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Cinnamaldehyde (CA) has been known to exhibit anti-inflammatory and anticancer effects. Although numerous pharmacological effects have been demonstrated, regulatory effect of CA on the functional activation of monocytes and macrophages has not been fully elucidated yet. To evaluate its monocyte/macrophage-mediated immune responses, macrophages activated by lipopolysaccharide (LPS), and monocytes treated with proaggregative antibodies, and extracellular matrix protein fibronectin were employed. CA was able to suppress both the production of nitric oxide (NO) and upregulation of surface levels of costimulatory molecules (CD80 and CD69) and pattern recognition receptors (toll-like receptor 2 (TLR2) and complement receptor (CR3)). In addition, CA also blocked cell-cell adhesion induced by the activation of CD29 and CD43 but not cell-fibronectin adhesion. Immunoblotting analysis suggested that CA inhibition was due to the inhibition of phosphoinositide-3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK)1 as well as nuclear factor-(NF-) κB activation. In particular, thiol compounds with sulphydryl group, L-cysteine and dithiothreitol (DTT), strongly abrogated CA-mediated NO production and NF-κB activation. Therefore, our results suggest that CA can act as a strong regulator of monocyte/macrophage-mediated immune responses by thiolation of target cysteine residues in PI3K or PDK1.
Monocytes/macrophages play a critical role in managing innate and adaptive immunity-including inflammatory processes by secreting proinflammatory molecules (eg. tumor necrosis factor (TNF)-α, and nitric oxide (NO)) . The activation of macrophages and monocytes is mediated by activation of various receptors including Toll like receptor-(TLR-) 4 and their counter molecules such as lipopolysaccharide (LPS) derived from bacteria or virus . In parallel, the activation of these cells triggers various cellular responses such as cell migration, adhesion, extravasation, and infiltration to induce effective movement of these cells into inflamed tissue by adhesion molecules such as β1 (CD18) or β2 (CD29) integrins and their ligands such as vascular cell adhesion molecule-(VCAM-) 1 or intercellular adhesion molecule-(ICAM-)1 . The molecular interaction between surface receptors and counter molecules seen in various cellular inflammatory responses generates a series of complex signaling events composed of numerous intracellular enzymes such as phosphoinositide-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 [4, 5] linked to actin cytoskeleton rearrangement for modulating cellular activation or the proinflammatory gene expression by mediating with transcription factors like NF-κB and AP-1 . Recently, inflammatory responses by monocytes and macrophages were reported to be a critical pathological event in triggering various acute or chronic diseases such as septic shock, cancer, autoimmune diseases, cardiovascular diseases, obesity, and diabetes [7, 8]. It is therefore considered that development of promising regulators of monocyte/macrophage-mediated inflammatory responses without side effects could be useful for prevention of, or as the therapeutic remedy for, various inflammation-mediated diseases .
Cinnamaldehyde (CA; Figure 1(a)), a major bioactive compound isolated from the leaves of Cinnamomum osmophloeum Kaneh [10, 11], has been known to trigger apoptosis through mitochondrial permeability transition in human promyelocytic leukemia HL-60 cells , by activating the proapoptotic Bcl-2 family proteins . Treatment of cultured mouse splenocytes with CA in a dose-dependent manner blocked the proliferation of lymphocytes induced by concanavalin A and LPS . This compound was also found to suppress NF-κB activation within macrophage-like RAW264.7 cells . It has been demonstrated that CA is capable of blocking inducible nitric oxide synthase (iNOS) and NO production by mediation of NF-κB activation blockade in LPS-stimulated RAW264.7 cells . Moreover, the production of PGE2 was also reduced by CA exposure in cultured rat cerebral microvascular endothelial cells . These results strongly suggested that CA can be applied as an anti-inflammatory drug. However, the pharmacological target and inhibitory mechanism of CA, and its activity on various cellular events such as cell adhesion and migration commonly seen in the functional activation of monocytes/macrophages, have not been examined yet. Thus, in this study, we investigated the detailed regulatory roles of CA on monocyte/macrophage-mediated immune responses and its potential target enzyme.
CA was kindly supplied from the Aging Tissue Bank (Pusan National University, Busan, South Korea). LPS, phorbol 12-myristate 13-acetate (PMA), FITC-dextran, 1,4-dithiolthreitol (DTT), L-cysteine, and TNF-α were obtained from Sigma Chemical Co. (St. Louis, MO). LY294002 and wortmannin and U0126 were from Calbiochem (La Jolla, CA). RAW264.7 and TLR4-expressing HEK293 cells were purchased from American Type Culture Collection (Rockville, MD) and Invivogen (San Diego, CA). All other chemicals were purchased from Sigma. Fibronectin was obtained from BD Biosciences (San Diego, CA). Phospho-specific antibodies to p85, PDK1, Akt, and I κ B αwere obtained from Cell Signaling (Beverly, MA). Cell-cell adhesion-inducing antibodies to CD29 (MEM 101A, purified IgG1), CD43 (161-46, ascites, IgG1), and P5D2 were used as reported previously [18, 19]. Antibodies to costimulatory (CD80, CD86, CD40, and CD69) and adhesion (CD29, CD43, and CD18) molecules were from BD Biosciences (San Diego, CA). Antibodies to pattern recognition receptors (dectin-1, TLR2, TLR4, SR, and CR3) were purchased from Serotec (Raleigh, NC). Suntide, a peptide sequence derived from Akt (protein kinase B) , was synthesized by Peptron (Daejeon, South Korea).
RAW264.7 and TLR4-expressing HEK293 cells were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY), glutamine, and antibiotics (penicillin and streptomycin), at 37°C with 5% CO2.
The inhibitory effect of CA on NO production was determined as previously described . CA solubilized with DMSO (100%) was diluted with RPMI1640. RAW264.7 cells (2 × 106 cells/ml) were incubated with LPS (1μg/ml) in the presence or absence of CA for 24h. Supernatants were assayed for NO and TNF-α contents using Griess reagent.
Since RAW264.7 cells are not easily transfected with certain types of DNA constructs, TLR4-expressing HEK293 cells (1 × 106 cells/ml) were used to be transfected with 1μg of plasmid with NF-κB and β-galactosidase by using the calcium phosphate method in a 12-well plate. The cells were used for experiments 48h after transfection. Luciferase assays were performed using the Luciferase Assay System (Promega) .
To measure the phagocytic activity of RAW264.7 cells, a previously reported method was used with slight modifications . RAW264.7 (5 × 104) cells pretreated with CA were resuspended in 100μl PBS containing 1% human serum and incubated with FITC-dextran (1mg/ml) at 37°C and 0°C for 30min. The incubation was stopped by the addition of 2ml ice-cold PBS containing 1% human serum and 0.02% sodium azide. The cells were washed three times with cold PBS-azide and analyzed by flow cytometry.
U937 cell adhesion assay was performed as previously reported [24, 25]. Briefly, U937 cells maintained in complete RPMI1640 medium (supplemented with 100U/ml of penicillin 100 μg/ml of streptomycin, and 10% FBS) were preincubated with CA for 1h at 37°C and further incubated with aggregation-inducing (agonistic) antibodies (1μg/ml) in a 96-well plate. After a 50minute incubation, cell-cell clusters were determined by homotypic cell-cell adhesion assay using a hemocytometer  and analyzed with an inverted light microscope equipped with a COHU high-performance CCD (Diavert) video camera. For the cell-fibronectin adhesion assay, CA-treated U937 cells (5 × 105 cells/well) were seeded on a fibronectin-coated (50μg/ml) plate and incubated for 3h . After removing unbound cells with PBS, the attached cells were treated with 0.1% crystal violet for 15min. The OD value at 540nm was measured by a Spectramax 250 microplate reader.
RAW264.7 cells (2 × 106 cells/ml) were incubated with CA for 30min. After scratching the cultured cells with a pipette, the cells were further incubated for 48h. The images of the cells in culture were obtained using an inverted phase contrast microscope attached to a video camera.
Surface levels of adhesion molecules (CD29, CD43, and CD18) in U937 and co-stimulatory molecules (CD69, CD80, CD40, and CD86) and pattern recognition receptors (dectin-1, TLR2, TLR4, SR, and CR3) in RAW264.7 cells were determined by flow cytometric analysis as reported previously . Stained cells were analyzed on a FACScan device (Becton-Dickinson, San Jose, CA).
Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously .
The total RNA from the CA and LPS-treated RAW264.7 cells was prepared by adding TRIzol Reagent (Gibco BRL), according to manufacturer's protocol. Semiquantitative RT reactions were conducted using MuLV reverse transcriptase as reported previously . The primers (Bioneer, Daejeon, South Korea) were used as previously reported .
RAW264.7 cells (5 × 106 cells/ml) were washed 3 times in cold PBS with 1mM sodium orthovanadate and lysed in lysis buffer (20mM Tris-HCl, pH 7.4, 2mM EDTA, 2mM ethyleneglycotetraacetic acid, 50mM β-glycerophosphate, 1mM sodium orthovanadate, 1mM dithiothreitol, 1% Triton X-100, 10% glycerol, 10μg/ml aprotinin, 10μg/ml pepstatin, 1mM benzimide, and 2 mM PMSF) for 30min with rotation at 4°C. The lysates were clarified by centrifugation at 16,000 × g for 10min at 4°C and stored at −20°C until needed. Whole cell lysates were then analyzed by immunoblotting. Proteins were separated on 10% SDS-polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride (PVDF) membrane. Membranes were blocked for 60min in Tris-buffered saline containing 3% bovine serum albumin, 20mM NaF, 2mM EDTA, and 0.2% Tween 20 at room temperature. The membrane was incubated for 60min with specific primary antibody at 4°C, washed 3 times with the same buffer, and incubated for additional 60min with horse radish peroxidase-(HRP-) conjugated secondary antibody. The total and phosphorylated levels of p85, PDK1, Akt, IκBα, and β-actin were visualized using the ECL system (Amersham, Little Chalfont, Buckinghamshire, UK).
α Cyano-4-hydroxycinnamic acid (20mg) (Bruker Daltonics, Bremen, Germany) was dissolved in 1ml acetone:ethanol (1:2, v/v), and 0.5μl of the matrix solution was mixed with an equivalent volume of sample. Analysis was performed using an Ultraflex TOF/TOF system (Bruker Daltonics). The Ultraflex TOF/TOF system was operated in positive ion reflect mode. Each spectrum was the cumulative average of 250–450 laser shots. Mass spectra were first calibrated in the closed external mode using the peptide calibration standard II (Bruker Daltonics), sometimes using the internal statistical mode to achieve maximum calibration mass accuracy.
The Student's t-test and one–way ANOVA were used to determine the statistical significance between values of the various experimental and control groups. P values of.05 or less were considered to be statistically significant.
Monocytes/macrophages are the prime immune cells managing inflammatory responses, which contribute to development of number of diseases such as cancer, diabetes, and atherosclerosis [30, 31]. This view led us to develop novel immunoregulatory drugs based on the functional activation of monocytes and macrophages without side effects to prevent such diseases. In this context, medicinal plants that have traditionally been used for long time are considered as attractive biopharmaceutical candidates. With this goal, therefore, we have attempted to develop macrophage function regulators using naturally occurring compounds or plants for a decade.
The regulatory effect of CA on LPS-induced macrophage immune responses was initially examined. Upon nontoxic concentrations (0 to 40μM) (Figure 1(b)), CA strongly suppressed the production of NO (Figure 2(a)) and the surface upregulation of costimulatory (CD80 and CD69) and pattern recognition (TLR2 and CR3) molecules (Figure 2(b)). Moreover, CA protected cells from LPS-induced cytotoxicity and apoptosis, mainly induced by the NO produced (Figure 2(c)). The inhibition of NO release occurred at the transcriptional levels, according to Figure 3. Thus, CA blocked mRNA expression of iNOS as well as other proinflammatory cytokines such as TNF-α and IL-1β as much as 80 to 95% (Figure 3). Because transcriptional downregulation of inflammatory mediators by CA has been reported to inhibit NF-κB activation [16, 32], reporter gene assay for NF-κB and immunoblotting analysis of upstream signaling were further conducted. As Figure 4(a) shows, CA blocked NF-κB-mediated luciferase activity induced by LPS treatment, similar to previous papers [15, 32]. Interestingly, CA also blocked a series of NF-κB activation signaling pathways. This compound suppressed the phosphorylation of IκBα, Akt, and PDK1 but not p85, a regulatory subunit of PI3K (Figure 4(b)), suggesting that the pharmacological target of CA may be PI3K or PDK1 in LPS-mediated macrophage immune responses. Unlike LPS-induced inflammatory responses, FITC-dextran-induced phagocytic uptake of RAW264.7 cells, a major response found in innate immunity, was not negatively modulated by this compound (Figure 5), which suggests that CA cannot regulate all macrophage functions but modulates mostly LPS-mediated immune responses in macrophages.
Meanwhile, CA also strongly suppressed cell-cell adhesions induced by proaggregative antibodies to CD29 and CD43 up to 85% (Figures 6(a) and 6(b)). However, adhesion of U937 cells to fibronectin, an extracellular matrix protein acting as a CD29 ligand, was not suppressed by this compound (Figure 6(c)), indicating that adhesion between cells but not cell and extracellular matrix could be blocked by CA. However, CA did not suppress the surface levels of adhesion molecules such as CD18, CD29, and CD43 even at 40μM (Figure 6(d)), while CA diminished the migration of RAW264.7 cells in an in vitro wound healing assay, compared to normal (Figure 6(e)). Considering that cell-fibronectin adhesion only requires simple activation of CD29, and while intracellular signaling (ERK, p38, and protein kinase Cδ) and actin cytoskeleton change are important factors in cell-cell adhesion events [24, 25], CA seems to modulate intracellular signaling events rather than the blockade of a direct interaction between CD29 and fibronectin. In particular, these signaling events targeted to CA seem to be involved in modulating cell migration commonly seen in both cell-cell adhesion (Figure 6(a)) and wound healing assays (Figure 6(e)). CD29-mediated cell-cell adhesion is an essential phenomenon for survival and activation of immune cells, particular for an interaction between antigeny presenting cells (APC) and T lymphocytes or NK cells. Therefore, antiaggregative effect of CA may contribute to the regulation of monocyte/macrophage roles as APC requiring their adhesion responses. Similarly, since PI3K/Akt inhibition by LY294002 and wortmannin suppressed CD29-mediated cell-cell adhesion, CA inhibition of adhesion and migration events seems to occur at the level of PI3K/PDK1, as in the case of LPS signaling.
Since thiol compounds such as DTT and L-cysteine are reported to block CA inhibition , we finally examined whether this pattern can be observed under the same conditions. As expected, pretreatment of L-cysteine (200μM) or DTT (300μM) before CA treatment abrogated the inhibitory activity of CA in both NO production (Figure 7(a)) and NF-κB-mediated luciferase activity (Figure 7(b)), suggesting that thiolation is the major chemical mechanism of CA inhibition. Up to date, we have not been able to locate the exact thiolation site on the target protein by CA. However, recent findings revealing that an adduct formation of hydroquinone with the sulfhydryl group of Cys-310 in Akt is able to block the phosphorylation of both Thr-308 and Ser-473  seem to suggest that a target cysteine sequence of PI3K or PDK1 can serve as a thiolation site affecting their phosphorylation and activation. Indeed, we failed to detect an adduct formation between CA and Suntide, a peptide fragment containing Cysteine-310 designed according to Akt amino acid sequence (Figure 7(c)). Because identification of CA target thiolation site is an important step to understand the exact molecular mechanism of CA inhibition, we are currently undertaking further analysis using other peptide sequences containing cysteine residues from PI3K and PDK1.
In conclusion, we found that CA was able to suppress the production of NO and upregulation of surface levels of costimulatory molecules such as the surface upregulation of both costimulatory (CD80 and CD69) and pattern recognition molecules (TLR2 and CR3). In addition, CA also blocked both cell migration and cell-cell adhesion induced by CD29 and CD43, but not cell-fibronectin adhesion. The CA inhibition was likely due to the inhibition of PI3K and PDK1, important for NF-κB activation of signaling components, according to immunoblotting analysis. In particular, L-cysteine and DTT strongly interfered CA-mediated inhibition of NO production and NF-κB activation. Therefore, our results suggest that CA can act as a strong regulator of monocyte/macrophage-mediated immune responses, possibly by the induction of thiolation at cysteine residues in the target enzyme (PDK1 or PI3K). To prove a detailed inhibitory mechanism, identification of molecular targets of CA will be investigated in our next series of experiments.
This study was financially supported by a grant (2008–2011) supported from MEST and KOTEF through the Human Resource Training Project for Regional Innovation. B. H. Kim and Y. G. Lee equally contributed to this work.