Department of Radiotherapy Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing, Jiangsu 210009, P.R. China.
Int J Oncol. 2018 Jun;52(6):1853-1862. doi: 10.3892/ijo.2018.4349. Epub 2018 Mar 29.
Autophagy inhibition is crucial for the improvement of the efficacy of radiotherapy in cancer. The aim of the present study was to determine the potential therapeutic value of autophagy and its correlation with mitochondria in human esophageal carcinoma cells following treatment with ionizing radiation (IR). Autophagy in Eca‑109 cells was induced under poor nutrient conditions. The formation of autophagic vacuoles was monitored using electron microscopy. In addition, cell apoptosis after IR and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. LC3, beclin‑1, cytochrome c and apoptosis-related proteins were assayed by western blotting. A nude mouse xenograft model was also employed to verify the biological effects and mechanisms underlying autophagy in vivo. The formed autophagic vesicles and increased LC3 II/LC3 I ratio indicated marked induction of autophagy by Earle's balanced salt solution (EBSS) in Eca‑109 cells. 3‑Methyladenine or LY294002 significantly antagonized EBSS-induced autophagy and increased apoptosis of irradiated cells, suggesting that autophagy inhibition conferred radiosensitivity in vitro. Notably, IR induced prominent release of cytochrome c and Bax activation, and decreased Bcl-2 and MMP expression in Eca‑109 cells under poor nutrient conditions. Of note, these changes were more prominent following pretreatment with autophagy inhibitors. In vivo, IR treatment mildly delayed tumor growth, but the radiotherapeutic effect was improved significantly by abolishing autophagy. Furthermore, mitochondrial signaling was investigated in the Eca‑109 xenograft nude mice model, and the results were consistent with the in vitro study. Therefore, the mitochondrial pathway may be associated with improvement of radiosensitivity in Eca‑109 cells.
自噬抑制对于提高癌症放射治疗的疗效至关重要。本研究旨在确定自噬及其与人类食管癌细胞接受电离辐射（IR）后线粒体之间的相关性，从而评估其潜在的治疗价值。在营养条件较差的情况下诱导 Eca-109 细胞中的自噬。通过电子显微镜监测自噬小体的形成。此外，通过流式细胞术分析 IR 后细胞凋亡和线粒体膜电位（MMP）。通过蛋白质印迹法测定 LC3、beclin-1、细胞色素 c 和凋亡相关蛋白。还建立了裸鼠异种移植模型以验证自噬在体内的生物学效应和机制。形成的自噬小泡和 LC3 II/LC3 I 比值增加表明 Earle 平衡盐溶液（EBSS）在 Eca-109 细胞中明显诱导了自噬。3-甲基腺嘌呤或 LY294002 显著拮抗 EBSS 诱导的自噬并增加照射细胞的凋亡，表明自噬抑制在体外赋予放射敏感性。值得注意的是，IR 在营养条件差的情况下诱导细胞色素 c 明显释放和 Bax 激活，并降低 Bcl-2 和 MMP 表达。值得注意的是，在用自噬抑制剂预处理后，这些变化更为明显。在体内，IR 治疗轻度延迟肿瘤生长，但通过消除自噬可显著改善放射治疗效果。此外，还在 Eca-109 异种移植裸鼠模型中研究了线粒体信号通路，结果与体外研究一致。因此，线粒体信号通路可能与 Eca-109 细胞放射敏感性的提高有关。
