Department of Bone Metabolism, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, Shandong Key Laboratory of Oral Tissue Regeneration, Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, China.
Department of Bone Metabolism, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, Shandong Key Laboratory of Oral Tissue Regeneration, Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan 250012, China.
Cell Signal. 2021 Feb;78:109841. doi: 10.1016/j.cellsig.2020.109841. Epub 2020 Nov 18.
Eldecalcitol (ED-71) is a new type of vitamin D analog, and vitamin D has been reported to have therapeutic effects in infectious disease, autoimmune disease, and cancer. However, the anti-cancer effect of ED-71 remains unclear. The objective of this study was to explore the anti-cancer effect of ED-71 in human osteosarcoma cells and to identify the related mechanism. The CCK8 assay results showed that ED-71 inhibited MG-63 cell viability in dose and time dependent manners. Cloning and Transwell invasion assays showed that ED-71 inhibited clonal and invasion ability of MG-63 cells. Flow cytometry results showed ED-71 the G2/M cycle arrest rate, apoptosis, and intracellular ROS. Western blot was used to detect cleaved-caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and P62 levels and the mTOR pathway. The increase of LC3-II and P62 indicated that ED-71 induced the formation of autophagosomes and inhibited autophagy flux. Furthermore, ED-71-induced apoptosis was weakened after adding 3-methyladenine and ED-71-induced early autophagy was weakened by caspase-3 inhibitor (Z-VAD-FMK), which indicated the two processes active each other in the presence of ED-71. Furthermore, N-acetylcysteine (NAC) pretreatment reversed the ED-71-treatment outcomes, including increased apoptosis and autophagy and inhibition of the PI3K/Akt/mTOR pathway. In conclusion, our results reveal that ED-71 induced G2/M arrest, apoptosis and autophagy in MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway.
艾地骨化醇（ED-71）是一种新型维生素 D 类似物，已有研究报道维生素 D 在感染性疾病、自身免疫性疾病和癌症中具有治疗作用。然而，ED-71 的抗癌作用尚不清楚。本研究旨在探讨 ED-71 对人骨肉瘤细胞的抗癌作用及其相关机制。CCK8 检测结果表明，ED-71 呈剂量和时间依赖性抑制 MG-63 细胞活力。克隆形成和 Transwell 侵袭实验表明 ED-71 抑制 MG-63 细胞的克隆和侵袭能力。流式细胞术结果显示 ED-71 可使细胞周期阻滞于 G2/M 期，促进细胞凋亡，增加细胞内 ROS。Western blot 检测结果显示，ED-71 可上调 cleaved-caspase-3、Bax、Bcl-2、LC3-II/LC3-I 和 P62 的蛋白表达，同时抑制 mTOR 通路。LC3-II 和 P62 的增加表明 ED-71 诱导自噬体的形成并抑制自噬流。此外，加入 3-甲基腺嘌呤后，ED-71 诱导的细胞凋亡减弱，caspase-3 抑制剂（Z-VAD-FMK）减弱 ED-71 诱导的早期自噬，表明在 ED-71 存在的情况下，这两个过程相互作用。进一步的研究发现，N-乙酰半胱氨酸（NAC）预处理逆转了 ED-71 处理后的结果，包括增加细胞凋亡和自噬以及抑制 PI3K/Akt/mTOR 通路。综上所述，我们的研究结果表明 ED-71 通过 ROS 积累诱导 MG-63 细胞发生 G2/M 期阻滞、凋亡和自噬，从而抑制 PI3K/Akt/mTOR 信号通路。
