Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats
Takuji Tanaka5,
Hiroyuki Kohno1,
Reona Shimada,
Seiko Kagami,
Fumio Yamaguchi2,
Shigehiro Kataoka2,
Toshiaki Ariga2,
Akira Murakami3,
Koich Koshimizu3 and
Hajime Ohigashi4
Department of Pathology and
1 Department of Serology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293,
2 Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037,
3 Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Iwade-Utita, Wakayama 649-6493 and
4 Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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Abstract
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The modifying effects of dietary feeding of a polyisoprenylated benzophenone, garcinol, isolated from Garcinia indica fruit rind on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of garcinol on proliferating cell nuclear antigen (PCNA) index in ACF and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in liver. In addition, we examined the effects of garcinol on 12-O-tetradecanoylphorbol-13-acetate-induced O2 generation in differentiated human promyelocytic HL-60 cells and lipopolysaccharide (LPS)- and interferon (IFN)-
-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Western blotting analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression was done in LPS- and IFN-
-treated mouse macrophage RAW 264.7 cells. Rats were given subcutaneous injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received the experimental diet containing 0.01 or 0.05% garcinol for 5 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 97 ± 15 ACF/rat at the end of the study (week 5). Dietary administration of garcinol caused significant reduction in the frequency of ACF: 72 ± 15 (26% reduction, P < 0.01) at a dose of 0.01% and 58 ± 8 (40% reduction, P < 0.001) at a dose of 0.05%. Garcinol administration significantly lowered PCNA index in ACF. Feeding of garcinol significantly elevated liver GST and QR activities. In addition, garcinol could suppress O2 and NO generation and expression of iNOS and COX-2 proteins. These findings might suggest possible chemopreventive ability of garcinol, through induction of liver GST and QR, inhibition of O2 and NO generation and/or suppression of iNOS and COX-2 expression, on colon tumorigenesis.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; CDNB, 1-chloro-2,4-dinitrobenzene; COX-2, cyclooxygenase-2; EGCG, ()-epigallocatechin gallate; GST, glutathione S-transferase; INF, interferon; iNOS, inducible nitric oxide synthase; IR, inhibitory rate; LPS, lipopolysaccharide; NO, nitric oxide; O2, superoxide anion; PCNA, proliferating cell nuclear antigen; QR, quinone reductase; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction
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Dietary factors play an important role in human health and in the development of certain chronic diseases including cancer (1,2). Some foods contain antitumor compounds as well as mutagens and/or carcinogens (3). Recent research has also focused on the presence of minor constituents or non-nutrients, which possess antimutagenic and anticarcinogenic properties, in diets (4). Such compounds are candidates for chemopreventive agents against cancer development in humans. We have previously reported inhibitory effects of natural non-nutritive compounds on the development of aberrant crypt foci (ACF) (57), which are precursor lesions for colon carcinoma (810).
A number of compounds including flavonoids are potential antioxidants and could scavenge free radicals and chelate metal ions (11). Oxidative stress is closely associated with carcinogenic processes by its capacity for causing DNA mutation, protein modification and, thereby, cellular damage. In particular, carcinogenesis related to chronic inflammation caused by bacterial or viral infection in certain organs might be due to the excessive and prolonged oxidative insults of neutrophils and macrophages (3). Superoxide anion (O2) generation from leukocytes depends on the action of multi-component NADPH oxidase (12) and nitric oxide (NO) on inducible nitric oxide synthase (iNOS) (13). In colon carcinogenesis, increased expression of iNOS was found in both human and rat colonic tumors (14,15). Recently, it was reported that an iNOS-selective inhibitor effectively inhibits colonic ACF (16). Cyclooxygenase-2 (COX-2), induced by several stimuli associated with inflammation, is involved in carcinogenesis, including colon tumorigenesis of human (17) and rodents (18). Increased COX-2 expression was reported in the polyps of a mouse familial adenomatous polyposis model (19). When COX-2 was inactivated, the number and size of the intestinal polyps were dramatically reduced in this model (19). Furthermore, COX-2 specific inhibitors could inhibit chemically induced colon carcinogenesis (20,21). A recent in vitro study by Suh et al. (22) revealed differentiating, antiproliferative and anti-inflammatory effects of a novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, on several cultured cell lines. The compound could suppress the abilities of various inflammatory cytokines, including interferon (INF)-
, interleukin-1 and tumor necrosis factor-
to induce de novo formation of iNOS and COX-2. Thus, it is possible that certain compounds with suppressive effects on O2 and NO generation and expression of iNOS and COX-2 are chemopreventive agents against colon carcinogenesis.
A polyisoprenylated benzophenone, garcinol (Figure 1
), also named camboginol (23), is present in Guttiferae (Garcinia indica, Garcinia huillkensis and Garcinia cambogia). Garcinia is a rich source of secondary metabolites including xanthones, flavanoids, benzophenones, lactones and phenolic acids (24 and references therein). Garcinol is known to be antioxidant (25).
In the present study, with a short-term rat ACF bioassay using the colon carcinogen azoxymethane (AOM), we assessed the possible inhibitory action of garcinol in ACF formation. Since certain phase II detoxifying enzyme inducers are considered to be promising chemopreventive agents against cancer (26), effects of garcinol on liver glutathione S-transferase (GST) and quinone reductase (QR) activities were also assayed. We report here the inhibitory effects of garcinol on ACF development through increase in liver GST and QR activities and/or suppression of cell proliferation activity cell in colonic crypts. In addition, garcinol suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced O2 generation in differentiated human promyelocytic HL-60 cells and lipopolysaccharide (LPS)- and IFN-
-induced NO generation in mouse macrophage RAW 264.7 cells.
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Materials and methods
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Animals, chemicals, diets and cells
Male F344 rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan) aged 5 weeks were used for an ACF assay. The animals were maintained in the Kanazawa Medical University Animal Facility according to the Institutional Animal Care Guidelines. All animals were housed in plastic cages (four or five rats/cage) with free access to drinking water and a basal diet, CE-2 (CLEA Japan Inc., Tokyo, Japan), under controlled conditions of humidity (50 ± 10%), lighting (12 h light/dark cycle) and temperature (23 ± 2°C). They were quarantined for 14 days and randomized by body weight into experimental and control groups. AOM for ACF induction was purchased from Sigma Chemical Co. (St Louis, MO, USA). Garcinol (>98% purity) purified from G.indica dried fruit rind (`Kokum', purchased from Indo World Trading Co., New Delhi, India) was provided from Kikkoman Corporation (Noda City, Japan). Powdered CE-2 diet was used as basal diet throughout the study. Experimental diet was made by mixing garcinol in powdered basal diet CE-2 at a concentration (w/w) of 0.01 or 0.05%. TPA was obtained from Research Biochemicals International (Natick, MA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Gibco BRL. LPS (Escherichia coli serotye 0127, B8) was purchased from Difco Labs (Detroit, MI) and IFN-
from Genzyme (Cambridge, MA). Cytochrome c was obtained from Sigma Chemical Co. All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). HL-60 cells (no. CCL 240) were purchased from American Type Culture Collection. RAW 264.7 cells were kindly donated by Otsuka Pharmaceutical Co. (Otsu, Japan).
Experimental procedure for ACF assay
Thiry-three male F344 rats were divided into four experimental and control groups. Groups 13 were initiated with AOM by three weekly s.c. injections (15 mg/kg body wt). AOM injections were performed between 10:00 a.m. and 11:00 a.m. Rats in groups 2 and 3 were fed the diets containing garcinol at 0.01 and 0.05%, respectively, for 5 weeks, starting 1 week before the first dosing of AOM. Group 4 was given the diet containing 0.05% garcinol alone. Group 5 served as an untreated control. Rats were killed at week 5 by CO2 asphyxiation, to assess the incidences of colonic ACF. They underwent careful necropsy, with emphasis on the colon, liver, kidney, lung and heart. All grossly abnormal lesions in any tissue, and the organs such as liver, kidney, lung and heart were fixed in 10% buffered formalin solution.
Determination of ACF
The frequency of ACF was determined according to the method described in our previous report (6). At necropsy, the colons were flushed with saline, excised, cut open longitudinally along the main axis and then washed with saline. They were cut and fixed in 10% buffered formalin for at least 24 h. Fixed colons were dipped in a 0.5% solution of methylene blue in distilled water for 30 s, briefly washed with the distilled water and placed on a microscope slide with the mucosal surface up. Using a light microscope at a magnification of x40, ACF were distinguished from the surrounding `normal-appearing' crypts by their increased size (8).
Proliferating cell nuclear antigen (PCNA) immunohistochemistry
Immunohistochemical staining for PCNA was performed by the avidinbiotin complex method (Vecstain Elite ABC kit; Vector, Burlingame, CA). Tissue sections were deparaffinized with xylene, hydrated through a graded ethanol series, immersed in 0.3% hydrogen peroxide in absolute methanol for 30 min at room temperature to block endogenous peroxidase activity and then washed in phosphate-buffered saline (pH 7.2). Following incubation with normal rabbit serum at room temperature for 10 min to block background staining, the sections were incubated with an anti-PCNA antibody (mouse monoclonal PC10; Dako, Kyoto, Japan; a 1:100 dilution) for 12 h in a humidified chamber at room temperature. They were then reacted with 3,3'-diaminobenzidine and counterstained with Harris' hematoxylin. For determination of PCNA-positive index, 10 full-length crypts (aberrant crypts, `normal-appearing' crypts or normal crypts) of each colon were examined. The number of PCNA positively stained nuclei in each crypt column was recorded. The PCNA-positive index (number of positive stained nucleix100/total number of nuclei counted) was then calculated. The scorer was unaware of the group to which the specimens belonged.
Assay of GST and QR activities
To determine whether garcinol can modify liver GST and QR activities, livers were excised immediately from all rats at necropsy. The livers were perfused with saline to remove blood and minced into small pieces. Aliquots from minced livers were processed to obtain the cytosolic fraction as described (27). The activities of GST with 1-chloro-2,4-dinitrobenzene (CDNB) and/or 1,2-dichloro-4-nitrobenzene (DCNB) as substrates, and QR with NADH and menadione as substrates, were determined as described (28,29). All assays were performed by spectrophotometer at 340 nm and all samples were measured in triplicate. One unit of enzyme activity is the amount of enzyme catalyzing the conversion of 1 µmol of substrate to produce per min at 25°C. Cytosolic protein concentrations were determined by the Bradford method (30) using bovine serum albumin (BSA) as the standard.
O2 generation test
Inhibitory tests of TPA-inducd O2 generation were performed as reported previously, with slight modifications (31). Differentiated HL-60 cells, suspended in 1 ml Hank's buffer, were treated with each test compound or the vehicle. After preincubation at 37°C for 15 min, the suspension was centrifuged and the extracellular compounds were removed by washing with 1% BSA twice. Then, the cells were suspended in 1 ml Hank's buffer, and incubated with 100 nM TPA or vehicle and 1 mg/ml cytochrome c at 37°C for 30 min. The reaction was terminated by adding an O2 dismutase solution (10 000 U/ml) and being placed on ice. After centrifugation, the level of extracellular O2 was measured by the cytochrome c reduction method, in which reduced cytochrome c was quantified by measuring the visible absorption of the supernatant at 550 nm (Abs550). Cells treated with the compound, cytochrome c, and vehicle without TPA, and cells with the vehicle without the compound, cytochrome c, or TPA were used as negative and positive controls, respectively. Cells treated with the vehicle without the compound, cytochrome c or vehicle without TPA were used as blanks. Inhibitory rates (IRs) were calculated by the following formula: {1 [(compound, Abs550) (negative, Abs550)/(positive, Abs550) (blank, Abs550)]}x100 (%). Cell viability was determined by a trypan-blue dye exclusion test. Each experiment was done independently in triplicate, and the data are shown as means ± SD. A green tea polyphenol, ()-epigallocatechin gallate (EGCG), was used as a positive control (32).
NO generation test
Inhibitory tests of LPS/INF-
-induced NO generation were performed as reported previously (33). Murine macrophage cell line RAW 264.7 cells, grown confluent in 2 ml of DMEM medium on a 6-well plate, were treated with LPS (100 ng/ml), tetrahydrobiopterin (10 mg/ml), INF-
(100 U/ml), L-arginine (2 mM) and the test compound or vehicle. After 18 h, the levels of nitrite (NO2) were measured by Griess assay. Cells treated with the compound and vehicle without stimulation, and cells with the vehicle without the compound or stimulation were used as negative and positive controls, respectively. Cells treated with the vehicle without the compound, and the vehicle without stimulation, were used as blanks. IRs were calculated by the following formula: {1 [(compound, Abs530) (negative, Abs530)/(positive, Abs530) (blank, Abs530)]}x100 (%). Cytotoxicity was measured by a 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide assay. Each experiment was done independently in triplicate, and the data are shown as means ± SD. In this assay, EGCG was used as a positive control, since it inhibits NO generation (32,34).
Western blotting
Western blotting was performed as reported previously (33). Confluent RAW 254.7 cells were stimulated and incubated in the same manner as described above. After cells were washed, a boiling lysis solution (1% SDS, 1 mM sodium vanadate, and 10 mM Tris buffer, pH 7.4) was added to the cells which were then scraped from the dish, sonicated and boiled for 10 min. Protein concentrations were determined using a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) and BSA was employed as the standard. Proteins (10 mg) were separated on 10% polyacrylamide gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, MA). After blocking, the membranes were incubated with a primary anti-mouse iNOS antibody (1:1000 dilution; Affinity Bioreagents, Golden, CO) and then a secondary antibody (peroxidase-conjugated swine anti-rabbit IgG, 1:1000 dilution; Dako, Glostrup, Denmark). The blots were developed using an ECL detection kit (Amersham Life Science, Little Chalfont, UK). The antibodies were stripped and the blots were successively reprobed with each primary antibody. The first incubation was with a goat anti-rat COX-2 (cross-reacts with mouse counterparts, 1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal anti-ß-actin antibody (1:1000 dilution; Biochemical Technologies, Stoughton, MA). Each membrane was then incubated with a corresponding secondary antibody, peroxidase-conjugated rabbit anti-goat IgG (1:1000 dilution; Dako) or peroxidase-conjugated swine anti-rabbit IgG (1:1000 dilution; Dako). The levels of iNOS or COX-2 bands were corrected using those of ß-actin as an internal standard. Each experiment was done independently in triplicate, and the data are expressed as means ± SD. EGCG was used as a positive control.
Statistical evaluation
Where applicable, data were analyzed using Student's t-test or Welch's t-test with P < 0.05 as the criterion of significance.
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Results
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General observation
All animals remained healthy throughout the experimental period. Body weight gain and food consumption of rats in various groups are shown in Table I
. Food intake and body weight gain did not differ significantly among the groups, suggesting that diets containing 0.01 or 0.05% garcinol were well tolerated and supported normal growth in rats without any adverse effects. At the end of the study, mean body weights of all groups were almost comparable (Table II
). The mean liver weight and relative liver weight (liver weight/100 g body wt) in group 3 given AOM and 0.05% garcinol were significantly lower than those of group 1 (P < 0.05 and P < 0.02).
Frequency of ACF
Table III
summarizes the data on colonic ACF formation. All rats belonging to groups 13, initiated with AOM, developed ACF. In group 1, AOM induced 97 ± 15 ACF per rat. In groups 2 and 3, the dietary administration of garcinol caused significant inhibition in the ACF incidence: 72 ± 15 at the dose of 0.01% (P < 0.01) and 58 ± 8 at the dose of 0.05% (P < 0.001). Furthermore, there was a significant decrease in the number of ACF per cm2 (P < 0.05 and P < 0.002), the total number of crypts per colon (P < 0.001), or the number of crypts per focus (P < 0.05 and P < 0.02) in these groups. Also, the percentages of ACF consisting of more than four crypts in groups 2 and 3 were significantly smaller than that of group 1 (P < 0.05 and P < 0.02, respectively). In group 4, which was given a garcinol diet alone, and group 5, which was untreated, there were no microscopically observable changes, including ACF, in colonic morphology.
PCNA-labeling index in ACF and `normal-appearing' colonic crypts
The PCNA-labeling indices in ACF and `normal-appearing' crypts are presented in Table IV
. The mean PCNA-labeling indices in ACF of groups 2 and 3 were significantly lower than that of group 1 (P < 0.005 and P < 0.001, respectively). The PCNA-labeling indices in `normal-appearing' crypts of these groups were also significantly decreased by feeding of garcinol-containing diets (P < 0.01 and P < 0.005, respectively). Garcinol diet (0.05%) did not affect the PCNA-labeling index in normal crypts of rats in group 4, when compared with that of untreated rats (group 5).
Liver GST and QR activities
Liver GST and QR activities at the end of the study are shown in Table V
. AOM treatment (group 1) significantly elevated liver GST using CDNB as a substrate (P < 0.05) and QR (P < 0.001) activities when compared with those of untreated rats (group 5). GST activities in groups 2 and 3 were significantly greater than that of group 1 (P < 0.005). In rats given a 0.05% garcinol diet alone (group 4), GST and QR activities were significantly elevated when compared with those in an untreated control (P < 0.001 and P < 0.005, respectively).
Effect of garcinol on O2 generation, NO generation and LPS- and INF-
-induced expression of iNOS and COX-2 proteins.
As summarized in Table VI
, notable cytotoxicity caused by garcinol was not observed at a concentration of 10 µM. The difference of cell viabilities between the treatment of 10 mM (80.2 ± 4.5%) and 0.1 mM garcinol (90.9 ± 6.3%) was not statistically significant (P < 0.07 by Student's t-test). The IR of O2 generation by 0.1 µM garcinol (5.6 ± 3.0%) was comparable with that of 1 µM EGCG (3.0 ± 1.0%). The inhibitory activities (5.6100%) of garcinol with various doses were greater than those of EGCG (055.6%). As shown in Table VII
, no cytotoxicity caused by garcinol was found at a concentration of 2.5 µM. The IRs of NO generation (3.596.5%) by garcinol with various concentrations were slightly higher than those of EGCG (36.586.7%). As illustrated in Figure 2
, suppressive effects of 2.5 µM garcinol on expression of iNOS and COX-2 proteins were greater than that of EGCG.

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Fig. 2. Suppressing effects of garcinol and EGCG on LPS- and IFN- -induced iNOS and COX-2 expression in RAW 264.7 cells.
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Discussion
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The results described here clearly indicate that dietary administration of garcinol from G.indica significantly inhibits AOM-induced ACF formation in male F344 rats without causing any adverse effects. Moreover, percentages of large ACF consisting of four or more aberrant crypts were reduced by feeding of garcinol. These findings may suggest that dietary garcinol suppresses chemically induced colon carcinogenesis, since the number of ACF consisting of four or more aberrant crypts is correlated with the incidence of colonic adenocarcinoma induced by a colonic carcinogen, AOM (10).
Epidemiological studies indicate that diets and life-style are important environmental factors associated with the development of most chronic diseases, including cardiovascular diseases and certain types of cancer (1). Whereas genetic and environmental factors have been recognized as the causatives in the etiology of colonic carcinoma, it is the carcinoma most related to diet (35). International dietary guidelines for the prevention of chronic diseases recommend increased consumption of plant foods, including fruits, vegetables, cereal, etc. Such plant foods contain the traditional macronutrients and a wide variety of physiologically active phytochemicals, including phenolics. In the present study, rats fed the diets containing 0.01 and 0.05% garcinol showed no adverse effects on food consumption and growth rate. These findings are important, since long-term oral administration of tested compounds is needed for human clinical trials.
Little is known about biological activities of garcinol. Dried rind of G.indica (`Kokum') containing garcinol (23%, w/w) is used as a garnish for curry and in traditional medicine (a vermifuge and heart medicine) in India. Although a yellow pigment garcinol has a weak antioxidant effect (25) and antibacterial/antifungal activities (36), its other biological activities are not well known. Structurally related compounds, guttiferones, inhibit the cytopathic effects of in vitro HIV infection (24). Recently, Iimura et al. found that garcinol acts as DNA topoisomerases I and II inhibitor (Patent Abstract of Japan: Publication no. H10-2039688), suggesting possible anticancer agent of garcinol.
Several explanations for the inhibitory effects of garcinol on ACF formation by AOM are considered. In addition, several natural compounds could induce detoxification enzymes (37). In the present study, dietary administration of garcinol significantly elevated GST and QR activities in liver, as did auraptene (6) and organosulfur compounds (38). Such biological action may contribute to its `blocking' effect (39) on AOM-induced ACF formation.
Activated leukocyte-derived O2 and NO generation have both been reported to be involved in colon carcinogenesis (15,40). Increased COX-2 expression also involves colon carcinogenesis (17,18). In the current study, garcinol inhibited TPA-induced O2 generation in human promyelocytic HL-60 cells. The inhibition was greater than EGCG. The results may indicate that garcinol has a marked potential for the suppression of activated leukocyte-induced oxidative stress by attenuating O2 generating systems, such as NADPH oxidase. Garcinol also had inhibitory effects on LPS- and IFN-
-induced NO generation in mouse macrophage RAW 264.7 cells. NO is known to be generated in conjunction with the conversion of L-arginine to the stoichiometric amount of L-citrulline. While measurement of NO2 by the Griess assay allows detection of the sum of the NO generation inhibitory and NO scavenging effects, the L-citrulline measurement strictly reflects the former. Thus, the data on NO generation show the suppressive effect on NO generation from RAW 264.7 cells, i.e. inhibition of the de novo synthesis and/or catalytic activity of iNOS. Western blot analysis revealed that the inhibition of NO generation is due to suppression of iNOS protein expression. Whereas EGCG suppresses NO generation by inhibiting the transcriptional activity of nuclear factor
B (34), thereby attenuating iNOS protein expression, the action mechanisms of garcinol remain to be addressed. Also, we should confirm whether garcinol inhibits iNOS activity or perturbs NO2/NO3 conversion. Garcinol could suppress COX-2 levels in LPS- and IFN-
-treated mouse macrophage RAW 264.7 cells. The results on iNOS and COX-2 expression are interesting, since resveratrol (41) and other natural compounds (16,42) are well-known suppressors of the de novo formation of both iNOS and COX-2 and exert chemopreventive effects against carcer development. The role of both iNOS and COX-2 as enhancers of carcinogenesis in many organs including colon (15,19,4345) is receiving increasing attention and, thus, suppression of either the synthesis or the activity of these enzymes is currently a target for cancer chemoprevention (16,46). Recently, Suh et al. synthesized interesting synthetic analogs (22,47), based on triterpenoids, biosynthesized in plants by the cyclization of squalene, which are used for medicinal purposes in many Asian countries and some (ursolic and oleanolic acids) are known to be anti-inflammatory and anticarcinogenic compounds (48,49). The synthetic oleanane triterpenoid compound, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, possesses differentiating, antiproliferative and anti-inflammatory activities and can suppress the ability of inflammatory cytokines to induce de novo formation of iNOS and COX-2 in mouse peritoneal macrophages (22).
In the current study, the PCNA-labeling index in ACF and `normal-appearing' crypts was decreased by dietary feeding of garcinol. Zheng et al. (50) recently reported similar findings that retinoids have the ability to reduce the PCNA-labeling index in ACF and in `normal-appearing' crypts with their ability to prevent ACF. Cell proliferation plays an important role in multistage carcinogenesis with multiple genetic changes (51). Eicosanoids, the metabolites of arachidonic acid through the lipoxygenase and cyclooxygenase pathways, possess a variety of biological activities. Some metabolites of both pathways cause hyperproliferative responses (52). In fact, several reports have shown that cyclooxygenase inhibitors suppress colon carcinogenesis (19,53). Thus, the inhibitory effect of garcinol may be due, in part, to modification of cell proliferation through the above mechanisms.
In conclusion, the results of this study suggest that dietary garcinol has a beneficial effect on chemically induced colonic preneoplastic progression in rats that provides an effective dietary chemopreventive approach to disease management. However, a long-term bioassay needs to confirm the results, which is now being planned in our laboratory, since a recent study indicated that certain compounds, which could reduce the number of ACF and reduce proliferation, did not inhibit the formation of colon tumors (54).
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Notes
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5 To whom correspondence should be addressed Email: takutt{at}kanazawa-med.ac.jp 
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Acknowledgments
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The authors thank Ayumi Nagaoka for secretarial assistance. We also express thanks to the staff of the Research Animal Facility. This research was supported by grants for Project Research (P99-1) and Collaborative Research (C99-1) from Kanazawa Medical University, by a Grant-in Aid for the second term for a Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, by a Grant-in-Aid from the Ministry of Health and Welfare of Japan, by Grants-in-Aid for Scientific Research (nos10671782 and 11138255) from the Ministry of Education, Science, Sports and Culture of Japan, and a grant (HS-52260) for Comprehensive Research Project on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation.
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Received January 18, 2000;
revised February 29, 2000;