Li Yiquan, Zhu Yilong, Fang Jinbo, Li Wenjie, Li Shanzhi, Liu Xing, Liu Zirui, Song Gaojie, Shang Chao, Cong Jianan, Bai Bing, Sun Lili, Jin Ningyi, Li Xiao
Academician Workstation of Jilin Province, Changchun University of Chinese Medicine, Changchun, China.
Institute of Military Veterinary Medicine, Academy of Military Medical Science, Changchun, China.
Front Oncol. 2020 Jun 25;10:1026. doi: 10.3389/fonc.2020.01026. eCollection 2020.
Apoptin is a protein that specifically induces apoptosis in tumor cells. The anti-tumorigenic functions of Apoptin, including autophagy activation and its interaction with apoptosis, have not been precisely elucidated. Here we investigate the main pathways of apoptin-mediated killing of human liver cancer cells, as well as its putative role in autophagy and apoptosis. The anti-proliferative effect of apoptin in liver cancer cells was analyzed by crystal violet staining and MTS detection, and also using a tumor-based model. The main pathway related to apoptin-induced growth inhibition was evaluated by flow cytometry and fluorescence staining. The relationship between apoptosis and autophagy on apoptin-treating cells was analyzed using apoptosis and autophagy inhibitors, mitochondrial staining, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. The effect of ROS toward the apoptosis and autophagy of apoptin-treating cells was also evaluated by ROS detection, Annexin V-FITC/PI flow detection, LC3 staining, and western blotting. Inhibition of apoptosis in apoptin-treating liver cancer cells significantly reduced the autophagy levels . The overall inhibition increased from 12 h and the effect was most obvious at 48 h. Inhibition of autophagy could increase apoptin-induced apoptosis of cells in a time-dependent manner, reaching its peak at 24 h. Apoptin significantly alters ROS levels in liver cancer cells, and this effect is directly related to apoptosis and autophagy. ROS appears to be the key factor linking apoptin-induced autophagy and apoptosis through the mitochondria in liver cancer cells. Therefore, evaluating the interaction between apoptin-induced apoptosis and autophagy is a promising step for the development of alternate tumor therapies.
凋亡素是一种能特异性诱导肿瘤细胞凋亡的蛋白质。凋亡素的抗肿瘤功能，包括自噬激活及其与凋亡的相互作用，尚未得到确切阐明。在此，我们研究凋亡素介导的人肝癌细胞杀伤的主要途径，以及其在自噬和凋亡中的假定作用。通过结晶紫染色和MTS检测，以及使用基于肿瘤的模型，分析凋亡素在肝癌细胞中的抗增殖作用。通过流式细胞术和荧光染色评估与凋亡素诱导的生长抑制相关的主要途径。使用凋亡和自噬抑制剂、线粒体染色、膜联蛋白V-FITC/PI流式检测、LC3染色和蛋白质免疫印迹分析凋亡素处理细胞时凋亡与自噬之间的关系。还通过ROS检测、膜联蛋白V-FITC/PI流式检测、LC3染色和蛋白质免疫印迹评估ROS对凋亡素处理细胞凋亡和自噬的影响。抑制凋亡素处理的肝癌细胞中的凋亡显著降低了自噬水平。总体抑制从12小时开始增加，在48小时时效果最明显。抑制自噬可使凋亡素诱导的细胞凋亡呈时间依赖性增加，在24小时时达到峰值。凋亡素显著改变肝癌细胞中的ROS水平，且这种作用与凋亡和自噬直接相关。ROS似乎是通过线粒体连接凋亡素诱导的肝癌细胞自噬和凋亡的关键因素。因此，评估凋亡素诱导的凋亡与自噬之间的相互作用是开发替代肿瘤治疗方法的有前景的一步。
