Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, P.R. China.
Oncol Rep. 2018 Oct;40(4):2260-2268. doi: 10.3892/or.2018.6619. Epub 2018 Aug 1.
Osteosarcoma is the most common primary malignant tumor of the bone found predominantly in children and teenagers and results in early metastasis and poor prognosis. The present study primarily focused on the impact of celastrol on apoptosis and autophagy of osteosarcoma HOS cells, as well as the related mechanisms. Following the appropriate treatment, the human osteosarcoma cell line HOS was assessed for viability, Ca2+ in cells, apoptosis and changes in cell morphology using Cell Counting Kit-8, flow cytometry, inverted phase contrast microscope, Hoechst staining and transmission electron microscopy. The expression levels of various proteins, including endoplasmic reticulum stress (ERS)-related proteins (Bip, PERK, p-PERK, IRE1α, calnexin, PDI and Erol‑Lα), apoptosis-related proteins (CHOP, cleaved caspase‑12), mitochondrial apoptosis-related proteins (Bax, Bcl-2 and cytochrome c), cleaved caspase-3, and autophagy-related proteins (LC3-Ⅰ, LC3-Ⅱ and P62) and β-actin, were assessed with western blotting. Celastrol significantly inhibited the viability of HOS cells in a dose-dependent manner and promoted the expression of ERS-related, apoptosis-related and mitochondrial apoptosis-related proteins. The ERS inhibitor tauroursodeoxycholate promoted celastrol-induced autophagy and apoptosis of HOS cells. Pretreatment with the PERK inhibitor GSK2656157 significantly promoted celastrol-induced death and attenuated HOS cell autophagy. Our results indicated that the ERS pathway and the mitochondrial pathway were involved in celastrol-induced apoptosis of HOS cells. The ERS/PERK pathway may protect HOS cells from apoptosis by celastrol and may play a complicated role in the process of autophagy.
骨肉瘤是最常见的原发性骨恶性肿瘤，主要发生在儿童和青少年，早期转移和预后不良。本研究主要集中在 Celastrol 对骨肉瘤 HOS 细胞凋亡和自噬的影响及其相关机制。通过适当的处理，使用细胞计数试剂盒-8、流式细胞术、倒置相差显微镜、Hoechst 染色和透射电子显微镜评估人骨肉瘤细胞系 HOS 的细胞活力、细胞内 Ca2+、细胞凋亡和细胞形态变化。使用 Western blot 检测内质网应激（ERS）相关蛋白（Bip、PERK、p-PERK、IRE1α、calnexin、PDI 和 Erol-Lα）、凋亡相关蛋白（CHOP、cleaved caspase-12）、线粒体凋亡相关蛋白（Bax、Bcl-2 和细胞色素 c）、cleaved caspase-3 和自噬相关蛋白（LC3-Ⅰ、LC3-Ⅱ 和 P62）以及β-肌动蛋白的表达水平。Celastrol 显著抑制 HOS 细胞的活力，并呈剂量依赖性，促进 ERS 相关、凋亡相关和线粒体凋亡相关蛋白的表达。ERS 抑制剂牛磺熊去氧胆酸促进 Celastrol 诱导的 HOS 细胞自噬和凋亡。PERK 抑制剂 GSK2656157 的预处理显著促进 Celastrol 诱导的 HOS 细胞死亡，并减弱 HOS 细胞自噬。我们的结果表明，ERS 通路和线粒体通路参与了 Celastrol 诱导的 HOS 细胞凋亡。ERS/PERK 通路可能通过 Celastrol 保护 HOS 细胞免于凋亡，并在自噬过程中发挥复杂作用。
