Abstract
Ellagic acid (EA) and some derivatives have been reported to inhibit cancer cell proliferation, induce cell cycle arrest, and modulate some important cellular processes related to cancer. This study aimed to identify possible structure-activity relationships of EA and some in vivo derivatives in their antiproliferative effect on both human colon cancer and normal cells, and to compare this activity with that of other polyphenols. Our results showed that 4,4′-di-O-methylellagic acid (4,4′-DiOMEA) was the most effective compound in the inhibition of colon cancer cell proliferation. 4,4′-DiOMEA was 13-fold more effective than other compounds of the same family. In addition, 4,4′-DiOMEA was very active against colon cancer cells resistant to the chemotherapeutic agent 5-fluoracil, whereas no effect was observed in nonmalignant colon cells. Moreover, no correlation between antiproliferative and antioxidant activities was found, further supporting that structure differences might result in dissimilar molecular targets involved in their differential effects. Finally, microarray analysis revealed that 4,4′-DiOMEA modulated Wnt signaling, which might be involved in the potential antitumor action of this compound. Our results suggest that structural-activity differences between EA and 4,4′-DiOMEA might constitute the basis for a new strategy in anticancer drug discovery based on these chemical modifications.
Introduction
According to the American Cancer Society, cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells (http://www.cancer.org). Colorectal cancer (CRC) is one of the most common cancers worldwide. According to the European Colorectal Cancer Patient Organization, CRC represents the second most common malignant tumor in Europe, killing 230,000 people each year (http://www.europacolon.com). There are different factors associated with a high risk of developing CRC, such as obesity, physical inactivity, diet high in red or processed meat, alcohol consumption, and long-term smoking. Interestingly, a diet rich in vegetables and fruits was demonstrated to be significantly associated with a reduced risk of developing colon cancer (American Cancer Society, 2011, 2012).
Chemotherapy administration is one of the most important decisions to make in the management of cancer patients (American Cancer Society, 2012). The vast majority of chemotherapeutic treatments produce adverse side effects that habitually persist after a long-term period. The antimetabolite 5-fluorouracil (5-FU) is the most commonly used chemotherapeutic agent in CRC. In addition to its side effects, resistance to 5-FU is relatively frequent and new strategies to overcome resistance are urgently needed to gain effectiveness of the treatment. Consequently, identification of new compounds and structures with anticancer properties with the aim of reducing adverse events and/or useful in overcoming drug resistance is necessary for patients and current health systems.
Nature constitutes an important source of cancer chemopreventive compounds. Indeed, a number of important chemotherapeutic drugs have been obtained from natural sources or derived from natural structures (González-Vallinas et al., 2013a). In fact, chemoprevention through dietary intervention is an emerging option to delay or reduce the mortality of cancer and minimize the adverse effects of chemotherapeutic treatments (Coates et al., 2007; González-Vallinas et al., 2013b). Berries, pomegranates, muscadine grapes, walnuts, almonds, and pecans contain bioactive compounds that are directly related to cancer prevention, including polyphenols with interesting properties modulating cell signaling cascades (Espin et al., 2013). Ellagitannins are a class of hydrolysable tannins that have been reported to exert antioxidant, anti-inflammatory, and antitumorigenic properties and to inhibit angiogenesis and prevent the genomic instability that leads to cancer development (Stoner et al., 2007; Umesalma and Sudhandiran, 2011).
Ellagitannins are found naturally in foods as hexahydroxydiphenoyl-glucose esters, whose hydrolysis releases ellagic acid (EA) that is poorly absorbed in the stomach and small intestine but is highly metabolized by the intestinal microbiota to produce urolithin A (Uro-A; 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one) and urolithin B (Uro-B; 3-hydroxy-6H-dibenzo[b,d]pyran-6-one) (Cerdá et al., 2004; Sharma et al., 2010). These two in vivo metabolites are then conjugated with glucuronic acid and/or methyl ethers and are the main products absorbed and detected in plasma, urine, and prostate tissue (González-Sarrías et al., 2010a; Larrosa et al., 2010a). Whereas conjugated metabolites are more abundant in the systemic circulation, EA derivatives aglycones, including urolithins, can reach relevant concentrations in the colonic mucosa of patients with CRC (Nuñez-Sánchez et al., 2014).
EA exerts antiproliferative and antioxidant properties as described in a multitude of in vitro and in vivo studies and in different cancer cell lines (Narayanan et al., 1999; Seeram et al., 2005; González-Sarrías et al., 2009; Chung et al., 2013; Qiu et al., 2013; Santos et al., 2013; Vanella et al., 2013; Umesalma et al., 2014; Zhang et al., 2014). The antiproliferative action of EA could be mediated for its ability to directly inhibit the DNA binding of certain carcinogens, including nitrosamines (Mandal et al., 1988; Mandal and Stoner, 1990) and polycyclic aromatic hydrocarbons (Teel et al., 1986).
Likewise, urolithins (mainly Uro-A) inhibit colon cancer cell proliferation, induce cell cycle arrest, and modulate some important cellular processes involved in colon cancer development such as the inflammatory process, transformation, hyperproliferation, initiation of carcinogenesis, angiogenesis, and metastasis (Aggarwal and Shishodia, 2006; Larrosa et al., 2010b; Li et al., 2012).Therefore, EA and derivatives (including urolithins) available in natural compounds contribute to colon cancer chemoprevention and might constitute a complementary therapeutic approach for the treatment of colon cancer (González-Sarrías et al., 2009, 2010b).
We compared the antiproliferative properties of different polyphenols, including EA and in vivo derivatives with reported biologic properties, which can exert potential beneficial effects by inhibiting cancer cell growth (Table 1). Because EA has been described as an antioxidant and anti-inflammatory compound (Huang et al., 2012), we also analyzed the potential correlation between this biologic activity and the inhibition of colon cancer cell growth. This study aimed to reveal the structural modifications that result in a potentiation of the activity of this compound in colon cancer prevention, as well as to gain new insights regarding its mechanism of action.
Phenolic compounds and in vivo derivatives included in the study and the main reported properties related to antitumor potential.
Materials and Methods
Phenolic Compounds and Derived Metabolites
EA, resveratrol, homovanillic acid, dihydrocaffeic acid, gallic acid, 4-O-methylgallic acid, and 3-O-methylgallic acid were purchased from Sigma-Aldrich (St. Louis, MO). 3,3′-Di-O-methylellagic acid (3,3′-DiOMEA; 99% purity) and 4,4′-di-O-methylellagic acid (4,4′-DiOMEA; 99% purity) were purchased from Bertin Pharma (Montigny le Bretonneux, France). Uro-A and Uro-B, both with purity higher than 95%, were provided by CEBAS-CSIC (Murcia, Spain).
Cell Culture
SW-620 and HT-29 human colon cancer cells, as well as CCD18Co normal human colon cells, were obtained from American Type Culture Collection (Manassas, VA) and were maintained in their respective culture media at 37°C with 5% CO2 and 95% humidity. SW-620-5FuR corresponds to a cell line derived from SW-620, in which acquired resistance to 5-FU was reached by continuously exposing cells to increasing concentrations of 5-FU up to 150 µM over 15 months, as previously described (González-Vallinas et al., 2013b). SW-620, SW-620-5FuR, and HT-29 were cultured in Dulbecco’s modified Eagle’s medium (Gibco/Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco/Invitrogen), 2 mM glutamine (BioWhittaker; Lonza Group, Basel, Switzerland) and 1% antibiotics/antifungal agents (containing 10,000 U/ml penicillin base, 10,000 µg/ml streptomycin base, and 25,000 ng/ml amphotericin B; Gibco/Invitrogen). CCD18Co cells were cultured in Eagle’s minimal essential medium (American Type Culture Collection) supplemented with 10% fetal bovine serum (Gibco/Invitrogen) and 1% antibiotics/antifungal agents (containing 10,000 U/ml penicillin base, 10,000 µg/ml streptomycin base, and 25,000 ng/ml amphotericin B; Gibco/Invitrogen). Cells were kept subconfluent, and media were changed every day. Stock solutions for each phenolic and derived metabolite were prepared by dissolving the corresponding compound in dimethylsulfoxide (DMSO).The maximum final concentration of DMSO in all culture media was 0.2 µl/ml, including nontreated cells.
Cell Viability Assay
SW-620, SW-620-5FuR, HT-29, and CCD18Co cells were seeded in 24-well plates in the exponential growth phase using 500 µl cell suspension per well at a density between 15 × 103 and 60 × 103 cells. After 24 hours, media were replaced with 500 µl media containing serial concentrations of each polyphenol. After 72 hours of treatment, cells were subjected to an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with 50 µl/well of MTT (5 mg/ml in phosphate-buffered saline) (Sigma-Aldrich). After 3 hours of incubation at 37°C, MTT-containing media were removed and the MTT reduced to purple formazan by living cells was solubilized by the addition of 200 µl/well of DMSO. After 1 hour of incubation, quantities of the formazan product, which are directly related to the number of viable cells, were measured at 560 nm using a scanning spectrophotometer microplate reader (Biochrom Asys UVM 340 Microplate Reader; ISOGEN, De Meern, The Netherlands). At least two independent experiments each performed in triplicate were conducted in each case.
Determination of Antioxidant Capacity
Ferric Reducing Antioxidant Power Assay.
The ferric reducing antioxidant power (FRAP) assay was used for determining the antioxidant capacity. One hundred-fifty microliters of FRAP reagent prepared daily and preincubated at 37°C 10 minutes was mixed with 40 µl of test sample, standards, or methanol (for the reagent blank). The standard curve was constructed using serial dilutions of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX) in DMSO between 0 and 500 µM. The FRAP reagent was prepared from 300 mM sodium acetate buffer (pH 3.6), 20 mM ferric chloride, and 10 mM ferric-2,4,6-trypyridyl-s-triazine (Sigma-Aldrich) made up in 40 mM hydrochloric acid. All three of the above solutions were mixed together in a 10:1:1 (v/v/v) ratio. The absorbance of the reaction mixture was measured spectrophotometrically at 550 nm after incubation at 37°C for 15 minutes. Experiments were repeated three times and all measurements were taken in triplicate. Values were derived from the TROLOX standard curve.
2,2-Diphenyl-1-Picrylhydrazyl Assay.
Scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals is the basis of this antioxidant capacity assay. DPPH was purchased from Sigma-Aldrich, and a working solution at 200 µM as well as dilutions of the assayed polyphenols were prepared in methanol. The standard curve was constructed using serial dilutions of TROLOX in methanol between 0 and 100 µM. Fifty microliters of each concentration of TROLOX and polyphenols was put in each 96-well plate in triplicate and then 200 µl DPPH was added. The absorbance of the reaction mixture was measured at 520 nm after 15 minutes of incubation at room temperature in the dark. Experiments were repeated three times and all measurements were taken in triplicate. Values were derived from the TROLOX standard curve.
RNA Isolation
SW-620 colon cancer cells (1.7 × 105 cells per well) were seeded in six-well plates and maintained under standard culture conditions. After overnight incubation, cells were treated with different concentrations of 4,4′-DiOMEA [0 (nontreated), 5, 20, and 50 µM] with three replicates per test concentration. After 72 hours of treatment, the culture medium was discarded and total RNA was isolated from each plate using the RNeasy Mini Kit (Qiagen, Germantown, MD) following the manufacturer’s instructions. RNA quantity and quality were checked by UV spectroscopy (NanoDrop 2000 Spectrophotometer; Thermo Scientific, Waltham, MA).
The experiment was independently repeated four times in the same conditions and total RNA from each experiment was independently analyzed.
Gene Expression Assays
A comparative microarray gene expression analysis between nontreated (control) and 5 µM 4,4′-DiOMEA–treated SW-620 colon cancer cells for 72 hours was performed at the Genomic Service Facility at the Spanish National Center for Biotechnology (Madrid, Spain). RNA integrity was determined using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and 200 ng total RNA from each sample was reverse transcribed and fluorescently labeled using the one-color Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s protocol. The complementary RNAs were prepared for hybridization in an Agilent SurePrint G3 Human 8 × 60 K (Whole Human Genome Microarray Kit) platform using the one-color gene expression system following the manufacturer’s protocol (Agilent Technologies).
Quantitative Real-Time Polymerase Chain Reaction Analysis
Validation of microarray data was performed using quantitative real-time polymerase chain reaction (PCR) analysis for measuring the transcript levels in the selected group of differentially regulated genes. Total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions, and 1 μg total RNA was reverse transcribed by a High Capacity cDNA Archive Kit (Applied Biosystems) for 2 hours at 37°C. TaqMan assays for gene expression (Applied Biosystems, Foster City, CA), which contain the specific primer and TaqMan probe for each gene, were used. Quantitative PCR was performed in real time and in triplicate on the 7900HT Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase gene expression in each sample was used as an endogenous reference for the relative quantification of transcripts. RQ Manager software (Applied Biosystems) was used for data analysis. To calculate the relative expression of each gene, we applied the 2-∆∆ threshold cycle method as previously described (Ramírez de Molina et al., 2007, 2008).
Statistical Analysis
Dose-response curves of the cell viability assays were analyzed by analysis of variance with Bonferroni and Tukey post hoc tests. Data were presented as means ± S.E.M. of at least two independent experiments each performed in triplicate. Statistical significance was defined as P < 0.05. The statistical analyses were performed with R statistical software (version 2.15, http://www.r-project.org; R Project for Statistical Computing, Vienna, Austria).
Data from microarray analysis were extracted and analyzed with FIESTA viewer software (version 1.0, http://bioinfogp.cnb.csic.es/tools/FIESTA; Centro Nacional de Biotecnología, Madrid, Spain). Statistical significance to determine differences in gene expression between groups (nontreated and 5 µM 4,4′-DiOMEA–treated cells in four independent experiments) was determined by the Limma package (linear models for microarray data) (Smyth, 2004), using a P value <0.05 as the level of significance. We set a minimum change of gene expression (either overexpression or repression) of 2-fold of the control (nontreated cells) to define that a gene is differentially regulated. Differentially expressed genes were classified and used for computational analysis to identify potential functional pathways and networks using Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA) as well as the Gene Ontology, KEGG Pathway, and Gene Set Enrichment Analysis databases. The results are presented as P values of the hypergeometric test and P values of the hypergeometric test adjusted by false discovery rate correction, using <0.05 as the level of significance.
Results
4,4′-DiOMEA Strongly Inhibits Colon Cancer Cell Growth.
We studied the growth inhibitory effect of 11 different phenolic compounds and derived metabolites (Table 1) in a preliminary screening on HT-29 cells based on the MTT assay. Results showed that 6 of the 11 compounds (homovanillic acid, dihydrocaffeic acid, gallic acid, 4-O-methylgallic acid, 3-O-methylgallic acid, and Uro-B) had no effect on cell growth in HT-29 colon cancer cells at the assayed concentrations (1–100 µM) (Table 2).
Sensitivity of different human cell lines to selected compounds
Data are presented as IC50 values (concentration of an inhibitor that is needed for 50% inhibition of cell proliferation at 72 hours), with means ± S.E.M. of at least two independent experiments each performed in triplicate.
By contrast, 5 of 11 the analyzed compounds (EA, 3,3′-DiOMEA, 4,4′-DiOMEA, Uro-A, and resveratrol) displayed antiproliferative activity in the HT-29 colon cancer cell line under these assay conditions (Fig. 1; Table 2).
Effects of different phenolic compounds on colon cancer cell viability. Cell proliferation assay of resveratrol (A), EA (B), Uro-A (C), Uro-B (D), 3,3′-DiOMEA (E), and 4,4′-DiOMEA (F) in a representative experiment of at least two independent assays performed in HT-29 and SW-620 cells.
Interestingly, the effects of EA, 3,3′-DiOMEA, and 4,4′-DiOMEA on colon cancer cell viability were significantly different despite belonging to the same family of polyphenols (Table 2). The structural variation of 4,4′-DiOMEA was related to the highest antiproliferative activity, (IC50 of 7.6 ± 1.5 µM in HT-29 cells), which was 12-fold higher than that of its EA precursor. These results suggest that 4,4′-DiOMEA is the most effective agent against colon cancer cells within the members of the EA family tested, which was confirmed using an additional human colon cancer–derived cell line (SW-620) (Fig. 2; Table 2), in which the growth inhibitory effect of this compound was around 13-fold higher than that exerted by the precursor EA.
4,4′-DiOMEA induces human colon cancer cell growth inhibition. Dose-response curves of the cell viability assays after 72 hours treatment of SW-620 colon cancer cells with increasing concentrations of EA and its derivatives (3,3′-DiOMEA and 4,4′-DiOMEA). Data represent means ± S.E.M. of at least two independent experiments each performed in triplicate. Asterisks indicate statistically different values in treated cells with respect to the control (nontreated cells); **P < 0.01. The double line indicates the ratio of viable cells at time zero.
4,4′-DiOMEA Is an Effective Agent against Colon Cancer Cells Resistant to the Chemotherapeutic Drug 5-FU.
With the aim of determining whether 4,4′-DiOMEA could be helpful in the management of chemoresistance of colon cancer cells, its effect on the proliferation of SW-620 cells resistant to 5-FU (SW-620-5FuR) was evaluated. Cell sensitivity of SW-620 colon cancer cells and SW-620-5FuR cells with acquired resistance to 5-FU treatment was assayed. SW-620-5FuR resistance to 5-FU was previously verified. We observed that the IC50 value of SW-620-5FuR for 5-FU was higher than 5000 µM, whereas the IC50 value of parental SW-620 was 7.1 ± 1.3 µM (Table 2). Our results showed that SW-620-5FuR cells were sensitive to 4,4′-DiOMEA (Table 2), which suggests that this compound might be helpful in treatment strategies aimed at overcoming 5-FU resistance.
Furthermore, to determine the potential specificity of this compound against cancer cells, its antiproliferative action on normal human colon epithelial CCD18Co cells was also determined. Table 2 shows that normal colon cells were not affected under conditions in which cancer cell growth was totally abrogated by 4,4′-DiOMEA. Thus, EA and 3,3′-DiOMEA displayed relatively low sensitivity against colon cancer cells (IC50 > 70 µM) and their growth inhibitory activity was almost 2-fold higher than that for normal cells (IC50 = approximately 40 µM). By contrast, 4,4′-DiOMEA displayed high activity against both HT-29 and SW-620 colon cancer cells (IC50 ≤ 10 µM) under conditions in which normal cells were almost not affected by this compound (IC50 ≥ 55 µM) (Table 2), which further pointed to this EA derivate as a promising agent in colon cancer therapy.
The Antiproliferative Activity of 4,4′-DiOMEA Is Not Related to Its Antioxidant Activity and Might Be Mediated by Wnt Signaling Inhibition.
To evaluate whether the reported antioxidant activity of these compounds was related to their antiproliferative activity against colon cancer cell growth, the antioxidant capacity of EA, 3,3′-DiOMEA, and 4,4′-DiOMEA was determined by the FRAP assay at the same concentration in which antiproliferative activity was observed. Figure 3A shows that EA and 3,3′-DiOMEA exhibited a dose-dependent effect, but 4,4′-DiOMEA did not show antioxidant activity at any assayed concentration.
(A) FRAP assay after addition of EA and its derivatives. (B) Validation of the antioxidant capacity of EA and its derivatives by DPPH assay. Data represent means ± S.E.M. of the equivalent of TROLOX (Eq TROLOX) from three independent experiments, with three replicates per test concentration. Asterisks indicate statistically different values of the equivalent of TROLOX in polyphenol concentrations of 25, 50, and 100 µM with respect to the lowest concentration (10 µM); **P < 0.01.
In fact, the ferric reducing power of EA was higher than that exerted by the other compounds, whereas its antiproliferative activity against colon cancer cells was similar to that of 3,3′-DiOMEA (with almost 100-fold lower antioxidant power) and was more than 10-fold lower than that of 4,4′-DiOMEA (with no antioxidant activity). To validate these results, the antioxidant capacity of these polyphenols was further determined by the DPPH assay. The antioxidant capacity of these three polyphenols was confirmed by this alternative method, showing a dose-dependent effect for EA and lower antioxidant power for 3,3′-DiOMEA, whereas 4,4′-DiOMEA did not show activity at any concentration tested (Fig. 3B).
To gain insight in the molecular mechanisms involved in the antiproliferative activity of 4,4′-DiOMEA, a comparative microarray gene expression analysis between nontreated (control) and 5 µM 4,4′-DiOMEA–treated SW-620 colon cancer cells was performed. Our results showed that only 11 genes were differentially expressed between nontreated and 4,4′-DiOMEA–treated cells; of these genes, 5 were downregulated and 6 were upregulated (Table 3).
Genes differentially expressed after 4,4′-DiOMEA treatment
Microarray data of differentially expressed genes after treatment of human colon cancer SW-620 cells with 5 μM 4,4′-DiOMEA for 72 hours (conditions in which antiproliferative activity is observed). The experiment was repeated four times, each performed in triplicate per test concentration. Genes showing a statistical significant differential expression (P < 0.05) and more than 2-fold absolute change variation are shown. Presented data include fold changes, intensity values (control, logControl; treated, logExperiment), S.E.M. values, and P values for the Limma package (linear models for microarray data).
Gene Ontology analysis of differentially expressed genes was performed to identify the most relevant networks and cellular functions involved in the antiproliferative activity of this molecule (Table 4). This analysis showed that Wnt signaling was the most relevant pathway in cancer modulated by this compound, which could be mediating its antiproliferative effect in colon cancer cells. WNT16 was highlighted as the main differentially expressed gene after 4,4′-DiOMEA treatment. This gene is involved in important processes such as the response to oxidative stress and pathways related to cancer, including both Wnt and Hedgehog signaling pathways (Table 4). The modulation of WNT16 by 4,4′-DiOMEA was validated by quantitative real-time PCR (Fig. 4) in which a dose-dependent downregulation of WNT16 by 4,4′-DiOMEA was observed, showing decreased levels of WNT16 mRNA versus nontreated cells by 36, 50, and 81% after 5, 20, and 50 µM 4,4′-DiOMEA treatment, respectively.
Gene Ontology analysis of differentially expressed genes after 4,4′-DiOMEA treatment
Analysis of biologic processes and pathways significantly altered by 4,4′-DiOMEA in SW-620 colon cancer cells. P value (Hyp) represents the P value of the hypergeometric test used in this analysis. Data were obtained from the Gene Ontology, KEGG pathways, and Gene Set Enrichment Analysis databases.
WNT16 mRNA expression in human SW-620 colon cancer–derived cells treated with different concentrations of 4,4′-DiOMEA. Relative quantification for WNT16 in SW-620 cells treated with 5, 20, and 50 µM 4,4′-DiOMEA in relation to nontreated cells normalized to the endogenous control (glyceraldehyde 3-phosphate dehydrogenase). Four independent experiments were performed (each concentration in triplicate), and all samples were amplified in triplicate. Means ± S.E.M. are shown. Asterisks indicate statistically different values in treated cells (5, 20, and 50 µM) with respect to the control (nontreated cells; 0 µM); **P < 0.01.
Discussion
Ellagitannins constitute a diverse group of polyphenols with known biologic activity (Larrosa et al., 2010a). However, this activity is greatly affected by the low bioavailability of both ellagitannins and their hydrolysis product EA, which is further metabolized to urolithins by the colon microbiota to yield urolithins (Espin et al., 2013). In fact, the occurrence of urolithins, EA, and a number of derivatives, including dimethylellagic acid, was recently described in both normal and malignant colon tissues from patients with CRC after consumption of pomegranate extracts (Nuñez-Sánchez et al., 2014). This study suggested that these metabolites could be the real active molecules involved in the reported biologic effects for ellagitannins and EA, especially those effects related to gastrointestinal pathologies such as CRC (Nuñez-Sánchez et al., 2014). In addition, this study established the basis for the investigation of different EA-derived metabolites, including urolithins, as possible antitumor compounds. In this regard, our study considers EA and in vivo metabolites with the aim of elucidating possible structure-activity relationships that could be involved in the antiproliferative effect of these molecules on colon cancer cells, an approach that could be useful in the design and development of new antitumor agents. Our results showed strong differences within the EA family of compounds regarding effects on colon cancer cell viability, with 4,4′-DiOMEA being the most effective compound and inducing cell growth inhibition in a dose-dependent manner (Fig. 2). To our knowledge, these results are the first to show the potent antiproliferative activity of 4,4-DiOMEA as a promising chemotherapeutic drug and also confirm previous studies regarding the effect of EA and Uro-A on the growth rate of premalignant cells in different types of cancer (Stoner et al., 2007; González-Sarrías et al., 2009; Li et al., 2012; Chung et al., 2013; González-Vallinas et al., 2013b; Qiu et al., 2013; Santos et al., 2013; Vanella et al., 2013; Umesalma et al., 2014; Zhang et al., 2014). In this regard, a recent study reported that Uro-A exerted the highest antiproliferative activity on a panel of colon cancer cell lines, followed by Uro-C, Uro-D, and Uro-B. This study also reported that HT-29 cells were able to partially overcome the effects after 48 hours, which was related to the complete glucuronidation of urolithins that exerted lower anticancer activity (González-Sarrías et al., 2014). Therefore, this supports our results regarding the lack of an antiproliferative effect of Uro-B on HT-29 cells.
It is important to highlight that the differential hydroxyl (–OH) substitution of Uro-A and Uro-B resulted in drastically different antiproliferative activity of these compounds (Fig. 5), suggesting that additional –OH at the 8 position in Uro-A is essential for this biologic activity.
Rank of the order of antiproliferative activity against colon cancer cells of EA and derivatives from lowest to the highest regarding their chemical structures.
In this regard, a previous study also supported the potential role of –OH groups in urolithins in the interaction with breast cancer resistant protein transporter BCRP. This study suggested that the presence of an –OH group at the 8 position, but not at the 3 position, might favor the interaction with BCRP (González-Sarrías et al., 2013). It is important to take into account that urolithins are dibenzopyran-6-one derivatives produced by the opening and decarboxylation of one of the lactone rings of EA and the sequential removal of different hydroxyls. Since Uro-A was more active than both EA and its 3,3′-DiOMEA derivative, this result suggests that the presence of a lactone ring of EA is not relevant for its antiproliferative activity in cancer cells.
Regarding DiOMEA derivatives, these compounds have a methoxy group replacing an alcohol group with respect to EA structure, and this change produces a decrease in the molecular polarity (Cichocki et al., 2008; Paul et al., 2009). The significant increase in the activity of 4,4′-DiOMEA with respect to the 3,3′ derivative further confirmed the important role of –OH groups in these positions, which could establish the basis for structural-based EA drug design with enhanced anticancer activity (Fig. 5). In addition, results showed that CCD18Co normal human colon epithelial cells were more sensitive to EA and 3,3′-DiOMEA treatments than cancer cells. On the contrary, 4,4′-DiOMEA was more selective against colon cancer cells, which further supported its use as a potential cancer chemopreventive agent. It is also important to note that 4,4′-DiOMEA exerted significant antiproliferative activity in SW-620-5FuR cells, a cell line that does not respond to 5-FU treatment, which is one of the most commonly used treatments for CRC. Clinical studies have demonstrated that only 10–15% of patients with advanced CRC respond to administration of 5-FU alone; these response rates modestly increase to near 50% when this drug is combined with other antitumoral agents (Zhang et al., 2008). Consequently, drug resistance represents one of the main problems of current chemotherapy failure. In this regard, anticancer therapies based on the combination of agents targeting different molecules, either within the same signaling pathway or involved in different pathways, may more likely avoid resistance to therapy. Our results indicate that 4,4′-DiOMEA might constitute a promising coadjuvant agent in CRC therapy, although future additional preclinical and clinical experiments will be required.
EA has been reported to exert antioxidant effects (Huang et al., 2012), which could be mediating its anticancer activity. Thus, we studied the potential correlation between the antiproliferative and antioxidant activities for EA and its derivatives 3,3′-DiOMEA and 4,4′-DiOMEA. The results showed that EA exerted the highest antioxidant activity using the FRAP method. The activity of 3,3′-DiOMEA was 100-fold lower than that of EA at the maximum concentration assayed, whereas 4,4′-DiOMEA did not show activity at any concentration tested. These results of antioxidant capacity were further confirmed by using the DPPH assay, an alternative method for determination of antioxidant power. This suggests that the antioxidant activity of these EA derivatives does not seem to be directly related to their antiproliferative effects on colon cancer cell lines; however, additional research is needed, including other related methods such as lipid peroxidation, to further understand the molecular events leading to the potential antitumor action of this compound and the putative involvement of its additional biologic activities. In this sense, microarray analysis revealed that modulation of Wnt signaling might be involved in the antiproliferative action of this compound. This result is in agreement with other studies in which Wnt and Hedgehog pathway regulation by Uro-A and EA has been observed in cancer cells (Anitha et al., 2013; Espin et al., 2013). In this sense, Wnt proteins are reported to be extensively involved in oncogenesis and their expression is regulated by the nuclear factor κ-light-chain-enhancer of activated B cells after DNA damage. Specifically, the overexpression of WNT16 in nearby normal cells was suggested to be responsible for the development of chemotherapy resistance in cancer cells (Sun et al., 2012). The expression of WNT16 in the tumor microenvironment attenuates the cytotoxic effects of chemotherapy in vivo, promoting tumor cell survival and disease progression. This suggests a mechanism by which consecutive cycles of genotoxic chemotherapy might increase drug resistance in subsequent treatment in the tumor microenvironment (Sun et al., 2012), further supporting the potential interest in its downregulation by 4,4′-DiOMEA in colon cancer therapy.
In conclusion, we report here that the most effective compound in colon cancer cell growth inhibition of this family of polyphenols was 4,4′-DiOMEA, which was also effective in colon cancer cells resistant to the chemotherapeutic agent 5-FU and had almost imperceptible activity on normal cells. Our study reveals that the small structural variations of EA conducting to the 4,4′-DiOMEA derivative results in a promising strategy to develop new structural-based EA anticancer drugs for CRC. The antiproliferative activity observed does not seem to be related to the antioxidant power of this compound; rather, it seems to be related to the modulation of Wnt signaling pathways.
Authorship Contributions
Participated in research design: Ramírez de Molina, Vargas, Reglero.
Conducted experiments: Vargas, Molina, Sánchez, Martínez-Romero, González-Vallinas, Martín-Hernández, Gómez de Cedrón.
Contributed new reagents or analytic tools: Dávalos, Calani, Del Rio, González-Sarrías, Espín, Tomás-Barberán.
Performed data analysis: Ramírez de Molina, Vargas, Molina, Martínez-Romero, Martín-Hernández, Del Rio, González-Sarrías, Espín, Tomás-Barberán, Reglero.
Wrote or contributed to the writing of the manuscript: Ramírez de Molina, Vargas, Dávalos, González-Sarrías, Espín, Tomás-Barberán, Reglero.
Footnotes
- Received December 6, 2014.
- Accepted February 27, 2015.
A.R.d.M. and T.V. contributed equally to this work.
This research was supported by the Ministerio de Economía y Competitividad del Gobierno de España [Plan Nacional I+D+i AGL2013-48943-C2-2-R and IPT-2011-1248-060000], Comunidad de Madrid [Grant P2013/ABI-2728 ALIBIRD-CM], and European Union Structural Funds.
Abbreviations
- 3,3′-DiOMEA
- 3,3′-di-O-methylellagic acid
- 4,4′-DiOMEA
- 4,4′-di-O-methylellagic acid
- 5-FU
- 5-fluorouracil
- CRC
- colorectal cancer
- DMSO
- dimethylsulfoxide
- DPPH
- 2,2-diphenyl-1-picrylhydrazyl
- EA
- ellagic acid
- FRAP
- ferric reducing antioxidant power
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PCR
- polymerase chain reaction
- TROLOX
- 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
- Uro-A
- urolithin A, 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one
- Uro-B
- urolithin B, 3-hydroxy-6H-dibenzo[b,d]pyran-6-one
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
