Received date: October 25, 2012; Accepted date: November 20, 2012; Published date: 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.
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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.
Polyphenols; Biphasic; Proliferation; Apoptosis; Cell cycle; Gene expression
GSSG: Oxidized glutathione; GSH: Reduced glutathione; ROS: Reactive Oxygen Species
Dietary consumption of fruits is associated with a lower incidence of chronic and degenerative disorders . 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 . 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 . 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 . Low concentration of blueberry extract (12.5 μg/ml) was also shown to increase proliferation of murine pancreatic β cells . Similarly, low concentration of resveratrol (10 μM) increased proliferation of a breast carcinoma cell line, T47D , while higher concentration (32 μM) increased cell death in these cells . Low concentrations of cocoa procyanidins (5-10 μg/ml) attenuated 4-hydroxynonenal-induced apoptosis of a neuron-like cell line (PC12) , and 20 μM of caffeoylquinic acid increased cell viability of a neuroblastoma cell line (SH-SY5Y), and reversed the effect of β-amyloid . 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 .
Other studies have indicated that bioavailability of phenolic compounds from fruits is far below the amounts that have been orally ingested . 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 . 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 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 . 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 . 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 . Strawberry extracts from 8 different cultivars reduced HepG2 cell viability with IC50s of 20-40 mg/ ml  and chlorogenic acid, a coffee constituent, decreased viability of a long cancer cell line, A549, with an EC50 of 0.47 mM .
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) .
All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) if not otherwise specified.
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 . 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) 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 . 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).
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 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.
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).
Colorimetric caspase-3 activity
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).
Fluorescence ROS detection
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.
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 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).
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.
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 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.
|Gene name and symbol||Category||Function||Reference|
|Glutathionreductase (GSR)||Regulators of oxidative stress||Recycles oxidized glutathione (GSSG)|||
|Glutathion S-transferase α 1 (GSTA1)||Mitigates electrophiles and products of peroxidation|||
|Nuclear factor (erythroid-drived 2)-like 2 (NFE2L2/ NRF2)||Activates antioxidant genes that contain antioxidant response element (ARE) on their promoters|||
|Catalase (CAT)||Converts hydrogen peroxide to H2O and O2|||
|NAD(P)H dehydrogenase, quinone 1 (NQO1)||Reduces quinones to hydroquinones|||
|Gamma-glutamylcysteinesynthetase (GCLM)||Modifier subunit of glutamate cystein ligase|||
|Glutamate-cysteine ligase, catalytic subunit (GCLC)||Catalytic subunit of glutamate cysteine ligase|||
|DNA methyltransferase 3 α (DNMT3A)||Modifiers of epigenome||Functions in de novo methylation of DNA|||
|DNA methyltransferase 3 β (DNMT3B)||Functions in de novo methylation of DNA|||
|DNA methyltransferase 1 (DNMT1)||Maintains DNA methylation after DNA replication|||
|Sirtuin 1 (SIRT1)||Homolog to the yeast Sir2 that functions as deacetylase|||
|Cyclin-dependent kinase inhibitor 1 A (CDKN 1A/p21 Waf1)||Regulators of cell cycle progression||Inhibits cyclin-dependent kinases|||
|Cell division cycle 23 homolog (CC23)||A component of anaphase-promoting complex|||
|Cyclin D 1 (CCND1)||Involves in G1/S cell cycle transition||[38,56]|
|CDyclin E 1 (CCNE1)||Involves in G1/S cell cycle transition|||
|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|||
Table 1: The list of genes selected to be investigated in response to blueberry polyphenols at 96 hours of exposure (real-time RT-PCR).
Statistical analysis of gene expression
For each sample, the Ct values of target genes were normalized to Ct values of 18Sr RNA to compute ΔCt 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 , 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.
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<0.001). Because of the unexpected, bell-shaped, dose-dependent curve for cell proliferation, the same experiment was performed two more times, with similar results, indicating that the concentration of 25 μg/mL induced the maximal cell proliferation rate among all treatments at 96 h of exposure.
Figure 1: Blueberry polyphenolic enriched extract alters the proliferation of HepG2 cells.
Cells were treated with vehicle or 6.25-100 μg/mL polyphenolic enriched extract for 96 h and absorbance was measured at 490 nm using a proliferation MTS assay. Data are reported as mean ± SEM for n=6 independent experiments (for each time point and each dose, respectively). Values that do not share the same letters are significantly different from each other (p<0.001, by Tukey’s test).
Cell-cycle alterations were dependent on concentrations of polyphenolic extract
In order to validate our initial findings with an independent test, phosphorylated histone H3 labeling (pH3) was used to describe changes in cell proliferation that are specific to the mitotic phase of the cell cycle, as previously described . Again, the concentration of 25 μg/mL phenolic extract induced the highest proliferation rate, expressed as percent pH3 positive cells (1.853 ± 0.023 vs. control 1.460 ± 0.020, p<0.001) while the higher concentrations registered a lower rate of proliferation, which for 100 μg/mL equaled the control values at 96 h exposure (Figure 2).
Figure 2: Blueberry polyphenolic enriched extract increased proliferation of HepG2 cells differentially based on the concentration. Cells were treated with vehicle or polyphenolic extract (6.25-100 μg/ml) for 96 h and then stained for phospho-histoneH3. Nuclei were stained with DAPI. A) Depicts the percent positive cells. Data are presented as mean ± SEM of percent positive cells of 3 independent experiments (b: p< 0.001). Means that do not share letters are significantly different from each other. B) Representative images of immunofluorescent slides.
Based on the preliminary results in cell proliferation, we further hypothesized that the concentration of 25 μg/mL induces the maximal proliferation rate for HepG2 cells. Therefore, in all the subsequent experiments we investigated the differential effects of the smaller concentration range (12.5-50 μg/ml) of polyphenols upon apoptosis, cell cycle, ROS generation, and gene expression at 96 h of exposure.
Low polyphenol concentrations did not alter caspase-3 dependent apoptosis in HepG2 cells
A colorimetric activity assay kit for activated caspase-3 was used to evaluate the effect of blueberry polyphenolic-extract concentrations on apoptosis of HepG2 cells. HepG2 cells were treated with vehicle or 12.5- 50 μg/ml blueberry polyphenols for 96 h and cell extracts were analyzed for caspase-3 activity. Results indicated no change in caspase activity for any treatment group as compared to control (Figure 3). These data indicated that blueberry polyphenols in the range of concentrations used in this study do not alter apoptosis rates of HepG2 cells.
Figure 3: Blueberry polyphenolic enriched-extract did not affect caspase-3 activity in HepG2 cells. Cells were treated with vehicle or 12.5- 50 μg/ml of polyphenolic extract for 96 h and absorbance of cell lysates containing caspase-3 substrate were measured at 405 nm. Absorbance was converted to μM using a caspase-3 standard curve. Samples activities were normalized to their protein contents. Data are reported as the mean ± SEM of 3 independent experiments. Means that share letters are not significantly different from each other (p<0.05).
Differential alterations to cell cycle phases were induced by different concentrations of polyphenols
In order to determine which cell-cycle phases are altered in association with the described outcomes in cell proliferation, HepG2 cells were analyzed by flow-cytometry. Results indicated that all treatments significantly increased the percent of cells in the G0/G1-phase when compared to control (Figures 4A and table 2). The percent of cells in S-phase was significantly decreased only by the 25 μg/mL concentration, which reduced it by 19% as compared to the control group (Figures 4C and table 2). The percent of cells in G2/M-phase was significantly reduced (by 20%) only in the 50 μg/ml treatment group as compared to control whereas all other concentrations of polyphenols did not show any effect (Figures 4B and table 2).
Figure 4: Different concentrations of blueberry polyphenols affected different phases of cell cycle. Cells were treated with vehicle or 12.5-50 μg/ ml of polyphenols for 96 h. DNA was stained with propidium iodide and cell cycle distribution was detected using flow cytometry. A)Indicates the effect on G0/G1-phase (b:p<0.05, c:p<0.001, cb:p<0.001). B) Depicts changes on S-phase (p<0.001). C) Represents the effect on G2/M-phase (P<0.01). Data are reported as the mean ± SEM of 5 independent experiments. Means that do not share letters are significantly different from each other.
|Treatment group||Percent cells in each cell cycle phase|
|Control||59.35 ± 0.60a||12.55 ± 0.45a||28.10 ± 0.59a|
|12.5 µg/mL||61.61 ± 0.33b||12.09 ± 0.56a||26.30 ± 0.72a|
|25 µg/mL||64.29 ± 0.47c||12.73 ± 0.49a||22.98 ± 0.60b|
|50 µg/mL||63.21 ± 0.36bc||10.00 ± 0.31b||26.79 ± 0.18a|
Table 2: Tabular presentation of cell cycle distribution.
Expression of regulators of cell-cycle, oxidative stress, and epigenome related to cell proliferation were differentially regulated by different polyphenolic extract concentrations
Real-time RT-PCR was employed to assess the expression level of 17 genes involved in cellular antioxidant mechanisms, epigenetic modifications, and cell cycle regulation (Table 1) in response to exposure to low blueberry polyphenol concentrations. HepG2 cells were treated with vehicle or 12.5-50 μg/mL blueberry polyphenols for 96 h and their mRNA levels were measured (Figure 5). Treatment with 12.5 μg/mL did not change the transcript levels for any gene while the 25 μg/mL decreased the expression of 4 genes involved in DNA methylation (with 28% for DNMT1, and 22% for DNMT3A) and cell cycle progression (with 37% for CCND1, and with 29% for CDK1A) when compared to the control group (Figures 5A and 5B). The expression of more genes was altered by the treatment with 50 μg/ mL polyphenolic extract: DNMT1 (39% decrease), DNMT3A (33% decrease), and SIRT1 (25% decrease); cell cycle regulators: CCND1 (60% decrease), CDKN1A/P21Waf1 (39% decrease), and PCNA (26% decrease); antioxidant regulators: GSLM (26% decrease) and GSR (23% decrease) when compared to control (Figures 5A-5C).
Figure 5: Different concentrations of polyphenols differentially altered expression of oxidative stress, cell cycle, and epigenome regulators. Cells were treated with vehicle or 12.5-50 μg/ml of polyphenols for 96 h and RNA was extracted. The mRNA levels were measured using real-time PCR as described in materials and methods. Data are reported as the mean ± SEM of relative gene expression normalized to control values of 3 independent experiments, each performed in triplicate. Means that do not share letters are significantly different from each other (FDR< 0.05). A) Represents expression of the genes involved in cell cycle progression; B) Depicts expression of the epigenome modifiers; and C) Illustrates gene expression of the oxidative stress regulators.
Low concentrations of polyphenolic extract did not affect intracellular concentration of ROS
An oxidation-sensitive fluorescent probe was used to investigate whether polyphenols affect intracellular ROS concentration since many dietary polyphenols have been shown to have antioxidant properties . HepG2 cells were treated with vehicle or 12.5-50 μg/ml blueberry polyphenols for 96 h and the cellular ROS concentration was measured. The results indicated no significant changes by any treatment (Figure 6). Thus, it was concluded that intracellular ROS are not affected by the polyphenol concentrations used in our experiments.
Figure 6: Blueberry polyphenols did not affect the intracellular concentration of reactive oxygen species in HepG2 cells. Cells were treated with vehicle or 12.5-50 μg/ml of polyphenols for 96 h and intracellular ROS concentration was measured as described in material and methods. Fluorescence intensities were converted to nM using a specific ROS standard curve. Data are reported as the mean ± SEM, n=8. Means that do not share letters are not significantly different from each other at P ≤ 0.05.
Low concentrations of polyphenolic extract did not affect cellular redox state
Since it has been shown that fruit polyphenols can alter the cellular redox state in vitro at higher concentrations , a luminescent GSH/ GSSG assay was employed to examine this outcome in our system. HepG2 cells were treated with vehicle or 12.5-50 μg/ml blueberry polyphenols for 96 h and their GSH/GSSG status was determined. The results indicated no significant alteration of cellular redox state by any polyphenol concentration (Figure 7).
Figure 7: Blueberry polyphenolic enriched-extract did not affect the intracellular redox balance in HepG2 cells. Cells were treated with vehicle or 12.5-50 μg/ml of polyphenols for 96 h and intracellular concentration of GSH, GSSG and their ratio (GSH/GSSG) were measured as described in Materials and Methods. Data are reported as the mean ± SEM of three independent experiments, each performed in triplicates. Means that do not share letters are not significantly different from each other at P ≤ 0.05.
Plant polyphenols have a broad range of reported beneficial effects from protection of neuronal cells against apoptosis [20,35], to anticancer properties by inhibiting cell proliferation [26,27]. This broad range of outcomes might be due to their concentration-specific effect. Because of potential biphasic effects described for some compounds , it is not always clear about the role that different concentrations might play upon cellular phenotypes. Such a “dose response biphasic effect” has been characterized as low dose stimulatory, with a maximum of 30-60% induction over control level, and high dose-inhibitory effect [14,36]. In cancer research, an exhaustive number of studies have focused on apoptotic effects of higher dose ranges (>200 μg/ml) of plant extracts. In this study we selected the HepG2 hepatocarcinoma cell line, in order to investigate the potential impact of lower wide range of concentrations of blueberry polyphenolic extract (6.25-100 μg/ml) upon cell proliferation, apoptosis, oxidation potential, and gene expression related to cell proliferation, and in order to delineate the molecular basis for this possible concentration-dependent effects of these compounds. This cell line was previously studied in response to higher concentrations of blueberry polyphenolic extracts (0.2-1.6 mg/ ml) .
Since the doubling time of HepG2 cells is approximately 48 h , the 96 h time exposure was selected in order to provide full coverage for all cells to undergo at least one proliferation cycle under the exposure to polyphenolic extract. The results of our MTS assay revealed a nonlinear response in cell proliferation with a maximum of 44% increase over control level at 25 μg/ml, while higher concentrations (up to 100 μg/ml) registered a lower rate of proliferation, similar to that of the control (Figure 1). This result is consistent with the maximum increase induced by low concentrations of natural compounds described by the biphasic dose response phenomenon [14,36]. Thus, we were prompted to investigate in more detail the underlying alterations of cell cycle and apoptosis that could be responsible for the observed outcome.
In order to independently validate the results of the MTS proliferation assay, we used another marker of cell proliferation, phosphorylated histone H3 (pH3), which is specific to late G2-phase of the cell cycle, and its abundance increases during the M-phase (until anaphase) . The pH3 assessment also indicated that the concentration of 25 μg/ml induced maximal proliferation, with a subsequent decrease to control values for higher concentrations (Figure 2). As indicated in figure 3, we did not detect any differences in caspase-dependent apoptosis rates under the various concentrations of polyphenolic extract. Therefore, it was concluded that the changes in proliferation were exclusively the result of higher mitotic rates, with no contribution from differences in apoptosis. Although none of the concentrations we have used decreased the proliferation below the control level or increased apoptosis, previously published data indicated a strong apoptotic activity induced by higher concentrations (0.2-1.6 mg/ml, EC50= 0.563 mg/ml) of blueberry extract in the same cell line, HepG2 .
In order to investigate the alterations in cell-cycle phases, we performed flow-cytometry analysis at 96 h on cells treated with concentrations of polyphenols from 12.5 to 50 μg/ml (Figure 4 and table 2). As indicated in the results section, the percent of cells in G0/G1 registered a continued increase up to the concentration of 25 μg/ml, with no further differences at 50 μg/ml. Interestingly, however, the percent of cells in S-phase was decreased only for the blueberry polyphenolic extract concentration of 25 μg/ml, while no changes were reported for the 50 μg/ml when compared to control. The effect on the S-phase, induced by different concentrations, resembled an inverted image of cell proliferation, suggesting that the maximum cell proliferation at 25 μg/ml could be, in part, the result of a relative decrease in the duration of S-phase. Changes in the percent of cells in G2/M-phase were specific to the highest concentration only (50 μg/ml), which did not reflect our findings when using the pH3 marker. However, it should be pointed out that phosphorylation of histone H3 occurs in the pericentromeric heterochromatin in late G2, and disappears prior to chromosome decondensation in telophase . Therefore, while flow-cytometry gives an assessment of the total percent of cells in G2/M, the pH3 assay only indicates the percent of cells in late G2 and in most of the M-phase sub stages (except telophase), thus being more specific for the M-phase.
Of further interest to us was characterizing the expression of genes that are involved in cell-cycle progression, anti-oxidant metabolism, and epigenetic events associated with cell proliferation (Table 1). At 25 and50 μg/ml, the expression of CCND1 and CDKN1A/P21Waf1 was reduced as compared to control levels (Figure 5A). CCND1 promotes cell cycle progression at G0/G1  by several mechanisms, including activation of cyclin-dependent kinases (CDKs) . Interestingly, while CCND1 over expression in G1 promotes the transition to S-phase, its expression is down-regulated in S-phase in order to allow for DNA synthesis . In our study, no changes in G0/G1 phase were observed between 25 and 50 μg/ml, and because of a possible decrease of the S-phase duration, one may speculate that the decreased expression of CCND1 could occur due to a faster transition of cells through the S-phase. At concentrations of 25 and 50 μg/ml, CDKN1A/P21 Waf1expression was also down regulated, with lowest levels for the highest concentration of polyphenols. Since CDKN1A/P21 Waf1 inhibits cell-cycle progression , it is surprising that its decreased expression was not associated with an increased cell proliferation at 50 μg/ml. This could be explained by the significant decrease in PCNA expression level at 50 μg/ml since this protein regulates the processivity rate of DNA replication during S-phase. The significant decrease in PCNA expression at 50 μg/ml could have reduced DNA replication rate during S-phase, suggesting the activation of a second mechanism that inhibits cell proliferation and, thus, may be responsible for the decreased cell proliferation at higher concentration of polyphenols (50 μg/ml relevant to 25 μg/ml).
The effect of polyphenols on the expression of genes involved in epigenetic modifications was also concentration-specific (Figure 5B). While 25 μg/ml reduced only the expression of two methyltransferases (DNMT1 and DNMT3A), the treatment with 50 μg/ml of polyphenols reduced the expression of SIRT1, a member of the sirtuin family of class III histone deacetylases, as well. Reduced expression of DNA methyltransferases may alter DNA methylation and, hence, expression of genes involved in cell cycle progression or signaling pathways leading to cell survival and growth . Sirtuins regulate many cellular processes, including inflammation, senescence, and cell cycle regulation . SIRT1 has multiple targets including histones, transcription factors, and molecules that modulate energy metabolism, stress response, and cell survival . Collectively, these results indicated that increasing amounts of polyphenols affect expression of more genes, as is evident by the number of genes affected by 50 μg/ ml vs. 25 μg/ml, involved in the regulation of global gene expression and/or energy metabolism, thereby potentially altering cell survival and growth.
We also examined the expression of six genes involved in response to oxidative stress, which may be involved in the pathogenesis of cancer. We explored whether the changes in the observed cell proliferation were correlated to oxidative metabolism. Both antioxidant and prooxidant activity of polyphenols have been reported [45-47] in different systems. In our study, the effect of polyphenols on the expression of these genes was also concentration-dependent. While 12.5 and 25 μg/ml did not have significant effects on the expression of any of these genes, the 50 μg/ml concentration reduced the expression of glutathione reductase (GSR) and gamma-gluytamyl cysteine synthetase (GCLM). The reduction of the expression of these genes by the exposure to 50 μg/ml blueberry polyphenols suggested an alteration of cellular redox status. However, this was not confirmed by the intracellular GHS/GSSG quantification. This discrepancy might be caused by the differences between the sensitivities of detection of the two techniques we have employed. Real-time PCR is a very sensitive assay that accurately and quantitatively measures small changes in gene expression that may result in biological alterations during time, while less sensitive assays such as GSH/GSSG may not be able to detect the small resulting enzymatic activities caused by reduction of the expression of these genes. Thus, based on our relative gene expression data, the possibility of alteration of redox system by 50 μg/ml cannot be entirely excluded. Because polyphenols have been shown to exhibit direct antioxidant activity by scavenging reactive oxygen species or chelating transition metals, independent of the action of detoxifying enzymes, we examined intracellular ROS concentration in response to polyphenol exposure. Our results indicated no significant effect on cellular ROS concentration under the exposure to different polyphenol concentrations. These data indicate that the increased proliferation of HepG2 cells exposed to blueberry polyphenols in the range we have used here is independent of their antioxidant system.
Here we indicated, for the first time to our knowledge, that the exposure of HepG2 cells to lower concentrations of blueberry phenolic extract (6.25-100 μg/ml) for 96 h induced an increase in cell proliferation with a significant peak at 25 μg/ml, with lower proliferation (comparable to the level of control) at concentrations up to 100 μg/ml. The increase was associated with a reduction of the percent of cells in S-phase, and by an increased percent of cells that underwent histone H3 phosphorylation. These changes were not related to alterations in production of ROS or GSH oxidation. Gene expression assessment revealed that several genes related to cell-cycle progression and associated epigenetic machinery were variably altered by specific concentrations of polyphenols. While CCND1 and CDKN1A were under-expressed at 25 and 50 μg/ml, the latter concentration also induced the repression of PCNA and SIRT1, suggesting that a second mechanism responsible for cell-cycle alterations was induced at the higher concentration, potentially offsetting the increased proliferation observed for 25 μg/ml. Taken together, our in vitro data from HepG2 cells support the concept of biphasic dose response effect of blueberry polyphenols. This differential dose response effect of chemical compounds deserves more attention since it is important to specify doses for maximizing their efficacies toward better targeting different phenotypes and avoiding the potential unexpected effects that may be associated with improper doses, and in the context of establishing dietary recommendations . More studies using additional cancer lines are necessary in order to further elucidate the potential mechanism(s) responsible for this differential dose dependent effect of blueberry polyphenolic extracts on cell growth and proliferation since each cancer line has its own characteristics that stem from the molecular abnormalities that may alter its response to various treatments. It is also essential to follow up these studies using in vivo models for liver cancer to determine the proliferative and/or apoptotic concentrations of polyphenols in liver carcinoma.
In conclusion, our study indicated that, in contrast to the higher concentrations usually involved in cancer research, blueberry polyphenols at lower concentrations (specifically 25 μg/ml) exerted an increase in HepG2 cell proliferation. This increase was independent of the oxidative status, but was associated with changes in the expression of genes controlling cell-cycle progression and epigenetic machinery.
This work was supported by funds from USDA-ARS through project nos. 0204- 41510-001-24S and 58-6250-6-003. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.