Nitric oxide production inhibition and mechanism of phenanthrene ...

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2521–2525

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Nitric oxide production inhibition and mechanism of phenanthrene analogs in lipopolysaccharide-stimulated RAW264.7 macrophages Lian-qi Chen a,b, Xiao-fei Shen b, Bo-yang Hu b, Yuan Lin b, Ighodaro Igbe b,d, Cheng-gang Zhang c, Guo-lin Zhang b, Xiao-hong Yuan a,⇑, Fei Wang b,⇑ a

School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, China Key Laboratory of Natural Medicine and Clinical Translation, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China College of Chemistry and Materials, Sichuan Normal University, Chengdu, China d Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria b c

a r t i c l e

i n f o

Article history: Received 27 December 2015 Revised 7 March 2016 Accepted 25 March 2016 Available online 25 March 2016 Keywords: Phenanthrene Anti-inflammatory activity p38 IjBa Macrophage

a b s t r a c t Natural phenanthrene derivatives are considered to be important resource for the anti-inflammatory therapeutics, but their structure–activity relationship and mechanisms are still unknown. In this study we evaluated 20 synthesized phenanthrene analogs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Compounds 10, 11 and 17 were found to inhibit the production of nitric oxide (NO) with IC50 values of 37.26 lM, 5.05 lM and 20.31 lM, respectively. Compound 11 decreased LPS-induced expression of inducible NO synthase (iNOS), inhibited phosphorylation of p38 mitogen-activated protein kinase (MAPK) and serine/threonine kinase Akt. It also suppressed the phosphorylation and degradation of inhibitory kappa B-a (IjBa). Data obtained suggest that compound 11 exerts anti-inflammatory effects by inhibiting p38 MAPK and nuclear factor jB (NF-jB) pathways, which warrants further investigation as a new anti-inflammatory pharmaceutical tool. Ó 2016 Elsevier Ltd. All rights reserved.

Inflammation acts as a trigger and a complication in many human diseases, such as diabetes, cardiovascular diseases, cancer and sepsis. Lipopolysaccharide (LPS), a component of Gramnegative bacteria’s cell wall, can be recognized by toll-like receptor 4 (TLR4) and induce acute or chronic inflammatory responses, which leads to incidence of sepsis and other inflammatory diseases.1 Once LPS binds to TLR4, it will activate nuclear factor jB (NF-jB) pathway and MAPK pathways, leading to the expression of interleukins (ILs), nitric oxide (NO), interferons (IFNs) and some other inflammatory factors.2,3 Thus, crucial proteins in these inflammatory signaling pathways are potential targets in drug discovery for the new anti-inflammatory therapeutics. Phenanthrene derivatives are widely existed in plant kingdom and they are considered to be important resource for the discovery of new natural medicines because of their relatively simple structure but broad spectrum bio-activities, including antimicrobial, Abbreviations: LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor jB; NO, nitric oxide; TLR4, toll-like receptor 4; IL, interleukin; IFN, interferon; ERK, signal-regulated protein kinase; IKK, IjB kinase; iNOS, inducible NO synthase; IjBa, inhibitory kappa B-a; JNK, c-Jun N-terminal kinase. ⇑ Corresponding authors. E-mail addresses: [email protected] (X.-h. Yuan), [email protected] (F. Wang). http://dx.doi.org/10.1016/j.bmcl.2016.03.088 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

spasmolytic, anti-inflammatory, antiplatelet aggregation and antiallergic activities.4 Some natural phenanthrene derivatives isolated from Dendrobium nobile, Aristolochia manshuriensis and Eulophia ochreata have been reported to exhibit the anti-inflammatory effects with relatively unknown mechanism.5–7 1,2-Diphenylethylene, also named stilbene, shares similar structural properties with phenanthrene. Resveratrol is one of the most outstanding stilbenoid because of its well-known use as cardioprotective, anti-aging, anti-cancer and anti-inflammatory agents.8,9 Combretastatin A-4, another stilbene derivative, has been shown to be a potent inhibitor of the tubulin protein in preclinical trials for tumor vasculature treatment, and more effective and specific analogs are also being developed.10 Therefore, understanding of structure– activity relationship of phenanthrene derivatives and their mechanisms of action play an important role in the discovery of novel anti-inflammatory drugs. In our previous study, some phenanthrene derivatives isolated from Dendrobium denneanum exert were shown to exert their anti-inflammatory activity by inhibiting MAPK and NF-jB pathways.11 In the present study, we further evaluated a group of synthesized phenanthrene analogs (Fig. 1) to determine their structure–activity relationship with respect to their anti-inflammatory effects, and the purity and structural assignments of these

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Figure 1. The structures of 20 phenanthrene analogs.

synthesized compounds were determined by 1H NMR, 13C NMR, and MS spectral analysis (Analytical Data in Supplementary Material).12,13 Murine macrophage RAW264.7 cells were treated with 20 compounds at the concentration of 50 lM for the measurement of NO production and cell viability assay. As shown in Table 1, compounds 10, 11 and 17 were found to most potently inhibit the NO production induced by LPS without affecting the viability of RAW264.7 cells and the half maximal inhibitory concentrations (IC50) of these three compounds were 37.26 lM, 5.05 lM and 20.31 lM, respectively (Fig. 2). Our results showed

Table 1 Effects of 20 compounds on LPS-induced NO production and cell viability

*

Compounds

NO production at 50 lM (% of LPS)

Cell viability (% DMSO)

DMSO LPS BAY 11–7082* No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 No. 11 No. 12 No. 13 No. 14 No. 15 No. 16 No. 17 No. 18 No. 19 No. 20

3.6 100.0 1.2 84.6 48.8 79.0 109.2 75.0 53.1 86.0 106.9 83.0 45.5 3.9 120.7 46.7 64.3 45.3 78.2 39.8 92.7 108.8 98.7

100 — — 76.5 99.3 56.1 88.0 101.2 106.9 122.6 102.8 99.7 66.7 114.4 106.5 96.7 120.6 101.2 100.9 94.7 111.1 102.4 91.1

BAY 11-7082 used at the final concentration of 10 lM.

that the anti-inflammatory effects of these compounds were highly dependent on their core structure and the substituents. Those compounds (2, 6, 10, 11, 15 and 17) that exhibited anti-inflammatory activity were phenanthrene and cis-stilbene derivatives and possessed two benzene rings which were at the same side of the center conjunct double bond. In comparing the structure–activity relationship between compounds 10, 11 versus 4 and compounds 2, 15 versus 5; the introduction of 1,3-dioxolane on the benzene ring dramatically decreased the anti-inflammatory effect in both compounds 4 and 5, whereas the introduction of methoxy groups on the benzene rings increased the antiinflammatory effect in compounds 10, 11, 2 and 15. The methyl formate group at the center conjunct double bond (compounds 10, 11, 2, and 15) also enhanced anti-inflammatory activity compared with those compounds with formic acid group (compounds 4 and 16). Previous studies4–7 has also shown that natural phenanthrene derivatives with potent anti-inflammatory effect generally have methoxy groups on the benzene rings, thus suggesting that the core structure and the methoxy groups on the benzene rings play important roles in their anti-inflammatory functions. To further study the mechanism of these compounds-mediated inhibition of NO production, the expression of iNOS, the major enzyme catalyzing the formation of NO,14 was examined by using compound 11, methyl (E)-2,3-bis(3,4-dimethoxyphenyl)acrylate. LPS treatment significantly increased the mRNA expression of iNOS, which could be inhibited by BAY 11-7082, a NF-jB inhibitor, used as a positive control (Fig. 3A).15 Pretreatment with compound 11 significantly blocked LPS-induced iNOS expression in a concentration-dependent manner. Consistent with its inhibitory effect on the mRNA expression of iNOS, compound 11 also significantly decreased the protein expression of iNOS in a concentrationdependent manner (Fig. 3B). These results indicated that compound 11 could decrease NO production in LPS-activated RAW264.7 cells by inhibiting iNOS expression. Other pro-inflammatory cytokines, such as IL-1b and IL-6, are also produced in LPS-activated macrophages.16 We therefore


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Figure 2. Effect of compounds 10, 11 and 17 on NO production. (A–C) The structures of compounds 10, 11 and 17, respectively. (D–F) RAW264.7 cells were pretreated with three compounds at various concentrations for 2 h, then stimulated with LPS (0.5 lg/mL) for 24 h. NO production was measured by Griess Reagent. All the data points represent means ± SD of triplicate times.

Figure 3. Inhibitory effect of compound 11 on LPS-induced iNOS expression. (A) RAW264.7 cells were pre-treated with compound 11 at the indicated concentrations or 10 lM BAY 11-7082 (BAY), respectively, for 2 h, then stimulated with LPS (0.5 lg/mL) for 24 h. Total RNAs were extracted and the mRNA levels of iNOS were measured by qRT-PCR, GAPDH served as the internal control. **p < 0.01, ***p < 0.001 compared to LPS treatment alone. (B) RAW264.7 cells were seeded in 6-well plate and pretreated with compound 11 (1, 10, 50 lM) and BAY 11-7082 (10 lM) for 2 h before stimulation with LPS (0.5 lg/mL) for 24 h. Cell lysates were immunoblotted with anti-iNOS antibody. GAPDH staining is shown as a loading control.

examined the effect of compound 11 on expression of IL-1b and IL-6. The mRNA expression of these two cytokines was significantly increased after stimulation by LPS but where potently inhibited by compound 11 in a concentration-dependent manner (Fig. 4A and B). This inhibition was more potent than the NF-jB

inhibitor BAY 11-7082 (10 lM). The protein expression of IL-6 was also inhibited by compound 11 (Fig. 4C). These results indicated that compound 11 inhibits the expression of proinflammatory mediators at the transcriptional level. In unstimulated RAW264.7 cells, the transcriptional factor NF-jB is inhibited by binding with its inhibitory protein IjB as a complex. Once upstream signals activate IjB kinases (IKKs), the activated IKKs can phosphorylate IjB and lead to its ubiquitylation and subsequent degradation. The dissociated NF-jB then translocate into the nucleus and start the transcription of inflammatory factors.17 In this study, we examined the effect of compound 11 on IjBa, the key factor in the mediation of NF-jB activation. The phosphorylation and degradation of IjBa occurred in 5 min after RAW264.6 cells exposure to LPS, which was significantly inhibited by compound 11 (Fig. 5A). The serine/threonine kinase Akt is reported to act as an activator of IKKs, leading to the degradation of IjB.18 Hence, the effect of compound 11 on Akt was then examined. Stimulation of RAW264.7 cells with LPS resulted in the increased activation of Akt, which was abolished by compound 11 treatment (Fig. 5B). These results suggest that compound 11 may decrease the expression of pro-inflammatory cytokines by inhibiting the activation of NF-jB mediated by phosphorylation of IjBa and Akt. MAPK pathway is involved in multiple cellular responses, including the inflammatory reactions.19 In mammals, MAPKs can be grouped into three subfamilies: the extracellular signalregulated protein kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 MAPKs.20 The activation of TLR4 by LPS can lead to the phosphorylation of MAPKs. Therefore, we examined the effect of compound 11 on these MAPKs. As shown in Figure 6, ERK1/2, JNK1/2 and p38 was phosphorylated in RAW264.7 cells after the treatment with LPS. Pretreatment with compound 11 significantly inhibited the phosphorylation of p38 MAPK. In contrast, the phosphorylation of ERK1/2 and JNK1/2 were not affected by compound 11. Previously we found that the natural phenanthrene derivatives with anti-inflammatory effect were found to inhibit the NF-jB pathway and JNK and p38 MAPK pathways.11 Another three similar

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Figure 4. Compound 11 inhibited the expression of LPS-induced inflammatory factors. RAW264.7 cells were seeded in 6-well plates and pretreated with compound 11 (1, 10, 50 lM) and BAY 11-7082 (BAY, 10 lM) for 2 h before stimulation with LPS (0.5 lg/mL) for 24 h. The mRNA levels of IL-1b (A) and IL-6 (B) were measured by qRT-PCR with GAPDH as an internal control. (C) The concentrations of IL-6 in the supernatants were measured by ELISA. Data shown are the mean ± standard deviation (SD) of three experiments performed in triplicate. **p < 0.01 and ***p < 0.001 compared to LPS treatment alone.

Figure 5. Inhibitory effect of compound 11 on LPS-activated NF-jB pathway. (A) RAW264.7 cells were pre-treated with compound 11 before stimulated with LPS (0.5 lg/mL) for 5 min or 30 min (phosphorylated-Akt and Akt). (A) Cell lysates were immunoblotted with anti-phospho-IjBa and anti-IjBa antibodies. GAPDH staining is shown as a loading control. (B) RAW264.7 cells were pre-treated with compound 11 before stimulated with LPS (0.5 lg/mL) for 30 min. Cell lysates were immunoblotted with anti-phospho-Akt and anti-Akt antibodies. GAPDH staining is shown as a loading control.

Figure 6. Effects of compound 11 on MAPK pathway in LPS-induced RAW264.7 cells. RAW264.7 cells were treated with compound 11 at the indicated concentrations for 2 h, and then stimulated with LPS (0.5 lg/mL) for 30 min. Cell lysates were immunoblotted with the indicated antibody, respectively. The total ERK1/2, JNK, or p38 staining is shown as a loading control, respectively.

phenanthrene derivatives from Dendrobium nobile also exert the anti-inflammatory effects by inhibiting NF-jB pathway and ERK, JNK and p38 MAPK pathways.5 Phenanthrene-based tylophorine1 could inhibit the lung cancer cell growth by suppressing the NF-jB pathway and Akt phosphorylation.21 Denbinobin inhibits TNF-a and PMA-induced NF-jB activation and induce apoptosis via inducing intracellular reactive oxygen species (ROS) generation and sustained activation of the ERK1/2, p38 and JNK1/2 MAPKs in human leukemic cells.22 These findings suggest that NF-jB pathway is the main target that phenanthrene and cis-stilbene derivatives interfere with. Their effects on the subfamilies of MAPK signaling pathway are different even though they have methoxy groups in their benzene rings. The inhibition of p38 MAPK resulted in the suppression of transcriptional factors AP-1 and NF-jB, which are necessary for expression of pro-inflammatory cytokines.23 Compound 11 inhibited activation of p38 MAPK without affecting ERK and JNK MAPKs. This suggests that compound 11 maybe specific in exerting its anti-inflammatory function and has reduced off-target effect on other cell physiological

activities when compared with other anti-inflammatory compounds.11,24–26 This resulting observation warrants the development of compound 11 as a potential anti-inflammatory agent with lesser side-effects. In conclusion, we evaluated the anti-inflammatory effect of 20 synthesized phenanthrene analogs in LPS-stimulated RAW264.7 cells, and the structure–activity relationship analysis revealed that the core structure and the methoxy groups on the benzene rings were the most important elements for their anti-inflammatory functions. The compound 11, methyl (E)-2,3-bis(3,4-dimethoxyphenyl)acrylate, produced the most effective inhibition of NO production with IC50 value of 5 lM. In determining their mechanisms of action, compound 11 decreased the expression of pro-inflammatory cytokines by inhibiting IjBa/Akt/p38-mediated activation of NF-jB pathway. Our study gives the reference information to the development of this compound and it analogs into anti-inflammatory agents. Compound 11 warrants further investigation as a new pharmaceutical tool for the prevention and treatment of inflammatory diseases.


Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 91413108, 21272189, 21561142003), Sichuan Youth Science & Technology Foundation (No. 2014JQ0028), the West Light Foundation of the Chinese Academy of Sciences. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03. 088. References and notes 1. 2. 3. 4. 5.

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