Benja-ummarit (BU) 是一种传统的泰国草药配方，已被泰国传统医生开出治疗肝癌的处方。 BU 的临床试验表明，肝细胞癌 (HCC) 患者的总生存期有所增加，包括 1-3 期（有或没有既往标准化疗）和末期。临床结果不同于其他基于细胞凋亡的传统化疗。 BU 抗癌特性的分子机制尚不清楚。通过 HCC 细胞系和 HCC 大鼠组织的形态学和分子分析来研究 BU 诱导的铁死亡。BU 提取物在 HepG2 和 HuH-7 细胞中的细胞毒性，无论有或没有 LX与索拉非尼相比，分别通过 MTT 测定和观察球体形成来确定 2D 和 3D 培养物中的 -2。分别通过光学显微镜和透射电子显微镜（TEM）评估处理细胞的形态变化和细胞超微结构。此外，分别使用蛋白质印迹分析和免疫组织化学染色测定了细胞系和大鼠肝组织中铁死亡蛋白标记物的变化。为了研究介导铁死亡的途径，用铁螯合剂预处理细胞以确认 BU 提取物诱导的铁依赖性铁死亡。使用扫描离子电导显微镜 (SICM) 测量细胞内 ROS（铁死亡的介质）。 SICM 还用于测定细胞硬度。使用 LC-MS/MS 研究 BU 处理细胞的脂质谱。BU 提取物在所有 HCC 细胞培养条件下诱导细胞死亡。通过 MTT 测定，HepG2 和 HuH-7 中的 BU-IC50 分别为 31.24 ± 4.46 μg/mL 和 23.35 ± 0.27 μg/mL。在与 LX-2 共培养中，BU 在 HepG2 和 HuH-7 细胞中表现出相似的细胞毒性趋势。光学显微镜显示细胞膨胀特征且质膜完整，TEM 显微镜显示 BU 处理的细胞中线粒体肿胀且线粒体嵴减少。 BU 通过增加 DMT1 和 NCOA4 表达并减少 FTH1 表达来促进细胞内铁水平。 BU 还通过降低 CD98、NRF2 和 GPX4 表达以及促进 KEAP1 表达来抑制细胞抗氧化系统。 HCC大鼠肝组织IHC结果显示肿瘤区域DMT1缺失，GPX4高表达。在BU处理组中，用铁螯合剂预处理部分恢复了细胞活力，并将细胞死亡模式转变为更像细胞凋亡的形态。 SICM 显示 BU 处理后 24 小时细胞内 ROS 水平和细胞硬度增加。更详细地了解 BU 介导的铁死亡，细胞脂质分析显示，在 BU 处理的细胞中，3 种多不饱和脂质的表达增加，这些脂质对脂质过氧化非常敏感。细胞内铁水平、ROS 水平和细胞脂质组成的变化先前已被研究过。在癌细胞中有报道。因此，通过 BU 诱导的铁死亡来靶向铁依赖性 ROS 途径和多不饱和脂质可能比基于细胞凋亡的癌症药物更具癌症特异性。这些观察结果与 BU 的临床结果一致。 BU 的铁死亡诱导机制使其成为治疗 HCC 的极有前景的新型候选药物。版权所有 © 2024。由 Elsevier B.V. 出版。

Benja-ummarit (BU), a traditional Thai herbal formula, has been prescribed by traditional Thai practitioners for the treatment of liver cancer. Clinical trials of BU have shown an increase in overall survival in hepatocellular carcinoma (HCC) patients, including stage 1-3 (with or without prior standard chemotherapy) and terminal stage. The clinical outcomes differ from those of other apoptosis-based conventional chemotherapies. The molecular mechanisms underlying the anti-cancer properties of BU remain unclear.To investigate BU-induced ferroptosis through morphological and molecular analyses of HCC cell lines and HCC rat tissues.Cytotoxicity of BU extract in HepG2 and HuH-7 cells, with or without LX-2 in 2D and 3D cultures, was determined through MTT assay and by observing spheroid formation, respectively, as compared to sorafenib. Morphological changes and the cellular ultrastructure of the treated cells were evaluated by light microscopy and transmission electron microscopy (TEM), respectively. In addition, alterations in ferroptosis protein markers in both cell lines and rat liver tissue were determined using western blot analysis and immunohistochemical staining, respectively. To investigate the pathways mediating ferroptosis, cells were pretreated with an iron chelator to confirm the iron-dependent ferroptosis induced by the BU extract. Intracellular ROS, a mediator of ferroptosis, was measured using a scanning ion conductance microscope (SICM). SICM was also used to determine cellular stiffness. The lipid profiles of BU-treated cells were studied using LC-MS/MS.The BU extract induced cell death under all HCC cell culture conditions. The BU-IC50 in HepG2 and HuH-7 were 31.24 ± 4.46 μg/mL and 23.35 ± 0.27 μg/mL, respectively as determined by MTT assay. In co-culture with LX-2, BU exhibited a similar trend of cytotoxicity in both HepG2 and HuH-7 cells. Light microscopy showed cell ballooning features with intact plasma membranes, and TEM microscopy showed mitochondrial swelling and reduced mitochondrial cristae in BU-treated cells. BU promotes intracellular iron levels by increasing DMT1 and NCOA4 expression and decreasing FTH1 expression. BU also suppressed the cellular antioxidant system by lowering CD98, NRF2, and GPX4 expression, and promoting KEAP1 expression. IHC results of HCC rat liver tissues showed the absence of DMT1 and high expression of GPX4 in the tumor area. Pre-treatment with an iron chelator partially restored cell viability and shifted the mode of cell death to a more apoptosis-like morphology in the BU-treated group. The SICM showed increased intracellular ROS levels and cellular stiffness 24 h after BU treatment. In more detail of BU-mediated ferroptosis, cellular lipid profiling revealed increased expression of 3 polyunsaturated lipids, which are highly susceptible to lipid peroxidation, in BU-treated cells.Alterations in intracellular iron levels, ROS levels, and cellular lipid composition have been previously reported in cancer cells. Therefore, targeting the iron-dependent ROS pathway and polyunsaturated lipids via BU-induced ferroptosis may be more cancer-specific than apoptosis-based cancer drugs. These observations are in accordance with the clinical outcomes of BU. The ferroptosis-inducing mechanism of BU makes it an extremely promising novel drug candidate for the treatment of HCC.Copyright © 2024. Published by Elsevier B.V.