Journal of Cancer Prevention 2013; 18(2): 186-191
Published online June 30, 2013
https://doi.org/10.15430/JCP.2013.18.2.186
© Korean Society of Cancer Prevention
Joon-Yeop Yang1, Xiancai Zhong1, Hye-Won Yum1, Hyung-Jun Lee1, Joydeb Kumar Kundu2, Hye-Kyung Na3, and Young-Joon Surh1
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 2College of Pharmacy, Keimyung University, Daegu, 3Department of Food and Nutrition, Sungshin Women’s University, Seoul, Korea
Correspondence to :
Young-Joon Surh, College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea Tel: +82-2-880-7845, Fax: +82-2-872-1795, E-mail: surh@snu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Turmeric (
Keywords: Colitis, Curcumin, Dextran sulfate sodium, STAT3, Mouse colon
Colorectal cancer (CRC), the third most common malignant neoplasm with the second leading cause of death worldwide, is related to inflammatory bowel disease (IBD) including ulcerative colitis and Crohn’s disease.1 As chronic inflammation has been recognized as an essential risk factor for a variety of human malignancies,2 alleviating inflammation is an important strategy for chemoprevention. Chemoprevention is a strategy of use of nontoxic chemicals such as phytochemicals to inhibit, retard, or reverse the process of multistage carcinogenesis.3
Curcumin (Fig. 1A), the major yellow colouring pigment found in the household spice turmeric (
The transcription factor STAT3 is has been reported to play a role in colonic inflammation.6 STAT3 is a transcription factor activated by a variety of cytokines and growth factors.7,8 Upon activation, STAT3 translocates to the nucleus, where it regulates genes involved in apoptosis (e.g., Bcl-X1), cell cycle regulator (e.g., cyclin D1), migration, and survival depending on the cell types.9,10
Although the inhibitory effect of curcumin on DSS-induced mouse colitis has been reported,11 the detailed molecular mechanism is still unclear. This prompted us to investigate the effects of curcumin on activation of STAT3, a key transcription factor involved in inflammatory signaling, in the colon of mice challenged with DSS.
Curcumin was obtained from LKT laboratories (Minneapolis, MN, USA). DSS with a molecular weight of 36,000-50,000 was obtained from MP Biomedical Inc. (Solon, OH, USA). Rabbit polyclonal COX-2 antibody was the product of Cayman Chemical Co. (Ann Arbor, MI, USA). Primary antibodies for pSTAT3, STAT3 and cyclinD1 were procured from Cell Signaling Technology (Danvers, MA, USA). p53 and CDK4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-actin was purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were products of Zymed Laboratories (San Francisco, CA, USA). Oligonucleotide probe containing the STAT3 consensus sequence (5′-GAT CCT TCT GGG AAT TCC TAG ATC-3′) was purchased from Santa Cruz Biotechnology.
Polyvinylidine difluoride (PVDF) membrane was supplied by Gelman Laboratory (Ann Arbor, MI, USA). BCA reagent is a product of Pierce (Rockford, USA). The enhanced chemiluminescence (ECL) detection kit and [
2. Animal treatment
Male Institute of Cancer Research (ICR) mice (5 weeks of age) were purchased from Central Lab. Animal Inc. (Seoul, Korea). The animals were housed in climate-controlled quarters (24°C at 50% humidity) with a 12-h light/12-h dark cycle. Male ICR mice were divided into 4 groups (Fig. 1B). Curcumin suspended in 0.05% carboxymethyl cellulose was given orally (0.1 or 0.25 mmol/kg body weight daily) for 7 days. After one week of treatment with either vehicle or curcumin, animals were given 3% DSS in drinking water, except those of the control group, for 7 days with or without curcumin treatment. Control mice were treated with vehicle (0.05% CMC) only.
3. Western blot analysis
Collected colon tissue was homogenized in ice-cold lysis buffer [150 mM NaCl, 0.5% Triton-X 100, 50 mM, Tris-HCl (pH 7.4), 20 mM ethylene glycol tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 1 mM Na3VO4 and protease inhibitor cocktail tablet] and lysed for 30 min at 0°C followed by centrifugation at 14,000 rpm for 15 min. Supernatant was collected and total protein concentration was quantified by using the BCA protein assay kit. Cell lysates (30-50
4. Preparation of cytosolic and nuclear extracts from mouse colon
The nuclear extract from mouse colon was prepared by following procedure. In brief, collected colon of mice was homogenized in hypotonic buffer A [10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA and 0.1 mM phenylmethylsulfonylfluoride (PMSF)]. 10% No-nidet P-40 (NP-40) solution was added into homogenate and the mixture was then centrifuged for 2 min at 14,000 rpm. The supernatant was collected as a cytosolic fraction. The precipitated nuclei were washed twice with buffer A plus 40
5. Electrophoresis mobility shift assay (EMSA)
The EMSA for STAT3 DNA binding was performed using a DNA-protein binding assay kit, according to the manufacturer’s protocol (Gibco BRL, Grand Island, NY, USA). Briefly, STAT3 oligonucleotide probe 5′-GAT CCT TCT GGG AAT TCC TAG ATC-3′ was labeled with [
6. Statistics
Values were expressed as the mean±SEM of at least three independent experiments. Statistical significance was determined by Student's t-test and P<0.05 was considered to be statistically significant.
Curcumin inhibits DSS-induced DNA binding of STAT3 in mouse colon
STAT3 contributes to colitis-associated colorectal cancer through transcriptional activation of diverse pro-inflammatory and proliferative genes.12Our study revealed that the DNA binding of STAT3 was markedly increased in colon tissue of mice challenged with DSS alone, while administration of curcumin by gavage significantly negated DSS-induced STAT3 DNA binding (Fig. 2A). Mechanistically, STAT3 is activated through phosphorylation of tyrosine 705 residue by a variety of protein tyrosine kinases.13 Western blot analyses revealed that curcumin suppressed DSS-induced phosphorylation of STAT3 in mouse colon (Fig. 2B).
Curcumin attenuates DSS-induced expression of cyclin D1 and CDK4 in mouse colon
Colitis, which often turns into colorectal cancer, is associated with increased cell proliferation. One of the cell cycle regulatory proteins is cyclin D1, which by forming a complex with Cdk4, promotes cell cycle progression. The expression of both Cyclin D1 and Cdk4 is transcriptionally regulated by STAT3. DSS treatment elevated the expression of cyclin D1 in mouse colon, which was attenuated by administration of curcumin (Fig. 3). Likewise, curcumin inhibited DSS-induced expression of CDK4 in mouse colon (Fig. 3).
Curcumin inhibits DDS-induced expression of p53
p53 is a key biosensor of inflammatory stress. Since p53 is activated by phosphorylation during inflammation stress,14 we investigated the level of p53 to measure the degree of acute inflammation. Expression levels of p53 and its target protein p21 were elevated by DSS treatment and it was inhibited by curcumin (Fig. 4).
Despite many anticancer treatments have been developed, cancer is still a global health problem with severe morbidity and mortality. As cancer is a multi-step process typically occurring over an extended period beginning with initiation followed by promotion and progression, the preventive strategy to reduce the risk of cancer development is important.15 One such example is chemoprevention. The goals of chemoprevention are to block, retard or reverse the process of carcinogenesis, using non-toxic substances of natural or synthetic origin.15,16 Dietary phytochemicals such as capsicin, resveratrol, curcumin have been known to prevent the initiation and promotion of multistep carcinogenesis.15,16
Curcumin is a polyphenol and pharmacologically active compound of the perennial herb
Recently, STAT3 has been reported to be strongly phosphorylated in patients with IBD such as ulcerative colitis and Crohn’s disease25 as well as in DSS-induced colitis in mice.26 STAT3 is reported to be involved in colonic inflammation and play an important role in colitis-associated cancer.6,12 During the early stage of colitis-associated tumorigenesis, the activation of STAT3 as well as NF-
p53 is a key biosensor of inflammatory stress and activated by phosphorylation during inflammation.14 Therefore, we investigated the level of p53 to measure the degree of acute inflammation. p53 was induced by DSS treatment and it was inhibited by curcumin in a dose-dependent manner.
Taken together, the above findings suggest that oral administration of curcumin provokes anti-inflammatory effects by inhibiting the STAT3. In addition, curcumin also elicits anti-proliferative effects through suppression cyclin D1, CDK4 expression, which is likely to be mediated by blocking STAT3 signaling. Considering STAT3 as a potential molecular link between inflammation and cancer and the role of aberrantly overexpressed cyclin D1 in tumor promotion, the present study provides the molecular basis for the previously reported chemopreventive effects of curcumin.
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