肝细胞癌（HCC）是癌症相关死亡的主要原因之一。尽管多激酶抑制剂可以延长晚期HCC患者的总体生存期，但耐药性的出现削弱了这些益处，最终导致治疗失败。因此，迫切需要新型有效的药物来阻止肝癌的进展。本研究采用浓度梯度增量法建立获得性索拉非尼或瑞戈非尼耐药的SNU-449细胞。使用细胞计数试剂盒8测定法评估细胞活力。一个由 793 个与代谢相关的生物活性小分子组成的库，筛选出针对亲代细胞和耐药细胞的化合物。筛选出的化合物将被添加到HCC亲本细胞和耐药细胞中，然后进行全面评估。使用试剂盒对细胞内三磷酸腺苷 (ATP) 水平进行定量。应用流式细胞术评估细胞凋亡和活性氧（ROS）。实时定量 PCR 研究相对基因表达，蛋白质印迹分析评估 HCC 亲代细胞和耐药细胞中的蛋白质表达变化。体内异种移植模型评估了Mito-LND和(E)-Akt抑制剂-IV对肝肿瘤的作用，用苏木精和伊红染色观察组织结构，免疫组化染色观察内质网应激蛋白表达。从化合物库中，我们筛选出了两种新化合物 Mito-LND 和 (E)-Akt 抑制剂-IV，可以有效杀死亲代细胞和耐药细胞。 Mito-LND 可通过上调糖酵解中间体并下调三羧酸 (TCA) 循环中间体，从而减少 ATP 产生并增加 ROS，从而显着抑制 HCC 亲代细胞和耐药细胞的增殖并诱导细胞凋亡。 (E)-Akt 抑制剂-IV 通过减少糖酵解中间体、增加 TCA 循环中间体以及降低 ATP 合成和 ROS 水平，取得了类似的结果。这两种化合物通过 AMPK/MAPK 通路和内质网应激反应的相互作用触发 HCC 细胞凋亡。体内实验还表明，这两种化合物能够显着抑制HCC细胞的生长并诱导内质网应激。通过高通量筛选，我们鉴定出Mito-LND和(E)-Akt抑制剂-IV是两种针对亲代细胞的新型化合物。和耐药 HCC 细胞，这可以为 HCC 患者提供新的策略。© 2024。作者。

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Although multi-kinase inhibitors can prolong the overall survival of late-stage HCC patients, the emergence of drug resistance diminishes these benefits, ultimately resulting in treatment failure. Therefore, there is an urgent need for novel and effective drugs to impede the progression of liver cancer.This study employed a concentration gradient increment method to establish acquired sorafenib or regorafenib-resistant SNU-449 cells. Cell viability was assessed using the cell counting kit-8 assay. A library of 793 bioactive small molecules related to metabolism screened compounds targeting both parental and drug-resistant cells. The screened compounds will be added to both the HCC parental cells and the drug-resistant cells, followed by a comprehensive assessment. Intracellular adenosine triphosphate (ATP) levels were quantified using kits. Flow cytometry was applied to assess cell apoptosis and reactive oxygen species (ROS). Real-time quantitative PCR studied relative gene expression, and western blot analysis assessed protein expression changes in HCC parental and drug-resistant cells. A xenograft model in vivo evaluated Mito-LND and (E)-Akt inhibitor-IV effects on liver tumors, with hematoxylin and eosin staining for tissue structure and immunohistochemistry staining for endoplasmic reticulum stress protein expression.From the compound library, we screened out two novel compounds, Mito-LND and (E)-Akt inhibitor-IV, which could potently kill both parental cells and drug-resistant cells. Mito-LND could significantly suppress proliferation and induce apoptosis in HCC parental and drug-resistant cells by upregulating glycolytic intermediates and downregulating those of the tricarboxylic acid (TCA) cycle, thereby decreasing ATP production and increasing ROS. (E)-Akt inhibitor-IV achieved comparable results by reducing glycolytic intermediates, increasing TCA cycle intermediates, and decreasing ATP synthesis and ROS levels. Both compounds trigger apoptosis in HCC cells through the interplay of the AMPK/MAPK pathway and the endoplasmic reticulum stress response. In vivo assays also showed that these two compounds could significantly inhibit the growth of HCC cells and induce endoplasmic reticulum stress.Through high throughput screening, we identified that Mito-LND and (E)-Akt inhibitor-IV are two novel compounds against both parental and drug-resistant HCC cells, which could offer new strategies for HCC patients.© 2024. The Author(s).