AZD3582 increases heme oxygenase-1 expression and antioxidant activity in vascular endothelial and gastric mucosal cells

European Journal of Pharmaceutical Sciences 25 (2005) 229–235

AZD3582 increases heme oxygenase-1 expression and antioxidant activity in vascular endothelial and gastric mucosal cells Georg Berndt a , Nina Grosser a , Janet Hoogstraate b , Henning Schr¨oder a,∗ a

Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle-Wittenberg, 06099 Halle (Saale), Germany b AstraZeneca RandD S¨ odert¨alje, S-151 85 S¨odert¨alje, Sweden Received 2 August 2004; received in revised form 18 February 2005; accepted 23 February 2005 Available online 29 March 2005

Abstract AZD3582 [4-(nitrooxy)-butyl-(2S)-2-(6-methoxy-2-naphthyl)-propanoate] is a COX-inhibiting nitric oxide donator (CINOD). Incubation of human endothelial cells (derived from umbilical cord) with AZD3582 (10–100 ␮M) led to increased expression of heme oxygenase (HO)-1 mRNA and protein. Heme oxygenase-1 (HO-1) is a crucial mediator of antioxidant and tissue-protective actions. In contrast, naproxen (a nonselective NSAID) and rofecoxib (a selective inhibitor of COX-2), did not affect HO-1 expression. Pre-treating endothelial cells with AZD3582 at concentrations that were effective at inducing HO-1 also reduced NADPH-dependent production of oxygen radicals. Antioxidant activity in the endothelial cells persisted after AZD3582 had been washed out from the incubation medium. When added exogenously to the cells at low micromolar concentrations, the HO-1 metabolite, bilirubin, virtually abolished NADPH-dependent oxidative stress. AZD3582-induced blockade of free-radical formation was reversed in the presence of the HO-1 inhibitor, tin protoporphyrin-IX (SnPP). Similar results were obtained in human gastric mucosal cells (KATO-III). Our results demonstrate that HO-1 is a novel target of AZD3582. © 2005 Elsevier B.V. All rights reserved. Keywords: AZD3582; Nitric oxide; Endothelium; Gastric mucosa; Non-steroidal anti-inflammatory drugs; COX-2

1. Introduction Non-selective, non-steroidal anti-inflammatory drugs (NSAIDs) are effective in the treatment of pain and inflammation. Their analgesic and anti-inflammatory effects arise from the blockade of prostaglandin synthesis through inhibition of cyclooxygenase (COX)-1 and COX-2. However, their long-term use is compromised by significant gastrotoxicity manifesting as intestinal bleeding and/or the development or Abbreviations: CINOD, COX-inhibiting nitric oxide donator; HO, heme oxygenase; NO, nitric oxide; AZD3582, 4-(nitrooxy)-butyl-(2S)-2-(6methoxy-2-naphthyl)-propanoate; NSAID, non-steroidal anti-inflammatory drug; COX, cyclooxygenase; ROS, reactive oxygen species; SnPP, tin protoporphyrin-IX ∗ Corresponding author. Present address: Research DMPK, AstraZeneca R&D S¨odert¨alje, S-151 85 S¨odert¨alje, Sweden. Tel.: +46 8553 25810; fax: +46 8553 21650. E-mail address: [email protected] (H. Schr¨oder). 0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.02.015

exacerbation of peptic ulcer disease (Soll et al., 1991; Cryer and Feldman, 1999). These gastrotoxic effects are thought to be due to topical irritation of the epithelium, as well suppression of COX-mediated endogenous prostaglandin synthesis (Schoen and Vender, 1989; Soll et al., 1991). In the stomach, prostaglandins exert a protective effect by stimulating mucus production and increasing bicarbonate production (Wolfe et al., 1999). Nitric oxide (NO) is also recognized as a critical mediator of many of the gastrointestinal processes that contribute to the protection of the gastric mucosa and exerts many of the same physiologically protective actions as prostaglandins (Shanbhag et al., 1992). In particular, NO functions as a smooth-muscle relaxing agent and is thus thought to counteract the reduction in gastric blood flow caused by inhibitors of prostaglandin synthesis, such as NSAIDs (Kitagawa et al., 1990; Whittle et al., 1990; Wallace, 1996). Cytoprotective effects by NO may also occur in-

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dependently of vasodilation through maintenance of mucus and bicarbonate secretion (Wallace and Miller, 2000), and decreased leucocyte adherence (Lopez-Belmonte and Whittle, 1995). A therapeutic goal has been to maintain the analgesic and anti-inflammatory efficacy of non-selective NSAIDs while improving the gastrointestinal safety. Recognition of the protective effects of NO in the gastric mucosa led to the development of the CINOD (COX-inhibiting nitric oxide donator) class of compounds (Wallace, 2001). The mechanism of action of CINODs involves COX inhibition and NO donation. The donated NO is expected to counter the detrimental effects of prostaglandin deficiency, while the analgesic and anti-inflammatory effects come from the inhibition of both COX isoforms. AZD3582 [4-(nitrooxy)butyl-(2S)-2-(6-methoxy-2-naphthyl)propanoate] was the first CINOD to be studied in both animals and in large-scale clinical trials. AZD3582 is metabolized to yield naproxen and NO (Berndt et al., 2004); Adding et al., manuscript in preparation). In various models of pain and inflammation, it has demonstrated similar analgesic and anti-inflammatory activity to naproxen (Wallace et al., 1999; Fiorucci et al., 2001; Wallace, 2001; Berge et al., 2002), while exhibiting reduced gastrointestinal toxicity (Wallace, 2001; Ojteg et al., 2002; Hawkey et al., 2003). The donation of NO by AZD3582 may have functional consequences beyond the gastrointestinal system. In addition to its known vasodilatory and platelet inhibitory actions, NO has recently been demonstrated to provide antioxidant protection in various cell types and organs, including endothelium, nerve and kidney (Polte et al., 2000, 2002; Mohanakumar et al., 2002; Oberle et al., 2002). At least some of these effects are thought to be due to induction of the heme oxygenase (HO)-1 gene by NO. Indeed, NO is an important endogenous inducer of HO-1 (Yee et al., 1996; Polte et al., 2000). In recent years, heme oxygenase-1 (HO-1) has emerged as a crucial mediator of antioxidant and tissue-protective actions. HO-1 anti-sense and knock-out studies as well as clinical investigations of HO-1 promoter polymorphisms have clearly shown that HO-1 assumes a central role in cellular antioxidant defense and, specifically, in vascular protection (Chen et al., 2002a; Otterbein et al., 2003; Yet et al., 2003). Cytoprotective and anti-inflammatory actions of HO-1 outside the vasculature have also been documented in various tissues including heart, kidney, neuronal cells and gastric tissue (Immenschuh and Ramadori, 2000; Otterbein and Choi, 2000; Polte et al., 2000, 2002; Baranano et al., 2002; Ryter et al., 2002; Grosser et al., 2003; Becker et al., 2003). Among other agents, activators of cGMP formation, such as NO or atrial natriuretic peptide, were reported to act as HO-1 inducers in the endothelium and to prevent oxidant injury via this pathway (Immenschuh et al., 1998; Immenschuh and Ramadori, 2000; Polte et al., 2000, 2002; Grosser et al., 2003; Kiemer et al., 2003).

HO-1 is an inducible enzyme that catalyzes the degradation of heme. This process leads to generation of bilirubin, iron and carbon monoxide. Bilirubin exerts strong antioxidant effects at physiologic plasma concentrations. High-normal plasma levels of bilirubin were reported to be inversely related to atherogenic risk and to provide protection against endothelial damage. Risk reduction by bilirubin was comparable with that of HDL cholesterol (Hopkins et al., 1996; Mayer, 2000; Baranano et al., 2002). Carbon monoxide has likewise been shown to produce anti-apoptotic and cytoprotective actions (Otterbein and Choi, 2000; Otterbein et al., 2003). Our aim was to determine whether AZD3582 increases HO-1 expression through donation of NO, and consequentially enhances cellular antioxidant activity. The nonselective NSAID naproxen and the COX-2-selective NSAID rofecoxib were used as comparators. Human endothelial cells were used as a non-gastric model system and results are compared with those obtained in a gastric mucosal cell line.

2. Materials and methods 2.1. Materials Fetal bovine serum, cell culture media and penicillinstreptomycin were obtained from Gibco (Eggenstein, Germany). The Chemiluminescence Western Blotting Kit was from Amersham (Freiburg, Germany) and the Random Primed DNA Labeling Kit was from Roche (Penzberg, Germany). HO-1 primary antibody, anti-rabbit peroxidaseconjugated secondary antibody and tin protoporphyrin-IX (SnPP) were obtained from Alexis (Gr¨unberg, Germany). AZD3582 was provided by AstraZeneca (S¨odert¨alje, Sweden). Rofecoxib was provided by MSD (Haar, Germany). Naproxen, reduced nicotinamide adenine dinucleotidephosphate (NADPH) and lucigenin were purchased from Sigma (Deisenhofen, Germany). For HO-1 probes, the template was an EcoRI restriction fragment of the human HO-1 cDNA (clone 2/10), which was kindly provided by Dr. Rex Tyrrell, School of Pharmacy and Pharmacology, University of Bath, UK (Keyse and Tyrrell, 1989). 2.2. Cell culture Human endothelial cells derived from umbilical cord were obtained as a cell line (ECV304) from the European Collection of Cell Cultures (Suda et al., 2001). ECV304 endothelial cells were grown in M199 containing 10% fetal bovine serum, streptomycin (100 ␮g ml−1 ) and penicillin (100 U ml−1 ). Gastric epithelial cells derived from human gastric adenocarcinoma were obtained as a cell line (KATOIII) from the American Type Culture Collection (Sekiguchi et al., 1978). KATO-III cells were grown in RPMI 1640 containing 20% fetal bovine serum, streptomycin (100 ␮g ml−1 ), penicillin (100 U ml−1 ) and 4 mM glutamine. Both cell types

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Fig. 1. Effect of AZD3582 (A) and naproxen (B) on HO-1 mRNA expression in endothelial cells. Incubations, mRNA isolation and Northern blot analysis were performed as described under Section 2. The blots shown are representative of three experiments with similar results. The densitometric data are mean ± S.E.M. of n = 3 separate experiments.* P < 0.05, treatment vs. control (CON), one-way ANOVA and Bonferroni’s multiple comparison test.

were grown in a humidified incubator at 37 ◦ C (95% room air, 5% CO2 ). 2.3. HO-1 mRNA analysis Sub-confluent endothelial cells in 150 mm culture dishes were incubated for 8 h in the presence of control (culture) media, AZD3582 (up to 100 ␮M), naproxen (up to 300 ␮M) or rofecoxib (up to 300 ␮M). Total RNA was extracted using Trizol reagent (Gibco, Eggenstein, Germany) according to the instructions of the supplier. Briefly, samples containing equal amounts of RNA (20–30 ␮g) were separated in a 1% denaturing formaldehyde gel. Separated RNA was transferred onto a positively charged Nylon membrane by vacuum transfer (500 mbar). The transferred RNA was fixed by bak-

ing at 80 ◦ C for 30 min. After 32 P-labeling of a human HO1 cDNA probe with a Random Primed DNA Labeling Kit, the membranes were hybridized overnight at 65 ◦ C. Equal loading was assessed by a second hybridization using a 32 Plabeled ␤-actin cDNA probe. The established inducer of HO1, cadmium chloride (CdCl2 ), was used as positive control. Quantitation of HO-1 mRNA content was performed using computer-assisted videodensitometry. 2.4. HO-1 protein analysis Endothelial cells or gastric epithelial cells were cultured in 150 mm dishes as described above. After 24 h incubation with control (culture) media or AZD3582 (100 ␮M–1 mM), cells were washed and extracted as described previously (Polte

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et al., 2000). Protein (100 ␮g) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, protein was transferred to a nitrocellulose membrane, and a polyclonal antibody to human HO-1 was used to identify HO-1 protein content. Antigen-antibody complexes were visualized with the horseradish peroxidase chemiluminescence system, according to the manufacturer’s instructions (Amersham, Freiburg, Germany). 2.5. Formation of reactive oxygen species NADPH-dependent reactive oxygen species (ROS) formation was measured by monitoring lucigenin-derived chemiluminescence at 37 ◦ C using the Berthold LB96V luminometer according to previously published protocols (Li et al., 1998; Siegert et al., 1999; M¨unzel et al., 2002). Cells were cultured in six-well plates, pre-treated with AZD3582 or naproxen (up to 300 ␮M) for 24 h, subsequently suspended in phosphate-buffered saline (PBS) and incubated with lucigenin (50 ␮M) and NADPH (100 ␮M). SnPP was added 15 min prior to AZD3582; bilirubin was added exogenously to the suspended cells. Chemiluminescence was measured in relative light units (RLU) every 5 min over a period of 30 min. Data are presented as the mean of peak values during the 30 min measurement and expressed as the percentage of maximal light emission (%RLUmax ) of NADPH-treated control cells.

Fig. 3. Effect of AZD3582 on HO-1 protein expression in endothelial (A) and gastric mucosal cells (B). Incubations, protein isolation and Western blot analysis were performed as described under Section 2. The blots shown are representative of three experiments with similar results.

3. Results Incubation of endothelial cells with AZD3582 (10–100 ␮M) produced concentration-dependent increases in HO-1 mRNA (Fig. 1A), whereas naproxen (up to 300 ␮M; Fig. 1B) and rofecoxib (up to 300 ␮M; Fig. 2) had no effect under these conditions. HO-1 mRNA induction by AZD3582 in endothelial cells was associated with elevated HO-1 protein levels (Fig. 3A). Similar increases in HO-1 protein were detected in the gastric mucosal cell line, KATO-III (Fig. 3B). In endothelial (Fig. 4A) and gastric mucosal cells (Fig. 4B), NADPHdependent ROS formation was inhibited after pre-treatment

Fig. 2. Effect of rofecoxib on HO-1 mRNA expression in endothelial cells. Incubations, mRNA isolation and Northern blot analysis were performed as described under Section 2. The blots shown are representative of three experiments with similar results.

Fig. 4. Effect of AZD3582 and naproxen on NADPH-dependent reactive oxygen species formation in endothelial (a) and gastric mucosal cells (b). Incubations and measurements of lucigenin-enhanced chemiluminescence were performed as described under Section 2. * P < 0.05, treatment vs. control (CON), one-way ANOVA and Bonferroni’s multiple comparison test. All data shown are means ± S.E.M. of n = 3 independent observations in separate cell culture wells.

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with AZD3582, but not after naproxen pre-treatment. The HO-1 metabolite, bilirubin, when added exogenously to endothelial (Fig. 5A) or gastric mucosal cells (Fig. 5B), profoundly reduced NADPH-dependent oxidative stress with ROS formation nearing that of cells not treated with NADPH (Fig. 5A and B). AZD3582-induced blockade of ROS formation was prevented by the HO-1 inhibitor, SnPP (Appleton et al., 1999; Polte et al., 2000) (Fig. 6). SnPP alone had no significant effect on ROS formation under these conditions (data not shown).

4. Discussion

Fig. 5. Effect of bilirubin on NADPH-dependent reactive oxygen species formation in endothelial (A) and gastric mucosal cells (B). Incubations and measurements of lucigenin-enhanced chemiluminescence were performed as described under Section 2. * P < 0.05, treatment vs. control (CON), oneway ANOVA and Bonferroni’s multiple comparison test. All data shown are means ± S.E.M. of n = 3 independent observations in separate cell culture wells.

Fig. 6. Modulation of AZD3582-induced antioxidant protection by SnPP in endothelial cells. Incubations were carried out as described under Section 2. * P < 0.05, treatment vs. control (CON). * P < 0.05, treatment vs. AZD3582 alone. All data shown are means ± S.E.M. of n = 3 independent observations in separate cell culture wells.

The present study demonstrated that, in vitro, AZD3582 induces the antioxidant defence protein, HO-1, in endothelial cells. In contrast, naproxen and rofecoxib did not alter HO1 expression in vitro. As naproxen is the main metabolite of AZD3582, these data suggest that the increases in HO1 expression observed with AZD3582 were attributable to released NO and not due to COX-1 or COX-2 inhibition. The results also suggest that HO-1 is induced by AZD3582 even though the COX-inhibiting moiety, naproxen, inhibits the synthesis of prostaglandins, which are known to have a permissive function in HO-1 induction in some biological systems (Koizumi et al., 1995; Otterbein and Choi, 2000; Chen et al., 2002b). The increases in HO-1 mRNA levels were detected at micromolar concentrations of AZD3582 (10–100 ␮m) that are considerably higher than anticipated tissue or plasma levels of AZD3582 during oral therapy with AZD3582 (Fiorucci et al., 2001; Hawkey et al., 2003). Further studies would be needed to determine whether the concentrations of AZD3582 achieved in vivo would donate sufficient NO for induction of HO-1. The biological activity of NO is very much dependent on the cellular or chemical environment (e.g. concentration of hemoglobin or thiols). In vitro, it is possible that disproportionately higher levels of AZD3582 may be required to induce an effect due to the greater scavenging of NO by hemoglobin or albumin, for example. Indeed, a previous study showed that higher concentrations of AZD3582 were necessary to release NO, as measured by cGMP, in these cellular systems (Berndt et al., 2004), than were required for NO release in vivo (Adding et al., manuscript in preparation). Induction of the HO-1 gene through AZD3582, demonstrated in the current study at the mRNA and protein level, may be of clinical relevance if it were also to occur in vivo after AZD3582 administration, since HO-1 has been shown to play a crucial role in protecting various tissues against oxidant and inflammatory injury. Increased HO-1 expression leads to degradation of heme and accumulation of iron, bilirubin and carbon monoxide (CO), followed by reduced sensitivity of tissues to oxidant damage (Maines, 1997; Platt and Nath, 1998; Suttner et al., 1999; Yang et al., 1999). Of these metabolites, bilirubin acts as a direct antiox-

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idant (Stocker et al., 1987), whereas CO may exert tissueprotective actions, primarily through its vasodilatory and antiplatelet effects (Maines, 1997; Coceani, 2000). However, recent evidence points to direct anti-inflammatory properties of CO (Otterbein et al., 2000), which may complement and support the cytoprotective and antioxidant actions of bilirubin. In the current study, after pre-treatment with AZD3582 and subsequent washout of the CINOD, ROS formation brought about by the addition of NADPH was inhibited. Based on these findings, AZD3582 activates anti-oxidant signaling pathways, and it is plausible that these pathways may remain active even after removal of AZD3582. That HO-1 could be a mediator of the actions of AZD3582 under these conditions is possibly explained by the direct radical scavenging effect of bilirubin. The HO-1 metabolite, bilirubin, when added exogenously to the cells, profoundly reduced NADPHdependent oxidative stress with ROS formation nearing that of cells not treated with NADPH. This effect was seen at low micromolar concentrations of bilirubin, which are in the upper range of the reference interval for plasma levels. Furthermore, the induction of HO-1 by AZD3582 was abrogated in the presence of the HO-1 inhibitor SnPP. Our findings thus also lend support to the concept of bilirubin as a biologically important antioxidant (Stocker et al., 1987). We observed increases in HO-1 expression in vitro caused by AZD3582, not only in endothelial cells, but also in a gastric mucosal cell line. As in endothelial cells, this genomic action was associated with a strong antioxidant effect of AZD3582 that was mimicked by exogenous bilirubin. These findings are supported by previous studies demonstrating gastroprotective functions of HO-1 (Colpaert and Lefebvre, 2000; Becker et al., 2003). HO-1 induction by AZD3582 may provide another mechanism of gastric protection in addition to earlier reported pathways, such as the maintenance of gastric blood flow, mucus and bicarbonate secretion (Wallace and Miller, 2000) or decreased leucocyte adherence (Lopez-Belmonte and Whittle, 1995). In summary, we have shown that AZD3582 increases expression of the antioxidant defence protein HO-1 in endothelial and gastric cells. In contrast, expression of HO1 mRNA was not induced by the NSAIDs naproxen and rofecoxib, suggesting that the HO-1 induction was activated by NO-dependent mechanisms. HO-1 induction by AZD3582 was followed by inhibition of ROS formation in both cell types. The clinical significance of HO-1 induction by AZD3582, with subsequent increases in bilirubin and CO formation, are not yet known, but may have a positive effect on endothelial function if sufficient NO donation is achieved in vivo. If other members of the CINOD class also have the ability to induce HO-1, this property could distinguish CINODs from non-selective and COX-2-selective NSAIDs (FitzGerald and Patrono, 2001; Wallace and Del Soldato, 2003), although its clinical significance would have to be established.

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