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Department of Food Science and Human Nutrition, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611-0370;
* Department of Food Science and Technology, Ohio State University, Columbus, OH 43210-1007; and
College of Veterinary Medicine, Department of Pathobiology, University of Florida, Gainesville, FL 32611-0880
3To 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: • polyphenols • anticancer • synergy • signal transduction
Polyphenols, which are plant-derived antioxidants, occur in fruits and vegetables and have been examined extensively for their antiproliferative and proapoptotic effects in various cancer cell lines (1–4). Specifically, quercetin and ellagic acid (Fig. 1), 2 polyphenols present in muscadine grapes and other small fruits, were shown to exert antiproliferative and proapoptotic effects in several cancer cell lines (5–8). In a previous study, we demonstrated that quercetin and ellagic acid interact synergistically in the induction of apoptosis and reduction of proliferation, but not apparently for cell cycle kinetics in human MOLT-4 leukemia cells (9). Quercetin and ellagic acid, incubated individually with various cancer cell lines, were shown to increase protein levels and mRNA expression of p21waf1/cip1, as the underlying mechanism for apoptosis (5,6,10,11). Moreover, quercetin was shown to induce p53 protein levels and to modulate phosphorylation and activation of extracellular signal-regulated protein kinase (ERK)4 1,2, p38 and c-jun N-terminal (JNK)1,2 mitogen-activated protein (MAP) kinases in different cell lines, again, as possible apoptosis-inducing mechanisms (7,10–12). The effects of ellagic acid on these stress-induced MAP kinases have not been reported, nor has any synergy among polyphenols. Because our previous study showed a concentration-dependent reduction in proliferation and the induction of apoptosis by quercetin, as well as a synergistic interaction of quercetin and ellagic acid for both end-points, the specific objective of this study was to explore the signaling mechanisms that may explain the synergy between quercetin and ellagic acid. In view of the fact that both compounds were shown previously to activate p21waf1/cip1, we hypothesized that the synergy would be apparent in the p21waf1/cip1-pathway in MOLT-4 cells. We investigated the activation of p53 and the MAP kinases, p38 and JNK1,2, by both compounds alone and in combination, to determine whether the synergistic interaction was reflected in these pathways and whether these pathways were involved in the activation of p21cip1/waf1. In addition, the intracellular generation of reactive oxygen species (ROS) and the stability of quercetin were examined as possible underlying redox-based mechanisms for the stimulation of these stress-activated pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-pifithrin, camptothecin, 2,7 dichlorofluorescein diacetate (DCFH-DA) (Sigma Chemical); Western blot reagents and iTAQTM DNA polymerase components for real-time PCR, RNA extraction kit AurumTM Total RNA Mini (Bio-Rad); SuperScript TM III RNase H– reverse transcriptase (Invitrogen); probes and primers for real-time PCR (Integrated DNA Technologies); primary and secondary anti-rabbit antibodies for Western blotting (Cell Signaling Technology); secondary anti-mouse antibodies (Upstate, Cell Signaling Solutions). Cell culture. MOLT-4 cells (American Type Culture Collection), derived from an acute lymphoblastic leukemia T precursor, were maintained in RPMI-1640 medium containing 10% FBS, 2mmol/L L-glutamine, 100,000 U/L penicillin, 0.1 g/L streptomycin, 0.25 mg/L fungizone, and 0.05 g/L gentamycin. Ellagic acid, quercetin, and camptothecin, a potent topoisomerase-I-inhibitor and inducer of apoptosis, were dissolved in DMSO and added to 5 x 108 cells/L with a maximum final DMSO concentration of 2 mL/L. Effects of DMSO alone at 0.5% were tested in all assays.
Western blotting analysis. After the cells were harvested, cellular extractions and Western blotting were performed. In brief, cells were extracted in lysis buffer, diluted in loading buffer, and boiled for 3 min. All buffers were prepared according to the ECL Plus Western Blotting Kit manual (Amersham Bioscience). Equal amounts of protein were loaded onto a 10% SDS polyacrylamide gel and separated electrophoretically at 75 V in a Mini-PROTEAN III system (Bio-Rad). Protein then was blotted to a polyvinylidene fluoride membrane at 15 V overnight. The membrane was blocked and incubated with specific antibodies against p21waf1/cip1, p53, and phosphorylated p53 at serine-15, phosphorylated forms of p38 and JNK1,2, according to the manufacturer’s protocol. After incubation with the horseradish peroxidase–linked secondary antibody, protein signals were developed using the ECL Plus chemiluminescence detection reagents. Membranes were exposed to a Kodak X-OMAT AR film and developed.
Real-time PCR. After the cells were harvested, they were washed twice in PBS. Total RNA of 5 x 106 cells was extracted using the RNA extraction kit AurumTM Total RNA Mini. RNA was reversed transcribed with the SuperScript TM III RNase H– reverse transcriptase, according to the manufacturer’s instructions. Real-time PCR was performed in 25 µL total volume containing iTAQTM DNA polymerase components, 0.1 µmol/L probe, 0.5 µmol/L of each primer, and cDNA of each sample. The primers and probe for p21waf1/cip1 were sense: 5'-CTGGAGACTCTCAGGGTCGAA-3', antisense: 5'-GGCGTTTGGAGTG-GTAGAAATCT-3' probe: 5'-6FAM-ACGGCGGCAGACCAGCATGA-3BHQ_1 (13). Human ß-actin was used as an endogenous loading control gene for the normalization of p21waf1/cip1 mRNA. Primers and probes were: Sense: 5'-AGCCTCGCCTTTGCCGA, antisense: 5'-CTGGTGCCTGGGGCG, probe: 5'-CY5-CCGCCGCCCGTCCACACCCGCC-3BHQ_2 (14). Real-time PCR was performed in a Smart Cycler System II (Cepheid) under the following conditions: 2 min at 95°C followed by 40 cycles of 15 s at 95°C, 30 s at 58°C (60°C for ß-actin) and 15 s (20 s for ß-actin) at 72°C. The amplified products were separated on a 1% agarose gel and stained with ethidium bromide to confirm the length of the amplicon and to exclude the amplification of side products. Relative quantification was performed by normalizing p21waf1/cip1 mRNA concentrations to those of the endogenous control ß-actin.
DCFH-DA oxidation assay. To determine the amount of intracellular ROS induced by quercetin and ellagic acid, a microplate method using DCFH-DA was adapted from Wang and Joseph (15). MOLT-4 cell concentrations were adjusted to 1 x 109 cells/L. Cells were washed twice with PBS and incubated with 10 µmol/L DCFH-DA for 30 min at 37°C to preload cells with DCFH-DA substrate. After cells were washed twice, quercetin, ellagic acid, and combinations of both were added to cells in a 96-well plate. Fluorescence was determined after 20 min of incubation with polyphenols using an F-max spectrofluorometer (Molecular Devices) at 538 nm excitation and 485 nm emission wavelengths.
HPLC analysis of quercetin. To determine quercetin stability in cell culture medium and cytosol, cells and cell culture medium without cells were incubated with quercetin, with and without ellagic acid for 0–6 h. Cell culture medium without cells was acidified 1:1 with 0.5 mol/L HCl in methanol. Cells were pelleted by centrifugation (12,000 x g for 10 min) and the supernatant acidified 1:1 with 0.5 mol/L HCl in methanol. The cytosolic content was extracted with a cell lysis buffer containing 1% Triton X 100 and acidified 1:1 with 0.5 mol/L HCl in methanol. All samples were analyzed immediately by HPLC. Separation of phenolics was performed on a Waters 2695 Alliance HPLC system using a Waters 996 PDA detector, and compounds separated using a Waters Nova-Pak C18 column (300 x 3.9 mm) with a C18 guard column. Mobile phases consisted of water (phase A) and 60% methanol (phase B), both adjusted to pH 2.4 with o-phosphoric acid. A gradient solvent program ran phase B from 0 to 30% in 3 min, 30–50% in 2 min, 50–70% in 5 min, and 70–100% in 2 min, and held for 5 min, all at 1 mL/min according to Talcott and Lee (16). Compounds were identified and quantified using authentic standards of each compound and compared with the retention time and UV spectra for each standard.
Statistical analysis. Data were analyzed by 1-way ANOVA with the JMP-software (SAS Institute). Differences were deemed significant at P < 0.05 using a Tukey-Kramer HSD comparison for all pairs for the quantification of p21waf1/cip1 protein levels, steady-state mRNA levels, and DCF-assay. The calculated values based on the additive effects of the single compounds were determined using the following formula: Calculated Effect[QE] = (Effect[Q] + Effect[E]) –Effect[Control] and were compared with the experimental effects of the same concentrations used for the corresponding combination of quercetin and ellagic acid. Quercetin stability data were analyzed with a 2-way ANOVA with time and the presence or absence of ellagic acid as factors. Differences were considered significant at P < 0.05 using a Tukey-Kramer HSD comparison for all pairs.
The term "more than additive effect" was used when the experimentally observed values were higher than the calculated value, whereas the term "potentiating" describes a more than additive interaction in which one of the compounds does not have an effect by itself.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Western blot analysis of p21waf1/cip1 protein levels.
Western blot analysis demonstrated a concentration- and time-dependent increase in p21waf1/cip1 protein levels after 1, 6, and 10 h of incubation for quercetin at concentrations 
5 µmol/L; (data for 6 and 10 h shown in Fig. 2A). Ellagic acid did not increase p21waf1/cip1 protein levels by itself, but appeared to potentiate the effects of quercetin at both ratios of 1:1 and 1:4 after 1, 6, and 10 h. PMA was used as a positive control for the induction of p21waf1/cip1 protein levels at 0.5 µmol/L. The quantification of protein levels after 10 h (Fig. 2B) demonstrates that the calculated values for the combinations of quercetin and ellagic acid were significantly lower than the corresponding experimental value, which confirms the potentiating effect of ellagic acid on quercetin. This potentiating effect was significant when ellagic acid reached a concentration 20 µmol/L.
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Western blot analysis of p53 protein levels and p53 phosphorylation at Ser 15. The level of p53 protein was increased by quercetin at 20 and 50 µmol/L, but there was no further increase in protein levels with the addition of ellagic acid to quercetin-treated cells. (Fig. 3A). Quercetin increased the phosphorylation of p53 in a concentration-dependent manner after 10 h, whereas ellagic acid did not increase phosphorylation at any concentration (Fig. 3B). The phosphorylation of p53 was synergistically increased by quercetin and ellagic acid (Fig. 3B) similar to the changes seen in p21waf1/cip1 protein levels (Fig. 2A, 2B, and Fig. 3C).
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-pifithrin was used to determine the role of p53 in the activation of p21waf1/cip1 (Fig. 3C). In treatments with 20 µmol/L quercetin alone or in combination with ellagic acid at 20 µmol/L, the inhibitor prevented the increase in the expression of p21waf1/cip1 compared with control cells after 6 h. p53 phosphorylation was also decreased in treatments containing the inhibitor. These data show that phosphorylated p53 is involved in the activation of p21waf1/cip1. Western blot analysis of JNK1,2 and p38 phosphorylation. The phosphorylation of JNK1,2 and p38 was determined after 6 h for quercetin, ellagic acid, and the 1:1 combination at a 20 µmol/L concentration (Fig. 3D). Ellagic acid did not increase phosphorylation of JNK1,2 or p38. Quercetin induced an increase in p38 phosphorylation, but not JNK1,2 phosphorylation, whereas the combination of polyphenols increased phosphorylation of both MAP kinases above that elicited by quercetin alone. Because the combination of polyphenols was the only way to activate the JNK1,2 pathway in addition to greater p38 phosphorylation, this suggests that both MAP kinases may be involved in the potentiating effects of ellagic acid on quercetin in the induction of p21waf1/cip1 expression and p53 activation.
Intracellular ROS. The DCF-assay was performed to determine whether the potentiating influence of ellagic acid on quercetin in intracellular signal transduction was due to the generation of intracellular ROS. Quercetin induced a concentration-dependent increase in the generation of ROS at concentrations of 20 and 50 µmol/L, whereas treatment with ellagic acid did not induce an increase in ROS compared with the control (Fig. 4). The combination of ellagic acid and quercetin had no further potentiating effect. Only the combination of quercetin and ellagic acid at 30 µmol/L each induced a response significantly higher than the control. The induction of ROS by quercetin may be involved in the onset of apoptosis in MOLT-4 cells among other mechanisms; however, ellagic acid neither contributes to nor inhibits ROS generation due to the presence of quercetin.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In other studies, quercetin was shown to induce apoptosis through the induction of p53, p21waf1/cip1, and MAP kinases in several cell culture models (7,10,11,17,18). In liver cells, quercetin induced an increase of p21waf1/cip1 protein levels and mRNA within 2 h, whereas p53 protein levels and mRNA were not affected during the same time period. Phosphorylation of p53 was not investigated in that study (10). In endometrial cancer cells, quercetin did not induce protein levels of p53 or p21waf1/cip1 (19). In human aortic smooth muscle cells, quercetin increased p21waf1/cip1 but not p53 protein levels (20). Quercetin increased p21waf1/cip1 mRNA levels in osteoblastic rat cells and protein levels in human epidermoid carcinoma A431 cells (11,17). In lung carcinoma cells, quercetin induced phosphorylation of the MAP kinases, JNK1,2 and p38 (7). In our MOLT-4 cells, quercetin increased p21waf1/cip1 protein levels, mRNA levels, and p53 phosphorylation at serine 15 in a time- and concentration-dependent manner, without increasing p53 protein levels. Quercetin induced p38 but not JNK1,2.
Ellagic acid was shown to induce p21waf1/cip1 and p53 in colon carcinoma cells (5) and cervical carcinoma cells at 100 µmol/L (6). We did not show an effect of ellagic acid on p21waf1/cip1 or p53, which may be due to the low levels that we used, i.e., a more physiological level. In our study, ellagic acid potentiated the effects of quercetin on p21waf1/cip1 protein and mRNA levels and on p53 phosphorylation when added at this lower concentration. Ellagic acid enhanced quercetin’s effect on p38, whereas both compounds were necessary for JNK1,2 phosphorylation. The potentiating effects of ellagic acid on the signal transduction–inducing activity of quercetin was not demonstrated previously.
This effect of ellagic acid and quercetin on the MAP kinases suggests a possible mechanism for the synergistic induction of apoptosis. It appears from these studies that quercetin can induce apoptosis when the p38 pathway is activated, but JNK1,2 is not required for induction of apoptosis in the presence of quercetin. In the presence of both quercetin and ellagic acid, both the p38 and JNK1,2 pathways are activated, and p53 phosphorylation is subsequently more than additively increased, resulting in induction of p21 levels to a more than additive extent, which finally culminates in a more than additive effect on apoptosis.
ROS. In this study, the addition of quercetin to cells in culture was shown to result in the generation of intracellular ROS. Other studies also reported that quercetin induced the generation of ROS in HL-60 leukemia cells (21), and in human lymphocytes in a concentration-dependent manner (22). In our study, the generation of intracellular ROS by quercetin in a concentration-dependent manner was not altered by the addition of ellagic acid. Therefore, ROS are not responsible for the synergy observed between ellagic acid and quercetin.
HPLC analysis of quercetin. In this study, it was demonstrated that quercetin is unstable in cell culture medium with or without cells and also intracellularly over a 6-h period. Quercetin was shown in other studies to be unstable under cell culture conditions and may be oxidized to quinones and other oxidative metabolites that could potentially affect MAP kinase activity (21,23). Ellagic acid apparently did not have an influence on the stability of quercetin under the given conditions. We conclude that the potentiation demonstrated in the presence of both compounds is not due to stabilization of quercetin and a prolongation of quercetin’s effect. This is not to say that oxidative degradation of quercetin leading to the generation of ROS could have been a potential contributor to the proapoptotic effects (21); however, we can rule out that ellagic acid potentiates quercetin’s effects via stabilization of quercetin.
In conclusion, this study showed that ellagic acid potentiates the effect of quercetin on p21waf1/cip1 protein levels, p53 phosphorylation, and to a lesser extent, on p21waf1/cip1 mRNA levels. The activation of p21waf1/cip1 by quercetin and quercetin/ellagic acid combination was p53 dependent. The potentiating effects of ellagic acid on quercetin also are apparent as a nonsignificant trend (P = 0.069) in the upregulation of p21waf1/cip1 mRNA. The activation of p53 through phosphorylation by the combination of ellagic acid and quercetin coincides with the activation of both p38 and JNK1,2. (Fig. 6). Possibly, the activation of JNK1,2 and p38 may be involved in the synergistic activation of p53 by both quercetin and ellagic acid, resulting in synergistic expression of p21waf1/cip1 and finally, apoptosis through the activation of caspase-3 as previously described (9).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the Viticulture Advisory Council, Tallahassee, FL, Florida Department of Agriculture and Consumer Services and by the U.S. Department of Agriculture CREES NRI.
4 Abbreviations used: DCF, 2',6'-dichlorofluorescein; DCFH-DA, 2',7'-dichlorofluorescein diacetate; DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; HSD, honestly significant difference; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinases; PMA, phorbol 12-myristate13-acetate; ROS, reactive oxygen species.
Manuscript received 9 September 2004. Initial review completed 12 October 2004. Revision accepted 28 December 2004.
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0.05. Values are means ± SEM, n = 3.
