title: The proteasome inhibitor lactacystin enhances GSH synthesis capacity by increased expression of antioxidant components in an Nrf2-independent, but p38 MAPK-dependent manner in rat colorectal carcinoma cells.
abstract: 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.
authors.affiliation: a Tumor Biology Research Group, Department of Medical Biology, Faculty of Health Sciences , University of Tromsø , Tromsø , Norway.
authors.forename: Nils-Erik
authors.initials: NE
authors.lastname: Huseby
authors.affiliation: a Tumor Biology Research Group, Department of Medical Biology, Faculty of Health Sciences , University of Tromsø , Tromsø , Norway.
authors.forename: Chandra
authors.initials: C
authors.lastname: Ravuri
authors.affiliation: b Molecular Inflammation Research Group, Department of Medical Biology, Faculty of Health Sciences , University of Tromsø , Tromsø , Norway.
authors.forename: Ugo
authors.initials: U
authors.lastname: Moens
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D005978:Glutathione / Q000096:biosynthesis* / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051267:NF-E2-Related Factor 2 / Q000096:biosynthesis / Q000235:genetics; D018384:Oxidative Stress / Q000187:drug effects*; D061988:Proteasome Inhibitors / Q000494:pharmacology*; D051381:Rats; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D061988:Proteasome Inhibitors; C067713:lactacystin; D048051:p38 Mitogen-Activated Protein Kinases; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine
keywords: Cystine/glutamate transporter; glutamate cysteine ligase; glutathione regulation; oxidative stress; proteasomal inhibitors; γ-glutamyltransferase