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Bioactive Constituents, Metabolites, and Functions
Pterostilbene inhibits adipocyte conditioned-medium-induced colorectal cancer cell migration through targeting FABP5-related signaling pathway Yu-Hsuan Hsiao, Nien-Chi Chen, Yen-Chun Koh, Kalyanam Nagabhushanam, Chi-Tang Ho, and Min-Hsiung Pan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03997 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019
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Graphic Abstract
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Pterostilbene inhibits adipocyte conditioned-medium-induced colorectal cancer cell migration through targeting FABP5-related signaling pathway
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Yu-Hsuan Hsiao,§# Nien-Chi Chen,§# Yen-Chun Koh§, Nagabhushanam Kalyanam, Υ
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Chi-Tang Ho,† and Min-Hsiung Pan§,£,¶*
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§Institute
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Taiwan
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ΥSabinsa
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†Department
of Food Science, Rutgers University, New Brunswick, NJ 08901, USA
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£Department
of Medical Research, China Medical University Hospital, China Medical
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University, Taichung, Taiwan
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¶Department
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Taiwan
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of Food Sciences and Technology, National Taiwan University, Taipei 106, Corporation, 20 Lake Drive, East Windsor, NJ 08520, USA
of Health and Nutrition Biotechnology, Asia University, Taichung,
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#These
authors contributed equally.
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*Correspondence
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Min-Hsiung Pan
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Institute of Food Science and Technology
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National Taiwan University
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No. 1, Sec. 4, Roosevelt Rd., Taipei, 10617, Taiwan
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Tel: +886 2 33664133; E-mail:
[email protected] 23
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ABSTRACT
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Pterostilbene (PTS) is a phenolic compound with diverse pharmacologic activities.
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However, its potential for inhibiting obesity-related colorectal cancer (CRC) remains
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unclear. Our study evaluated the mechanism of inhibitory effects of PTS on adipocyte
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conditioned-medium (aCM)-induced malignant transformation in HT-29 colorectal
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adenocarcinoma cells. The results demonstrated that PTS could downregulate the
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expression of aCM-induced fatty acid-binding protein 5 (FABP5) and pro-metastasis
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factors such as vascular endothelial growth factor (VEGF), matrix metalloproteinase-2
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(MMP2), MMP9, and extracellular tumor necrosis factor alpha (TNF-α) via inhibiting
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aCM-induced nuclear factor-kappa B (NF-κB), β-catenin, and peroxisome proliferator-
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activated receptor γ (PPAR-γ). Moreover, PTS can suppress aCM-stimulated
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phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), p38 mitogen-activated
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protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-
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terminal kinases 1/2 (JNK 1/2) signaling pathways activation that are upstream of NF-
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B, β-catenin, and PPAR-γ. Therefore, we suggest that PTS could alleviate adiposity-
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induced metastasis in CRC via inhibiting cell migration through downregulating
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FABP5 gene expression.
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Key words: colorectal cancer (CRC), obesity, fatty acid-binding protein 5 (FABP5),
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pterostilbene (PTS), adipocyte conditioned-medium (aCM)
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Abbreviation: aCM, adipocyte conditioned-medium; Akt, protein kinase B; ERK,
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extracellular signal-regulated kinase; CRC, colorectal cancer; FABPs, fatty acid
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binding proteins; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein
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kinase; MMP, matrix metalloproteinase; NF-B, nuclear factor B; PI3K, 2 ACS Paragon Plus Environment
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phosphoinositide 3-kinase; PPAR, peroxisome proliferator-activated receptor; PTS,
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pterostilbene; TNF-, tumor necrosis factor alpha; VEGF, vascular endothelial growth
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factor.
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INTRODUCTION
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Colorectal cancer (CRC) is one of the most common gastrointestinal malignant
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tumors in the world, and the incident cases of CRC ranked number three in 2018.1 CRC
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is also termed a lifestyle disease associated with unhealthy diet, tobacco smoking,
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excess alcohol consumption, physical inactivity, and obesity. It is known that obesity
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and inflammatory bowel disease (IBD) will increase the risk of CRC.2 Worldwide,
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obesity is currently becoming epidemic and the data showed that 2.7 billion adults
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globally are expected to suffer from obesity and overweight by 2025 (World Obesity
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Federation). Raised body mass index (BMI) also increases the risk of cancer of the
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breast, colon, prostate, endometrium, kidney, and gall bladder in both men and women.
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Furthermore, the adipocytes around the cancer cells then provided sufficient energy
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sources when obesity was occurred.3 Being obese, the dynamic situation of adipose
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tissue will be disrupted and the hypertrophic adipocyte stimulates secretion of several
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signaling cytokines, such as insulin, leptin, IL-6, and tumor necrosis factor alpha (TNF-
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α).4 This unique adipose tissue microenvironment not only favors both tumor initiation
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and progression but also promotes epithelial mesenchymal transition (EMT) and
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metastasis.5,6 Understanding the interaction between adipocytes and cancer cells will
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be important for developing prevention and treatment of obesity-associated colon
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cancer.
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The fatty acid-binding protein (FABP) family is a group of proteins with small
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molecular weight around (14-15 kDa) that function to bind with some hydrophobic
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ligands, such as fatty acids and eicosanoids, and are implicated for the fatty acid
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transportation to specific cell organelles. The importance of theirs in fatty acid
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transportation are shown in regulating lipid-mediated transcriptional in nucleus,
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facilitating lipid oxidation in the mitochondrion or peroxisome, mediating lipid
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synthesis or controlling trafficking around the endoplasmic reticulum, and also 4 ACS Paragon Plus Environment
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modulating of enzyme activity and cytoplasmic lipid storage.7,8 The FABP family have
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been identified with tissue-specific distribution: L-FABP/FABP1 found in liver tissue
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but also , I-FABP/FABP2 locating in intestinal, heart specific protein H-FABP/FABP3,
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A-FABP/FABP4/aP2 that mainly found in adipose tissue, epidermal specific protein E-
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FABP/FABP5/mal1, the ileal Il-FABP protein derived from FABP6 gene, and lastly B-
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FABP/FABP7, M-FABP/FABP8, and T-FABP/FABP9 which can be commonly found
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in brain, myelin and testis, respectively.9,10 Among them, FABP5 is a 15 kDa protein
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involved in the uptake and transport of long-chain fatty acids, and its function is to
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activate this downstream signal transduction and intermediate metabolic pathway via
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ligand binding, fatty acid transporting within cytoplasm and nuclear hormone receptor
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interacting.8,11 High expression of FABP5 protein level was reported to be significant
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and positively correlated to the incidence of some metabolic alteration, such as obesity,
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type II diabetes and/or atherogenic dyslipidemia.12 In addition, FABP5, a cancer-
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promoting gene, is expressed in abnormal skin, adipose tissue, intestine, lung, brain,
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stomach, heart, skeletal muscle, endothelial cells, and mammary cells and is
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upregulated in bladder cancer, breast cancer, cervical cancer, oral squamous cell
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carcinoma, prostatic carcinomas, and colorectal cancer.8,10,13 It is well understood that
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involvement of fatty acids is required for structural membrane bilayer formation, and
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acting as cellular energy source and cellular signaling molecules especially during cell
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proliferation. Therefore, the role of FABPs during cancer development is strikingly
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sight catching, may be crucial and needed to be corroborated.
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Pterostilbene (trans-3,5-dimethoxy-4’-hydroxystilbene), a natural polyphenolic
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compound belonging to stilbene phytoalexin and a dimethylated analog of resveratrol,
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was found in two cultivars of Vaccinium ashei and in V. stamineum at levels of 99–520
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ng/g dry sample and has diverse pharmacologic activities including anti-inflammation, 5 ACS Paragon Plus Environment
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antioxidant, antiproliferative, analgesic, anti-obesity, and anticancer activity.14-17 One
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study demonstrated that the bioavailability of pterostilbene was approximately 3- to 4-
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fold higher than the bioavailability of resveratrol.18 Various bioactivity studies of
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pterostilbene have been reported; however, the potential effects of pterostilbene on
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obesity-related CRC have not been studied yet. Hence, in this research, we investigated
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the inhibitory effects of pterostilbene on adiposity-induced CRC cell migration through
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targeting the FABP5-related signaling pathway.
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MATERIALS AND METHODS
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Chemicals and reagents
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Pterostilbene was provided by Sabinsa Corporation (East Windsor, NJ, USA). Being
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analyzed by high-performance liquid chromatography (HPLC), the purity of
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pterostilbene determined as up to 99.2%. For cell culturing, Dulbecco’s modified
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Eagle’s medium (DMEM), fetal bovine serum (FBS), fetal calf serum (FCS), and
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penicillin−streptomycin were purchased from Gibco BRL (Grand Island, NY, USA).
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The β-actin antibody (mouse monoclonal) was purchased from Sigma-Aldrich (USA).
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The β-catenin, Akt, phospho-Akt, NF-κB p65, phospho-NF-κB p65, ERK 1/2,
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phospho-ERK 1/2, JNK, phospho-JNK, MMP2, p38 MAPK, phospho-p38 MAPK,
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PPAR- antibodies were purchased from Cell Signaling Technology (Beverly, MA,
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USA). The FABP5, MMP9, PI3K, phospho-PI3K, TNF- antibodies were obtained
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from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals used were
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in the purest form available commercially.
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Cell culture and adipocyte differentiation
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COLO205, HCT116, SW480 cells were grown in RPMI containing 10% fetal bovine
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serum, 1% penicillin-streptomycin and HT-29 cells were grown in RPMI or DMEM in 6 ACS Paragon Plus Environment
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a humidified atmosphere containing 5% CO2 at 37 ℃. 3T3-L1 mouse preadipocyte was
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purchased from the American Type Culture Collection (Rockville, MD, USA) and were
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cultured in DMEM at the same condition as mentioned while confluence. The
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differentiated of 3T3-L1 preadipocyte was based on our previously study.19
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Adipocyte conditioned-medium (aCM) preparation
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When 3T3-L1 cells were fully differentiated (Day 8), the medium was replaced with
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DMEM without serum. After 24 h, the medium was collected and stored at -20 ℃.
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Oil Red O stain
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The stain method was mentioned in our previous study.19 First, removed the medium
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and washing by PBS for twice, adding 4% formaldehyde overnight for cell fixing.
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Second, removed formaldehyde and PBS washing, adding 200 μL Oil Red O at room
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temperature for 5 min to stained cell. After incubated, cell was washed by PBS for twice
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then moved to a microscopy and photographed by a digital camera at 100×
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magnifications. A semi-quantification could be decided by adding 100 μL isopropanol
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to elute Oil Red O then read the absorbance at 510 nm.
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MTT assay
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Approximately 2 x 105 cells were plated in each well of a 96-well plate containing 200
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μL medium and were cultured at 37 °C under a humidified 5% CO2 atmosphere for 24
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h. Then, the medium was replaced by 25% aCM without serum and different
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concentrations of PTS. After 24 and 48 h, the medium was removed and the cells were
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incubated with 0.5 mg/mL, 100 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide) solution for 50 min. Metabolic activity was quantified
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by measuring the absorbance at 570 nm by an ELISA Reader. The percentage was 7 ACS Paragon Plus Environment
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calculated as Sample OD570nm/ Control OD570 nm.
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Trypan blue assay
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Approximately 2 x 105 cells were seeded in each well of a 24-well plate containing 1
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mL medium and were cultured at 37 °C under a humidified 5% CO2 atmosphere for 24
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h. At 24 h, the medium was replaced by 25% aCM without serum and PTS at doses of
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5 and 20 μM. Cells were collected 24 hours later followed by centrifugation at 1,200 g
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for 10 min. The cells were counted under a microscope after being stained by trypan
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blue dye.
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Preparation of proteins and Western blot
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Extraction of protein then analysis by Western blot were presented in our previous
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study.19 Scape off adherent cells then suspension in gold lysis buffer on ice for 1h, then
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centrifuge at 16,500 g for 1 h at 4 °C, collect the supernatant and determined the protein
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concentrations by using the Bradford assay.20 Loading 50 μg of each protein sample
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into sodium dodecyl sulfate (SDS)−polyacrylamide gel for electrophoresis, and it is
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followed by protein transfer to PVDF membranes. PVDF membranes were soaked in
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blocking solution for 1h before being incubated in primary antibody at 4 °C for 12 h,
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afterward incubated with conjugated secondary antibody in blocking solution at room
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temperature for 1 h. Last, enhanced-chemiluminescence was used to obtain image,
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images are imprinted on the film due to varying amounts of ambient light that reach the
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film surface. The density of the protein bands was quantified using ImageJ imaging
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software.
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Gelatin zymography
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For the detection of proteolytic enzyme activity, gelatin zymography was selected in 8 ACS Paragon Plus Environment
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order to understand its capability to degrade gelatin from different biological source.
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It enables identification of gelatinolytic activity in culture media and cell extracts by
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using SDS-polyacrylamide gels that co-polymerized with gelatin. By electrophoresing
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the samples under non-reducing condition, the enzymatic activity could be maintained.
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Using Triton X-100 to removed SDS then incubate with buffer containing calcium to
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renatured the enzymes activity, protease could able to hydrolyze the gelatin to produce
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a hole. Then stained the gelatin gel by Coomassie Blue, distained by a solution
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containing 8% acetic acid and 5% methanol. After distaining, quantified the band by
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ImageJ to analyzed the activity of MMP2 and MMP9.21 HT-29 cells were incubated in
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DMEM without serum in the presence of 25% aCM with or without PTS (5 and 20 μM)
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for 24 h. The unboiled samples (concentrated supernatant) were measured by using the
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Bradford assay.20 Equal amounts of protein for each sample were separated by 0.1%
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gelatin and 10% SDS–polyacrylamide gel electrophoresis. After electrophoresis, gels
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were soaked by washing buffer containing 4% Triton X-100 twice for 30 min, then
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incubated in another buffer containing 1% Triton X-100, 50 mM Tris–HCl, 5 mM
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CaCl2, and 0.02% NaN3 at 37 ℃ for 18 h. After staining by Coomassie Blue R-250 and
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destained by a solution with 50% methanol and 10% acetic acid, the density of the
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protein bands related to enzyme activities was quantified using ImageJ imaging
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software.6
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Wound healing assay
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The wound healing assay is a standard in vitro technique for probing collective cell
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migration. First, a cell-free area is created by removing the cells from the area through
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physical exclusion, we used 10 μL pipette tip to did this step. Then cells were washed
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twice with PBS to remove floating cells and then added medium without serum. These
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structure and function. After 24 and 48 h, using a magnitude microscope equipped for
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live-cell, the photos of the wound were taken using a 40× then quantified the cell
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migration percentage by ImageJ software.
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Reverse transcription-PCR
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Total RNA was prepared from HT-29 cells using TRIZOL reagent according to the
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supplier’s protocol. One μg of total RNA was added with 4 μL TransAMP Buffer and
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1 μL Reverse Transcriptase. The complementary DNA was synthesized by a
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polymerase chain reaction (PCR) Thermal Cycler under the condition of 25 ℃ for 10
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min, 42 ℃ for 15 min, 85 ℃ for 5 min, and then kept at 4 ℃. The cDNA (1 μL) was
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amplified
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GGTGCATTGGTTCAGCATCAGG-3#
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GTCACCTGTACTCGGATCTATGA-3#
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TGCACCACCAACTGCTTAG-3# (sense) and 5#-GAGGCAGGGATGATGTTC-3#
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(anti-sense). The PCR reaction consisted of 3 μL ddH2O, 5 μL SensiFAST SYBR No-
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ROX Mix, 0.5 μL 10 μM forward primer and reverse primer, and 1 μL cDNA in a total
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volume of 10 μL. Cycling was performed using a Real-Time PCR Detection System: 2
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min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 15 s at 65 °C, and 10 s at 72 °C,
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followed by a final incubation at 95 ℃ for 10 s.
by
PCR
with
the
following
primers:
FABP5
(sense), (anti-sense);
5#5#-
GAPDH
5#-
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Quantification of TNF alpha and IL-6 in mouse serum
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The TNF-α and IL-6 levels of aCM were assayed by means of the Mouse TNF-α ELISA
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Kit (ab100747) and Mouse IL-6 ELISA Kit (ab100712) (Abcam, Cambridge, UK). Ten
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mL of aCM were centrifuged as 200 μL prior to testing.
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Statistical analysis 10 ACS Paragon Plus Environment
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Data were expressed as the means ± SD for the indicated number of independently
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performed experiments. The statistical evaluation was performed by one-way analysis
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of variance (ANOVA) followed by Tukey’s HSD tests. A probability value of p < 0.05
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was considered statistically significant.
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RESULTS
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FABP5 expression in colon cancer cell lines
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First, to choose a suitable CRC cancer cell line, the FABP5 basal level of HT-29,
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SW480, COLO205, and HCT115 was screened by western blot analysis. Results
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demonstrated that FABP5 is expressed in all of these four CRC cancer lines (Fig. 1A).
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Among them, FABP5 expression is markedly upregulated in HCT116 compared to HT-
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29 (Fig. 1B). The model of this research is under the induction of conditioned-medium
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from normal 3T3-L1 adipocytes (Fig. 1C) to mimic the obese microenvironment. Along
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with the development of CRC, adipocytes will interact with cancer cells and can be
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reprogrammed to produce a cancer-associated adipocyte. Therefore, we chose HT-29,
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a Grade 2 colorectal adenocarcinoma, to do the following analysis.
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The inhibitory effect of PTS on HT-29 viability and migration induce by adipocyte
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conditioned-medium (aCM)
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As illustrated in Figure 2, the cytotoxicity of aCM and PTS was evaluated by MTT
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assay, Trypan blue assay, and microscopy examination. However, the result of the MTT
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assay demonstrated that 20 μM PTS inhibited the metabolic activity of HT-29 to 80.2
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± 5.9% (Fig. 2A). To ensure that this inhibition was caught by suppression of growth
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or death, we used the Trypan blue assay to evaluate the viability of HT-29 and examine
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the morphology of HT-29 by microscopy. As the results illustrated, there were no
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cytotoxic effects of PTS at 20 μM in HT-29 statistically (Fig. 2B), and no change was
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found in terms of the morphology of HT-29 (Fig. 2C). We performed a wound-healing 11 ACS Paragon Plus Environment
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experiment to determine the inhibitory effect of PTS on migration ability of colon
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cancer cell induced by aCM. As shown in Figure 2D, migration of HT-29 cells was
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significantly increased after being incubated with 25% aCM for 48 h and it was
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successfully inhibited by treatment with 20 μM PTS (Fig. 2D). Quantitative data
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derived from three independent experiments demonstrated that PTS effectively (p
