肝细胞癌（HCC）是一种高度致命的癌症，其特征是有优势的驱动突变，包括p53。因此，迫切需要寻找新的治疗HCC的药物。临床可用的抗炎植物化学药物Andrographolide（Andro）已显示出对多种癌症，包括HCC的抑制作用。然而，其作用的分子机制仍知之甚少。本研究旨在使用体外和体内模型，探索p53和p62如何共同影响Andro诱导的HCC细胞死亡的分子机制。进行了体外细胞实验，检测Andro对细胞存活率的影响，并阐明其作用机制。体内异种移植实验进一步验证了Andro的抗癌效果。Andro能够引起剂量和时间依赖的HCC细胞死亡，同时不损伤正常的HL-7702肝细胞。此外，Andro通过产生反应性氧化物（ROS）引起DNA损伤，这是导致细胞死亡的关键事件。值得注意的是，p53表达的HCC细胞相对于p53缺失的细胞对Andro诱导的细胞死亡表现出更大的耐受性，这可能是由于p53能够引起G2/M期细胞周期阻滞的能力。此外，Andro诱导的p62聚集导致RAD51和53BP1这两个参与DNA损伤修复的关键蛋白的泛素依赖性降解。因此，p62的沉默或基因敲除促进了DNA损伤修复并保护了HCC细胞。重要的是，p53或p62的干扰并不影响另一个蛋白的表达。这些发现进一步得到了通过p62基因敲除的HCC细胞形成的异种移植瘤对Andro治疗表现出增加抵抗能力的观察结果的支持。本研究阐明了Andro诱导的HCC细胞死亡的机制基础。它为将Andro重新用于HCC的治疗提供了宝贵的见解，而不论功能性p53是否存在。版权所有 © 2023 Elsevier GmbH。保留所有权利。

Hepatocellular carcinoma (HCC) is a highly lethal cancer characterized by dominant driver mutations, including p53. Consequently, there is an urgent need to search for novel therapeutic agents to treat HCC. Andrographolide (Andro), a clinically available anti-inflammatory phytochemical agent, has shown inhibitory effects against various types of cancer, including HCC. However, the underlying molecular mechanisms of its action remain poorly understood.This study aims to investigate the molecular mechanisms by which p53 and p62 collectively affect Andro-induced HCC cell death, using both in vitro and in vivo models.In vitro cellular experiments were conducted to examine the effects of Andro on cell viability and elucidate its mechanisms of action. In vivo xenograft experiments further validated the anti-cancer effects of Andro.Andro induced dose- and time-dependent HCC cell death while sparing normal HL-7702 hepatocytes. Furthermore, Andro caused DNA damage through the generation of reactive oxygen species (ROS), a critical event leading to cell death. Notably, HCC cells expressing p53 exhibited greater resistance to Andro-induced cell death compared to p53-deficient cells, likely due to the ability of p53 to induce G2/M cell cycle arrest. Additionally, Andro-induced p62 aggregation led to the proteasomal degradation of RAD51 and 53BP1, two key proteins involved in DNA damage repair. Consequently, silencing or knocking out p62 facilitated DNA damage repair and protected HCC cells. Importantly, disruption of either p53 or p62 did not affect the expression of the other protein. These findings were further supported by the observation that xenograft tumors formed by p62-knockout HCC cells displayed increased resistance to Andro treatment.This study elucidates the mechanistic basis of Andro-induced HCC cell death. It provides valuable insights for repurposing Andro for the treatment of HCC, regardless of the presence of functional p53.Copyright © 2023 Elsevier GmbH. All rights reserved.