Wataru Tomisato

Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis

AbstractApoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.

Geranylgeranylacetone protects cultured guinea pig gastric mucosal cells from indomethacin

One of the major side effects of nonsteroidal antiinflammatory drugs, such as indomethacin, is gastropathy. The purpose of this study was to search for a therapeutic drug to prevent this side effect in vitro. We found that geranylgeranylacetone, a unique antiulcer drug with a heat-shock protein-inducing ability, protected cultured guinea pig gastric mucosal cells from cell damage caused by indomethacin. This cytoprotective effect of geranylgeranylacetone required concentrations of more than 10−6 M and incubation periods of longer than 2 hr. Pretreatment of cells with an inhibitor of protein synthesis completely abolished the cytoprotective effect of geranylgeranylacetone, suggesting that some proteins induced by the drug are responsible for the cytoprotection. Since pretreatment of cells with low concentrations of ethanol, which also induced the heat-shock proteins, made cells resistant to indomethacin, heat-shock proteins are candidates for the proteins that are involved in the cytoprotective effect of geranylgeranylacetone against indomethacin.

Geranylgeranylacetone Protects Guinea Pig Gastric Mucosal Cells from Gastric Stressor-Induced Apoptosis

Various stressors induce apoptosis in gastric mucosal cells, which may cause gastric mucosal lesions in vivo. We recently reproduced gastric stressor-induced apoptosis in vitro, using primary cultures of guinea pig gastric mucosal cells. Geranylgeranylacetone is an antiulcer drug with heat-shock protein-inducing properties. The purpose of this study is to examine the effect of geranylgeranylacetone on gastric stressor-induced apoptosis in vitro. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, apoptotic DNA fragmentation. Pretreatment of cells with geranylgeranylacetone inhibited the apoptotic DNA fragmentation caused by each of these gastric stressors. Pretreatment of cells with a low concentration of ethanol, a procedure that is also known to induce heat-shock proteins, made cells resistant to the apoptotic DNA fragmentation. These results suggest that heat-shock proteins could be at least partly involved in the inhibitory effect of geranylgeranylacetone against apoptosis of gastric mucosal cells caused by these gastric stressors.

Effects of Prostaglandins on Spontaneous Apoptosis in Gastric Mucosal Cells

Prostaglandins have cytoprotective effects on gastric mucosa via the influence of various mechanisms. The purpose of this study is to examine the effects of prostaglandins on maturation-dependent spontaneous apoptosis in gastric mucosal cells in vitro, which mimics the apoptosis of gastric mucosal cells related with a rapid cell turnover rate in vivo. Both prostaglandin E1 and E2 inhibited spontaneous apoptosis in a dose-dependent manner and increased the viability of gastric mucosal cells in culture. A number of antiulcer drugs presently in clinical use were shown to increase the concentrations of prostaglandins in cells. All of the drugs tested clearly inhibited the spontaneous apoptosis in a dose-dependent manner. Based on these results, we propose that the cytoprotective effects of prostaglandins on gastric mucosa in vivo can be partially explained by an increase in the number of gastric mucosal cells present as a result of the inhibition of maturation-dependent spontaneous apoptosis.

Maturation-associated increase in sensitivity of cultured guinea pig gastric pit cells to hydrogen peroxide.

Guinea pig gastric pit cells in primary culture undergo serum-dependent cell maturation, which mimics their maturation in vivo. In this study we compared the sensitivities of these cells, as a function of early- and late-stage cell maturation, to various gastric stressors. Gastric pit cells in the early stage of maturation (two days of culture) were more resistant to apoptotic cell death induced by exposure to hydrogen peroxide than those cells in late-stage maturation (three days of culture). This maturation-associated difference was not observed for the sensitivities of cells to necrotic cell death induced by hydrogen peroxide, nor for necrotic and apoptotic cell death induced by other gastric stressors (ethanol and hydrochloric acid). The activities of catalase and glutathione peroxidase were specifically decreased in cells at the late stage of maturation compared to those at the early stage. The relative gastric pit cell phenotype sensitivity to hydrogen peroxide at the late stage of maturation may be due to a decrease in the activities of catalase and glutathione peroxidase, which are antioxidants for hydrogen peroxide.

Transforming growth factor-β1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-β1 (TGF-β1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-β1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-β1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-β1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.

Adaptive cytoprotection induced by pretreatment with ethanol protects against gastric cell damage by NSAIDs

In this study, we examined adaptive cytoprotection against NSAIDs in human gastric carcinoma cells in culture. Pretreatment of cells with low (nontoxic) concentrations of ethanol protected cells from cell death induced by subsequent exposure to NSAIDs. The adaptive cytoprotection against NSAIDs induced by ethanol was not attenuated by pretreatment of cells with inhibitors of protein synthesis or prostaglandin synthesis, thus inferring that neither newly synthesized proteins nor prostaglandins are involved in this process. Furthermore, treatment of cells with the low concentration of ethanol did not affect the synthesis and secretion of mucin. In in vivo experiments on rats, oral preadministration of a low dose of ethanol protected the gastric mucosa from gastric lesions induced by subsequent oral administration of NSAIDs. One possible explanation for this in vivo phenomenon is that the adaptive cytoprotection induced by ethanol protects the gastric mucosa from the direct cytotoxic effect of NSAIDs.

Low direct cytotoxicity and cytoprotective effects of nitric oxide releasing indomethacin

Nitric oxide (NO) releasing non-steroidal anti-inflammatory drugs (NSAIDs) have shown a marked reduction of gastrointestinal side effects and we here examined the cytotoxicity of NCX 530 (NO-indomethacin). Under conditions where indomethacin clearly induced both necrosis and apoptosis, NCX 530 induced neither. NCX 530 protected cells from celecoxib-induced necrosis and apoptosis. NCX 530 partially suppressed celecoxib-dependent membrane permeabilization and an inhibitor for guanylate cyclase suppressed the cytoprotective effect of NCX 530 against celecoxib. In vivo, NCX 530 alone produced fewer gastric lesions in rats than did indomethacin. A combination of the oral administration of celecoxib together with the intraperitoneal administration of indomethacin, but not of NCX 530, clearly resulted in the production of gastric lesions. The low direct cytotoxicity and the cytoprotective effect of NCX 530 observed in vitro may also act in vivo, thus ensuring that NCX 530 is safe for use on the gastric mucosa.

Molecular mechanism of adaptive cytoprotection induced by ethanol in human gastric cells.

Adaptive cytoprotection is the process by which the pretreatment of cells with low concentrations of a noxious agent prevents the damage caused by a subsequent exposure of those cells to higher concentrations of that same agent. In this study, a human gastric carcinoma cell line was used to examine the molecular mechanism of adaptive cytoprotection induced by ethanol. Pretreatment of cells with 1%–4% ethanol made cells resistant to a subsequent exposure to 8% ethanol. This adaptive cytoprotection was accompanied by an increase in prostaglandin E2 synthesis and was partially inhibited by inhibitors of cyclooxygenase-2, but not by an inhibitor of cyclooxygenase-1. Furthermore, the adaptive cytoprotection was not dependent on newly synthesized proteins and was inhibited by a protein tyrosine kinase inhibitor. Based on these results, it is proposed that the stimulation of cyclooxygenase-2-dependent prostaglandin E2 synthesis, which is regulated post-translationally by protein tyrosine phosphorylation, plays an important role in adaptive cytoprotection induced by ethanol in gastric cells.

Low direct cytotoxicity of nabumetone on gastric mucosal cells

Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa.