Javamide-II Found in Coffee Is Better than Caffeine at Suppressing

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Bioactive Constituents, Metabolites, and Functions

Javamide-II found in coffee is better than caffeine in suppressing TNF-alpha production in PMA/PHA-treated lymphocytic Jurkat cells. Jae B. Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01885 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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Journal of Agricultural and Food Chemistry

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Javamide-II found in coffee is better than caffeine in suppressing TNF-alpha production in PMA/PHA-treated lymphocytic Jurkat cells.

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Jae B. Park*

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Diet, Genomics, and Immunology Laboratory, Bldg. 307C, Rm. 131, BHNRC, ARS, USDA, Beltsville, MD 20705

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Running Head: TNF-alpha inhibition by javamide-II in lymphocytic Jurkat cells *Corresponding author: Tel: 301-504-8365; Fax: 301-504-9456; E-mail: [email protected].

ACS Paragon Plus Environment


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ABSTRACT

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Recent studies suggested positive benefits of coffee consumption on inflammation-related

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diseases such as liver diseases and diabetes where activated lymphocytes and TNF-alpha are

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critically implicated. Interestingly, some reports suggested that javamide-II found in coffee may

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have anti-inflammation activity greater than caffeine, but there is limited information about its

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effect on TNF-alpha production by activated lymphocytes. Therefore, the inhibitory effect of

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javamide-II on TNF-alpha was investigated in PMA/PHA-treated lymphocytic Jurkat cells. At 5

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µM, javamide-II, not caffeine, inhibited TNF-alpha production in the cells (45±4 %; P < 0.001).

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To elucidate the underlying mechanism, the phosphorylation of MAP kinases (ERK, p38, JNK)

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was investigated in the Jurkat cells. Javamide-II had little effect on JNK or p38 phosphorylation,

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but javamide-II (< 20 µM) decreased ERK phosphorylation, consequently reducing TNF-alpha

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mRNA expression in the cells (P < 0.001). The involvement of ERK phosphorylation was also

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confirmed by a ERK1/2 inhibitor (SCH772984). Furthermore, javamide-II was also found to

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inhibit IL-2 production up-regulated by ERK phosphorylation in the cells (P < 0.001). These data

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suggested that javamide-II may be a potent compound to suppress TNF-alpha production more

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efficiently than caffeine by inhibiting ERK phosphorylation in the Jurkat cells.

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Key words: Javamide-II, Coffee, TNF-alpha, IL-2, ERK, lymphocytic Jurkat cells.


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Introduction

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Tumor necrosis factors (TNF) consist of a group of cytokines that can induce inflammation

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and/or cell death. There are two types of TNF in the group; TNF-alpha and TNF-beta.1-3

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Especially, TNF-alpha is a potent inflammatory cytokine, significantly involved in the initiation

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and/or progression of acute/systemic inflammation.1-3 Because TNF-alpha is critically involved

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in inflammation processes,3-5 its deregulation is considered to be a major cause for progressing

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several human chronic diseases such as diabetes, liver disease, rheumatoid arthritis, psoriasis,

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inflammatory bowel disease and cancer.6-9 In humans, TNF-alpha can be produced in many

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different cell types (lymphocytes, macrophages, natural killer cells), and its expression is up-

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regulated by various stimulants.1-4 Although TNF-alpha is produced by several cell types, the

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production from activated T-lymphocytes is closely associated with inflammation in several

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organs including the intestine and liver.1-3 In addition, recent studies suggest that T-

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lymphocytes activated in the gut may play critical roles in the progression of chronic

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inflammation in the liver.10,11

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For years, there has been accumulating information suggesting that diet components may play

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significant roles in regulating immune functions, especially related to inflammation.12-13 Coffee

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is one of most consumed drinks worldwide.14, 15 Several studies suggested health benefits of

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coffee consumption on fat liver disease, diabetes and degenerative diseases, possibly by

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suppressing inflammation-related processes.14-16 In fact, caffeine has been considered as a major

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contributing component related to inflammation. However, its efficacy is often challenged by the

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requirement of relatively high doses in the experiments.17 Therefore, there have been continuing

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research efforts to find other bioactive coffee chemicals which are responsible for inhibiting



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inflammation and its related molecules. Javamide-II (Table 1) is a phenolic amide found in

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coffee.18-20 Interestingly, its analogues were reported to have several biological activities

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including anti-inflammation activity.18-20 However, potential effect of javamide-II has not been

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investigated related to TNF-alpha production from activated T-lymphocytes which are believed

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to be critically involved in chronic inflammation in several organs.10,11 Therefore, in this study,

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potential effect of javamide-II on TNF-alpha production was investigated in the PHA/PMA-

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treated Jurkat cells, because the PHA/PMA-treated Jurkat cells share several biological

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properties similar to activated T-lymphocytes.21 Especially, in this paper, potential effects of

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javamide-II on both TNF-alpha and cAMP production was examined in comparison with

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caffeine, because caffeine was previously reported to inhibit TNF-alpha and increase cAMP

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production,22, 23 Also, the phosphorylation of MAP kinases, ERK (Extracellular signal-regulated

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kinase), JNK (c-Jun N-terminal kinase) and p38 MAP kinase (p38 mitogen-activated protein

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kinase), was investigated as an underlying mechanism of TNF-alpha inhibition in PHA/PMA-

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treated Jurkat cells. Lastly, the effect of javamide-II on interleukin-2 (IL-2) was investigated in

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the same cells, because ERK activation was also reported to play a significant role in IL-2

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production in the cells. 24


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MATERIALS AND METHODS

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Chemicals

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Javamide-II was prepared as reported previously.18 Briefly, caffeic acid was dissolved in

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dimethyl sulfoxide (DMSO) and mixed with 1,3-diisopropylcarbodiimide (DIC). Tryptophan

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modified with phenylpropanol was added to the reaction mixture, and incubated at room

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temperatures with a gentle stirring for 12 hr. The synthesized products were recovered and the

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phenylpropanol moiety was removed under an alkaline condition. Javamide-II was purified by

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HPLC (Waters, Milford, MA) with purity more than 97% as reported previously.18 Forskolin and

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other chemicals were purchased from Sigma Chemical Co. (St. 116 Louis, MO).

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Materials

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Jurkat cells were purchased from ATCC (Manassas, VA). Erk1/2 (catalog number 9102),

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phospho-Erk1/2 (catalog number 9101), p38 MAPK (catalog number 9212), phospho-p38

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MAPK (catalog number 9211), JNK (catalog number 9252) and phospho-JNK (catalog number

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4668) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

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Cell culture

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Jurkat cells were grown in RPMI 1640 medium with L-glutamine containing 10% FBS, 100

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units/ml penicillin, and 100 units/ml streptomycin. Jurkat cells were maintained at 37 °C in a

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humidified atmosphere of 5% CO2. Jurkat cells (1 × 106 cells/well) were treated with javamide-II

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for 10 min with several different concentrations, then followed by the treatment of PHA

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(Phytohemagglutinin) (10ug/mL) and PMA (12-O-tetradecanoylphorbol 13-acetate) (10 ո g/ml)



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for 24, 48 and 72 h to induce TNF-alpha and IL-2, unless specified otherwise. Media were saved

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for the determination of TNF-alpha and IL-2 proteins.

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Measurement of TNF-alpha and IL-2

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TNF-alpha levels in the media samples were determined using Human TNF-alpha ELISA kit

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from R&D systems (Minneapolis, MN) and IL-2 levels in the samples were using IL-2

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Quantikine ELISA kit from R&D systems (Minneapolis, MN) following manufacturer’s

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protocols.

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cAMP assay

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Jurkat cells (1X106) were treated with javamide-II (5-20 µM) and forskolin (20 µM) for 30 min,

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to induce cAMP production. The amounts of cAMP in the cells was determined using Direct

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cyclic AMP kit (Cayman Chemical, Ann Arbor, MI). All cAMP measurements were performed

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according to the kit’s protocol.

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Western blot analysis

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For the Western blot of ERK, p38 MAPK and JNK samples were prepared using the cell extracts

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of the Jurkat cells treated with javamide-II (0, 10, 20 µM) for 10 min, then followed by

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PHA/PMA treatment for 45min. The Western blots were generated using Novex 4-12% Tris-

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Glycine Mini Gel and XCell II™ Blot Module kit (LifeTechnologies, Cambridge, MA, USA)

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with phospho-ERK (Catalog number 9101), ERK (Catalog number 9102), phospho-p38 MAPK

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(Catalog number 9211), p38 MAP kinase (Catalog number 9212), JNK (Catalog number 9252)


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and phospho-JNK (Catalog number 4668) antibodies (Cell Signal, Danvers, MA, USA). For the

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blot, the amounts of protein in the samples were determined using Bio-Rad protein assay kit

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(Hercules, CA, USA), and ERK, p38 MAP kinase and JNK antibodies (Cell Signal, Danvers,

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MA, USA) were used as control samples for the blot.

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RNA isolation and real time RT-PCR quantization of TNF-alpha mRNA

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Total RNA was isolated from the PHA/PMA-treated Jurkat cells treated with javamide-II (0-40

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µM) for 4 hr using TRIzol™ reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized using

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the AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies, Inc., Santa

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Clara, CA) according to the manufacturer’s protocol. Real time semi-quantitative PCR was

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carried out QuantStudio 12K Flex Real-Time PCR System (ThermoFisher Scientific, Waltham,

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MA) using the manufacturer’s protocol. Human TNF/TNF-alpha (assay ID: Hs99999043_m1)

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TaqMan probe was used as reported previously25 and GAPDH (assay ID: Hs02786624_g1)

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TaqMan probe was used as an internal control (For more information;

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http://www.thermofisher.com).

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Statistical analysis

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All statistic analyses were performed with the SigmaPlot 11.0 (Chicago, IL). P value was

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calculated using one-way ANOVA with Holm-Sidak method, and P < 0.05 was considered as

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statistically significant. Data points in all figures were represented as the mean ± SD (n=3 or 5).



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RESULTS

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Effect of javamide-II on the TNF-alpha expression in Jurkat cells.

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To determine the effect of javamide-II on TNF-alpha production, the TNF-alpha levels were

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measured in PHA/PMA-treated Jurkat cells with or without the treatment of javamide-II at 24,

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48, and 72 h. As shown in Figure2A, TNF-alpha production was increased over time, and the

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production was inhibited by javamide-II (10 µM) (P < 0.001). Since javamide-II was able to

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inhibit TNF-alpha production in the Jurkat cells, dose-dependent effect of javamide-II on TNF-

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alpha production was investigated in the Jurkat cells. As shown in Figure 2B, javamide-II

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inhibited TNF-alpha production in a dose-dependent manner in the cells (P < 0.001). The

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inhibitory effect was observed even at 1 µM and highest at the concentration of 10 µM (Figure

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2). However, there was no additional increase even at higher than 50 µM (The data not shown

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here), suggesting that the inhibition effect on TNF-alpha reached a maximal plateau

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approximately at 10 µM.

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Effect of javamide-II on cAMP production in Jurkat cells.

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Because the increased cAMP production was previously reported to be accountable for TNF-

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alpha inhibition in cells,23 the effect of javamide-II on cAMP production was investigated in the

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Jurkat cells. As shown in Figure 3, javamide-II increased intracellular cAMP levels in the cells.

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Even at 5 µM, javamide-II was able to increase cAMP production significantly compared to

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control in the cells (P < 0.05). Although caffeine increased cAMP in the cells as reported

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previously,26 high doses of caffeine (˃300 µM) were required in order to achieve similar levels

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of cAMP induced by javamide-II (5-20 µM) (The data not shown here). These data clearly


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suggest that javamide-II can increase cAMP production at concentrations much lower than

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caffeine in the Jurkat cells. To investigate the involvement of cAMP in the inhibition of TNF-

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alpha, the effect of forskolin on TNF-alpha was investigated in the Jurkat cells, because forskolin

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was known to have adenylyl cyclase activating property to increase cAMP in the cells.27-29 The

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data showed that that forskolin was able to reduce TNF-alpha production significantly in the

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Jurkat cells (P < 0.001) (Figure 4), suggesting that cAMP is involved in the inhibition of TNF-

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alpha in the cells.

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Effects of javamide-II on ERK, JNK and p38 MAPK phosphorylation

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Because the phosphorylation of ERK is likely to be one of main signaling transduction pathways

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induced by PHA/PMA in the Jurkat cells,24, 30 the effect of javamide-II on ERK phosphorylation

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was investigated in the cells. As shown in Figure 5, the treatment of javamide-II (10, 20 µM)

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inhibited ERK phosphorylation in PHA/PMA-treated Jurkat cells (P < 0.005). Also, forskolin

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was found to inhibit ERK phosphorylation in the cells (P < 0.001) (Figure. 6) to support the

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involvement of cAMP in reducing ERK phosphorylation. In fact, these data are in line with a

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previous report about inhibitory role of cAMP in ERK phosphorylation.31 Although ERK is a

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most likely signaling transduction pathway induced by PHA/PMA treatment,24, 30 the

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phosphorylation of JNK and p38 MAPK was also investigated in this study, because they are

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often involved in regulating numerous inflammatory cytokines including TNF-alpha.32 The

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treatment of javamide-II (10, 20 µM) did not decrease the phosphorylation of JNK in

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PHA/PMA-treated Jurkat cells (Figure. 7). Likewise, javamide-II (10, 20 µM) had little effect

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on p38 phosphorylation in the Jurkat cells (Figure. 8). These data suggest that javamide-II is

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most likely to suppress ERK phosphorylation via increasing cAMP in PHA/PMA-treated Jurkat



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cells. Additionally, a ERK inhibitor (SCH772984) was tested in inhibiting TNF-alpha production

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in the Jurkat cells, because javamide-II was found to inhibit TNF-alpha probable by suppressing

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ERK activation in the cells. As expected, SCH772984 inhibited the production of TNF-alpha as

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much as javamide-II (P < 0.001) (Figure 9), suggesting that ERK pathway is certainly involved

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in TNF-alpha production in the PHA/PMA-treated Jurkat cells.

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Effects of javamide-II on TNF-alpha mRNA in Jurkat cells.

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Because the data showed that javamide-II could suppress the phosphorylation of ERK, the effect

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of javamide-II on TNF-alpha mRNA expression was investigated in PHA/PMA-treated Jurkat

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cells. As shown in Figure 10, javamide-II could suppress the expression of TNF-alpha mRNA

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significantly even at < 20 µM (P < 0.001), suggesting that javamide-II may be a potent anti-

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inflammatory compound able to suppress the expression of TNF-alpha mRNA and protein by

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inhibiting ERK phosphorylation in the Jurkat cells.

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Effect of javamide-II on the IL-2 expression in Jurkat cells.

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Because ERK phosphorylation was also reported to be significantly involved in IL-2 production

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in the Jurkat cells,24, 30 the effect of javamide-II on interleukin-2 (IL-2) was investigated to

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further substantiate its purported inhibition of ERK phosphorylation. As shown in Figure 11, IL-

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2 production was inhibited considerably by the treatment of javamide-II (

Javamide-II Found in Coffee Is Better than Caffeine at Suppressing

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