Huseby Nils-Erik, Ravuri Chandra, Moens Ugo
a Tumor Biology Research Group, Department of Medical Biology, Faculty of Health Sciences , University of Tromsø , Tromsø , Norway.
b Molecular Inflammation Research Group, Department of Medical Biology, Faculty of Health Sciences , University of Tromsø , Tromsø , Norway.
Free Radic Res. 2016;50(1):1-13. doi: 10.3109/10715762.2015.1100730. Epub 2015 Nov 4.
Proteasome inhibitors may induce ER stress and oxidative stress, disrupt signaling pathways, and trigger apoptosis in several cancer cells. However, they are also reported to increase glutathione (GSH) synthesis and protect cells from oxidative stress. In the present study, we showed that the proteasome inhibitor lactacystin increased reactive oxygen species (ROS) and GSH levels after the treatment of HT-29 colorectal cancer cells. The increased GSH depended upon the activity of glutamate cysteine ligase (GCL), uptake of cystine/cysteine via the cystine/glutamate transporter [Formula: see text], and the activity of γ-glutamyltransferase (GGT). Increased transcription levels of the catalytic subunit of glutamate cysteine ligase (GCLC), the catalytic subunit xCT of [Formula: see text], and GGT were induced by lactacystin, although with different kinetics and stoichiometry. Lactacystin treatment also augmented protein levels of GCLC, xCT, and GGT, but significant levels were not detected until 48 h after initiation of lactacystin treatment. These increases in protein levels were dependent on the p38 MAPK pathway. Studies in cells transfected with siRNA against the transcription factor Nrf2 demonstrated that the promoter activities of xCT and GCLC, but not of GGT, depended on Nrf2. However, depletion of Nrf2 had no effect on lactacystin-induced upregulation of the GGT, GCLC, and xCT mRNA levels. Taken together, our results suggest that oxidative stress provoked by proteasomal inhibition results in the elevation of cellular GSH levels due to increased synthesis of GSH and uptake of cystine/cysteine. Following treatment with lactacystin, enhanced expression of antioxidant components involved in GSH homeostasis is p38 MAPK-dependent, but Nrf2-independent, resulting in increased GSH synthesis capacity.
蛋白酶体抑制剂可能会诱导内质网应激和氧化应激，破坏信号通路，并在多种癌细胞中引发凋亡。然而，也有报道称它们会增加谷胱甘肽（GSH）的合成并保护细胞免受氧化应激。在本研究中，我们发现蛋白酶体抑制剂乳胞素在处理HT - 29结肠癌细胞后会增加活性氧（ROS）和GSH水平。GSH的增加依赖于谷氨酸 - 半胱氨酸连接酶（GCL）的活性、通过胱氨酸/谷氨酸转运体[公式：见正文]摄取胱氨酸/半胱氨酸以及γ - 谷氨酰转移酶（GGT）的活性。乳胞素诱导了谷氨酸 - 半胱氨酸连接酶催化亚基（GCLC）、[公式：见正文]的催化亚基xCT和GGT转录水平的增加，尽管其动力学和化学计量不同。乳胞素处理还提高了GCLC、xCT和GGT的蛋白质水平，但直到乳胞素处理开始后48小时才检测到显著水平。这些蛋白质水平的增加依赖于p38丝裂原活化蛋白激酶（MAPK）途径。对用针对转录因子Nrf2的小干扰RNA（siRNA）转染的细胞进行的研究表明，xCT和GCLC的启动子活性依赖于Nrf2，而GGT的启动子活性则不依赖。然而，Nrf2的缺失对乳胞素诱导的GGT、GCLC和xCT mRNA水平上调没有影响。综上所述，我们的结果表明，蛋白酶体抑制引发的氧化应激导致细胞GSH水平升高，这是由于GSH合成增加和胱氨酸/半胱氨酸摄取增加所致。用乳胞素处理后，参与GSH稳态的抗氧化成分的表达增强是p38 MAPK依赖性的，但不依赖于Nrf2，从而导致GSH合成能力增加。
