BackgroundIntrahepatic cholangiocarcinoma (iCCA) has a poor prognosis and limited treatment options. Aprepitant, a selective NK‐1R antagonist, can inhibit the growth of various tumours in vitro and in vivo. However, it remains unclear whether aprepitant has cytotoxic effects on iCCA.MethodsWe measured the expression of SP/NK‐1R in clinical samples of iCCA by immunohistochemistry. Then, we detected the cytotoxic effects of aprepitant on iCCA cells via MTT, EdU and colony formation assay. We constructed a subcutaneous xenograft model of BALB/c nude mice by using HCCC‐9810 and RBE cell lines to explore the effects of aprepitant in vivo. To elucidate the potential mechanisms, we explored the pro‐apoptotic effect of aprepitant by flow cytometric, western blotting, ROS detection and JC‐1 staining. Furthermore, we detected the autophagic level of HCCC‐9810 and RBE by western blotting, mRFP‐eGFP‐LC3 adenovirus transfection and electron microscope.ResultsSP/NK‐1R is significantly expressed in iCCA. Aprepitant inhibited human iCCA xenograft growth and dose‐dependently decreased the viability of RBE and HCCC‐9810 cells. Aprepitant‐induced mitochondria‐dependent apoptosis through ROS/JNK pathway. Additionally, pretreatment with z‐VAD‐fmk partly reversed the effect of aprepitant on cell viability, while NAC completely attenuated the cytotoxic effects of aprepitant in vitro. Furthermore, we observed the dynamic changes of autophagosome in RBE and HCCC‐9810 cells treated with aprepitant.ConclusionSP/NK‐1R signalling is significantly activated in iCCA and promotes the proliferation of iCCA cells. By contrast, aprepitant can induce autophagy and apoptosis in iCCA cells via ROS accumulation and subsequent activation of JNK.

中文翻译：

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神经激肽-1受体拮抗剂阿瑞匹坦通过ROS/JNK调节肝内胆管癌自噬和凋亡

背景肝内胆管癌（iCCA）预后不良，治疗选择有限。阿瑞吡坦是一种选择性 NK-1R 拮抗剂，可在体外和体内抑制多种肿瘤的生长。然而，阿瑞匹坦对iCCA是否具有细胞毒作用尚不清楚。方法采用免疫组化法检测iCCA临床样本中SP/NK-1R的表达。然后，我们通过MTT、EdU和集落形成实验检测了阿瑞匹坦对iCCA细胞的细胞毒作用。我们利用HCCC-9810和RBE细胞系构建BALB/c裸鼠皮下异种移植模型，以探讨阿瑞匹坦的体内作用。为了阐明潜在的机制，我们通过流式细胞术、蛋白质印迹、ROS检测和JC-1染色探讨了阿瑞吡坦的促凋亡作用。此外，我们通过western blotting、mRFP-eGFP-LC3腺病毒转染和电镜检测HCCC-9810和RBE的自噬水平。结果SP/NK-1R在iCCA中显着表达。阿瑞匹坦抑制人 iCCA 异种移植物生长，并剂量依赖性地降低 RBE 和 HCCC-9810 细胞的活力。阿瑞吡坦通过 ROS/JNK 途径诱导线粒体依赖性细胞凋亡。此外，z-VAD-fmk预处理部分逆转了阿瑞匹坦对细胞活力的影响，而NAC在体外完全减弱了阿瑞匹坦的细胞毒性作用。此外，我们还观察了阿瑞匹坦处理后的RBE和HCCC-9810细胞中自噬体的动态变化。结论SP/NK-1R信号在iCCA中显着激活，促进iCCA细胞的增殖。相比之下，阿瑞匹坦可以通过 ROS 积累和随后的 JNK 激活诱导 iCCA 细胞自噬和凋亡。