Zhu Chunyang, Chen Shuming, Lu Yangyang, Song Jialin, Wang Shasha, Guo Jing, Han Xiaoxi, Fang YuanYuan, Zhang Siyi, Qiu Wensheng, Qi Weiwei
Department of Oncology, The Affiliated Hospital of Qingdao University, Qingdao, PR China.
Department of Oncology, The Affiliated Hospital of Qingdao University, Qingdao, PR China.
Transl Oncol. 2025 Jul 29;60:102481. doi: 10.1016/j.tranon.2025.102481.
Gastric cancer (GC) is a prevalent malignant tumor worldwide, with limited treatment targets. Timosaponin AIII (Tim AIII) is the naturally steroid saponin isolated from Anemarrhena, while this study initially confirms the anti-GC effect of Tim AIII.
MTT assay, cell cycle analysis, and wound healing assay were used to evaluate the inhibitory effects of Tim AIII on GC cells (AGS and HGC27). Evaluate the oxidative stress (OS) by measuring reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear factor erythroid-derived 2-like 2 (Nrf2) pathway. RNA sequencing and proteomics analysis were utilized to investigate deeper molecular mechanisms. To track the autophagic flux using transmission electron microscope, detecting changes in autophagy-related pathway proteins, staining with LC3B and lysosome. Experiments related to cell viability, OS, and autophagy levels were performed on normal gastric mucosal epithelial cells (GES-1) as parallel controls. Finally, Nude mouse subcutaneous tumor model to evaluate the anti-GC ability in vivo.
Tim AIII inhibits the viability, proliferation, and migration of GC cells. Tim AIII causes OS in GC cells by the increasing intracellular ROS and MDA levels and inhibiting the Keap1-Nrf2 pathway. RNA sequencing and proteomics analysis mainly focused on the autophagy-associated pathways and lysosome in GC cells. Tim AIII activates autophagy, as indicated by an increase in the number of autophagosomes, inhibition of the PI3K-AKT pathway, and activation of the AMPK pathway in GC cells. However, Tim AIII inhibits autophagy-lysosome fusion and impairs lysosomal function, which results in autophagic flux blockage in GC cells. The Tim AIII concentration that significantly inhibited GC cells in this study was applied to GES-1 cells. The results showed that at this concentration, Tim AIII exhibited no significant cytotoxic effects on GES-1 cells, did not induce OS, and had no impact on autophagy. Finally, Tim AIII also has the ability to inhibit tumor growth in vivo.
In summary, the results of our study indicate Tim AIII as a novel late-stage autophagy inhibitor, which may provide novel medical possibilities for GC.
胃癌（GC）是全球范围内一种常见的恶性肿瘤，治疗靶点有限。知母皂苷AIII（Tim AIII）是从知母中分离出的天然甾体皂苷，而本研究首次证实了Tim AIII的抗胃癌作用。
采用MTT法、细胞周期分析和伤口愈合试验评估Tim AIII对胃癌细胞（AGS和HGC27）的抑制作用。通过测量活性氧（ROS）和丙二醛（MDA）以及kelch样ECH相关蛋白1（Keap1）-核因子红细胞衍生2样2（Nrf2）通路来评估氧化应激（OS）。利用RNA测序和蛋白质组学分析来研究更深层次的分子机制。使用透射电子显微镜追踪自噬通量，检测自噬相关通路蛋白的变化，用LC3B和溶酶体进行染色。在正常胃黏膜上皮细胞（GES-1）上进行与细胞活力、OS和自噬水平相关的实验作为平行对照。最后，利用裸鼠皮下肿瘤模型评估其体内抗胃癌能力。
Tim AIII抑制胃癌细胞的活力、增殖和迁移。Tim AIII通过增加细胞内ROS和MDA水平并抑制Keap1-Nrf2通路在胃癌细胞中引起OS。RNA测序和蛋白质组学分析主要聚焦于胃癌细胞中的自噬相关通路和溶酶体。Tim AIII激活自噬，表现为胃癌细胞中自噬体数量增加、PI3K-AKT通路受抑制以及AMPK通路激活。然而，Tim AIII抑制自噬-溶酶体融合并损害溶酶体功能，导致胃癌细胞中自噬通量受阻。本研究中显著抑制胃癌细胞的Tim AIII浓度应用于GES-1细胞。结果表明，在此浓度下，Tim AIII对GES-1细胞无明显细胞毒性作用，不诱导OS，且对自噬无影响。最后，Tim AIII在体内也具有抑制肿瘤生长的能力。
总之，我们的研究结果表明Tim AIII是一种新型的晚期自噬抑制剂，这可能为胃癌提供新的医学治疗可能性。
