. Additionally, AO staining was employed to assess autophagic induction.The combination of infigratinib and PKI-402 showed a remarked synergistic suppression in cell viability in both CCA cell lines compared to treatment with single inhibitors. This antiproliferative effect was associated with cell cycle arrest in the G2-M phase and a decrease in the expression of cyclin A and cyclin B1 in CCA cells. Furthermore, the combination treatment induced apoptotic cell death to a greater extent than treatment with a single inhibitor. Infigratinib enhanced the induction of autophagy by PKI-402, as evidenced by marked increases of autophagic vacuoles stained acridine orange, levels of LC3B-II and suppression of levels of p-mTOR and. Notably, inhibition of autophagic flux by chloroquine prevented cell death induced by the combination treatment.This study demonstrated that concurrent inhibition of the key FGFR/PI3K/mTOR pathways in CCA carcinogenesis enhances the suppression of CCA cells. The present findings indicate potential clinical implications for using combination treatment modalities in CCA therapy.© 2024 The Authors.