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Atrovirinone inhibits proinflammatory mediator synthesis through disruption of NF-[kappa]B nuclear translocation and MAPK phosphorylation in the murine monocytic macrophage RAW 264.7.


In a previous communication we showed that atrovirinone, a 1,4-benzoquinone isolated from the roots of Garcinia atroviridis, was able to inhibit several major proinflammatory mediators of inflammation. In this report we show that atrovirinone inhibits NO and [PGE.sub.2] synthesis through inhibition of iNOS and COX-2 expression. We also show that atrovirinone inhibits the secretion of IL-1[beta] and IL-6 in a dose dependent fashion whereas the secretion of IL-10, the anti-inflammatory cytokine, was enhanced. Subsequently we determined that the inhibition of proinflammatory cytokine synthesis and inducible enzyme expression was due to a dose-dependent inhibition of phosphorylation of p38 and ERK1/2. We also showed that atrovirinone prevented phosphorylation of I-[kappa]B[alpha], which resulted in a reduction of p65NF-[kappa]B nuclear translocation as demonstrated by expression analysis. We conclude that atrovirinone is a potential anti-inflammatory drug lead that targets both the MAPK and NF-[kappa]B pathway.

[C]2010 Elsevier GmbH. All rights reserved.




Garcinia atroviridis







During inflammatory disease the macrophage produces excess amounts of mediators such as nitric oxide (NO), prostanoids and pro-inflammatory cytokines (Laskin and Laskin, 2001; Fitzpatrick, 2001). It is also evident that several signalling pathways control the synthesis of these mediators (Hanada and Yoshimura, 2002; Lewis and Manning, 1999) and therefore it is not surprising that anti-inflammatory drugs, either steroidal or non-steroidal, act to a large extent upon key molecules of these signal transduction pathways (Lewis and Manning, 1999). Probably the most pivotal enzymes involved in maintaining inflammation are the inducible enzymes; inducible nitric oxide synthase (iNOS/NOS2) and cyclooxygenase-2 (COX-2), which are responsible for the catalysis of NO and prostaglandin [E.sub.2] ([PGE.sub.2]) respectively (Surh et al. 2001). Continuous production of these molecules in chronic inflammation (Motilva et al. 2005) has been linked to development of autoimmune disorders, coronary artery disease, and cancer (Kubatka et al. 2003).

The ubiquitous NF-[kappa]B signalling pathway plays a crucial role in regulating inflammation through transcription of COX, iNOS and cytokine genes. Found in the cytoplasm of resting cells, dimer NF-[kappa]B is normally confined to an inactive cytoplasmic complex through binding to an inhibitory protein, I-[kappa]B, which masks its nuclear localization signal (Makarov, 2000). Exposure of cells to external proinflammatory stimuli such as mitogens, inflammatory cytokines and bacterial lipopolysaccharides (LPS) (Abate et al. 1998) causes rapid I-[kappa]B phosphorylation at Ser-32 and Ser-36 by I-[kappa]B kinase (IKK) followed by proteosomal degradation (Pando and Verma, 2000; Nasuhara et al. 1999; Griscavage et al. 1996). This causes dissociation of I-[kappa]B from NF-[kappa]B and subsequent nuclear translocation via specific machinery (Abate et al. 1998). In the nucleus, NF-[kappa]B induces the transcription of a large variety of target genes, by binding to the cis-acting [kappa]B element. The target genes are those that normally encode cytokines (Kiemer et al. 2003), cell adhesion molecules, and inflammatory enzymes including COX-2 (Abate et al. 1998) and iNOS (Lee et al. 2000).

The mitogen-activated protein kinases (MAPKs) are a group of signaling molecules that play a critical role in the regulation of cell growth and differentiation, as well as in the control of cellular responses to cytokines and stresses. The MAPKs that have been shown to play pivotal roles in proinflammatory signaling are extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) (Davis, 1994). A critical component of production of NO and proinflammatory cytokines in activated macrophages is via the phosphorylation of MAPKs (Ajizian et al. 1999; Carter et al. 1999; Chan and Riches, 1998). Furthermore specific MAPK inhibitors suppress the expression of the iNOS gene (Carter et al. 1999; Chan and Riches, 1998). The complexity of the pro-inflammatory signaling pathway is further compounded by the fact that the MAPK and NF-[kappa]B pathways converge and cross-talk (Carter et al. 1999).

Atrovirinone (2-[1'"-methoxycarbonyl-4"',6'"-dihydroxyphenol]-3-methoxy-5,6-di-[3-methyl-2-butenyl]-l,4-benzoquinone) (Fig. 1) is a prenylated benzoquinone isolated from the roots of Garcinia atroviridis in our laboratory (Permana et al. 2001). In general, benzoquinones are toxic to cells and are mostly sought for their cytotoxic properties against tumor cells (Permana et al. 2001; Podolak et al. 2005). However our preliminary studies suggest that atrovirinone has anti-inflammatory properties at non-cytotoxic concentrations (Syahida et al. 2006). The present study demonstrates that atrovirinone exhibits its antiinflammatory activity via suppression COX-2 and iNOS expression through disruption of p65NF-[kappa]B nuclear translocation, ERK 1/2 and p38 phosphorylation.


Materials and methods

Cell culture

RAW 264.7 cells were purchased from the European Collection of Cell Cultures (CAMR, UK) and maintained in DMEM supplemented with 10% foetal calf serum (FCS), 4.5 g/l glucose, sodium pyruvate (1 mM), L-glutamine (2mM), streptomycin (50 [micro]g/ml) and penicillin (50 U/ml). Cells at a confluency of 80-90% were scraped out and centrifuged at 110g at 4 []C for 10 min. The concentration was then adjusted to 1 x [10.sup.6] cells/ml and cell viability was always > 95% as determined by trypan blue dye exclusion.

Cytokine immunoassays

Following 18 h incubation with varying concentrations of atrovirinone spent media was collected for cytokine immunoassay. Supernatants of spent cell culture media were stored at -80[degrees]C prior to assay. Spent media was analyzed for IL-1[beta], IL-6 and IL-10 by enzyme-linked immunosorbent assay (ELISA) using commercial kits (OptEIA[TM], BD Pharmingen, USA) according to the manufacturer's instructions.

Preparation of whole cell extract

Cells were scraped out of culture flasks and rinsed 3 times with ice-cold Tris-buffered sucrose. The pellet was resuspended in 30 [micro]l of lysis buffer (0.5% Triton X-100, 2 mM EDTA, 2 mM PMSF, in 20 mM Tris-HCl, 2 ng/[micro]l Pepstatin A, pH 7.5) and incubated in ice for 30 min. Following incubation, the cells were disrupted by sonication at 20 Hz for 20 min on ice and centrifuged at 25150 g, 4 [degrees]C, for 20 min. The supernatant was collected and protein content was measured using the BCA assay (Pierce, USA).

Preparation of cytosolic extract

This procedure was carried out by following Protocol # PT3612-1 Version # PR16623 by BD Mercury TransFactor Kits (BD Biosciences, USA). Treated RAW 264.7 cells were collected and rinsed with 20 ml of cold PBS (58 mM [Na.sub.2][HPO.sub.4], 17 mM [NaH.sub.2][PO.sub.4], 68 mM NaCl, pH 7.5). Lysis buffer (supplied with kit) was added to the pellet and was left in ice for 15 minutes. Next, the cell suspension was centrifuged at 420g, 4 [degrees]C for five min, and the supernatant was discarded. Lysis Buffer was added to the remaining pellet. The cells were ruptured by rapid strokes of a 27 gauge needle and syringe and centrifuged for 20 min at 4 [degrees]C, at 11 000g. The supernatant collected was snap-frozen in liquid nitrogen. Protein content was measured using BCA assay (Pierce, USA).

Preparation of nuclear extract

The pellet acquired from cytosolic protein extraction was added with extraction buffer (supplied with kit). As described in cytosolic protein extraction procedure, a 27 gauge needle and syringe was used to disrupt the cell nuclei, followed by centrifugation of the disrupted nuclei at 21 000g for five min. The nuclear protein in the supernatant was snap-frozen in liquid nitrogen for storing, and measured for protein content using BCA assay (Pierce, USA).

Western blot analysis

RAW 264.7 cells were induced with a combination of 200 U/ml IFN-[gamma] and 10 [micro]g/ml LPS as described earlier and treated with atrovirinone for 18 hours for COX-2 and iNOS detection. For the detection of p65NF-[kappa]B, I-[kappa]B[alpha], p-I[kappa]B[alpha], p38, p-p38, ERK1/2 and p-ERK1/2 the cells were only treated for two hours. Whole protein extract was used to analyse all proteins except p65. Analysis for cytosolic and nuclear p65NF-[kappa]B was carried out with cytosolic and nuclear extracts respectively. Equal amounts of protein (50 [micro]g) were electrophoresed on a 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane (Osmonics, USA). The membrane was incubated for an hour in blocking buffer (5% BSA in Tris-buffered saline (TBS)-Tween 20 (0.05%). Detection involved incubation of the membrane in primary antibodies for two hours. The primary antibodies used were: rabbit polyclonal antibodies raised against iNOS (1:5000) and COX-2 (1:2500) (Cayman Chemicals, USA); rabbit polyclonal anti-p-I-[kappa]B[alpha] (1:1000) (Santa Cruz Biotechnology, USA); and mouse monoclonal [IgG.sub.1] antibodies raised against p65NF-[kappa]B (1:500) and I-[kappa]B[alpha] (1:750) (Santa Cruz Biotechnology, USA); mouse monoclonal [IgG.sub.1] antibodies raised against p38 (1:500) and phospho-p38 (1:1000) and ERK1/2 (1:500) and phospho-p38 (1:1000) (Santa Cruz Biotechnology, USA). The same membrane was stripped and re-probed with HRP-conjugated mouse anti-mouse [beta]-actin (1:10000) or TFIIB (1:5000). After washing three times with TBS-Tween, the membrane was hybridized with HRP-conjugated donkey anti-rabbit secondary antibody (1:5000) or HRP-conjugated donkey anti-mouse secondary antibody (1:5000) for 2 h and washed three times with TBS-Tween. The proteins were detected with Enhanced Chemiluminescence Western Blotting Reagent (Amersham Bioscience, UK) according to the manufacturer's instructions. The image was captured using a Chemi-Smart 3000 (Vilber Lourmat, France) imaging device. Band intensities were quantified by Image J Java-based image processing program and normalized by comparison to [beta]-actin/TFIIB.

Statistical analysis

All experiments were repeated at least three times. Results are expressed as means [+ or -] SEM. Statistical analysis was performed using ANOVA followed by LSD post hoc comparisons. Differences were considered to be significant at P < 0.05.


Effect of atrovirinone on cytokine secretion

Fig. 2 shows the effect of atrovirinone upon secretion of cytokines. Atrovirinone demonstrated a dose-dependent inhibitory effect upon IL-1[beta] secretion (Fig. 2a). However despite significant inhibition atrovirinone did not strongly inhibit IL-1[beta] secretion and none of the doses used could induce an inhibitory percentage of more than 50%, therefore the [IC.sub.50] was not able to be calculated. The inhibitory effect upon IL-6 secretion was more pronounced with an [IC.sub.50] of 5.29 [+ or -] 0.28 [micro]M (Fig. 2b). Atrovirinone demonstrated a slight trend of enhancement of IL-10 secretion whereby the highest dose caused significant increase in secretion (Fig. 2c).


Atrovirinone inhibits both iNOS and COX-2 expression

Immunoblots demonstrated that iNOS protein was absolutely undetectable in inactive cells, but appeared in high amounts following induction. Atrovirinone displayed a significant dose-dependent down-regulatory effect upon iNOS protein expression (Fig. 3a). Incubation of cells with IFN-[gamma]/LPS for 18 hours led to the expression of COX-2 protein. However, when atrovirinone was incorporated with the inducers in the culture media, COX-2 expression decreased in a dose-dependent fashion (Fig. 3b).


Atrovirinone inhibits nuclear translocation of p65

Since the NF-[kappa]B pathway is one of the major signalling pathways leading to the activation of cytokine, iNOS and COX genes we evaluated the effect of atrovirinone upon the cellular distribution of the p65NF-[kappa]B protein. Fig. 4 shows that atrovirinone prevented nuclear translocation of cytosolic p65NF-[kappa]B since increasing amounts of p65NF-[kappa]B were retained in the cytosol in a dose-dependent manner (Fig. 4a) and correspondingly decreasing amounts were found in the nuclear extract (Fig. 4b). These results suggest that atrovirinone may be interfering with the dissociation of I-[kappa]B from the NF-[kappa]B/I-[kappa]B cytosolic complex, hence inhibiting nuclear translocation of p65NF-[kappa]B.


Atrovirinone inhibits phosphorylation of I-[kappa]B[alpha]

Since the nuclear translocation of p65NF-[kappa]B is regulated via phosphorylation of its inhibitor I-[kappa]B[alpha], we decided to determine whether atrovirinone had any effect upon the phosphorylation of this molecule. Fig. 5a shows a dose-dependent accumulation of I-[kappa]B[alpha] in IFN-[gamma]/LPS- induced cells. Fig. 5b confirms that the accumulation of 1-[kappa]B[alpha] was due to a dose-dependent inhibition of phosphorylation by atrovirinone and subsequent degradation of I-[kappa]B[alpha].


Atrovirinone inhibits phosphorylation of p38 and ERK1/2

To determine the effect of atrovirinone upon MAPK expression we examined the effect of atrovirinone in comparison to the MEK inhibitor, PD98059 and the p38 inhibitor, SB203580. Stimulation of RAW 264.7 cells with IFN-[gamma]/LPS elicited and increased the level of phosphorylation of both p38 and ERK1/2 (Figs. 6a and b), with untreated control cells displaying only a weak signal. Atrovirinone suppressed the expression of phosphorylated forms of p38 and ERK1/2 significantly. Non-phosphorylated forms were not altered by treatments. The inhibitory effect upon phospho-ERK1/2 was extremely strong.



In a previous communication we have demonstrated that atrovirinone inhibits the secretion of NO, TNF-[alpha] and [PGE.sub.2] in RAW 264.7 cells and human blood (Syahida et al. 2006). We now demonstrate that in addition to those mediators atrovirinone also inhibits the secretion of IL-1[beta] and IL-6 and enhances the secretion of IL-10 at the highest non-toxic dose used. These inhibitory effects are due to disruption of the NF-[kappa]B and MAPK signalling pathways. The mechanism involves the inhibition of p65NF-[kappa]B nuclear translocation due to prevention of I-[kappa]B[alpha] phosphorylation. During macrophage activation, nuclear translocation of NF-[kappa]B is preceded by phosphorylation of IKK by NF-[kappa]B inducing kinase (NIK) (Kang et al. 2003; Jiang et al. 2003), followed by rapid phosphorylation of I-[kappa]B by IKK (Jijon et al. 2004) and degradation of phosphorylated I-[kappa]B by the proteosome complex (Liang et al. 1999). Phosphorylation of I-[kappa]B is generally regarded as the rate-limiting step in the liberation of NF-[kappa]B resulting in transcription of specific pro-inflammatory genes [Jijon et al. 2004; Liang et al. 1999; Griscavage et al. 1996; Pan et al. 2000]. Thus, the ability of a compound to inhibit the phosphorylation and degradation of I-[kappa]B[alpha] will cause the accumulation of both I-[kappa]B[alpha] and p65NF-[kappa]B in the cytosol, thereby reducing the amount of p65NF-[kappa]B that can enter the nucleus. The influence of atrovirinone on this pathway could be attributed to many factors; such as obstruction of IKK (Griscavage et al. 1996), proteasome (Liang et al. 1999), or upstream kinase activities; or down-regulation of kinase expression.

Additionally atrovirinone may exert its effects via a redox mechanism. It has been shown that activation of NF-[kappa]B by LPS and cytokines involves several redox systems (Lee et al. 2005; Okamoto et al. 1992). Reactive oxygen intermediates (ROI) are able to activate protein kinases that phosphorylate I[kappa]B. Furthermore, the oxidized form of NF-[kappa]B is reduced by thioredoxin, which is an important step in NF-[kappa]B activation. Benzoquinones are potent electrophiles and acceptors in the Micheal reaction and can readily react with thiol enzymes such as thioredoxin and glutathione in cells (Hayashi et al. 1993). Atrovirinone has significant inhibitory activity in intracellular oxidative stress (Syahida et al. 2006). It is therefore plausible to suggest that the benzoquinone moiety of atrovirinone acts as an intracellular antioxidant leading to inhibition of NF-[kappa]B pathway activation and subsequent suppression of inducible enzyme genes. Although Rel A/NF-[kappa]B1 (p65/p50) is the most commonly found heterodimer in activated cells, often referred to as the 'classic' NF-[kappa]B (Makarov, 2000), we focused our attention solely upon translocation of the p65 subunit since it is the rate limiting molecule as opposed to p50 which is constitutively expressed in the nucleus and has a low affinity for I-[kappa]B. Studies on the effect of benzoquinones upon iNOS in rat C6 glia cells have shown similar effects whereby NO inhibition and down regulation of iNOS mRNA expression were associated with inhibition of p65 but not p50 nuclear translocation (Brunmark and Cadenas, 1988).

Inhibition of the MAPK pathway may also lead to disruption of proinflammatory mediator synthesis. Closs et al. (Closs et al. 1996) showed that arginine is taken up into cells by cationic amino acid transporter (CAT)-2B systems, which are regulated by both ERK1/2 and p38 MAPK (Forsythe et al. 2001). Since arginine acts as a substrate for iNOS in the generation of NO it is possible that the inhibitory action upon MAPK may lead to reduced substrate availability and thus reduced NO synthesis. COX-2 synthesis has also been shown to be dependent upon ERK 1/2 and p38 thus it is conceivable that the inhibition of these signaling molecules results in the inhibition of COX-2 expression and PGE2 synthesis. There is ample evidence that demonstrates that both the MAPK and NF-[kappa]B pathways are responsible for the generation of proinflammatory cytokine synthesis. In particular p38 MAPK activates the synthesis of major proinflammatory cytokines (Kaminska, 2005). Our findings show that the inhibition of ERK 1/2 and p38 phosphorylation possibly accounts for the dose-dependent inhibition of cytokine synthesis. The differences in the extent of inhibition whereby atrovirinone seems to be more suppressive towards IL-6 secretion in comparison to the other cytokines remains to be elucidated.

In conclusion we have shown that atrovirinone, a naturally-occuring benzoquinone from the roots of G. atroviridis, strongly inhibits the synthesis of major proinflammatory mediators in RAW 264.7 cells via interruption of both the NF-[kappa]B and MAPK pathways. We have shown that atrovirinone acquires this effect by inhibiting the phosphorylation of I-[kappa]B[alpha], ERK 1/2 and p38, however further dissection of the mechanism may provide more accurate information regarding the precise target molecule involved.


We thank Ms Norazren Ismail and Mr Zulkhairi Zainol for excellent technical assistance. This investigation was financially supported by the Research University Grant Scheme (RUGS 04/01/07/0065RU), Universiti Putra Malaysia and Science Fund (02-01-04-SF00665), Ministry of Science, Technology & Innovation, Malaysia.


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Syahida, A., Israf, D.A., Permana, D., Lajis, N.H., Khozirah, S., Afiza, A.W., Khaizurin, T.A., Somchit, M.N., Sulaiman, M.R., Nasaruddin, A.A., 2006. Atrovirinone inhibits pro-inflammatory mediator release from murine macrophages and human whole blood. Immunol. Cell. Biol. 84, 1-9.

Abbreviations: NO. nitric oxide; [PGE.sub.2], prostaglandin [E.sub.2]; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; NF-[kappa]B, nuclear factor-[kappa]B; I-[kappa]B, inhibitory-[kappa]B; IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular-regulated kinase

* Corresponding author. Tel./fax: +603 8947 2337.

E-mail address: (D.A. Israf).

DA Israf (a), *, C.L. Tham (a), A. Syahida (b), N.H. Lajis (b), M.R. Sulaiman (a), A.S. Mohamad (a), Z.A. Zakaria (a)

(a) Department of Biomedical Science, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia

(b) Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Malaysia

doi: 10.1016/j.phymed.2010.02.006
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Author:Israf, D.A.; Tham, C.L.; Syahida, A.; Lajis, N.H.; Sulaiman, M.R.; Mohamad, A.S.; Zakaria, Z.A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Aug 1, 2010
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