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

50 Pages·2008·3.56 MB·English

JPET Fast Forward. Published on August 26, 2008 as DOI: 10.1124/jpet.108.141887
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JPET #141887
Clotrimazole ameliorates intestinal inflammation and
abnormal angiogenesis by inhibiting IL-8 expression through
κ
a NF- B-dependent manner

Dinesh Thapa, Jong Suk Lee, Su-Young Park, Yun-Hee Bae, Soo-Kyung Bae, Jun Bum Kwon, D
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Kyoung-Jin Kim, Mi-Kyoung Kwak, Young-Joon Park, Han Gon Choi, Jung-Ae Kim loa
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College of Pharmacy, Yeungnam University, Gyeongsan 712–749, South Korea (D.T., J.S.L., tjo
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S.Y.P., J.B.K., K.J.K., M.K.K., H.G.C., J.A.K.), School of Medicine, Pusan National University, ls.o
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Pusan 602-739 , South Korea (Y.H.B., S.K.B.), and Research Center, Samil Pharmaceutical Co. t A
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Ltd., Ansan 425-852, South Korea (Y.J.P.) T
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Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
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: [phone]
Fax: [phone]
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Number of text pages: 35 (excluding Tables and Figures) fro
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Number of tables: 3 et.a
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Figures: 6 tjo
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References: 40 ls.o
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Number of words in the Abstract: 226 t A
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Number of words in the Introduction: 443 T
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Number of words in the Discussion: 1416 rna
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ABBREVIATIONS: 5-ASA, 5-aminosalicylic acid; CAM, Chick chorioallantoic membrane; ry
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CLT, Clotrimazole; DMSO, dimethyl sulfoxide; ELISA, Enzyme-Linked Immunosorbent , 2
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Assay; IBD, inflammatory bowel disease; IL, interleukin; MPO, myeloperoxidase; MTT, 3- 3
κ κ
(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide; NF- B, nuclear factor- B;
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
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TNBS-induced weight loss, colon ulceration, and myeloperoxidase (MPO) activity increase. In n
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the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and d fro
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nuclear factor (NF)- B activation. In a cellular model of colitis using tumor necrosis factor jp
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(TNF)- -stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed e
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TNF- -mediated IL-8 induction and NF- B transcriptional activity revealed by a luciferase a
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reporter gene assay. Furthermore, co-treatment with CLT and PDTC, a NF- B inhibitor, a
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synergistically reduced the NF- B transcriptional activity as well as IL-8 expression. In an in PE
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vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation and urn
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invasion of human umbilical vein endothelial cells (HUVECs). The in vivo angiogenesis assay n
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using chick chorioallantoic membrane (CAM) also showed that CLT significantly inhibited the a
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IL-8-induced formation of new blood vessels. Collectively, these results suggest that CLT may 0
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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
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changes associated with IBD include disruption of epithelial barrier, infiltration of dysregulated n
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immune cells, and production of pro-inflammatory mediators including chemokines. d fro
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Chemokines are essential for activating and recruiting inflammatory cells. Interleukin jp
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(IL)-8, a multifunctional member of the chemotactic CXC cytokines, has been reported to be e
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upregulated in the colon of IBD patients (Banks et al., 2003; Subramanian et al., 2008). a
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Furthermore, it has been shown that the severity of inflammation in the colon is congruent with a
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the IL-8 expression (Mazzucchelli et al., 1994; Daig et al., 1996). In normal tissues, IL-8 PE
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expression is low or undetectable, whereas upon stimulation by various stimuli including tumor urn
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necrosis factor (TNF)- , IL-8 expression can be induced up to 100-fold (Baggiolini et al., 1994). n
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In contrast to the constitutively expressed chemokines which direct baseline leukocyte a
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trafficking, the inducible chemokines such as IL-8 further attracts leukocytes to local sites and 0
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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 D
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for more than 20 years. Recent studies have shown that CLT inhibits proliferation of human loa
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cancer cells (Khalid et al., 2005) and vascular endothelial cells, leading to inhibition of growth fro
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factor-stimulated angiogenesis (Takahashi et al., 1998), and thus, tumor growth (Belo et al., et.a
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2004). However, it has not been reported whether CLT modulates inflammatory angiogenesis or tjo
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not. In this study, we investigated that CLT suppresses intestinal inflammation via down- ls.o
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regulating pro-inflammatory cytokine IL-8 expression and blocking the epithelial and t A
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endothelial responses to IL-8. T
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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, D
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trypsin/EDTA and trypsin neutralizing solution (TNS) were purchased from Clonetics, Inc. loa
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(Walkersville, MD, USA). The Matrigel was purchased from BD Biosciences (Bedford, MA, fro
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USA). Trinitrobenzene sulfonic acid (TNBS), pyrrolidine dithiocarbamate (PDTC), 3-(4,5- et.a
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dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT), sodium pyruvate, dimethyl tjo
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sulfoxide (DMSO), protease inhibitor cocktail, sodium dodecyl sulfate (SDS) were obtained ls.o
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from Sigma Chemical Co. (St. Louis, MO, USA). 5-aminosalicylic acid (5-ASA) was t A
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purchased from Sigma where as Clotrimazole (1-[o-chloro- alpha-,alpha-diphenylbenzyl]- T
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imidazole) was supplied from Samil Pharmaceutical Co. Ltd. (Ansan, Korea) and Korea United rna
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Pharm. Inc. (Seoul, Korea). J
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TNBS-induced experimental colitis 3
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 D
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histologic examination as well as myeloperoxidase (MPO) activity measurement. Animal loa
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experiments were performed according to the institutional guidelines of the National Institute of fro
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Health and the University for the care and use of laboratory animals. et.a
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Myeloperoxidase (MPO) activity measurement rg
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MPO serves as a marker for tissue neutrophil infiltration. To measure MPO activity, we used a P
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MOP assay kit (ASA-001, Cytostore, Canada). The 1 cm segment of dissected colon tissues ou
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were washed in cold PBS (pH 7.4), weighed, suspended in 0.5 ml of ice-cold sample buffer per o
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50 mg of tissue, and homogenized 30 s using a tissue homogenizer at 4°C (Biospec Products ua
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Inc., Switzerland). The homogenized sample was mixed with an additional sample buffer to , 2
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give a final concentration of 50 mg/ml and centrifuged at 10000 rpm for 5 min twice. The level
μ
of MPO activity in the tissue was measured spectrophotometrically. Each 20 l of sample
μ μ
solution was mixed with 200 l of development reagent (12.5 l of H O containing chromogen
2 2
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
μ
required for converting 1 M of H O 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
μ
HT29 cells were resuspended in 50 l cytoplasmic extraction reagent I containing protease
inhibitor cocktail, and then, homogenized by vigorous vortexing for 15 s and left on ice (4°C).
μ
After 10 min, 2.3 l of cytoplasmic extraction reagent II was added and vortexed on the highest D
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setting for 5 s. The homogenates were then centrifuged at 14000 rpm for 5 min. Supernatant loa
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(cytoplasmic fraction) was transferred to a fresh tube and stored at -70°C. To lyse the pellet fro
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containing nuclear fraction, 30 µl aliquot of nuclear extraction reagent and protease inhibitor et.a
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cocktail were added. The pellets were vortexed for 1 min, left on ice (4°C) for 40 min, and tjo
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centrifuged for 10 min (4°C, 14000 rpm) to collect the supernatant (nuclear fraction). ls.o
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The soluble protein concentrations in cytoplasmic or nuclear lysates were determined by BCA t A
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protein assay kit (Pierce, Rockford, USA). The equal amount of proteins (15 µg regardless of T
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type of lysate) was separated on SDS-PAGE, and transferred to a Hybond ECL nitrocellulose rna
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membrane (Amersham Life Science, Buckinghamshire, UK) at 200 mA for an hour. The J
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membranes were blocked in a 5% skim milk in Tris-buffered saline-Tween 20 (TBST) (20 nM ry 6
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Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween 20) at room temperature for 1 h. The membrane 23
κ
was incubated with antibodies specific to NF- B (Santa Cruz Biotechnology, Santa Cruz, CA,
κ α κ α
USA), I- B- (Santa Cruz Biotechnology), phospho-I- B- (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 D
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histological lesions on the prepared cross trimmed H&E stained histological samples were loa
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evaluated by a pathologist, who was blinded to the experimental groups, according to previously fro
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established criteria (Peran et al., 2005) as shown in Table 1. et.a
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For immunohistochemistry, colon tissues were prepared for frozen section in 7 m thickness, tjo
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and incubated with rabbit anti-von Willebrand Factor (vWF) (Chemicon, Temecula, CA, USA) ls.o
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for overnight at 4°C. FITC-conjugated goat anti-rabbit IgG (Molecular Probe, Eugene, OR, t A
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USA) was used as a secondary antibody at 1:500 dilutions and incubated for 1 h at room T
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temperature. After mounting with the media containing DAPI (Vector Laboratories, Burlingame, rna
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CA, USA) for nuclear counterstaining, tissue slides were visualized under a fluorescent J
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microscope (Olympus). ry 6
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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 D
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at 95°C for 5 s, annealing at 55°C for 10 s, and extension at 72°C for 20 s using primer sets loa
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specific for human IL-8 (sense 5′-CACCCCAAATTTATCAAAGA-3′ and antisense 5′- d fro
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TCAAAAACTTCTCCACAACC-3), and GAPDH (provided from Qiagen). The real time PCR et.a
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product was normalized using GAPDH as an internal control. The fold change over control tjo
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level was determined according to the comparative cycle threshold method. ls.o
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IL-8 Enzyme-Linked Immunosorbent Assay (ELISA) T
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The secreted IL-8 level in culture supernatant was quantified by an ELISA kit (R&D) according rna
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to the manufacturer’s protocol. The measurements were made in three independent experiments J
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with duplicates. ry 6
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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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