Effects of Tea Polyphenols on Arachidonic Acid Metabolism in Human

Chapter 4

Effects of Tea Polyphenols on Arachidonic Acid Metabolism in Human Colon

Downloaded by FUDAN UNIV on April 8, 2017 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch004

Jungil Hong and Chung S. Yang* Laboratory for Cancer Research, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8520

Tea has been suggested to be a possible cancer chemopreventive agent, and tea polyphenols are believed to be responsible for its anti-carcinogenic effect. Since modulation of arachidonic acid metabolism has been demonstrated to be an important target for cancer chemoprevention, the effects of green and black tea polyphenols on human colonic enzymes involved in arachidonic acid metabolism were investigated. A t a concentration of 30 μg/mL, (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin-3gallate (ECG) from green tea and theaflavins from black tea inhibited lipoxygenase (LOX)-dependent activity by 30 to 75%. Tea polyphenols (30 μg/mL) inhibited cyclooxygenae (COX)-dependent arachidonic acid metabolism in microsomes from normal colon mucosa by 37 to 62%. E G C G and E C G (50 μΜ) inhibited cytosolic phospholipase A2 (cPLA2) activity by 15 and 30%, respectively. Theaflavins (50 μΜ) inhibited c P L A activity by 10-45%. The inhibition of arachidonic acid metabolism at multiple levels by tea polyphenols may decrease the risk of human colon cancer. 2

© 2003 American Chemical Society Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction Environmental factors are important in the causation of cancers. Diet is believed to account for about one-third of cancer cases in the United States (/). Dietary modification has been suggested as an important way for cancer chemoprevention. Many dietary factors have been studied as possible cancer chemopreventive agents. Among them, tea polyphenols have shown some promise. Tea (Camellia sinensis) is one of the most commonly consumed beverages in the world. Green tea contains polyphenols including (-)-epigallocatechin-3gallate (EGCG), (-)-epigallocatechin (EGC), epicatechin-3-gallate (ECG) and ()-epicatechin (EC), which account for up to 30 % of dry weight of the water extractable material in brewed tea. In black tea, through an oxidation and polymerization process, a significant portion of the catechins is converted to theaflavins (TFs) such as TF, TF monogallates (TF3-G, and TF3'-G), TF digallate (TFdi-G), and higher molecular weight polymers which are responsible for the dark brown color of the black tea (2). Among the many biological effects of tea, the anticarcinogenic effect has been extensively investigated. The inhibitory action of tea and tea constituents against chemically-induced carcinogenesis has been demonstrated in many animal models including those for the skin, lung, esophagus, stomach, liver, small intestine, pancreas, colon, bladder, prostate, and mammary glands (3-7). Several epidemiological studies also support a protective role of tea against development of colorectal, uterine, and gastric cancers (3,8,9). A variety of mechanisms have been suggested for the anticarcinogenic effect of tea polyphenols, including antioxidative activities, inhibition of many enzymes related to the tumor promotion such as ornithine decarboxylase, protein kinase C, cycloxygenase and lipoxygenase, inhibition of activator protein-1, and the inhibition of angiogenesis (3,10-15). Colorectal cancer is one of the most frequent types of cancer in the Western countries, and remains the second leading cause of cancer death in the United States (16). The importance of archidonic acid metabolism in colorectal carcinogenesis has been demonstrated by the observation that individuals regularly using nonsteroidal anti-inflammatory drugs (NSAIDs), which are inhibitors of C O X , showed significantly low incidence and mortality rates of colorectal cancer (17). Arachidonic acid is metabolized by three types of enzymes; COXs, LOXs and cytochromes P450. Arachidonic acid metabolism is frequently elevated in various tumors and its inhibitors effectively suppressed tumorigenesis. Consequently, the modulation of arachidonic acid metabolism has become a potentially important approach for cancer chemoprevention. The overall scheme of arachidonic acid metabolism is shown in Figure 1. In this

Shahidi et al.; Food Factors in Health Promotion and Disease Prevention ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Membrane phospholipids ^ Phospholipase Â 2 S

-

COOH

Arachidonic acid Cyclooxygenases


Lipoxygenase^ 5-, 12-, 15HETEs

I Leukotrienes Lipoxin

Prostaglandin Kb

P450 Oxidized metabolites

Prostaglandins Thromboxanes Prostacyclins

Figure 1. The overall scheme of arahcidonic acid metabolism. study, the effects of tea polyphenols on several important enzymes involved in arachidonic acid metabolism in human colon have been investigated.

Results and Discussion

Tea polyphenols and Lipoxygenase L O X s catalyze the oxygenation of arachidonic acid to produce hydroxyeicosatetraenoic acids (HETEs). 5-LOX, 12-LOX and 15-LOX have been identified in humans. HETEs and their metabolites are reported to be important regulators in proliferation and apoptosis in cancer cell lines (18-20). The effects of tea polyphenols and nordihydroguiaretic acid (NDGA) on L O X - dependent arachidonic acid metabolism in human colon are shown in


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Figure 2. In the current assay system, 15, 12, and 5-HETEs were produced during incubation of human colon cytosol with [ C]-arachidonic acid, which were inhibited almost completely by N D G A , a general L O X inhibitor, implying that the metabolites are LOX-dependent. Tea polyphenols (30 μg/mL) inhibited LOX-dependent arachidonic acid metabolism by 30-74%. E C G showed the most potent inhibitory effects, whereas black tea TFs displayed relatively weaker activity on a weight basis. Tea polyphenols inhibited the formation of 5, 12, and 15-HETEs to about the same extent.


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Figure 2. The inhibitory effect of tea polyphenols and nordihydorguiaretic acid (NDGA) on LOX-dependen arachidonic acid metabolites in cytosols from human colon. The incubation mixture contained 0.6 mg cytosolicprotein, 20 μΜ (0.22 μΟί) arachidonic acid and 2 mM CaCl with or without 30 μg/mL tea polyphenols or 20 μΜ NDGA in 100 mM Tris-HCl buffer, pH 7.4. The reactions (200 μί) were performed at 37° C for 30 min. The reaction products were analyzed by HPLC. Percent inhibition was calculated based on the sum of the total metabolites. The data are the mean ±S.E. from 3 independent experiments. The result of NDGA is the mean of duplicate experiments (Data from reference 13). 2


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Although there are some conflicting reports regarding the role of 15-LOX in carcinogenesis (27), inhibition of LOXs is generally considered to decrease tumor formation (22). LOXs are regulated at several different stages such as expression, up-regulation by cellular signal cascades, and activation by 5-LOX activating protein (FLAP). In addition to the catalytic inhibition observed herein, tea polyphenols may interrupt the multiple steps in the regulation of L O X s .

Tea polyphenols and Cycloxygenases C O X metabolizes arachidonic acid to prostaglandin G (PGG ), which is subsequently reduced to prostaglandin H (PGH ). P G H is further metabolized to eicosanoids such as prostaglandins, prostacyclin, and thromboxane by prostaglandin synthase, prostacyclin synthase and thromboxane synthase, respectively. Two C O X isoforms, COX-1 and COX-2, have been identified. Overexpression of COX-2, an inducible C O X isoform, has been observed in various types of cancers, including colorectal cancer (25-27). Prostaglandin E (PGE ), a major metabolite derived from C O X - catalyzed arachidonic acid metabolism, was reported to be involved in cell hyperproliferation, mitogenesis, tumor cell invasiveness, and angiogenesis as well as in the inhibition of apoptosis (28-31). Many experimental animal studies using NSAIDs also support the idea that inhibition of C O X is an important approach for prevention of cancers. As shown in figure 3, tea polyphenols (30 μg/mL) inhibited C O X dependent arachidonic acid metabolism in microsomes from normal colon mucosa by 37-62 %. Among the metabolites, thromboxane and 12hydroxyheptadecatrienoic acid were inhibited to a greater extent. E C G also exhibited strongest inhibitory action (on a weight basis), displaying non competitive inhibition with a Ki value of 16.9±1.3 μΜ by using ovine COX-1 (13). The IC o values (in μΜ) of each tea polyphenol in COX-dependent arachidonic acid metabolism in normal colon mucosa follow the order of TFdiG (41.8) < E C G (54.9) < E G C G (72.1) < TFs (92.8) < E G C (152.8). The inhibitory effect of tea polyphenols was generally less pronounced in colon tumor tissues (Figure 3). Black tea polyphenols with T F backbone enhance P G E formation in tumor (not normal) microsomes (73). The enhancement appears to be dependent on COX-2, which is frequently overexpressed in tumor tissues. Since all TFs showed the inhibitory effect on isolated ovine COX-2, the increase in P G E might not be the result of direct activation of COX-2 but due to the stimulation of an interaction between COX-2 and other microsomal factors. Black tea has shown relatively weaker anti-carcinogenic effects compared to green tea or no effects (32). The enhancing effect on the formation of P G E , one 2


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2

2

2

2

5

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2

2


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100

α Q


ο ο

EGCG

EGC

ECG

TFs

TFdiG

IM

Figure 3. The effects of tea polyphenols and Indomethacin (IM) on COXdependent metabolism of arachdonic acid in human colon. Products were analyzed from the reaction with 0.1 mg microsomal protein, 20 μΜ (0.22 μ€ΐ) arachidonic acid, and 1 mM glutathione, 1 mM epinephrine and 10 mMEDTA with or without 30 μ^πιΐ tea polyphenols or 5 μΜ IM in 100 mM Tris-HCl buffer, pH 7.4. The reaction was carried out at 37 °C for 30 min. Percent inhibition was calculated based on the sum of the total metabolites. The data are the mean ±S.E.from 4 or 5 independent experiments. The result of indomethacin is the mean of duplicate experiments. ' Significantly different effects (p

Effects of Tea Polyphenols on Arachidonic Acid Metabolism in Human

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