Li Xia, Jiang Zhiming, Feng Jianguo, Zhang Xiaoying, Wu Junzhou, Chen Wei
Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, Zhejiang Cancer Center, Hangzhou, Zhejiang 310022, China.
Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus), Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022, China.
Oncotarget. 2017 Jun 27;8(37):61846-61860. doi: 10.18632/oncotarget.18705. eCollection 2017 Sep 22.
Esophageal squamous cell carcinoma (ESCC) is a highly malignant cancer with poor response to both of chemotherapy and radiotherapy. 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino) phenyl carbamoylsulfanyl] propionic acid (2-AAPA), an irreversible inhibitor of glutathione reductase (GR), is able to induce intracellular oxidative stress, and has shown anticancer activity in many cancer cell lines. In this study, we investigated the effects of 2-AAPA on the cell proliferation, cell cycle and apoptosis and aimed to explore its mechanism of action in human esophageal cancer TE-13 cells. It was found that 2-AAPA inhibited growth of ESCC cells in a dose-dependent manner and it did not deplete reduced glutathione (GSH), but significantly increased the oxidized form glutathione (GSSG), resulting in decreased GSH/GSSG ratio. In consequence, significant reactive oxygen species (ROS) production was observed. The flow cytometric analysis revealed that 2-AAPA inhibited growth of esophageal cancer cells through arresting cell cycle in G/M phase, but apoptosis-independent mechanism. The G/M arrest was partially contributed by down-regulation of protein expression of Cdc-25c and up-regulation of phosphorylated Cdc-2 (Tyr15), Cyclin B1 (Ser147) and p53. Meanwhile, 2-AAPA-induced thiol oxidative stress led to increased protein -glutathionylation, which resulted in α-tubulin -glutathionylation-dependent depolymerization of microtubule in the TE-13 cells. In conclusion, we identified that 2-AAPA as an effective thiol oxidative stress inducer and proliferation of TE-13 cells were suppressed by G/M phase cell cycle arrest, mainly, through α-tubulin -glutathionylation-mediated microtubule depolymerization. Our results may introduce new target and approach for esophageal cancer therapy through generation of GR-mediated thiol oxidative stress.
食管鳞状细胞癌（ESCC）是一种高度恶性的癌症，对化疗和放疗的反应均较差。2-乙酰氨基-3-[4-(2-乙酰氨基-2-羧乙基硫代羰基氨基)苯基氨基甲酰硫基]丙酸（2-AAPA）是谷胱甘肽还原酶（GR）的不可逆抑制剂，能够诱导细胞内氧化应激，并已在多种癌细胞系中显示出抗癌活性。在本研究中，我们研究了2-AAPA对细胞增殖、细胞周期和凋亡的影响，旨在探讨其在人食管癌TE-13细胞中的作用机制。结果发现，2-AAPA以剂量依赖性方式抑制ESCC细胞的生长，且不消耗还原型谷胱甘肽（GSH），但显著增加氧化型谷胱甘肽（GSSG），导致GSH/GSSG比值降低。因此，观察到活性氧（ROS）的大量产生。流式细胞术分析显示，2-AAPA通过使细胞周期停滞在G/M期抑制食管癌细胞的生长，但不依赖凋亡机制。G/M期停滞部分归因于Cdc-25c蛋白表达下调以及磷酸化Cdc-2（Tyr15）、细胞周期蛋白B1（Ser147）和p53表达上调。同时，2-AAPA诱导的硫醇氧化应激导致蛋白质-谷胱甘肽化增加，并导致TE-13细胞中微管的α-微管蛋白-谷胱甘肽化依赖性解聚。总之，我们确定2-AAPA是一种有效的硫醇氧化应激诱导剂，TE-13细胞的增殖主要通过α-微管蛋白-谷胱甘肽化介导的微管解聚而被G/M期细胞周期停滞所抑制。我们的结果可能通过产生GR介导的硫醇氧化应激为食管癌治疗引入新的靶点和方法。
