Department of Endocrinology and Metabolism, the First Affiliated Hospital, China Medical University, Shenyang 110001, China.
J Clin Endocrinol Metab. 2011 May;96(5):E763-71. doi: 10.1210/jc.2010-2642. Epub 2011 Feb 23.
Our previous data showed that reactive oxygen species generation might be ascribed to the cytotoxic response of thyroid cancer cells to proteasome inhibition and the ability of cancer cells to induce catalytic subunit for glutamate cysteine ligase (GCLC) and subsequent production of glutathione, thereby scavenging reactive oxygen species was partly ascribed to the cytotoxic responses of thyroid cancer cells to proteasome inhibition. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for transcriptional activation of various cytoprotective genes including GCLC.
The purpose of this study was to determine the involvement of Nrf2 in GCLC induction and cytotoxicity of thyroid cancer cells mediated by proteasome inhibition.
The effects of proteasome inhibition on the expression and distribution of Nrf2 were analyzed using immunocytochemistry and Western blot. To ascertain the effect of Nrf2 and p38 MAPK, cells were transfected with Nrf2 plasmid or small interfering RNA against Nrf2 or p38 MAPK. Apoptotic cells, production of glutathione, and induction of GCLC mediated by proteasome inhibition were investigated using flow cytometry, spectrophotometry, and real-time RT-PCR, respectively.
Proteasome inhibition caused accumulation and nuclear translocation of Nrf2, which compromised the cytotoxic effects of proteasome inhibition, at least in part, via induction of GCLC. In addition, nuclear translocation of Nrf2 was p38 MAPK dependent, and p38 MAPK inhibition augmented the cytotoxic effects of proteasome, at least partly, via suppression of transactivation of Nrf2.
These studies support the hypothesis that proteasome inhibitors activate an antiapoptotic survival program through p38 MAPK that involves transcriptional activity of Nrf2.
我们之前的数据表明，活性氧的产生可能归因于甲状腺癌细胞对蛋白酶体抑制的细胞毒性反应，以及癌细胞诱导谷氨酸半胱氨酸连接酶（GCLC）催化亚基的能力和随后产生谷胱甘肽，从而清除活性氧部分归因于甲状腺癌细胞对蛋白酶体抑制的细胞毒性反应。核因子红细胞 2 相关因子 2（Nrf2）是一种转录因子，负责转录激活各种细胞保护基因，包括 GCLC。
本研究旨在确定 Nrf2 是否参与蛋白酶体抑制介导的甲状腺癌细胞中 GCLC 的诱导和细胞毒性。
使用免疫细胞化学和 Western blot 分析蛋白酶体抑制对 Nrf2 的表达和分布的影响。为了确定 Nrf2 和 p38 MAPK 的作用，用 Nrf2 质粒或针对 Nrf2 或 p38 MAPK 的小干扰 RNA 转染细胞。使用流式细胞术、分光光度法和实时 RT-PCR 分别研究蛋白酶体抑制介导的细胞凋亡、谷胱甘肽产生和 GCLC 诱导。
蛋白酶体抑制导致 Nrf2 的积累和核转位，这至少部分地通过诱导 GCLC 来减轻蛋白酶体抑制的细胞毒性作用。此外，Nrf2 的核转位依赖于 p38 MAPK，p38 MAPK 抑制至少部分地通过抑制 Nrf2 的转录激活增强了蛋白酶体的细胞毒性作用。
这些研究支持这样的假设，即蛋白酶体抑制剂通过 p38 MAPK 激活一种抗凋亡存活程序，该程序涉及 Nrf2 的转录活性。
