Reactive oxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells

https://doi.org/10.1016/S0014-2999(99)00599-3Get rights and content

Abstract

We previously reported the induction of apoptotic DNA fragmentation by nonsteroidal anti-inflammatory drugs (NSAIDs) in cultured rat gastric cells, and indicated that prostaglandin-synthesis is only marginally involved in the apoptotic process. In the present study, we examined whether the generation of reactive oxygen species is critically involved in NSAID-induced apoptosis. Indomethacin, sodium diclofenac, flurbiprofen, zaltoprofen, etodolac, but not mofezolac, enhanced apoptotic DNA fragmentation and mRNA expression for cyclooxygenase-2 in AGS cells, a cell line derived from human gastric epithelium. The apoptotic effect of indomethacin was then confirmed by fluorescent staining of the cells with annexin V. Apoptotic DNA fragmentation induced by indomethacin and flurbiprofen was suppressed by incubation of the cells with the anti-oxidants pyrrolidine dithiocarbamate, diphenyleneiodonium chloride, and N-acetyl-l-cysteine. These two NSAIDs also enhanced release from the cells of 8-isoprostane, a nonenzymatic product by free-radical-mediated peroxidation of arachidonic acid. Further, lucigenin chemiluminescence showed that the intracellular production of reactive oxygen species increased in cells treated with indomethacin. The present data thus indicate a crucial association between the generation of reactive oxygen species and NSAID-induced apoptosis in gastric epithelial cells.

Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) suppress cellular formation of prostaglandins through inhibition of cyclooxygenase, a rate-limiting enzyme in the biosynthesis pathway (Vane, 1971). Although their favorable anti-inflammatory, anti-pyretic, and analgesic properties have placed them in wide clinical use, their oral administration is accompanied by a high incidence of gastrointestinal side-effects. Given that prostaglandins confer cytoprotective effects in the gastrointestinal toxicity, this may largely result from the suppression of gastrointestinal prostaglandin synthesis Wallace, 1994, Vane and Botting, 1996. Against this, however, gene disruption of cyclooxygenase-1, a constitutive isoform normally present in gastric tissue which possibly contributes to the production of cytoprotective prostaglandins, did not induce gastric ulcers in mice (Langenbach et al., 1995). The gastrointestinal toxicity of NSAIDs therefore requires further investigation, especially with regard to the mechanisms by which it is induced.

We recently reported the apoptotic effect of NSAIDs in cultured rat gastric mucosal cells (Kusuhara et al., 1998). Induction of apoptotic DNA fragmentation was accompanied by enhanced expression of mRNA for cyclooxygenase-2, an inducible isoform, and suppressed by caspase inhibitors. In contrast, the apoptotic effect showed little connection with NSAID's inhibitory activity against cyclooxygenase-1, and was not prevented by exogenous supplementation of 16, 16-dimethyl prostaglandin E2. NSAID-induced apoptosis is thus considered unrelated to the suppressive effect of these drugs on cellular prostaglandin synthesis. Further, the apoptotic effect of NSAIDs indomethacin and sodium diclofenac, with a high incidence of gastrointestinal toxicity, was much stronger than that of less toxic drugs, indicating that apoptotic effect of NSAIDs is closely related to gastrointestinal toxicity. However, the signaling pathway leading to apoptotic cell death remains unclear.

Apoptosis induced by anti-cancer drugs including camptothecin and etoposide was accompanied by the generation of reactive oxygen species in several cell lines Verhaegen et al., 1995, Shimizu et al., 1998. Generation of reactive oxygen species was also involved in the apoptosis induced by glycochenodeoxycholate, a hydrophobic bile salt, in cultured hepatocytes (Patel and Gores, 1997). Together, these findings indicate that oxidative stress through the generation of reactive oxygen species is a key mediator of apoptosis induced by various chemicals and toxicants. Furthermore, reactive oxygen species have also been suggested to play a critical role in the receptor-mediated apoptosis induced by transforming growth factor β1 and tumor necrosis factor-α Cossarizza et al., 1995, Lafon et al., 1996. The recent discovery that the anti-apoptotic bcl-2 gene product has anti-oxidant properties lends further support to the importance of oxidative events in apoptosis Hockenbery et al., 1993, Kane et al., 1993. Reactive oxygen species have thus been shown to play an essential role in apoptotic pathways.

The purpose of the present study is to investigate whether reactive oxygen species are also involved in the apoptosis induced by NSAIDs, using six kinds of NSAIDs in clinical use as test drugs and AGS cells, a cell line derived from the human gastric epithelium, as target cells of apoptosis.

Section snippets

Reagents

Mofezolac (3,4-bis(4-methoxyphenyl)isoxazole-5-acetic acid) and 1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid (etodolac) were chemically synthesized, and 2-(10,11-dihydro-10-oxodibenzo[b,f]thiepin-2-yl)propionic acid (zaltoprofen) was extracted and purified from commercially available tablets at Yoshitomi Pharmaceutical Industries (Fukuoka, Japan). 1-(p-Chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid (indomethacin), sodium 2-(2,6-dichloroanilino)phenylacetic acid (sodium

Induction of apoptotic DNA fragmentation by NSAIDs in AGS cells

The effect of six kinds of NSAIDs at concentrations from 10 μM to 1 mM on apoptotic DNA fragmentation in AGS cells is shown in Fig. 1. Among these, indomethacin, sodium diclofenac, and flurbiprofen were found to significantly induce DNA fragmentation within 30 min after the onset of incubation. This fragmentation was exacerbated when the incubation period was prolonged to 6 h. Zaltoprofen and etodolac caused almost no fragmentation within 30 min, but did induce moderate fragmentation on

Discussion

In the present study, the participation of reactive oxygen species in the apoptotic effect of NSAIDs was examined in cultured human gastric epithelial cells. DNA fragmentation, a characteristic feature of cells undergoing apoptotic cell death Carson and Ribeiro, 1993, Williams and Smith, 1993, was significantly induced in AGS cells by incubation with indomethacin, sodium diclofenac, flurbiprofen, zaltoprofen, and etodolac. These apoptotic effects were incubation time- and drug

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