Research Institute of Life Science, College of Veterinary Medicine, Gyeongsang National University, Gazwa, Jinju 52828, Republic of Korea.
Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University, School of Medicine, 90 Chilam-dong, Jinju 52727, Republic of Korea.
Am J Chin Med. 2020;48(3):679-702. doi: 10.1142/S0192415X20500342. Epub 2020 Apr 24.
Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.
自噬是一种主动程序性细胞死亡的过程，其中垂死的细胞会诱导自噬体，然后被降解机制所调控。本研究旨在探讨柚皮苷类黄酮诱导 AGS 癌细胞发生自噬性细胞死亡的机制。AGS 细胞的生长抑制显示，柚皮苷处理后下调了 PI3K/Akt/mTOR 信号通路。透射电子显微镜观察显示，肿胀的线粒体和靠近核周区的溶酶体与自噬小泡融合。用雷帕霉素预处理柚皮苷处理的 AGS 细胞，发现 mTOR 磷酸化显著减少，LC3B 激活增加。随着柚皮苷时间依赖性处理，观察到 mTOR 磷酸化的减少与溶酶体功能的激活有关。通过 LAMP1 激活观察到溶酶体膜通透性（LMP）的诱导，导致溶酶体细胞死亡，溶酶体腔中的组织蛋白酶 D 释放到细胞质中。用柚皮苷处理的 AGS 细胞显示 BH3 结构域 Bad 上调，Bcl-xL 下调，Bad 磷酸化和线粒体荧光强度表达显著。通过个人系数相关性检查，观察到 AGS 细胞中溶酶体和 LC3B 的显著定位和激活。在柚皮苷处理过程中观察到 ERK1/2-p38 MAPKs 的激活和细胞内 ROS 的产生。还阐明了 NAC 的预处理抑制了线粒体-LC3B 共定位，其中 ROS 作为 ERK1/2-p38 MAPKs 激活的上游。通过 BAF A1 预处理、抑制 LAMP1、组织蛋白酶 D、ROS 和阻断 AGS 细胞死亡中的自噬溶酶体，评估了溶酶体细胞死亡的参与。综上所述，这些发现表明，柚皮苷诱导的自噬细胞死亡涉及 LMP 介导的溶酶体损伤和 BH3 蛋白 Bad 在 AGS 癌细胞中的激活。
