Nutritional Disorders & Therapy
Shafiee-Kermani et al., J Nutr Disorders Ther 2013, 3:1 http://dx.doi.org/10.4172/2161-0509.1000120
Research Article
Open Access
Lower Concentrations of Blueberry Polyphenolic-Rich Extract Differentially Alter HepG2 Cell Proliferation and Expression of Genes Related to Cell-Cycle, Oxidation and Epigenetic Machinery Farideh Shafiee-Kermani1, Michael A Grusak2, Sally J Gustafson1, Mary Ann Lila3 and Mihai D Niculescu4* USDA-ARS at North Carolina Research Campus, Kannapolis, NC, USA USDA-ARS Children’s Nutrition Research Center, Houston, TX, USA 3 Plants for Human Health Institute, North Carolina State University Kannapolis, NC, USA 4 Department of Nutrition, University of North Carolina at Chapel Hill, and UNC Nutrition Research Institute, Kannapolis, NC, USA 1 2
Abstract In vitro cancer models have been used to study the effect of relatively high concentrations (>200 µg/ml) of phenolic plant extracts upon cell proliferation. In this study we report that the treatment of human hepatocarcinoma, HepG2, cells with lower concentrations of blueberry phenolic extract (6.5-100 µg/mL) for 96 h induced a non-linear response in cell proliferation, with a significant peak at 25 µg/mL and lower proliferation observed at higher concentrations, while no differences in apoptosis were present across groups. Flow cytometry analysis indicated a reduction of almost 19% of cells in S-phase for 25 µg/mL, as compared to control, while, no changes were observed for other concentrations. The percent of cells in G2/M phase was reduced at 50 µg/ml, while all other concentrations increased the percent of cells in G0/G1 phase. Gene expression analysis revealed concentration-specific changes for several genes involved in cell-cycle regulation (cyclin D1, cyclin-dependent kinase inhibitor 1A, and proliferating cell nuclear antigen, PCNA), antioxidant metabolism (glutamate-cysteine ligase catalytic subunit and glutathione reductase), and epigenetic machinery related to cell-cycle progression (DNA-methyltransferase 1, DNA-methyltransferase 3a, and Sirtuin 1). Neither the generation of reactive oxygen species (ROS) nor the intracellular redox status was affected by any treatment. Taken together, these data indicated that lower concentrations of blueberry phenolic extracts induce differential effects upon cell proliferation and the expression of genes involved in cell-cycle progression and epigenetic machinery in HepG2 cells. These findings provide insight into the molecular mechanisms associated with concentration-specific alterations induced by blueberry polyphenols upon cell growth and proliferation in these cells.
Keywords: Polyphenols; Biphasic; Proliferation; Apoptosis; Cell cycle; Gene expression
Abbreviations: GSSG: Oxidized glutathione; GSH: Reduced glutathione; ROS: Reactive Oxygen Species
Introduction Dietary consumption of fruits is associated with a lower incidence of chronic and degenerative disorders [1]. This protective effect has been largely attributed to their phenolic content [2,3]. Clinical, in vitro, and in vivo studies utilizing fruit extracts have demonstrated protective effects against many chronic disorders including cancer, neurodegenerative diseases, atherosclerosis/cardiovascular, obesity, insulin resistance, and bone loss [4-9]. Although the precise mechanisms underlying the onset and progression of these disorders are not completely understood, increasing evidence strongly suggests oxidative stress as a major contributor in their pathogenesis [1,10-12]. One of the important aspects related to the molecular alterations induced by phenolic compounds, which should be considered when discussing their effectiveness against a wide range of metabolic processes, may be their possible biphasic effects [13]. The biphasic dose responses of other natural compounds, characterized by a low dose stimulatory and high dose inhibitory effect, have been reported since 1943 [14,15]. In rats, lower dietary concentrations (2.5-5 mg/kg body weight) of resveratrol, a polyphenol extracted from grape, reduced post-ischemic myocardial infarct size and cardiomyocyte apoptosis [16]. In contrast, higher concentrations (25-50 mg/kg body weight) of resveratrol in the same system increased myocardial infarct size and the number of apoptotic cells [16]. Low concentration of blueberry extract (12.5 µg/ml) was also shown to increase proliferation of murine pancreatic β cells [17]. Similarly, low concentration of resveratrol (10 µM) increased proliferation of a breast carcinoma cell line, T47D [18], while higher concentration (32 µM) increased cell death in these J Nutr Disorders Ther ISSN: 2161-0509 JNDT, an open access journal
cells [19]. Low concentrations of cocoa procyanidins (5-10 µg/ml) attenuated 4-hydroxynonenal-induced apoptosis of a neuron-like cell line (PC12) [20], and 20 µM of caffeoylquinic acid increased cell viability of a neuroblastoma cell line (SH-SY5Y), and reversed the effect of β-amyloid [21]. Finally, lower concentration (10 µM) of capsaicin, a phenolic compound extracted from vanilla bean induced proliferation of LNCaP cells, an androgen-sensitive cancer cell line, while at 200 µM it increased apoptosis in these cells [22]. Other studies have indicated that bioavailability of phenolic compounds from fruits is far below the amounts that have been orally ingested [23]. For instance, Wu et al. fed 690 mg of blueberry anthocyanins to 60-70 year-old women, and found the total urinary excretion during the first 6 h after consumption to be 23.2 µg, which was equivalent to 0.004 % of intake [24]. In cancer research extensive information is available about the inhibitory effects of high concentrations (>200 µg / ml) of plant phenolic extracts on cell growth and proliferation in vitro. For example, chlorogenic acid, a coffee constituent, decreased viability
*Corresponding author: Mihai D Niculescu, Department of Nutrition, University of North Carolina at Chapel Hill, and UNC Nutrition Research Institute, 500 Laureate Way, Kannapolis, NC 28081, USA, Tel: +1 704 250 5029; Fax: +1 704 250 5001; E-mail:
[email protected] Received October 25, 2012; Accepted November 20, 2012; Published November 22, 2012 Citation: Shafiee-Kermani F, Grusak MA, Gustafson SJ, Lila MA, Niculescu MD (2013) Lower Concentrations of Blueberry Polyphenolic-Rich Extract Differentially Alter HepG2 Cell Proliferation and Expression of Genes Related to Cell-Cycle, Oxidation and Epigenetic Machinery. J Nutr Disorders Ther 3:120. doi:10.4172/2161-0509.1000120 Copyright: © 2013 Shafiee-Kermani F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Volume 3 • Issue 1 • 1000120
Citation: Shafiee-Kermani F, Grusak MA, Gustafson SJ, Lila MA, Niculescu MD (2013) Lower Concentrations of Blueberry Polyphenolic-Rich Extract Differentially Alter HepG2 Cell Proliferation and Expression of Genes Related to Cell-Cycle, Oxidation and Epigenetic Machinery. J Nutr Disorders Ther 3:120. doi:10.4172/2161-0509.1000120 Page 2 of 10
of a lung cancer cell line, A549, with an EC50 of 0.47 mM. Anthocyanins and their respective anthocyanin-pyruvic acid adducts (250 µg/ml) extracted from blueberry demonstrated anticancer properties by inhibiting cell proliferation and invasion of breast cancer cell lines MDA-MB-231 and MCF7 [25]. Crude (1-7 mg/ml) and phenolic acid (0.5-3 mg/ml) fractions from muscadine grape decreased viability and increased apoptosis in colon cancer cell lines HT-29, and Caco-2 [26]. Anthocyanins from bog bilberry (a member of the blueberry genus) decreased viability in a hepatocarcinoma cell line (HepG2) with an IC50 of 0.563 mg/ml; in a colon cancer cell line (Caco-2) with an IC50 of 0.390 mg/ml; and in a nonmalignant embryonic murine fibroblast line (3T3-L1) with an IC50 of 0.214 mg/ml [27]. Strawberry extracts from 8 different cultivars reduced HepG2 cell viability with IC50s of 20-40 mg/ ml [28] and chlorogenic acid, a coffee constituent, decreased viability of a long cancer cell line, A549, with an EC50 of 0.47 mM [29]. In contrast, to the best of our knowledge, no information is available about the effects of a lower concentration range of blueberry polyphenolic extracts on cell viability and apoptosis of the above cell lines. To gain benefit from natural plant-based compounds, it is important to fully understand their roles across a wide-range of concentrations and doses in order to maximize their efficacy toward better targeting different cellular phenotypes, and preventing the potential undesirable effect that may be associated with their improper dosage. In order to explore the basis for the molecular mechanisms underlying the possible concentration-specific effect of blueberry polyphenols, this study aimed to examine the effect of lower concentration range of blueberry phenolic extracts (6.25-100 µg/ml) upon cell proliferation, oxidative metabolism, and gene expression related to cell-cycle progression, oxidative metabolism, and the epigenetic machinery, using HepG2 cells as an in vitro model of hepatocarcinoma. We selected this cell line since it was previously shown to be responsive to higher concentrations of blueberry genus extract (0.2-1.6 mg/ml) [27].
Materials and Methods All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) if not otherwise specified.
Blueberry source Individually quick-frozen, whole blueberries (Vaccinium angustifolium Aiton) were obtained from the Wild Blueberry Association of North America (Old Town, ME, USA). The blueberries were a composite of fruits from all major growing sites including Prince Edward Island, Quebec, New Brunswick, Nova Scotia, and Maine. The composite was harvested in the fall 2010, flash-frozen by Cherryfield Foods, Inc., ME USA, at -15°C, and subsequently stored at -80°C.
Preparation of the polyphenolic-rich extracts and stock solutions Extraction of wild blueberry polyphenolic-rich extract, quantification of its phenolic constituents and their effect in vivo were previously described [30]. Briefly, the whole frozen blueberry fruit (1 kg) were blended (Waring, Inc., Torrington, CT, USA) with methanol, acidified with 0.3% TFA (2 L/kg fruit), and filtered first through multiple layers of muslin sheets, and then on Whatman’s filter paper # 4 (Florham Park, NJ, USA) with the aid of suction. The collected hydro-alcoholic extract was evaporated to about 500 ml using a rotary evaporator at a temperature not exceeding 40°C. The obtained aqueous concentrated extract was partitioned against ethyl acetate (4×500 ml) J Nutr Disorders Ther ISSN: 2161-0509 JNDT, an open access journal
to remove lipophilic material. After evaporation of remaining EtOAc, the aqueous layer (500 ml) was loaded on an Amberlite XAD-7 column (30×10 cm) and preconditioned with acidified water (0.3% TFA). The resin was washed thoroughly with acidified water (0.3% TFA, 3 L) to remove free sugars and phenolic acids. The phenolic mixture was then eluted with 1 L of methanol (0.3% TFA), and the eluate was evaporated, and freeze-dried to yield 7.5 g of polyphenolic-rich extract. The polyphenolic-rich extract contained 702.0 ± 19.24 mg/g total phenolics as measured using the Folin-Ciocalteu method [30]. The total anthocyanins content of the extract was 261.2 mg/g as calculated from the sum of total anthocyanin peak areas measured by HPLC (calculated as cyanidin-3-O-glucoside equivalents). Stock solutions were prepared by dissolving the extracts in DMSO (1000x the highest treatment concentration) and kept at -20°C. Prior to use, the stock was diluted in cell culture medium and sterilized using a 0.2 µm Nalgene nylon membrane (NalgeNunc International, NY, USA). Dilutions were made in such a way that for every treatment, the final medium contained 0.1% DMSO. The same amount of DMSO was added to control medium (vehicle).
Cell culture Human hepatocarcinoma HepG2 cells were obtained from ATCC (Manassas, VA, USA). Cells were grown until reaching 80% confluency in 75 cm2 flasks containing minimum essential medium Eagle (ATCC) supplemented with 10% fetal bovine serum (Lonza, Walkersville, MD, USA) and 1% penicillin/streptomycin, referred to as complete medium throughout the paper, under 5% CO2 in a humidified incubator at 37°C.
MTS cell proliferation assay HepG2 cells were cultured in 96 well plates containing 100 µl complete medium at a density of approximately 5×103/well. Cells were allowed to attach for 48 h before treatment. Cells were then treated with the vehicle (control) or different concentrations of blueberry polyphenolic extract (6.5-100 µg/mL) for 96 h. Culture medium containing vehicle or treatments were replaced every 24 h. After 96 h of treatment, 20 µl of MTS assay reagent (Promega Corporation WI, USA) was added to each well and plates were returned to the incubator. After 2 h, absorbance at 490 nm in each well was measured using a multi-mode microplate reader (BioTek Instruments, Inc. Vermont, USA). The readings were adjusted for background.
Immunofluorescence proliferation assay HepG2 cells were cultured in 6 well plates at a density of approximately 100×103/well. Cells were maintained and treated as above. After 96 h of treatment, medium was aspirated and cells were detached by trypsinization. Cells were centrifuged at 130 rcf for 7 min and then resuspended and washed two times in phosphate buffer saline (PBS), and fixed in 4% formaldehyde for 30 min at room temperature. After two washes in PBS, they were stored at 4°C overnight. Cells were then spun onto glass slides (50-70×103/slide) using a Cyto-Tek centrifuge (Sakura Finetek, CA, USA) at 500 rpm for 6 min. Fixed slides were blocked in blocking solution (1X PBS, 0.1% TritonX-100, 5% goat serum) for 2 h at room temperature, followed by incubation in blocking solution containing a polyclonal anti-phospho-histone H3 (pH3 Ser10, 1:2000 dilution, Millipore Corporation, CA, USA) at 4×C with slow shaking over night. After 3 washes in blocking solution, slides were incubated in PBS containing a Cyanine Dye (Cy3)-conjugated goat anti-rabbit IgG (1:2000 dilution, Millipore Corporation) in dark and at room temperature, with slow shaking for 2 h. After 3 washes in blocking solution, slides were incubated in PBS containing 0.1 µg/ml diamino-phenylindole (DAPI) in dark and at room temperature, with
Volume 3 • Issue 1 • 1000120
Citation: Shafiee-Kermani F, Grusak MA, Gustafson SJ, Lila MA, Niculescu MD (2013) Lower Concentrations of Blueberry Polyphenolic-Rich Extract Differentially Alter HepG2 Cell Proliferation and Expression of Genes Related to Cell-Cycle, Oxidation and Epigenetic Machinery. J Nutr Disorders Ther 3:120. doi:10.4172/2161-0509.1000120 Page 3 of 10
slow shaking for 5 minutes. Slides were then washed briefly in PBS and mounted in an aqueous medium (Fluoromount) with glass cover slips. The slides were used for image analysis.
Image analysis The analysis of slides containing immuno-labeled cells was performed using a Zeiss Axion Imager A1 microscope (Carl Zeiss, NY, USA) with plan-Neofluar 10X objectives. A total of 15 equal fields from corners and the middle of each slide were selected and images were saved. Positive cells for phosphor-H3 were counted and expressed as percent number of cells from the total number of cells (DAPI positive), using ImageJ (NIH, available for free at http://rsbweb.nih.gov/ij/index. html).
were consecutively added to all the samples and luminescence was read using a multi-mode microplate reader (Enspire, PerkinElmer, Waltham, MA, USA). Readings were corrected for background and converted to µM concentrations using a glutathione standard curve. GSH/GSSG ratio was calculated based on the recommended conversion formula (one mole of GSSG generates two moles of GSH, according to manufacturer’s protocol).
Flow cytometry
HepG2 cells were cultured in 6 well plates containing complete medium, at a density of 200×103/well. Cells received 4 groups of treatments: vehicle, 12.5, 25, or 50 µg/mL polyphenolic extract, for 96 h. Caspase-3 activity was measured according to manufacturer protocol (Chemicon International, Millipore, Billerica, MA, USA). Briefly, cells were harvested by trypsinization and 1.5×106 cells were centrifuged. The pellet from each well was resuspended in ice-cold lysis buffer and incubated on ice for 10 min. Cells were then centrifuged at 1×105 rcf and the supernatant was used to prepare an assay mixture containing the caspase-3 substrate in a 96 well plate, according to the procedure provided by manufacturer. Samples were incubated in 37°C for 2 h and then their absorbance intensity at 405 nm was recorded using a multimode microplate reader (Enspire, Perkin Elmer,Waltham MA,USA). Readings were adjusted for background and converted to µM activity/h using a caspase-3 standard curve. The activity in each sample was normalized to its protein content using the Bradford assay (Pierce, Rockford, IL, USA).
HepG2 cells were cultured in 25 ml culture flasks containing complete medium at a density of 3×105/flask. Cells were maintained and treated as above. At the end of 96 h of treatment, cells were detached by trypsinization and prepared to single cell suspension by pipetting. Cells were then centrifuged at 130 rcf for 6 min. Each cell pellet was resuspended in 5 mL PBS and then centrifuged at 200 rcf. The pellets were thoroughly resuspended in 0.5 mL PBS. 5 mL 70% ice-cold ethanol was added slowly to the cell suspension. Cells were fixed in ethanol for 2 h at 4°C and then stored at -20°C for few days. Prior to flow-cytometry assessment, the fixed cells were centrifuged at 200 rcf for 6 min and then thoroughly resuspended in 5 mL PBS. The cell concentration was adjusted to 106 cells in 0.5 mL propidium iodide (PI) staining buffer (10 ml of 0.1% TritonX-100 in PBS, 2 mg DNAsefree RNAse, 200 µL of 1 mg/mL PI in distilled water). Stained cells were incubated at room temperature for 30 min and stored in 4°C overnight, protected from light. Flow cytometry assessment of cell-cycle phases was performed at the UNC Flow Cytometry Facility (Chapel Hill, USA) using a 488 nm laser on a Beckman Coulter CyAn analyzer (Brea, California, USA). PI fluorescence was measured with a 613/20 filter. The raw data were analyzed using ModFit Software (Verity Software House, Topsham, ME, USA). Data were expressed as percent cells in each cell-cycle phase. More than 10,000 events were counted for each sample.
Fluorescence ROS detection
Real-time RT-PCR
HepG2 cells were cultured in 96 well collagen-treated, black/clear plates (Becton Dickinson, Bedford, MA, USA) at a density of 3×103 cells/well. Cells received 4 groups of treatments: vehicle, 12.5, 25, or 50 µg/mL phenolic extract. After 96 h of treatment, intracellular ROS concentration was measured according to manufacturer protocol (Cell Biolabs Inc. San Diego, CA, USA). Briefly, the cell culture medium was replaced with 100 µL 1X DCFH-DA (2’, 7’-dichlorodihydrofluorescein diacetate) solution, and plates were incubated at 37°C for 60 min. Cells were then washed three times with PBS and treated again using the same concentrations of blueberry polyphenols for 1 h in 37°C. Fluorescence measurement was performed at 480 nm excitation and 530 nm emission using a multi-mode microplate reader (Enspire, PerkinElmer,Waltham, MA,USA). Readings were adjusted for background and then converted to nM concentrations using a DCF (2’, 7’-dichlorodihydrofluorescein) standard curve.
HepG2 cells were cultured in 6 well plates containing complete medium at a density of 1.5×105/well. Cells received 4 groups of treatments, vehicle, 12.5, 25, or 50 µg/mL polyphenolic extract, for 96 h. After 96 h of treatment medium was removed and cells were harvested by scraping in lysis buffer. Cell lysates were homogenized using QIAshredder (Qiagen,Valencia, CA, U.S.A.). RNA was isolated using RNeasy mini kit (Qiagen, Valencia, CA, USA). As Per manufacturer suggestion, genomic DNA was degraded using an on column DNase I digestion. The RNA concentration was measured using NanoDrop 8000 (Thermo Scientific, Wilmington, DE, USA). First strand cDNA synthesis was performed using the RT2 First Strand kit (SABiosciences, Frederick, MD, U.S.A.) with 500 ng RNA/sample. Gene expression was measured using SYBR green master mix and primers (SABiosciences, Frederick, MD, USA) for an array of 18 selected humans genes: GSR (NM_000637), GSTA1 (NM_145740), NFE2L2 (NM_006164), CAT (NM_001752), NQO1 (NM_000903), GCLM (NM_002061), GCLM (NM_002061), GCLC (NM_001498), DNMT3A (NM_022552), DNMT3B (NM_006892), DNMT1 (NM_001379), SIRT1 (NM_012238), CDKN1A (NM_000389), CDC 23 (NM_004661), CCND1 (NM053056), CCNE1 (NM_001238), CCNB1 (NM_031966), PCNA (NM_182649), and 18SrRNA (X03205) as internal control for normalizing expression of target genes. A complete description of these genes is summarized in table 1. Real-time PCR was performed using an Eppendorf Mastercycler ep realplex machine with silver thermal block. PCR conditions were identical for all genes and consisted of: initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec, annealing and extension at 60°C for 1 min, followed by
Colorimetric caspase-3 activity
Luminescence GSH/GSSG assay HepG2 cells were cultured in 96 well collagen-treated, white/ clear plates (Corning, Corning, NY) at a density of 600-750 cells/well. Cells were maintained and treated as above. After 96 h of treatment, total or oxidized glutathione was assayed according to the procedure provided by the manufacturer (Promega, Madison, WI, USA). Briefly, total glutathione lysis reagent (reducing reagent) was added to half of the samples to reduce GSSG for measuring total glutathion. Oxidized lysis reagent (oxidizing reagent) was added to other half of the samples to block GSH and reduce GSSG to GSH for measuring oxidized glutathione. Luciferin generation and luciferin detection reagents J Nutr Disorders Ther ISSN: 2161-0509 JNDT, an open access journal
Volume 3 • Issue 1 • 1000120
Citation: Shafiee-Kermani F, Grusak MA, Gustafson SJ, Lila MA, Niculescu MD (2013) Lower Concentrations of Blueberry Polyphenolic-Rich Extract Differentially Alter HepG2 Cell Proliferation and Expression of Genes Related to Cell-Cycle, Oxidation and Epigenetic Machinery. J Nutr Disorders Ther 3:120. doi:10.4172/2161-0509.1000120 Page 4 of 10 Gene name and symbol Glutathionreductase (GSR)
Category
Function
Reference
Regulators of oxidative stress Recycles oxidized glutathione (GSSG)
[48]
Glutathion S-transferase α 1 (GSTA1)
Mitigates electrophiles and products of peroxidation
[49]
Nuclear factor (erythroid-drived 2)-like 2 (NFE2L2/ NRF2)
Activates antioxidant genes that contain antioxidant response element (ARE) on their promoters
[50]
Catalase (CAT)
Converts hydrogen peroxide to H2O and O2
[51]
NAD(P)H dehydrogenase, quinone 1 (NQO1)
Reduces quinones to hydroquinones
[52]
Gamma-glutamylcysteinesynthetase (GCLM)
Modifier subunit of glutamate cystein ligase
[53]
Glutamate-cysteine ligase, catalytic subunit (GCLC)
Catalytic subunit of glutamate cysteine ligase
[53]
DNA methyltransferase 3 α (DNMT3A)
Functions in de novo methylation of DNA
[54]
DNA methyltransferase 3 β (DNMT3B)
Functions in de novo methylation of DNA
[54]
DNA methyltransferase 1 (DNMT1)
Maintains DNA methylation after DNA replication
[54]
Sirtuin 1 (SIRT1)
Homolog to the yeast Sir2 that functions as deacetylase
[44]
Inhibits cyclin-dependent kinases
[41]
Cell division cycle 23 homolog (CDC23)
A component of anaphase-promoting complex
[55]
Cyclin D 1 (CCND1)
Involves in G1/S cell cycle transition
[38, 56]
Cyclin E 1 (CCNE1)
Involves in G1/S cell cycle transition
[56]
Cyclin B 1 (CCNB1)
Involves in regulation of G2/M cell cycle transition
[56, 57]
Proliferating cell nuclear antigen (PCNA)
Regulates the processivity of DNA replication in S-phase
[58]
Cyclin-dependent kinase inhibitor 1 A (CDKN 1A/p21 Waf1)
Modifiers of epigenome
Regulators of cell cycle progression
Table 1: The list of genes selected to be investigated in response to blueberry polyphenols at 96 hours of exposure (real-time RT-PCR).
95°C for 15 sec, 60°C for 15 sec, and a final determination of specificity using melting curve (slow temperature increase from 60°C to 95°C for 20 min), and 95°C for 15 sec. Each amplification was performed in triplicate, and threshold values (Ct) were averaged across triplicates for each sample and gene.
Statistical analysis of gene expression For each sample, the Ct values of target genes were normalized to Ct values of 18Sr RNA to compute ∆Ct values. Due to the high number of variables (17 genes across four treatment groups), the statistical analysis was performed adjusting for a False Discovery Rate (FDR) of below 5%, using the SAM method with the MeV software [31], and adjusting for delta values accordingly.
Statistical analysis of experiments other than gene expression Statistical calculations were performed using Prism version 5 (GraphPad software, Inc., San Diego, CA). When more than two means were compared, one-way ANOVA followed by Tukey’s multiple comparison test were used. Data are reported as means ± SEM throughout the paper, and results considered significant for p-values below 0.05.
Results Proliferation of HepG2 cells was differentially affected by different concentrations of polyphenolic extract The MTS assay was used to investigate the potential effect of the wild blueberry polyphenolic extract upon HepG2 cells proliferation at different concentrations (0, 6.25, 12.5, 25, 50, and 100 µg/mL) for 96 h (Figure 1). Results revealed a non-linear response in proliferation with a significant peak at 25 µg/mL when compared to control (1.10 ± 0.024Ab vs. 0.762 ± 0.061Ab, p
