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Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells

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Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells Published OnlineFirst January 24, 2012; DOI: 10.1158/1541-7786.MCR-11-0359 Molecular Cancer DNA Damage and Cellular Stress Responses Research Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells Elena V. Sheveleva1, Terry H. Landowski1, Betty K. Samulitis1, Geoffrey Bartholomeusz2, Garth Powis2, and Robert T. Dorr1 Abstract Oxidative protein folding in the endoplasmic reticulum (ER) requires strict regulation of redox homeostasis. Disruption of the lumenal redox balance induces an integrated ER stress response that is associated with reduced protein translation, increased chaperone activity, and ultimately cell death. Imexon is a small-molecule chemo- therapeutic agent that has been shown to bind glutathione (GSH) and induce oxidative stress in tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, we investigate the effects of imexon on the integrated ER stress response in pancreatic carcinoma cells. Acute exposure to imexon induces an ER stress response characterized by accumulation of the oxidized form of the oxidoreductase Ero1a, phosphorylation of eIF2a, and inhibition of protein synthesis. An RNA interference chemosensitization screen identified the eukaryotic translation initiation factor eIF2B5 as a target that enhanced imexon-induced growth inhibition of MiaPaCa-2 pancreatic cancer cells, but did not significantly augment the effects of imexon on protein synthesis. Concurrent reduction of intracellular thiols with N-acetyl cysteine reversed imexon activity, however cotreatment with superoxide scavengers had no effect, suggesting thiol binding may be a primary component of the oxidative effects of imexon. Moreover, the data suggest that disruption of the redox balance in the ER is a potential therapeutic target. Mol Cancer Res; 10(3); 392–400. Ó2012 AACR. Introduction overall protein translation including reduced synthesis of the Imexon is a thiol-reactive small molecule that has shown tumor survival protein hypoxia-inducible factor-1a (9). The preclinical activity in pancreatic cancer (1, 2) and was mechanism for the global inhibition of protein synthesis by evaluated in patients with advanced pancreatic cancer in imexon in pancreatic cancer cells was not established. combination with gemcitabine (3). The mechanism of Whereas the previous mechanistic studies focused largely action is believed to involve binding to reduced sulfhydryls on the effects of imexon-induced ROS on the mitochondria, such as cysteine and glutathione (GSH; ref. 4). In cell line no studies had been carried out on possible oxidative effects models, the biologic consequences of this binding include a in the endoplasmic reticulum (ER), an organelle which is known to be highly dependant on proper redox balance to decrease in GSH levels, the accumulation of reactive oxygen fi species, loss of the mitochondrial membrane potential, allow for proper protein folding (10). Disul de bond for- fi mation in the ER is catalyzed by the transfer of electrons cytochrome C leakage from the mitochondria, and nally, fi activation of caspases-3 and -9 of the intrinsic pathway of from substrate proteins to oxidized protein disul de isom- apoptosis, and caspase-8 of the extrinsic pathway of apo- erase (PDI), resulting in the reduction of conserved thior- – edoxin motifs (CXXC). Oxidized PDI is then regenerated by ptosis (5 8). In human pancreatic cancer cells, similar effects fl were seen involving the accumulation of reactive oxygen the thiol oxidase avoprotein Ero1a. Protein assembly and species (ROS), loss of the mitochondrial membrane poten- folding in the ER requires coordinated oxidation and reduc- tion processes, and we hypothesized that those redox pro- tial, cell-cycle arrest in G2 phase, and activation of caspases-3, -8, and -9 leading to apoptosis (1). Pancreatic cancer cells cesses could be disrupted by the pro-oxidant effects of treated with imexon also showed a marked reduction in imexon. In this study, we provide evidence that imexon interferes with redox balance in the ER, inducing an ER stress response characterized by reduced protein translation Authors' Affiliations: 1Arizona Cancer Center, University of Arizona, Tuc- and inhibition of cell growth. son, Arizona; and 2Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas Materials and Methods Corresponding Author: Robert T. Dorr, Arizona Cancer Center, University Cell lines of Arizona, 1515 N. Campbell Avenue, Tucson, AZ 85724. Phone: 520-626- Three human pancreatic cancer cell lines were obtained 7892; Fax: 520-626-2751; E-mail: [email protected] from low-passage seed stocks at the American Type Culture doi: 10.1158/1541-7786.MCR-11-0359 Collection. A range of differentiation phenotypes was used, Ó2012 American Association for Cancer Research. including the poorly differentiated BxPC3 (CRL-1687), the 392 Mol Cancer Res; 10(3) March 2012 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst January 24, 2012; DOI: 10.1158/1541-7786.MCR-11-0359 Imexon Induces Oxidative ER Stress relatively gemcitabine-resistant Panc-1 cells (CRL-1469), Protein synthesis assay and the largely undifferentiated MiaPaCa-2 cells (CRL- Total protein synthesis was analyzed by 14C valine incor- 1420). All cell lines were maintained at 37C in humidified poration studies. Cells were plated in 96-well plates, trans- 5% CO2 in RPMI-1640 supplemented with penicillin (100 fected with nontargeting siRNA control (siCT) or si-eIF2B5 U/mL), streptomycin (100 mg/mL), and L-glutamine (2 overnight and exposed to imexon for a total of 24 hours, with mmol/L; Invitrogen), and 10% bovine calf serum 0.5 mCi/mL of 14C valine (Amersham Biosciences) added (Hyclone). Cell line identity was verified by short tandem during the last 6 hours of imexon exposure. Cells with repeat analysis by the University of Arizona Genomics incorporated radioactivity were harvested with a Packard Core (UAGC, Tucson, AZ). Imexon (4-imino-1,3-dia- FilterMate Harvester (Perkin Elmer). Data are expressed as zabicyclo[3.1.0.]-hexan-one) was provided by the Pharma- percent of control in which the control is untreated cells ceutical Resources Branch of the National Cancer Institute transfected with siCT (mean Æ SEM, n ¼ 7). Data were under a Rapid Access to Intervention in Development analyzed by a 2 sample Wilcoxon rank-sum test. Analysis of (RAID) grant to R.T. Dorr. the dose effect within each siRNA treatment group was carried out by ANOVA, with Dunnett's correction for siRNA synthetic lethal screen multiple comparisons against a control. An siRNA human library comprising 4 siRNA duplexes for each of 21,000 target genes was obtained from Dhar- Reversal of oxidative effects of imexon by N-acetyl macon. Transfection efficiency and target gene silencing cysteine were optimized using a panel of 9 noncoding and 4 cell death MiaPaCa-2 cells were pretreated overnight with 10 mmol/ inducing sequences (siTOX, siKIF11, siPLK-1, and L N-acetyl cysteine (NAC), followed by incubation with 0, siCOPB2; ref. 11). For each target, the 4 siRNA duplexes 250, or 500 mmol/L of imexon for a total of 24 hours. provided in the library were combined and arrayed in a 384- During this 24-hour period, NAC was either maintained well format. MiaPaCa-2 cells were transfected with the ("continuous") or washed out and replaced with fresh media siRNA library for 48 hours using Lipofectamine RNAiMax ("pretreated only"). Alternatively, imexon was added 2 hours (Invitrogen), at which time 90 mmol/L imexon (72 hours before the addition of NAC ("post-imexon"). Protein syn- 3 IC10) was added and the plates were incubated an additional thesis was measured by incorporation of 500 mCi/mL of H- 48 hours. Genes identified in the high-throughput screening leucine (Perkin Elmer) for 18 hours. Data are expressed as were validated using Dharmacon ON-TARGET plus percent of control in which the control is untreated cells SMARTpool siRNAs, which were distinct sequences from (mean Æ SEM, n ¼ 20). Statistical significance was evaluated those used in the screening process. Nontargeting siRNA by ANOVA. sequences were also used to ensure no off-target effects. The efficiency of RNA interference (RNAi) was measured by Reversal of oxidative effects of imexon by superoxide immunoblotting. scavengers MiaPaCa-2 cells were pretreated for 2 hours with the Growth inhibition analysis superoxide scavengers, 100 U/mL PEGylated superoxide Cell survival for the siRNA screening experiments were dismutase (PEG-SOD; Sigma Chemical Co.), 200 U/mL calculated by the conversion of resazurin to resorufinby PEGylated catalase (PEG-CAT; Sigma Chemical Co.), or metabolically active cells resulting in a fluorescent product 100 mmol/L 1-oxyl-2,2,6,6-tetramethyl-4-hydroxy-piperi- (em. 490; ref. 12; Promega, Inc.). Confirmatory growth dine (Tempol, Axxora, LLC), followed by incubation with 0, inhibition assays with eIF2B5 silencing were done with the 100, 250, or 500 mmol/L of imexon for a total of 24 hours. MTT assay as previously described (13). Cell growth inhi- Protein synthesis was measured by incorporation of 3H- bition data are expressed as percent survival compared with leucine (Perkin Elmer) for 18 hours. Data are expressed as fi untreated cells. The IC50 is de ned as the drug concentration percent of control in which the control is untreated cells required to produce 50% growth inhibition. (mean Æ SEM, n ¼ 18). Statistical significance was evaluated by ANOVA. Colony-forming cell assay Colony-forming assays were conducted on MiaPaca-2 Measurement of ER stress cells transfected with eIF2B5 or nontargeting siRNA (Dhar- Transcriptional activity of the ER stress element (ERSE) macon ON-TARGET plus SmartPool siRNA), followed by was measured with the Cignal ERSE Reporter Luciferase exposure to imexon for an additional 72 hours. Imexon was Assay (SA Bioscience Qiagen). The ERSE reporter assay is a removed and cells were then plated in MethoCult (StemCell mixture of an ERSE-responsive luciferase construct and a Technologies, Inc.) following the manufacturer's instruc- constitutively expressed Renilla luciferase construct (40:1).
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