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Carcinogenesis Advance Access originally published online on March 28, 2008
Carcinogenesis 2008 29(5):1057-1063; doi:10.1093/carcin/bgn080
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Suppressive effects of nobiletin on hyperleptinemia and colitis-related colon carcinogenesis in male ICR mice

Shingo Miyamoto, Yumiko Yasui1, Takuji Tanaka1, Hajime Ohigashi2 and Akira Murakami*

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
1 Department of Oncologic Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan
2 Present address: Faculty of Biotechnology, Fukui Prefectural University, Fukui 910-1195, Japan

* To whom correspondence should be addressed. Tel: +81 75 753 6282; Fax: +81 75 753 6284; Email: cancer{at}kais.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Adipocytokines are a group of adipocyte-secreted proteins that have significant effects on the metabolism of lipids and carbohydrates, as well as numerous other processes. A number of recent studies have indicated that some adipocytokines may significantly influence the proliferation of malignant cells in vitro, whereas it remains unclear whether they have similar roles in vivo. In this study, we determined serum levels of adipocytokines in mice with azoxymethane (AOM)- and dextran sulfate sodium (DSS)-induced colon carcinogenesis. Five-week-old ICR mice were given a single intraperitoneal injection of AOM followed by 1% DSS in drinking water for 7 days. Nobiletin (NOB), a citrus flavonoid, was given in the diet (100 p.p.m) for 17 weeks. Thereafter, the incidence and number of colon tumors and serum concentration of adipocytokines were determined at the end of week 20. The serum leptin level in AOM/DSS-treated mice was six times higher than that in untreated mice, whereas there were no significant differences in the levels of triglycerides, adiponectin and interleukin-6. Feeding with NOB abolished colonic malignancy and notably decreased the serum leptin level by 75%. Further, NOB suppressed the leptin-dependent, but not independent, proliferation of HT-29 colon cancer cells and decreased leptin secretion through inactivation of mitogen-activated protein kinase/extracellular signaling-regulated protein kinase, but not that of adiponectin in differentiated 3T3-L1 mouse adipocytes in a dose-dependent manner. Taken together, our results suggest that higher levels of leptin in serum promote colon carcinogenesis in mice, whereas NOB has chemopreventive effects against colon carcinogenesis, partly through regulation of leptin levels.

Abbreviations: AOM, azoxymethane; DMEM, Dulbecco's modified Eagle medium; DSS, dextran sulfate sodium; eIF4B, eukaryotic initiation factor 4B; ERK, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; IL-6, interleukin-6; MEK, mitogen-activated protein kinase/extracellular signaling-regulated protein kinase kinase; mTOR, mammalian target of rapamycin; NOB, nobiletin; Ob-R, leptin receptor; TNF-{alpha}, tumor necrosis factor-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Colorectal cancer was seen in about 1 million new cases throughout the world in 2002, with similar numbers for men and women. In terms of incidence, colorectal cancer ranks fourth in frequency in men and third in women (1). Epidemiological studies have provided abundant evidence that environmental factors, rather than genetic variations between populations, are of prime importance in the etiology of this disease (2,3). One of the most influential factors is obesity, whose prevalence has markedly increased over the past two decades, especially in industrialized countries (4). Obesity is known to increase the risk of several different chronic diseases, such as coronary heart disease, stroke and cancer (5,6). Further, the results of case–control and prospective studies suggest that obesity is a strong risk factor for colorectal cancer, especially in men (7–10). More recently, a prospective population-based study of ~90 000 subjects conducted by the American Cancer Society confirmed that obesity is directly associated with an increased risk of death from colon cancer (11). Animal studies have confirmed that finding and also showed that obesity enhances tumor development (12), whereas calorie restriction inhibits a broad range of spontaneous, transplanted and chemically induced tumors (13). However, the mechanism underlying the development of obesity-associated colon cancer has not been fully elucidated.

Until the discovery of adipocytokines, adipose tissue was only thought to have passive functions as an energy storage depot and mechanical barrier. Adipocytokines are a group of adipose tissue-secreted hormones that were initially reported in the early 1990s when leptin was described (14). Later, it was shown that leptin, resistin, plasminogen activator inhibitor-1, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) had positive relationships to adiposity (15). Since they have some crucial roles in immune regulation, vascular function and adipocyte metabolism, adipocytokines are considered to be central players in the pathogenesis of metabolic syndrome, a cluster of clinical symptoms that include obesity and insulin resistance. Consequently, the regulation of body weight and obesity-related pathology is rapidly becoming a critical concern for public health experts and medical scientists worldwide (16). Most of the studies on the relationship of obesity and colorectal carcinogenesis are using obese animals (e.g. db/db, ob/ob or high-fat diet consumption mice). However, it is difficult to determine which adipocytokine is involved in colon carcinogenesis in obese animals because, in addition to several adipocytokines, there are a number of altered physiological factors in obese individuals.

Thus, in the present study, we decided to use chemically induced colon carcinogenesis in mice to quantify the serum levels of adipocytokines (17). In addition, the effects of dietary citrus nobiletin (NOB, Figure 1), a candidate chemopreventive agent against cancer in the colon (18,19), toward colon carcinogenesis and the serum level of adipocytokines in mice were investigated to elucidate its regulatory activities.


Figure 1
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  		Fig. 1. Chemical structure of NOB.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Mice
Male Crj: CD-1 (ICR) mice (Charles River Japan, Tokyo, Japan) were obtained at 5 weeks old and maintained at Kanazawa Medical University Animal Facility according to the Institutional Animal Care Guidelines. On arrival, all mice were randomized and transferred to plastic cages (five mice per cage) and given free access to drinking water and a pelleted basal diet (CRF-1, Oriental Yeast, Tokyo, Japan) under controlled conditions of humidity (50 ± 10%), light (12/12 h light/dark cycle) and temperature (23 ± 2°C). All mice were quarantined for 1 week before starting the experiments.

Chemicals
Azoxymethane (AOM), a colonic carcinogen, was purchased from Sigma Chemical Co. (St Louis, MO). Dextran sulfate sodium (DSS) with a molecular weight of 36 000–50 000 was purchased from ICN Biochemicals (Aurora, OH), dissolved in distilled water at a concentration of 1% (wt/vol) and then used to induce colitis. NOB (>98% purity) was obtained from Nard Chemicals (Hyogo, Japan). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and bovine serum were purchased from Gibco BRL (Grand Island, NY). Human recombinant leptin was obtained from R&D Systems (Minneapolis, MN). Antibodies directed against Pi-mitogen-activated protein kinase/extracellular signaling-regulated protein kinase kinase (MEK)1/2 (Ser217/221, #9121), Pi-extracellular signaling-regulated protein kinase (ERK)1/2 (Thr202/Tyr204), Pi-mammalian target of rapamycin (mTOR) (Ser2448, #2971), Pi-S6 (Ser240/244, #2215), Pi-eukaryotic initiation factor 4B (eIF4B) (Ser422, #3591), as well as horseradish peroxidase-conjugated anti-rabbit antibody (#7074), were obtained from Cell Signaling Technology (Beverly, MA). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan) unless specified otherwise.

Animal treatment
A total of 80 male ICR mice were divided into one control and three experimental groups (Figure 2). Group 1 served as an untreated control. Group 2 mice were given NOB (100 p.p.m.) in their diet starting from week 3 of the experiment. Mice in groups 3 and 4 were given a single intraperitoneal injection of AOM (10 mg/kg body wt) at the beginning of the experiment and then received 1% DSS in drinking water for 7 days starting 1 week after the injection. Further, group 3 mice were maintained on the basal diet throughout the study, whereas those in group 4 were given the same diet as group 2. The dose of NOB used was determined on the basis of our previous studies (18,19). The animals were sequentially euthanized on weeks 5, 10 and 20 as follows. Three mice each from groups 1 and 2 and five mice each from groups 3 and 4 were euthanized on weeks 5 and 10, whereas nine mice each from groups 1 and 2 and 15 mice each from groups 3 and 4 were euthanized on week 20. The mice were killed under ether anesthesia, and blood samples were immediately collected from the abdominal aorta, after which all organs were removed, with the colons flashed with phosphate-buffered saline, excised, measured in the length (from the ileocecal junction to the anal verge), cut open longitudinally along the main axis and then washed again with phosphate-buffered saline. The colons were macroscopically inspected, and whole colons were processed for paraffin embedding after being cut and fixed in 10% buffered formalin for at least 24 h. Histopathological examinations were then done on paraffin-embedded sections after hematoxylin and eosin staining. Colonic neoplasms were diagnosed according to the description by Ward (20). Tissues other than the colon were also evaluated histopathologically.


Figure 2
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  		Fig. 2. Experimental protocol for AOM/DSS-induced carcinogenesis. Mice in groups 3 and 4 were given a single intraperitoneal injection of AOM (10 mg/kg body wt). Starting 1 week after the injection, they were administered 1% DSS in drinking water for 7 days (filled rectangle). Mice in groups 2 and 4 were given NOB (100 p.p.m.) in the diet from week 3.

 
Clinical chemistry
The collected blood samples were used for clinical chemistry with measurements for triglycerides (Triglyceride E-test, Wako Pure Chemical Industries), adiponectin (Mouse/Rat Adiponectin ELISA Kit, Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan), leptin (Quantikine Mouse leptin, ELISA/Assay Kit, R&D Systems), TNF-{alpha} (Quantikine Mouse leptin, TNF-{alpha}, ELISA/Assay Kit, R&D Systems) and IL-6 (Quantikine Mouse IL-6 ELISA Kit, R&D Systems, respectively) performed. Collected serum samples were examined without dilution to measure triglycerides, TNF-{alpha} and IL-6, whereas they were diluted 20- and 2000-fold for leptin and adiponectin measurements, respectively.

Cell culture
HT-29 human colon cancer cells and 3T3-L1 mouse pre-adipocytes were obtained from American Type Culture Collection (Manassas, VA). HT-29 and 3T3-L1 cells were maintained in DMEM supplemented with 10% FBS (HT-29) or 10% bovine serum (3T3-L1), as well as 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in a humidified 5% CO2 atmosphere.

Cell proliferation
HT-29 cells (5 x 103/200 µl per well) were seeded into 96-well plates under the growth conditions described above. Twenty-four hours after seeding, the cells were serum starved for 24 h and then treated with leptin (0.01–10 nM) for various time periods (0–72 h), according to a method reported previously by Ogunwobi et al. (21), with some modifications. For suppressive experiments, cells were pretreated for 1 h with NOB (0, 10 and 100 µM) before leptin exposure. At various time points, cell proliferation was assessed using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions.

Intracellular lipid accumulation and adipocytokines secretion
The 3T3-L1 cells (1 x 104/200 µl per well) were seeded into 96-well plates under the growth conditions described above. After reaching confluence, they were incubated for an additional 24 h (designated as day 0). Then, adipocyte differentiation was induced by treatment with a mixture of methylisobutylxanthine (0.5 mM), dexamethasone (1 µM) and insulin (10 µg/ml), components of an Adipogenesis Assay Kit (Chemicon International, Temecula, CA), in DMEM containing 10% FBS for 48 h. The medium was then replaced by DMEM containing 10% FBS and insulin (5 µg/ml) and changed to fresh medium every 2 days, according to a method reported previously by Maeda et al. (22), with some modifications. On day 2, NOB (0, 10 and 100 µM) was dissolved in dimethyl sulfoxide and then added to DMEM containing FBS and insulin. The final concentration of dimethyl sulfoxide was 0.1%, which was found not to affect cell growth (data not shown). After 12 days, the medium was collected and subjected to enzyme-linked immunosorbent assay to determine the levels of leptin, adiponectin, IL-6 and TNF-{alpha}. The cells were stained with the Oil Red-O component of an Adipogenesis Assay Kit according to the manufacturer's instructions. Stained cells were viewed using an inverted microscope (Leica Microsystems, Tokyo, Japan) (original magnification 1:200) and images were captured with a digital camera system. Stained oil droplets in 3T3-L1 cells were extracted with dye extraction solution and then the absorbance of the extracts were measured at 490 nm.

Western blotting
Following treatment with NOB, 3T3-L1 cells were washed with phosphate-buffered saline twice and lysed in lysis buffer [10 nM Tris, pH 7.4, 1% sodium dodecyl sulfate, 1 mM sodium metavanadate (V)] and centrifuged at 3200g for 5 min. Denatured proteins (40 µg) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and then transferred onto Immobilon-P membranes (Millipore, Billerica, MA). After blocking with Block Ace (Snow Brand Milk Products, Tokyo, Japan) for 1 h, the membranes were reacted with the appropriate specific primary antibody (1:1000) followed by the corresponding horseradish peroxidase-conjugated secondary antibody (1:1000). The blots were developed using ECL western blotting detection reagents.

Statistical analysis
Where applicable, data were analyzed using a Tukey–Kramer multiple comparison test (GraphPad Instat version 3.05, GraphPad Software, San Diego, CA), Fisher's exact probability test and Student's t-test (two sided), with P < 0.05 as the criterion of significance.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
General observations of mice
Throughout the study, dietary feeding with NOB did not cause clinically harmful symptoms including toxicity. The intake of water and food consumption (grams per day per mice) did not significantly differ among the four groups, and there were no marked changes in the mean relative liver weights and colon lengths (data not shown). In contrast, the mean body weight of group 3 was significantly higher (P < 0.05) than that of the groups 1 and 2, and NOB in the diet suppressed that increase by 56%, which was statistically significant (Table I). In parallel, the AOM/DSS treatment led to a notable increase in epididymal fat by 1.6-fold, whereas NOB tended to suppress that increase.


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  		Table I. Body and epididymal fat weights and colon tumor formation in male ICR mice

 
Incidence and multiplicity of colonic neoplasms
We reported previously the significant effects of NOB toward AOM-induced aberrant crypt foci formation (18) and carcinogenesis (19) in rats. In the present study, we attempted to confirm its preventive ability in inflammation-associated colon carcinogenesis model mice and also examined its effects on the serum levels of adipocytokines. Macroscopically, nodular and polypoid colonic tumors were observed in the middle and distal colon of mice in groups 3 and 4, which were shown to be tubular adenomas and adenocarcinomas in histopathological findings. As summarized in Table I, the mice in groups 1 and 2 did not develop neoplasms in any of the organs examined, including the colon. In contrast, group 3 had a 50% incidence of colonic tumors and 40% incidence of adenocarcinomas. In group 4, which received AOM/DSS and 100 p.p.m. of NOB in the diet, only a single colonic tumor developed in one mouse, which was shown in histopathological findings to be a tubular adenoma. Thus, the incidence of adenocarcinoma in group 4 (0%) was significantly lower than that in group 3 (P < 0.05). The multiplicity of colon adenomas in group 4 was also extremely lower than that in group 3.

Serum levels of leptin, adiponectin, IL-6, TNF-{alpha} and triglycerides
We assessed the serum levels of triglycerides and adipocytokines based on a previous report of a positive association of colon cancer with hypertriglycemia in Apc knockout mice (23). Interestingly, the serum concentrations of triglycerides, IL-6 and TNF-{alpha} in group 3 were elevated by 1.5–1.6-fold as compared with those in group 1, though the differences were not significant. Of note, the serum level of leptin in group 3 increased by 3.1–5.7-fold in a time-dependent manner (from 5 to 20 weeks) and was markedly suppressed by NOB (75–84%). When NOB was given by itself (group 2), it did not affect the level of leptin as compared with the control group.

Effects of leptin and NOB on cell proliferation
Leptin treatment (0.1–10 nM) for 24 h significantly increased HT-29 cell proliferation by 1.3–1.6-fold (Figure 4A) in a time-dependent manner (Figure 4B), which was consistent with previously reported findings (24,25). We also examined the effects of NOB on leptin-dependent and -independent cell growth. As shown in Figure 4C, NOB (10 or 100 µM) abolished leptin-enhanced cell growth, whereas it had no effects on cell proliferation when leptin was not added.

Effects of NOB on Oil Red-O staining and secretion of adipocytokines
We treated differentiated 3T3-L1 adipocytes with NOB (0.1, 1, 10 and 100 µM) to determine its effects on intracellular lipid accumulation and secretion of adipocytokines. Differentiated 3T3-L1 cells were notably loaded with lipid, as detected by Oil Red-O staining. NOB (100 µM) reduced the Oil Red-O staining level of 3T3-L1 cells to 40% (Figure 5A). Further, the flavonoid (1–100 µM) significantly reduced leptin secretion (61–100%, P < 0.05, Figure 5B) in a dose-dependent manner. However, it had no effect on the secretion of adiponectin, whereas IL-6 secretion was slightly increased by 10 µM of NOB. The level of TNF-{alpha} in media was not detectable following any of the treatments (data not shown).

NOB inhibited leptin secretion partly through suppressed MEK1/2 phosphorylation
The mTOR, a Ser/Thr kinase, is considered to play a crucial role as the regulator of differentiation (26) and leptin secretion (27). We investigated the effects of NOB and rapamycin, an mTOR inhibitor, on the leptin secretion and phosphorylation status of molecules (Figure 6) involved in insulin-signaling pathway (mTOR, eIF4B, S6, Raf, MEK1/2 and ERK1/2). NOB (10 and 100 µM) significantly reduced leptin secretion as well as rapamycin (data not shown). The phosphorylation state of eIF4B and S6, both of which are substrates of mTOR, was abolished in rapamycin-treated cells, whereas NOB selectively decreased the phosphorylation state of only eIF4B. The differing results obtained with NOB and rapamycin led us to examine whether NOB affects the Raf/MEK/ERK pathway. Interestingly, the phosphorylation state of MEK1/2 and ERK1/2, but not Raf, was notably decreased in NOB-treated cells, whereas rapamycin did not affect those of both Raf and MEK1/2 but dramatically increased ERK1/2 phosphorylation. Proposed molecular mechanisms by which NOB suppresses leptin secretion are shown in Figure 7.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
In the present study, we demonstrated for the first time that serum leptin levels are profoundly increased in mice with chemically induced colon carcinogenesis. In addition, a citrus flavonoid, NOB, lowered not only those levels but also reduced colon tumor development, whereas other adipocytokines (TNF-{alpha}, IL-6 and adiponectin) and triglycerides did not significantly alter by the treatment. Further, NOB abolished leptin-stimulated human colon cancer cell proliferation and leptin secretion from insulin-treated adipocytes in vitro as well as the flavonoid rutin (28). Together, these results led us to hypothesize that an increased level of leptin promotes colon carcinogenesis in mice and that NOB is able to inhibit that, at least in part, through regulation of leptin levels.

Recently, obesity has become a point of focus in investigations conducted to identify dietary and lifestyle factors related to an increased risk of colorectal cancer (29). Metabolic stress resulting from obesity has been shown to be associated with increased levels of oxidative stress (30), inflammatory cytokines, insulin (31) and lipids (32,33). Of interest, Niho et al. (23,34) revealed a hyperlipidemic state in Apc gene-deficient mice, used as a model of human familial adenomatous polyposis, as compared with their wild-type counterparts. Our present findings regarding the serum level of triglycerides (Figure 3) are similar to those, though the difference did not reach statistical significance. This discrepancy may attribute to the differences in genetic backgrounds of the mice used and/or the experimental protocols. Obesity is driven by white adipose tissue, from which excess or reduced levels of adipocytokines are secreted (35). Adiponectin is the most abundant cytokine in adipocytes and has been reported to have antidiabetic and anti-inflammatory properties (36), and low levels of adiponectin have been shown to be associated with an increased risk of colorectal cancer in humans (37). Several classical proinflammatory cytokines, e.g. TNF-{alpha} and to a large extent IL-6, are also secreted from adipocytes (38) and may participate in the regulation of obesity (39). In addition, epidemiological studies have revealed the roles of TNF-{alpha} and IL-6 in the onset of several types of cancer (40). However, there have been no results published regarding the hormonal role of leptin in chemically induced carcinogenesis in rodents.


Figure 3
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  		Fig. 3. Levels of serum leptin, triglycerides, IL-6, TNF-{alpha} and adiponectin in mice. Serum adipocytokine (leptin, IL-6, TNF-{alpha} and adiponectin) levels were quantified by enzyme-linked immunosorbent assay and triglycerides by a Triglyceride E-test. Values are shown as the mean ± SD (n = 3–15). Statistical analysis was performed using a Tukey–Kramer multiple comparison test and the data not sharing a letter, P < 0.05.

 
Leptin is a 16 kDa protein encoded by the ob gene and was first revealed in 1994 as a regulator of body weight and energy balance, with its activities displayed in the hypothalamus (14). It is well known that serum leptin levels are highly elevated in obese individuals (41,42) and that leptin is secreted mainly by white adipocytes (43). C57BL/KsJ-db/db (db/db) mice have a defect in the leptin receptor (Ob-R) gene (44), which leads to leptin regulatory impairments of food intake thereby resulting in hyperinsulinemia, hyperglycemia and hyperleptinemia in subjects with extreme obesity (45). In the present study, the mean body and epididymal fat weights in AOM/DSS-treated mice were greater than those in control mice while NOB feeding decreased those (Table I). These results raise the possibility that the elevation of serum leptin levels seen in carcinogenesis model mice is in part due to increases in body and fat weights, though the underlying mechanism is unclear. On the other hand, several studies have reported that mesenteric adipose tissue in inflammatory bowel disease patients overexpress leptin mRNA (46,47). A DSS-induced colitis animal model is considered to be very reliable and useful for elucidating the mechanism underlying the onset of inflammatory bowel disease (48), thus DSS treatment may be associated with elevated serum leptin levels in AOM/DSS-treated mice. This issue is now being addressed in our laboratory. Our results (Figure 4) as well as those of several other studies (21,24,25) indicate that leptin acts as a mitogenic factor in cultured human colon cancer cells. However, it is well known that obese animals with elevated leptin levels, e.g. wild mice fed a high-fat diet and db/db mice, are highly susceptible to chemically induced carcinogenesis (49,50). Collectively, it is considered that obesity-associated colon carcinogenesis is partly mediated through a leptin-involved mechanism.


Figure 4
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  		Fig. 4. Effects of leptin on proliferation of HT-29 human colon cancer cells and suppression by NOB. Twenty-four hours after seeding, cells were serum starved for 24 h and then treated with (A) leptin (0.01–10 nM) for 24 h (B) dimethyl sulfoxide (filled triangle) or 1 nM leptin (filled square) for different time periods (0–72 h) or (C) leptin in the presence of NOB (0, 10 and 100 µM) for 24 h. Cell numbers were determined using a Cell Counting Kit-8. Values are shown as the mean ± SD (n = 3). Statistical analysis was performed using a Student's t-test and the data not sharing a letter (a and b), P < 0.05 (at the same time point in panel B).

 
NOB (Figure 1), a polymethoxylated flavonoid predominant in citrus fruit peels (Figure 1) (51), has been reported to inhibit the proliferation of a variety of human cancer cell lines (52) and suppress colon carcinogenesis in rats (18,19). Although several reports have implied the preventive mechanism of NOB toward colon carcinogenesis, those results are not definitive. For example, NOB inhibited inducible nitric oxide synthase and cyclooxygenase-2 expression in macrophages (53) and reduced prostaglandin E2 levels in rat colonic mucosa treated with AOM (18,19). In the present study, we showed that dietary NOB decreased body and epididymal fat weights, which had been increased by treatment with AOM/DSS. These effects may contribute to its colon cancer preventive activities. In accordance with this notion, Saito et al. (54) recently reported that NOB enhanced both the differentiation and the lipolysis of adipocytes via activation of signaling cascades mediated by cyclic adenosine 3',5'-monophosphate and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein. Our findings showing that NOB reduced the accumulation of intracellular lipids (Figure 5A) were similar to those in that study.


Figure 5
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  		Fig. 5. Effects of NOB on Oil Red-O staining (A) and secretion of leptin, IL-6 and adiponectin from differentiated 3T3-L1 cells (B). 3T3-L1 mouse preadipocytes were induced to adipocyte differentiation with a mixture of methylisobutylxanthine (0.5 mM), dexamethasone (1 µM) and insulin (10 µg/ml) in DMEM containing 10% FBS for 48 h. Differentiated 3T3-L1 cells were treated with dimethyl sulfoxide alone or various concentrations of NOB for 12 days and then the supernatants were removed for measurements of adipocytokines. The cells were washed twice with phosphate-buffered saline and stained with Oil Red-O. Stained cells were then viewed using an inverted microscope (Leica Microsystems) (original magnification 1:200). Leptin, IL-6 and adiponectin secretion were quantified by enzyme-linked immunosorbent assay. Values are shown as the mean ± SD (n = 6). Statistical analysis was performed using a Student's t-test and the data not sharing a letter, P < 0.05.

 
In addition, it should be pointed out that NOB inhibited leptin-stimulated but not basal HT-29 colon cancer cell proliferation, though the mechanism is not fully understood. The biological activities of leptin are mediated through its receptor Ob-R, which consists of six splicing variants (Ob-Ra through Ob-Rf) (55). The long (Ob-Rb) and short (Ob-Ra) isoforms transduced leptin signals through ERK1/2- and c-Jun NH2-terminal kinase 1/2-dependent pathways in human Kupffer and peripheral blood mononuclear cells (56). Also, leptin was reported to stimulate HT-29 cell proliferation through the activation of ERK1/2 and c-Jun NH2-terminal kinase 1/2 (21,25). Importantly, we recently reported that NOB suppressed phorbol ester-induced activation of ERK1/2, c-Jun NH2-terminal kinase 1/2 and c-jun in THP-1 human monocytic cells (57). Thus, this flavonoid may inhibit leptin-induced cell proliferation by disrupting mitogen-activated protein kinase pathways.

In adipocytes, mTOR is a master regulator of protein synthesis (58), adipose tissue morphogenesis (59) and leptin synthesis/secretion (60). Consistent with previous study, rapamycin significantly suppressed leptin secretion from 3T3-L1 cells (data not shown), and the suppressive effect may be due to the suppression of S6 and eIF4B, the substrates of mTOR (Figure 6). Meanwhile, in the present study, NOB suppressed the activation of eIF4B but not S6. Kawabata et al. (61) recently showed that eIF4B phosphorylation is dependent not only on mTOR but also on mitogen-activated protein kinase pathway. In this study, NOB notably suppressed the phosphorylation of MEK1/2 and ERK1/2, but not Raf (Figure 6). The MEK1/2 activation may be regulated not only by a conventional Ras/Raf signal pathway but also by their autophosphorylation (62). Furthermore, our findings are consistent with those by Miyata et al. (63) who reported that NOB inhibited the auto-phosphorylation of MEK1/2 without the affecting Ras and Raf activity in HT-1080 colon cancer cells. Together, it is likely that NOB inhibits phosphorylation of MEK1/2 and eIF4B for decreasing leptin release. Based on the different mode of actions between NOB and rapamycin, their combination may lead to additive or synergistic leptin suppression.


Figure 6
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  		Fig. 6. Effects of NOB or rapamycin on mTOR-signaling pathway in differentiated 3T3-L1 cells. 3T3-L1 mouse pre-adipocytes (1 x 105 cells in 35 mm dish) were induced to adipocyte differentiation with a mixture of methylisobutylxanthine (0.5 mM), dexamethasone (1 µM) and insulin (10 µg/ml) in DMEM containing 10% FBS for 48 h. Differentiated 3T3-L1 cells were treated with dimethyl sulfoxide alone, various concentrations of NOB or 100 nM rapamycin for 12 days and then the supernatants were removed for measurements of adipocytokines. The cells were washed twice with phosphate-buffered saline and analyzed by western blotting using specific antibodies. RAP, rapamycin.

 


Figure 7
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  		Fig. 7. Proposed schema of molecular mechanisms by which NOB suppresses leptin secretion from 3T3-L1 cells. Differentiation and protein translation are regulated by both Akt/mTOR and MEK/ERK-signaling pathway. mTOR and the downstream factors, such as S6 and eIF4B, are phosphorylated in a constitutive manner. Rapamycin inhibits mTOR and thereby suppressing the activity of downstream molecules for blocking leptin secretion. Meanwhile, NOB induces the dephosphorylation of MEK1/2 without affecting mTOR and Raf and reduces phosphorylation of eIF4B.

 
In conclusion, the present results suggest that the level of leptin in serum is related to colon carcinogenesis and dietary NOB suppresses carcinogenesis partly through regulation of this hormone. Although additional studies are necessary to confirm our speculation, synthetic drugs or food ingredients targeting leptin secretion and activities may be useful for regulating obesity-associated colorectal cancer development.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Grant-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan (A.M., T.T.).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Parkin D, et al. Global cancer statistics, 2002. CA Cancer J. Clin. (2005) 55:74–108.[Abstract/Free Full Text]
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Received October 15, 2007; revised February 15, 2008; accepted March 12, 2008.


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M. Shimizu, Y. Shirakami, H. Sakai, S. Adachi, K. Hata, Y. Hirose, H. Tsurumi, T. Tanaka, and H. Moriwaki
(-)-Epigallocatechin Gallate Suppresses Azoxymethane-Induced Colonic Premalignant Lesions in Male C57BL/KsJ-db/db Mice
Cancer Prevention Research, September 1, 2008; 1(4): 298 - 304.
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