Ellagic Acid Potentiates the Effect of Quercetin on p21waf1/cip1, p53, and MAP-Kinases without Affecting Intracellular Generation of Reactive Oxygen Species In Vitro1,2

1. Susanne U. Mertens-Talcott,
2. Joshua A. Bomser^*,
3. Carlos Romero^†,
4. Stephen T. Talcott, and
5. Susan S. Percival³

+ Author Affiliations

Abstract

Anticarcinogenic effects attributed to polyphenols in fruits may be based on synergistic, additive, or antagonistic interactions of many compounds. In a previous study, it was demonstrated that quercetin and ellagic acid interacted synergistically in the induction of apoptosis in the human leukemia cell line, MOLT-4. To investigate possible cellular mechanisms, this study evaluated whether synergistic effects might be detectable within proapoptotic or antiproliferative signal transduction pathways. We found that quercetin and combinations of quercetin and ellagic acid nonsynergistically increased p53 protein levels. In contrast, ellagic acid potentiated the effects of quercetin for p21^cip1/waf1 protein levels and p53 phosphorylation at serine 15, possibly explaining the synergistic effect observed in apoptosis induction. Phosphorylation of the mitogen-activated protein (MAP) kinases, c-jun N-terminal (JNK)1,2 and p38, was also increased by the combination of ellagic acid and quercetin, whereas quercetin alone induced only p38. We further evaluated whether the generation of reactive oxygen species (ROS) and/or quercetin stability were influenced by interactions of ellagic acid with quercetin. Quercetin increased the generation of ROS, which was neither potentiated nor inhibited by ellagic acid. The stability of intracellular and extracellular quercetin was not influenced by the presence of ellagic acid. In summary, quercetin and ellagic acid combined increase the activation of p53 and p21^cip1/waf1 and the MAP kinases, JNK1,2 and p38, in a more than additive manner, suggesting a mechanism by which quercetin and ellagic acid synergistically induce apoptosis in cancer cells.

• 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 p21^waf1/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 p21^waf1/cip1, we hypothesized that the synergy would be apparent in the p21^waf1/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 p21^cip1/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.

Chemical structure of (A) ellagic acid and (B) quercetin

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MATERIALS AND METHODS

Reagents.

Cell culture supplies and reagents were obtained from the following sources: RPMI-1640 medium and fetal bovine serum (FBS) (Gibco BRL); l-glutamine (BioWhittaker); fungizone, gentamycin, streptomycin, penicillin (Gibco); quercetin, ellagic acid, dimethyl sulfoxide (DMSO), α-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 Aurum™ Total RNA Mini (Bio-Rad); SuperScript ™ 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 × 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 p21^waf1/cip1, p53, and phosphorylated p53 at serine-15, phosphorylated forms of p38 and JNK_1,2, 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 × 10⁶ cells was extracted using the RNA extraction kit Aurum™ Total RNA Mini. RNA was reversed transcribed with the SuperScript ™ III RNase H⁻ reverse transcriptase, according to the manufacturer’s instructions. Real-time PCR was performed in 25 μL total volume containing iTAQ™ 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 p21^waf1/cip1 were sense: 5′-CTGGAGACTCTCAGGGTCGAA-3′, antisense: 5′-GGCGTTTGGAGTG-GTAGAAATCT-3′ probe: 5′-6FAM-ACGGCGGCAGACCAGCATGA-3BHQ_1 (13). Human β-actin was used as an endogenous loading control gene for the normalization of p21^waf1/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 β-actin) and 15 s (20 s for β-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 p21^waf1/cip1 mRNA concentrations to those of the endogenous control β-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 × 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 × 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 × 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 p21^waf1/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

Control samples containing up to 0.5 mL/L DMSO vehicle in RPMI medium did not have an effect on the outcome in any of the experiments.

Western blot analysis of p21^waf1/cip1 protein levels.

Western blot analysis demonstrated a concentration- and time-dependent increase in p21^waf1/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 p21^waf1/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 p21^waf1/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.