Anacardic acid suppresses fibroblast-like synoviocyte proliferation and invasion and ameliorates collagen-induced arthritis in a mouse model

Cytokine 111 (2018) 350–356

Contents lists available at ScienceDirect

Cytokine journal homepage: www.elsevier.com/locate/cytokine

Anacardic acid suppresses fibroblast-like synoviocyte proliferation and invasion and ameliorates collagen-induced arthritis in a mouse model Guo-hui Yanga,1, Chi Zhangb,1, Nan Wanga, Yu Menga, Yi-sheng Wangb, a b

T

⁎

Department of Emergency Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Akt Fibroblast-like synoviocyte microRNA Rheumatoid arthritis

Anacardic acid, which is abundant in nutshell of Anacardium occidentale, has multiple pharmacological activities. In this study, we examined the therapeutic potential of anacardic acid in treating rheumatoid arthritis (RA). We explored the effects of anacardic acid on collagen-induced arthritis (CIA) in mice and on the proliferation and invasion of RA fibroblast-like synoviocytes (RA-FLSs). The underlying molecular mechanism was investigated. Anacardic acid treatment markedly suppressed paw swelling, joint destruction, and arthritis scores in CIA mice. The serum levels of tumor necrosis factor alpha (TNF- α) and interleutkin-1beta (IL- 1β) were significantly lowered by anacardic acid. In vitro assays demonstrated that anacardic acid impaired the proliferation and invasion abilities of RA-FLSs in the presence of TNF- α or IL- 1β. Western blot analysis revealed the reduction of Akt protein expression and phoshporylation in RA-FLSs by anacardic acid. However, the mRNA level of Akt remained unchanged. Anacardic acid treatment significantly increased the expression of miR-633 in RA-FLSs. Akt was identified as a novel target of miR-633. Overexpression of miR-633 significantly inhibited the proliferation and invasion of RA-FLSs, which was rescued by enforced expression of Akt. Depletion of miR-633 prevented anacardic acid-mediated suppression of proliferation and invasion of RA-FLSs, which was accompanied by increased expression of Akt protein. In conclusion, anacardic acid may serve as a promising agent in the treatment of RA.

1. Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation, cartilage destruction, and bone erosion [1]. Inflammatory cytokines especially tumor necrosis factor alpha (TNF-α) and interleutkin-1beta (IL-1β) play an essential role in the development of synovial inflammation in RA [2]. Hyperplasia of fibroblast-like synoviocytes (FLSs) is causally linked to the pathogenesis of RA [3]. RA-FLSs exhibit tumor- like aggressive phenotype including overproliferation and invasion [4]. RA-FLSs can produce inflammatory mediators and matrix degrading enzymes, causing cartilage and bone destruction. Therefore, FLSs serve as an important therapeutic target for the treatment of RA. microRNAs (miRs) are a large class of small non-coding regulatory RNAs and repress the expression of multiple genes via cleavage of target mRNAs or inhibition of protein translation [5]. miRs typically interact with the 3′-untranslated region (UTR) of target mRNAs. Various biological processes are regulated by miRs, such as proliferation, survival,

differentiation, invasion, and metastasis [6,7]. Aberrantly expressed miRs have been found in patients with RA, suggesting their involvement in this disease [8]. It has been documented that miR-125b induces inflammation in RA by activation of NF-κB pathway [9]. Another study reported that miR-192 inhibits the proliferation of RA-FLSs by repressing caveolin 1 [10]. Anacardic acid, which is abundantly detected in nutshell of Anacardium occidentale, is a general term applied to a family of 6-alkyl salicyclic acids having varying saturation degrees in the 15-carbon alkyl chain [11]. Anacardic acid is attracting increasing attention due to its multiple pharmacological activities including anticancer [12], cardioprotective [13], and antibacterial [14] properties. Anacardic acid acts as an inhibitor of histone acetyltransferase and shows the ability to potentiate apoptosis in tumor cells [15]. Several signaling pathways such as NF-κB [15], MAPK [16], and Akt [17] are regulated by anacardic acid. Since these signaling pathways are involved in the pathogenesis of RA [18], we hypothesized that anacardic acid may have therapeutic effects on RA. In this study, we

⁎

Corresponding author. E-mail address: [email protected] (Y.-s. Wang). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.cyto.2018.09.008 Received 1 June 2018; Received in revised form 1 September 2018; Accepted 13 September 2018 Available online 28 September 2018 1043-4666/ © 2018 Elsevier Ltd. All rights reserved.

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

Fig. 1. Anacardic acid attenuates collagen-induced inflammatory arthritis in mice. (A) Assessment of the effect of anacardic acid on the severity of arthritis induced by collagen in mice. *P < 0.05 vs. the CIA group. (B) Shown are representative photographs of knee joint sections stained with H&E. Arrow indicates cellular infiltration. Scale bar = 50 μm. (C) Histological scores were determined for each group. *P < 0.05 vs. the CIA group. (D) Measurement of serum TNF-α and IL-1β levels. *P < 0.05. AA = anacardic acid.

2.2. Histological assessment

explored the effects of anacardic acid on collagen-induced arthritis (CIA) in vivo and on the biological behaviors of RA-FLSs in vitro. The molecular mechanism underlying in the action of anacardic acid was examined.

Ankle joints were routinely fixed, decalcified in 5% formic acid, and embedded in paraffin. Tissue sections were subjected to hematoxylin and eosin (H&E) staining and evaluated in a blind manner by experienced pathologists. The degree of synovitis, pannus formation, bone erosion, and cartilage destruction was scored according to the following criteria: 0, normal; 1, mild changes; 2, moderate changes, and 3, severe changes.

2. Materials and methods 2.1. Mice and CIA model Animal studies were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Zhengzhou, China). Male DBA/1J mice (6 week old) were obtained from Laboratory Animal Resources, Chinese Academy of Sciences (Shanghai, China). For establishment of CIA model [19], mice were immunized with 100 μg chicken type II collagen (Sigma-Aldrich, St. Louis, MO, USA), which was emulsified with an equal volume (0.1 mL) of complete Freund’s adjuvant supplemented with 4 mg/mL heat-killed mycobacterium (Chondrex, LLC, Seattle, WA, USA). A booster immunization was performed on day 21 with 100 μg chicken type II collagen emulsified with an equal volume of incomplete Freund’s adjuvant. To determine therapeutic effects, anacardic acid (≥97% in purity; Tocris, Minneapolis, MN, USA) was administered via intraperitoneal injection at a dose of 1 or 5 mg/kg body weight twice per week for 3 weeks, starting from the day of booster immunization. Arthritis severity was evaluated twice per week in a blinded manner according to the following criteria [19]: 0 = no signs; 1 = mild swelling of the wrist or ankle; 2 = moderate swelling extending from the ankle to the tarsal bones; 3 = moderate swelling extending from the ankle to the metatarsal joints; 4 = severe swelling of the wrist and ankle including all digits. On day 42, animals were sacrificed and serum was collected. The hindlimbs were dissected and subjected to histological analysis.

2.3. Serum cytokine levels quantified by ELISA Serum samples were collected from experimental mice. The serum levels of TNF-α and IL-1β were determined using mouse specific ELISA kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. 2.4. Cell culture and treatment FLSs from RA patients were purchased from Cell Applications Inc. (San Diego, CA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich). The medium was changed every 3 days. For anacardic acid treatment, RA-FLSs were incubated for 48 h with various concentrations of anacardic acid (5, 30, and 60 μM), in the presence of recombinant human TNF-α (10 ng/mL; R&D Systems) or IL1β (10 ng/mL; R&D Systems) [20]. In some experiments, MK-2206 (1 μM; Selleck Chemicals, Houston, TX, USA) was added together with TNF-α (10 ng/mL) or IL-1β (10 ng/mL) to the culture medium and incubated for 48 h before measurement of cell proliferation and invasion. 2.5. EdU incorporation assay RA-FLSs were seeded onto 24-well plates (2 × 104 cells/well) and 351

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

upper chamber. The culture medium containing 10% FBS was added to the lower chamber. After incubation for 48 h, the invaded cells in the lower chamber were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The number of invaded cells was determined under a microscope. 2.7. Western blot analysis Cells were lysed in lysis buffer containing phophotase and protease inhibitors (Pierce Biotechnology, Rockford, IL, USA) and cleaned by centrifugation at 14,000g for 15 min at 4 °C. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies against phospho-Akt (Ser473) (1:500), Akt (1:1,000), phospho-p38 (1:500), p38 (1:1,000), phospho-ERK (1:500), ERK (1:1,000), phosphoSTAT3 (1:500), STAT3 (1:1,000), phospho-NF-κB p65 (1:500), NF-κB p65 (1:1,000), phospho-mTOR (1:200), mTOR (1:1000), phospho-eIF4E (1:500), eIF4E (1:1000), and β-actin (1:2,000). All of the antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were then incubated with horseradish peroxidaseconjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:5,000). Protein bands were visualized using the enhanced chemiluminescence technique (ECL, Amersham Biosciences, Little Chalfont, UK) and quantified using Quantity One software (BioRad Laboratories, Hercules, CA, USA). 2.8. Real-time PCR analysis Total RNA was extracted using TRIzol (Sigma- Aldrich) and reverse transcribed to cDNA using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories). Quantitative real-time PCR analysis of the Akt mRNA level was performed using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) with specific primers described in [21]. The expression of Akt mRNA was normalized to that of β-actin. For quantification of miR-633 levels, the TaqMan MicroRNA Assay kit (Applied Biosystems, Carlsbad, CA, USA) was used. U6 was used as a normalization control. 2.9. Plasmids and cell transfection Fig. 2. Anacardic acid suppresses the proliferation and invasion of RA-FLSs. RA-FLSs were treated with different concentrations of anacardic acid in the presence of TNF- α or IL- 1β and tested for proliferation and invasion. (A) EdU incorporation assay was used to determine cell proliferation. (B) Transwell invasion assay was performed to assess cell invasion. Representative images show the proliferation and invasion of TNF- α- treated cells. *P < 0.05 vs. control cells without anacardic acid treatment. AA = anacardic acid.

miR-633 mimic, anti-miR-633, and negative control oligonucleotides were purchased from Thermo Fisher Scientific (Chicago, IL, USA) and transfected at a concentration of 40 nM using Lipofectamine 3000 (Thermo Fisher Scientific). A fragment of the Akt 3′-UTR carrying the putative miR-633 binding site was amplified by PCR and inserted into the pGL3 luciferase reporter vector (Promega, Madison, WI, USA). Mutation of the miR-633 binding site was achieved using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). A fragment containing the Akt-encoding region without its 3′UTR was amplified by PCR and cloned into pcDNA3.1(+) vector. In some experiments, the Akt-expressing construct was co-transfected together with miR-633 mimic into RA-FLSs.

treated with TNF-α (10 ng/mL) or IL-1β (10 ng/mL) for 48 h. Afterwards, cells were incubated with 20 μM EdU (Sigma-Aldrich) for 2 h and fixed with 4% paraformaldehyde. The cells were permeabilized with 0.1% Triton X-100 for 10 min and incubated with the reaction cocktail (Tris–HCl, pH 8.5, 100 mM; CuSO4, 1 mM; fluorescent azide, 100 mM; ascorbic acid, 100 mM) for 30 min. After washing, the cells were counterstained with Hoechst 33,342 (5 mg/mL). The EdU-positive cells were examined under a microscope, and at least 300 cells were counted per well.

2.10. Luciferase reporter assay HEK293T cells purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) were co-transfected with Akt 3′UTR reporters and miR-633 mimic or control miRNA together with the Renilla luciferase-encoding plasmid pRL-TK (Promega). Forty-eight hours later, cells were lysed and luciferase activities were measured using the Dual-Luciferase Assay System (Promega) following the manufacturer’s protocol. The activity of firefly luciferase was normalized to that of Renilla luciferase.

2.6. Transwell invasion assay Matrigel-coated Transwell chambers (BD Biosciences, San Jose, CA, USA) were used in Transwell invasion assays. In brief, cells suspended in serum-free medium containing different concentrations of anacardic acid and TNF-α (10 ng/mL) or IL-1β (10 ng/mL) were seeded into the 352

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

Fig. 3. Anacardic acid represses Akt expression in RA-FLSs by upregulating miR-633. (A) Western blot analysis of indicated proteins in RA-FLSs after treatment with anacardic acid. Numbers indicate normalized protein levels. (B) Real-time PCR analysis of the mRNA expression level of Akt. N.S. means no significance relative to control cells without anacardic acid treatment. (C) Real-time PCR analysis of miRNA levels in RA-FLSs treated with vehicle or anacardic acid. *P < 0.05 vs. vehicle-treated cells. N.S. indicates no significance. (D) Luciferase reporter assay performed in HEK293T cells shows that transefection with miR633 mimic significantly suppressed the expression of the reporter gene harboring the 3′-UTR of Akt. (E) Left, Western blot analysis of Akt protein expression in RA-FLSs transfected with miR-633 mimic or negative control miR. Right, real-time PCR analysis of Akt mRNA levels. *P < 0.05; N.S. indicates no significance.

proliferation in RA-FLSs. Moreover, the anti-proliferative effect of anacardic acid was in a concentration-dependent manner. Transwell invasion assays further demonstrated that TNF- α- and IL- 1β- induced cell invasion was impaired in the presence of anacardic acid (Fig. 2B).

2.11. Statistical analysis Data are expressed as the mean ± standard deviation of three independent experiments. The student’s t-test was used to determine differences between two groups and one-way analysis of variance followed by Dunnett’s post hoc test was used for multiple comparisons. P < 0.05 was considered statistically significant.

3.3. Anacardic acid represses Akt expression in RA-FLSs by upregulating miR-633

3. Results

To determine the mechanism underlying the activity of anacardic acid, we examined several signaling pathways involved in the progression of RA. Western blot analysis demonstrated that anacardic acid treatment significantly suppressed the protein expression and phosphorylation of Akt (Fig. 3A), but exerted no effect on the phosphorylation of p38, ERK, STAT3, and NF-κB in RA-FLSs (data not shown). Examination of mTOR and eIF4E, two downstream genes regulated by Akt [22] showed that anacardic acid treatment did not alter the expression and phosphorylation of mTOR and eIF4E (Fig. 3A), suggesting that other Akt target genes likely mediate the activity of anacardic acid. Real-time PCR analysis revealed that the mRNA expression level of Akt was not altered by anacardic acid treatment (Fig. 3B). These data suggest that anacardic acid inhibits the expression of Akt at the posttranscriptional level. Next, we tested if induction of key miRs is responsible for the downregulation of Akt by anacardic acid. Bioinformatic analysis using the miRDB algorithm predicted 22 miRs with the potential to target Akt mRNA, including miR-1249-5p, miR-656-3p, miR-149-3p, miR-633, miR-302e, and miR-548a (data not shown). Anacardic acid-treated RAFLSs showed 4.8-fold higher expression levels of miR-633 than control cells (P < 0.05; Fig. 3C). However, the other miRs tested including miR-1249-5p, miR-656-3p, miR-149-3p, miR-302e, and miR-548a remained unchanged. To validate the activity of miR-633 to

3.1. Anacardic acid attenuates inflammatory arthritis in mice Administration of anacardic acid markedly diminished the severity of arthritis in mice exposed to collagen (Fig. 1A). The high dose of anacardic acid (5 mg/kg body weight) resulted in a more profound amelioration of disease than a lower dose of 1 mg/kg body weight. As revealed by histopathology (Fig. 1B), CIA mice displayed severe synovial inflammation, pannus formation, cartilage damage, and bone erosion. Anacardic acid treatment led to a remarkable improvement in histopathological findings (Fig. 1B) and scores (Fig. 1C). Analysis of inflammatory mediators revealed that the levels of TNF-α and IL-1β were significantly higher in sera from CIA mice than those from control mice (Fig. 1D). The elevation in the inflammatory cytokines was significantly attenuated in anacardic acid-treated groups (Fig. 1D). 3.2. Anacardic acid suppresses the proliferation and invasion of RA-FLSs Since RA-FLSs are a key player in the pathogenesis of RA [3], we sought to explore the effect of anacardic acid on the behaviors of RAFLSs. As determined by EdU incorporation assays (Fig. 2A), anacardic acid treatment significantly blocked TNF- α- and IL- 1β- induced 353

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

Fig. 4. miR-633 overexpression suppresses the proliferation and invasion of RA-FLSs by targeting Akt. RA-FLSs were transfected with miR-633 mimic alone or together with the Akt-expressing plasmid or empty vector and examined for proliferation (A) and invasion (B). Inserts in (A) are representative blots for Akt expression detected in RA-FLSs transfected with the Akt-expressing plasmid or empty vector. (C and D) RA-FLSs were treated with MK-2206 (1 μM) in the presence of TNF-α or IL-1β for 48 h and assayed for proliferation (C) and invasion (D). *P < 0.05.

activity against RA-FLSs is dependent on induction of miR-633. To address this question, we performed miR-633 knockdown experiments. It was found that depletion of miR-633 rendered RA-FLSs more resistant to anacardic acid-mediated suppression of proliferation (Fig. 5A) and invasion (Fig. 5B). In addition, downregulation of miR-633 reversed anacardic acid-mediated inhibition of Akt expression in RA-FLSs (Fig. 5C).

downregulate Akt, we performed luciferase reporter assays. The results showed that overexpression of miR-633 significantly reduced the activity of luciferase reporter harboring Akt 3′-UTR (Fig. 3D). Consistently, miR-633 overexpression significantly repressed the protein expression of Akt in RA-FLSs, without altering the levels of Akt transcripts (Fig. 3E). 3.4. miR-633 overexpression suppresses the proliferation and invasion of RA-FLSs by targeting Akt

4. Discussion

Similar to anacardic acid treatment, overexpression of miR-633 significantly restrained the proliferation (Fig. 4A) and invasion (Fig. 4B) of RA-FLSs. Rescue experiments revealed that enforced expression of Akt led to restoration of proliferation and invasion in miR633-overexpressing RA-FLSs (Fig. 4A and 4B). Pharmacological inhibition of Akt activity also caused a suppression of cell proliferation and invasion in RA-FLSs exposed to TNF- α and IL- 1β (Fig. 4C and 4D).

In this study, we showed that anacardic acid had antiarthritic activity in a mouse model. Administration of anacardic acid led to a dosedependent inhibition of synovial inflammation induced by collagen. TNF-α and IL-1β are important inflammatory mediators contributing to the pathogenesis of RA [23]. They can be produced by RA-FLSs and modulate the biological behaviors of RA-FLSs. Anacardic acid treatment significantly suppressed the serum levels of TNF-α and IL-1β in CIA mice, confirming its anti-inflammatory activity. Anacardic acid has shown anti-proliferative activity in prostate cancer [17] and breast cancer [24] cells. Anacardic acid can elicit apoptosis through a caspase-independent but rather p53-dependent manner [17,25,26]. However, in some types of cells such as ovarian

3.5. Knockdown of miR-633 confers resistance to anacardic acid-mediated suppressive activity Next, we explored whether anacardic acid-mediated suppressive 354

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

FLSs, but did not affect the mRNA abundance of Akt. In contrast to Akt signaling, the MAPK, STAT3, and NF-κB pathways were not altered by anacardic acid. These findings suggest that anacardic acid inhibits the expression of Akt at a post-transcriptional level. Previous studies have indicated that Akt signaling plays an important role in the pathogenesis of RA [18]. Inhibition of Akt leads to a reduction in RA-FLS proliferation and invasion [28,29]. Therefore, we suggest that anacardic acidmediated antiarthritic activity is, at least partially, ascribed to suppression of Akt signaling. miRs are well known as a regulator of target genes at the posttranscriptional level [5]. It has been documented that miR26a/b inhibits the translation of ERBB2 mRNA in estrogen receptor-positive breast cancer cells [30]. miR-9 was found to induce post-transcriptional inhibition of FOXO1 expression through binding to FOXO1 3′-UTR [31]. In this study, we identified miR-633 as a negative regulator of Akt in RA-FLSs. Overexpression of miR-633 inhibited the Akt protein expression but not the mRNA expression in RA-FLSs, indicating the posttranscriptional regulation of Akt. In addition, anacardic acid treatment significantly enhanced the expression of miR-633. These results suggest that miR-633 may serve as a functional mediator of anacardic acid by targeting Akt. In line with this hypothesis, ectopic expression of miR633 or pharmacological inhibition of Akt significantly inhibited the proliferation and invasion of RA-FLSs. In contrast, depletion of miR-633 or overexpression of Akt rescued RA-FLSs from anacardic acid-mediated growth and invasion suppression. Taken together, anacardic acid suppresses the aggressive phenotype of RA-FLSs largely by downregulating Akt via induction of miR-633. In conclusion, we provide first evidence for the potential antiarthritic activity of anacardic acid. Anacardic acid-mediated suppression of RA-FLS proliferation and invasion is causally linked to induction of miR-633 and downregulation of Akt expression. These findings suggest that anacardic acid may provide therapeutic benefits in the treatment of RA. References [1] Y. Zhuang, J. Liu, P. Ma, J. Bai, Y. Ding, H. Yang, Y. Fan, M. Lin, S. Li, Q. Hou, Tamarixinin A alleviates joint destruction of rheumatoid arthritis by blockade of MAPK and NF-κB Activation, Front Pharmacol. 8 (2017) 538. [2] L.J. Zhu, T.C. Yang, Q. Wu, L.P. Yuan, Z.W. Chen, M.H. Luo, H.O. Zeng, D.L. He, C.J. Mo, Tumor necrosis factor receptor-associated factor (TRAF) 6 inhibition mitigates the pro-inflammatory roles and proliferation of rheumatoid arthritis fibroblast-like synoviocytes, Cytokine 93 (2017) 26–33. [3] E. Choy, Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis, Rheumatology (Oxford) 51 (Suppl 5) (2012) v3–v11. [4] M. Huang, L. Wang, S. Zeng, Q. Qiu, Y. Zou, M. Shi, H. Xu, L. Liang, Indirubin inhibits the migration, invasion, and activation of fibroblast-like synoviocytes from rheumatoid arthritis patients, Inflamm. Res. 66 (5) (2017) 433–440. [5] P.Y. Yue, W.Y. Ha, C.C. Lau, F.M. Cheung, A.W. Lee, W.T. Ng, R.K. Ngan, C.C. Yau, D.L. Kwong, H.L. Lung, N.K. Mak, M.L. Lung, R.N. Wong, MicroRNA profiling study reveals miR-150 in association with metastasis in nasopharyngeal carcinoma, Sci. Rep. 7 (1) (2017) 12012. [6] H.L. Wang, R. Zhou, J. Liu, Y. Chang, S. Liu, X.B. Wang, M.F. Huang, Q. Zhao, MicroRNA-196b inhibits late apoptosis of pancreatic cancer cells by targeting CADM1, Sci. Rep. 7 (1) (2017) 11467. [7] L. Song, H.S. Lin, J.N. Gong, H. Han, X.S. Wang, R. Su, M.T. Chen, C. Shen, Y.N. Ma, J. Yu, J.W. Zhang, microRNA-451-modulated hnRNP A1 takes a part in granulocytic differentiation regulation and acute myeloid leukemia, Oncotarget. 8 (33) (2017) 55453–55466. [8] S. Sujitha, M. Rasool, MicroRNAs and bioactive compounds on TLR/MAPK signaling in rheumatoid arthritis, Clin. Chim. Acta. 473 (2017) 106–115. [9] B. Zhang, L.S. Wang, Y.H. Zhou, Elevated microRNA-125b promotes inflammation in rheumatoid arthritis by activation of NF-κB pathway, Biomed. Pharmacother. 93 (2017) 1151–1157. [10] S. Li, Z. Jin, X. Lu, MicroRNA-192 suppresses cell proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes by downregulating caveolin 1, Mol. Cell. Biochem. 432 (1–2) (2017) 123–130. [11] A. Hollands, R. Corriden, G. Gysler, S. Dahesh, J. Olson, S. Raza Ali, M.T. Kunkel, A.E. Lin, S. Forli, A.C. Newton, G.B. Kumar, B.G. Nair, J.J. Perry, V. Nizet, Natural product anacardic acid from cashew nut shells stimulates neutrophil extracellular trap production and bactericidal activity, J. Biol. Chem. 291 (27) (2016) 13964–13973. [12] D.J. Schultz, P. Muluhngwi, N. Alizadeh-Rad, M.A. Green, E.C. Rouchka, S.J. Waigel, C.M. Klinge, Genome-wide miRNA response to anacardic acid in breast cancer cells, PLoS ONE 12 (9) (2017) e0184471.

Fig. 5. Knockdown of miR-633 confers resistance to anacardic acid-mediated suppressive activity. RA-FLSs were transfected with anti-miR-633 or anti-control and treated with or without anacardic acid. After incubation for 48 h, the cells were tested for proliferation (A), invasion (B), and Akt expression (C). * P < 0.05.

cancer cells [27], anacardic acid exerts an opposite effect on cell proliferation. In this study, we demonstrated that anacardic acid treatment significantly restrained the proliferation of RA-FLSs induced by TNF-α and IL-1β. Moreover, the invasion capacity of RA-FLSs was reduced after exposure to anacardic acid. The in vitro data confirming the antiarthritic activity of anacardic acid. Anacardic acid has the capacity to orchestrate multiple signaling pathways in different biological contexts [15–17]. For instance, it was reported that anacardic acid inhibited both inducible and constitutive NF-κB activation in chronic myeloid leukemia cells, consequently downregulating a number of target genes including cyclin D1, cyclooxygenase-2, Bcl-2, Bcl-xL, cFLIP, cIAP-1, survivin, matrix metalloproteinase-9, intercellular adhesion molecule-1, and vascular endothelial growth factor [15]. Anacardic acid-mediated inhibition of NFκB and MAPK signaling contributes to the re-sensitization of TRAILresistant cancer cells to TRAIL [16]. Our data showed that anacardic acid treatment selectively inhibited the protein expression of Akt in RA-

355

Cytokine 111 (2018) 350–356

G.-h. Yang et al.

(7) (2007) 1261–1274. [23] W.X. Sun, Y. Liu, W. Zhou, H.W. Li, J. Yang, Z.B. Chen, Shikonin inhibits TNF-α production through suppressing PKC-NF-κB-dependent decrease of IL-10 in rheumatoid arthritis-like cell model, J. Nat. Med. 71 (2) (2017) 349–356. [24] D.J1. Schultz, N.S. Wickramasinghe, M.M. Ivanova, S.M. Isaacs, S.M. Dougherty, Y. Imbert-Fernandez, A.R. Cunningham, C. Chen, C.M. Klinge, Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation, Mol. Cancer Ther. 9 (3) (2010) 594–605. [25] S. Sukumari-Ramesh, N. Singh, M.A. Jensen, K.M. Dhandapani, J.R. Vender, Anacardic acid induces caspase-independent apoptosis and radiosensitizes pituitary adenoma cells, J. Neurosurg. 114 (6) (2011) 1681–1690. [26] J. Tan, B. Chen, L. He, Y. Tang, Z. Jiang, G. Yin, J. Wang, X. Jiang, Anacardic acid (6-pentadecylsalicylic acid) induces apoptosis of prostate cancer cells through inhibition of androgen receptor and activation of p53 signaling, Chin. J. Cancer Res. 24 (4) (2012) 275–283. [27] Y. Zhao, W.F. Gou, S. Chen, Y. Takano, H.C. Zheng, Anacardic acid enhances the proliferation of human ovarian cancer cells, Xiu YL, PLoS One. 9 (6) (2014) e99361. [28] S. Li, J.W. Chen, X. Xie, J. Tian, C. Deng, J. Wang, H.N. Gan, F. Li, Autophagy inhibitor regulates apoptosis and proliferation of synovial fibroblasts through the inhibition of PI3K/AKT pathway in collagen-induced arthritis rat model, Am. J. Transl. Res. 9 (5) (2017) 2065–2076. [29] V.S. Clarimundo, M. Farinon, R.T. Pedó, V.O.N. Teixeira, C. Nör, P.S. Gulko, R.M. Xavier, P.G. de Oliveira, Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway, Peptides 95 (2017) 57–61. [30] S. Tan, K. Ding, Q.Y. Chong, J. Zhao, Y. Liu, Y. Shao, Y. Zhang, Q. Yu, Z. Xiong, W. Zhang, M. Zhang, G. Li, X. Li, X. Kong, A. Ahmad, Z. Wu, Q. Wu, X. Zhao, P.E. Lobie, T. Zhu, Post-transcriptional regulation of ERBB2 by miR26a/b and HuR confers resistance to tamoxifen in estrogen receptor-positive breast cancer cells, J. Biol. Chem. 292 (33) (2017) 13551–13564. [31] D.Z. Liu, B. Chang, X.D. Li, Q.H. Zhang, Y.H. Zou, MicroRNA-9 promotes the proliferation, migration, and invasion of breast cancer cells via down-regulating FOXO1, Clin. Transl. Oncol. 19 (9) (2017) 1133–1140.

[13] C. Peng, X. Luo, S. Li, H. Sun, Phenylephrine-induced cardiac hypertrophy is attenuated by a histone acetylase inhibitor anacardic acid in mice, Mol. Biosyst. 13 (4) (2017) 714–724. [14] S. Muzaffar, B.B. Chattoo, Apoptosis-inducing factor (Aif1) mediates anacardic acidinduced apoptosis in Saccharomyces cerevisiae, Apoptosis 22 (3) (2017) 463–474. [15] B. Sung, M.K. Pandey, K.S. Ahn, T. Yi, M.M. Chaturvedi, M. Liu, B.B. Aggarwal, Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-kappaB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-kappaBalpha kinase, leading to potentiation of apoptosis, Blood 111 (10) (2008) 4880–4891. [16] M. Harsha Raj, B. Yashaswini, J. Rssler, B.P. Salimath, Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways, Apoptosis 21 (5) (2016) 578–593. [17] J. Tan, X. Jiang, G. Yin, L. He, J. Liu, Z. Long, Z. Jiang, K. Yao, Anacardic acid induces cell apoptosis of prostatic cancer through autophagy by ER stress/DAPK3/ Akt signaling pathway, Oncol. Rep. 38 (3) (2017) 1373–1382. [18] H.R. Kim, M.L. Cho, K.W. Kim, J.Y. Juhn, S.Y. Hwang, C.H. Yoon, S.H. Park, S.H. Lee, H.Y. Kim, Up-regulation of IL-23p19 expression in rheumatoid arthritis synovial fibroblasts by IL-17 through PI3-kinase-, NF-kappaB- and p38 MAPK-dependent signalling pathways, Rheumatology (Oxford) 46 (1) (2007) 57–64. [19] W. Tang, Y. Lu, Q.Y. Tian, Y. Zhang, F.J. Guo, G.Y. Liu, N.M. Syed, Y. Lai, E.A. Lin, L. Kong, J. Su, F. Yin, A.H. Ding, A. Zanin-Zhorov, M.L. Dustin, J. Tao, J. Craft, Z. Yin, J.Q. Feng, S.B. Abramson, X.P. Yu, C.J. Liu, The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice, Science 332 (6028) (2011) 478–484. [20] D. Chen, S. Zeng, M. Huang, H. Xu, L. Liang, X. Yang, Role of protein arginine methyltransferase 5 in inflammation and migration of fibroblast-like synoviocytes in rheumatoid arthritis, J. Cell Mol. Med. 21 (4) (2017) 781–790. [21] G.M. Zhang, C.Y. Bao, F.N. Wan, D.L. Cao, X.J. Qin, H.L. Zhang, Y. Zhu, B. Dai, G.H. Shi, D.W. Ye, MicroRNA-302a suppresses tumor cell proliferation by inhibiting AKT in prostate cancer, PLoS ONE 10 (4) (2015) e0124410. [22] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129

356