Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting IL-8

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Clotrimazole ameliorates intestinal inflammation and

abnormal angiogenesis by inhibiting IL-8 expression through

a NF-κB-dependent manner

Dinesh Thapa, Jong Suk Lee, Su-Young Park, Yun-Hee Bae, Soo-Kyung Bae, Jun Bum Kwon,

Kyoung-Jin Kim, Mi-Kyoung Kwak, Young-Joon Park, Han Gon Choi, Jung-Ae Kim

College of Pharmacy, Yeungnam University, Gyeongsan 712–749, South Korea (D.T., J.S.L.,

S.Y.P., J.B.K., K.J.K., M.K.K., H.G.C., J.A.K.), School of Medicine, Pusan National University,

Pusan 602-739 , South Korea (Y.H.B., S.K.B.), and Research Center, Samil Pharmaceutical Co.

Ltd., Ansan 425-852, South Korea (Y.J.P.)

JPET Fast Forward. Published on August 26, 2008 as DOI:10.1124/jpet.108.141887

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on August 26, 2008 as DOI: 10.1124/jpet.108.141887

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Running Title: Inhibition of intestinal inflammation via IL-8 suppression

Corresponding author: Jung-Ae Kim, Ph. D.

Address correspondence to: Dr. Jung-Ae Kim, College of Pharmacy, Yeungnam University,

214-1 Dae-Dong, Yeungnam University, Gyeongsan 712-749, Korea.

E-mail: [email protected]

Tel: +82-53-810-2816

Fax: +82-53-810-4654

Number of text pages: 35 (excluding Tables and Figures)

Number of tables: 3

Figures: 6

References: 40

Number of words in the Abstract: 226

Number of words in the Introduction: 443

Number of words in the Discussion: 1416

ABBREVIATIONS: 5-ASA, 5-aminosalicylic acid; CAM, Chick chorioallantoic membrane;

CLT, Clotrimazole; DMSO, dimethyl sulfoxide; ELISA, Enzyme-Linked Immunosorbent

Assay; IBD, inflammatory bowel disease; IL, interleukin; MPO, myeloperoxidase; MTT, 3-

(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide; NF-κB, nuclear factor-κB;

PDTC, pyrrolidine dithiocarbamate; STAT, signal transducer and activator of transcription;

TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor

Section: GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL


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ABSTRACT

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of

neutrophils, but also in inducing exaggerated angiogenesis at the inflammed site. In the present

study, we investigated that clotrimazole (CLT) inhibits intestinal inflammation, and the

inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene

sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the

TNBS-induced weight loss, colon ulceration, and myeloperoxidase (MPO) activity increase. In

the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and

nuclear factor (NF)-κB activation. In a cellular model of colitis using tumor necrosis factor

(TNF)-α-stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed

TNF-α-mediated IL-8 induction and NF-κB transcriptional activity revealed by a luciferase

reporter gene assay. Furthermore, co-treatment with CLT and PDTC, a NF-κB inhibitor,

synergistically reduced the NF-κB transcriptional activity as well as IL-8 expression. In an in

vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation and

invasion of human umbilical vein endothelial cells (HUVECs). The in vivo angiogenesis assay

using chick chorioallantoic membrane (CAM) also showed that CLT significantly inhibited the

IL-8-induced formation of new blood vessels. Collectively, these results suggest that CLT may

prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression,

but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells

during pathogenesis of intestinal inflammation.


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INTRODUCTION

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the

gastrointestinal tract. The IBD patients suffer from the rectal bleeding, severe diarrhea,

abdominal pain, and weight loss. Although the etiology of IBD has not been fully elucidated,

many studies have suggested that multiple factors, genetic, immunologic, and environmental

factors, all contribute to the pathogenesis of IBD (Fiocchi, 1998). The common pathological

changes associated with IBD include disruption of epithelial barrier, infiltration of dysregulated

immune cells, and production of pro-inflammatory mediators including chemokines.

Chemokines are essential for activating and recruiting inflammatory cells. Interleukin

(IL)-8, a multifunctional member of the chemotactic CXC cytokines, has been reported to be

upregulated in the colon of IBD patients (Banks et al., 2003; Subramanian et al., 2008).

Furthermore, it has been shown that the severity of inflammation in the colon is congruent with

the IL-8 expression (Mazzucchelli et al., 1994; Daig et al., 1996). In normal tissues, IL-8

expression is low or undetectable, whereas upon stimulation by various stimuli including tumor

necrosis factor (TNF)-α, IL-8 expression can be induced up to 100-fold (Baggiolini et al., 1994).

In contrast to the constitutively expressed chemokines which direct baseline leukocyte

trafficking, the inducible chemokines such as IL-8 further attracts leukocytes to local sites and

aggravates inflammation (Laing and Secombes, 2004).

Angiogenesis, the process of new capillary formation from preexisting vessels, is

thought to be intrinsic to inflammation. Inflammatory mediators either directly or indirectly

promote angiogenesis, which, in turn, contributes to inflammatory pathology by allowing

further recruitment of inflammatory cells (Jackson et al., 1997). Recent studies showing that

excessive angiogenesis in IBD is particularly prominent in areas of active inflammation


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indicates that angiogenesis is an integral component of IBD pathogenesis (Saito et al., 2003;

Danese et al., 2007). In addition to its pro-inflammatory action, IL-8 also acts as an angiogenic

factor by directly enhancing endothelial cell survival, proliferation, and matrix

metalloproteinase production (Heidemann et al., 2003; Li et al., 2003). Thus, the development

of strategies to block IL-8 production or IL-8-induced actions may hold a promise in treating

chronic inflammatory diseases like IBD.

Clotrimazole (CLT) is an antifungal imidazole derivative that has been in clinical use

for more than 20 years. Recent studies have shown that CLT inhibits proliferation of human

cancer cells (Khalid et al., 2005) and vascular endothelial cells, leading to inhibition of growth

factor-stimulated angiogenesis (Takahashi et al., 1998), and thus, tumor growth (Belo et al.,

2004). However, it has not been reported whether CLT modulates inflammatory angiogenesis or

not. In this study, we investigated that CLT suppresses intestinal inflammation via down-

regulating pro-inflammatory cytokine IL-8 expression and blocking the epithelial and

endothelial responses to IL-8.


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METHODS

Materials

The recombinant human IL-8 (also called CXCL8) was purchased from R&D Systems

(Minneapolis, MN, USA). An endothelial growth medium (EGM)-2 bullet kit and EGM-2

SingleQuots (hydrocortisone, hFGF, VEGF, R3-IGF-1, ascorbic acid, hEGF, heparin,

gentamicin and FBS) were purchased from Cambrex (San Diego, CA, USA). HEPES,

trypsin/EDTA and trypsin neutralizing solution (TNS) were purchased from Clonetics, Inc.

(Walkersville, MD, USA). The Matrigel was purchased from BD Biosciences (Bedford, MA,

USA). Trinitrobenzene sulfonic acid (TNBS), pyrrolidine dithiocarbamate (PDTC), 3-(4,5-

dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT), sodium pyruvate, dimethyl

sulfoxide (DMSO), protease inhibitor cocktail, sodium dodecyl sulfate (SDS) were obtained

from Sigma Chemical Co. (St. Louis, MO, USA). 5-aminosalicylic acid (5-ASA) was

purchased from Sigma where as Clotrimazole (1-[o-chloro- alpha-,alpha-diphenylbenzyl]-

imidazole) was supplied from Samil Pharmaceutical Co. Ltd. (Ansan, Korea) and Korea United

Pharm. Inc. (Seoul, Korea).

TNBS-induced experimental colitis

Sprague Dawly rats (7–8-week-old) were purchased from Samtaco bio Korea (Osan, Korea).

Rats were kept fasting (able to drink ad libidum) for 24 h prior to induction of colitis. Then,

they were anesthetized lightly using diethyl ether. Using a polyethylene catheter fitted onto a 1

ml syringe, rats were slowly injected with 0.8 ml of 5% TNBS in 50% (v/v) ethanol into the

lumen of the colon (8 cm proximal to the anus through the rectum), and they were kept in a

vertical position for 60 s before being returned to their cages. Rats in the control group were


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handled similarly, but 50% (v/v) ethanol alone was administrated instead. To investigate the

effect of drugs, rats were administrated CLT (10–50 mg/kg/day) through intraperitoneally for 5

days after the TNBS administration. In our experiments, 5-ASA, an active metabolite of

sulfasalazine which is a commonly used drug for IBD, was used as a positive control.

All rats were sacrificed on day 7 after the TNBS administration. The macroscopic ulceration and

severity of colitis was evaluated by two independent investigators who were blinded to the

treatment. The colon tissues from 5 to 7 cm proximal to the rectum were cut out, and used for

histologic examination as well as myeloperoxidase (MPO) activity measurement. Animal

experiments were performed according to the institutional guidelines of the National Institute of

Health and the University for the care and use of laboratory animals.

Myeloperoxidase (MPO) activity measurement

MPO serves as a marker for tissue neutrophil infiltration. To measure MPO activity, we used a

MOP assay kit (ASA-001, Cytostore, Canada). The 1 cm segment of dissected colon tissues

were washed in cold PBS (pH 7.4), weighed, suspended in 0.5 ml of ice-cold sample buffer per

50 mg of tissue, and homogenized 30 s using a tissue homogenizer at 4°C (Biospec Products

Inc., Switzerland). The homogenized sample was mixed with an additional sample buffer to

give a final concentration of 50 mg/ml and centrifuged at 10000 rpm for 5 min twice. The level

of MPO activity in the tissue was measured spectrophotometrically. Each 20 μl of sample

solution was mixed with 200 μl of development reagent (12.5 μl of H2O2 containing chromogen

powder in phosphate buffer 2.5 ml), and then the absorbance at 450 nm was measured

immediately and at 1 min intervals using a spectrophotometer (Molecular Devices, Versa MAX,

CA, USA). One unit of MPO activity was defined by Bradley et al. (1982) as the activity

required for converting 1 μM of H2O2 at 25°C for 7 min, and expressed as Units/g of tissue.


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Western blot analysis

Cytoplasmic or nuclear extracts were collected using an NE-PER (Pierce, Rockford, USA) kit

as per the instructions of the manufacturer. Briefly, colon tissue homogenate and collected

HT29 cells were resuspended in 50 μl cytoplasmic extraction reagent I containing protease

inhibitor cocktail, and then, homogenized by vigorous vortexing for 15 s and left on ice (4°C).

After 10 min, 2.3 μl of cytoplasmic extraction reagent II was added and vortexed on the highest

setting for 5 s. The homogenates were then centrifuged at 14000 rpm for 5 min. Supernatant

(cytoplasmic fraction) was transferred to a fresh tube and stored at -70°C. To lyse the pellet

containing nuclear fraction, 30 µl aliquot of nuclear extraction reagent and protease inhibitor

cocktail were added. The pellets were vortexed for 1 min, left on ice (4°C) for 40 min, and

centrifuged for 10 min (4°C, 14000 rpm) to collect the supernatant (nuclear fraction).

The soluble protein concentrations in cytoplasmic or nuclear lysates were determined by BCA

protein assay kit (Pierce, Rockford, USA). The equal amount of proteins (15 µg regardless of

type of lysate) was separated on SDS-PAGE, and transferred to a Hybond ECL nitrocellulose

membrane (Amersham Life Science, Buckinghamshire, UK) at 200 mA for an hour. The

membranes were blocked in a 5% skim milk in Tris-buffered saline-Tween 20 (TBST) (20 nM

Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20) at room temperature for 1 h. The membrane

was incubated with antibodies specific to NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA,

USA), I-κB-α (Santa Cruz Biotechnology), phospho-I-κB-α (Cell Signaling Technology,

Beverly, MA, USA), COX-1, COX-2 (Cayman Chemical Co., Ann Arbor, MI, USA),

pSTAT3(Tyr705) and STAT3 (Cell Signaling Technology) in skim milk-TBS at 4°C overnight.

Then, the membrane was washed three times with TBST, and incubated with horseradish

peroxidase-conjugated secondary antibody in skim milk-TBS for 1 h at room temperature. The


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immunoreactive proteins were visualized with an ECL kit (Amersham Life Science,

Buckinghamshire, UK).

Histology and Immunohostochemical examination

For histological examination, colon tissues from 6 to 7 cm proximal to the rectum were cut out

and fixed in 4% paraformaldehyde/PBS. The paraffin-embedded tissue sections (3 µm

thickness) were stained with hematoxylin and eosin (H&E) using standard techniques. The

histological lesions on the prepared cross trimmed H&E stained histological samples were

evaluated by a pathologist, who was blinded to the experimental groups, according to previously

established criteria (Peran et al., 2005) as shown in Table 1.

For immunohistochemistry, colon tissues were prepared for frozen section in 7 μm thickness,

and incubated with rabbit anti-von Willebrand Factor (vWF) (Chemicon, Temecula, CA, USA)

for overnight at 4°C. FITC-conjugated goat anti-rabbit IgG (Molecular Probe, Eugene, OR,

USA) was used as a secondary antibody at 1:500 dilutions and incubated for 1 h at room

temperature. After mounting with the media containing DAPI (Vector Laboratories, Burlingame,

CA, USA) for nuclear counterstaining, tissue slides were visualized under a fluorescent

microscope (Olympus).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA from rat colon tissues and HT29 cells was extracted by using a RNeasy kit (Qiagen

GmbH, Hilden, Germany) according to the manufacturer's instruction, and reverse transcribed

by using a Reverse Transcription kit (Qiagen GmbH). Two microliters of cDNA from rat colon

and HT29 cells were used for conventional PCR and real-time PCR, respectively.

The predicted size of PCR products for rat IL-8 (primer sets sense 5′-


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ACGCTGGCTTCTGACAACACTAGT-3′ and antisense 5′-

CTTCTCTGTCCTGAGACGAGAAGG-3′) was 496 bp. For equal loading normalization,

GAPDH (sense 5′-ACCACAGTCCATGCCATCAC-3′ and antisense 5′-

TCCACCACCCTGTTGCTGTA-3′) was used. PCR products were separated on 1.8% agarose

gels and visualized by UV transillumination. Real-time PCR was carried out with a PCR kit

(Qiagen GmbH, Quantitect SYBR green) using a Rotor-Gene 6000 (Corbett Life Science,

Mortlake, Australia) in the condition of 95°C for 15 min followed by 45 cycles of denaturation

at 95°C for 5 s, annealing at 55°C for 10 s, and extension at 72°C for 20 s using primer sets

specific for human IL-8 (sense 5′-CACCCCAAATTTATCAAAGA-3′ and antisense 5′-

TCAAAAACTTCTCCACAACC-3′), and GAPDH (provided from Qiagen). The real time PCR

product was normalized using GAPDH as an internal control. The fold change over control

level was determined according to the comparative cycle threshold method.

IL-8 Enzyme-Linked Immunosorbent Assay (ELISA)

The secreted IL-8 level in culture supernatant was quantified by an ELISA kit (R&D) according

to the manufacturer’s protocol. The measurements were made in three independent experiments

with duplicates.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego,

CA, USA). HUVECs were maintained in cell culture flasks coated with gelatin 0.2%, and

cultured with EBM-2 containing hydrocortisone, hFGF, VEGF, R3-IGF-1, ascorbic acid, hEGF,

heparin, gentamicin and 2% FBS. HUVECs and maintained in EBM-2 (Sigma, St. Louis, MO,

USA). Confluent cultures of HUVECs were serially passaged until sixth passages.


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The HT29 human colon cancer cell line and U937 human leukemia cell line were purchased

from American Type Culture Collection (Rockville, MA, USA), and the cells were grown in

RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal

bovine serum (FBS), 1% penicillin/streptomycin, 1 mM sodium pyruvate, and 2 mM glutamine.

Culture medium was replaced every other day.

Transient transfection and luciferase assay

NF-κB transactivation was studied using luciferase reporter plasmids from Stratagene (La Jolla,

CA, USA) according to the manufacturer’s instruction. Briefly, vector DNA (pNF-κB-

Luciferase plasmid) was allowed to form complexes with GeneJammer (Stratagene) at a 0.003%

(v/v) final concentration. The DNA-GeneJammer mixture was added to 40 to 50% confluent

HT29 cells which were already washed once in an antibiotic-free complete medium. The

transfection was continued at 37°C for 20 h, and then, the medium was changed to normal

growth medium. After the cells were allowed to grow for another 24 h, the transfected cells

were pretreated with CLT or PDTC for 1 h followed by TNF-α for 3 h. The cell lysates was

used for luciferase assay using a dual luciferase reporter assay system (Promega Corporation,

Madison, WI, USA), and the emitted light was measured with a luminometer (Turner

Biosystems, CA, USA).

Monocyte Adhesion assay

The monocyte-epithelial cell adhesion was evaluated using U937 monocytic cells and HT29

colonic epithelial cells as previously described (Kobayashi et al., 2006). U937 cells were

prelabeled with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester

(BCECF/AM, 10 µg/ml) for 1 h at 37°C. HT29 cells in 48-well plate was pretreated with CLT


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or 5-ASA for 1 h and then stimulated with IL-8 for 3 h. Then, the cells were co-incubated with

BCECF/AM-prelabeled U937 cells (5 × 105 cells/well) for 40 min at 37°C. The plates were

washed twice with PBS to remove non-adhering U937 cells. A set of cells representing each

group was taken and imaged by fluorescence microscopy connected to a digital camera

(TE2000-U, Nikon, Japan). The cells in the other sets were lysed with 0.1% Triton X-100 in 0.1

M Tris, and the BCECF fluorescence was analyzed by using a Fluostar optima microplate reader

(BMG Labtech GmbH, Germany) using excitation at 485 nm and emission at 520 nm.

HUVEC proliferation assay

HUVECs plated at a density of 5 × 104 cells/well were incubated in serum-reduced (1% FBS)

EBM-2 for 24 h, and then, co-treated with VEGF (20 ng/ml) in the absence or presence of CLT

for 48 h. After incubation, the viable cell number was measured by MTT assay. The cells were

incubated with 10 μl of 5 mg/ml of MTT solution added to the medium for 4 h at 37°C. Then,

the culture medium was removed, and 200 μl of DMSO was added to each well to dissolve

formazan. Absorbance was measured at 540 nm using a microplate reader (Molecular Devices,

Versa MAX Sunnyvale, CA, USA). The viable cell number was expressed as a percent of the

control culture.

Tube formation assay

The tube formation assays were performed on 48-well plates coated with 100 μl of Matrigel per

well and polymerized at 37°C for 30 min. HUVECs were suspended in EBM-2 medium

containing 1% FBS were plated on matrigel at a density of 1 × 104 cells per well, and they were

then treated with test compounds. After 14 h, four fields of each culture well were randomly

selected and photographed with a microscope attached to a CCD camera (TE2000-U, Nikon,


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Japan).

Matrigel-Invasion assay

In vitro invasion assay was performed using transwell inserts (Corning, NY, USA), which

consisted of a 24-well companion plate with 12 cell culture inserts containing 8 μm pore size

filters. Briefly, the upper and lower parts of transwell inserts were coated with 10 µl of Matrigel

(0.5 mg/ml) and 40 µl of type I collagen (0.5 mg/ml), respectively. HUVECs (5 × 104 cells/100

μl) were added to each insert (upper chamber), and the chemoattractant (1% FBS) was placed in

each well. The cells were incubated for 24 h at 37°C. After incubation, the upper surface of the

transwell was wiped out with a cotton swab to remove non-invading cells. The cells that had

invaded to the lower surface of the membrane were fixed with methanol and stained with H&E.

Invasion was expressed as the number of invaded cells per microscopic field by counting the

cells in randomly chosen microscopic fields. At least five microscopic fields were counted per

group, and three independent experiments were performed.

Chick chorioallantoic membrane (CAM) Assay

In vivo neovascularization was examined by using Chick chorioallantoic membrane (CAM).

Briefly, 10-day-old embryos were purchased from Baek-ja farm (Cheongsong, Korea) and were

incubated at 37°C with 55% relative humidity. A small hole was punctured in the shell

concealing the air sac using a hypodermic needle. A second hole was punctured in the shell on

the broadside of the egg directly over avascular portion of the embryonic membrane, as

observed during candling. A false air sac was created beneath the second hole by the application

of negative pressure to the first hole, causing the CAM to be separated from the shell. A

window, approximately 1 cm2, was cut in the shell over the dropped CAM with the use of a


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small crafts grinding wheel (Dremel, Racine WI, USA). Sterile filter disks (Whatman No.1 filter

paper, UK) were soaked in 3 mg/ml cortisone acetate in a solution of 90% ethanol and water

and subsequently air dried under sterile conditions. IL-8 was used to stimulate vessel growth on

the CAMs of 10-day-old chick embryos. Sterile filter disks absorbed with IL-8 (10 ng/disk)

dissolved in PBS containing 0.1% BSA were placed on the growing CAMs. Then, test

compounds or a vehicle was added directly to CAMs topically. After 72 h, CAM tissue directly

beneath the disk was resected from the embryo, and was harvested under light microscopy

(Leica, Germany). The number of vessel branch points contained in a circular region equal to

the area of filter disk was counted for each section.

Data Analysis

The data are expressed as mean ± S.E.M. of three independent experiments. Statistical analysis

was done with student’s t test to calculate differences between groups. For histological

assessment, a multiple comparison tests for different dose groups were conducted using Levene

test and one way ANOVA test. Statistical analysis was performed using SPSS for window

(Release 14.0K, SPSS Inc., USA). P values less than 0.05 were considered statistically

significant.


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RESULTS

CLT ameliorates TNBS-induced rat colitis

The rats treated with TNBS developed significant signs of colitis, bloody diarrhea and wasting

conditions with sluggish and weak movement. In addition, TNBS induced stagnated body

weight in contrast to the weight gain in vehicle-treated control group (Fig. 1A). The colon

length distal to cecum was shortened by TNBS (Figs. 1B and 1C), whereas the weight of colon

tissue was increased by TNBS (Fig. 1D). However, the rats administered with CLT (10 to 50

mg/kg) or 5-ASA (100 mg/kg), an active metabolite of sulfasalazine that is a commonly used

drug for IBD, for 5 days significantly recovered the body and colon weight (Figs. 1A and 1D).

To confirm the TNBS-induced colitis, we measured colonic MPO activity, which serves as a

marker for tissue neutrophil infiltration, and COX-2 induction. In addition, since many studies

have elucidated that NF-κB is actively involved in the regulation of inflammatory genes

(Baeuerle and Henkel, 1994; Tak and Firestein, 2001; Blackwell and Christman, 1997), we

examined the activation of inflammatory transcription factor, NF-κB. TNBS-induced increase in

MPO activity (Fig. 2A) and COX-2 and nuclear NF-κB was significantly reduced by CLT (Fig.

2B). The cytosolic I-κB-α level was decreased by TNBS, which was blocked by CLT

administration. Such inhibitory effects of CLT at 10 mg/kg dose were comparable to 100 mg/kg

of 5-ASA, and high dose of CLT (50 mg/kg) almost completely suppressed the MPO activity.

Next, we investigated whether the protective effects of CLT in TNBS-induced colitis is

associated with the inhibition of pathogenic angiogenesis and IL-8 reduction by examining

histological changes. In histomorphometrical examinations as shown in Table 2, TNBS induced

significant increase of thickness of mucosa, submucosa and total colonic walls, and number of

infiltrated inflammatory cells in the both mucosa and submucosa. However, such


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histopathological changes were dramatically suppressed by the treatment with 5-ASA and CLT.

In addition, the quantitation of microscopic scores of the colon tissues revealed that TNBS

induced ulceration, mucus cell depletion, inflammatory cell infiltration and edematous changes

whereas treatment with CLT or 5-ASA reduced the TNBS-induced changes, which were

summarized in Table 3. The lesion site in TNBS-treated rat colon showed that the swollen

submucosal layer contained increased number of blood vessels which was detected by using

anti-vWF antibody (Fig. 2C). The mucosa and submucosal blood vessels in response to the

stimulation with TNBS was recovered by the administration of CLT. In addition, IL-8 tissue

level was highly increased in TNBS-treated inflamed colon, and such abnormal increase was

significantly suppressed by the administration of CLT and 5-ASA (Fig. 2D). Since STAT3, a

transcriptional regulator of several cytokines including IL-8 (Yeh et al., 2004), is reported to be

upregulated in IBD, we also examined any changes in STAT3. As depicted in Fig. 2E, STAT3

was highly phosphorylated in TNBS-treated inflamed colon, and such abnormal

phosphorylation was significantly suppressed by the administration of CLT and 5-ASA.

CLT inhibits TNF-α-induced IL-8 expression via suppression of NF-κB activation in HT29

cells

To examine the mechanism by which CLT regulates IL-8 expression in colon epithelial cells, we

used an in vitro model of colitis. Treatment of HT29 colon epithelial cells with TNF-α induced a

dramatic increase in IL-8 mRNA expression measured by real time PCR (Fig. 3A) and protein

secretion detected by ELISA (Fig. 3B). However, CLT significantly suppressed the TNF-α-

induced IL-8 expression in a concentration-dependent manner. Furthermore, the effective

concentration of CLT was almost 1000 times lower than 5-ASA. Since IL-8 expression has been

reported to be related to the activation of NF-κB, we also examined whether the inhibitory effect


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of CLT on the expression of IL-8 is mediated through the regulation of NF-κB activation. As in

TNBS-induced rat colitis, TNF-α increased NF-κB nuclear translocation and I-κB-α

phosphorylation in along with I-κB-α decrease in HT29 cells (Fig. 3C). Furthermore, TNF-α

stimulation (10 ng/ml) led to 7 fold increase in NF-κB transcriptional activity in the cells

transfected with NF-κB-Luc plasmid (Fig. 3D). Treatment of the cells with CLT, however,

concentration-dependently inhibited the TNF-α-induced transcriptional activity of NF-κB. Such

effect of CLT (5 μM) was similar to that of PDTC (50 μM), a NF-κB inhibitor. Moreover, the

combinated treatment with CLT (5 μM) and PDTC (50 μM) synergistically suppressed the NF-

κB activation. Similarly, co-treatment with PDTC and CLT also synergistically inhibited the

TNF-α-induced IL-8 secretion (Fig. 3E).

Inhibitory effects of CLT on IL-8-induced monocyte adhesion to colon epithelial cells

Since IL-8 is known as a chemokine playing an important role in the pathogenesis of IBD

(Mazzucchelli et al., 1994), we examined whether CLT inhibits IL-8-induced monocyte

adhesion to colon epithelial cells, which is an initial event of colon inflammation. As shown in

Fig. 4, CLT significantly reduced the IL-8-induced U937 cell adhesion to HT29 cells.

Importantly, such inhibitory action of CLT was effective at the concentrations between 1 and 10

μM, which was comparable to the effect of 20 mM of 5-ASA.

CLT inhibits the IL-8-induced angiogenesis in vitro and in vivo

IL-8 is also known to play a pro-angiogenic factor in inflamed site. Since we found that CLT

suppressed the TNBS-induced abnormal angiogenesis in rat colitis model, we also examined a

potential that CLT inhibits IL-8-induced neovascularization by analyzing each step of

angiogenesis, endothelial proliferation, tube formation and migration, using HUVECs.


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Treatment with CLT concentration-dependently inhibited the IL-8-induced endothelial cell

proliferation (Fig. 5A). When HUVECs were placed on a Matrigel-coated plate in the presence

of IL-8 (10 ng/ml), they rapidly aligned with one another and formed tube-like structures within

14 h, and CLT prevented the IL-8-stimulated tube formation in a concentration-dependent

manner (Fig. 5B). In an invasion assay using a Matrigel-coated transwell insert, treatment of the

cells with IL-8 (10 ng/ml) increased the number of invaded cells into the lower compartment

through the Matrigel by more than 3-fold compared to the control in which only low level of

FBS (1%) was present as a chemoattractant (Fig. 5C). However, CLT significantly suppressed

the IL-8-induced invasion of the cells. To further confirm the anti-angiogenic effects of CLT, we

performed CAM assay which is an in vivo model of angiogenesis. IL-8 increased the number of

newly formed blood vessel which was assessed as the number of vessel branch points. Such IL-

8-induced neovascularization was significantly suppressed by the treatment with CLT in a dose-

dependent manner (Fig. 6).


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DISCUSSION

CLT, an anti-mycotic agent, has been reported as a potent growth inhibitor of tumor

(Khalid et al., 2005) and vascular endothelial cells (Takahashi et al., 1998). In the present study,

we demonstrated that CLT also suppresses intestinal inflammation by inhibiting pro-

inflammatory cytokine expression and pathogenic angiogenesis.

In TNBS-induced colitis which is a well established model of intestinal inflammation

with some resemblances to human IBD, the histopathological lesions of the colon are generally

documented as ulceration, mucus cell depletion, inflammatory cell infiltration and edematous

changes. In the present study, the inhibition of the histopathological changes (Tables 2 and 3)

against TNBS-induced colitis by CLT was considered as direct evidence that CLT ameliorated

or treated the TNBS-induced colitis. In addition, similar or more favorable inhibitory effects

were detected in CLT (50 mg/kg) treated group compared to 5-ASA (100 mg/kg) group,

indicating that CLT may be a better drug for IBD treatment.

Many evidences have implicated that the pathogenesis of chronic inflammatory disease

including IBD involves angiogenesis (Pousa et al., 2008). Based on these notions,

antiangiogenic approach is suggested as a new therapeutic intervention for chronic

inflammatory diseases (Danese et al., 2007). Inflammatory cells release pro-inflammatory

cytokines and chemokines which activate directly or indirectly vascular endothelial cells to

make new blood vessels at the inflammatory sites. On the other hand, pathogenic angiogenesis

during chronic inflammation may aggravate the inflammatory response by inducing enhanced

supply of nutrients and oxygen, abnormal and dysfunctional vessel architecture, and enhanced

recruitment of inflammatory cells (Chidlow et al., 2007). There is a common molecule involved

in the process of inflammation and angiogenesis. NF-κB is one of the critical molecules


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regulating the expression and function of various genes involved not only in inflammation but

also in cell survival, growth, and migration. The excess or inappropriate NF-κB activation has

been documented in the colon tissue of IBD patients and animal models of IBD, mainly in

lamina propria mononuclear cells and epithelial cells of the inflamed gut (Rogler et al., 1998;

Sánchez-Hidalgo et al., 2007). The importance of NF-κB activity in the pathogenesis of IBD has

also been hinted by the fact that anti-inflammatory drugs commonly used for IBD inhibit NF-κB

(Wahl et al., 1998). Corticosteroids have been shown to block nuclear translocation of NF-κB

with increased level of I-κB, the chief endogenous inhibitor of NF-κB (Scheinman et al., 1995).

Other IBD drugs such as sulfasalazine and 5-ASA have also been shown to block NF-κB

activation through inhibiting the activity of the IKK complex (Gan et al., 2005) and blocking

functionally important posttranslational modifications of the p65 NF-κB subunit. In the present

study, we showed that CLT suppressed both rat colitis and nuclear translocation and

transcriptional activity of NF-κB induced by TNBS (Fig. 2B). These results are in agreement

with a previous report that blocking the p65 NF-κB subunit in a TNBS-induced mouse colitis

model abrogates the signs of colitis (Neurath et al., 1996). This inhibitory effect of CLT on NF-

κB activity was well correlated not only with the inhibition of inflammation (Fig. 1) but also

with the suppression of abnormal angiogenesis (Fig. 2C). These results indicate that CLT may

regulate the genes involved in both inflammation and angiogenesis.

The expression of COX-2, one of the NF-κB-dependent inflammatory genes, is also

known to be associated with enhancement of angiogenesis (Rhodes and Campbell, 2002). In the

present study, we showed that TNBS enhanced COX-2 expression, and such increase was

blocked by the administration of CLT. Although there is a report showing no significant increase

in COX-2 expression in a 2,4-dinitrobenzenesulfonic acid (DNBS)-induced model of IBD

(Antonioli et al., 2007), our results are consistent with previous findings (Sánchez-Hidalgo et al.,


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2007). Since one of the complications of IBD is an increased risk for development of colon

cancer which is closely related to the increased expression of COX-2, the blocking effect of

CLT on TNBS-induced COX-2 implicates that CLT may prevent the occurrence of colitis-

associated colon cancer.

Among many factors involved in both inflammation and angiogenesis, IL-8 is recently

highlighted. In fact, IL-8 increase has been observed in inflamed intestinal mucosa of patients

with chronic IBD, implying an important role in the pathogenesis of IBD (Williams et al., 2000;

Daig et al., 1996). In agreement with previous other reports for IL-8 induction (Murayama et al.,

1997; Brasier et al., 1998), our result also demonstrated that IL-8 expression was hardly

detectable in vehicle-treated HT29 cells or rat colon tissue, and TNBS (Fig 2D) and TNF-α

(Figs. 3A and 3B) remarkably increased IL-8 expression. Such increased expression of IL-8 was

significantly decreased by CLT treatment. More importantly, the inhibitory effect of CLT on IL-

8 expression was well correlated with the inhibitory actions of CLT on NF-κB transcriptional

activity. Furthermore, the NF-κB inhibiting activity of CLT was comparable to that of PDTC, a

NF-κB inhibitor, in TNF-α-treated HT29 cells (Figs. 3D and 3E), representing that the

molecular target of CLT action is NF-κB. These findings were further supported by the

synergistic effect of CLT in combination with PDTC on the suppression of IL-8 increase.

It is well known that TNF-α preferentially induce activation of NF-κB whereas many

other cytokines activate STAT family. Phosphorylated STAT proteins by kinases such as Janus

kinases in response to cytokine or growth factor receptor activation migrate into the nucleus and

regulate the transcription of cytokine genes. STAT3 activation has been observed in many

colitis models and in the colonic mucosa of patients with active IBD (Mitsuyama et al., 2007).

In the present study, CLT was able to suppress TNBS-induced phosphorylation of STAT3 in the

rat colon tissue. Thus, inhibition of STAT3 activation seems likely to be involved in


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amelioration of TNBS-induced colitis by CLT. Furthermore, recent reports suggest that STAT-3

is involved in the synthesis of IL-8, a pro-inflammatory and pro-angiogenic factor (Yeh et al.,

2004). Our results showing that CLT suppressed both STAT-3 phosphorylation and IL-8

expression by TNBS suggest that regulatory effect of CLT on IL-8 level is associated with

suppression of STAT-3 phosphorylation.

In addition to the well-characterized role of IL-8 in the chemoattraction and

recruitment of leukocytes into the intestinal mucosa, IL-8 is also known to exert effects on

vascular endothelial cells to promote in vitro tube formation (Heidemann et al., 2003) and

pathological angiogenesis (Koch et al., 1992). Especially, such angiogenic action of IL-8 is well

documented in tumor-associated angiogenesis and tumor progression (Chen et al., 2003).

Recently, IL-8 has been also reported to induce inflammatory angiogenesis by regulating

endothelial cell proliferation, survival, migration and MMP-2 production (Koch et al., 1992; Li

et al., 2005). Similarly, our results showed that IL-8 induced endothelial cell proliferation, tube

formation, and migration. Importantly, CLT suppressed the IL-8 induced angiogenesis in vitro

as well as in vivo (Figs. 5 and 6). Like TNF-α that is an inducer of NF-κB and also a NF-κB-

dependent gene product, it has been suggested that IL-8 expression is NF-κB-dependent, and its

action not only in inflammation but also in angiogenesis is dependent on NF-κB activity

(Manna and Ramesh, 2005). Based on these reports and our results of inhibitory action of CLT

in NF-κB translocation and transcriptional activity, the action of CLT in IL-8-induced

angiogenesis is also believed to be mediated through suppression of NF-κB.

Although we clearly demonstrated that CLT down-regulate the IL-8 expression and

inflammatory angiogenesis in colon tissue of rats, we can not eliminate the possibilities that

other cytokines are possibly the prime target of CLT action. TNBS-induced colitis is a model of

Th1-type colitis in which many other important cytokines such as TNF-α, IFN-γ, IL-2, or IL-6


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are increased. However, although we did not analyze them in this study, suppression of NF-κB

activation by CLT is consistently observed both in vivo and in vitro, indicating the possible

regulation of CLT on the NF-κB-dependent cytokines including TNF-α and IL-8. In addition,

we also noted the inhibitory effect of CLT on STAT-3 phosphorylation in TNBS-induced

colitis. These results indicate that CLT ameliorate TNBS-induced colitis via NF-κB and STAT-

3 pathways.

Finally, we found that the inhibitory action of CLT in NF-κB activation, inflammation,

and abnormal angiogenesis was achieved at much lower concentrations than 5-ASA in both in

vivo TNBS-treated rat colitis and in vitro HT29 cells or HUVECs. These results suggest that

CLT may be a more effective therapeutic agent for IBD than a conventional drug, 5-ASA. In

summary, our results provide strong evidence that CLT is able to ameliorate intestinal

inflammation by inhibiting abnormal angiogenesis and blocking the production and action of

IL-8 via inhibition of NF-κB activation.


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ACKNOWLEDGMENTS

We would like to thank Prof Sae Kwang Ku (Department of Anatomy and Histology, Daegu

Hany University, South Korea) for his expert support in histological evaluation of colon tissues.

We appreciate Samil Pharmaceutical Co. Ltd. (Ansan, Korea) and United Pharm. Inc. (Seoul,

Korea) for providing CLT.


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FOOTNOTES

This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant

funded by the Korea government (MOST) (No. R11-2007-040-02004-0) and by a grant No.

RTI04-01-04 from the Regional Technology Innovation Program of the Ministry of Commerce,

Industry, and Energy (MOCIE).

Reprint request: Jung-Ae Kim, College of Pharmacy, Yeungnam University, 214-1 Dae-dong,

Gyeongsan 712–749, South Korea

e-mail: [email protected]

The first two authors (D.T. and J.S.L.) have equally contributed to this work.


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LEGENDS FOR FIGURES

Fig. 1. CLT improves the clinical and morphological features of TNBS-induced colitis in

rats. Colitis was induced by rectal administration of TNBS. The control group received 50%

ethanol as a vehicle. (A) The body weight was recorded daily from day 1 to day 6. The

macroscopic appearance (B), the mean values of the colon length distal to cecum (C) and the

wet weight of colon (distal 5 to 6 cm portion) (D) were analyzed. The data represents the mean

± S.E.M. for 5 rats per group. *p<0.05, compared to vehicle-treated control. # p<0.05, compared

to TNBS-treated group.

Fig. 2. CLT suppresses the TNBS-induced inflammatory response and pathogenic

angiogenesis. (A) The MPO activity of colon tissue was measured by using MPO assay kit, and

expressed as mean ± S.E.M. for 5 rats per group. (B) The expression level of COX-1, COX-2,

and NF-κB was detected by Western blotting. The western blot is a representative of three

independent experiments. In the bar graph, relative expression of COX-2 and NF-κB

normalized to Actin level was expressed as mean ± S.E.M. (C) The colon section of each

experimental group was stained with H&E (first column) or incubated with primary rabbit anti-

vWF antibody for overnight at 4°C. FITC-conjugated goat anti-rabbit IgG was used as a

secondary antibody (middle column). DAPI was used for nuclear counterstaining (third column).


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(magnification 200×). (D) Total RNA was extracted from the colon tissue and the level of IL-8

mRNA was determined by RT-PCR. The equal loading of RNA was confirmed using GAPDH

as a reference. (E) The phospho-STAT-3 and STAT-3 expressions were detected by Western

blotting. In the bar graph, relative expression of phospho-STAT-3 normalized to STAT-3 level

was expressed as mean ± S.E.M. *p<0.05, compared to vehicle-treated control. #p<0.05,

compared to TNBS-treated group.

Fig. 3. Inhibitory effect of CLT on TNF-α induced IL-8 expression is mediated through

suppression of NF-κB activation in HT29 cells. HT29 cells were pretreated with various

concentrations of CLT or 20 mM of 5-ASA for 1 h, and then, stimulated with TNF-α (10 ng/ml).

(A) Total RNA was extracted from the cells 3 h after TNF-α stimulation, and IL-8 mRNA

expression was quantified by real-time PCR. (B, E) IL-8 protein secretion in culture supernatant

was quantified by using an ELISA kit. (C) Proteins were extracted from the cells stimulated

with TNF-α (10 ng/ml) for 15 min. Nuclear extracts were analyzed for NF-κB (p65) using anti-

human NF-κB antibody. Cytosolic extracts were analyzed by using anti-human I-κBα and

phospho-I-κBα antibodies. In the bar graph, the relative expression of NF-κB and I-κB-α

normalized to Actin level was expressed as mean ± S.E.M. (D) HT29 cells were transfected with

NF-κB luciferase (firefly) construct together with pRL-TK (renilla luciferase) as a transfection


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control using GeneJammer transfection reagent. After transfection, the cells were stimulated

with TNF-α in the presence or absence of CLT and/or PDTC. Luciferase activities were

determined using a dual-luciferase reporter assay kit and normalized to those of control values.

Data are means ± S.E.M. from three independent experiments. *p<0.05, compared to vehicle-

treated control. # p<0.05, compared to TNF-α-treated group. ‡ p<0.05, compared to TNF-α and

CLT-treated group

Fig. 4. Inhibitory effects of CLT on the IL-8-induced adhesion of U937 cells to HT29 cells.

HT29 cells were pretreated with various concentrations of CLT and 20 mM of 5-ASA for 1 h,

and then, stimulated by 10 ng/ml of TNF-α. After 3 h, HT29 cells were co-cultured with U937

cells which were already labeled with BCECF-AM (10 μg/ml). The adhesion of BCECF

fluorescence-labeled U937 cells to HT29 colon epithelial cells was captured by using a

fluorescence microscope (A) and quantitated by using a fluorescence-detecting microplate

reader (Fluostar Optima, BMG Labtech GmbH, Germany) using excitation at 485 nm and

emission at 520 nm (B). Data are means ± S.E.M. from three independent experiments. *p<0.05,

compared to vehicle-treated control. # p<0.05, compared to IL-8-treated group.

Fig. 5. CLT inhibits IL-8-induced angiogenesis in vitro. (A) Serum starved HUVECs were


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pretreated with CLT for 1 h prior to IL-8 treatment for 48 h. The cell proliferation was assessed

by MTT assay. (B) HUVECs seeded in matrigel coated 48-well plate were incubated with IL-8

and CLT for 14 h, and then, digital images were taken under the microscope (40×) (C) The

lower and upper parts of transwell insert were coated with collagen and matrigel, respectively.

HUVECs were treated with various concentrations of CLT. After 24 h, the cells on the bottom

side of the filter were fixed, stained and counted under the microscope. The data represent the

means ± S.E.M. of at least three independent experiments. *p<0.05, compared to untreated

control. # p<0.05 compared to IL-8-stimulated group.

Fig. 6. CLT inhibits IL-8-induced angiogenesis in vivo. The 10 day-old chick embryos were

treated with IL-8 in the presence or absence of CLT (1, 10, or 20 μg/CAM) by means of filter

disks. After 3 days of incubation, the CAM tissue directly beneath each filter disk was resected

and digital image was captured. The images were imported into image analysis program to

quantitate the number of new branches formed. The data represent the means ± S.E.M. of at

least six chick embryos. *p<0.05, compared to untreated control, # p<0.05 compared to IL-8-

treated group.


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TABLES

Table 1. Criteria for Assessment of Microscopic Colonic Damage

Mucosal epithelium

1. Ulceration: none (0); mild - surface (1); moderate(2); extensive-full thickness (3)

Crypts

2. Mitotic activity: lower third (0); mild mid-third(1); moderate mid-third (2); upper third (3)

3. Mucus depletion: none (0); mild (1); moderate (2); severe (3)

Lamina propria

4. Inflammatory cell infiltrate: none (0); mild (1); moderate (2); severe (3)

5. Vascularity: none (0); mild (1); moderate (2); severe (3)

Submucosal

6. Inflammatory cell infiltrate: none (0); mild (1); moderate (2); severe (3)

7. Edema: none (0); mild (1); moderate (2); severe (3)

Maximum score: 21. Modified from Peran et al. (2005).


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Table 2. Effects of CLT treatment on the colon morphological changes in TNBS-induced rat colitis

Histomorphometry

Groups

Thicknesses (μm/cross trimmed sections)

Mucosa Submucosa Total colonic walls

Control 254.30±54.09 193.14±14.86 661.18±65.00 TNBS 481.97±45.21a 522.37±88.35a 1110.56±96.92a TNBS+5-ASA 303.28±16.09c 261.00±57.73c 801.38±91.29bc TNBS+CLT10 352.42±42.09ac 242.97±52.91c 830.02±66.51ac TNBS+CLT50 290.95±40.46c 199.52±42.36c 688.43±86.99c

Values are expressed as Mean ± S.D. of five rats (1 field/head); a p<0.01 and b p<0.05 compared with vehicle-treated control; c p<0.01 compared with TNBS-treated group.

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

JPET

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Table 3. Effects of CLT treatment on the extent of the inflammatory lesion in the TNBS-treated rat colon

Regions

Groups

Epithelium Crypts Lamina propria Submucosa Totals

(Max = 21) Ulceration Mitotic activity

Mucus depeletion

Inflammory cells infiltered

Vascularity Inflammory

cells infiltered Edema

Control 0.00±0.00 0.40±0.55 0.00±0.00 0.40±0.55 0.00±0.00 0.00±0.00 0.60±0.55 1.40±0.89 TNBS 2.20±1.10a 1.00±0.71 2.40±0.89a 2.20±0.84a 0.60±0.55 2.60±0.55a 2.60±0.55a 13.60±3.85a TNBS+5-ASA 1.20±0.45a 0.80±0.84 1.80±0.45 1.20±0.45d 0.40±0.55 0.00±0.00c 1.40±0.55bc 6.80±1.64ac TNBS+CLT10 0.40±0.55d 0.40±0.55 2.00±0.71a 1.80±0.84a 0.60±0.45 1.40±0.55ad 1.80±0.45ad 9.00±2.12a TNBS+CLT50 0.40±0.55d 0.00±0.00d 1.20±0.84bd 0.60±0.89ac 0.20±0.45 0.40±0.89d 1.20±0.45c 4.00±1.41bc

Values are expressed as Mean ± S.D. of five rats (1 field/head). ap<0.01 and bp<0.05 compared with vehicle-treated control; cp<0.01 and dp<0.05 compared with TNBS-treated group.

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Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting IL-8

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