Apple Polyphenol Phloretin Inhibits Colorectal ... - ACS Publications

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Apple polyphenol phloretin inhibits colorectal cancer cell growth through inhibition of type 2 glucose transporter and activation of p53-mediated signaling proteins Sheng-Tsai Lin, Shih-Hsin Tu, Po-Sheng Yang, Sung-Po Hsu, Wei-Hwa Lee, ChiTang Ho, Chih-Hsiung Wu, Yu-Hsin Lai, Ming-Yao Chen, and Li-Ching Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02861 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 22, 2016

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Journal of Agricultural and Food Chemistry

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Title:

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The apple polyphenol phloretin inhibits colorectal cancer cell growth via inhibition of

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the type 2 glucose transporter and activation of p53-mediated signaling

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Authors:

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Sheng-Tsai Lin§† Shih-Hsin Tu&,#,¥,; Po-Sheng Yang※％@ †; Sung-Po Hsu『, Wei- Hwa

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Lee◎, ¢; Chi-Tang Ho【; Chih-Hsiung Wu#"; Yu-Hsin Lai§ ; Ming-Yao Chen§*; and

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Li-Ching Chen&,¥,£*

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Affiliations

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§

Division of Gastroenterology and Hepatology, Department of Internal Medicine,

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Shuang Ho Hospital, new Taipei city, Taiwan

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&

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#

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University, Taipei, Taiwan

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¥

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TMU Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan

Department of Surgery, School of Medicine, College of Medicine, Taipei Medical

Breast Medical Center, Taipei Medical University Hospital, Taipei, Taiwan

※

Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan

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％Department

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@

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『

of Medicine, Mackay Medical College, New Taipei City. Taiwan

Mackay Junior College of Medicine, Nursing, and Management, Taipei, Taiwan

Department of physiology, Taipei Medical University, Taipei, Taiwan.

ACS Paragon Plus Environment


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◎Department

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University, Taiwan

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¢

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【Department

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08901, USA

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"

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£

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University, Taipei, Taiwan

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†Dr. Sheng-Tsai Lin and Dr. Po-Sheng Yang contributed equally to this manuscript.

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*Correspondence:

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Li-Ching Chen, Ph.D., Graduate Institute of Clinical Medicine, College of Medicine,

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Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone:

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+886-2-2736-1661 ext. 3328; Fax: +886-2-2739-3422; E-mail: [email protected];

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Ming-Yao Chen, M.D., Division of Gastroenterology, Department of Internal Medicine,

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Shuang Ho Hospital, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei 110,

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Taiwan. Phone: +886-2-2249-0088 ext. 8810; Fax: +886-2-2739-3422; E-mail:

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[email protected]

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Keywords

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Glucose transporter 2, Colon cancer, Phloretin, p53, Apple polyphenol.

of Pathology, School of Medicine, College of Medicine, Taipei Medical

Department of Pathology, Taipei Medical University-Shuang Ho Hospital, of Food Science, Rutgers University, New Brunswick, New Jersey

Department of Surgery, En Chu Kong Hospital, New Taipei City 237, Taiwan Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical


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Abstract

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Glucose transporters (GLUTs) are required for glucose uptake in malignant cells, and

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they can be used as molecular targets for cancer therapy. An RT-PCR analysis was

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performed to investigate the mRNA levels of 14 subtypes of GLUTs in human

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colorectal cancer (COLO 205 and HT-29) and normal (FHC) cells. RT-PCR (n=27)

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was used to assess the differences in paired tissue samples (tumor vs. normal) isolated

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from colorectal cancer patients. GLUT2 was detected in all tested cells. The average

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GLUT2 mRNA level in 12 of 27 (44.4%) cases were 2.4-fold higher in tumor

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compared to normal tissues (*p=0.027). Higher GLUT2 mRNA expression was

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preferentially detected in advanced-stage tumors (stage 0 vs. 3 = 16.38-fold, 95% CI =

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9.22 to 26.54-fold, *p = 0.029). The apple polyphenol phloretin (Ph) and siRNA

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methods were used to inhibit GLUT2 protein expression. Ph (0-100 µM, for 24 h)

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induced COLO 205 cell growth cycle arrest in a p53-dependent manner, which was

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confirmed by pre-treatment of the cells with a p53-specific dominant negative

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expression vector. Hepatocyte nuclear factor 6 (HNF6), which was previously

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reported to be a transcription factor that activates GLUT2 and p53, was also induced

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by Ph (0-100 µM, for 24 h). The anti-tumor effect of Ph (25 mg/kg or DMSO twice a

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week for six weeks) was demonstrated in vivo using BALB/c nude mice bearing COLO

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205 tumor xenografts. In conclusion, targeting GLUT2 could potentially suppress

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colorectal tumor cell invasiveness.



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Introduction

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Glucose, a vital source of energy for cell, transport across the cell membrane by

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specific glucose transporter (or sensor) proteins, which is the rate-limiting step for its

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subsequent utilization.1 The mammalian glucose transporter family (GLUT1–12, 14)

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was identified in human tissues, and the protein structure was recently elucidated.2

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Among these GLUTs, the type 2 glucose transporter (GLUT2) is predominantly

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expression in liver, pancreas, intestine and kidney tissue 3 and is influenced by the

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extracellular glucose level and insulin.4 The role of other glucose transporters in

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colorectal cancer was not investigated. Targeting GLUT1 with specific antibodies

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significantly inhibited cell growth proliferation and induced cell apoptosis in in vitro,

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providing evidence for their anti-tumor activity.5 Inhibition of glucose uptake and

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glycolysis also elevated intracellular oxidative stress, which triggers stress-activated

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pathways that result in cancer cell autophagy or apoptosis.6 In contrast, increased

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glucose uptake promoted invasion and metastasis of colon cancer cells.7 However, the

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molecular mechanisms in cancer cells adapt to glucose deprivation remain poorly

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understood.

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Increased consumption of apple and its derivatives has been shown to be associated

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with the prevention of colorectal cancer.8 Most previous reports focused on apple

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polysaccharides that affected colon cancer cell growth or induced apoptosis.9-12


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However, many studies have reported that the phytochemicals produced by apples have

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function as antioxidants and anti-proliferative effects on cancer cells.13, 14

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Dihydrochalcone phlorizin (phloretin 2'-glucoside) and its precursor phloretin (Ph)

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contribute to the important health benefits of apple fruit and its processed products,15, 16

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and in our previous studies, it was shown to suppress transmembrane glucose

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transportation.17 Moreover, Ph is a specific inhibitor of GLUT2 in human liver cancer

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cells.17 Previous studies also demonstrated that Ph is an inhibitor that blocks glucose

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transmembrane transport, which leads to tumor cell growth arrest and induction of

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apoptotic cell death.17 In vivo studies demonstrated that Ph suppress the growth of

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xenograft tumors, such as bladder and liver cancer.17-19 These findings suggest that Ph

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potential effects on anti-tumor activity. However, the mechanisms of Ph in human

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colon cancer cells is still limited.

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In this study, GLUT2 was detected in colon cancer cells. We demonstrated that high

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levels of GLUT2 in human colon cancer tissues are associated with advanced stages

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(stage 3 and 4) and poor prognosis. Ph significantly inhibited colon cancer cell growth

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in an in vivo xenograft mouse model. Our results showed that apple polyphenols inhibit

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GLUT2 and can be effectively used for colon cancer chemoprevention.

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Materials and Methods

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Patient samples

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All participants (n =27) in this study provided written informed consent. The study and

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consent procedure were permitted by the Research Ethics Committee of Taipei Medical

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University Hospital. Human colon tumor and adjacent normal epithelial paired

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tissues were obtained from anonymous donors following a protocol approved by the

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Institutional Review Board (TMU-JIRB, No: 201410010). All clinical investigations

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were conducted according to the regulations expressed in the Declaration of

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Helsinki. Histological analysis showed that each patient’s tissue sample was more than

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80% tumor. All samples were collected and grouped according to their clinical

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characteristics.

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Cell lines and cell culture

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The HT-29 and COLO 205 cell lines were isolated from human colon adenocarcinomas

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(American Type Culture Collection, ATCC-CCL-221, -HTB-38 and -CCL-222). The

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FHC cell line (CRL-1831; ATCC) was a primary cell line isolated from epithelial cell

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cultures of human fetal normal colonic mucosa.20 Hep 3B and Hep G2 cells were

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established from human hepatocellular carcinoma (HB-8064 and HB-8065; ATCC).21

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COLO 205 and Hep G2 cells express wild-type p53 gene.22, 23 The p53 gene of the

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HT-29 cells, on the other hand, is mutated at codons 27324 and 241.25 In Hep 3B cells,


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the p53 gene is partially deleted.23 The cell lines were growth in medium as the same in

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our previous study.26

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Determination of cell growth curves

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A total of 1 x 106 cells (human colon cancer and non-cancer FHC cells) was plated into

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a 100 mm petri dish. Ph in 0.05% dimethyl-sulfoxide (DMSO) was treated with an

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indicated doses. For control groups, 0.05% DMSO without Ph was added. Media were

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changed daily until the cells counted.

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Protein extraction, Western blotting analysis and antibodies

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Cells treated with DMSO and Ph were harvested for immunblotting analysis. Cell

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lysates (100 µg protein per lane) were separated by 12% sodium dodecyl

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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a

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PVDF membrane (Millipore) using standard techniques.27 Primary antibodies were

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purchased from multiple sources. Antibodies against WAF1/Cip (p21) and E-cadherin

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were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti- Kip1 (p27)

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was purchased from BD Bioscience pharmingen (San Diego, CA). Anti-GAPDH

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(ab9485) antibodies was purchased from Abcam (Cambridge, UK). Antibodies against

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GLUT2 (H-67), p53 (DO-1), and PARP (F2), and E-cadherin (67A4) were obtained

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from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

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Flow cytometry analysis



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The populations of different cell cycle phases in cells treated with Ph or DMSO were

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analysed by flow cytometry.28 The population of different phase of the cell cycle was

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evaluated using FACSCAN laser flow cytometric analysis software (Becton Dickenson,

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CA, USA).

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In vitro apoptosis assay

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DNA fragmentation assay and Annexin V staining flow cytometric assay were both

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used to evaluate cell apoptosis.28 The apoptotic cells were detected using the

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commercial Annexin V detection kit and according to the Muse® Annexin V and Dead

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Cell assay kit manufacturer’s procedure (Merck Millipore, Darmstadt, Germany).

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Immunocytochemical staining analysis

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The paraffin-embedded tissue section slides were washed with PBS and incubated 16

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hrs at 4°C with the anti-GLUT2 antibody (1:50, Alpha Diagnostic, San Antonio, TX).

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The slides were incubated with a biotinylated anti-mouse antibody and detected by

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peroxidase-conjugated streptavidin (DAKO LSAB+ kit; Dako Corp., Carpinteria, CA).

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27

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RNA isolation and real-time PCR analysis

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Total RNA from human tissue samples was isolated using TRIzol reagent (Invitrogen,

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CA, USA) according to our previous paper. Real-time PCR reaction was performed on

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the LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim,


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Germany).27 The forward and reverse primers sequences were designed as follows:

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GLUT2-f (5ˈ-AGTTAGATGAGGAAGTCAAAGCAA-3ˈ) and GLUT2-r

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(5ˈ-TAGGCTGTCGGTAGCTGG-3ˈ).19 The β-glucuronidase (GUS)-specific PCR

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products from the same RNA samples were amplified and served as internal controls.

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Primers GUS-f (5ˈ-AAACAGCCCGTTTACTTGAG-3ˈ) and GUS-r

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(5ˈ-AGTGTTCCCTGCTAGAATAGATG-3ˈ) were used for PCR amplification of

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GUS. The GLUT2 mRNA fluorescence intensity was neutralized with GUS using

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Roche LightCycler Software. Each of the GLUTs subunit–specific primers are shown

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in Supplementary Table 1.

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In vitro invasion assays

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In vitro invasion assays were performed using Transwell chambers (Corning Costar,

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Cambridge, MA, USA) according to our previous paper.29

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Treatment of COLO 205-derived xenografts in vivo

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COLO 205 cells (5 x 106) were transplanted into BALB/c nude mice (6–7 weeks of

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age).26, 28Tumor size was measured after transplantation using calipers and estimated

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according to the following formula: tumor volume (mm3) = L x W2/2, where L is the

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length and W is the width.26 Mice treated with DMSO or a dose of 25 mg/kg thrice daily

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for 6 weeks when the tumors were reached a mean volume of more than 200 mm.3 The

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study was approved by the Association for Assessment and Accreditation of



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Laboratory Animal Care, and the all processes were performed based on the Taipei

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Medical University animal care and use rules (licenses No. LAC-2015-0098).

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RNA interference

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GLUT2 expression was inhibited in COLO 205 cells and established at least two

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independent siRNA clones. Scrambled sequences in accordance to each siRNA were

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used as controls (Supplementary Table 1). In addition, Basic Local Alignment Search

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Tool (BLAST) analysis to avoid the sequence homology with other genes, the

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designated primer sequences were introduced into the pSUPER vector (OligoEngine

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Co., Seattle, WA, USA) digested with BglII and HindIII restriction enzymes to create

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the pSUPER-GLUT2-Si and pSUPER-scramble plasmid. The procedure of cell

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transfection was based on the previous study.27

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Generation of stable GLUT2-siRNA-expressing cell lines

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The pSUPER-GLUT2-si and pSUPER-scramble plasmids were transfected into the

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cells, selected with G418 (4 mg/mL) selection medium, and two G418-resistant clones

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(GLUT2-Si-1 and GLUT2-Si-2) were isolated.

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Statistical methods

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All results were expressed as the mean value of at least three experiments with 95%

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confidence intervals (CIs), unless otherwise stated. A paired t-test was used to compare


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GLUT2 mRNA expression in paired normal vs. tumor tissues from colon cancer

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patients. Annexin V apoptosis staining, invasion assays, the MTT test, flow cytometry

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analysis, and fold ratios of GLUT2 mRNA expression in tumors vs. normal samples

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were compared based on the Mann-Whitney U test. The statistical software of

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SigmaPlot graphing (San Jose, CA, USA) and Statistical Package for the Social

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Sciences, v. 16.0 (SPSS, Chicago, IL, USA) were performed to compare the control and

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the study groups . When a p-value of 0.05 or less was considered statistically

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significant.

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Results

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The expression profiles of GLUTs in human colon cancer and normal cells

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RT-PCR analysis was performed to investigate the overall expression profiles of the

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GLUTs in human colon cancer (COLO 205 and HT-29) and normal (FHC) cells. The

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mRNA of human hepatoma (Hep G2, Hep 3B) cells was used as a positive control.

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We found that a large variety of GLUTs were detected with at least 10 different

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subtypes in the HT-29 cells (Figure 1A). In contrast, only four GLUT subtypes

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(GLUT1, 2, 12, 14) were detected in the COLO 205 cells. FHC, a primary cell line

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derived from long-term epithelial cell cultures of human fetal normal colonic mucosa,

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was selected as a normal control.20 Interestingly, we found that GLUT2 and GLUT12

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were detected in all of the cells (Figure 1A). Our previous study indicated that



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targeting GLUT2 in human liver cancer had significant anti-tumor effects.17, 19 These

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results suggests that GLUT2 is important for colon cancer cell growth.

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GLUT2 mRNA and protein expression in advanced-stage human colon tumor

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tissues

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We then performed real-time RT-PCR analysis to study the mRNA levels of GLUT2 in

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paired tumor (denoted as T) vs. normal (denoted as N) colon tissue (Figure 1B, n=27).

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The PCR amplification curves were “left-shifted” in the tumor (Figure 1B, red lines)

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comparative to normal tissues (green lines). The real-time PCR results were used to

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subdivide the samples into two groups according to their GLUT2 mRNA expression

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patterns. As shown in Figure 1C, 44.4% of cases (12 of 27) fell into the T > N group, in

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which expression of GLUT2 was higher in tumor than in normal tissue (*p=0.027), and

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55.5% of cases (15 of 27) fell into the N>T group (p=0.207) (Figure 1C, right panel).

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In the T>N group, the average GLUT2 mRNA expression in tumor cells was 2.4-fold

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higher than that in normal cells (copy number for tumor cells = 497,655 vs. normal

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cells = 73,638; difference = 424,017, 95% CI = 285,647 to 709,664, *p= 0.027).

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However, in the N>T group, a less than 1.5-fold difference in GLUT2 mRNA

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expression was detected (Figure 1C, right panel, bars 3 vs. 4, p= 0.207).

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After histological inspection, all samples (each paired tumor vs. normal tissues) were

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collected and categorized based on other clinical information (Table 1). We found that


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advanced-stage tumors were associated with substantially higher levels of GLUT2

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mRNA expression (stage 0 vs. 3 = 16.38-fold, 95% CI = 9.22 to 26.54-fold, *p = 0.029).

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To confirm this observation, we performed immunohistochemical staining (Figure

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1D), and the results show that higher GLUT2 protein levels were detected in stages

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3-4 compared to those of earlier stages and normal cells. In conclusion, higher

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GLUT2 mRNA and protein expression levels were detected in advanced-stage colon

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tumor tissues (Figure 1D, and Table 1, *p=0.029).

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GLUT2 involvement in advanced-stage colon cancer cell growth

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We then compared the GLUT2 protein expression in human colon cancer (COLO 205

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vs. HT-29) and normal (FHC) cells (Figure 2A). The results demonstrated that the

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highest level of GLUT2 protein was detected in COLO 205 cells compared to that of

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the HT-29 and FHC cells. The COLO 205 cells were derived from metastatic ascites

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sample (ATCC-CCL-222),30 indicating that GLUT2 was preferentially expressed in

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the advanced-stage colorectal cancer cells. To confirm this hypothesis, we used an

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apple polyphenol, Ph, which was identified as an inhibitor of GLUT2,17, 19 to evaluate

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the effects of GLUT2 on cancer cell proliferation. As shown in Figure 2A, cells were

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treated with Ph (50-200 µM for 24-48 h) and evaluated using the MTT method. We

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found that Ph significantly suppressed cell proliferation in COLO 205 cells compared

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to that of HT-29 cells (> 100 µM, *p100

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µM) significantly inhibited p53wt COLO 205 cell growth (Figure 3A, upper panel,

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bars 3 and 4 vs. bar 1, *p 100 µM for 24 h).



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HNF6 has also been reported to activate the p53 promoter and therefore increase the

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p53 protein level.34 Overexpression of HNF6 significantly suppressed the

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epithelial-mesenchymal transition through p53-mediated signals. These results

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suggested that Ph, as a functional inhibitor of GLUT2, should induce HNF6-mediated

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p53 activation and suppress tumor cell invasiveness. To test this hypothesis, we

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treated the COLO 205 cells with Ph (50-200 µM for 24 h). The results demonstrated

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that inhibition of GLUT2 by Ph induced HNF6-mediated p53 signaling activation

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(Figure 5A, left). We further tested this hypothesis by stable inhibition of GLUT2

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mRNA expression by siRNA, and GLUT2 (Si) cells were established (Figure 5A,

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right). The results demonstrated that stable inhibition of the GLUT2 mRNA

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expression in COLO205 cancer cells significantly induced HNF6-mediated p53 and

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p27/Kip1 protein expression compared to that of the control (Sc) cells (Figure 5A,

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right panel).

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These results indicate that HNF6-induced p53 protein expression plays a critical role

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in GLUT2-silenced cells as the COLO 205 cells were derived from metastatic

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ascites.30 To test whether GLUT2 inhibition in COLO 205 cells decreases invasive

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activity, we used a matrix gel invasion assay, and the results indicated that treatment

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with a low dose of Ph (50 µM for 24 h) significantly inhibited COLO 205 cell invasion

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(Figure 5B, black bars, *p50 and 100 µM) in both COLO205 and HT-29 cells; however, the cell growth

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inhibition was only observed in the p53wt COLO 205 cancer cells treated with 100

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µM Ph. Similar results were shown in prostate cancer; Ph modified glucose entry into

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the cells by either modulating GLUT (GLUT1 and GLUT4) protein expression or

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altering glucose binding.37 More importantly, they demonstrated that the GLUTs can

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easily be induced in the LNCaP (with p53wt)38 cells compared to the p53-null PC339

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prostate cancer cells after Ph treatment.37 The p53wt gene is required for some forms

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of apoptosis with increased frequency in advanced and hormone-resistant human

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prostate cancers (such as PC-3) cells.38 In the same study,38 PNT1A, which is an

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immortalized non-tumorigenic prostate cell line, was more resistant to Ph treatment


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than the LNCaP and PC-3 tumor cells. All these results implied that the phenotypic

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characteristics of cancer cells are responsible for the Ph-induced effects in glucose

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uptake and GLUT expression. Previous studies have also demonstrated that

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Ph-induced cancer cell apoptosis was observed in B16 melanoma and HL60 leukemia

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cells via the p53-dependent pathway.40 Our study is the first to demonstrate that

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p53wt is involved in GLUT2 inhibition, which induced colorectal cancer cell growth

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inhibition.

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In human breast cancer (MCF-7) cells, prominent upregulation of p53 and Bax and

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cleavage of poly (ADP)-ribose polymerase were detected in the Ph-treated cells.41

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Previous reports have also found decreased transport and metabolism in

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glucose-induced apoptotic cell death.42 Glucose deprivation-induced ATP depletion43

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initiated the mitochondrial death pathway cascade44 and increased oxidative stress,

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which triggered p53-associated apoptosis.45 The therapeutic importance of the

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Warburg effect is increasingly recognized, and blocking glucose transporters has

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become a common anticancer strategy. We previously identified Ph as a novel small

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compound that inhibits the glucose transport of hepatoma cells and reduces cancer

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cell growth through a glucose deprivation-like mechanism.17, 19 We hypothesized that

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compounds targeting GLUT2 should be efficacious in vivo as anticancer agents. Here,

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we report that Ph inhibited not only cell growth in colon cancer cell lines but also



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cancer growth in a nude mouse model. A previous study found that treatment with

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streptozotocin, which can induce type-1 diabetes in animals, significantly upregulated

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GLUT2 expression in liver cells.46 These observations suggested that for increased

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uptake of glucose into cells, upregulation of the GLUTs was required. Similarly, as

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shown in Figure 2C, the GLUT2 protein level was upregulated in the Ph-treated

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COLO 205 cells, and we hypothesized there was a compensatory feedback effect due

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to inhibition of glucose uptake.

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Phloretin is found in apples and its-derived products, it is conjugated to glucosidic to

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form phloridzin (phloretin 2'-O-glucose).47 It is also produced from Erwinia herbicola

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Y46, which degrades phloridzin to yield Ph.48 However, the highest levels of

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phloretin glycosides were found in apple purees and commercial juices as a

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consequence of the processing conditions.49 Apples also contain other phytochemicals

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or polysaccharides with chemopreventive activity in colon cancer.8, 50 A previous

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study demonstrated that Ph was affect like a non-steroid estrogen through competition

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for the binding of 17β-estradiol to estrogen receptor.51 Another study found that Ph

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suppresses cancer cell growth in B16 melanoma and K-562 erythroblasts cells.52 In

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the present study, Ph-induced COLO 205 cell growth inhibition was observed in

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high-dose group. It is implied that Ph may inhibit COLO 205 cell growth through

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different mechanisms.


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In this study, the transcription factor hepatocyte nuclear factor 6 (HNF6 or

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ONECUT-1) was induced by Ph treatment in the COLO 205 cells.53 Ectopic

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expression of HNF4 alpha inhibited different kinds of colon cancer cells such as

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HT-29, LoVo, and SW480 growth, and its malignancy effects such as migration and

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invasion. HNF4 also induced G2/M phase arrest and promoted apoptosis in the cells

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described above.54 HNF6 has also been reported to activate the promoter activity of

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p53 and therefore increase the p53 protein level.34 Overexpression of HNF6

442

significantly suppressed the epithelial-mesenchymal transition through p53-mediated

443

signals.34 These results indicated that Ph, as a functional inhibitor of GLUT2, should

444

induce HNF6-mediated p53 activation and suppress tumor cell invasiveness (Figure

445

6).



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Disclosure of Potential Conflicts of Interest

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The authors declare that no financial competing interests or financial relationships

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exist with other people or organizations involved in this study

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Author contributions

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Conception and design (project conception, development of overall research plan, and

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study oversight): S.T. Lin, P.S. Yang, M.Y. Chen, P.S. Yang, L.C. Chen

452

Development of methodology: S.P. Hsu, Y.H. Lai

453

Acquisition of data:: (provided animals, acquired and managed patients, performed the

454

in vitro cell experiments, provided facilities, compounds, etc.): S.H. Tu, W.H. Lee,

455

C.H. Wu, P.S. Yang, C.T. Ho

456

Analysis and interpretation of data:: (e.g., statistical analysis, biostatistics,

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computational analysis): S.T. Lin, M.Y. Chen, Y.H. Lai

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Writing, review, and/or revision of the manuscript: M.Y. Chen, L.C. Chen

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Study supervision: C.H. Wu

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Disclaimer::

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The authors have no relevant affiliations or financial involvement with any

462

organization or entity with a financial interest in or financial conflict with this research.

463

Acknowledgments:


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This study was supported by the Health and Welfare Surcharge on tobacco products

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(MOHW105-TDU-B-212-134001) and by the Ministry of Science and Technology

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(MOST 104-2320-B-038-066-MY2).

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Grant Support:

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This study was supported by the Health and Welfare Surcharge on tobacco products

469

(MOHW105-TDU-B-212-134001) and by the Ministry of Science and Technology

470

(MOST 104-2320-B-038-066-MY2).

471

Abbreviations:

472

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose transporters (GLUTs),

473

hepatocyte nuclear factor 6 (HNF6), phloretin (Ph), type 2 glucose transporter

474

(GLUT2)



475

References

476 477 478

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645


Page 32 of 45

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646

Figure legends

647

Figure 1. Glucose transporter expression profile in human colorectal tumor and

648

normal cells.

649

(A) Detection of GLUT subtypes by RT-PCR in cancerous human colorectal (HT-29,

650

COLO 205) and liver (Hep G2, Hep 3B) cell lines. The FHC cells were considered

651

normal human colorectal cells. (B) Relative mRNA expression of GLUT2 in paired

652

normal and tumor tissues isolated from colorectal cancer patients was detected by

653

RT-PCR (upper panel) and real-time PCR (lower panel). (C) GLUT2 mRNA

654

expression levels were calculated from the real-time PCR data. Copy numbers (x105

655

per µg mRNA) were calculated from mean real-time PCR data; error bars indicate the

656

95% confidence intervals. Data were analyzed with paired t-tests, and two-sided

657

p-values are presented. (D) Immunolocalization of the GLUT2 protein in human

658

colorectal tumor tissues. The tumor tissues were cut into 8 µm serial sections and

659

probed with antibodies specific to human GLUT2. N, normal; T, tumor; I.H.C.,

660

immunohistochemistry stain; H.E., hematoxylin and eosin stain. The malignant colon

661

cancer cells are indicated by red arrows. Scale bar = 200 µm.

662

Figure 2. Effects of Ph-induced GLUT2 and G0/G1 phase cell cycle regulatory

663

proteins in colon cancer cells. (A) GLUT2 was involved in advanced-stage colon

664

cancer cell growth. The protein expression of GLUT2 was detected by



665

immunoblotting analysis in the FHC, COLO 205 and HT-29 cells. In each case,

666

GADPH expression served as a control. Cell proliferation in cells treated with DMSO

667

vs. Ph (50-200 µM) was measured by MTT assays, in which a greater number of cells

668

was reflected by an increased OD540 nm at the indicated time points. The experiment was

669

repeated four times with duplicate samples. Data points represent the mean; error bars

670

indicate 95% confidence intervals. The data were analyzed by nonparametric two-sided

671

tests (Kruskal-Wallis and Mann-Whitney tests). Over 48 h in both COLO 205 and

672

HT-29 cells, the mean OD540 nm of DMSO-treated cells was significantly different than

673

that for Ph-treated cells (*p = 0.05 for all comparisons). (B) Flow cytometry (FACS)

674

analysis of DNA was conducted after COLO205, HT-29, and FHC cells were

675

synchronized by 0.04% FCS for 24 h and then switched to culture media supplemented

676

with 10% FBS containing 0.05% DMSO (control) or Ph (100 to 200 µM in 0.05%

677

DMSO) for an additional 16 h. Lower panel, flow cytometry chart of all these cells. The

678

data were analyzed by nonparametric two-sided tests (Kruskal-Wallis and

679

Mann-Whitney tests, *p = 0.05 for all comparisons). (C) The COLO 205 and HT-29

680

cells were treated with or without Ph (10-200 µM) for 16 h. The protein levels of

681

GLUT2, p53, p21/Cip1, p27/Kip1 and PARP were determined by immunoblotting

682

analysis. In each case, GADPH expression served as a control. (D) The COLO 205

683

cells were pre-treated with a p53-specific dominant negative expression vector


Page 34 of 45

Page 35 of 45


684

(indicated as p53DN cells) for 24 h. Both the wild type (p53wt) and p53DN cells were

685

treated with Ph (50-200 µM) for 16 h, and the proteins were harvested for detection of

686

p27/Kip1 and PARP proteins by immunoblotting analysis. Membranes were also

687

probed with anti-GADPH antibodies to correct for differences in protein loading.

688

Figure 3. The Ph-induced cell growth inhibition was attenuated through

689

inhibition of p53-mediated signals.

690

(A) Both COLO 205 (p53wt and p53DN) cell lines were treated with Ph (50-200 µM)

691

for 24 h, and cell growth was calculated by the MTT method. (B) Both of the cell

692

lines were also treated with a high concentration (200 µM) of Ph for different times,

693

and the cells were analyzed by flow cytometry. The data were analyzed by paired

694

t-tests. The mean of DMSO-treated cells was significantly different from that of

695

Ph-treated cells (*p = 0.05 for all comparisons).

696

Figure 4. Ph-induced apoptosis mediated by p53 was observed in the COLO 205

697

cells

698

(A) Both COLO 205 (p53wt and p53DN) cell lines were treated with Ph (50-200 µM)

699

for 24 h. After Ph treatment, the apoptotic cells were stained by Annexin V-specific

700

antibodies, and the cells were assessed by flow cytometry. The data were analyzed by

701

paired t-tests. The mean of DMSO-treated cells was significantly different from that

702

of Ph-treated cells (*p = 0.05 for all comparisons). (B) Both of the COLO205 (p53wt



703

and p53DN) cell lines were treated with Ph (200 µM) for different times, and a DNA

704

fragmentation assay was performed.

705

Figure 5. The GLUT2 inhibition-induced HNF6 protein expression was involved

706

in the activation of p53-mediated anti-tumor effects in COLO 205 cells.

707

(A) The COLO205 cells were treated with the GLUT2 inhibitor Ph (50-200 µM) for

708

24 h, and the protein and mRNA levels were assessed by RT-PCR and

709

immunoblotting analysis (left panel). We also established cells with stable inhibition

710

of GLUT2 mRNA expression by siRNA (right panel). The protein levels of GLUT2,

711

HNF6, p53 and p27/Kip1 were detected by immunoblotting analysis. Membranes

712

were also probed with anti-GADPH antibodies to correct for differences in protein

713

loading. (B) The invasion assay was performed in both p53wt and p53DN COLO 205

714

cells treated with Ph (50-200 µM) for 24 h. The data were analyzed by paired t-tests.

715

The mean of DMSO-treated cells was significantly different from that of Ph-treated

716

cells (*p = 0.05 for all comparisons). (C) The in vivo anti-tumor effect of Ph was tested

717

by treating mice bearing COLO 205 tumor xenografts. After transplantation, tumor size

718

was measured using calipers, and tumor volume was estimated according to the

719

following formula: tumor volume (mm3) = L x W2/2, where L is the length and W is the

720

width. Once tumors reached a mean volume of 200 mm3, the animals received

721

intraperitoneal injections of either DMSO or 25 mg/kg Ph three times per week for 6


Page 36 of 45

Page 37 of 45


722

weeks. All animal studies were performed according to the local guidelines for animal

723

care and protection. (D) After the mice were sacrificed, the protein lysates were isolated

724

from the COLO 205 xenograft tumors, and the p53-mediated signaling proteins were

725

detected by immunoblotting analysis.

726

Figure 6. Mode of GLUT2 inhibition-induced anti-tumor effects in human COLO

727

205 cancer cells.

728

In response to Ph treatment, cell glucose uptake was suppressed through GLUT2.

729

Subsequently, low concentrations of intracellular glucose transcriptionally activated

730

HNF6 protein expression (data shown in Figure 5A). The overexpressed GA was

731

reported to be a transcription factor33 that induced GLUT2 compensatory upregulation

732

in response to glucose deprivation (seen in Figure 2C). HNF6 also acts as a

733

transcription factor34 to induce p53-mediated signals and trigger G0/G1 cell cycle

734

arrest, which then caused the in vivo anti-tumor effects in the COLO 205 xenograft

735

mice.


Figure 1 revised.

FH C C O H LO T 2 H 29 05 ep H G2 ep FH 3B C C O L H O2 T 2 05 H 9 ep H G2 ep 3B

C.

5

(10x /µg mRNA)

Copy number

3.5 3.0

o

x

o

GLUT1

o

o

o

o

o

GLUT2

x

x

o

o

o

GLUT3

x

x

x

x

o

GLUT4

x

x

x

o

x

GLUT5

x

x

o

o

o

GLUT6

x

x

o

x

x

GLUT7

x

x

o

o

o

GLUT8

x

x

o

o

o

GLUT9

x

x

o

o

o

GLUT10

x

x

o

o

o

GLUT11

x

o

o

o

o

GLUT12

o

o

o

o

o

GLUT14

o

o

o

o

o

GUS

o

o

o

o

o

β-Actin

2.5

2.5 1.5 1.0

GUS GLUT2 GUS

6

Normal Tumor n= 27

5 4 3 2 1 0

1

* p =0.027

10 20 30 x Cycles

p =0.207

1.5 1.0

0.5

0.5

0

0

Normal Tumor

Stage-1

T>N

N>T

(n=12)

(n=15)

Tumor Stage-2B Stage-3B

IHC

H.E.

Normal

GLUT2

2.0

2.0

(n=27)

D.

3.0

N TN TN TN TN T

Fluorescence (folds)

o

* p =0.029

Page 38 of 45

B.

x

Fold

A.



Stage-4A

40

Figure revised. Page 39 2, of 45

6

9 T2

H

C

FH

C O LO

20

5

A.

GLUT2 GAPDH

B. % of control

100 80 60

Cell number (x 10 )


HT 29 *

3.0 2.5 2.0

HT 29 0 µM 50 µM 100 µM 200 µM

1.5

0.5 Hours 0

24

48

COLO 205 * *

FHC

* *

1.0

0 µM 100 µM 200 µM

COLO 205

* 0 0 µM 100 µM 200 µM

24

48

0

FHC

48

0 µM 100 µM 200 µM

40 20 0

sub-G1 G0/G1 S G2/M

C. 0

sub-G1 G0/G1

COLO 205 10 50 100 200 0

S G2/M

sub-G1 G0/G1

HT 29 10 50 100 200 (Ph µM) GLUT2 GAPDH

0

COLO 205 50 100

200

0

HT 29 50 100

200 (Ph µM) p21 p27 p53 PARP GAPDH

D.

24

COLO 205 (p53 wt) 0 50 100 200

COLO 205 (p53 DN) 0 50 100 200 (Ph µM) p27 PARP


GAPDH

S G2/M

Figure 3, revised.


Page 40 of 45

A. 6

Cell number (x 10 )

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

6

Cell number (x 10 )

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

* *

0

0

100

50

100

200 µM

0 µM

COLO 205 (p53 wt) G1 G2M S Sub G1

80 60

50 µM

100 µM

200 µM

50 µM

100 µM

200 µM

*

Treated with Ph (200 µM)

*

40 20 0

3 hr 3

100 % of control

50

0 µM 200 µM

COLO 205 (p53 DN)

100

% of control

B.

COLO 205 (p53 wt)

6 12 24 hr COLO 205 (p53 DN)

6 hr

12 hr

24 hr

Treated with Ph (200 µM)

80 60 40 20 0

3 hr 3

6

12

6 hr

24 hr


12 hr

24 hr

Figure Page 41 4, of revised. 45


A. p53wt (Ph µM)

0

50

0

50

100

200

100

200

% of apoptosis (late + early)

100

p53wt *p=0.002

80 60 40

p53DN

*

20

0 (ph µM) 0 50 100 200

0 50 100 200

0 50 100 200

0 50 100 200

Death

Live

Death

Live

B.

p53DN (Ph µM)

COLO 205 (p53wt) 24 hr

36 hr

48 hr

0 200 0 200 0 200

COLO 205 (p53DN) 24 hr

36 hr

48 hr

0 200 0 200 0 200 (Ph µM)


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Figure 6

Glucose

Phloretin

Phloretin

Glucose

Cell growth arrest Cell growth arrest



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Page 44 of 45

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Page 45 of 45


Table of Contacts Table of Contacts 338x190mm (96 x 96 DPI)


Apple Polyphenol Phloretin Inhibits Colorectal ... - ACS Publications

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