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. 2022 Mar 23;31(4):433–441. doi: 10.1007/s10068-022-01052-9

Resveratrol analogue, HS-1793, inhibits inflammatory mediator release from macrophages by interfering with the TLR4 mediated NF-κB activation

Wol Soon Jo 1, Sung Dae Kim 2, Soo Kyung Jeong 1,3, Su Jung Oh 1, Moon Taek ParK 1, Chang Geun Lee 1, Young- Rok Kang 1, Min Ho Jeong 3,✉
PMCID: PMC8994813  PMID: 35464242

Abstract

Resveratrol is known to have anti-inflammatory properties. However, high-dose resveratrol is required for optimal anti-inflammatory effects. HS-1793 is a derivative designed to be metabolically stable and more effective than resveratrol. We tested whether HS-1793 also has anti-inflammatory activity. HS-1793 effectively inhibited the mRNA and protein expression of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in macrophages. Therefore, the production of nitric oxide (NO) and prostaglandin E2 (PGE2) was significantly attenuated. In addition, HS-1793 completely suppressed the production of inflammatory cytokines enhanced by LPS treatment along with a decrease in Toll-like receptor 4 (TLR4) expression. At the same time, the expression of myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase 1 (IRAK1), and TNF receptor-associated factor 6 (TRAF6) signaling molecules and the nuclear translocation of nuclear factor kappa B (NF-κB)/p65 were also downregulated. We conclusively suggest that HS-1793 also exhibits anti-inflammatory properties by effectively inhibiting TLR4-mediated NF-κB activation.

Keywords: Resveratrol analogue, HS-1793, Macrophage, Anti-inflammation, TLR4·NF-κB

Introduction

Inflammation usually occurs in response to nonself, such as microbial infection and foreign body invasion, but also in response to self, such as cell transformation and tissue injury. Harm-recognizing cells secrete a variety of chemicals, triggering an immune system response. In general, inflammatory reaction proceeds acutely, and the whole process usually lasts for a few hours or days (Raziyeva et al., 2021). As the injurious agents causing the acute inflammatory response are eliminated, inflammatory mechanisms activate the processes of repair and regeneration. However, when inflammatory reaction dose not subside and rather gets turned up too high and lasts for a long time, the inflammation proceeds to a chronic course. Chronic inflammation eventually attacks and damages nearby healthy tissues and organs causing several diseases and conditions (Chen et al., 2018; Serhan, 2017). It can also underlie all major stages in cancer formation including initiation, progression, metastasis, and drug resistance (Nakamura and Smyth, 2017).

In order to reduce the risks associated with chronic inflammation, long-term and continuous therapeutic approaches are required to control and reduce inflammatory mechanism. Medicines such as glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs), while useful, cause other problems as a result of side effects when used for long periods. This requires new approaches to overcome inflammation mediated diseases and certain natural products could be safer yet still effective sources for the management of chronic inflammation. One of these agent is resveratrol (trans-3, 4', 5-tri-hydroxystilbene) found in various foods. The plants that source these foods make resveratrol as a defense mechanism against stress and injury such as fungal infection and UV radiation. The preventive and therapeutic effects of resveratrol in various diseases have been described in several studies, all of which are related by demonstrations of its significant anti-inflammatory activity (de Sá Coutinho et al., 2018). However, because resveratrol has inherent biologic drawbacks, the practical use of these prophylactic and therapeutic effects is limited. Resveratrol is extremely photosensitive and poorly water soluble (Vian et al., 2005). Furthermore, the absorbed resveratrol in the body is rapidly metabolized and excreted, making it difficult to maintain sufficient blood concentration (Walle, 2011). As a result of these limitations, high concentrations of resveratrol have commonly been used in most laboratory studies, and these are at levels which are not physiologically relevant. Accordingly, a novel synthetic resveratrol derivative [4-(6-hydroxy-2-naphthyl)-1,3-benzenediol, HS-1793] was developed that improves solubility and prevents degradation while maintaining the biological activity of the original molecule. Unlike resveratrol, HS-1793 has no unstable double bond, instead having an aromatic ring with two different hydroxyl positions. These structural changes make it less photosensitive, more stable metabolically, and more powerful than resveratrol (Song et al., 2007).

Bacterial LPS, the major component of Gram-negative bacterial cell wall, has been commonly used to establish an experimental model of inflammatory because it plays a crucial role in the innate immunity by stimulating many inflammatory cells. Among inflammatory cells, macrophages, in particular, are specialized cells that recognize, phagocytose and destroy bacteria and other harmful organisms, and play a critical role in all inflammatory processes from initiation to maintenance and resolution. One of the established cell lines that retain the characteristics of macrophages is the RAW264.7 cell line transformed by Abelson leukemia virus, and LPS-stimulated RAW264.7 macrophage is a commonly used model for investigating anti-inflammatory effect. In addition, as an important sensor for LPS, TLR4 activation and downstream signaling events that potentiate the production of inflammatory mediators and cytokines are also well established. Recently, the protective effect and molecular mechanism of resveratrol in LPS-induced inflammatory response has been reported (Tong et al., 2020). Resveratrol decreased the production of pro-inflammatory mediators while increasing the expression of anti-inflammatory mediator IL-10 through suppression of the TLR4-NF-kB/mitogen-activated protein kinases (MAPKs)/interferon regulatory factor 3 (IRF3) signaling cascades in LPS-stimulated RAW264.7 cells. HS-1793, a synthetic derivative of resveratrol, is also presumed to have a similar function, however, the evaluation of anti-inflammatory effect following HS-1793 treatment has not been performed. Therefore, in this study, we have investigated the inhibitory effect and molecular mechanism of HS-1793 in LPS-induced inflammatory reaction using RAW264.7 macrophage model as in resveratrol.

Materials and methods

Preparation of HS-1793

HS-1793 was synthesized in the same manner as described previously (Jeong et al., 2012). It was made of a 50 mM stock solution in ethanol and diluted in saline or culture media for all experiments. Ethanol equivalent to the HS-1793 stock solution was diluted in the same manner and used as a vehicle control.

Cell and reagents

RAW264.7 macrophage cell line used in the experiments was purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). The complete culture medium was Dulbecco’s modified Eagle’s medium (DMEM, Wel gene, Daegu, Korea) containing 10% fetal bovine serum (FBS, Gibco Ltd., Grand Island, NY, USA), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were cultured in an incubator under humidified conditions at 37 °C supplied with 5% CO2. Cells after 10–15 passages were discarded and no longer used for these assays. LPS, L-NG-methyl-L-arginine acetate (L-NMMA, NO synthase inhibitor), S-methylisothiourea sulfate (SMT, iNOS inhibitor), N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398, COX-2 inhibitor) and(E)-3-[(4-methylphenylsulfonyl]-2-propenenitrile (BAY11-7082, IκB/IKK Inhibitor) used in this study were products of Sigma Chemicals (St. Louis, MO, USA).

Cell viability assay

MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was performed to investigate the cytotoxicity of HS-1793 (Jo et al., 2010). RAW264.7 cells were seeded in 96-well tissue culture plates (2 × 104 cells/well) and incubated overnight. The next day, cells were treated with HS-1793 at the indicated concentrations for 24 h with or without LPS (500 ng/ml). MTT assay was started by adding 10 μl of MTT solution (10 mg/mL) to each well of a 96-well plate, and the plates were further incubated for 4 h. The supernatant was removed, and the formazan crystals produced were dissolved in 200 μl of dimethylsulfoxide. Absorbance values at 550 nm were determined with an automated microplate reader (OpsysMR, DYNEX. Ltd).

Measurement of NO production

Nitrite accumulation, an indicator of NO production, was measured using the Griess reagent. RAW264.7 cells were seeded in 24-well plates (1 × 105 cells/well) and pre-treated with HS-1793, L-NMMA (100 µM) or SMT (50 µM) for 1 h. The cells were then stimulated with LPS (500 ng/ml) and further cultured for 24 h. After treatment, the culture supernatants were collected and mixed with an equal volume of Griess reagent (mixture at 1:1 of 0.1% naphthylethylenediamine dihydrochloride and 1% sulphanilamide in 5% H3PO4). The mixture was incubated at room temperature for 10 min, and the absorbance at 550 nm was measured in an automated microplate reader. Nitrite concentration (μM) was calculated from a NaNO2 standard curve.

Measurement of PGE2 and cytokines level

RAW264.7 cells were seeded in 24-well plates (1 × 105 cells/well) and pre-treated with HS-1793, NS-398 (50 µM) or BAY11-7082 (20 µM) for 1 h. The cells were then stimulated with LPS (500 ng/ml) and further cultured for 24 h. After treatment, the culture supernatants were collected. The amount of PGE2, interleukin (IL)-6, IL-8, tumor necrosis factor alpha (TNF-α) and IL-1β released from the cells was analyzed using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instruction for PGE2 (R&D Systems, Minneapolis, MN, USA) and cytokines (BD Biosciences, Franklin Lakes, NJ, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RAW264.7 cells were seeded in 6-well (6 × 105cells/ml) plates and pretreated with HS-1793, SMT (50 µM) or NS-938 (50 µM) for 1 h prior to adding 500 ng/ml LPS. The cells were further cultured for 12 h, and then harvested by centrifugation at 300 g for 5 min at 4 °C. RT-qPCR was performed using the SYBR Green reporter method. One microgram of total RNA was isolated using an RNeasy Mini kit (Qiagen, Valencia, CA, USA), which was subsequently reverse transcribed to cDNA with Transcriptor First Strand cDNA Synthesis Kit (Roche Nutley, New Jersey,USA). Two microliter of cDNA were added to the reaction mixture containing 6 µl of ddH2O, 10 µl of 2 × SYBR Green Master Mix (Bio-Rad, Hercules, USA) and 1 µl of each primer. The primers were as follows: iNOS forward (5′-GAG ACA GGG AAG TCT GAA GCA C-3′) and reverse (5′-CCAGCAGTAGTTGCTCCTCTTC-3′), COX-2 forward (5′-GCG ACA TA C TCA AGC AGG AGC A-3′) and reverse (5′-AGT GGT AAC CGC TCA GGT GTT G-3′), TLR4 forward (5′-CCT GCG TGA GAC CAG AAA G-3′) and reverse (5′-TTCAGCTCCATGCATTGATAA-3′), MyD88 forward (5′-CTG CTC GAG CTG CTT ACC A-3′) and reverse (5′-CTT CAA GAT ATA CTT TTG GCA ATC C-3′), TRAF6 forward (5′-TTT TGG TTG CCA TGA AAA GA-3′) and reverse (5′-CTC ATG TGT GAC TGG GTG TTC-3′), IRAK1 forward (5′-AGT TCT AA C GTG CTT CTG GA-3′) and reverse (5′-TCA CTA CTA GAG GCT GCC AT-3′), and GAPDH forward (5′-CAT CAC TGC CAC CCA GAA GAC TG-3′) and reverse (5′-ATG CCA GTG AGC TTC CCG TTC AG-3′). The GAPDH was used as a control to normalize the expression values of iNOS, COX-2, TLR4, MyD88, TRAF6 and IRAK1. The reaction consisted of an initial activation step (30 s at 95 °C) and 40 cycling steps (denaturation for 5 s at 95 °C, annealing and extension for 30 s at 60 °C), and then melt curve analysis was performed. Detection of the quenched probe, calculation of threshold cycles, and further analysis were done using CFX manager software (version 2.1; Applied Bio Rad). The relative gene expression levels were calculated using the 2−△△Ct method (Jeong et al., 2017).

Western blot analysis

Cells were lysed in Protein Extraction Solution (Elpis Biotech, Daejeon, Korea) for 20 min on ice. Nuclear protein was fractionated using Nuclear/Cytosol Fractionation Kit (BioVision Inc., California, USA) according to the manufacturer's instruction. Protein concentrations were determined with Bradford assay kit (Bio-Rad, Hercules, USA), and protein sizes were identified with protein standard marker (Bio-Rad, Hercules, USA). Protein samples were separated on 4 ~ 15% Tris Glycine gel and transferred to a PVDF membrane. The membrane was incubated in blocking buffer (5% nonfat skim milk), followed by probing with the corresponding primary antibodies and visualization with horseradish peroxidase (HRP)-conjugated secondary antibodies. The membrane was developed with a light-emitting nonradioactive method using the Amersham Biosciences ECL system (Amersham Biosciences, Piscataway, NJ, USA). Antibodies against TLR4, MyD88, TRAF 6 and IRAK1 were purchased from Abcam (Cambridge, MA, USA). Antibodies against iNOS, COX-2, NF-κB p65, histone and GADPH were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG antibodies were purchased from Cell Signaling Technology and HRP-conjugated goat anti-mouse IgG antibodies were obtained from Santa Cruz Biotechnologies. The optical density of all Western blots was quantified using Image J (NIH, Bethesda, MD, USA) and normalized to GADPH or Histone H3.

Data analysis

Prior to any statistical analysis, data were tested for normal distribution and homogeneity of variance using the Kolmogorov–Smirnov test and Levene’s test, respectively. The Statistical Package for the Social Sciences statistical software for Windows, Ver. 18.0 (SPSS Inc., Chicago, USA) was used in statistical analysis of the data, which are presented as mean ± standard error (SE). Comparisons between two groups were assessed by Student's t-test, and multiple comparisons were evaluated by one-way ANOVA with Dunnett's or Tukey's post-hoc test. A value of p < 0.05 was considered significant.

Results and discussion

Macrophages are a key cell type of immune cells that develop from hematopoietic stem cells during hematopoiesis. They are a part of white blood cells derived from common myeloid progenitor, but undergo a different differentiation process from granulocytes. They are produced by the differentiation of monocytes in tissues and found in essentially all tissue as part of the mononuclear phagocytic system. Macrophages respond to infection or other insults as the expert phagocytes. Besides phagocytosis, they play important and diverse roles throughout most stages of inflammation especially in tissue regeneration and repair. On the other hand, they also play a crucial role in the development of pathological lesion caused by chronic inflammation and excessive scarring (Oishi and Manabe, 2018; Watanabe et al., 2019). Macrophages are known to actively secrete cytotoxic and pro-inflammatory mediators in the acute response to tissue injury (Jou et al., 2013). Among these mediators, PGE2 is probably the best studied inflammatory prostaglandin in association with human pathological and physiological conditions Goetzl et al., 1995). PGE2 is a prostanoid fatty acid derivative of arachidonic acid generated from cell membrane phospholipids. In activated macrophages, arachidonic acid is converted to prostaglandins by the enzyme cycloogygenase. Among the COX isoforms, the inducible isoform of COX-2 is considered the most active in the development of an inflammatory response. Another enzyme that works most actively in inflammatory conditions is NOS which produces NO (Moncada and Bolaños. 2006). iNOS is also the most active pro-inflammatory NOS isoform. Indeed, the amount of pro-inflammatory mediators, especially endotoxin-induced NO and PGE2 can be used as a surrogate for the progression of inflammation and provide markers that are useful for investigating the effect of pharmacological interventions (Nunes Et al., 2018; Moussa et al., 2017). In this study, HS-1793 was tested for cytotoxicity on RAW 264.7 cells. The cytotoxicity test was conducted in the concentration range (0–5 μM) for 24 h used in our previous studies, and no cytotoxic effects were observed with any of the tested concentrations. Therefore, concentrations from 0.63 to 5 μM were further analyzed for the anti-inflammatory effects of HS-1793 (Fig. 1). In previous reports on the anti-inflammatory propety of resveratrol, the concentration, which inhibits the expression of pro-inflammatory mediators and cytokines, was mostly 5 μM or more (Bigagli et al., 2017; Schwager et al., 2017; Zong et al., 2012; Jakusa et al., 2013). In the present study, first, the pro-inflammatory mediators secreted in the culture supernatant by stimulating RAW 264.7 cells with LPS were measured. As expected, LPS-induced NO production was suppressed more effectively by SMT, a preferential inhibitor of iNOS, than by L-NMMA, a relatively non-selective inhibitor of all NOS isoforms (Fig. 2A). HS-1793 also reduced LPS-induced NO production in a concentration-dependent manner, and at a concentration of 5 µM, it was more effective than the positive control. RAW 264.7 cells stimulated with LPS, produced significant amounts of PGE2 as well as NO (Fig. 2B). LPS-induced PGE2 production was also effectively suppressed by HS-1793 in a concentration-dependent manner at levels that were comparable to NS-398, a COX-2 inhibitor. LPS-induced increases in NO and PGE2 production in RAW 264.7 cells were parallel to both mRNA and protein expression levels of iNOS and COX, respectively (Fig. 3). HS-1793 pre-treatment on macrophages significantly reduced mRNA and protein expression levels of them in a pattern similar to that of the inhibition of NO and PGE2, respectively, which was comparable with the response to specific enzyme inhibitors. These findings indicate that HS-1793 contributed to the inhibitory effects of on the secretion of pro-inflammatory mediators in macrophages by suppressing the enzymatic activities at both mRNA and protein expression levels.

Fig. 1.

Fig. 1

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(A) Chemical structure of HS-1793. (B) Effect of HS-1793 on viability of RAW 264.7 cells. Cells were treated with different concentrations of HS-1793 for 24 h with or without LPS. Viability was measured by using MTT assay and expressed as mean ± SE from three independent experiments

Fig. 2.

Fig. 2

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Effect of HS-1793 on NO and PGE2 production in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentrations of HS-1793, L-NMMA (500 µM), SMT (50 µM) and NS-398 (50 µM) for 1 h before LPS (500 ng/mL) treatment. (A) Nitrite content was measured using the Griess reaction and (B) PGE2 concentration was measured by ELISA. Data are presented as mean ± SE from three independent experiments by one-way analysis of variance followed by Student’s t-test or Dunnett’s multiple comparison test. *p < 0.05 compared to untreated cells (control) versus LPS alone and #p < 0.05 compared to LPS alone versus HS-1793, L-NMMA, SMT or NS-398 treated groups, respectively

Fig. 3.

Fig. 3

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Effect of HS-1793 on iNOS and COX-2 expression in LPS-stimulated RAW 264.7 cells. Total RNA was used to perform RT-PCR, and the relative fold changes of (A) iNOS and (B) COX-2 mRNA expression are indicated. (C) Whole-cell extracts were prepared, and the expression levels of iNOS and COX-2 were determined by Western blotting. Cells were pretreated with the indicated concentrations of HS-1793, SMT (50 µM) and NS-398 (50 µM) for 1 h before LPS (500 ng/mL) treatment. The optical density of all Western blots was quantified using Image J and normalized to GADPH or Histone H3. Data are presented as mean ± SE from three independent experiments by one-way analysis of variance followed by Student’s t-test or Dunnett’s multiple comparison test. *p < 0.05 compared to untreated cells (control) versus LPS alone and #p < 0.05 compared to LPS alone versus HS-1793, SMT or NS-398 treated group, respectively

Cytokines are a large group of small proteins that are released by specific cells of immune system and involved in signaling of immune response and regulation. One structurally related subgroup known as chemokines function primarily in the migration of leukocytes although some of them also possess a variety of other functions. During inflammatory response of different types, numerous cytokines and chemokines are synthetized and released. Therefore, the amount of cytokines, as well as pro-inflammatory mediators, are useful markers reflecting the degree of inflammation and investigating pharmacological effects. Among them, pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 including a chemokine IL-8, are produced mainly by activated macrophages and are known to be deeply involved in both the initiation as well as the progression of many human pathologic conditions. Moreover, as previously reported, the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 is required to synergistically induce the production of pro-inflammatory mediators such as NO and PGE2 (Zimmermann-Franco et al., 2018; Švajger and Jeras, 2012). As shown in Fig. 4 in this study, LPS treatment significantly enhanced the secretion of TNF-α, IL-1β, IL-6, and IL-8 by RAW 264.7 cells, whereas HS-1793 markedly attenuated this response.

Fig. 4.

Fig. 4

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Effect of HS-1793 on pro-inflammatory cytokines in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentrations of HS-1793 and BAY11-7082 (20 µM) for 1 h before LPS (500 ng/mL) treatment. Supernatants were obtained and the amounts of (A) IL-6, (B) IL-8, (C) TNF-α, and (D) IL-1β were measured by ELISA. Data are presented as mean ± SE from three independent experiments by one-way analysis of variance followed by Student’s t-test or Dunnett’s multiple comparison test. *p < 0.05 compared to untreated cells (control) versus LPS alone and #p < 0.05 compared to LPS alone versus HS-1793 or BAY11-7082 treated group, respectively

Cytokines are involved in endocrine, autocrine and paracrine signaling and perform their function by activating downstream pathways via binding to surface receptors on target cells. As well as alterations in downstream pathways, upstream signaling pathways that control the cytokine production in cells have an important role in immunomodulation and cancer progression (Guven-Maiorov et al., 2014). Monocytes/macrophages express several families of pattern recognotion receptors (PRRs) including Toll-like receptors (TLRs). PRRs bind pathogen-associated molecular patterns (PAMPs) that trigger cellular responses. LPS is a conserved bacterial PAMP and LPS stimulation induces the TLR4 complex on the cell surface resulting in the activation of a series of downstream signaling events. Because TLR4 is unique in that it can be found not only on the plasma membraned but also in endosomes, TLR4 signaling processes not only activate NF-κB and MAP kinase pathways, but also trigger activation of interferon regulatory factors (IRFs). It has been reported that resveratrol exhibits anti-inflammatory effect through suppressing all these signaling pathways in LPS-induced inflammatory model in RAW264.7 cells (Tong et al., 2020). However, key importance for activating the transcription factors that induce the expression of many inflammatory genes is the NF-κB signaling pathway, a shared component included in all TLRs. Several reports have confirmed that resveratrol has anti-inflammatory activity on RAW 264.7 cells via suppression of TLR4-NF-κB pathway following LPS stimulation (Zong et al., 2012; Capiralla et al., 2012; Jakus et al., 2013). The present study also showed that the enhanced secretion of cytokines in the LPS stimulated cells was completely inhibited following treatment with BAY11-7082, a selective inhibitor of NF-κB kinase (Fig. 4). Thus, along with the attenuating effect of HS-1793 on the levels of cytokine secretion, the effect of HS-1793 on changes in upstream signaling molecules related to the TLR4-NF-κB pathway was investigated. LPS triggers receptor dimerization of TLR4 and activate specific intracellular pathways, which induces recruitment of adapter molecules such as MyD88. MyD88 recruits another adaptor molecules such as IRAK1 and TRAF6, which leads activation of IKK through TAK1. IKK phosphorylates IκBα, resulting in its dissociation from NF-κB and subsequent degradation. The free NF-κB migrates into the nucleus, binds to DNA, and activates many genes (Guven et al., 2013). In the present study, TLR4 expression was significantly increased in RAW 264.7 cells stimulated with LPS compared to untreated cells, which was accompanied by up-regulation of MyD88, TRAF6 and IRAK1 at both the mRNA and protein levels. HS-1793 pretreatment in LPS-stimulated macrophages effectively blocked the upregulation of all these upper signaling pathways. It was also confirmed that HS-1793 concentration-dependently downregulated the nuclear translocation of transcription factor NF-κB through Western blot analysis of NF-κB/p65 protein in nuclear fractions of LPS-stimulated RAW 264.7 cells (Fig. 5). These findings suggest that the anti-inflammatory activity of HS-1793 are related to the suppression of the initiation of TLR4 signaling cascades and subsequent inhibition of NF-κB activation, the upstream pathways controlling the concentration of pro-inflammatory cytokines and mediators in macrophages. Excessive production of these cytokines and pro-inflammatory mediators has been linked to the development of chronic inflammatory diseases (Aldawsari et al., 2016). Thus, HS-1793, like resveratrol, could be a good candidate material to be developed as a successful and safe strategy to treat chronic inflammatory conditions.

Fig. 5.

Fig. 5

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Effect of HS-1793 on TLR4 mediated NF-κB activation in LPS-stimulated RAW 264.7 cells. Cells were pretreated with the indicated concentrations of HS-1793 for 1 h before LPS (500 ng/mL) treatment. (A) Total RNA was used to perform RT-PCR, and the relative fold changes of TLR4, MyD88, TRAF6 and IRAK1 mRNA expression are shown. (B) Whole-cell or nuclear extracts were prepared, and the expression levels of TLR4, MyD88, TRAF6, IRAK1 and NF-κB/p65 were determined by Western blotting. The optical density of all Western blots was quantified using Image J and normalized to GADPH or Histone H3. Data are presented as mean ± SE, from three independent experiments by one-way analysis of variance followed by Student’s t-test or Dunnett’s multiple comparison test. *p < 0.05 compared to untreated cells (control) versus LPS alone and #p < 0.05 compared to LPS alone versus HS-1793 treated group

Macrophages are also an important cellular component within the tumor stroma, and various pro-inflammatory bioactive molecules produced by macrophages in chronic inflammatory conditions provide a mutagenic environment in the sub-epithelial stroma. Once transformed, these neoplastic cells maintain intricate interactions with macrophages producing an inflammatory tumor microenvironment (Greten and Grivennikov, 2019). Tumor-associated macrophages do not primarily exert an immune function but rather favor the development of cancer and promotion of all stages of tumorigenesis (Poh and Ernst, 2018). Constitutive activation of NF-κB, the main signaling pathway that secretes pro-inflammatory cytokines, leads to chronic inflammation, which can also underlie the critical events of cancer initiation and progression (Trinchieri, 2012). As the inactivation of NF-κB switches inflammation-induced tumor growth to regression, the modulation of NF-κB has been suggested as an attractive target in cancer therapy (Luo et al., 2004). In previous studies, we have shown that HS-1793 induces a strong anti-cancer activity in various cancer cell lines (Jeong et al., 2009, 2011; Kim et al., 2012). Furthermore, we have found that HS-1793 stimulates a potent anti-tumor immunity by reducing regulatory T cells in a mouse breast tumor model (Choi et al., 2012; Jeong et al., 2017). Therefore, given that tumor progression is profoundly influenced by cancer cells and anti-tumor immunity as well as interactions between cancer cells and their surrounding stroma, the data of our previous and present studies suggest that HS-1793 may be a useful natural therapeutic and prophylactic anti-cancer agent.

Extensive research on resveratrol has improved understanding of how it modulates important signaling pathways in inflammatory cells and its anti-inflammatory effects. Resveratrol derivative, HS-1793 was synthesized to have a more stable structure by eliminating the chemically unstable double bond of resveratrol. As resveratrol is used as a safe functional food ingredient derived from natural products, it is estimated that HS-1793 would have a similar degree of safety. In our previously performed toxicity tests, HS-1793 is non-genotoxic at the dose tested in three standard tests as expected (Jung et al.). In conclusion, the present study findings demonstrate HS-1793 as a promising new option for improved therapeutic management of patients with chronic inflammatory diseases while preserving the pharmacological effect of resveratrol. Given that macrophages play a major role in tumor progression through inflammation in the tumor microenvironment, HS-1793 may also be a useful natural therapeutic and prophylactic anti-cancer agent.

Acknowledgements

This work was supported by the Dong-A University Research Fund.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Wol Soon Jo, Email: sailorjo@dirams.re.kr.

Sung Dae Kim, Email: kim79sd@knu.ac.kr.

Soo Kyung Jeong, Email: soo87@dirams.re.kr.

Su Jung Oh, Email: osj10050@dirams.re.kr.

Moon Taek ParK, Email: mtpark@dirams.re.kr.

Chang Geun Lee, Email: cglee@dirams.re.kr.

Young- Rok Kang, Email: yeongrok@dirams.re.kr.

Min Ho Jeong, Email: mhjeong@dau.ac.kr.

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