Abstract
The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.
Highlights
The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs
Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase C (PKC)–p38 MAPK (p38)– dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis
Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury
Summary
Indomethacin (an NSAID) interferes with human AGS proliferation and induces mitochondrial hyper-fission. The level of pDRP1Ser-616, a marker for fissogenic activation of DRP1, was found to be significantly increased in the indomethacin-treated cells compared with control. Data indicated that mitochondrial metabolic parameters were severely compromised upon indomethacin treatment compared with control (Table 2). Data indicated that DRP1 activation already started 6 h after indomethacin treatment, and it further increased until 24 h (Fig. 2C) as evident from immunoblot data performed using total cell lysates. The protective effect of SB203580 and siDRP1 pretreatment on the indomethacin-induced metabolic crisis was further reflected from the preservation of dehydrogenase activity, mitochondrial respiration, oxygen consumption, and ATP production (Table 2). Immunoblot analysis indicated that indomethacin-induced OPA1 depletion and increased p38 phosphorylation were both rectified by SB203580 pretreatment or PKC–PSI pretreatment (Fig. 4, A and B). 144.28 Ϯ 5.9 28.04 Ϯ 3.4*** 93.34 Ϯ 3.9### 120.29 Ϯ 2.6* 74.21 Ϯ 6.9### 99.61 Ϯ 4.6**
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