Modulation of arachidonic acid metabolism by curcumin and related ß-diketone derivatives: effects on cytosolic phospholipase A2, cyclooxygenases and 5-lipoxygenase

Jungil Hong, Mousumi Bose, Jihyeung Ju, Jae-Ha Ryu, Xiaoxin Chen, Shengmin Sang, Mao-Jung Lee and Chung S. Yang1

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA

1 To whom correspondence should be addressed Email: csyang{at}rci.rutger.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aberrant arachidonic acid metabolism is involved in the inflammatory and carcinogenic processes. In this study, we investigated the effects of curcumin, a naturally occurring chemopreventive agent, and related ß-diketone derivatives on the release of arachidonic acid and its metabolites in the murine macrophage RAW264.7 cells and in HT-29 human colon cancer cells. We also examined their effects on the catalytic activities and protein levels of related enzymes: cytosolic phospholipase A2 (cPLA2), cyclooxygenases (COX) as well as 5-lipoxygenase (5-LOX). At 10 µM, dibenzoylmethane (DBM), trimethoxydibenzoylmethane (TDM), tetrahydrocurcumin (THC) and curcumin effectively inhibited the release of arachidonic acid and its metabolites in lipopolysaccharide (LPS)-stimulated RAW cells and A23187-stimulated HT-29 cells. Inhibition of phosphorylation of cPLA2, the activation process of this enzyme, rather than direct inhibition of cPLA2 activity appears to be involved in the effect of curcumin. All the curcuminoids (10 µM) potently inhibited the formation of prostaglandin E2 (PGE2) in LPS-stimulated RAW cells. Curcumin (20 µM) significantly inhibited LPS-induced COX-2 expression; this effect, rather than the catalytic inhibition of COX, may contribute to the decreased PGE2 formation. Without LPS-stimulation, however, curcumin increased the COX-2 level in the macrophage cells. Studies with isolated ovine COX-1 and COX-2 enzymes showed that the curcuminoids had significantly higher inhibitory effects on the peroxidase activity of COX-1 than that of COX-2. Curcumin and THC potently inhibited the activity of human recombinant 5-LOX, showing estimated IC50 values of 0.7 and 3 µM, respectively. The results suggest that curcumin affects arachidonic acid metabolism by blocking the phosphorylation of cPLA2, decreasing the expression of COX-2 and inhibiting the catalytic activities of 5-LOX. These activities may contribute to the anti-inflammatory and anticarcinogenic actions of curcumin and its analogs.

Abbreviations: COX, cyclooxygenase; cPLA2, cytosolic PLA2; DBM, dibenzoylmethane; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; LOX, lipoxygenase; LPS, lipopolysaccharides; LTB4, leukotriene B4; MAPK, mitogen activated protein kinase; MEK, MAPK kinase; PLA2, phospholipase A2; PG, prostaglandin; PGE2, prostaglandin E2; TDM, trimethoxydibenzoylmethane; THC, tetrahydrocurcumin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Curcumin (diferuloyl methane) is a major constituent found in the spice turmeric, which is a dried powder from the rhizomes of Curcuma longa L.. This spice has been used as a traditional medicine for treatment of inflammation, gastrointestinal disorders, hepatic disorders, diabetic wounds, skin wounds, rheumatism, sinusitis and other disorders (1). Curcumin is also a food coloring and flavoring agent approved by the Food and Drug Administration.

Studies using chemically induced animal carcinogenesis models demonstrated the chemopreventive effect of curcumin in the colon, duodenum, forestomach, mammary glands, oral cavity and skin (27). A variety of mechanisms have been suggested for the anticarcinogenic effect of curcumin, including antioxidative activities, modulation of the cell cycle, inhibition of the enzymes related to tumor promotion such as ornithine decarboxylase, protein kinase C and inducible nitric oxide synthase, inhibition of epidermal growth factor receptor tyrosine kinases, inhibition of activator protein-1 (AP-1) and nuclear factor-{kappa}B (NF-{kappa}B), and inhibition of angiogenesis (812). The anticarcinogenic action of curcumin derivatives with ß-diketone structure, such as tetrahydrocurcumin (THC) and dibenzoylmethane (DBM), has also been reported (6,13).

Many studies have demonstrated the involvement of aberrant arachidonic acid metabolism in carcinogenesis. Membrane phospholipids, the major source of arachidonic acid are hydrolyzed by phospholipase A2 (PLA2); the released arachidonic acid is further metabolized by three different types of oxygenases: cyclooxygenase (COX), lipoxygenase (LOX) and cytochromes P450. Modulation of arachidonic acid metabolism by inhibiting these enzymes has been considered as an effective mechanism for chemoprevention. Inhibition of arachidonic acid metabolism by curcumin has been suggested to be a key mechanism for its anticarcinogenic action (1416). Curcumin has been reported to inhibit COX-2 expression in gastrointestinal cancer cells and mouse skin (1719). Several previous studies have also indicated that curcumin affects the formation of COX- and LOX-dependent metabolites and decreases activities of PLA2 and PLC{gamma}1 (20,21). However, the precise mechanisms involved in the decreased enzyme activities and metabolite formation are not clear. The inhibition could be due to the direct inhibition of the enzyme activities, to the decrease of protein levels of these enzymes or to altered molecular regulation. In the present study, we analyzed the effect of curcumin and related ß-diketone analogs on the release of arachidonic acid and its metabolites in the intact cells as well as catalytic activities and protein levels of related enzymes, cytosolic PLA2 (cPLA2), COX-1, COX-2 and 5-LOX, in the murine macrophage RAW264.7 cells and HT-29 human colon cancer cells. Our results suggest that curcumin and related ß-diketone derivatives are potent modulators of arachidonic acid metabolism. The results also indicate that curcumin can affect arachidonic acid metabolism by inhibiting the phosphorylation of cPLA2, expression of COX-2 and the enzyme activity of 5-LOX. Their inhibitory effect may be an important anti-inflammatory mechanism and may contribute to their anticarcinogenic actions.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and cell lines
[5,6,8,9,11,12,14,15-3H](N) Arachidonic acid and 1-palmitoyl 2-[1-14C] arachidonyl sn-glycero-3-phosphorylcholine were purchased from NEN Life Science (Boston, MA). Arachidonic acid metabolite standards, ovine COX-1 and COX-2, human recombinant 5-LOX and human COX-2 monoclonal antibody were from Cayman Chemical Company (Ann Arbor, MI). Phospho-cPLA2 (Ser505) and cPLA2 antibody were from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Anti-5-LOX antibody was obtained from Transduction Laboratories (Lexington, KY). Curcumin and related ß-diketone derivatives were generously provided by Dr M.-T.Huang of our department. The purity of curcumin and DBM was determined to be >95% by high performance liquid chromatography (HPLC), whereas THC and trimethoxydibenzoylmethane (TDM) was ~70% pure. Structures of these compounds are shown in Figure 1. Murine macrophage RAW264.7 and the HT-29 human colon cancer cell line were obtained from American Type Culture Collection (Rockville, MD). RAW264.7 and HT-29 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing L-glutamine, glucose and sodium bicarbonate, and McCoy 5A medium, respectively, supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin, at 37°C in 95% humidity and 5% CO2. All other chemicals were purchased from Sigma Chemical (St Louis, MO).



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Fig. 1. Structures of curcumin and related ß-diketone derivatives used in the present study.

 
Release of arachidonic acid and its metabolites in intact cell system
RAW264.7 and HT-29 cells were plated into a 24-well plate at ~3.5 x 105 and 2.0 x 105 cells/well, respectively, in the growth media. After 24 h, the media were removed and replaced with 1 ml of serum free DMEM for RAW cells or Ham's F-12 media for HT-29 cells containing 0.1 µCi/ml [5,6,8,9,11,12,14, 15-3H](N) arachidonic acid. The cells were incubated overnight, resulting in >90% of arachidonic acid incorporated into the cell membrane. The cells were washed twice with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin to remove unabsorbed arachidonic acid. RAW cells were stimulated with 2 µg/ml lipopolysaccharides (LPS) (from Escherichia coli, serotype 055:b5) for 1 h, and then incubated with fresh medium containing tested compounds or vehicle (final concentration, 0.1% DMSO) for 18 h. HT-29 cells were incubated with 10 µM A23187 for 20 min, and then were treated with tested compounds or vehicle in fresh medium for 90 min. After incubation, the culture medium was collected, and centrifuged for 10 min at 10 000 g. Radioactivity of the cell culture media was measured by a scintillation counter (Model LS3801, Beckman Coulter, Fullerton, CA). For analyzing prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels, RAW cells were treated in a similar manner as above without using radiolabeled arachidonic acid. After incubation, the culture medium was collected and the PGE2 and LTB4 levels were analyzed by an enzyme immunoassay (Cayman, Ann Arbor, MI).

Western blotting
RAW264.7 cells were plated into a 6-well plate at ~2 x 106 cells/well. After 24 h, the media were replaced with serum free DMEM for 24 h, and the cells were treated as described above. The cells were washed with ice cold PBS twice and lysed with cell lysis buffer (1 mM PMSF, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na2VO4, in 20 mM Tris, pH 7.4). The cell lysate was sonicated and centrifuged at 10 000 g for 15 min at 4°C. The supernatant containing 30–50 µg protein was loaded onto 10% or 4–15% gradient sodium dodecyl sulfate (SDS)–polyacrylamide gel. After electrophoresis, the proteins were transferred onto PVDF membrane and probed with antibodies for cPLA2, phospho-cPLA2, COX-2 and 5-LOX. The western blot was visualized using an ECL detection kit (Amersham, Arlington Heights, IL) and the densitometry was quantified using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). Protein concentration in the cell lysates was determined by the method of Bradford (Bio-Rad, Hercules, CA).

cPLA2 assays
For preparing the substrate solution, 1-palmitoyl 2-[1-14C]arachidonyl sn-glycero-3-phosphorylcholine (hot:cold, 1:3) and phosphoinositides were dried under a stream of N2 (g). Triton X-100 (2 mM) in 100 mM Tris–HCl buffer, pH 7.4, was added to the dried lipids, and the substrate micelles were prepared by sonification in a bath-sonicator for 3 min. cPLA2 activity was assayed at 37°C in a reaction mixture (100 µl) consisting of 20 µg microsomal protein from HT-29 cells (the enzyme source), 20 µM substrate, 5 µg/ml phosphoinositides, 100 µM CaCl2 and 200 µM Triton X-100 in 100 mM Tris–HCl buffer, pH 7.4. The reaction was initiated by adding 10 µl of substrate solution after a 5 min pre-incubation, and terminated after 30 min of incubation by the addition of 10 µl of 0.5 N HCl. Modified Dole's method was used for the extraction of free arachidonic acid (22). In brief, 5 vol (550 µl) of Dole solution (2-propanol:heptane:1 N H2SO4, 78:20:2) was added to the reaction mixture. After 10 min, arachidonic acid was extracted by adding 2 vol (220 µl) of water and 3 vol (330 µl) of heptane. The upper heptane layer was collected, and contaminated phospholipids were removed from the organic layer by treatment of silicic acid (30 mg, 100–200 mesh) twice. After separation of the heptane layer from silicic acid by centrifugation, 200 µl of the heptane layer was collected and measured for radioactivity in 4 ml scintillation cocktail.

COX and LOX assays
COX-dependent activity was measured at 37°C for 30 min in a reaction mixture (100 µl) consisting of 50 µg of RAW cell lysates, 20 µM (0.1 mCi) [3H]arachidonic acid, 1 mM reduced glutathione and 1 mM epinephrine in a 100 mM Tris–HCl buffer, pH 7.4. For the 5-LOX assay, the reaction mixture (100 µl), containing 0.5 U human recombinant 5-LOX, 20 µM (0.1 µCi) [3H]arachidonic acid, 2 mM CaCl2 and 1 mM ATP in a 100 mM Tris–HCl buffer, pH 7.4, was incubated at 37°C for 10 min with tested compounds. The reactions were terminated by the addition of 15 µl of 0.5 N HCl. An equal volume of ice cold acetonitrile was added to the reaction mixture and centrifuged at 10 000 g for 10 min. COX and 5-LOX metabolites in the supernatant were analyzed by a reverse phase HPLC system (23). For peroxidase assay with isolated ovine COX-1 and COX-2, each enzyme (25 U) was incubated at an ambient temperature in a reaction mixture (200 µl) consisting of 100 µM arachidonic acid, 170 µM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and 1 µM bovine hemin in a 100 mM Tris–HCl buffer, pH 7.4 (24). The absorbance change, at 590 nm due to oxidation of TMPD during the initial 5 min reaction, was analyzed in a microplate reader.

Data analysis
Statistical significance was evaluated using the Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of curcuminoids on the release of arachidonic acid and its metabolites in intact cells
The release of pre-labeled arachidonic acid and its metabolites from RAW264.7 murine macrophages to the culture media increased >2-fold after stimulation by LPS. The released radioactivity accounted for 3–5% of total labeled arachidonic acid during an 18-h incubation with the stimulated cells. HPLC profile showed that most of the released radioactivity was due to metabolites; arachidonic acid accounted for <3% of the total radioactivity in the media (data not shown). The time-dependent release of metabolites and its inhibition by curcumin are shown in Figure 2A. All the curcuminoids (at 10 µM) significantly decreased the release of arachidonic acid metabolites from RAW264.7 cells during the 18-h incubation (Figure 2B). TDM, THC and curcumin similarly inhibited the release by >50%, whereas DBM was slightly less effective. To study their effects on specific arachidonic acid metabolites, released PGE2 and LTB4 in the media from LPS-stimulated RAW cells were investigated. PGE2 formation was markedly increased after stimulation with LPS, and all curcuminoids (10 µM) strongly inhibited the formation of PGE2, with TDM showing the most potent inhibitory effect (Figure 2C). There was no significant change in the level of LTB4 after stimulation of RAW cells by LPS. Curcumin and THC also significantly decreased the level of LTB4 in the culture medium; TDM, however, increased the LTB4 level (Figure 2D).



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Fig. 2. Effects of curcuminoids on the release of arachidonic acid and its metabolites. RAW264.7 and HT-29 cells labeled with 0.1 µCi/ml [5,6,8,9,11,12,14, 15-3H](N) arachidonic acid were stimulated with 2 µg/ml LPS for 1 h and 10 µM A23187 for 20 min, respectively. Fresh medium containing 10 µM of curcuminoids or vehicle (final concentration, 0.1% DMSO) was then added. (A) Time-dependent release of arachidonic acid metabolites from RAW cells and its inhibition by curcumin. The radioactivity in culture medium was analyzed. (B) Inhibitory effects on the release of arachidonic acid and its metabolites by RAW264.7 cells after an incubation of 18 h. (C and D) Effects of curcuminoids on PGE2 and LTB4 levels in culture media after a 18-h incubation with RAW cells. The metabolites were analyzed by enzyme immunoassay. (E) Inhibitory effects of curcuminoids on A23187-stimulated arachidonic acid release from HT-29 cells. After a 90-min incubation with HT-29 cells, the radioactivity in the culture media was analyzed. (F) Effects of MAPK inhibitors (10 µM; PD, PD98059, and SB, SB203587) on the release of arachidonic acid and metabolites from RAW and HT-29 cells. Each bar represents the mean±SD (n = 4 in the case of A, B, E and F, n = 8 in the case of C and D). *,**Significantly different from control (*P<0.05; **P < 0.01).

 
A23187, a calcium ionophore, markedly increased arachidonic acid release in HT-29 cells, and all curcuminoids also significantly inhibited this event (Figure 2E). Curcumin, which showed the most potent inhibitory effect, inhibited the release in a concentration-dependent manner with an estimated IC50 of 4.4 µM, and the inhibition was significant even at a 1-µM concentration (by ~17%) in HT-29 cells (data not shown). Curcumin also significantly inhibited A23187-induced arachidonic acid release in other types of cancer cells and normal immortalized cells including HCT-116 human colon adenocarcinoma cells, KYSE-150 and KYSE-450 human esophageal squamous carcinoma cells, and IEC-6 immortalized rat intestinal cells to a similar extent (data not shown). To explore the involvement of mitogen activated protein kinases (MAPKs) in the release of arachidonic acid and metabolites from RAW or HT-29 cell system, the effects of PD98059, a MEK (MAPK kinase) inhibitor, or SB203587, a p38 and c-jun N-terminal kinase (JNK) inhibitor, were investigated. PD98059 significantly inhibited the release from LPS-stimulated RAW264.7 cells, but SB203587 was not effective (Figure 2F). Both inhibitors did not show any appreciable inhibition in A23187-stimulated HT-29 cells.

Effects on cPLA2
Among several types of PLA2, cPLA2 has been reported to play a major role in releasing arachidonic acid from membrane phospholipids (25). In order to evaluate the potential of curcuminoids for the inhibition of catalytic activity of cPLA2, the microsomal fraction from HT-29 cells was used as an enzyme source. The activity was almost exclusively due to cPLA2, because (i) the microsomal fraction showed a 7.7-fold increase of PLA2 activity in the presence of 5 µg/ml phosphoinositides and 100 µM of calcium (data not shown), and (ii) it was almost completely abolished by methyl arachidonyl fluorophosphate (a cPLA2 inhibitor) and EGTA (a calcium chelator), but not significantly affected by bromoenol lactone (an iPLA2 inhibitor) and dithiothreitol (a sPLA2 inhibitor) (data not shown). Among the curcuminoids (50 µM), only THC inhibited cPLA2 activity (by 36%), but curcumin slightly increased the activity (Figure 3A). To account for the strong inhibition of the release of arachidonic acid and its metabolites in intact cells, we investigated the effect of curcumin on the protein level and phosphorylation of cPLA2. Treatment of RAW264.7 cells with LPS induced the phosphorylation of cPLA2 at Ser505 without changing the protein level. Incubation of curcumin for different periods of time with LPS-stimulated cells decreased the level of phospho-cPLA2 and the effect was concentration-dependent (Figure 3). Significant inhibition was observed even with 5 µM of curcumin.



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Fig. 3. Effects of curcuminoids on cPLA2 activity from microsomal fraction of HT-29 cells and on the levels of cPLA2 and phospho-cPLA2 in LPS-stimulated RAW264.7 cells. (A) The reaction mixture (100 µl) consisted of 20 µg microsomal protein from HT-29 cells, 20 µM (0.0024 µCi) 1-palmitoyl 2-[1-14C]arachidonyl sn-glycero-3-phosphorylcholine, 5 µg/ml phosphoinositides, 100 µM CaCl2 in 100 mM Tris–HCl buffer, pH 7.4, with 5(hollow bar) or 50(filled bar) µM of curcuminoids. The reactions were carried out at 37°C for 30 min. The results are the mean ± SD (n = 3). (B and C) Time- and concentration-dependent effects of curcumin on levels of total cPLA2 and phospho-cPLA2 in LPS-stimulated RAW264.7 cells. Cells were incubated with 20 µM of curcumin or vehicle (DMSO) for different time periods or with different concentrations of curcumin for 18 h followed by treatment with 2 µg/ml LPS for 1 h. Western blot analysis was performed on cell lysates with antibodies against cPLA2 or phospho-cPLA2 (Ser505). The results are representative of two (B) or three (C) independent experiments. Lower panel in (C) shows the densitometry quantification of phospho-cPLA2 level normalized to each control (mean ± SD, n = 3). *,**Significantly different from control (*P < 0.05; **P < 0.01).

 
Effects on COX
RAW264.7 cell lysates were used as an enzyme source for COX-dependent activity. Without LPS stimulation, the lysate did not have measurable COX-dependent activity. The lysate from LPS-stimulated RAW cells, however, actively catalyzed arachidonic acid metabolism (Figure 4). The formation of arachidonic acid metabolites was almost completely inhibited by 20 µM indomethacin, a COX inhibitor (Table I), whereas nordihydroguaiarectic acid, a LOX inhibitor, did not affect the reaction (data not shown). The western blot analysis showed that the COX-2 protein level was not clearly detected in non-stimulated RAW cells, and COX-2 was markedly increased by LPS-stimulation (Figure 4). The results indicate that the metabolites were produced mainly by COX-2. After a 30-min incubation of arachidonic acid with the lysates of LPS-stimulated RAW cell, several products were formed, including PGF2{alpha}, PGE2, PGD2, 2-hydroxyheptadecatrienoic acid (HHT) and hydroxyeicosatetraenoic acid (HETEs) (Figure 4B). All curcuminoids (50 µM) inhibited the COX-dependent arachidonic acid metabolism by 8–32% when the sum of the metabolites was compared. THC showed the most potent inhibitory effect, followed by curcumin, showing 32 and 23% inhibition, respectively; DBM and TDM showed only ~10% inhibition. Among the COX-dependent metabolites, inhibition of PGD2 formation by curcumin was most pronounced, but PGE2 formation was slightly increased (Table I).



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Fig. 4. COX-2 levels and chromatograms of [3H]arachidonic acid metabolites in lysates from unstimulated RAW264.7 cells (A) and LPS-stimulated (18 h incubation) RAW264.7 cells (B). COX-2 levels were determined by western blot. The incubation mixture (100 µl) consisted of RAW264.7 cell lysates (50 µg protein), 20 µM (0.1 µCi) arachidonic acid, 1 mM glutathione and 1 mM epinephrine in 100 mM Tris–HCl buffer, pH 7.4. The reactions were carried out at 37°C for 30 min. (HHT, 2-hydroxyheptadecatrienoic acid; AA, arachidonic acid).

 

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Table I. Effects of curcuminoids (50 µM) and indomethacin (20 µM) on COX-dependent arachidonic acid metabolites by LPS-stimulated RAW264.7 cell lysates

 
In order to determine the selectivity of curcuminoids in inhibiting the two COX isoforms, their effects on the peroxidase activity of isolated ovine COX-1 and -2 were analyzed. All curcuminoids generally showed more potent inhibition against COX-1 than COX-2 (Figure 5). Curcumin showed the strongest inhibitory effect on the peroxidase activity of ovine COX-1 with an estimated IC50 of ~50 µM (Figure 5). Other curcuminoids (50 µM) all inhibited COX-1 by ~20%.



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Fig. 5. Different effects of curcuminoids on ovine COX-1 and COX-2. The incubation mixture (200 µl) consisted of 25 U of ovine COX-1 or COX-2, 100 µM arachidonic acid, 1 µM hemin and 170 µM TMPD with curcumin in 100 mM Tris–HCl buffer, pH 7.4, with or without of curcuminoids. The reactions were carried out at an ambient temperature for 5 min. The peroxidase activity of COX was analyzed using microplate reader at 590 nm. **Significantly different between the effects on COX-1 and COX-2 (P < 0.01).

 
Stimulation of RAW264.7 cells with LPS increased the COX-2 protein level within 6 h, and the level was sustained until 24 h (Figure 6A). Curcumin (20 µM) significantly decreased the LPS-induced COX-2 expression in an 18-h treatment (Figure 6B). In another experiment, the time-dependent induction of COX-2 by LPS was seen, and at each time point marked inhibition by curcumin was observed (Figure 6C). Interestingly, curcumin induced COX-2 expression in RAW264.7 cells in the absence of LPS-stimulation, and the COX-2 protein level peaked at 14 h (Figure 6C), whereas resting cells did not show any appreciable COX-2 expression during the 24-h incubation time (data not shown).



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Fig. 6. Effects of curcumin on COX-2 expression in RAW264.7 cells. RAW264.7 cells were incubated with 20 µM of curcumin or vehicle (DMSO) for different time periods (A) or for 18 h (B), followed by treatment with 2 µg/ml LPS for 1 h. Western blot analysis was performed on cell lysates with antibodies against COX-2. (C) RAW264.7 cells were incubated with curcumin (30 µM), LPS or curcumin and LPS for the time periods indicated. Densitometry quantification of COX-2 level was normalized to each LPS control (mean ± SD) (B), or to unstimulated control (C). The results are representative of three (B) or two (A and C) independent experiments. *Significantly different from control (P < 0.05).

 
Effects on 5-LOX
Since curcumin and THC significantly decreased LTB4 level (Figure 2D), the effect of curcumin and THC on the 5-LOX pathway was investigated. When human recombinant 5-LOX was incubated with arachidonic acid, a major peak with the same retention time as 5-HETE (~57 min) and several unidentified peaks were produced (Figure 7). AA-861, a specific 5-LOX inhibitor, selectively inhibited the formation of the major peak. Significant inhibition of this metabolite by curcumin (2 µM) was also observed. Inhibitors for 5-LOX activating protein (MK-886) and LTA4 hydrolase (bestatin) did not affect the reaction (data not shown).



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Fig. 7. Effects of curcumin and THC on 5-LOX activity. Chromatograms of [3H]arachidonic acid metabolites by human recombinant 5-LOX preparation were shown from a reaction with 5-LOX (A), in the presence of 20 µM AA-861, a 5-LOX inhibitor (B) or in the presence of 2 µM curcumin (C). (D) Concentration-dependent inhibitory effect of curcumin (circle) and THC (square) on the formation of 5-HETE by human recombinant 5-LOX. The incubation mixture (100 µl) contained 0.5 U of 5-LOX, 20 µM (0.1 µCi) arachidonic acid, 2 mM CaCl2 and 1 mM ATP with different concentrations of curcumin or THC in 100 mM Tris–HCl buffer, pH 7.4. The reactions were carried out at 37°C for 10 min, and the products were analyzed by HPLC. Each symbol represents the mean of duplicates. (E) Effects of curcumin on 5-LOX protein level in LPS-stimulated RAW264.7 cells. Cells were incubated with different concentrations of curcumin or vehicle (DMSO) for 18 h followed by treatment with 2 µg/ml LPS for 1 h. Western blot analysis was performed on cell lysates with an antibody against 5-LOX. Lower panel in (E) shows the quantification of 5-LOX level normalized to each control (mean ± SD, n = 3). *Significantly different from control (P < 0.05).

 
Curcumin and THC inhibited the formation of 5-HETE by human recombinant 5-LOX dose-dependently. The IC50 values of curcumin and THC were calculated to be 0.69 and 2.99 µM, respectively (Figure 7D). The effect of curcumin on the 5-LOX protein level in LPS-stimulated RAW cells was also investigated. The protein level of 5-LOX was not significantly changed after stimulation with LPS, and curcumin down-regulated 5-LOX protein at 20 µM (by 38%) after 18 h of treatment.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aberrant arachidonic acid metabolism occurs in inflammatory and carcinogenic processes, and modulation of arachidonic acid metabolism has been suggested to be an important strategy for cancer prevention (2628). The present study demonstrates that curcumin and related ß-diketone derivatives effectively inhibit the release of arachidonic acid and its metabolites from murine macrophage RAW264.7 cells and HT-29 cells. The release of arachidonic acid by PLA2 from membrane phospholipids is usually the rate-limiting step for further arachidonic acid metabolism. Among several types of PLA2, cPLA2 plays a major role in catalyzing the release of arachidonic acid in most tissues (25,29). Our observation that curcuminoids are not effective in inhibiting the catalytic activity of cPLA2 suggests that other mechanisms may be involved. Indeed, the results in Figure 3 demonstrate that curcumin inhibited the LPS-induced phosphorylation of cPLA2 without affecting its protein level. Therefore, inhibition of cPLA2 phosphorylation appears to be an important mechanism for decreasing arachidonic acid release by curcumin. This mechanism may also account for the observation by Rao et al. that oral administration of curcumin significantly decreased the PLA2 activity by ~50% in the colonic mucosa and tumors of azoxymethane-treated rats (21).

Several previous studies indicated that LPS stimulates MAPKs, including extracellular signal-regulated kinases 1/2, p38 and JNK in cancer cells and inflammatory cells (3032). The MAPKs also induce phosphorylation of cPLA2 either directly or indirectly (3336). Curcumin is reported to inhibit the MAPKs (19,37,38). In our RAW cell system, PD98059, an MEK inhibitor, significantly inhibited arachidonic acid release, whereas SB203587, a p38 and JNK inhibitor, did not show the inhibition. Therefore, it is probable that curcumin also inhibits a MEK-related pathway, resulting in the inhibition of cPLA2 phosphorylation. Nevertheless, this mechanism may not apply to the effect of curcuminoids on A23187-induced arachidonic acid release in HT-29 cells, since inhibition of MAPKs had no effect in this system. Stimulation with A23187 is known to induce a sustained calcium mobilization, but only weak activation of MAPKs (36). The modulation of calcium mobilization by curcuminoids may also be important in the inhibition of arachidonic acid release, and this mechanism remains to be investigated.

In LPS-stimulated RAW cell lysates, the COX-dependent activity is believed to be due mainly to COX-2. The inhibition of THC and curcumin on COX activity appears to be higher than that of DBM and TDM (Table I), suggesting that the phenolic group of THC and curcumin is important for the inhibition. Considering the effect of curcuminoids on the catalytic activity of COX, it may not be a major mechanism for inhibition of PGE2 formation in intact cells (Figure 2C). Their inhibition might be due mostly to modulation of expression of COX-2 as illustrated by the data in Figure 6. The possible effects of curcuminoids on the down-stream enzyme PGE synthase and others also remain to be further investigated.

Apparently, COX-2 is less susceptible to inhibition by curcuminoids than COX-1 (Figure 5). Huang et al. reported that curcumin (100 µM) almost completely inhibited the formation of COX-dependent metabolites by mouse epidermis (20). Our results are somewhat different, showing that curcumin (50 µM) inhibited the activity by <25%. It is possible that the major source of COX isoform from mouse epidermis in the previous study was COX-1. It was reported that curcumin also decreased the level of COX-dependent metabolites in colonic mucosa and tumors of rats (21). Since our results indicate that curcumin inhibits COX-1 and shows similar inhibitory effects on arachidonic acid release in both normal immortalized and cancer cells, there is no selective advantage in its action against cancer cells versus normal cells. In addition to the inhibition of COX activity by curcumin, the decrease of COX-2 protein level, as observed herein, could play an even more important role. The decrease of COX-2 expression at the protein or mRNA levels by curcumin has been observed in several gastrointestinal cells and mouse skin (1719). The mechanism involved in the repression has been suggested to be due to its inhibitory effect of curcumin on AP-1 and NF-{kappa}B. Curcumin, however, increased the COX-2 protein level in RAW cells without LPS-stimulation. It is possible that curcumin may act as a stress factor to activate related signaling, including AP-1 and NF-{kappa}B in the cells under resting status.

Metabolites of 5-LOX are reported to be important regulators in the proliferation and apoptosis of cancer cell lines (39,40). They are believed to be involved in tumor development by affecting cell proliferation, inflammation, apoptosis and angiogenesis (27). We found that curcumin and THC showed potent inhibitory effects on the catalytic activity of 5-LOX. It was reported that curcumin can bind to the active site of the soybean LOX, inhibit the enzyme activity competitively and become oxygenated (41,42). Since curcumin has a potent inhibitory effect on 5-LOX, this activity may contribute significantly to its anticarcinogenic activities. Considering the effects of DBM and TDM on LTB4 formation in RAW cells, the phenolic group and the size of the molecule appear to be more important than the ß-diketone structure in the inhibition of 5-LOX.

The overall schemes of arachidonic acid metabolism modulated by curcumin are illustrated in Figure 8. Curcumin can modulate arachidonic acid metabolism at several targets, including inhibition of phosphorylation of cPLA2, inhibition of COX-2 protein expression and catalytic activity (although weakly), and inhibition of 5-LOX activity. Dual inhibition of 5-LOX/COX has been suggested to be a desirable approach in the development of new drugs for anti-inflammation and chemoprevention (43,44). Curcumin and THC are demonstrated herein to fit in this category of agents. They can be utilized as chemopreventive agents or for therapeutic purposes if a high enough concentration of the agents can be delivered to the target sites.



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Fig. 8. Proposed targets for modulation of arachidonic acid metabolism by curcumin in cells. Curcumin modulates arachidonic acid metabolism at different stages by inhibiting phosphorylation of cPLA2 (the activation process of the enzyme), inhibiting COX-2 protein expression and catalytic activity, and inhibiting 5-LOX activity (T, possible inhibitory targets of curcumin).

 

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 Abstract
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 Materials and methods
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 References
 
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Received January 14, 2004; revised March 23, 2004; accepted April 3, 2004.