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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611
4To whom correspondence should be addressed. E-mail: percival{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • phytochemicals • polyphenols • anticancer • apoptosis • potentiation
It has been theorized that cancer risk reduction may be achieved by greater consumption of phytochemical-rich fruits and vegetables. Fresh and processed fruits contain high concentrations of diverse phytochemical compounds such as polyphenols, anthocyanins, flavonols and flavan-3-ols. Suggested anticancer mechanisms include antioxidant, anti-inflammatory and antiproliferative activities, inhibition of bioactive enzymes and induction of detoxification enzymes (1,2).
Of particular interest to this laboratory are the potential health benefits of the muscadine grape (Vitis rotundifolia) compared with other Vitis species (3). One distinctive aspect of this fruit is its high level of ellagic acid. Ellagic acid has been shown to exhibit anticarcinogenic properties such as induction of cell cycle arrest and apoptosis (4), as well as the inhibition of tumor formation and growth in animals (5,6). Muscadine grapes also contain a moderate amount of quercetin. Quercetin is another common flavonoid; like ellagic acid, it affects cell cycle kinetics, proliferation and the induction of apoptosis in cell culture (7,8). Hertog et al. (9) considered quercetin to be the "most important" flavonoid due to its ubiquity and concentration in the diet. Blueberries, red raspberries and strawberries contain both quercetin and ellagic acid (Table 1), but this combination is unique to the muscadine grape species among the other Vitis species. Both of these compounds, although structurally different (Fig. 1), have been shown to have anticancer activity. Evaluating the effects of these compounds individually and in combination may help identify the possible benefits suggested by this grape’s unique composition.
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The objective of this study was to evaluate the interaction between ellagic acid and quercetin on cellular growth variables in the MOLT-4 human leukemia cell line. These interactions were evaluated within a range of physiological concentrations in blood plasma after consumption of foods, as has been determined in studies measuring quercetin levels after consumption of yellow onions (13,14). Bioavailability data for ellagic acid in humans are limited, yet intestinal absorption is assumed to occur on the basis of animal models in which various cellular variables were affected by oral ellagic acid (6,15–17). Because of their structural differences, we hypothesized that there would be a significant interaction on cellular growth variables when the two compounds were combined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture supplies and reagents were obtained from the following sources: RPMI-1640 medium and fetal bovine serum (FBS) , (Gibco BRL, Grand Island, NY); L-glutamine (BioWhittaker, Grand Island, NY); gentamycin, streptomycin, penicillin (Gibco, Walkersville, MD); phycoerythrin-conjugated active caspase-3 antibody apoptosis kit 1 (BD PharMingen, San Diego, CA); quercetin, ellagic acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), camptothecin, trypan blue, ethidium bromide, acridine orange, propidium iodide (Sigma Chemical, St. Louis, MO).
Cell culture.
The MOLT-4 cell line, derived from an acute lymphoblastic leukemia T precursor, was maintained in RPMI-1640 medium containing 10% FBS, 2 mmol/L L-glutamine, 100,000 U/L penicillin, 0.1 g/L streptomycin, 0.25 mg/L fungizone and 0.05g/L gentamycin. Quercetin, ellagic acid and camptothecin were dissolved in DMSO and added to 5 x 108 cells/L with a maximum final DMSO concentration of 2 mL/L. Final concentrations used in the culture were camptothecin at 0.05 µmol/L, ellagic acid at 5 µmol/L or 10 µmol/L (E5 or E10), quercetin at 5 µmol/L or 10 µmol/L (Q5 or Q10), and combinations of flavonoids at 5 µmol/L each (E5Q5) or 10 µmol/L each (E10Q10). Cells were incubated from 12 to 48 h. A control culture containing DMSO at 2 mL/L was included in all assays. Camptothecin (Cam), a potent topoisomerase-I-inhibitor, was used as a positive control for apoptosis. Viability and cell counts were performed by trypan blue dye exclusion in a Neubauer hemacytometer.
Colorimetric MTT (tetrazolium) assay.
Mitochondrial activity. MTT was dissolved in PBS at 5 g/L, sterile-filtered and added 1:1 (v:v) to 100 µL cell suspension and incubated at 37°C for 3.5 h in a 96-well plate. MTT is cleaved by active mitochondria into blue formazan crystals. After incubation, 100 µL 2-propanol containing 0.04 mol/L HCl were added to each well to dissolve the formazan crystals. The concentration of formazan was quantified spectrophotometrically using a test wavelength of 540 nm with a reference wavelength of 630 nm on a UV-max kinetic microplate reader (Molecular Devices, Menlo Park, CA).
Isobolographic analysis.
To determine formally whether quercetin and ellagic acid interact synergistically, we performed an isobolographic analysis and calculation of the combination index (CI) on data derived from the MTT assay after 48 h (18,19). The concentration of compound that caused a 40% effect (ED40) was determined empirically for ellagic acid and quercetin on the basis of a range of concentrations from 1 to 40 µmol/L for ellagic acid and 1 to 10 µmol for quercetin. The isobologram was generated by combining quercetin and ellagic acid at ratios of 1:1 and 1:4. The CI for both combination ratios (1:1 and 1:4) was calculated according to the following equation: CI = [(D)E/(Dx)E] + [(D)Q/(Dx)Q] + [(D)E(D)Q/(Dx)E(Dx)Q], whereas (D)E and (D)Q are the ED40 with the combination of ellagic acid and quercetin and (Dx)E and (Dx)Q are the ED40 of ellagic acid and quercetin as single compounds. CI = 1 represents the conservation isobologram and indicates additive effects. CI values < 1 indicate a more than expected additive effect (synergy).
Cell cycle kinetics.
Treated cells were harvested, washed twice in PBS and fixed in 50% ethanol (v:v in PBS) at -20°C for at least 30 min. Debris and ethanol were removed by underlying samples with FBS and centrifuging at 300 x g for 3min. Cells were treated with 125 µL of 500,000 U/L ribonuclease in 38 mmol/L sodium citrate buffer for 15 min, and stained with propidium iodide (0.05 g/L in 38 mmol/L sodium citrate buffer). Analysis was conducted by flow cytometry at 488 nm excitation and 620 nm emission wavelengths, by fluorescence-activated cell sorting (FACS) analysis on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA).
The resulting histogram consisted of two main peaks (G0/G1- and G2/M-phase DNA) with the S-phase DNA between the two major peaks. Histograms were analyzed after gating out the debris (<3% in all samples) and the percentage of cells in the G0/G1 and G2/M peak established with markers. The S phase was calculated by difference.
Fluorescent microscopy.
To determine whether the reduced viability observed in treated cells was based on apoptosis or necrosis, cells were analyzed qualitatively by fluorescent microscopy. Cells were washed twice, resuspended in PBS and stained with 20 µmol/L ethidium bromide and 12 µmol/L acridine orange. Cells were analyzed by fluorescent microscopy (excitation 470 nm and emission fluorescence 515 nm). Acridine orange penetrates intact membranes and fluoresces green, whereas ethidium bromide penetrates disrupted membranes and fluoresces orange. Cells having small nuclear bodies indicate DNA fragmentation and apoptosis. Green nuclear bodies indicate that the cell is in an early state of apoptosis, with an intact membrane, and orange color indicates that a cell has a disrupted membrane and is in a later stage of apoptosis.
Active caspase-3 assay.
Cells were stained with phycoerythrin-conjugated active caspase-3 antibody according to the manufacturer’s protocol to quantify those cells undergoing apoptosis (BD BioSciences, San Jose, CA). The antibody specifically targets the active form of caspase-3 and does not bind to the pro-caspase form according to the BD PharMingen Technical Data Sheet. Analysis was performed by flow cytometry on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The histograms consisted of two peaks, active caspase-3-negative and -positive cells. Data are expressed as the percentage of active caspase-3-positive cells of total cells.
Statistical analysis.
Statistical analysis was performed with a two way-ANOVA with the JMP-software (SAS Institute, Cary, NC). Differences were deemed significant at P < 0.05 using a Student’s post-hoc t test. The additive effect of the combined compounds was estimated by the determination of calculated values (cE5Q5 and cE10Q10), which were then compared with the actual values obtained in samples treated with a combination of 5 or 10 µmol/L each of ellagic acid and quercetin. The calculated values were determined using the following formulas:
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Experimental effects that were significantly different from the calculated value suggested a synergistic interaction of the two compounds. The observed synergistic interaction was confirmed by isobolographic analysis of data derived from the MTT assay analysis at 48 h.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The DMSO vehicle did not influence any growth variable. In contrast, the 0.05 µmol/L Cam treatment, which was used as a positive control, significantly reduced cell numbers, cell viability, the induction of G0/G1 phase arrest and apoptosis at all time points. Unless presented, data acquired at 12 h of incubation did not differ among the treatments.
Overall, cell growth occurred between 24 and 48 h from the initial count of 5 x 108 cells/L (Table 2). The growth variables of total cell number, viability and mitochondrial activity were all highly correlated. For example, mitochondrial activity at 48 h correlated with viability (r2 = 0.97, P = 0.003) and with cell number (r2 = 0.99, P = 0.002) as well as with the percentage of cells having active caspase-3 (r2 = 0.96, P = 0.05).
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Cell cycle kinetics.
Cell cycle kinetics were examined after 12, 24 and 48 h (Fig. 2). As with the cellular growth variables, ellagic acid had a modest but significant influence on the cell cycle at both concentrations (E5 and E10) compared with the control. Compared with the untreated control cells, ellagic acid treatment at 5 µmol/L induced a minor but significant increase in the G2/M phase after 12, 24 and 48 h, a decrease in the G0/G1 phase at 24 and 48 h and a small significant increase in the S phase after 48 h. At 10 µmol/L, a slight but significant increase in the percentage of cells in the G2/M phase at 12 h, significantly fewer cells in the G2/M phase at 24 h and a profile similar to control cells by 48 h were observed.
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Combining quercetin and ellagic acid (Q + E) resulted in a cell cycle distribution very different from that seen with the single compounds. At 12 h, significantly more cells were in the G0/G1 phase and significantly fewer cells were in the G2/M phase when incubated with 10 µmol/L of each compound. By 24 h, there were significantly fewer cells in G0/G1 phase and significantly more cells in the S phase in cells incubated with the 5 µmol/L combination as well as the 10 µmol/L combination. At 24 h, the cells incubated with the 10 µmol/L Q + E combination were still able to make the transition from G0/G1 to S; however, by 48 h, the peaks were indistinguishable by flow cytometry, due to extensive cell damage. Cam-treated cells were also severely damaged by 48 h, which made it impossible to assess quantitatively the distribution of cell cycle phases in these two samples.
Fluorescent microscopy.
Fluorescent microscopy was employed to qualitatively visualize apoptosis after 48 h of incubation (Fig. 3). Control cells and cells treated with E10 did not show fragmented nuclei. Cells incubated with camptothecin- and quercetin-containing treatments showed fragmented nuclei, with nuclear morphology indicative of early (kidney-shaped nuclei) to late stages of apoptosis (nuclei with fragmented DNA). E10Q10 caused the most apoptosis among quercetin-containing samples. Overall, the observation of apoptotic fragmentation of nuclei indicates that quercetin-containing treatments induce apoptosis in MOLT-4 cells.
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The activity of caspase-3 was assessed at 12 and 24 h as a quantitative counterpart to fluorescent microscopy (Fig. 4). At 12 h, caspase-3 activity did not differ from that at 24 h (data not shown). At 24 h, very few control, untreated cells had any active caspase-3. Ellagic acid at either 5 or 10 µmol/L had no significant effect on caspase-3 activation. Incubation with quercetin significantly increased the percentage of cells with active caspase-3. Both combined treatments (E5Q5 and E10Q10) increased caspase-3-activity significantly (12.8 and 23.8-fold, respectively) relative to the control. The combined treatments were significantly greater than the calculated values (cE5Q5 and cE10Q10). Therefore, the experimental effects of the combinations were greater than what was predicted from the additive calculation, suggesting potentiation or synergy.
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The isobolographic analysis was performed with the MTT assay after 48 h using the ED40 of both polyphenols (Fig. 5). Possible synergistic interaction between quercetin and ellagic acid was examined at concentration ratios of 1:1 and 1:4 (quercetin:ellagic acid). The straight, solid line drawn between the points of data acquired with quercetin alone and ellagic acid alone predicted the ED40 on mitochondrial activity if the combination of the two compounds caused an additive effect. The data that were derived empirically from combinations of quercetin and ellagic acid deviated toward the lower left from the hypotenuse. The CI for the ED40 was calculated as 0.67 and 0.49 for the ratios 1:1 and 1:4, respectively. A CI < 1 indicates a potentiating, synergistic interaction between the compounds.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The phenolic levels chosen for use in cell culture were derived from those that might reasonably be expected in the blood after food consumption. Plasma levels are not very well characterized due to chemical rearrangements that can occur in the intestine such as deglycosylation, sulfation, and methylation. For the purposes of this study, we assumed that the level of quercetin absorption would be 
50% (13), which is 15 mg if it is derived from a large (130 g) serving of onions (21). Assuming 5 L of blood, 10 µmol/L in cell culture would be a physiologic level potentially achievable in the blood. Others (22) have also suggested that micromolar concentrations are physiologically realistic (23).
Specifically, the assays chosen for this study determined the potential of ellagic acid and quercetin to induce cell death and suppress proliferation in leukemia cells. Ellagic acid did not affect cellular growth variables at most time points at these low concentrations. Although some of the changes due to ellagic acid were significant, the effects were minor compared with the changes that occurred when the cells were treated with equimolar concentrations of quercetin. By 48 h, all cell cycle profiles had changed toward that of the control cells, suggesting that the induced cell cycle arrest was transient.
Previous researchers have reported that ellagic acid caused a G0/G1-phase arrest, reduction in proliferation and induction of apoptosis at concentrations from 10-9 to 10-5 mol/L after 48 h in CaSki cells, a cervical epithelial carcinoma cell line (4). In colorectal adenocarcinoma cells, SW-480, ellagic acid induced a cell cycle arrest in the G0/G1 phase and induced apoptosis after 48 h of incubation (24). Another study showed that ellagic acid at 10 µmol/L did not affect DNA-fragmentation in L1210 leukemic cells after 24 h (25). Concentrations and incubation times in these reports are similar to those in this study, but in contrast to these studies, MOLT-4 leukemic cells showed only marginal changes compared with the control. Differences in susceptibility to ellagic acid may be due to differences in the nature of the cell lines. Cell lines derived from peripheral tissues might perceive and respond to compounds differently than would cells derived from the colon.
Quercetin reduced cell proliferation as shown by fewer cells as well as an accumulation of cells in the S and G2/M phase that may be unable to divide. Quercetin-treated cells were also dying as indicated by the reduced viability and decreased MTT activity. The data suggest that much of this cell death was via apoptosis as shown by caspase-3 activation and observations from the fluorescent micrographs at 48 h.
In previous reports, quercetin was shown to cause cell cycle arrest in the G0/G1 phase (26,27), in the G2/M-phase (28,29) or in the S-phase (30). These differences are most likely due to the specific cell type and/or the concentration used. Our data also suggest that cells undergo a transient cell cycle arrest when incubated with quercetin. Cells that undergo growth arrest may sometimes be protected from apoptosis, whereas our data showed apoptosis as well as growth arrest. It is possible that some of the cell population cannot repair polyphenol-induced DNA damage and therefore subsequently die via apoptosis. However, surviving cells of the population may be able to normalize their cell cycle as shown by data acquired at 48 h.
The two compounds together had a greater effect on the cells than could be explained by a simple additive effect. To test this statistically, a calculation of the additive effect was derived from the results of the single compounds in each experiment. This allowed for statistical comparison of total cell number, viability, mitochondrial activity and number of cells with active caspase-3 activity between the experimental and predicted (calculated) values. These additive values were significantly different from the experimental results obtained from the combination of compounds. In every case, the effect of the combined phenolics was significantly more detrimental to the cancer cells than the calculated additive effect. To formally confirm the observed synergistic interaction of quercetin and ellagic acid, an isobolographic analysis was performed. Because the MTT assay was highly correlated with cell counts, viability and caspase-3 activity and was simple to perform, we chose the MTT assay after 48 h of treatment with which to do the isobolographic analysis. The ED40 was chosen as a data point that lies within the linear range of response to the treatments. The synergistic effects also could be shown at ED30 and ED50 (data not shown). The results clearly indicate a synergistic interaction of quercetin and ellagic acid at the ratios 1:1 and 1:4. The CI for both ratios were <1 and the data points for the ED40 at both ratios were outside the 95% confidence interval, thus confirming synergy.
In this discussion, the terms synergy and potentiation are used synonymously. The "more than additive" effects caused by combinations of ellagic acid and quercetin, strictly speaking, should be termed potentiating in cases in which ellagic acid does not show a significant effect by itself, and synergy when both show an effect individually.
The cell cycle profile was vastly different when cells were incubated with two compounds together compared with the profile of the cells incubated with individual compounds. This, coupled with the observations of apoptosis in some of the cell population and of a transient cell cycle arrest, suggests that more than one cellular pathway may be involved. In human breast cancer cells treated with quercetin, a pathway leading to apoptosis and a pathway leading to cell cycle arrest were shown (29). Our data, taken together, lead us to hypothesize that ellagic acid and quercetin possibly work via different pathways, which is not unexpected considering their different chemical structures. The relationship between cell cycle arrest and apoptosis by quercetin and ellagic acid combined, as well as the mechanism by which they interact synergistically, remain to be investigated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the Viticulture Advisory Council, Tallahassee, FL.
3 This is Florida Agricultural Experiment Station Journal Series number R-09555.
5 Abbreviations used: C, control; Cam, camptothecin 0.05 µmol/L; cE10Q10, calculated additive value for E10 and Q10; CI, combination index; (D)E, ED40 of ellagic acid when used in combination with quercetin; (Dx)E, ED40 of ellagic acid when used alone; (D)Q, ED40 of quercetin when used in combination with ellagic acid; (Dx)Q, ED40 of quercetin when used alone; DMSO, dimethylsulfoxide; E10, ellagic acid 10 µmol/L; E10Q10, ellagic acid + quercetin 10 µmol/L each; E5Q5 ellagic acid + quercetin 5 µmol/L each; ED40, concentration of compound(s) causing 40% of the effect; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; G0/G1 phase, cell cycle phases in which cells contain one DNA copy; G2/M phase, cell cycle phase in which cells contain two DNA copies; M phase, mitosis phase; MTT, 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide; Q10, quercetin 10 µmol/L; S phase, cell cycle phase of DNA synthesis.
Manuscript received 10 March 2003. Initial review completed 29 March 2003. Revision accepted 24 May 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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S. M. Poulose, E. D. Harris, and B. S. Patil Citrus Limonoids Induce Apoptosis in Human Neuroblastoma Cells and Have Radical Scavenging Activity J. Nutr., April 1, 2005; 135(4): 870 - 877. [Abstract] [Full Text] [PDF]
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S. U. Mertens-Talcott, J. A. Bomser, C. Romero, S. T. Talcott, and S. S. Percival Ellagic Acid Potentiates the Effect of Quercetin on p21waf1/cip1, p53, and MAP-Kinases without Affecting Intracellular Generation of Reactive Oxygen Species In Vitro J. Nutr., March 1, 2005; 135(3): 609 - 614. [Abstract] [Full Text] [PDF]
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