肝细胞癌（HCC）的转移导致预后不良，其中Notch1的激活是一个重要因素。 Cepharanthine (Cep) 因其有效的抗病毒功能和多种细胞内靶点而被确定。我们前期的研究仅报道了Cep对肺癌的抗癌功效，并未进行深入探索。本研究旨在探讨Cep在HCC中的抗转移作用、涉及的靶点以及分子机制。与C5WBF344细胞相比，Notch1-N1ICD稳定过表达产生的C5WN1细胞。采用C5WN1细胞和携带C5WN1细胞的小鼠作为HCC模型。通过生物信息学分析、RNA测序、分子对接、细胞热位移测定（CETSA）、药物亲和力响应靶稳定性（DARTS）、微量热泳动（MST）和瞬时敲低技术来鉴定潜在靶点。细胞凋亡测定、免疫荧光染色、qRT-PCR、蛋白质印迹、Elisa、流式细胞仪、迁移和划痕实验、透射电子显微镜 (TEM)、激光扫描共聚焦显微镜 (LSCM)、显微计算机断层扫描 (micro-CT) 和通过组织病理学实验来分析其抗HCC的功效、功能和机制。Notch1在HCC中表达增加并促进转移。令人惊讶的是，Cep（体外 2 μg/ml，体内 5 mg kg-1）在 C5WN1 细胞和原位小鼠模型中表现出有效的 Notch1 信号通路抑制作用和抗转移功效，这一点通过减少 Notch1/MMP-2/MMP- 来证明。 9 表达、TGF-β 释放、细胞迁移减少、肺转移减少并延长生存期。 RNA测序显示Cep处理的HCC细胞的差异基因定位于内质网(ER)。分子对接、CETSA、DARTS和MST进一步鉴定Cep的可能靶点是分布在ER中的GRP78。正如预期的那样，Cep (2 μg/ml) 上调了 ER 应激的关键分子，例如 GRP78，诱导 β-淀粉样蛋白积累，并促进 HCC 中的钙爆发。相反，抑制 GRP78 可减弱 Cep 诱导的 ER 应激。此外，抑制ER应激可以减轻Cep诱导的Notch1失活和HCC细胞的迁移。综上所述，本研究发现Cep具有优异的抗HCC转移作用，其中GRP78可以被Cep直接结合并激活，从而导致ER压力和Notch1阻塞。该研究首次揭示了Cep介导的抗癌作用的效果、关键靶点和机制，为植物医学的分子靶向治疗提供了新的见解。版权所有©2024 Elsevier GmbH。版权所有。

The metastasis of hepatocellular carcinoma (HCC) leads to a poor prognosis, wherein the activation of Notch1 is an essential contributor. Cepharanthine (Cep) has been identified for its effective antiviral function and versatile intracellular targets. Our previous study has only reported the anti-cancer efficacy of Cep in lung cancer, without an in-depth exploration. Herein, the present study aims to investigate the anti-metastasis effect in HCC, the target involved, and the molecular mechanism of Cep.Stable over-expression of Notch1-N1ICD yielded C5WN1 cells compared with C5WBF344 cells. The C5WN1 cells and C5WN1 cell-bearing mice were applied as the HCC model. The bioinformatics analysis, RNA sequencing, molecular docking, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), microscale thermophoresis (MST), and transient knockdown techniques were carried out to identify the underlying target. The apoptosis assay, immunofluorescent staining, qRT-PCR, Western blots, Elisa, flow cytometry, migration and scratching experiments, Transmission electron microscopy (TEM), laser scanning confocal microscopy (LSCM), micro-computed tomography (micro-CT), and histopathological experiments were conducted to assay the anti-HCC efficacy, functions, and mechanism.Notch1 had an increased expression in HCC and contributed to metastasis thereupon. Surprisingly, Cep (2 μg/ml in vitro, 5 mg kg-1in vivo) presented potent Notch1 signaling pathway inhibitory effect and anti-metastasis efficacy in C5WN1 cells and in situ mice models as evidenced by reduced Notch1/MMP-2/MMP-9 expression, TGF-β release, decreased cell migration, diminished pulmonary metastases, and prolonged survival. RNA sequencing showed that the differential gene of Cep-treated HCC cells was positioned in the endoplasmic reticulum (ER). Molecular docking, CETSA, DARTS, and MST further identified that the possible target of Cep was GRP78, which was distributed in the ER. As expected, Cep (2 μg/ml) up-regulated the critical molecules of ER stress such as GRP78, induced β-amyloid accumulation, and promoted calcium burst in HCC. In contrast, suppression of GRP78 attenuated Cep-induced ER stress. Furthermore, inhibition of ER stress abated Cep-induced Notch1 inactivation and HCC cells' migration.Taken together, the present study finds that Cep possesses excellent anti-metastasis of HCC, wherein the GRP78 could be directly bound and activated by Cep, leading to ER stress and Notch1 blockage. This study reveals for the first time the effect, critical target, and mechanism of the Cep-mediated anti-cancer effect, providing novel insights into the molecular target therapy by phytomedicine.Copyright © 2024 Elsevier GmbH. All rights reserved.