Non-steroidal anti-inflammatory drugs inhibit calpain activity and membrane localization of calpain 2 protease

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are used frequently worldwide for the alleviation of pain despite their capacity to cause adverse gastrointestinal (GI) side effects. GI toxicity, once thought to be the result of non-specific inhibition of cyclooxegenase (COX) enzymes, is now hypothesized to have multiple other causes that are COX independent. In particular, NSAIDs inhibit intestinal epithelial restitution, the process by which barrier function in intestinal mucosa is restored at sites of epithelial wounds within hours through cell spreading and migration. Accordingly, recent evidence indicates that the expression of calpain proteases, which play a key role in cell migration, is decreased by NSAIDs that inhibit cell migration in intestinal epithelial cells (IEC). Here, we examine the effect of NSAIDs on calpain activity and membrane expression in IEC-6 cells. Indomethacin, NS-398, and SC-560 inhibited calpain activity and decreased expression of calpain 2 in total membrane fractions and in plasma membranes involved in cell attachment to the substrate. Additionally, we demonstrated that inhibition of calpain activity by NSAIDs or ALLM, a calpain inhibitor, limits cell migration and in vitro wound healing of IEC-6 cells. Our results indicate that NSAIDs may inhibit cell migration by decreasing calpain activity and membrane-associated expression of calpain 2. Our results provide valuable insight into the mechanisms behind NSAID-induced GI toxicity and provide a potential pathway through which these negative side effects can be avoided in future members of the NSAID class.

Figure 1

Inhibition of calpain activity by NSAIDs. Micrographs were taken of BOC-LM-CMAC fluorescence in IEC-6 cells cultured on collagen following 48 h of NSAID treatment (A). Calpain activity was assessed in IEC-6 cells following 6 (B), 12 (C), 24 (D), 48 (E), or 72 h (E) of treatment with vehicle control (0.1% DMSO), indomethacin (Indo, 100 μM), NS-398 (100 μM), or SC-560 (1 μM). * indicates a statistically significant difference from control (p < 0.05).

Figure 2

Calpain activity measured by BOC-LM-CMAC fluorescence in IEC-6 cells treated with a range of concentrations of the calpain inhibitor, ALLM (24 h). * indicates a statistically significant difference from control (p < 0.05).

Figure 3

Calpain 2 protein expression in membrane fractions isolated from IEC-6 cells treated for 72 h with vehicle control (0.1% DMSO), indomethacin (Indo, 100 μM), NS-398 (100 μM), or SC-560 (1 μM). * indicates a statistically significant difference from control (p < 0.05).

Figure 4

Calpain 2 protein expression in IEC-6 cell footprints. Micrographs (A) of cells treated for 48 h with vehicle control (0.1% DMSO), indomethacin (Indo, 100 μM), NS-398 (100 μM), or SC-560 (1 μM). Calpain 2 is in green, actin is in orange, and cell nuclei were stained with DAPI (blue). Absence of blue fluorescence confirms that cells were properly de-roofed. Photos were analyzed for calpain 2 expression (B). * indicates a statistically significant difference from control (p < 0.05).

Figure 5

Inhibition of IEC-6 cell migration by inhibitors of calpain activity. IEC-6 cells were treated with indomethacin (100 μM), NS–398 (100 μM), SC-560 (1 μM) or ALLM (50 mM) for 24 (A) or 48 h (B) prior to wounding. Migrating cells were then photographed at 4 h post wounding and analyzed by measuring the fraction of a specified area covered by migrating cells. Results were normalized to control and presented as mean ± se. * indicates a statistically significant difference from control (p < 0.05).