Drug-Induced Alterations to Gene and Protein Expression in Intestinal Epithelial Cell 6 Cells Suggest a Role for Calpains in the Gastrointestinal Toxicity of Nonsteroidal Anti-Inflammatory Agents

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-LeuNle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents despite their well documented gastrointestinal (GI) toxicity. Adverse gastrointestinal effects of NSAIDs in humans and other species include oral, gastric, duodenal, and colonic ulceration (Lichtenberger, 2001;Tomisato et al., 2004). Despite exhaustive investigation, the mechanisms responsible for NSAID-associated GI damage are not completely understood. Evidence gathered to date suggests that NSAIDs may promote ulcer formation not only by inhibiting mucosal cyclooxygenase (COX) and decreasing cytoprotective prostaglandins (PGs) but also by adversely influencing intestinal microflora, neutrophil recruitment, surface hydrophobicity, and epithelial restitution (Lichtenberger, 2001;Little et al., 2007). Although the inhibition of COX isoforms has received much attention and investigation as the basis of GI

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

“…Cells, cultured as described previously [15,41], were grown in DMEM supplemented with 5% fetal bovine serum, insulin (10 μg/mL), and gentamicin (50 μg/mL) at 37 °C with 5% CO 2 . Passages 15–20 were used for the purposes of this study.…”

“…Migration studies were performed as previously described [15,18,27]. Briefly, cells were seeded onto collagen I-coated 60 mm plates (Corning) and treated for 24 h with 10 nM MgTX prior to wounding.…”

“…Migration also occurs under normal conditions where epithelial cells migrate from the intestinal crypts where they originate and cell division is actively occurring, up the long axis of the villi to the apex whereupon apoptosis occurs and the cells slough off into the lumen [20–25]. Previous studies have established that treatment with NSAIDs inhibits cell migration and epithelial restitution through a variety of pathways, including depolarization of E m , disruption of calpain protease signaling, and suppression of expression of voltage-gated potassium channels (K v ) [14,15,17,18,26–28], and that this may be one mechanism through which NSAIDs cause GI toxicity.…”

“…Specifically, NSAIDs with ulcerogenic potential (indomethacin and NS-398), but not non-ulcerogenic NSAIDs (SC-560), decrease total and surface expression of K v 1.4 and surface expression of K v 1.6 in IEC-6 cell monolayers, and it was suggested that this loss of expression inhibited cell migration and caused depolarization of E m [18]. Further, reexamination of data from a previous study [15] which used microarray technology to identify novel targets of NSAID activity in cultured intestinal epithelial cells (IEC-6) suggested that NS-398 suppressed mRNA expression of K v 1.3 (−3.183 fold).…”

Early Epithelial Restitution by Nonsteroidal Anti-Inflammatory Drug–Activated Gene 1 Counteracts Intestinal Ulcerative Injuries

“…However, the therapeutic uses of NSAIDs are frequently limited by the significant negative side effects, including ulceration, most notably in the gastrointestinal tract. Increasing evidence indicates that the ulcerogenic properties of NSAIDs are not exclusively the result of inhibition of protective cyclooxygenase metabolites (3,9,10). NSAID-activated gene 1 has been investigated as a potent mediator of cyclooxygenase-independent pathogenesis by NSAIDs (11,12).…”