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Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line, RAW 264.7, by Mallotus japonicus phloroglucinol derivatives
Biochimica et Biophysica Acta 1571 (2002) 115 – 123 www.bba-direct.com
Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line, RAW 264.7, by Mallotus japonicus phloroglucinol derivatives Rie Ishii a, Masakazu Horie a, Koichi Saito a, Munehisa Arisawa b, Susumu Kitanaka c,* b
a Saitama Institute of Public Health, 639-1 Kamiokubo Saitama, Saitama 338-0824, Japan Laboratory of Herbal Garden, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama, Toyama 930-0194, Japan c College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
Received 28 August 2001; received in revised form 17 January 2002; accepted 4 February 2002
Abstract An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E2 production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE2 production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC50 of 1.0 AM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE2 production by activated macrophages. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Mallotus japonicus; Euphorbiaceae; Phloroglucinol derivative; Macrophage; Prostaglandin E2; Prostaglandin endoperoxide synthase
1. Introduction Mallotus japonicus Mueller Arg. (Euphorbiaceae) is a dioecious and deciduous tree that is distributed throughout tropical and temperate Asia. Parts of the tree have been used for a long time in folk medicine; the bark is used as a medication for ulcers and the treatment of cancer and the leaves are used as treatment for boils. An extract of the tree’s pericarps has been reported to exhibit anti-tumor activity [1,2], anti-tumor-promoting activity [3], and anti-viral activity [4,5]. We previously found an 80% aqueous acetone extract of the pericarps (MJE) to inhibit the production of nitric oxide (NO) by activated macrophages treated with lipopolysaccharide (LPS) and interferon-g; furthermore, several phloroglucinol derivatives were isolated from MJE
*
Corresponding author. Tel.: +81-47-465-5356; fax: +81-47-465-5440. E-mail address: [email protected] (S. Kitanaka).
and identified as its active compounds [6]. We theorized that MJE and its phloroglucinol derivatives might have antiinflammatory activity. Prostaglandins (PGs) are important mediators in a diverse range of biological processes, such as cell proliferation, inflammatory and immune responses, platelet aggregation, and the extracellular matrix biosynthesis [7,8]. Prostaglandin endoperoxide synthase (PGHS) is thought to be a key enzyme in the initiation of PG synthesis from arachidonic acid in various cells [9,10]. It is now well established that two genetically distinct isoforms of PGHS exist. PGHS-1 is constitutively expressed as a housekeeping enzyme in almost all tissues [11] and mediates physiological responses, including platelet regulation [12]. In contrast, PGHS-2 is induced by several kinds of stimuli such as endotoxins, cytokines and mitogens [13 – 15]. This occurs in a number of cells, including macrophages [14,16]. Moreover, it has been determined that the PGHS-2 isoform is primarily responsible for prostanoid synthesis in pathological processes, such as acute and
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 2 0 0 - 3
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chronic inflammatory responses [17,18]. It is thought that the observed side effects of nonselective inhibitors of PGHS-1 and -2, such as gastrointestinal toxicity [19], are due to the suppression of PGHS-1-derived prostanoids. It is further thought that the pharmacological action of these inhibitors can be attributed to their inhibition of PGHS-2 activity at sites of inflammation. Therefore, immense effort has been devoted to the development of selective inhibitors of PGHS2 [20,21]. In order to clarify the mechanism by which M. japonicus exerts its anti-inflammatory activity, we investigated the effects of MJE and its constituents on PGE2 production by activated macrophages. We further investigated its selectivity toward PGHS-2. The present paper describes our results and the mechanisms by which the extract and its constituents act.
2. Materials and methods 2.1. Materials The compounds, mallotophenone (1), mallotojaponin (2), butyrylmallotolerin (3), mallotochromanol (4), isobutyrylmallotochromanol (5), and isomallotochromanol (7) were isolated from MJE as described previously [1,4,5,22,23]. Isomallotochromene (6) was derived from 2 by cyclization of its side chain [4]. Structural formulae of these compounds are shown in Fig. 1. Ham’s F12 medium, aspirin, and indomethacin were obtained from Sigma (St. Louis, MO, USA). NS-398 from Cayman Chemical Company (Ann
Arbor, MI, USA), cycloheximide from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA), fetal bovine serum from GIBCO BRL (Grand Island, NY, USA), LPS (Escherichia coli, O55: B5) from Difco (Detroit, MI, USA), 125 I-labeled protein A and [1-14C] arachidonic acid were obtained from Amersham Pharmacia Biotech UK (Amersham, England). Polyclonal rabbit anti-PGHS-2 antibody from Oxford Biochemical Research (Oxford, MI, USA), polyclonal goat anti-PGHS-1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rabbit anti-goat IgG was purchased from Zymed Laboratories (Lexington, KY, USA). All other chemicals and reagents were of the purest commercial grade available. 2.2. Cell culture RAW 264.7 cells, a mouse macrophage-like cell line transformed with the Abelson leukemia virus, were obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained by culturing in Ham’s F12 medium supplemented 10% heat-inactivated fetal bovine serum in a CO2 incubator (5% CO2 –95% humidified air) at 37 jC. The cells were seeded onto plastic petri dishes (Falcon, #1001; Becton Dickinson) and passaged twice a week. 2.3. PGE2 production assay The cells were suspended in the medium at a density of 2.0 105 cells/ml. A 400-Al cell suspension was poured into each well of a 24-well microplate (Sumitomo Bakelite, #8024R; Tokyo, Japan), and then incubated at 37 jC for 2 h.
Fig. 1. Chemical structures of the phloroglucinol derivatives examined in the present study.
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MJE or a test compound and 100 ng/ml LPS were added to the culture medium, and the cells were incubated at 37 jC for 16 h. The medium were collected in a microfuge tube
Fig. 2. Inhibitory effects of MJE and its phloroglucinol derivatives on LPSinduced PGE2 production in RAW 264.7 cells. Cells were treated with various concentrations of MJE or one of its phloroglucinol derivatives in the presence of 100 ng/ml LPS at 37 jC for 16 h. The symbols correspond to: (A) MJE (-o-), 1 (-D-), 2 (-E-), and 6 (-.-), and (B) 3 (-5-), 4 (-y-), 5 (-.-), and 7 (-D-). The results are the means F S.E. for two independent experiments involving triplicate assays. Statistical significance: * P < 0.05, ** P < 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS).
Fig. 3. Effects of MJE and its phloroglucinol derivatives on the release of radioactivity from [14C]arachidonic acid-labeled macrophages. Cells were labeled with [14C]arachidonic acid at 37 jC for 18 h, washed three times, suspended in a fresh medium. The cells were reseeded, and treated with MJE or its phloroglucinol derivatives (compounds 2, 4, 6, and 7) in the presence of 100 ng/ml LPS, followed by incubation for 0 – 2 h. Radioactivity released into the culture supernatant was determined on a liquid scintillation analyzer. The results are the means F S.E. for two independent experiments involving duplicate assays. The symbols correspond to: (A) control cells (treated with LPS) (-n-), + NONE (absence of LPS) (- w -), 30 Ag/ml MJE (-o-), and 30 AM 2 (-E-), and (B) control cells (-n-), 30 AM 4 (-y-), 30 AM 6 (-.-), and 30 AM 7 (-D-). Statistical significance: ** P < 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS).
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and then centrifuged at 2800 g for 1 min. The supernatant was placed in a new microfuge tube to determine the amount of PGE2 using a PGE2 Enzyme-Immuno-Assay kit (Cayman Chemical). 2.4. Measurement of radioactivity released from [14C]arachidonic acid-labeled macrophages Cells were seeded at 6.0 105 cells/ml onto a plastic petri dish and then incubated at 37 jC for 2 h. The medium was replaced with a fresh medium containing 3.7 kBq of [1-14C]arachidonic acid per milliliter, and the cells were
incubated at 37 jC for 18 h. The cells were washed three times to remove unincorporated arachidonic acid, and suspended at 6.0 105 cells/ml in a fresh medium. A 500-Al cell suspension was poured into each well of a 24-well microplate and then incubated at 37 jC for 5 min to allow the cells to adhere to the plate. Then, MJE or a test compound and 100 ng/ml LPS were added to the culture medium. The cells were further incubated at 37 jC for 0 – 2 h. The culture supernatants were collected and centrifuged at 5500 g for 1 min, and 250 Al aliquots of the supernatants were mixed with ACS II (Amersham). Radioactivity was counted on a liquid scintillation analyzer (LSC-3500; Aloka, Tokyo, Japan).
Fig. 4. Direct effects of MJE and its phloroglucinol derivatives on PGHS-1 or PGHS-2 activities in macrophages. (A) Cells were pretreated with 1 AM cycloheximide for 4 h. Then, cells were washed twice, suspended with fresh medium containing 1 AM cycloheximide, and were further incubated for 10 min. MJE, compounds 2, 6, 7, indomethacin, or NS-398 were added to the cell culture in the presence of 100 AM arachidonic acid, followed by incubation for 3 h. (B) Cells were pretreated with 100 AM aspirin for 4 h. Then cells were washed twice, suspended with fresh medium, and were further incubated with 100 ng/ml LPS for 6 h. MJE, compounds 2, 6, 7, indomethacin, or NS-398 were added to the cell culture, followed by incubation for 30 min. PGE2 concentrations in the culture supernatant were determined by Enzyme-Immuno-Assay. The results are presented as the means F S.E. for two different experiments involving duplicate assays. Statistical significance: ** P < 0.01 vs. corresponding controls (cells treated with 100 AM arachidonic acid or 100 ng/ml LPS).
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2.5. Effect on PGHS-1 activity and PGHS-2 activity The inhibitory effects of MJE and the compounds on the PGHS-1 activity and PGHS-2 activity in intact cells were examined by a previously reported method [24] with slight modification. In brief, arachidonic acid concentration was modified from 10 to 100 AM in an assay to determine PGHS-1 activity, stimulant was converted from TPA to LPS in a PGHS-2 activity assay. 2.6. Western blot analysis of PGHS-1 and PGHS-2 The cells were plated at 1.2 106 cells/ml onto a 35 mm petri dish (Sumitomo Bakelite, #1135R), and then incubated at 37 jC for 2 h. MJE or a test compound and 100 ng/ml LPS were added to the culture medium. After incubation for 16 h, the cells were chilled on ice, scraped from the dish using a cell scraper, collected in a microfuge tube and then centrifuged at 2800 g for 1 min. The cells were washed twice with phosphate-buffered saline without divalent cations by repeated centrifugation at 2800 g. The final cell pellet was extracted with 35 Al of a lysis buffer, comprised of 1% (v/v) Triton X-100, 0.1 mM EDTA and 1% aprotinin (Sigma) in 20 mM HEPES –NaOH buffer (pH 7.5), at 4 jC for 30 min. The cell lysate was centrifuged at 5500 g for 1 min. A 30-Al resultant supernatant was placed in a new microfuge tube. A 60 Ag protein of cell extract was boiled in an SDS-sample buffer, loaded onto a 5– 20% gradient slab gel (Atto, Tokyo, Japan), and electrophoresed at 20 mA/gel.
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The proteins in the gel were electro-transferred onto a polyvinylidene fluoride membrane (Immobilon-PR; Millipore, Bedford, MA, USA). The proteins on the membrane were probed with a polyclonal goat anti-PGHS-1 antibody or a polyclonal rabbit anti-PGHS-2 antibody as primary antibodies. For the analysis of PGHS-1, rabbit anti-goat IgG was used as secondary antibody. Finally, the proteins on the membrane were reacted with 125I-labeled protein A. After air-drying, the membrane was closely placed on X-ray film, and the bands on the membrane were photographed. The radioactivity of the bands corresponding to PGHS-1 or -2 were quantitated on an auto well g-counter (ARC-300; Aloka). 2.7. Reverse transcription-polymerase chain reaction (RTPCR) analysis of PGHS-2 mRNA The cells were cultured at 1.2 106 cells/ml at 37 jC for 2 h, and then MJE or a test compound was added to the culture medium together with 100 ng/ml LPS, and the cells were incubated for 6 h. Total RNA was isolated from the cell pellet using an RNA isolation kit (Qiagen, Hilden, Germany). The final amount of RNA was determined by absorbance at 260 nm. Fifty nanograms of the RNA samples were reversetranscribed into cDNA by RT-PCR using oligo (dT)12 – 18 primer. The PCR samples contained 25 Al of the reaction mixture, comprised of 50 mM KCl, 5 mM MgCl2, 0.16 mM dNTP, 5.0 units of Taq DNA polymerase (Takara shuzo, Shiga, Japan), and 5 pmol of sense and antisense primers in
Fig. 5. Effects of MJE and its phloroglucinol derivatives on PGHS-1 protein level. Cells were treated with MJE, compounds 1, 2, 6, or 7 in the presence of 100 ng/ml LPS for 16 h, and then cell lysates were prepared and assayed as described in the text. The amounts of the PGHS-1 protein were calculated from the radioactivity of the bands corresponding to PGHS-1 protein. The results are expressed relative to the amount of PGHS-1 in 60 Ag of the control cell (100%: treated with LPS, but MJE of other compounds not added).
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10 mM Tris – HCl (pH 8.3). The sense primer for PGHS-2 was 5V-TCAAAAGAAGTGCTGGAAAAGGTT-3V, and the antisense primer was 5V-TCTACCTGAGTGTCTTTGACTGTG-3V[25]. The sense primer for h-actin was 5V-GTGGGCCGCTCTAGGCACCAA-3V, and the antisense primer was 5VCTCTTTGATGTCACGCACGATTTC-3V [26]. The PCR reaction was performed under the following conditions: 25 cycles of denaturation at 94 jC for 1 min, annealing at 57 jC for 1 min and extension at 72 jC for 1.5 min, using a thermal cycler (GeneAmpR PCR Systems 9700; PE Applied Biosystems, USA). The PCR products were run on a 2% agarose gel and visualized by ethidium bromide staining. The bands in the gel were photographed and their fluorescence intensity was analyzed using a densitometer (CS-930; Shimadzu Scientific Instrument and Equipment, Kyoto, Japan). 2.8. Protein assay Protein concentrations were determined according to Bradford, using bovine serum albumin as the standard [27].
2.9. Statistical analysis Results were analyzed for statistical significance by Dunnett’s test for multiple comparison.
3. Results 3.1. Inhibitory effect on PGE2 production by activated macrophages LPS caused a significant increase in PGE2 production in RAW 264.7 macrophages during incubation for 16 h. The addition of MJE resulted in a concentration-dependent inhibition of PGE2 production by LPS-activated macrophages (IC50: 17.1 Ag/ml) (Fig. 2). Seven phloroglucinol derivatives showed inhibitory activity toward PGE2 production. The degree of inhibition of these phloroglucinol derivatives on PGE2 production was as follows: 7 (IC50: 1.0 AM) > 6 (IC50: 6.0 AM)>2 (IC50: 7.2 AM)> 3 (IC50:
Fig. 6. Inhibitory activities of MJE and its phloroglucinol derivatives towards PGHS-2 protein induction. Cells were treated with various concentration of MJE, compounds 1, 2, 6, or 7 in the presence of 100 ng/ml LPS for 16 h, then cell lysates were prepared and assayed as described in the text. The amounts of the PGHS-2 protein were calculated from the radioactivity of the bands corresponding to PGHS-2 protein. The results are expressed relative to the amount of PGHS-2 in 60 Ag of the control cell (100%: treated with LPS, but MJE of other compounds not added). The symbols correspond to: (A) MJE (-o-), 1 (-D-), and 6 (-.-), (B) 2 (-E-), and 7 (-D-). The results are for a typical example of repeated experiments.
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23.8 AM) = 4 (IC50: 23.8 AM)> 5 (IC50: > 30 AM)> 1 (IC50: >30 AM). MJE (30 Ag/ml) and all of the compounds (30 AM) examined in the present study did not affect cellular viability, as determined by monitoring the mitochondrial reduction of tetrazolium to formazan and through morphological observations (data not shown). All experiments were performed under conditions that did not affect cell viability. 3.2. Effect on the release of radioactivity from [14C]arachidonic acid-labeled macrophages Time course experiments showed that LPS induced release of radioactivity within 30 min of LPS treatment (Fig. 3). However, MJE and its phloroglucinol derivatives did not affect LPS-induced release of radioactivity from [14C]arachidonic acid-labeled macrophages at concentrations of 30 Ag/ml or 30 AM, respectively. 3.3. Effect on PGHS-1 activity and PGHS-2 activity In an assay to determine PGHS-1 activity, indomethacin, a nonselective PGHS-1/PGHS-2 inhibitor, showed signifi-
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cant inhibition of PGE2 production (IC50: 0.14 AM) (Fig. 4(A)). In contrast, NS-398, a selective PGHS-2 inhibitor, elicited more than a 20-fold weaker inhibition than that observed with indomethacin (IC50: 2.15 AM). MJE and the phloroglucinol derivatives 2, 6, and 7, did not show inhibition of PGHS-1-dependent PGE2 production at concentrations 30 Ag/ml or 30 AM, respectively. In an assay to determine PGHS-2 activity, both indomethacin and NS398 showed significant concentration-dependent inhibition, with IC50’s of 0.12 and 0.23 AM, respectively (Fig. 4(B)). It was observed that NS-398 selectively inhibited PGHS-2 activity. MJE and the test compounds 2, 6, and 7, were not observed to inhibit PGHS-2-dependent PGE2 production. These results indicate that inhibition of LPS-induced PGE2 production by MJE and its phloroglucinol derivatives was not due to a direct inhibition of either PGHS-1 or PGHS-2. 3.4. Inhibitory effect on PGHS-1 and PGHS-2 protein Western blot analysis revealed that PGHS-1 protein ordinarily exists, and that treatment with LPS for 16 h had little or no effect the PGHS-1 protein levels of macrophages (Fig. 5). MJE and the test compounds 1, 2, 6, and 7, had no effect on
Fig. 7. Effect of MJE and its phloroglucinol derivatives on PGHS-2 mRNA expression. Cells were treated with various concentrations of MJE, compounds 2, 6, or 7 in the presence of 100 ng/ml LPS for 6 h, and the cells were subsequently prepared and assayed as described in the text. The symbols correspond to: (A) MJE (-o-), and 2 (-E-), (B) 6 (-.-), and 7 (-D-). The lower panel shows the PCR products corresponding to the PGHS-2 (296 bp) and h-actin (540 bp) bands. The amounts of PGHS-2 mRNA in (A) and (B) are shown as percentage values relative to h-actin, individually. The results are for a typical example of repeated experiments.
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PGHS-1 protein levels in the presence of LPS. PGHS-2 protein was not detected in RAW 264.7 macrophages (Fig. 6) when they were incubated in medium alone; however, 100 ng/ml of LPS induced dramatic production of PGHS-2 protein during incubation for 16 h. MJE showed significant inhibition of PGHS-2 protein levels. Compounds 7 (IC50: 0.8 AM), 6 (IC50: 1.9 AM), 2 (IC50: 4.3 AM), and 1 (inhibition at 30 AM: 19.4%) were found to remarkably reduce the amount of PGHS-2 protein. 3.5. Inhibitory effects on PGHS-2 mRNA expression Treatment with LPS for 6 h increased the level of PGHS-2 mRNA expression (Fig. 7). MJE and the test compounds 2, 6, and 7 were observed to decrease the level of induced PGHS-2 mRNA in a concentration-dependent manner. At 30 AM, compounds 6 and 7 almost totally blocked the expression of PGHS-2 mRNA, and a low-level inhibition of PGHS-2 mRNA was noted at concentrations as low as 1 AM. The inhibition of PGHS-2 mRNA expression paralleled the observed inhibition of PGHS-2 protein induction. Therefore, it appears that MJE and its phloroglucinol derivatives inhibit the induction of PGHS-2 by suppressing PGHS-2 mRNA expression.
4. Discussion In the present study, we investigated the effects of MJE and several of its phloroglucinol derivatives on PGE2 production in macrophages activated by LPS in order to clarify the anti-inflammatory activity of MJE and its phloroglucinol derivatives. MJE inhibited PGE2 production during macrophage activation, and seven of its phloroglucinol derivatives were also observed to inhibit PGE2 production in the following order: 7>6>2>3 = 4>5>1. Compounds 2 and 1 were studied in terms of structure – activity relationships. The presence of an isoprenyl side chain at C-3 position in 2 might explain why 2 was a stronger inhibitor than 1. Compounds 6 and 7 were the strongest inhibitors of all compounds tested. This indicates that cyclization at the para-position might be the most important factor contributing to inhibitory activity, because both 6 and 7 share this feature. Furthermore, 7 showed significantly more inhibition than 6, which further suggests that the presence of a free hydroxy group at the chroman ring, which is unique to 7, might lead to stronger inhibition. We previously reported that MJE inhibits NO production by macrophages activated with LPS and interferon-g, and that MJE’s phloroglucinol derivatives are responsible for the majority of this inhibition [6]. Compounds 6 and 7 also resulted in a significant inhibition of NO production. The observation that PGE2 production is inhibited by phloroglucinol derivatives in the present study parallels our previous observation that phloroglucinol derivatives inhibit NO production. Therefore, we speculate that the phloroglucinol derivatives may mainly mediate both NO
production and PGE2 production by the mechanism such as inhibition of the signaling pathways in macrophages triggered by LPS. MJE and its phloroglucinol derivatives were not observed to effect the release of radioactivity, which suggests that the inhibitory effect of MJE and its phloroglucinol derivatives on macrophage PGE2 production is not due to an inhibition of phospholipase A2. PGHS exhibits both a bis-oxygenase (cyclooxygenase) activity catalyzing PGG2 formation and a peroxidase activity catalyzing a two-electron reduction of PGG2 to PGH2. Both cyclooxygenase and peroxidase activities are associated with a single protein molecule [28,29]. MJE and its phloroglucinol derivatives did not inhibit either PGHS-1- or -2-dependent PGE2 production in macrophages. These results indicate that MJE and its derivatives do not directly inhibit these two activities of PGHS enzymes. The observed inhibition of PGE2 production in activated macrophages by MJE and its various phloroglucinol derivatives resulted from an alteration in PGHS-2 gene expression. Thus, MJE and its derivatives provide a distinct advantage over nonselective, PGHS-1-directed inhibitors. We suggest that MJE and its phloroglucinol derivatives might play a role in modulating inflammatory and immune responses by inhibiting the production of PGs. The properties of its phloroglucinol derivatives indicate that further studies on these molecules are needed, both to determine their molecular mechanisms of action and to evaluate them as a potentially useful agents for prevention or treatment of disease.
Acknowledgements This study was financially supported in part by a Grantin-Aid for Research on Eye and Ear Science, Immunology, Allergy and Organ Transplantation from the Health Sciences Research Grants, for Ministry of Health, Labour and Welfare, by the development of characteristic education and High-Tech Research Center from the Ministry of Education, Culture, Sports, Science to Nihon University and by Technology of Japan and for the Promotion and Mutual Aid Corporation for Private School of Japan to Nihon University.
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Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line, RAW 264.7, by Mallotus japonicus phloroglucinol derivatives Rie Ishii a, Masakazu Horie a, Koichi Saito a, Munehisa Arisawa b, Susumu Kitanaka c,* b
a Saitama Institute of Public Health, 639-1 Kamiokubo Saitama, Saitama 338-0824, Japan Laboratory of Herbal Garden, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani Toyama, Toyama 930-0194, Japan c College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
Received 28 August 2001; received in revised form 17 January 2002; accepted 4 February 2002
Abstract An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E2 production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE2 production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC50 of 1.0 AM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE2 production by activated macrophages. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Mallotus japonicus; Euphorbiaceae; Phloroglucinol derivative; Macrophage; Prostaglandin E2; Prostaglandin endoperoxide synthase
1. Introduction Mallotus japonicus Mueller Arg. (Euphorbiaceae) is a dioecious and deciduous tree that is distributed throughout tropical and temperate Asia. Parts of the tree have been used for a long time in folk medicine; the bark is used as a medication for ulcers and the treatment of cancer and the leaves are used as treatment for boils. An extract of the tree’s pericarps has been reported to exhibit anti-tumor activity [1,2], anti-tumor-promoting activity [3], and anti-viral activity [4,5]. We previously found an 80% aqueous acetone extract of the pericarps (MJE) to inhibit the production of nitric oxide (NO) by activated macrophages treated with lipopolysaccharide (LPS) and interferon-g; furthermore, several phloroglucinol derivatives were isolated from MJE
*
Corresponding author. Tel.: +81-47-465-5356; fax: +81-47-465-5440. E-mail address: [email protected] (S. Kitanaka).
and identified as its active compounds [6]. We theorized that MJE and its phloroglucinol derivatives might have antiinflammatory activity. Prostaglandins (PGs) are important mediators in a diverse range of biological processes, such as cell proliferation, inflammatory and immune responses, platelet aggregation, and the extracellular matrix biosynthesis [7,8]. Prostaglandin endoperoxide synthase (PGHS) is thought to be a key enzyme in the initiation of PG synthesis from arachidonic acid in various cells [9,10]. It is now well established that two genetically distinct isoforms of PGHS exist. PGHS-1 is constitutively expressed as a housekeeping enzyme in almost all tissues [11] and mediates physiological responses, including platelet regulation [12]. In contrast, PGHS-2 is induced by several kinds of stimuli such as endotoxins, cytokines and mitogens [13 – 15]. This occurs in a number of cells, including macrophages [14,16]. Moreover, it has been determined that the PGHS-2 isoform is primarily responsible for prostanoid synthesis in pathological processes, such as acute and
0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 2 0 0 - 3
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chronic inflammatory responses [17,18]. It is thought that the observed side effects of nonselective inhibitors of PGHS-1 and -2, such as gastrointestinal toxicity [19], are due to the suppression of PGHS-1-derived prostanoids. It is further thought that the pharmacological action of these inhibitors can be attributed to their inhibition of PGHS-2 activity at sites of inflammation. Therefore, immense effort has been devoted to the development of selective inhibitors of PGHS2 [20,21]. In order to clarify the mechanism by which M. japonicus exerts its anti-inflammatory activity, we investigated the effects of MJE and its constituents on PGE2 production by activated macrophages. We further investigated its selectivity toward PGHS-2. The present paper describes our results and the mechanisms by which the extract and its constituents act.
2. Materials and methods 2.1. Materials The compounds, mallotophenone (1), mallotojaponin (2), butyrylmallotolerin (3), mallotochromanol (4), isobutyrylmallotochromanol (5), and isomallotochromanol (7) were isolated from MJE as described previously [1,4,5,22,23]. Isomallotochromene (6) was derived from 2 by cyclization of its side chain [4]. Structural formulae of these compounds are shown in Fig. 1. Ham’s F12 medium, aspirin, and indomethacin were obtained from Sigma (St. Louis, MO, USA). NS-398 from Cayman Chemical Company (Ann
Arbor, MI, USA), cycloheximide from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA), fetal bovine serum from GIBCO BRL (Grand Island, NY, USA), LPS (Escherichia coli, O55: B5) from Difco (Detroit, MI, USA), 125 I-labeled protein A and [1-14C] arachidonic acid were obtained from Amersham Pharmacia Biotech UK (Amersham, England). Polyclonal rabbit anti-PGHS-2 antibody from Oxford Biochemical Research (Oxford, MI, USA), polyclonal goat anti-PGHS-1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rabbit anti-goat IgG was purchased from Zymed Laboratories (Lexington, KY, USA). All other chemicals and reagents were of the purest commercial grade available. 2.2. Cell culture RAW 264.7 cells, a mouse macrophage-like cell line transformed with the Abelson leukemia virus, were obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained by culturing in Ham’s F12 medium supplemented 10% heat-inactivated fetal bovine serum in a CO2 incubator (5% CO2 –95% humidified air) at 37 jC. The cells were seeded onto plastic petri dishes (Falcon, #1001; Becton Dickinson) and passaged twice a week. 2.3. PGE2 production assay The cells were suspended in the medium at a density of 2.0 105 cells/ml. A 400-Al cell suspension was poured into each well of a 24-well microplate (Sumitomo Bakelite, #8024R; Tokyo, Japan), and then incubated at 37 jC for 2 h.
Fig. 1. Chemical structures of the phloroglucinol derivatives examined in the present study.
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MJE or a test compound and 100 ng/ml LPS were added to the culture medium, and the cells were incubated at 37 jC for 16 h. The medium were collected in a microfuge tube
Fig. 2. Inhibitory effects of MJE and its phloroglucinol derivatives on LPSinduced PGE2 production in RAW 264.7 cells. Cells were treated with various concentrations of MJE or one of its phloroglucinol derivatives in the presence of 100 ng/ml LPS at 37 jC for 16 h. The symbols correspond to: (A) MJE (-o-), 1 (-D-), 2 (-E-), and 6 (-.-), and (B) 3 (-5-), 4 (-y-), 5 (-.-), and 7 (-D-). The results are the means F S.E. for two independent experiments involving triplicate assays. Statistical significance: * P < 0.05, ** P < 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS).
Fig. 3. Effects of MJE and its phloroglucinol derivatives on the release of radioactivity from [14C]arachidonic acid-labeled macrophages. Cells were labeled with [14C]arachidonic acid at 37 jC for 18 h, washed three times, suspended in a fresh medium. The cells were reseeded, and treated with MJE or its phloroglucinol derivatives (compounds 2, 4, 6, and 7) in the presence of 100 ng/ml LPS, followed by incubation for 0 – 2 h. Radioactivity released into the culture supernatant was determined on a liquid scintillation analyzer. The results are the means F S.E. for two independent experiments involving duplicate assays. The symbols correspond to: (A) control cells (treated with LPS) (-n-), + NONE (absence of LPS) (- w -), 30 Ag/ml MJE (-o-), and 30 AM 2 (-E-), and (B) control cells (-n-), 30 AM 4 (-y-), 30 AM 6 (-.-), and 30 AM 7 (-D-). Statistical significance: ** P < 0.01 vs. corresponding controls (cells treated with 100 ng/ml LPS).
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and then centrifuged at 2800 g for 1 min. The supernatant was placed in a new microfuge tube to determine the amount of PGE2 using a PGE2 Enzyme-Immuno-Assay kit (Cayman Chemical). 2.4. Measurement of radioactivity released from [14C]arachidonic acid-labeled macrophages Cells were seeded at 6.0 105 cells/ml onto a plastic petri dish and then incubated at 37 jC for 2 h. The medium was replaced with a fresh medium containing 3.7 kBq of [1-14C]arachidonic acid per milliliter, and the cells were
incubated at 37 jC for 18 h. The cells were washed three times to remove unincorporated arachidonic acid, and suspended at 6.0 105 cells/ml in a fresh medium. A 500-Al cell suspension was poured into each well of a 24-well microplate and then incubated at 37 jC for 5 min to allow the cells to adhere to the plate. Then, MJE or a test compound and 100 ng/ml LPS were added to the culture medium. The cells were further incubated at 37 jC for 0 – 2 h. The culture supernatants were collected and centrifuged at 5500 g for 1 min, and 250 Al aliquots of the supernatants were mixed with ACS II (Amersham). Radioactivity was counted on a liquid scintillation analyzer (LSC-3500; Aloka, Tokyo, Japan).
Fig. 4. Direct effects of MJE and its phloroglucinol derivatives on PGHS-1 or PGHS-2 activities in macrophages. (A) Cells were pretreated with 1 AM cycloheximide for 4 h. Then, cells were washed twice, suspended with fresh medium containing 1 AM cycloheximide, and were further incubated for 10 min. MJE, compounds 2, 6, 7, indomethacin, or NS-398 were added to the cell culture in the presence of 100 AM arachidonic acid, followed by incubation for 3 h. (B) Cells were pretreated with 100 AM aspirin for 4 h. Then cells were washed twice, suspended with fresh medium, and were further incubated with 100 ng/ml LPS for 6 h. MJE, compounds 2, 6, 7, indomethacin, or NS-398 were added to the cell culture, followed by incubation for 30 min. PGE2 concentrations in the culture supernatant were determined by Enzyme-Immuno-Assay. The results are presented as the means F S.E. for two different experiments involving duplicate assays. Statistical significance: ** P < 0.01 vs. corresponding controls (cells treated with 100 AM arachidonic acid or 100 ng/ml LPS).
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2.5. Effect on PGHS-1 activity and PGHS-2 activity The inhibitory effects of MJE and the compounds on the PGHS-1 activity and PGHS-2 activity in intact cells were examined by a previously reported method [24] with slight modification. In brief, arachidonic acid concentration was modified from 10 to 100 AM in an assay to determine PGHS-1 activity, stimulant was converted from TPA to LPS in a PGHS-2 activity assay. 2.6. Western blot analysis of PGHS-1 and PGHS-2 The cells were plated at 1.2 106 cells/ml onto a 35 mm petri dish (Sumitomo Bakelite, #1135R), and then incubated at 37 jC for 2 h. MJE or a test compound and 100 ng/ml LPS were added to the culture medium. After incubation for 16 h, the cells were chilled on ice, scraped from the dish using a cell scraper, collected in a microfuge tube and then centrifuged at 2800 g for 1 min. The cells were washed twice with phosphate-buffered saline without divalent cations by repeated centrifugation at 2800 g. The final cell pellet was extracted with 35 Al of a lysis buffer, comprised of 1% (v/v) Triton X-100, 0.1 mM EDTA and 1% aprotinin (Sigma) in 20 mM HEPES –NaOH buffer (pH 7.5), at 4 jC for 30 min. The cell lysate was centrifuged at 5500 g for 1 min. A 30-Al resultant supernatant was placed in a new microfuge tube. A 60 Ag protein of cell extract was boiled in an SDS-sample buffer, loaded onto a 5– 20% gradient slab gel (Atto, Tokyo, Japan), and electrophoresed at 20 mA/gel.
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The proteins in the gel were electro-transferred onto a polyvinylidene fluoride membrane (Immobilon-PR; Millipore, Bedford, MA, USA). The proteins on the membrane were probed with a polyclonal goat anti-PGHS-1 antibody or a polyclonal rabbit anti-PGHS-2 antibody as primary antibodies. For the analysis of PGHS-1, rabbit anti-goat IgG was used as secondary antibody. Finally, the proteins on the membrane were reacted with 125I-labeled protein A. After air-drying, the membrane was closely placed on X-ray film, and the bands on the membrane were photographed. The radioactivity of the bands corresponding to PGHS-1 or -2 were quantitated on an auto well g-counter (ARC-300; Aloka). 2.7. Reverse transcription-polymerase chain reaction (RTPCR) analysis of PGHS-2 mRNA The cells were cultured at 1.2 106 cells/ml at 37 jC for 2 h, and then MJE or a test compound was added to the culture medium together with 100 ng/ml LPS, and the cells were incubated for 6 h. Total RNA was isolated from the cell pellet using an RNA isolation kit (Qiagen, Hilden, Germany). The final amount of RNA was determined by absorbance at 260 nm. Fifty nanograms of the RNA samples were reversetranscribed into cDNA by RT-PCR using oligo (dT)12 – 18 primer. The PCR samples contained 25 Al of the reaction mixture, comprised of 50 mM KCl, 5 mM MgCl2, 0.16 mM dNTP, 5.0 units of Taq DNA polymerase (Takara shuzo, Shiga, Japan), and 5 pmol of sense and antisense primers in
Fig. 5. Effects of MJE and its phloroglucinol derivatives on PGHS-1 protein level. Cells were treated with MJE, compounds 1, 2, 6, or 7 in the presence of 100 ng/ml LPS for 16 h, and then cell lysates were prepared and assayed as described in the text. The amounts of the PGHS-1 protein were calculated from the radioactivity of the bands corresponding to PGHS-1 protein. The results are expressed relative to the amount of PGHS-1 in 60 Ag of the control cell (100%: treated with LPS, but MJE of other compounds not added).
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10 mM Tris – HCl (pH 8.3). The sense primer for PGHS-2 was 5V-TCAAAAGAAGTGCTGGAAAAGGTT-3V, and the antisense primer was 5V-TCTACCTGAGTGTCTTTGACTGTG-3V[25]. The sense primer for h-actin was 5V-GTGGGCCGCTCTAGGCACCAA-3V, and the antisense primer was 5VCTCTTTGATGTCACGCACGATTTC-3V [26]. The PCR reaction was performed under the following conditions: 25 cycles of denaturation at 94 jC for 1 min, annealing at 57 jC for 1 min and extension at 72 jC for 1.5 min, using a thermal cycler (GeneAmpR PCR Systems 9700; PE Applied Biosystems, USA). The PCR products were run on a 2% agarose gel and visualized by ethidium bromide staining. The bands in the gel were photographed and their fluorescence intensity was analyzed using a densitometer (CS-930; Shimadzu Scientific Instrument and Equipment, Kyoto, Japan). 2.8. Protein assay Protein concentrations were determined according to Bradford, using bovine serum albumin as the standard [27].
2.9. Statistical analysis Results were analyzed for statistical significance by Dunnett’s test for multiple comparison.
3. Results 3.1. Inhibitory effect on PGE2 production by activated macrophages LPS caused a significant increase in PGE2 production in RAW 264.7 macrophages during incubation for 16 h. The addition of MJE resulted in a concentration-dependent inhibition of PGE2 production by LPS-activated macrophages (IC50: 17.1 Ag/ml) (Fig. 2). Seven phloroglucinol derivatives showed inhibitory activity toward PGE2 production. The degree of inhibition of these phloroglucinol derivatives on PGE2 production was as follows: 7 (IC50: 1.0 AM) > 6 (IC50: 6.0 AM)>2 (IC50: 7.2 AM)> 3 (IC50:
Fig. 6. Inhibitory activities of MJE and its phloroglucinol derivatives towards PGHS-2 protein induction. Cells were treated with various concentration of MJE, compounds 1, 2, 6, or 7 in the presence of 100 ng/ml LPS for 16 h, then cell lysates were prepared and assayed as described in the text. The amounts of the PGHS-2 protein were calculated from the radioactivity of the bands corresponding to PGHS-2 protein. The results are expressed relative to the amount of PGHS-2 in 60 Ag of the control cell (100%: treated with LPS, but MJE of other compounds not added). The symbols correspond to: (A) MJE (-o-), 1 (-D-), and 6 (-.-), (B) 2 (-E-), and 7 (-D-). The results are for a typical example of repeated experiments.
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23.8 AM) = 4 (IC50: 23.8 AM)> 5 (IC50: > 30 AM)> 1 (IC50: >30 AM). MJE (30 Ag/ml) and all of the compounds (30 AM) examined in the present study did not affect cellular viability, as determined by monitoring the mitochondrial reduction of tetrazolium to formazan and through morphological observations (data not shown). All experiments were performed under conditions that did not affect cell viability. 3.2. Effect on the release of radioactivity from [14C]arachidonic acid-labeled macrophages Time course experiments showed that LPS induced release of radioactivity within 30 min of LPS treatment (Fig. 3). However, MJE and its phloroglucinol derivatives did not affect LPS-induced release of radioactivity from [14C]arachidonic acid-labeled macrophages at concentrations of 30 Ag/ml or 30 AM, respectively. 3.3. Effect on PGHS-1 activity and PGHS-2 activity In an assay to determine PGHS-1 activity, indomethacin, a nonselective PGHS-1/PGHS-2 inhibitor, showed signifi-
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cant inhibition of PGE2 production (IC50: 0.14 AM) (Fig. 4(A)). In contrast, NS-398, a selective PGHS-2 inhibitor, elicited more than a 20-fold weaker inhibition than that observed with indomethacin (IC50: 2.15 AM). MJE and the phloroglucinol derivatives 2, 6, and 7, did not show inhibition of PGHS-1-dependent PGE2 production at concentrations 30 Ag/ml or 30 AM, respectively. In an assay to determine PGHS-2 activity, both indomethacin and NS398 showed significant concentration-dependent inhibition, with IC50’s of 0.12 and 0.23 AM, respectively (Fig. 4(B)). It was observed that NS-398 selectively inhibited PGHS-2 activity. MJE and the test compounds 2, 6, and 7, were not observed to inhibit PGHS-2-dependent PGE2 production. These results indicate that inhibition of LPS-induced PGE2 production by MJE and its phloroglucinol derivatives was not due to a direct inhibition of either PGHS-1 or PGHS-2. 3.4. Inhibitory effect on PGHS-1 and PGHS-2 protein Western blot analysis revealed that PGHS-1 protein ordinarily exists, and that treatment with LPS for 16 h had little or no effect the PGHS-1 protein levels of macrophages (Fig. 5). MJE and the test compounds 1, 2, 6, and 7, had no effect on
Fig. 7. Effect of MJE and its phloroglucinol derivatives on PGHS-2 mRNA expression. Cells were treated with various concentrations of MJE, compounds 2, 6, or 7 in the presence of 100 ng/ml LPS for 6 h, and the cells were subsequently prepared and assayed as described in the text. The symbols correspond to: (A) MJE (-o-), and 2 (-E-), (B) 6 (-.-), and 7 (-D-). The lower panel shows the PCR products corresponding to the PGHS-2 (296 bp) and h-actin (540 bp) bands. The amounts of PGHS-2 mRNA in (A) and (B) are shown as percentage values relative to h-actin, individually. The results are for a typical example of repeated experiments.
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PGHS-1 protein levels in the presence of LPS. PGHS-2 protein was not detected in RAW 264.7 macrophages (Fig. 6) when they were incubated in medium alone; however, 100 ng/ml of LPS induced dramatic production of PGHS-2 protein during incubation for 16 h. MJE showed significant inhibition of PGHS-2 protein levels. Compounds 7 (IC50: 0.8 AM), 6 (IC50: 1.9 AM), 2 (IC50: 4.3 AM), and 1 (inhibition at 30 AM: 19.4%) were found to remarkably reduce the amount of PGHS-2 protein. 3.5. Inhibitory effects on PGHS-2 mRNA expression Treatment with LPS for 6 h increased the level of PGHS-2 mRNA expression (Fig. 7). MJE and the test compounds 2, 6, and 7 were observed to decrease the level of induced PGHS-2 mRNA in a concentration-dependent manner. At 30 AM, compounds 6 and 7 almost totally blocked the expression of PGHS-2 mRNA, and a low-level inhibition of PGHS-2 mRNA was noted at concentrations as low as 1 AM. The inhibition of PGHS-2 mRNA expression paralleled the observed inhibition of PGHS-2 protein induction. Therefore, it appears that MJE and its phloroglucinol derivatives inhibit the induction of PGHS-2 by suppressing PGHS-2 mRNA expression.
4. Discussion In the present study, we investigated the effects of MJE and several of its phloroglucinol derivatives on PGE2 production in macrophages activated by LPS in order to clarify the anti-inflammatory activity of MJE and its phloroglucinol derivatives. MJE inhibited PGE2 production during macrophage activation, and seven of its phloroglucinol derivatives were also observed to inhibit PGE2 production in the following order: 7>6>2>3 = 4>5>1. Compounds 2 and 1 were studied in terms of structure – activity relationships. The presence of an isoprenyl side chain at C-3 position in 2 might explain why 2 was a stronger inhibitor than 1. Compounds 6 and 7 were the strongest inhibitors of all compounds tested. This indicates that cyclization at the para-position might be the most important factor contributing to inhibitory activity, because both 6 and 7 share this feature. Furthermore, 7 showed significantly more inhibition than 6, which further suggests that the presence of a free hydroxy group at the chroman ring, which is unique to 7, might lead to stronger inhibition. We previously reported that MJE inhibits NO production by macrophages activated with LPS and interferon-g, and that MJE’s phloroglucinol derivatives are responsible for the majority of this inhibition [6]. Compounds 6 and 7 also resulted in a significant inhibition of NO production. The observation that PGE2 production is inhibited by phloroglucinol derivatives in the present study parallels our previous observation that phloroglucinol derivatives inhibit NO production. Therefore, we speculate that the phloroglucinol derivatives may mainly mediate both NO
production and PGE2 production by the mechanism such as inhibition of the signaling pathways in macrophages triggered by LPS. MJE and its phloroglucinol derivatives were not observed to effect the release of radioactivity, which suggests that the inhibitory effect of MJE and its phloroglucinol derivatives on macrophage PGE2 production is not due to an inhibition of phospholipase A2. PGHS exhibits both a bis-oxygenase (cyclooxygenase) activity catalyzing PGG2 formation and a peroxidase activity catalyzing a two-electron reduction of PGG2 to PGH2. Both cyclooxygenase and peroxidase activities are associated with a single protein molecule [28,29]. MJE and its phloroglucinol derivatives did not inhibit either PGHS-1- or -2-dependent PGE2 production in macrophages. These results indicate that MJE and its derivatives do not directly inhibit these two activities of PGHS enzymes. The observed inhibition of PGE2 production in activated macrophages by MJE and its various phloroglucinol derivatives resulted from an alteration in PGHS-2 gene expression. Thus, MJE and its derivatives provide a distinct advantage over nonselective, PGHS-1-directed inhibitors. We suggest that MJE and its phloroglucinol derivatives might play a role in modulating inflammatory and immune responses by inhibiting the production of PGs. The properties of its phloroglucinol derivatives indicate that further studies on these molecules are needed, both to determine their molecular mechanisms of action and to evaluate them as a potentially useful agents for prevention or treatment of disease.
Acknowledgements This study was financially supported in part by a Grantin-Aid for Research on Eye and Ear Science, Immunology, Allergy and Organ Transplantation from the Health Sciences Research Grants, for Ministry of Health, Labour and Welfare, by the development of characteristic education and High-Tech Research Center from the Ministry of Education, Culture, Sports, Science to Nihon University and by Technology of Japan and for the Promotion and Mutual Aid Corporation for Private School of Japan to Nihon University.
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