Electrochemical and Mechanistic Study of Reactivities of α-, ß-, γ-, and δ-Tocopherol toward Electrogenerated Superoxide in N,N-Dimethylformamide through Proton-Coupled Electron Transfer.

Scavenging of superoxide radical anion (O2•-) by tocopherols (TOH) and related compounds was investigated on the basis of cyclic voltammetry and in situ electrolytic electron spin resonance spectrum in N,N-dimethylformamide (DMF) with the aid of density functional theory (DFT) calculations. Quasi-reversible dioxygen/O2•- redox was modified by the presence of TOH, suggesting that the electrogenerated O2•- was scavenged by α-, ß-, γ-TOH through proton-coupled electron transfer (PCET), but not by δ-TOH. The reactivities of α-, ß-, γ-, and δ-TOH toward O2•- characterized by the methyl group on the 6-chromanol ring was experimentally confirmed, where the methyl group promotes the PCET mechanism. Furthermore, comparative analyses using some related compounds suggested that the para-oxygen-atom in the 6-chromanol ring is required for a successful electron transfer (ET) to O2•- through the PCET. The electrochemical and DFT results in dehydrated DMF suggested that the PCET mechanism involves the preceding proton transfer (PT) forming a hydroperoxyl radical, followed by a PCET (intermolecular ET-PT). The O2•- scavenging by TOH proceeds efficiently along the PCET mechanism involving one ET and two PTs.

Down-regulation of aquaporin 9 gene transcription by 10-hydroxy-2-decenoic acid: A major fatty acid in royal jelly.

10-Hydroxy-trans-2-decenoic acid (10H2DA) is a unique lipid component of royal jelly produced by worker honeybees that exerts insulin-like effects. We herein investigated the effects of 10H2DA on the gene expression of aquaporin 9 (AQP9), which functions as a glycerol transporter in the liver, to clarify whether 10H2DA modulates energy metabolism. 10H2DA suppressed AQP9 gene expression in HepG2 cells by promoting the phosphorylation of Akt and AMP-activated protein kinase (AMPK). This suppression was partially recovered by the treatment of cells with inhibitors for Akt and AMPK. Based on the result showing that leptomycin B partially recovered the suppression of AQP9 gene expression, 10H2DA inhibited the expression of Foxa2, a transcription factor for the AQP9 gene, and also induced its nuclear exclusion. Although 10H2DA up-regulated phosphoenolpyruvate carboxykinase and glucose-6-phosphatase gene expression, this was suppressed through the modulation of Foxa2 by insulin. These results suggest that 10H2DA suppresses AQP9 gene expression through the phosphorylation of Akt and AMPK and down-regulation of Foxa2 expression.

Determination of lamotrigine in human plasma using liquid chromatography-tandem mass spectrometry.

AIM: Lamotrigine (LTG) is a widely used anti-epileptic drug that is administered to avoid seizures and to maintain seizure-free status. However, several factors reportedly cause individual differences of plasma LTG levels, and the therapeutic target range of LTG varies between individuals. Thus, to optimize effective doses of LTG, we developed a rapid and simple method for determining plasma LTG concentrations. METHODS: Lamotrigine and the internal standard papaverine were extracted from human plasma using solid-phase extraction. After filtration, 5-µL aliquots of final samples were injected into the liquid chromatography-tandem mass spectrometry instrument and LTG and internal standard were separated using a Cadenza CD-C18 column (100 × 2 mm, 3 µm) with 0.1% formic acid in water/acetonitrile (2/1, v/v). RESULTS: The calibration curve was linear from 0.2 to 5.0 µg/mL, and assessments of recovery, intra- and inter-day precision and accuracy, matrix effects, freeze and thaw stability, and long-term stability demonstrated good reproducibility. Retention times of LTG and internal standard were 1.6 and 2.0 minutes, respectively, and the total run time was 3.5 minutes for each sample. CONCLUSION: We developed a rapid and simple method for determining plasma LTG concentrations. The present novel system could be used to inform LTG dose adjustments for individual patients.

Anti-Androgenic Activity of Icarisid II from Epimedium Herb in Prostate Cancer LNCaP Cells.

Anti-androgens are regarded as potential therapeutic agents for the treatment of prostate cancer. We determined that an epimedium herb (EH) extract exhibited anti-androgenic activity in a luciferase assay using androgen receptor-positive prostate cancer LNCaP cells. Nine EH-derived flavonoids were examined. The results identified icarisid II as a very potent anti-androgenic EH-derived flavonoid. A quantitative RT-PCR analysis confirmed that the flavonol suppressed the expression of the androgen-responsive KLK3 gene.

Effects of 14 frequently used drugs on prostate-specific antigen expression in prostate cancer LNCaP cells.

Prostate cancer occurs more frequently among older males and such elderly individuals often have chronic underlying disorders for which various drugs are administered for treatment. The levels of prostate-specific antigen (PSA), a widely used prostate cancer marker, are influenced by a number of drugs, such as non-steroidal anti-inflammatory drugs and statins. In the present study, the drugs prescribed to patients on a repeat prescription collected at the pharmacy of the Gifu Pharmaceutical University (Gifu, Japan) were examined for their effects on the levels of PSA expression in prostate cancer LNCaP cells. Among the 14 drugs investigated, betamethasone, an agonist of the glucocorticoid receptor, was found to increase the levels of PSA mRNA expression in the LNCaP cells. This betamethasone-induced expression was mediated, at least in part, through androgen receptor (AR) transcriptional activation. Dexamethasone, a typical agonist of the glucocorticoid receptor, was also found to stimulate the AR transcriptional activity, however, to a lesser extent than betamethasone. Therefore, it would be interesting to examine in future studies whether the serum PSA levels in prostate cancer patients are influenced by betamethasone.

Anti-androgenic activity of hydroxyxanthones in prostate cancer LNCaP cells.

Anti-androgens are used to treat prostate cancer. Here, we report that hydroxyxanthones from a plant extract act as anti-androgens in androgen receptor (AR)-positive prostate cancer LNCaP cells. Anti-androgenic activity of the ethanol extract from Garcinia subelliptica was observed in a luciferase assay using LNCaP/MMTV cells with a stably integrated mouse mammary tumor virus (MMTV) promoter. HPLC-based activity profiling followed by a chemical library-based assay strategy enabled the rapid identification of several active principles bearing a xanthone core substituted with hydroxyl and isoprenyl groups. Among the active compounds, 2-(1,1-dimethyl-allyl)-1,4,5,6-tetrahydroxyxanthone (subelliptenone F) was identified as a potent inhibitor of AR transcriptional activity. The structure-activity relationship of some substituents on the xanthone core was also determined using the chemical library-based bioassay. A quantitative RT-PCR analysis revealed that treatment with the compound resulted in a significant reduction in AR-induced gene (KLK3) expression. Hydroxyxanthone may be a possible candidate for the development of a new anti-androgenic molecule.

Suppression of metallothionein 3 gene expression by androgen in LNCaP prostate cancer cells.

Androgen deprivation therapy is the standard treatment for prostate cancer. However, tumors often progress towards a more aggressive phenotype despite treatment. Prostate tissue has a high zinc concentration, which may correlate with prostate cancer progression. Therefore, we investigated the effect of dihydrotestosterone (DHT) on the gene expression of metallothioneins (MTs) and zinc transporters in prostate cancer with quantitative real-time polymerase chain reaction (PCR). The MT3 gene expression in LNCaP cells was suppressed by DHT in a dose-dependent manner. However, it increased in a culture medium containing androgen-deficient charcoal-stripped fetal bovine serum (FBS). Bicalutamide, an androgen receptor antagonist, increased the gene expression of MT3 and partially reversed the suppression of MT3 gene expression induced by DHT. In PC-3 cells lacking androgen receptors, DHT and bicalutamide exerted no effect on MT3 gene expression. The reporter gene assay with a luciferase reporter plasmid containing the 5'-flanking region of MT3 demonstrated a decrease in luciferase activity caused by DHT that was reversed by bicalutamide. These results suggest that MT3 gene expression is downregulated by androgen.

Characterization of the low pH/low nutrient-resistant LNCaP cell subline LNCaP-F10.

Intratumoral regions of low extracellular pH and low nutrition are common features of solid tumors. Although cancer cells normally die when they are removed from their environment, a small population of cells survive. In the present study, the subline LNCaP-F10, of the prostate cancer cell line LNCaP, was isolated and its low pH/low nutrient-resistant properties were examined. LNCaP-F10 cells were grown under low-pH/low-nutrient conditions, which caused cell death of the LNCaP cells. The cell death was associated with oligonucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, indicating that low-pH/low-nutrient induced apoptosis in these cells. Significant differences in the expression of BCL2, BIRC5 and DAPK1 were detected between LNCaP-F10 and LNCaP cells. Tumor growth caused by implantation of LNCaP-F10 cells into the renal subcapsular space of nude mice in the absence or presence of prostate stromal cell stimulation was greater than that caused by implantation of LNCaP cells. LNCaP-F10 cells were resistant to apoptosis induced by an environment of low-pH/low-nutrient in vitro, and displayed malignant potential in vivo.

Antiandrogenic activity of resveratrol analogs in prostate cancer LNCaP cells.

The suppression of androgen signaling is a therapeutic target for the treatment of prostate cancer. Resveratrol (3,4',5-trihydroxystilbene) is known to inhibit the function of the androgen receptor (AR). In the present study, we investigated the antiandrogenic activities of resveratrol analogs in order to identify a potent antiandrogen compound. Resveratrol analogs were isolated from plants or were semisynthesized from resveratrol. AR transcriptional activity was measured in prostate cancer LNCaP cells using a luciferase assay with the MMTV-luc reporter plasmid. Among the resveratrol analogs tested, 4'-O-methylresveratrol (3,5-dihydroxy-4'-methoxystilbene) was the most effective inhibitor of AR transcriptional activity. Introduction of a methoxy group to the C-4' of resveratrol and its analogs increased their antiandrogenic activity compared with the unmodified counterparts. Conversely, modification of the 3- and/or 5-hydroxyl groups reduced the antiandrogenic activity. 4'-O-methylresveratrol was more effective than resveratrol in inhibiting Akt phosphorylation, which is related to AR signaling, in LNCaP cells. The hydroxyl groups in resveratrol play a key role in their antiandrogenic effect by modulating AR transcriptional activity.

Low androgen sensitivity is associated with low levels of Akt phosphorylation in LNCaP-E9 cells.

We previously characterized LNCaP-E9, a low-androgen-sensitive LNCaP cell subline. LNCaP-E9 cells exhibit lower expression of androgen-regulated genes, including prostate-specific antigen (PSA), FK506 binding protein 5 (FKBP5), and prostatic acid phosphatase (PAcP), compared with LNCaP cells after treatment with the synthetic androgen R1881, confirming that the cells have low sensitivity to androgens. To understand the mechanism underlying low androgen sensitivity of LNCaP-E9 cells, we examined the activities of the Akt, p44/42, and p38 mitogen-activated protein kinase signaling pathways, all of which are known to be linked to androgen receptor signaling. We found that the phosphorylation of Akt at Ser473 was markedly lower in LNCaP-E9 cells than in LNCaP cells. Inhibition of Akt phosphorylation by the phosphatidylinositol 3-kinase inhibitor LY294002 resulted in reduction of PSA expression in LNCaP cells. Conversely, activation of Akt by serum starvation led to the induction of PSA expression in LNCaP-E9 cells. These results suggest that the impaired Akt phosphorylation in LNCaP-E9 cells is associated with low androgen sensitivity.

AMP-activated protein kinase modulates the gene expression of aquaporin 9 via forkhead box a2.

Aquaporin 9 (AQP9) is permeable to glycerol, which is a source material in lipogenesis and gluconeogenesis in the liver. We investigated the transcriptional regulation of the AQP9 gene by AMP-activated protein kinase (AMPK), known as an energy sensor in cells since AMPK contributes to the metabolism of carbohydrate, lipid, and protein by regulating the expression of many enzymes and transcription factors in metabolic pathways. An AMPK activator, 5-aminoimidazole-4-carboxamide-1-ß-d-ribonucleoside (AICAR), was observed to suppress the expression of the AQP9 gene in HepG2 cells by promoting the phosphorylation of AMPK and AKT/PKB. Forkhead box a2 (Foxa2) was speculated to be one of the transcriptional regulators of AQP9 gene expression repressed by AICAR from the results of a reporter gene assay with a plasmid containing the promoter region of the AQP9 gene and knock-down of the Foxa2 gene by a specific siRNA. AICAR was determined to induce the phosphorylation and nuclear exclusion of Foxa2. Leptomycin B, inhibiting the binding of the nuclear exclusion signal sequence and chromosome region maintenance 1, prevented nuclear export of Foxa2 triggered by AICAR. These results suggest that the activated AMPK by AICAR causes suppression of the gene expression of AQP9 through transcriptional regulation by Foxa2.

Androgen receptor W741C and T877A mutations in AIDL cells, an androgen-independent subline of prostate cancer LNCaP cells.

The androgen-independent LNCaP (AIDL) cell line was generated by maintaining prostate cancer LNCaP cells in a hormone-deprived medium. Notably, synthetic androgen R1881-related gene response is attenuated in AIDL cells as compared to the parental LNCaP cells. The aim of this study was to clarify the mechanisms underlying androgen sensitivity in AIDL cells. We first examined the expression of androgen receptor (AR) and its co-regulators. However, no significant difference in mRNA expression was found between LNCaP and AIDL cells. Remarkably, AR protein levels were induced by R1881 and DHT in LNCaP cells, but not in AIDL cells. We next performed the cDNA sequencing to detect mutations in the AR gene. The T877A mutation was detected both in LNCaP and AIDL cells. Furthermore, AIDL cells harbored a missense substitution (TGG → TGT) in the AR gene, which caused a point mutation at codon 741 (W741C). Double T877A and W741C AR mutants have been previously reported to exhibit reduced androgen sensitivity. Hence, the low-androgen-sensitive responses of AIDL cells may be explained, at least in part, by AR gene mutations.

Castration- and aging-induced changes in the expression of zinc transporter and metallothionein in rat prostate.

Prostate tissue contains high concentrations of zinc. Zinc content in the prostate gland changes in prostatic disease, such as benign prostate hyperplasia and prostate cancer, which occur more frequently with increasing age. Prostate zinc content is also known to decrease after castration in animal models. It is not clear how prostate zinc content is regulated; therefore, to clarify the mechanisms underlying zinc homeostasis, we examined zinc content and the expression of zinc transporters and metallothioneins in the prostates of aged or castrated rats. Zinc concentration was measured by flame atomic absorption spectrometry. The mRNA expression of zinc transporters and metallothioneins was determined by real-time reverse transcriptase polymerase chain reaction analysis. The expression of the zinc transporter Slc30a2 (Znt2) in ventral prostate (VP) of aged rats (21 months) was approximately 21-fold higher than that in VP of young rats (4 months), and zinc levels in VP of young rats increased significantly compared with that in aged rats. Zinc content in lateral prostate (LP) and dorsal prostate did not differ between young and aged rats. Decreased metallothionein-3 (Mt3) expression was observed in LP of castrated rats, and this reduction was prevented by testosterone replacement. Zinc content and Mt3 expression levels correlated significantly in rat LP. Our findings suggest that Mt3 could play a critical role in zinc homeostasis in rat LP.

Up-regulation of the lysyl hydroxylase 2 gene by acetaminophen and isoniazid is modulated by transcription factor c-Myb.

OBJECTIVES: Lysyl hydroxylase 2 (LH2), an isoform of hydroxylase, catalyses the hydroxylation of lysine residues in the telopeptide of collagen to form stable and irreversible cross-linkages in collagen. Increased activity of this enzyme in activated stellate cells in human liver has been proposed to relate to the promotion of hepatic fibrosis. In the present study, we examined the regulation of LH2 expression in drug-induced liver injury in order to clarify the mechanisms behind the hepatic fibrosis caused by certain drugs. METHODS: The mRNA and protein expression of the target gene were detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) with specific primers and Western blotting with a specific antibody, respectively. KEY FINDINGS: The expression of LH2 was increased in HepG2 cells incubated with acetaminophen and isoniazid. This increase was accompanied by an increase in the expression of c-myeloblastosis viral oncogene homolog (Myb) mRNA. Over-expression of c-Myb in cells transfected with a c-Myb expression plasmid, pMbm I, caused an increase in the expression of LH2 mRNA. Mutation of the Myb-binding site in the promoter region of the LH2 gene resulted in a loss of transcriptional activation in the reporter gene assay. CONCLUSIONS: These results suggest that c-Myb modulates the expression of the LH2 gene in HepG2 cells incubated with drugs causing hepatic fibrosis.

Pamidronate inhibits antiapoptotic bcl-2 expression through inhibition of the mevalonate pathway in prostate cancer PC-3 cells.

Bisphosphonates are expected to be efficacious to prevent the growth of metastatic cancer in bone tissue. Bone metastases often occur in patients with various cancers, such as breast, lung and prostate cancer. Bcl-2 is a potent antiapoptotic protein and its expression is known to be closely related to its function. In this study, to investigate the effect of bisphosphonates on cancer cells, we focused on bcl-2 expression in bisphosphonate-treated prostate cancer cells. First, we observed that bcl-2 mRNA expression in PC-3 was significantly inhibited to 12% of the control level by treatment with 100 microM pamidronate for 12h. Inhibition was seen in cells treated with nitrogen-containing bisphosphonates, which have the ability to inhibit isoprenoid biosynthesis via the mevalonate pathway, but not in non-nitrogen-containing etidronate. Simultaneous treatment with geranylgeraniol, an intermediate of the mevalonate pathway, significantly blocked inhibition by pamidronate, and treatment with geranylgeranyl transferase inhibitor GGTI-286 also suppressed bcl-2 mRNA expression. Furthermore, pamidronate inhibited the translocation of Rap1 protein to the membrane fraction, suggesting that a change in posttranslational modification of Rap1 occurred in treated cells. Finally, knockdown of Rap1 by siRNA resulted in the inhibition of bcl-2 expression. These results strongly indicate that bcl-2 reduction in bisphosphonate-treated PC-3 cells is dependent on inhibition of the mevalonate pathway. The inhibitory effect of bisphosphonates on bcl-2 expression shown in prostate cancer cell line should be tested in animal experiments and clinical studies.

Protein kinase C is inhibited by bisphosphonates in prostate cancer PC-3 cells.

Bisphosphonates are expected to be effective at preventing tumor metastasis to bone tissue. Since protein kinase C (PKC) plays a crucial role in cancer progression, we examined the effect of bisphosphonates on PKC expression to clarify the mechanism behind the inhibition of the bone metastasis of prostate cancer by bisphosphonates. We found that pamidronate inhibits PKC protein expression and PKC activity in prostate cancer PC-3 cells. PKC protein expression was markedly reduced by treatment with 100 microM of pamidronate. The inhibitory effect of PKC expression by pamidronate was specific for PKCalpha and PKCzeta. Nitrogen-containing bisphosphonates are known to inhibit the mevalonate pathway, but the effect of pamidronate on PKC expression was not due to the inhibition of this pathway. Urokinase-type plasminogen activator (uPA) is one of the critical proteins in tumor metastasis and decreased in bisphosphonate-treated PC-3 cells. We also showed that uPA expression was suppressed by PKC inhibitors (calphostin C and staurosporine) and induced by a PKC activator (PMA) in PC-3 cells, suggesting that the inhibition of uPA by bisphosphonates is involved in PKC inhibition. This is the first finding that bisphosphonates suppress PKC expression in cancer cells. These results strongly suggest that one of the mechanisms behind the inhibitory effect of bisphosphonates on tumor bone metastasis is mediated by PKC inhibition.

Regulation of glyceraldehyde 3-phosphate dehydrogenase expression by metformin in HepG2 cells.

Biguanides are known to have a serious side effect, lactic acidosis. We previously reported that buformin suppressed the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPD) and suggested that this decrease was one of the causes of lactic acidosis. In this study, we examined the signaling pathway and regulatory factors for the expression of the GAPD gene triggered by metformin in HepG2 cells. The mRNA and protein expression of GAPD, detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively, decreased upon treatment of the cells with 10 mM metformin for 24 h. Under the conditions, metformin induced phosphorylation of AMP-activated protein kinase (AMPK). The expression of GAPD mRNA decreased on treatment with an activator for AMPK, 5-amino-imidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Inhibitors for signal transducers, Compound C, H-89, and MDL-12,330A, restored the level of GAPD mRNA. A luciferase reporter plasmid containing bp -1795 to +57 of the 5'-flanking region of the GAPD gene was constructed for a reporter gene assay. The luciferase activity in transfectants decreased on incubation with metformin. A mutant reporter plasmid with an altered cAMP-response element (CRE) counteracted the metformin-mediated repression of GAPD transcription. These results suggest that signal transducers, adenylate cyclase (AC), protein kinase A (PKA), and AMPK, are involved in the signaling pathway triggered by metformin and CRE-binding protein is one of the transcription factors for the GAPD gene down-regulated by metformin.

Overexpression of thymosin beta4 increases pseudopodia formation in LNCaP prostate cancer cells.

Thymosin beta4, a major G-actin-sequestering protein, is known to be involved in tumor metastasis. In the present study, we found that thymosin beta4 expression promotes the formation of actin-based pseudopodia-like extensions, associated with cell migration, in human prostate cancer LNCaP cells. Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and Cdc42/Rac1/RhoA inhibitor Clostridium difficile toxin B significantly reduced pseudopodia formation in thymosin beta4-overexpressing LNCaP cells, suggesting that the pseudopodia formation by thymosin beta4 is probably involved in PI3K and Rho family pathway. We recently reported that thymosin beta4 expression is upregulated by androgen deprivation in prostate cancer cells. The increase in thymosin beta4 may be one of the causes of prostate cancer progression after androgen ablation therapy.

Preventive effect of co-administration of water containing magnesium ion on indomethacin induced lesions of gastric mucosa in adjuvant-induced arthritis rat.

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and that rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions as compared with patients with other diseases. We demonstrate the preventive effect of the co-administration of bittern water (BW, nigari-sui in Japanese), which enables the effective intake of Mg(2+), on the ulcerogenic response to indomethacin in adjuvant-induced arthritis (AA) rats. Four kinds of BW with different Mg(2+) contents; ranging from 10-200 mg/l Mg(2+) (BW-10, 25, 50, 200) were used in this study. Arthritis was induced by the injection of 50 microl of a suspension of 10 mg/ml heat-killed butyricum (Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of rats. Oral administration of indomethacin (40 mg/kg) caused hemorrhagic lesions in the gastric mucosa of AA rats at 14 d after adjuvant injection, and the lesion score of AA rats administered indomethacin was significantly higher than that of normal rats administered indomethacin. The expression of the mRNA for inducible nitric oxide synthase (iNOS) mRNA expression and the production of nitric oxide (NO) in the gastric mucosa of AA rats were also increased by the administration of indomethacin. The co-administration of BWs decreased the ulcerogenic response to indomethacin in AA rats. In addition, the administration of BW attenuated the increase in iNOS mRNA expression and NO production in AA rats receiving indomethacin. The oral administration of Mg(2+) to AA rats had a potent preventive effect on the ulcerogenic response to indomethacin in AA rats, probably due to an inhibition in the rise in iNOS and NO levels in the gastric mucosa.

Involvement of interleukin 18 in indomethacin-induced lesions of the gastric mucosa in adjuvant-induced arthritis rat.

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) have significant side effects, such as gastroenteropathy, and rheumatoid arthritis patients taking NSAIDs are more susceptible to NSAIDs-induced gastric lesions in comparison with other patients. The pathogenic mechanism of these lesions is not fully understood. We demonstrate whether interleukin 18 (IL-18) expression relate the aggravation of gastric lesion in adjuvant-induced arthritis (AA) rats following the oral administration of indomethacin. Arthritis was induced by injecting 50 microl of a suspension of 10mg/ml heat-killed butyricum (Mycobacterium butyricum) in Bayol F oil into the plantar region of the right hind foot and tail of Dark Agouti rats resulting in an arthritis incidence of 100%. Two weeks after injection, the rats were administered indomethacin (40mg/kg) orally, and were killed under deep ether anesthesia 6h later. The gastric mucosa was then examined. Oral administration of indomethacin caused hemorrhagic lesions in the gastric mucosa of AA rats, and the lesion score for AA rats following indomethacin treatment was significantly higher than for normal rats administered indomethacin. The expression of the IL-18 mRNA and mature IL-18 protein in the gastric mucosa of AA rats administered indomethacin were also higher in comparison with normal rats receiving indomethacin. In addition, interferon-gamma and nitric oxide levels in the gastric mucosa of AA rats were increased by the oral administration of indomethacin. It is possible that IL-18 expression in AA rats is more sensitive to indomethacin, and the IL-18 may play a role in the aggravation of gastric lesions in AA rats treated with indomethacin.