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Carcinogenesis Advance Access originally published online on February 6, 2008
Carcinogenesis 2008 29(4):824-829; doi:10.1093/carcin/bgn028
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Plasminogen activator inhibitor-1 (Pai-1) blockers suppress intestinal polyp formation in Min mice

Michihiro Mutoh*, Naoko Niho, Masami Komiya, Mami Takahashi, Rina Ohtsubo, Kiyoshi Nakatogawa1, Kentaro Ueda1, Takashi Sugimura and Keiji Wakabayashi

Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
1 Shizuoka Coffein Co. Ltd, 129 Suidocho, Shizuoka-shi 420-0008, Japan

* To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 3543 9305;Email: mimutoh{at}gan2.ncc.go.jp


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Obesity and hyperlipidemia are known to increase colorectal tumor risk. We noticed that Min mice, featuring a defect in the adenomatous polyposis coli (Apc) gene, develop intestinal polyps along with high serum triglyceride (TG) levels up to 10-fold those observed in wild-type mice. In these mice, messenger RNA (mRNA) expression of lipoprotein lipase, which catalyzes hydrolysis of TG, is downregulated. In the present study, we focused on adipocytokines, especially plasminogen activator inhibitor-1 (Pai-1), which is involved in hyperlipidemic status and may promote intestinal polyp formation in Min mice. Serum Pai-1 levels in the 15-week-old male Min mice were eight times higher than in wild-type mice and hepatic Pai-1 mRNA levels were 11-fold increased. In addition, Pai-1 immunostaining was strong in small intestinal epithelial cells of Min mice. Administration of a PAI-1 inhibitor, SK-216, at 25, 50 and 100 p.p.m. doses in the diet for 9 weeks reduced serum Pai-1 levels and hepatic Pai-1 mRNA levels of Min mice to the wild-type levels. Moreover, SK-216 at 50 and 100 p.p.m. significantly reduced total numbers of intestinal polyps to 64 and 56% of the untreated group value, respectively. Serum TG levels were also decreased by 43% at the dose of 100 p.p.m. Administration of 50 p.p.m. SK-116, another PAI-1 inhibitor, for 9 weeks similarly reduced serum Pai-1 levels and total numbers of intestinal polyps to 70% of the untreated group value. These results indicate that Pai-1 induction associated with hypertriglyceridemia may contribute to intestinal polyp formation with Apc deficiency, and PAI-1 could thus be a novel target for colorectal chemopreventive agents.

Abbreviations: Ab, antibody; APC, adenomatous polyposis coli; LPL, lipoprotein lipase; mRNA, messenger RNA; Pai-1, plasminogen activator inhibitor-1; PCR, polymerase chain reaction; RT, reverse transcription; TG, triglyceride


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
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Colon cancer is one of the most common solid cancers, has an increasing incidence in developed countries and is now widely considered to be obesity associated (1,2). Hyperlipidemia, especially hypertriglyceridemia, is known to increase the risk of colorectal tumor (3–5) and recently we reported an age-dependent hyperlipidemic state in adenomatous polyposis coli (Apc)-deficient Min and Apc1309 mice, animal models of familial adenomatous polyposis (6–8). Familial adenomatous polyposis is characterized by the appearance of hundreds of adenomatous polyps in the colon and rectum due to germ line mutations of the APC gene. Truncating mutations in APC activate Wnt signaling to promote cell growth and are observed in almost 70% of the sporadic colorectal cancers (9).

Although the direct link between APC deficiency and hyperlipidemia has yet to be clarified, it is notable that serum triglyceride (TG) levels in Apc-deficient mice are almost 10-fold than those in wild-type littermates, this appearing due to low messenger RNA (mRNA) expression levels for lipoprotein lipase (LPL), which catalyzes hydrolysis of TG (6). We have demonstrated that induction of LPL mRNA by peroxisome proliferator-activated receptor-{alpha} and -{gamma} agonists and a selective LPL-inducing agent, NO-1886, which lacks potential for activating the peroxisome proliferator-activated receptor pathways, suppressed both the hyperlipidemic status and intestinal polyp formation (6–8). Adipocytokines upregulated in the metabolic syndrome, also associated with a hyperlipidemic status, may be factors impacting on intestinal polyp formation. During investigation of expression levels of adipocytokines in Min mice, we have occasionally detected remarkable elevation of hepatic plasminogen activator inhibitor-1 (Pai-1) expression.

PAI-1 is a direct binding primary inhibitor of plasminogen activators, uPA and tPA, and is known to be induced by TG, very low-density lipoprotein (TG-rich lipoprotein), transforming growth factor β, various growth factors, tumor suppressor p53 and Nuclear factor kappa B (NF{kappa}B) (10–12). Moreover, a very low-density lipoprotein response element has been identified in the promoter region of the PAI-1 gene locus that mediates very low-density lipoprotein-induced PAI-1 transcription in endothelial cells (13). PAI-1 can also inhibit activation of metalloproteinases via inhibition of plasmin production from plasminogen. Metalloproteinases degrade extracellular matrix proteins, which modulate cellular adhesion and migration (14,15). In contrast to its antiproteolytic activity, it has been reported that PAI-1 promotes cancer invasion and metastasis (16). Furthermore, PAI-1 could modulate cell proliferation and stimulate angiogenesis. In one experiment using Pai-1-deficient mice, Pai-1 deficiency abolished cancer invasion and angiogenic activity (17). In this context, it should be mentioned that PAI-1 is significantly upregulated in neoplastic tissue of the human colon (18). The PAI-1 promoter 4G/5G polymorphism, in which the 4G allele is associated with high PAI-1 expression, may influence the development of aggressive fibromatosis in familial adenomatous polyposis patients (19). Thus, it is conceivable that Pai-1 is one of the factors that explain linkages between hyperlipidemia and intestinal tumorigenesis.

In this study, we examined the effects of Pai-1 inhibitors, SK-216 and SK-116, in the diet on both hyperlipidemia and intestinal polyp formation in Min mice and demonstrated concomitant suppression of both. The possible mechanisms of its action in Apc-deficient mice and usage of PAI-1 inhibitors as possible candidates for colon cancer prevention are also discussed.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
Azoxymethane-induced F344 rat colon cancer-derived cell line RCN-9, which was obtained from RIKEN Bioresource Center (Tsukuba, Japan), was used. RCN-9 cells were grown in Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum at 37°C in a humidified incubator with 5% CO2.

Animals and chemicals
Male C57BL/6-ApcMin/+ mice (Min mice) were purchased from The Jackson Laboratory Maine, USA at 6 weeks of age and genotyped as previously reported (20). Heterozygotes of the Min strain and wild-type (C57BL/6J) mice were acclimated to laboratory conditions for 1 week. Five mice were housed per plastic cage with sterilized softwood chips as bedding in a barrier-sustained animal room at 24 ± 2°C and 55% humidity on a 12 h light/dark cycle. The PAI-1 inhibitors SK-216, disodium [5-[[6-[5-(1,1-dimethylethyl-2-benzoxazolyl)-2-naphthalenyl]oxy] pentyl] propanedioate, and SK-116, disodium [5-[[6-[4-phenyl-6-(phenylmethoxy)-2-pyrimidinyl]-2-naphthalenyl]oxy] pentyl]propanedioate, were chemically synthesized at Shizuoka Coffein Co. Ltd. Their structures are shown in Figure 1. In vitro data showed that these compounds reverse the inhibitory effects of PAI-1 against both tPA and urokinase using the method described by Charlton et al. (21). IC50 for SK-216 is 44 µM and that for SK-116 is 35 µM were reported in international patent WO04/010996 and WO04/011442, respectively. SK-216 was well mixed at concentrations of 25–100 p.p.m. in AIN-76A powdered basal diet (CLEA Japan, Tokyo, Japan). SK-116 was also well mixed at 50 p.p.m. in the same way.


Figure 1
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  		Fig. 1. Structures of SK-216 (A) and SK-116 (B).

 
Animal experiments
To investigate the effects of PAI-1 inhibitors on intestinal polyp formation, 10 male Min mice at 7 weeks of age were given SK-216 and SK-116 in the diet for 8 weeks. Food and water were available ad libitum. The animals were observed daily for clinical signs and mortality. Body weights and food consumption were measured weekly. Animals were anesthetized with ether and killed, and blood samples were collected from the abdominal aorta. Serum levels of TG and total cholesterol were measured as reported (6). The experiments were conducted according to the Guidelines for Animal Experiments in National Cancer Center of the Committee for Ethics of Animal Experimentation of the National Cancer Center.

The intestinal tract was removed, filled with 10% buffered formalin and separated into the small intestine, cecum and colon. The small intestine was divided into the proximal segment (4 cm in length) and then the proximal (middle) and distal halves of the remainder. All segments were opened longitudinally and fixed flat between sheets of filter paper in 10% buffered formalin. The numbers and sizes of polyps and their distributions in the intestine were assessed with a stereoscopic microscope (6).

To investigate the effects of PAI-1 inhibitors on Pai-1 expression levels in mouse intestinal mucosa, C57/BL6 mice were purchased from CLEA Japan at 6 weeks of age and were given either 0 or 100 p.p.m. SK-216 for 1 week. A day before killing, 200 µl soy oil (Wako Pure Chemical Industries, Ltd, Osaka, Japan) was given by gavage to the mice. The intestinal mucosa was removed by scraping for further reverse transcription (RT)–polymerase chain reaction (PCR) analysis.

Real-time PCR analysis and RT–PCR analysis
Tissue samples from liver of mice were rapidly deep-frozen in liquid nitrogen and stored at –80°C. Total RNA was isolated from tissues by using Isogen (Nippon Gene, Tokyo, Japan), treated with DNase (Invitrogen) and 3 µg aliquots in a final volume of 20 µl were used for synthesis of cDNA using an Omniscript RT Kit (Qiagen, Hilden, Germany) and an oligo(dT) primer. Real-time PCR was carried out using a DNA Engine Option TM 2 (MJ Japan Ltd, Tokyo, Japan) with SYBR Green Realtime PCR Master Mix (Toyobo Co., Osaka, Japan) according to the manufacturer's instructions. Primers for mouse Pai-1 (5'primer-GACACCCTCAGCATGTTCATC and 3'primer-AGGGTTGCACTAAACATGTCAG) and glyceraldehyde-3-phosphate dehydrogenase (5'primer-TTGTCTCCTGCGACTTCA and 3'primer-CACCACCCTGTTGCTGTA) were employed (22). To assess the specificity of each primer set, amplicons generated from the PCR reaction were analyzed for melting curves and also by electrophoresis in 2% agarose gels.

Tissue samples from intestinal mucosa were treated the same as liver samples and 1 µl of cDNA was included in a final volume of 10 µl with a PTC-200 DNA Engine (MJ Research, Waltham, MA) by using a Omniscript RT Kit (Qiagen). Cycling conditions were as follows: 94°C for 5 s, annealing temperature (60°C) for 30 s, 72°C for 60 s and 32 cycles after an initial step of 95°C for 3 min. A final elongation step of 72°C for 10 min completed the PCR. The products were then electrophoresed on 2% agarose gels.

Immunohistochemical staining
Small intestines were fixed, embedded and sectioned as Swiss rolls for further immunohistochemical examination with the avidin–biotin complex immunoperoxidase technique and polyclonal rabbit anti-Pai-1 antibodies (Abs) (Santa Cruz Biotechnology, Santa Cruz, CA) at 100x dilution. As the secondary Ab, biotinylated horse anti-rabbit IgG, affinity purified was employed at 200x dilution. Staining was performed using avidin–biotin reagents (Vectastain ABC reagents; Vector Laboratories), 3,3'-diaminobenzidine and hydrogen peroxide, and the sections were counterstained with hematoxylin to facilitate orientation. As a negative control, consecutive sections were immunostained without exposure to the primary Ab.

Enzyme-linked immunosorbent assay
The concentration of Pai-1 in the plasma was determined using a MOUSE PAI-1 Total Antigen ELISA Kit (Innovative Research, MI, USA) for five samples each from wild-type mice, untreated Min and PAI-1 inhibitor-treated Min mice, according to the manufacturer's protocol.

Western blot analysis
Protein expression was analyzed by western blot. Cells (2 x 105) were seeded in 24-well plates. After treatment, cells were lysed in 100 µl lysis buffer [0.0625 M Tris–HCl (pH 6.8), 20% 2-mercaptoethanol, 10% glycerol, 5% sodium dodecyl sulfate]. Equal amounts of protein were separated in 10% polyacrylamide gel electrophoresis–sodium dodecyl sulfate gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA). Abs against the Pai-1 (Santa Cruz Biotechnology) were used at a 1:2000 dilution. Peroxidase-conjugated secondary Abs for anti-rabbit IgG were obtained from GE Healthcare, Buckingham shire, UK. Blots were developed with enhanced chemiluminescence western blotting detection reagents (Amersham Biosciences, Buckingham shire, UK).

NF{kappa}B–DNA-binding activity assay
The activity of NF{kappa}B binding to oligonucleotides containing an NF{kappa}B consensus binding sites was measured by using TransAMTM NF{kappa}B Transcription Assay Kits according to the manufacturer's instructions (ActiveMotif, CA, USA). Briefly, RCN-9 cells treated with 50 µM SK-216 for 6 h were harvested from 24-well plates, and nuclear fractions were isolated by using a Nuclear Extract Kit from ActiveMotif. Nuclear fractions were applied to 96-well plates coated with oligonucleotides containing an NF{kappa}B consensus binding site. After treatment with anti-NF{kappa}B Ab, another peroxidase-linked Ab specific for NF{kappa}B was added. The substrate solution was added and optical density was assessed using the microplate reader set to 450 nm.

Statistical analysis
All the results are expressed as mean ± standard error values, with statistical analysis using Dunnett's test, except for the serum Pai-1 level investigation in Figure 2A. The Mann–Whitney test was used for statistical analyses of the serum Pai-1 level. Differences were considered to be statistically significant at P < 0.05.


Figure 2
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  		Fig. 2. Changes of serum Pai-1 level, liver Pai-1 mRNA and protein expression in the small intestine of Min mice. (A) Serum Pai-1 levels were detected by enzyme-linked immunosorbent assay using samples from Min mice (n = 5 each group) given diets containing SK-216 at doses of 25–100 p.p.m. for 9 weeks and also that from untreated wild-type mice (n = 5). Data are means ± standard errors *P < 0.05. (B) Quantitative real-time PCR analysis of Pai-1 mRNA expression in the livers of Min mice given diets containing SK-216 at doses from 25–100 p.p.m. for 9 weeks and also that in untreated wild-type mice. Data are mean of two mice of each group. (C) Immunohistochemistry of Pai-1 expression in normal parts of the small intestine of a Min mouse. (D) Immunohistochemistry of Pai-1 expression in a polyp part of the small intestine in a Min mouse. Bars represent 200 µm. Data are representative of six mice.

 

   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Expression of Pai-1 in serum, liver and small intestine
Serum Pai-1 was obviously increased (approximately eight times) in Min mice at 15 weeks of age as compared with wild-type littermates (Figure 2A). Pai-1 mRNA levels in the Min mice livers were also almost 11-fold elevated (Figure 2B). Furthermore, expression of Pai-1 was detected in small intestinal epithelial cells, but appeared weaker in stromal cells of Min mice (Figure 2C). Expression of Pai-1 was similar in both polyp and non-tumorous epithelial cells in the intestine (Figure 2D). Pai-1 immunostaining was strong in small intestinal epithelial cells of Min mice, but slightly weak in wild-type littermates, and no artificial stain was detected in controls using the second Ab only (data not shown).

Suppression of intestinal polyp formation in Min mice by Pai-1 inhibitors
Administration of SK-216 at 25, 50 and 100 p.p.m. for 9 weeks did not affect body weights, food intake or clinical signs of Min mice throughout the experimental period. Average daily food intake did not differ significantly among the groups, being 3.1, 3.4, 3.1 and 3.0 g per mouse per day for the 0, 25, 50 and 100 p.p.m. groups of Min mice, respectively. During the experiment, severe hemorrhage, which may occur with fibrinolysis, was not observed in both Pai-1 inhibitor-treated or -untreated mice. In addition, there were no changes observed in any organ weights that might have been attributable to toxicity.

Table I summarizes data for the number and distribution of intestinal polyps in the basal diet and SK-216 treated groups. Almost all polyps developed in the small intestine, with only a few in the colon (6). The total number of polyps was significantly decreased by administration of 50 and 100 p.p.m. SK-216 to 64 and 56% of the untreated control value, respectively. Reduction in the proximal, middle and distal parts was by 56, 48 and 28% with 50 p.p.m. and by 63, 59 and 28% with 100 p.p.m., respectively. Treatment with SK-216 did not affect the numbers of colon polyps. Administration of 50 p.p.m. SK-116 also decreased the total number of polyps to 70% of the untreated control value with significant reduction in the proximal section (Table II).


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  		Table I. Suppression of intestinal polyp development in Min mice by SK-216

 


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  		Table II. Suppression of intestinal polyp development in Min mice by SK-116

 
Figure 3 shows the size distributions of intestinal polyps in the basal diet, SK-216-and SK-116-treated groups. The maximal number of polyps was observed in the size range between 0.5 and 3.0 mm in diameter. Administration of 50 and 100 p.p.m. SK-216 reduced the numbers of polyps of all sizes (Figure 3A). Administration of 50 p.p.m. SK-116 reduced the numbers of polyps sized <1.5 mm in diameter (Figure 3B).


Figure 3
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  		Fig. 3. Effects of SK-216 and SK-116 on the size distribution of intestinal polyps in Min mice. (A) Min mice were fed a basal diet (black filled box) or a diet containing 25 p.p.m. (large dotted box), 50 p.p.m. (dotted box) or 100 p.p.m. (open box) SK-216 for 9 weeks. (B) Min mice were fed a basal diet (black filled box) or a diet containing 50 p.p.m. (dotted box) SK-116 for 9 weeks. The number of polyps per mouse in each size class is given as a mean ± standard error. *P < 0.05; **P < 0.01.

 
Reduction of serum Pai-1 levels and liver Pai-1 mRNA levels in Min mice by Pai-1 inhibitors
It has been recognized that Pai-1 inhibitors, SK-216 and SK-116, inhibit Pai-1 activity. The present study revealed that Pai-1 inhibitors suppress Pai-1 at both protein and mRNA levels. The highest dose used in this study, 100 p.p.m. SK-216, suppressed serum Pai-1 levels to the wild-type level (Figure 2A). Real-time PCR revealed that administration of 25, 50 and 100 p.p.m. SK-216 for 9 weeks suppressed increased hepatic Pai-1 mRNA levels (Figure 2B) in Min mice in a dose-dependent manner. Another Pai-1 inhibitor, SK-116, also reduced serum Pai-1 level in Min mice from 20.1 ± 6.7 ng/ml (0 p.p.m.) to 6.9 ± 4.1 ng/ml (50 p.p.m.). In the immunohistochemistry study, Pai-1 could be detected more weakly in non-tumorous and polyp epithelial cells of the small intestine in Min mice treated with 100 p.p.m. SK-216 compared with that of untreated Min mice (data not shown).

Improvement of serum lipid levels in Min mice by Pai-1 inhibitors
Consistent with our previous reports (6–8), serum TG levels in the Min mice fed the basal diet at 15 weeks of age were higher at 117 mg/dl than the 39.2 mg/dl in wild-type mice (Figure 4). Total cholesterol levels in Min mice were also increased 1.3-fold (92 versus 62 mg/dl) while free fatty acid levels were almost the same in both Min and wild-type mice. Administration of 50 and 100 p.p.m. SK-216 decreased serum levels of TG in Min mice to 74 and 57% of the untreated control value, respectively (Figure 4). Administration of 50 and 100 p.p.m. SK-216 also decreased serum levels of TG in wild-type mice from 39.2 to 18.4 and 17.6 mg/dl (P < 0.01), respectively. The levels of total cholesterol and free fatty acid were not decreased by SK-216 treatment.


Figure 4
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  		Fig. 4. Suppression of serum lipid levels in Min mice by SK-216. Values for serum levels of TG (A), total cholesterol (B) and free fatty acids (C) in Min mice given diet containing SK-216 at doses of 25–100 p.p.m. for 9 weeks are shown. Data are means ± standard errors. **P < 0.01.

 
Reduction in serum TG levels was also observed in the 50 p.p.m. SK-116-treated group. Administration of 50 p.p.m. SK-116 decreased serum levels of TG in Min mice to 76% of the untreated control value.

Decrease of Pai-1 mRNA levels and NF{kappa}B binding activity in intestinal mucosa cells by Pai-1 inhibitor
To investigate whether the Pai-1 inhibitors directly targeted the intestinal mucosa, SK-216 in diet was administered to C57/BL6 mice. As shown in Figure 5A, treatment with soy oil, consisting of TG as a major component, increased Pai-1 expression levels in the intestinal mucosa of two out of three mice. A weeklong treatment with 100 p.p.m. SK-216 reduced Pai-1 mRNA levels to lower than non-treated mice in two out of three mice. Similar results were obtained in an in vitro study. Treatment with 50 µM SK-216 for 17 h reduced basal Pai-1 protein levels in the colon cancer cell line RCN-9 (Figure 5B). Moreover, 50 µM SK-216 treatment for 6 h decreased NF{kappa}B activity (Figure 5C). These results suggest that decreased NF{kappa}B activity may be involved in both suppression of Pai-1 mRNA and inhibition of polyp formation in Min mice by SK-216 treatment.


Figure 5
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  		Fig. 5. Decrease of Pai-1 mRNA levels and NF{kappa}B-binding activity in intestinal mucosa/RCN-9 cells by SK-216 treatment. (A) RT–PCR for Pai-1 and GAPDH are shown. C57/BL mice (n = 3 for each group) were fed diet with or without 100 p.p.m. SK-216 for a week and gavaged with 200 µl soy oil 2 h before collecting intestinal mucosa. RT–PCR was performed as describe in Materials and Methods. (B) Western blotting for Pai-1 and beta-actin are shown. RCN-9 cells grown in 24-well plates were treated for 17 h with indicated doses of SK-216. Actin was used as a loading control. (C) RCN-9 cells grown in six-well plates were treated with and without 50 µM SK-216 for 6 h. Nuclear fractions of RCN-9 cells were isolated and analyzed for NF{kappa}B-binding activity as describe in Materials and Methods. Values represent means ± standard errors of three wells.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
This study provided evidence that administration of the PAI-1 inhibitors SK-216 and SK116, which also reduce Pai-1 mRNA and protein levels, suppresses intestinal polyp formation in Min mice. It is therefore speculated that Pai-1 activity itself may play an important role in intestinal polyp formation in Apc-deficient mice.

We previously reported markedly increased serum levels of TGs and low levels of LPL mRNA in liver and small intestine in Min mice compared with their wild-type counterparts (6–8). Thus, we hypothesized that hypertriglyceridemia is a leading cause of intestinal polyp formation. However, the molecular mechanisms could only be partially addressed since only little information is available as to effects of TG-rich lipoproteins (23,24). TG-rich lipoproteins from type IV hyperlipidemic patients induce phosphorylation of p38 mitogen-activated protein kinase, and CAMP response element binding protein Inhibitor of kappa B {alpha} and activate DNA-binding activity of transcriptional factors, CREB, NF{kappa}B and AP-1. TG-rich lipoproteins also upregulate the expression of proinflammatory and adhesion-related genes, monocyte chemoattractant protein-1, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and PAI-1. These mitogen-activated protein kinase pathways and molecules are well known to be involved in endothelial cell growth. Treatment of smooth muscle cells with low-density lipoprotein results in the activation of protein kinase C and mitogen-activated protein kinase as well as induction of the cell cycle-related genes c-fos, c-myc (24) and early growth response gene-1 (egr-1, 25). Thus, hypertriglyceridemia may also modify epithelial cell growth. To explore molecular mechanisms underlying the link between hypertriglyceridemia and polyp formation, we first selected candidate molecules from those which are increased with the metabolic syndrome (26). Focusing on adipocytokines, we selected Pai-1 among possible candidate molecules, including adiponectin, IL-1, IL-6, leptin and tumor necrosis factor {alpha}. Liver was used for the RT–PCR analysis because this is the major Pai-1-producing organ and expression may correlate with hyperlipidemic states in the mice. Moreover, Pai-1 immunostaining was strong in small intestinal epithelial cells of Min mice. The reason why PAI-1 inhibitors also reduced serum TG levels remains unclear and examination of Pai-1 effects on TG metabolism would appear warranted.

Regarding the mechanisms underlying suppression of intestinal polyp formation by PAI-1 inhibitor in Min mice, contrary reports should be noted (10,27). Inhibition of PAI-1 activation results in generation of active growth factors from inactivated forms like heparin-bound epidermal growth factor, hepatocyte growth factor, basic fibroblast growth factor or insulin-like growth factors (10). Moreover, suppression of PAI-1 enhances growth factor signaling through the phosphatidylinositol 3-kinase–protein kinase B route (27). These reports indicate that PAI-1 may inhibit cell proliferation. However, Li et al. (28) have reported that genetic Pai-1 deficiency reduced the number of aggressive fibromatosis tumors in Apc/Apc1638N mice. The data from Li et al. were partially consistent with our results. Especially in male Pai-1-null Apc/Apc1638N mice, the number of aggressive fibromatosis tumors was decreased. However, no significant difference was observed in the number of gastrointestinal tumors compared with that of Apc/Apc1638N mice (0.7 ± 0.5 versus 1.1 ± 0.4; P > 0.05). These results may be due to weak statistical power derived from the relatively few intestinal polyps that developed in Apc/Apc1638N mice. Pai-1–/– tumor cells demonstrated reduced proliferation and motility in vitro (28). In addition, our in vitro study demonstrated that Pai-1 inhibitor SK-216 also decreased NF{kappa}B activity. This decrease in NF{kappa}B activity may be involved in both suppression of Pai-1 levels and inhibition of polyp development in Min mice.

In conclusion, this study indicated that the PAI-1 inhibitors, SK-216 and SK-116, have potential benefit for suppression of intestinal polyp development. Thus, SK-216, SK-116 and related derivatives could be promising candidate chemopreventive agents for colon cancer. As it is becoming increasingly clear that hyperlipidemia is an important player in carcinogenesis, our observations may lead to a better understanding of the role of hyperlipidemia in colon carcinogenesis.


   Funding
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Grants-in-Aid for Cancer Research, for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour, and Welfare of Japan (H19-013).


   Acknowledgments
 
During the performance of this work, N.N. was the recipient of a Research Resident fellowship from the Foundation for Promotion of Cancer Research.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 10, 2007; revised January 23, 2008; accepted January 23, 2008.


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