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Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice
Author’s Accepted Manuscript Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice Eun-Ju Chung, Eun-ju Do, Sang-Yeob Kim, Eun A Cho, Dong-Hee Kim, Sehyung Pak, Sung Wook Hwang, Hyo Jeong Lee, Jeong-Sik Byeon, Byong Duk Ye, Dong Hoon Yang, Sang Hyoung Park, Suk-Kyun Yang, Jin-Ho Kim, Seung-Jae Myung
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S0014-2999(16)30703-8 http://dx.doi.org/10.1016/j.ejphar.2016.11.012 EJP70917
To appear in: European Journal of Pharmacology Received date: 30 June 2016 Revised date: 31 October 2016 Accepted date: 7 November 2016 Cite this article as: Eun-Ju Chung, Eun-ju Do, Sang-Yeob Kim, Eun A Cho, Dong-Hee Kim, Sehyung Pak, Sung Wook Hwang, Hyo Jeong Lee, Jeong-Sik Byeon, Byong Duk Ye, Dong Hoon Yang, Sang Hyoung Park, Suk-Kyun Yang, Jin-Ho Kim and Seung-Jae Myung, Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.11.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice
Eun-Ju Chung1*, Eun-ju Do2*, Sang-Yeob Kim2,3, Eun A Cho2, Dong-Hee Kim2, Sehyung Pak2, Sung Wook Hwang4, Hyo Jeong Lee1, Jeong-Sik Byeon4, Byong Duk Ye4, Dong Hoon Yang4, Sang Hyoung Park4, Suk-Kyun Yang4, Jin-Ho Kim4, and Seung-Jae Myung2,4 1
Health Screening & Promotion Center, Seoul, Korea; 2Asan Institute for Life Sciences, Asan
Medical Center, Seoul, Korea; 3Department of Medicine, University of Ulsan, College of Medicine, Seoul, Republic of Korea; 4Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;
*
Eun-ju Chung and Eun-ju Do contributed equally to this article.
Correspondence Seung-Jae Myung, MD, PhD, AGAF Department of Gastroenterology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-Ro, 43-Gil, Songpa-gu, Seoul, 138-736, Korea. Phone: +82-2-3010-3917, Fax: +82-2-476-0824 E-mail: [email protected]
Disclosure statement: The authors disclose no potential conflicts of interest.
1
Abstract Western-style diet (WD) and dysbiosis are known to be associated with colonic inflammation, which contributes to carcinogenesis. Metformin (Met) exerts anti-inflammatory effects to induce AMP-activated protein kinase (AMPK), resulting in suppressed protein synthesis and reduced cell proliferation. Probiotic VSL#3 (V) modifies microbial composition. We investigated
the
chemopreventive
mechanisms
of
Met
and
V
in
WD-induced
colitis-associated colon carcinogenesis. Male BALB/c mice were randomly divided into five groups: a control diet (CD) group, WD group, WD+ Met (250 mg/kg/day) group, WD+V (1.3 million bacteria/day) group, and WD+Met+V group. All mice were exposed to azoxymethane (10 mg/kg) followed by 2% dextran sodium sulfate (DSS) for 7 days. Using HCT-116 human colon cancer cell line, expression of AMPK, extracellular signal-regulated kinase (ERK), cyclin D1, and Bcl-2 was investigated and cell cycle arrest was assessed. WD enhanced the severity of colitis and tumor growth compared with CD. The combination of Met and V significantly ameliorated colitis and tumor growth by inhibiting macrophage infiltration and maintaining epithelial integrity. In vitro assays showed that the combination therapy promoted late apoptosis by inhibiting cyclin D1 and Bcl-2 and activating pro-apoptotic ERK. A combination therapy with Met and V attenuates tumor growth in a mouse model of WD-induced colitic cancer, suggesting that this strategy could be useful for the chemoprevention of colon cancer.
Key words: colon cancer; chemoprevention; metformin; VSL#3; Western-style diet
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1. Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in Western populations.(Siegel et al., 2015) Recently, the incidence of CRC in Asia has significantly increased, and one of the factors involved is westernization of the diet.(Sung et al., 2005) Dietary fats influence intestinal inflammation and dysbiosis, which may be associated with risk of inflammatory bowel disease (IBD).(Ananthakrishnan et al., 2014) Chronic inflammation predisposes the intestine to cancer development.(Han and Theiss, 2014) A Western-style diet (WD) contributes to colon inflammation and carcinogenesis via macrophage activation.(Kim et al., 2010a) It is reasonable to predict that switching dietary patterns from a WD to a healthier Mediterranean-style food would reduce the incidence of CRC.(O'Keefe et al., 2015) However, a safe drug that could inhibit the cancer promoting effects of a WD would also be beneficial for the at-risk populations. Therefore, our present study was designed to identify a suitable chemopreventive strategy to suppress the colitis-associated carcinogenesis (CAC) induced by a WD. Among the many known chemopreventive drugs, metformin (Met) and VSL#3 (V) were selected. Met is a widely prescribed antihyperglycemic agent of the biguanide family. Its potential benefits extend beyond its original usage, including reducing cancer risk and delaying aging.(Cabreiro et al., 2013; Dowling et al., 2011; Kasznicki et al., 2014) In mammals, its mode of action is partially mediated by AMP-activated protein kinase (AMPK) activation, which results in downregulation of the mTOR and IGF-1/AKT pathways.(Pierotti et al., 2013) Two studies have demonstrated that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis by down regulation of anti-apoptotic proteins as well as AMPK. (Zhuang et al., 2008; Queiroz et al., 2014) V (VSL#3 Pharmaceuticals, Gaithersburg, MD) is a mixture of eight strains of lactic acid-producing bacteria. It protected the epithelial barrier and increased the expression 3
of tight junction proteins by activating the p38 and extracellular signal-regulated kinase (ERK) in colitis.(Dai et al., 2012) However, studies of V in CRC development have yielded controversial findings.(Appleyard et al., 2011; Arthur et al., 2013) A study reported that V attenuated various inflammatory-associated parameters, delaying transition to dysplasia in a rat model of colitis-associated cancer.(Appleyard et al., 2011) Another recent study revealed that anti-inflammatory agent balsalazide and VLS#3 suppressed CAC thorough modulation of the interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) pathway.(Do et al., 2015) These lines of evidence suggest that a combination therapy of Met and probiotic V could represent an effective chemopreventive strategy for WD-induced CAC. In our current study, we provide evidence that combination therapy with Met and V yields chemopreventive effects against WD-induced CAC.
2. Materials and methods
2.1. Chemicals, Animals and experimental diets
AOM and Met was purchased from Sigma Aldrich Corp. (St. Louis, MO, USA). DSS (molecular weight 36,000-50,000) was purchased from MP Biomedicals (Aurora, OH). Male, four-week-old BALB/c mice (Charles River Laboratories Japan, Yokohama, Japan) were randomized to receive a CD (D10011), a WD (D16365B) or a WD with Met (D16365B is mixed with Met, 250 mg/kg/day) (Hosono et al., 2010) for 8 weeks (Research Diet, Inc., Bethlehem, PA), as shown in Table 1. We used BALB/c mice following preliminary study with WD of our laboratory. (Kim et al., 2010a)
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2.2. Induction of CAC in the mouse model
The experimental design is shown in Figure 1A, a total of 50 mice were randomly divided into the following five groups: CD group (n=10), mice were fed without Met or V (VSL Pharmaceuticals, Gaithersburg, MD); WD group (n=10), mice were fed without Met or V; WD+M group (n=10), fed a drug with Met (3~4g per mouse/day); WD+V group (n=10), fed a drug with V (1.3 million bacteria/day); and WD+M+V group (n=10) with both drugs, Met and V. An animal model of CAC was induced by the intraperitoneal injection of 10 mg/kg AOM at 6 weeks of age. One week later, 2% DSS was added to drinking water for 7 days. Drugs were administered from 1 week prior to AOM injection until death. Mice were observed daily for water and dietary intake, and they were killed at 13 weeks of age (Fig. 1A). All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences at the Asan Medical Center, consistent with the Institute of Laboratory Animal Resources (ILAR) guidelines.
2.3. Evaluation of colitis severity and tumor
The disease activity index (DAI) was calculated based on the cumulative score of the parameters (weight loss, rectal bleeding, and stool consistency).(Kim et al., 2010c) Mice were killed at the indicated times and the diameter and number of tumors (2–4 mm, >4 mm and Total) were measured by an independent observer who was unaware of the treatment.
2.4. Immunohistochemical analysis 5
Ki-67 (Abcam, Cambridge, UK, #ab15580) and F4/80 (Serotec, Raleigh, NC, # MCA497GA) antibodies were used. Immunohistochemistry was performed on 4 µm thick paraffin-embedded sections of the colon. For antigen retrieval, slides were immersed in citrate buffer (pH 6), heated in a decloaking chamber at 125°C for 3 min, and then cooled for 30–40 min. After the addition of 3% hydrogen peroxide, sections were incubated for 10 min. After washing with TBST (pH 7), the slides were stained with a primary antibody (F4/80, 1:100: Ki-67, 1:500) at 4°C overnight. A secondary antibody was then used from the VECTASTAIN ABC Kit (Vector, Burlingame, CA) in accordance with the manufacturer’s instructions. The slides were developed with 3, 3`-diaminobenzidine (DAB, Dako, Hamburg, Germany), and were counterstained with hematoxylin. A total of five randomly selected fields were photographed at 400 magnifications, and positively stained cells were counted and analyzed by two observers who were blinded to the study.
2.5. Immunofluorescence analysis
Frozen sections (4 µm) of colonic tissue collected on days 4 and 8 after colitis induction were collected and immunofluorescence staining was performed using anti-Claudin-1 antibody (Abcam, #ab15098). Nuclei were counterstained with DAPI (Sigma– Aldrich) and stained sections were examined using fluorescence microscopy (Olympus AX70, Olympus Optical Co., Tokyo, Japan).
2.6. Apoptosis and cell cycle analysis
A human colon adenocarcinoma cell line (HCT-116) was obtained from the 6
American Type Culture Collection (Manassas, VA). For the preparation of V medium, 0.01g probiotic bacteria were incubated overnight with RPMI 1640. Live bacteria were removed by filtration through a 0.2μm syringe filter and V medium was placed on HCT-116 monolayers (Ewaschuk et al., 2006). Cells were incubated in media containing defined concentrations of Met (5mM, Enzo Life Sciences, Farmingdale, NY) and/or V (20, 40 and 60%) for 24 hours. Cells treated with propidium iodide (PI) and RNase A can be utilized to assess cell cycle stages (G0/G1, S, G2/M), and to identify apoptotic cells (hypodiploid, sub G0 peak). Half a million cells were pelleted at 1000g for 5 min and cells were mixed with 5 μl PI staining solution and 5 μl Annexin V staining solution, following the manufacturer’s instructions (BD Pharmingen, San Jose, CA). Cells were analyzed using a Becton–Dickinson FACS Calibur flow cytometer.
2.7. Western blot analysis
Cultured cells treated with the Met and/or V for the indicated periods of time were washed with PBS. To obtain total cell extracts, the cells were harvested in lysis buffer (50 mM Tris–HCl, 100 mM NaCl, 5 mM EDTA, 1% TritonX-100) containing a cocktail of phosphatase inhibitor (GenDEPOT P3200-001, TX, USA) and a cocktail of protease inhibitors (Sigma–Aldrich P8340). Lysates were incubated on ice for 30 min and centrifuged at 15,000 g. Protein concentrations were determined using a Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA). The proteins were separated by SDS–PAGE (7.5–12.5% gels) and transferred onto a Hybond-P PVDF membrane (Amersham Biosciences, Little Chalfont, UK). After transfer, the membranes were blocked with Blocking One-P (Nacalai Tesque, Kyoto, Japan) and incubated with primary antibodies in TBST with 5% dry milk at 4°C, overnight.
7
The primary antibodies used included anti-pAMPK (Thr172, #2535), anti-AMPK (#5831), anti-p-pERK1/2 (Thr202/Tyr204, #9101), anti-pERK1/2 (#9102), anti-pAKT (Ser473, #4060), anti-AKT (#4685) (1:1000, Cell Signaling, Danvers, MA), anti-Cyclin D1 (92G2, #2978), and anti- Bcl-2 (83-8B, Enzo #ADI-AAM-072-E). As a loading control, β-actin (1:10,000, Sigma–Aldrich #A5441) was used. HRP-conjugated anti-rabbit IgG (1:7000, Cell Signaling #7074) and HRP-conjugated anti-mouse IgG (1:7000, Cell Signaling #7076) were used as secondary antibodies. Horseradish peroxidase-conjugated secondary antibodies and an ECL detection kit (Amersham Biosciences) were used to detect specific proteins. Densitometry analysis of the blots was performed using ImageJ software.
2.8. Statistical Analysis
Statistical analyses were performed using SPSS 14.0 (SPSS Inc., Chicago, IL). Data were expressed as a mean ± standard deviation. Significant differences were evaluated using the Student’s t-test or one-way ANOVA, where appropriate. A value of P<0.05 was considered statistically significant.
3. Results
3.1. WD exacerbates DSS-induced colitis, whereas combination therapy attenuates severity of colitis
Mice fed a WD exhibited marked weight loss and more bloody stools during or soon after the cessation of DSS administration. On day 5 after DSS, the DAI scores and
8
weight loss in the WD group became significantly higher than those in the CD group (Fig. 1B and 1C). Compared to the CD group, WD alone or a single drug-treated groups showed higher DAI scores during DSS-induced colitis. A total of 3 of 10 mice in the WD group died as a result of severe colitis. By contrast, DAI scores in the combination therapy group had significantly lower than those in the single drug-treated groups (P<0.05, Fig. 1B and 1C).
3.2. WD exacerbates tumor growth, whereas combination therapy reduced tumor formation
With regard to tumor formation, the effect of Met and/or V was assessed by quantifying the number of tumors within the colon and rectum of mice. All groups had lowto high-grade dysplasia at the end point. The total number of tumors in the Met+V-treated groups were significantly low compared to WD and single drug-treated groups (P<0.01, Fig. 2B). The WD group showed multiple and larger tumors and some tumors conglomerated from the middle colon to rectum; however, all tumors in CD group were smaller than 2 mm (Fig. 2A). Therefore, we had to divide the number of tumor based on the nodule size (2–4 mm and >4 mm). There was no significant difference in nodules sized 2–4 mm between any of the groups. The large tumors (>4 mm) in the WD group significantly increased compared to those in the CD group, and significantly decreased in the Met therapy and combination therapy groups (P<0.01; Fig. 2A, B). Interestingly, in the combination therapy group, most nodules were <4 mm and there were no tumors in 2 of 10 mice. Immunohistochemical staining showed that the WD group had significantly increased expression of Ki-67 at the base of the crypts and shortened and distorted crypt structures compared with the CD group. The combination therapy group showed significantly fewer Ki-67-positive cells than the WD group (P<0.01; Fig. 2C, D).
9
3.3. Combination therapy prevents macrophage infiltration and protects epithelial integrity during WD-induced CAC
The effects of Met and V on macrophage infiltration were examined during colitis induction by DSS. After 4 days of continuous DSS ingestion, F4/80-positive cells could be observed at the base of crypts in all groups of mice. On day 8, F4/80-positive macrophages increased and were distributed in both submucosal space and base of crypts, especially in WD group, whereas the combination therapy group showed few macrophages at the base of crypts (Fig. 3A, B). The combination therapy group showed significantly fewer F4/80-positive cells compared with the WD group on days 4 and 8 (P<0.05; Fig. 3A, B). Since epithelial tight junctions regulate gut permeability and integrity, we examined and compared the effects of Met and V on the expression of tight junction proteins and microscopic damage to crypt structures by western blotting and immunofluorescence microscopy, respectively. Cryostat sections of tissues from each group were stained with anti-Claudin-1 antibody on days 4 and 8. There were no prominent differences between the groups on day 4. On day 8, WD group and WD with single treatment groups showed very low Claudin-1 protein expression and severe crypt destruction; however, the combination therapy group showed intense reactivity with anti-Claudin-1 antibody at the epithelial surface and an intact crypt structure on day 8 (Fig. 3C). Here, we confirmed that WD promoted gut barrier alteration and the disruption of tight junctions, which induced macrophage infiltration; however, the combination therapy attenuated the structural damage and macrophage infiltration in WD-associated DSS-colitis through Claudin-1 tight junction protein expression.
3.4. Combination therapy promotes late apoptosis by inhibiting Cyclin D1 and Bcl-2 as well
10
as activating AMPK and ERK.
As shown Fig. 4A, we observed V activated AMPK and ERK in a dose-dependent manner, therefore, high dose V (60%) with Met promoted ERK phosphorylation more effectively than low dose (Fig. 4A). In addition, combination therapy with Met and V significantly inhibited anti-apoptotic protein and induced late apoptosis effectively. Thus, our results consistently showed that combination therapy with Met and V suppressed the expression of Cyclin D1 and Bcl-2. Cell cycle assay exhibited that Met with high dose V (60%) effectively induced G2/M arrest (Fig. 4B). Annexin V/PI staining data revealed that late apoptosis was induced by Met treatment and the effect was augmented when combined with high dose V (60%) (Fig. 4C). Our data indicate that the precise molecular mechanism of the chemopreventive effects of Met and V could be to induce growth arrest and to inhibit apoptosis by the sustained increase of ERK/AMPK activity.
4. Discussion
Excess fat intake is a major contributor to CAC, and high-fat diet-induced obesity leads to intestinal inflammation and changes in gut microbiota.(Lee et al., 2015) Met has been proposed as a promising therapeutic agent in the treatment of several types of cancer, and probiotic V has also been reported to modulate barrier function and reduce experimental colitis.(Uronis et al., 2011) Herein, we investigated the chemopreventive effects of Met and V in WD-induced CAC, which could be helpful in the development of chemopreventive strategies against CRC. In the current study, the cancer was exacerbated by WD and our goal was to test the efficacy of Met and V in WD-induced CAC. We induced tumors in the colon of mice fed 11
CD or WD using AOM/DSS regimen; however, mice fed WD received the preventive agents. Consistent with our expectations, a combination therapy with Met and V attenuated the severity of WD-induced DSS colitis and suppressed tumor growth. These effects were associated with drug-induced inhibition of macrophage activation and protection of epithelial integrity. Previously, we showed that WD increases susceptibility to AOM/DSS-induced tumorigenesis via effects on macrophage recruitment and subsequent inflammatory signal activation.(Kim et al., 2010a) Macrophage accumulation was found to be an important driver in the development of CAC, and these cells can produce a wide variety of inflammatory mediators.(Mantovani, 2009; Mantovani and Sica, 2010) In the biomedical literature, macrophages have been reported to be associated with altered immune responses within the tumor microenvironment, and several studies have shown that tumor-associated macrophages (TAMs) can exert pro-tumor functions.(Condeelis and Pollard, 2006; Kang et al., 2010) The anti-inflammatory effects of Met in CAC have been reported, and it was shown that Met could suppress NF-kB activation in intestinal epithelial cells and ameliorate colitis-associated tumorigenesis in mice.(Koh et al., 2014) In contrast, V did not reduce CAC in a study using an AOM–Il10-/- mouse model, although it modified the microbial composition.(Arthur et al., 2013) We observed in our study that Met partially contributed to the reduced severity of colitis and macrophage accumulation, but V did not show these effects. However, the combination significantly reduced the numbers of F4/80-positive macrophages and was associated with a lower DAI, suggesting an interaction between two drugs to suppress WD-induced inflammatory responses. Interestingly, these results were in accordance with tumor incidence, indicating that activation of inflammatory signals by macrophages and destruction of epithelial integrity are major components of colon carcinogenesis. To examine the molecular mechanism underlying the phenomenon, we performed 12
in vitro experiments with the HCT-116 cell. We found that inhibition of anti-apoptotic proteins and induction of late apoptosis were critical for the anti-tumor effect of Met with V. AMPK and ERK play an important role in regulating cancer cell proliferation, differentiation, and apoptosis. Met-induced glucose deprivation activates AMPK, and ERK exhibits pro-apoptotic effects in HCT-116 cells.(Kim et al., 2010b) Cyclin D1 is a key regulator of the cell cycle and acts as an oncogene in several types of cancer. Bcl-2 is also considered an important anti-apoptotic protein. Western blotting with several antibodies using HCT-116 cells exhibites interesting results (Fig. 4A). Met with high dose V (60%) inhibits cancer cell growth by downregulating Cyclin D1 and Bcl-2 and mediates cell cycle arrest by activating AMPK and ERK phosphorylation. Suppression of Cyclin D1 was prominent in Met and combination treatment, however, V treatment did not suppress Cyclin D1 or Bcl-2. Several reports support our data that cell cycle arrest induced Met requires AMPK activation and cyclin D1 downregulation.(Fang et al., 2014; Zhuang and Miskimins, 2008) A previous study reported that V could protect the intestinal epithelial barrier by activating the ERK signaling pathway.(Bassaganya-Riera et al., 2012; Dai et al., 2012) The ERK signaling pathway is a major determinant in the control of many cellular processes and activation of the ERK and/or phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathways is associated with neoplastic transformation in various human tumor cells. These pathways are often up-regulated in human cancers, and represent attractive targets for anticancer drugs.(Kohno and Pouyssegur, 2003; Song et al., 2005). A report also showed the pro-apoptotic potential of AMPK activated conditions, such as in AICAR (AMPK activator)-treated cells.(Chen et al., 2013) Another report suggested that AMPK can specifically antagonize ERK activity in HCT-116 cells and that ERK exhibits pro-apoptotic effects under conditions of glucose deprivation.(Kim et al., 2010b) However, the antagonizing effect between the ERK and AMPK was not evident in our 13
data. We thus surmise that combination therapy of Met and V induces late apoptosis of cancer cells. The critical mechanisms are down-regulating Cyclin D1 as well as activating AMPK and ERK. The additive effect of combination therapy could be induced via proapoptotic ERK signaling of high dose V (60%). CRC is a type of tumor with an increased rate of incidence in patients with type 2 diabetes and high-fat diet-induced obesity. Type 2 diabetes and obese patients should thus be considered as a risk group for this cancer and preventative measures could be helpful for this population. V combined with Met could be a safe and effective chemopreventive strategy to protect against CRC in the risk group. In summary, this is the first study to report the anti-tumor effects of a combination therapy with Met and V in vivo and in vitro. Our findings suggest that this combination therapy reduced the severity of colitis and attenuated tumor growth in a WD-induced colitic cancer by inhibiting macrophage accumulation and preventing epithelial barrier destruction. Cyclin D1 down-regulation by Met and pro-apoptotic ERK activation by high dose V induce late apoptosis of cancer cells, therefore, the mechanisms are involved in the beneficial effects of these two drugs. Therefore, combinations of Met and V might represent a safe and promising therapeutic approach for the chemoprevention of WD-induced colon cancer.
Acknowledgements: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C3078). No potential conflicts of interest were disclosed. This research was supported by a grant from Asan Institute for Life Sciences (No. 2016-559). This material is based upon work supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under Industrial Technology Innovation Program. No. 10063408, 'Development of channel-inserted convergence smart 14
endoscopic system based on multispectral fluorescence imaging for precise diagnosis of digestive diseases'.
Figure Legends Fig. 1. Experimental course and disease activity. (A) Schematic representation of the experimental course. Filled triangles ( ) indicate intraperitoneal injection of 10 mg/kg azoxymethane (AOM); DSS indicates a dose of 2% dextran sulfate sodium. Mice were fed a control diet (CD) or a WD for 8 weeks with or without metformin (M) and VSL#3 (V) treatment. (B, C) Disease activity index (DAI) scores and weight loss during the induction of murine colitis. DAI was calculated as a cumulative score of three parameters (weight loss, stool consistency, and anal bleeding); *P<0.05. Values indicate means + S.D. (n=10 per group).
Fig. 2. Tumor incidence and cell proliferation. (A and B) Tumor incidence based on nodule size (2–4 mm, >4 mm and Total) was evaluated. (C and D) Immunohistochemical staining for Ki-67. The WD group showed increased Ki-67 staining at the base of the crypts. The indices indicate the number of positively stained nuclei counted in the crypts of the colon. A total of five randomly selected fields were examined at 400 magnification. Scale bar: 50 μm. Each bar represents the mean +S.D.
Fig. 3. Macrophage accumulation and Claudin-1 expression in mice colon tissues. (A and B) Immunohistochemical staining for F4/80 and immunofluorescent staining for Claudin-1. Cryostat sections of tissues from each group were stained with anti-F4/80 or anti-Claudin-1 antibody on days 4 and 8 after 2% DSS administration. (C) F4/80-positive cells were counted
15
at each indicated time point after treatment with AOM and DSS. A total of five randomly selected fields were examined at 400 magnification. Scale bar: 50 μm. Each bar represents the mean + S.D.
Fig. 4. Pro-apoptotic effects of Met and/or V in HCT-116 colon cancer cell line. (A) Western blot analysis. (B) Cell cycle distribution in HCT-116 cells treated with Met and/or V for 24 hours. (C) The pro-apoptotic effects of Met and V, as demonstrated by the incorporation of Annexin V and/or propidium iodide (PI); different labeling patterns identify the different cell populations as follows: region R1, live cells, PI– Annexin V–; region R2, apoptotic cells PI– Annexin V+; region R3, damaged cells PI+ Annexin V–; and region R4, dead cells PI+ Annexin V+.
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inflammation and colitis-associated colon cancer. Journal of gastroenterology and hepatology 29, 502-510. Kohno, M., Pouyssegur, J., 2003. Pharmacological inhibitors of the ERK signaling pathway: application as anticancer drugs. Progress in cell cycle research 5, 219-224. Lee, D., Albenberg, L., 2015. Diet in the pathogenesis and treatment of inflammatory bowel diseases. Gastroenterology 148, 1087-1106. Lin, H.Y., Tang, H.Y., 2008. Resveratrol is pro-apoptotic and thyroid hormone is anti-apoptotic in glioma cells: both actions are integrin and ERK mediated. Carcinogenesis 29, 62-69. Mantovani, A., 2009. Cancer: Inflaming metastasis. Nature 457, 36-37. Mantovani, A., Sica, A., 2010. Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Current opinion in immunology 22, 231-237. Nguyen, T.T., Tran, E., 2004. The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis 25, 647-659. O'Keefe, S.J., Li, J.V., 2015. Fat, fibre and cancer risk in African Americans and rural Africans. Nat Commun 6, 6342. Pierotti, M.A., Berrino, F., 2013. Targeting metabolism for cancer treatment and prevention: metformin, an old drug with multi-faceted effects. Oncogene 32, 1475-1487. Randhawa, H., Kibble, K., 2013. Activation of ERK signaling and induction of colon cancer cell death by piperlongumine. Toxicology in vitro : an international journal published in association with BIBRA 27, 1626-1633. Siegel, R.L., Miller, K.D., 2015. Cancer statistics, 2015. CA Cancer J Clin 65, 5-29. Song, P., Wei, J., 2005. Distinct roles of the ERK pathway in modulating apoptosis of Ras-transformed and non-transformed cells induced by anticancer agent FR901228. FEBS letters 579, 90-94. Sung, J.J., Lau, J.Y., 2005. Increasing incidence of colorectal cancer in Asia: implications for screening. Lancet Oncol 6, 871-876. Uronis, J.M., Arthur, J.C., 2011. Gut microbial diversity is reduced by the probiotic VSL#3 and correlates with decreased TNBS-induced colitis. Inflammatory bowel diseases 17, 289-297. Vergara, D., Simeone, P., 2012. Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells. Molecular bioSystems 8, 1078-1087. Zhuang, Y., Miskimins, W.K., 2008. Cell cycle arrest in Metformin treated breast cancer cells involves 18
activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1. Journal of molecular signaling 3, 18-28.
Table 1. Experimental diet composition. Dietary component
Control diet
Western-style diet
(D10011)
(D16365B)
(AIN-76A diet)
(Newmark Stress Diet B)
Fat (corn oil), %
5
20
Corn starch, %
15
7.5
Calcium, mg/g
-
0.88
Monosodium phosphate, mg/g
-
7.98
Monopotassium phosphate, mg/g
-
7.91
Vitamin D3, IU/g
-
0.12
DL-Methionine, %
0.3
0.36
Choline bitartate, %
0.2
0.24
Fiber (cellulose), %
5
6
3.9
4.8
Kcal/g (approx.)
19
20
21
22
23
24
25
26
PII: DOI: Reference:
www.elsevier.com/locate/ejphar
S0014-2999(16)30703-8 http://dx.doi.org/10.1016/j.ejphar.2016.11.012 EJP70917
To appear in: European Journal of Pharmacology Received date: 30 June 2016 Revised date: 31 October 2016 Accepted date: 7 November 2016 Cite this article as: Eun-Ju Chung, Eun-ju Do, Sang-Yeob Kim, Eun A Cho, Dong-Hee Kim, Sehyung Pak, Sung Wook Hwang, Hyo Jeong Lee, Jeong-Sik Byeon, Byong Duk Ye, Dong Hoon Yang, Sang Hyoung Park, Suk-Kyun Yang, Jin-Ho Kim and Seung-Jae Myung, Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.11.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Combination of metformin and VSL#3 additively suppresses western-style diet induced colon cancer in mice
Eun-Ju Chung1*, Eun-ju Do2*, Sang-Yeob Kim2,3, Eun A Cho2, Dong-Hee Kim2, Sehyung Pak2, Sung Wook Hwang4, Hyo Jeong Lee1, Jeong-Sik Byeon4, Byong Duk Ye4, Dong Hoon Yang4, Sang Hyoung Park4, Suk-Kyun Yang4, Jin-Ho Kim4, and Seung-Jae Myung2,4 1
Health Screening & Promotion Center, Seoul, Korea; 2Asan Institute for Life Sciences, Asan
Medical Center, Seoul, Korea; 3Department of Medicine, University of Ulsan, College of Medicine, Seoul, Republic of Korea; 4Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;
*
Eun-ju Chung and Eun-ju Do contributed equally to this article.
Correspondence Seung-Jae Myung, MD, PhD, AGAF Department of Gastroenterology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-Ro, 43-Gil, Songpa-gu, Seoul, 138-736, Korea. Phone: +82-2-3010-3917, Fax: +82-2-476-0824 E-mail: [email protected]
Disclosure statement: The authors disclose no potential conflicts of interest.
1
Abstract Western-style diet (WD) and dysbiosis are known to be associated with colonic inflammation, which contributes to carcinogenesis. Metformin (Met) exerts anti-inflammatory effects to induce AMP-activated protein kinase (AMPK), resulting in suppressed protein synthesis and reduced cell proliferation. Probiotic VSL#3 (V) modifies microbial composition. We investigated
the
chemopreventive
mechanisms
of
Met
and
V
in
WD-induced
colitis-associated colon carcinogenesis. Male BALB/c mice were randomly divided into five groups: a control diet (CD) group, WD group, WD+ Met (250 mg/kg/day) group, WD+V (1.3 million bacteria/day) group, and WD+Met+V group. All mice were exposed to azoxymethane (10 mg/kg) followed by 2% dextran sodium sulfate (DSS) for 7 days. Using HCT-116 human colon cancer cell line, expression of AMPK, extracellular signal-regulated kinase (ERK), cyclin D1, and Bcl-2 was investigated and cell cycle arrest was assessed. WD enhanced the severity of colitis and tumor growth compared with CD. The combination of Met and V significantly ameliorated colitis and tumor growth by inhibiting macrophage infiltration and maintaining epithelial integrity. In vitro assays showed that the combination therapy promoted late apoptosis by inhibiting cyclin D1 and Bcl-2 and activating pro-apoptotic ERK. A combination therapy with Met and V attenuates tumor growth in a mouse model of WD-induced colitic cancer, suggesting that this strategy could be useful for the chemoprevention of colon cancer.
Key words: colon cancer; chemoprevention; metformin; VSL#3; Western-style diet
2
1. Introduction Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in Western populations.(Siegel et al., 2015) Recently, the incidence of CRC in Asia has significantly increased, and one of the factors involved is westernization of the diet.(Sung et al., 2005) Dietary fats influence intestinal inflammation and dysbiosis, which may be associated with risk of inflammatory bowel disease (IBD).(Ananthakrishnan et al., 2014) Chronic inflammation predisposes the intestine to cancer development.(Han and Theiss, 2014) A Western-style diet (WD) contributes to colon inflammation and carcinogenesis via macrophage activation.(Kim et al., 2010a) It is reasonable to predict that switching dietary patterns from a WD to a healthier Mediterranean-style food would reduce the incidence of CRC.(O'Keefe et al., 2015) However, a safe drug that could inhibit the cancer promoting effects of a WD would also be beneficial for the at-risk populations. Therefore, our present study was designed to identify a suitable chemopreventive strategy to suppress the colitis-associated carcinogenesis (CAC) induced by a WD. Among the many known chemopreventive drugs, metformin (Met) and VSL#3 (V) were selected. Met is a widely prescribed antihyperglycemic agent of the biguanide family. Its potential benefits extend beyond its original usage, including reducing cancer risk and delaying aging.(Cabreiro et al., 2013; Dowling et al., 2011; Kasznicki et al., 2014) In mammals, its mode of action is partially mediated by AMP-activated protein kinase (AMPK) activation, which results in downregulation of the mTOR and IGF-1/AKT pathways.(Pierotti et al., 2013) Two studies have demonstrated that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis by down regulation of anti-apoptotic proteins as well as AMPK. (Zhuang et al., 2008; Queiroz et al., 2014) V (VSL#3 Pharmaceuticals, Gaithersburg, MD) is a mixture of eight strains of lactic acid-producing bacteria. It protected the epithelial barrier and increased the expression 3
of tight junction proteins by activating the p38 and extracellular signal-regulated kinase (ERK) in colitis.(Dai et al., 2012) However, studies of V in CRC development have yielded controversial findings.(Appleyard et al., 2011; Arthur et al., 2013) A study reported that V attenuated various inflammatory-associated parameters, delaying transition to dysplasia in a rat model of colitis-associated cancer.(Appleyard et al., 2011) Another recent study revealed that anti-inflammatory agent balsalazide and VLS#3 suppressed CAC thorough modulation of the interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) pathway.(Do et al., 2015) These lines of evidence suggest that a combination therapy of Met and probiotic V could represent an effective chemopreventive strategy for WD-induced CAC. In our current study, we provide evidence that combination therapy with Met and V yields chemopreventive effects against WD-induced CAC.
2. Materials and methods
2.1. Chemicals, Animals and experimental diets
AOM and Met was purchased from Sigma Aldrich Corp. (St. Louis, MO, USA). DSS (molecular weight 36,000-50,000) was purchased from MP Biomedicals (Aurora, OH). Male, four-week-old BALB/c mice (Charles River Laboratories Japan, Yokohama, Japan) were randomized to receive a CD (D10011), a WD (D16365B) or a WD with Met (D16365B is mixed with Met, 250 mg/kg/day) (Hosono et al., 2010) for 8 weeks (Research Diet, Inc., Bethlehem, PA), as shown in Table 1. We used BALB/c mice following preliminary study with WD of our laboratory. (Kim et al., 2010a)
4
2.2. Induction of CAC in the mouse model
The experimental design is shown in Figure 1A, a total of 50 mice were randomly divided into the following five groups: CD group (n=10), mice were fed without Met or V (VSL Pharmaceuticals, Gaithersburg, MD); WD group (n=10), mice were fed without Met or V; WD+M group (n=10), fed a drug with Met (3~4g per mouse/day); WD+V group (n=10), fed a drug with V (1.3 million bacteria/day); and WD+M+V group (n=10) with both drugs, Met and V. An animal model of CAC was induced by the intraperitoneal injection of 10 mg/kg AOM at 6 weeks of age. One week later, 2% DSS was added to drinking water for 7 days. Drugs were administered from 1 week prior to AOM injection until death. Mice were observed daily for water and dietary intake, and they were killed at 13 weeks of age (Fig. 1A). All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences at the Asan Medical Center, consistent with the Institute of Laboratory Animal Resources (ILAR) guidelines.
2.3. Evaluation of colitis severity and tumor
The disease activity index (DAI) was calculated based on the cumulative score of the parameters (weight loss, rectal bleeding, and stool consistency).(Kim et al., 2010c) Mice were killed at the indicated times and the diameter and number of tumors (2–4 mm, >4 mm and Total) were measured by an independent observer who was unaware of the treatment.
2.4. Immunohistochemical analysis 5
Ki-67 (Abcam, Cambridge, UK, #ab15580) and F4/80 (Serotec, Raleigh, NC, # MCA497GA) antibodies were used. Immunohistochemistry was performed on 4 µm thick paraffin-embedded sections of the colon. For antigen retrieval, slides were immersed in citrate buffer (pH 6), heated in a decloaking chamber at 125°C for 3 min, and then cooled for 30–40 min. After the addition of 3% hydrogen peroxide, sections were incubated for 10 min. After washing with TBST (pH 7), the slides were stained with a primary antibody (F4/80, 1:100: Ki-67, 1:500) at 4°C overnight. A secondary antibody was then used from the VECTASTAIN ABC Kit (Vector, Burlingame, CA) in accordance with the manufacturer’s instructions. The slides were developed with 3, 3`-diaminobenzidine (DAB, Dako, Hamburg, Germany), and were counterstained with hematoxylin. A total of five randomly selected fields were photographed at 400 magnifications, and positively stained cells were counted and analyzed by two observers who were blinded to the study.
2.5. Immunofluorescence analysis
Frozen sections (4 µm) of colonic tissue collected on days 4 and 8 after colitis induction were collected and immunofluorescence staining was performed using anti-Claudin-1 antibody (Abcam, #ab15098). Nuclei were counterstained with DAPI (Sigma– Aldrich) and stained sections were examined using fluorescence microscopy (Olympus AX70, Olympus Optical Co., Tokyo, Japan).
2.6. Apoptosis and cell cycle analysis
A human colon adenocarcinoma cell line (HCT-116) was obtained from the 6
American Type Culture Collection (Manassas, VA). For the preparation of V medium, 0.01g probiotic bacteria were incubated overnight with RPMI 1640. Live bacteria were removed by filtration through a 0.2μm syringe filter and V medium was placed on HCT-116 monolayers (Ewaschuk et al., 2006). Cells were incubated in media containing defined concentrations of Met (5mM, Enzo Life Sciences, Farmingdale, NY) and/or V (20, 40 and 60%) for 24 hours. Cells treated with propidium iodide (PI) and RNase A can be utilized to assess cell cycle stages (G0/G1, S, G2/M), and to identify apoptotic cells (hypodiploid, sub G0 peak). Half a million cells were pelleted at 1000g for 5 min and cells were mixed with 5 μl PI staining solution and 5 μl Annexin V staining solution, following the manufacturer’s instructions (BD Pharmingen, San Jose, CA). Cells were analyzed using a Becton–Dickinson FACS Calibur flow cytometer.
2.7. Western blot analysis
Cultured cells treated with the Met and/or V for the indicated periods of time were washed with PBS. To obtain total cell extracts, the cells were harvested in lysis buffer (50 mM Tris–HCl, 100 mM NaCl, 5 mM EDTA, 1% TritonX-100) containing a cocktail of phosphatase inhibitor (GenDEPOT P3200-001, TX, USA) and a cocktail of protease inhibitors (Sigma–Aldrich P8340). Lysates were incubated on ice for 30 min and centrifuged at 15,000 g. Protein concentrations were determined using a Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA). The proteins were separated by SDS–PAGE (7.5–12.5% gels) and transferred onto a Hybond-P PVDF membrane (Amersham Biosciences, Little Chalfont, UK). After transfer, the membranes were blocked with Blocking One-P (Nacalai Tesque, Kyoto, Japan) and incubated with primary antibodies in TBST with 5% dry milk at 4°C, overnight.
7
The primary antibodies used included anti-pAMPK (Thr172, #2535), anti-AMPK (#5831), anti-p-pERK1/2 (Thr202/Tyr204, #9101), anti-pERK1/2 (#9102), anti-pAKT (Ser473, #4060), anti-AKT (#4685) (1:1000, Cell Signaling, Danvers, MA), anti-Cyclin D1 (92G2, #2978), and anti- Bcl-2 (83-8B, Enzo #ADI-AAM-072-E). As a loading control, β-actin (1:10,000, Sigma–Aldrich #A5441) was used. HRP-conjugated anti-rabbit IgG (1:7000, Cell Signaling #7074) and HRP-conjugated anti-mouse IgG (1:7000, Cell Signaling #7076) were used as secondary antibodies. Horseradish peroxidase-conjugated secondary antibodies and an ECL detection kit (Amersham Biosciences) were used to detect specific proteins. Densitometry analysis of the blots was performed using ImageJ software.
2.8. Statistical Analysis
Statistical analyses were performed using SPSS 14.0 (SPSS Inc., Chicago, IL). Data were expressed as a mean ± standard deviation. Significant differences were evaluated using the Student’s t-test or one-way ANOVA, where appropriate. A value of P<0.05 was considered statistically significant.
3. Results
3.1. WD exacerbates DSS-induced colitis, whereas combination therapy attenuates severity of colitis
Mice fed a WD exhibited marked weight loss and more bloody stools during or soon after the cessation of DSS administration. On day 5 after DSS, the DAI scores and
8
weight loss in the WD group became significantly higher than those in the CD group (Fig. 1B and 1C). Compared to the CD group, WD alone or a single drug-treated groups showed higher DAI scores during DSS-induced colitis. A total of 3 of 10 mice in the WD group died as a result of severe colitis. By contrast, DAI scores in the combination therapy group had significantly lower than those in the single drug-treated groups (P<0.05, Fig. 1B and 1C).
3.2. WD exacerbates tumor growth, whereas combination therapy reduced tumor formation
With regard to tumor formation, the effect of Met and/or V was assessed by quantifying the number of tumors within the colon and rectum of mice. All groups had lowto high-grade dysplasia at the end point. The total number of tumors in the Met+V-treated groups were significantly low compared to WD and single drug-treated groups (P<0.01, Fig. 2B). The WD group showed multiple and larger tumors and some tumors conglomerated from the middle colon to rectum; however, all tumors in CD group were smaller than 2 mm (Fig. 2A). Therefore, we had to divide the number of tumor based on the nodule size (2–4 mm and >4 mm). There was no significant difference in nodules sized 2–4 mm between any of the groups. The large tumors (>4 mm) in the WD group significantly increased compared to those in the CD group, and significantly decreased in the Met therapy and combination therapy groups (P<0.01; Fig. 2A, B). Interestingly, in the combination therapy group, most nodules were <4 mm and there were no tumors in 2 of 10 mice. Immunohistochemical staining showed that the WD group had significantly increased expression of Ki-67 at the base of the crypts and shortened and distorted crypt structures compared with the CD group. The combination therapy group showed significantly fewer Ki-67-positive cells than the WD group (P<0.01; Fig. 2C, D).
9
3.3. Combination therapy prevents macrophage infiltration and protects epithelial integrity during WD-induced CAC
The effects of Met and V on macrophage infiltration were examined during colitis induction by DSS. After 4 days of continuous DSS ingestion, F4/80-positive cells could be observed at the base of crypts in all groups of mice. On day 8, F4/80-positive macrophages increased and were distributed in both submucosal space and base of crypts, especially in WD group, whereas the combination therapy group showed few macrophages at the base of crypts (Fig. 3A, B). The combination therapy group showed significantly fewer F4/80-positive cells compared with the WD group on days 4 and 8 (P<0.05; Fig. 3A, B). Since epithelial tight junctions regulate gut permeability and integrity, we examined and compared the effects of Met and V on the expression of tight junction proteins and microscopic damage to crypt structures by western blotting and immunofluorescence microscopy, respectively. Cryostat sections of tissues from each group were stained with anti-Claudin-1 antibody on days 4 and 8. There were no prominent differences between the groups on day 4. On day 8, WD group and WD with single treatment groups showed very low Claudin-1 protein expression and severe crypt destruction; however, the combination therapy group showed intense reactivity with anti-Claudin-1 antibody at the epithelial surface and an intact crypt structure on day 8 (Fig. 3C). Here, we confirmed that WD promoted gut barrier alteration and the disruption of tight junctions, which induced macrophage infiltration; however, the combination therapy attenuated the structural damage and macrophage infiltration in WD-associated DSS-colitis through Claudin-1 tight junction protein expression.
3.4. Combination therapy promotes late apoptosis by inhibiting Cyclin D1 and Bcl-2 as well
10
as activating AMPK and ERK.
As shown Fig. 4A, we observed V activated AMPK and ERK in a dose-dependent manner, therefore, high dose V (60%) with Met promoted ERK phosphorylation more effectively than low dose (Fig. 4A). In addition, combination therapy with Met and V significantly inhibited anti-apoptotic protein and induced late apoptosis effectively. Thus, our results consistently showed that combination therapy with Met and V suppressed the expression of Cyclin D1 and Bcl-2. Cell cycle assay exhibited that Met with high dose V (60%) effectively induced G2/M arrest (Fig. 4B). Annexin V/PI staining data revealed that late apoptosis was induced by Met treatment and the effect was augmented when combined with high dose V (60%) (Fig. 4C). Our data indicate that the precise molecular mechanism of the chemopreventive effects of Met and V could be to induce growth arrest and to inhibit apoptosis by the sustained increase of ERK/AMPK activity.
4. Discussion
Excess fat intake is a major contributor to CAC, and high-fat diet-induced obesity leads to intestinal inflammation and changes in gut microbiota.(Lee et al., 2015) Met has been proposed as a promising therapeutic agent in the treatment of several types of cancer, and probiotic V has also been reported to modulate barrier function and reduce experimental colitis.(Uronis et al., 2011) Herein, we investigated the chemopreventive effects of Met and V in WD-induced CAC, which could be helpful in the development of chemopreventive strategies against CRC. In the current study, the cancer was exacerbated by WD and our goal was to test the efficacy of Met and V in WD-induced CAC. We induced tumors in the colon of mice fed 11
CD or WD using AOM/DSS regimen; however, mice fed WD received the preventive agents. Consistent with our expectations, a combination therapy with Met and V attenuated the severity of WD-induced DSS colitis and suppressed tumor growth. These effects were associated with drug-induced inhibition of macrophage activation and protection of epithelial integrity. Previously, we showed that WD increases susceptibility to AOM/DSS-induced tumorigenesis via effects on macrophage recruitment and subsequent inflammatory signal activation.(Kim et al., 2010a) Macrophage accumulation was found to be an important driver in the development of CAC, and these cells can produce a wide variety of inflammatory mediators.(Mantovani, 2009; Mantovani and Sica, 2010) In the biomedical literature, macrophages have been reported to be associated with altered immune responses within the tumor microenvironment, and several studies have shown that tumor-associated macrophages (TAMs) can exert pro-tumor functions.(Condeelis and Pollard, 2006; Kang et al., 2010) The anti-inflammatory effects of Met in CAC have been reported, and it was shown that Met could suppress NF-kB activation in intestinal epithelial cells and ameliorate colitis-associated tumorigenesis in mice.(Koh et al., 2014) In contrast, V did not reduce CAC in a study using an AOM–Il10-/- mouse model, although it modified the microbial composition.(Arthur et al., 2013) We observed in our study that Met partially contributed to the reduced severity of colitis and macrophage accumulation, but V did not show these effects. However, the combination significantly reduced the numbers of F4/80-positive macrophages and was associated with a lower DAI, suggesting an interaction between two drugs to suppress WD-induced inflammatory responses. Interestingly, these results were in accordance with tumor incidence, indicating that activation of inflammatory signals by macrophages and destruction of epithelial integrity are major components of colon carcinogenesis. To examine the molecular mechanism underlying the phenomenon, we performed 12
in vitro experiments with the HCT-116 cell. We found that inhibition of anti-apoptotic proteins and induction of late apoptosis were critical for the anti-tumor effect of Met with V. AMPK and ERK play an important role in regulating cancer cell proliferation, differentiation, and apoptosis. Met-induced glucose deprivation activates AMPK, and ERK exhibits pro-apoptotic effects in HCT-116 cells.(Kim et al., 2010b) Cyclin D1 is a key regulator of the cell cycle and acts as an oncogene in several types of cancer. Bcl-2 is also considered an important anti-apoptotic protein. Western blotting with several antibodies using HCT-116 cells exhibites interesting results (Fig. 4A). Met with high dose V (60%) inhibits cancer cell growth by downregulating Cyclin D1 and Bcl-2 and mediates cell cycle arrest by activating AMPK and ERK phosphorylation. Suppression of Cyclin D1 was prominent in Met and combination treatment, however, V treatment did not suppress Cyclin D1 or Bcl-2. Several reports support our data that cell cycle arrest induced Met requires AMPK activation and cyclin D1 downregulation.(Fang et al., 2014; Zhuang and Miskimins, 2008) A previous study reported that V could protect the intestinal epithelial barrier by activating the ERK signaling pathway.(Bassaganya-Riera et al., 2012; Dai et al., 2012) The ERK signaling pathway is a major determinant in the control of many cellular processes and activation of the ERK and/or phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathways is associated with neoplastic transformation in various human tumor cells. These pathways are often up-regulated in human cancers, and represent attractive targets for anticancer drugs.(Kohno and Pouyssegur, 2003; Song et al., 2005). A report also showed the pro-apoptotic potential of AMPK activated conditions, such as in AICAR (AMPK activator)-treated cells.(Chen et al., 2013) Another report suggested that AMPK can specifically antagonize ERK activity in HCT-116 cells and that ERK exhibits pro-apoptotic effects under conditions of glucose deprivation.(Kim et al., 2010b) However, the antagonizing effect between the ERK and AMPK was not evident in our 13
data. We thus surmise that combination therapy of Met and V induces late apoptosis of cancer cells. The critical mechanisms are down-regulating Cyclin D1 as well as activating AMPK and ERK. The additive effect of combination therapy could be induced via proapoptotic ERK signaling of high dose V (60%). CRC is a type of tumor with an increased rate of incidence in patients with type 2 diabetes and high-fat diet-induced obesity. Type 2 diabetes and obese patients should thus be considered as a risk group for this cancer and preventative measures could be helpful for this population. V combined with Met could be a safe and effective chemopreventive strategy to protect against CRC in the risk group. In summary, this is the first study to report the anti-tumor effects of a combination therapy with Met and V in vivo and in vitro. Our findings suggest that this combination therapy reduced the severity of colitis and attenuated tumor growth in a WD-induced colitic cancer by inhibiting macrophage accumulation and preventing epithelial barrier destruction. Cyclin D1 down-regulation by Met and pro-apoptotic ERK activation by high dose V induce late apoptosis of cancer cells, therefore, the mechanisms are involved in the beneficial effects of these two drugs. Therefore, combinations of Met and V might represent a safe and promising therapeutic approach for the chemoprevention of WD-induced colon cancer.
Acknowledgements: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C3078). No potential conflicts of interest were disclosed. This research was supported by a grant from Asan Institute for Life Sciences (No. 2016-559). This material is based upon work supported by the Ministry of Trade, Industry & Energy (MOTIE, Korea) under Industrial Technology Innovation Program. No. 10063408, 'Development of channel-inserted convergence smart 14
endoscopic system based on multispectral fluorescence imaging for precise diagnosis of digestive diseases'.
Figure Legends Fig. 1. Experimental course and disease activity. (A) Schematic representation of the experimental course. Filled triangles ( ) indicate intraperitoneal injection of 10 mg/kg azoxymethane (AOM); DSS indicates a dose of 2% dextran sulfate sodium. Mice were fed a control diet (CD) or a WD for 8 weeks with or without metformin (M) and VSL#3 (V) treatment. (B, C) Disease activity index (DAI) scores and weight loss during the induction of murine colitis. DAI was calculated as a cumulative score of three parameters (weight loss, stool consistency, and anal bleeding); *P<0.05. Values indicate means + S.D. (n=10 per group).
Fig. 2. Tumor incidence and cell proliferation. (A and B) Tumor incidence based on nodule size (2–4 mm, >4 mm and Total) was evaluated. (C and D) Immunohistochemical staining for Ki-67. The WD group showed increased Ki-67 staining at the base of the crypts. The indices indicate the number of positively stained nuclei counted in the crypts of the colon. A total of five randomly selected fields were examined at 400 magnification. Scale bar: 50 μm. Each bar represents the mean +S.D.
Fig. 3. Macrophage accumulation and Claudin-1 expression in mice colon tissues. (A and B) Immunohistochemical staining for F4/80 and immunofluorescent staining for Claudin-1. Cryostat sections of tissues from each group were stained with anti-F4/80 or anti-Claudin-1 antibody on days 4 and 8 after 2% DSS administration. (C) F4/80-positive cells were counted
15
at each indicated time point after treatment with AOM and DSS. A total of five randomly selected fields were examined at 400 magnification. Scale bar: 50 μm. Each bar represents the mean + S.D.
Fig. 4. Pro-apoptotic effects of Met and/or V in HCT-116 colon cancer cell line. (A) Western blot analysis. (B) Cell cycle distribution in HCT-116 cells treated with Met and/or V for 24 hours. (C) The pro-apoptotic effects of Met and V, as demonstrated by the incorporation of Annexin V and/or propidium iodide (PI); different labeling patterns identify the different cell populations as follows: region R1, live cells, PI– Annexin V–; region R2, apoptotic cells PI– Annexin V+; region R3, damaged cells PI+ Annexin V–; and region R4, dead cells PI+ Annexin V+.
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Table 1. Experimental diet composition. Dietary component
Control diet
Western-style diet
(D10011)
(D16365B)
(AIN-76A diet)
(Newmark Stress Diet B)
Fat (corn oil), %
5
20
Corn starch, %
15
7.5
Calcium, mg/g
-
0.88
Monosodium phosphate, mg/g
-
7.98
Monopotassium phosphate, mg/g
-
7.91
Vitamin D3, IU/g
-
0.12
DL-Methionine, %
0.3
0.36
Choline bitartate, %
0.2
0.24
Fiber (cellulose), %
5
6
3.9
4.8
Kcal/g (approx.)
19
20
21
22
23
24
25
26