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© 2007 American Society for Nutrition J. Nutr. 137:2622-2628, December 2007

Biochemical, Molecular, and Genetic Mechanisms

Annurca Apple Polyphenols Have Potent Demethylating Activity and Can Reactivate Silenced Tumor Suppressor Genes in Colorectal Cancer Cells1,2

Lucia Fini3,4, Michael Selgrad3, Vincenzo Fogliano5, Giulia Graziani5, Marco Romano6, Erin Hotchkiss3, Yahya A. Daoud7, Edward B. De Vol7, C. Richard Boland3,* and Luigi Ricciardiello3,*

3 GI Cancer Research Laboratory, Department of Internal Medicine, Sammons Cancer Center, and Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75246; 4 Internal Medicine Department, Catholic University of Sacred Heart Gemelli Hospital, 00168 Rome, Italy; 5 Department of Nutrition Sciences, University of Naples, 80138 Naples, Italy; 6 Division of Gastroenterology, Second University of Naples, 80138 Naples, Italy; and 7 Institute for Health Care Research and Improvement, Baylor Health Care System, Dallas, TX 75246

* To whom correspondence should be addressed. E-mail: luigir{at}baylorhealth.edu or rickbo{at}baylorhealth.edu.


   ABSTRACT
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Introduction
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The CpG island methylator phenotype is characterized by DNA hypermethylation in the promoters of tumor suppressor genes with silencing of transcription. Hypermethylation of the promoter of hMLH1 and subsequent microsatellite instability occurs in ~12% of sporadic colorectal cancers (CRC). Annurca apple, a variety of southern Italy, is rich in polyphenols that are associated with anticancer properties. Populations in southern Italy have lower incidences of CRC than elsewhere in the western world. We evaluated the mechanisms of putative anticancer effects of Annurca polyphenol extract (APE) in in vitro models of CRC. We extracted polyphenols from Annurca apples and treated RKO, SW48, and SW480 cells with APE and assessed the cell viability, apoptosis, and cell cycle. DNA methylation of selected tumor suppressor genes was evaluated after treatment with APE and was compared with the synthetic demethylating agent 5-aza-2'deoxycytidine (5-aza-2dC). DNA methyltransferase (DNMT)-1 and -3b levels were evaluated. Decreased cell viability and induction of apoptosis was evident after treatment. We found no significant changes in cell cycle dynamics. We observed significant increases of p53 protein expression in RKO after treatment. APE treatment strongly reduced DNA methylation in the promoters of hMLH1, p14ARF, and p16INK4a with consequent restoration of normal expression. These effects were qualitatively comparable with those obtained with 5-aza-2dC. We observed a significant reduction in expression of DNMT proteins after treatment without changes in messenger RNA. In conclusion, APE have potent demethylating activity through the inhibition of DNMT proteins. The lack of toxicity in Annurca extracts makes them excellent candidates for the chemoprevention of CRC.


   Introduction
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 ABSTRACT
 Introduction
Methods
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 LITERATURE CITED
 
Colorectal cancer (CRC)8 is the 4th most common cancer and the 3rd most common cause of cancer death in western countries (1). The Mediterranean area is characterized by a lower incidence of cancers, including CRC (2). Although the overall incidence of CRC in Italy is comparable to that in continental European countries and the United States, a significant geographical north-south gradient exists (3). Marked differences in the nutritional behavior occur across the country, in which the southern diet more fully reflects the Mediterranean model. This is compatible with the long-held belief that variations in nutrition are responsible for preventing cancer in certain geographical regions (4).

The Mediterranean diet, rich in fruits, vegetables, olive oil, and red wine, is associated with a lower incidence of cancer (2,4). Polyphenols are richly represented in many of the foods that form the basis of the Mediterranean diet and have a broad spectrum of properties (antineoplastic, antioxidant, and antiinflammatory), which could play an important role in this scenario (5).

In a recent report, Graziani et al. (6) demonstrated that polyphenols extracted from Annurca apples can prevent exogenous damage in vitro to human gastric epithelial cells and in vivo to rat gastric mucosa. The Annurca apple is a variety with a "Protected Geographical Indication" of the Campania region in southern Italy; these apples are extremely rich in catechin, epicatechin, and chlorogenic acid and display a stronger antioxidant activity compared with other varieties (7).

The development of CRC has been described as a multistep model where the accumulation of genetic and epigenetic events mediate the adenoma-carcinoma sequence (8). The accumulation of mutations is driven through distinct pathways by different types of genomic instability and the best characterized of these are called chromosomal instability (CIN) and microsatellite instability (MSI) (9). In addition, an epigenetic pathway has been proposed wherein tumor suppressor genes are inactivated by promoter methylation and the silencing of gene transcription (10). This pathway has been called the CpG island methylator phenotype (CIMP). Nevertheless, there is some degree of overlap in these pathways and DNA hypermethylation has been demonstrated as a common and early event in carcinogenesis (11–14).

The regulation of DNA methylation is not well understood, but it involves DNA methyl transferases (DNMT), which catalyze the transfer of methyl groups to the carbon-5 position of cytosines in CpG islands. DNMT-1 is largely responsible for maintaining methylation and it also contributes to de novo DNA promoter methylation in cancer (15). Some models suggest that DNMT-3b cooperates with DNMT-1 to maintain DNA methylation status (16). Because epigenetic changes are highly relevant to colon carcinogenesis, it represents a target for the novel strategies to prevent or treat cancer. Recently, 5-aza-cytidine and its metabolite 5-aza-2'deoxycytidine (5-aza-2dC) have been approved by the FDA for the treatment of myelodysplastic syndromes (17). However, serious side effects, including myelotoxicity, limit the use of these drugs in other clinical settings (18).

Interestingly, demethylating activities have been reported for certain tea catechins and soybean isoflavones in breast and esophageal squamous cell lines (19,20). The apparent safety and the easy access through the diet make these natural compounds attractive as chemopreventive and chemotherapeutic agents. Moreover, there is evidence that mixtures of bioactive compounds naturally present in foods may act synergistically and might be more active than the solitary compounds isolated from food (21).

In this study, we tested the anticancer properties of Annurca apple polyphenol extract (APE) in in vitro models of CRC.


   Methods
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    Cell culture and treatments. We purchased the human CRC cell lines RKO, SW48, and SW480 from the American Type Culture Collection. The cells were cultured in Iscove's Modified Dulbecco's Media supplemented with 10% fetal bovine serum (Life Technologies), 0.1 U/L penicillin G, and 0.1 mg/L streptomycin (GIBCO, Invitrogen). The cultures were maintained at 37°C in 5% CO2. We extracted APE from the frozen flesh of Annurca apples as previously described (the phenolic compounds present with their concentration are reported in Table 1) (6). To give APE (i.e. a mixture of different phenolic compounds with different molecular weight) a molar concentration, we decided to arbitrarily express APE concentration as catechin equivalents. To do this, the sum of the concentration of all the compounds present in the extract was considered and quantified using a catechin calibration curve. Then, the concentrations of the phenolic compounds were expressed in catechin equivalents. The concentrations (expressed in cathechin equivalent) measured by liquid chromatography-mass spectrometry (LC-MS) for each compound present in 2 µmol/L of APE were as follows: chlorogenic acid, 0.43 µmol/L; caffeic acid, 0.1 µmol/L; catechin, 0.70 µmol/L; epicatechin, 0.63 µmol/L; rutin, 0.01µmol/L; and phlorizin 0.07 µmol/L.


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  		TABLE 1 Main phenolic compound constituents in Annurca apple flesh

 
5-aza-2dC was purchased from Sigma Aldrich. Cells were treated with APE or 5-aza-2dC; 5-aza-2dC treatment was performed at a final concentration of 5 µmol/L for 96 h, changing the conditioned media each day. We performed the APE treatment based on the IC50 results by changing the conditioned media every 48 h.

    Cell viability [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cells were seeded at a density of 3000 cells per well in 96-well plates. The next day, cells were treated with concentrations ranging from 0 to 10 µmol/L APE dissolved in methanol. We used the appropriate amounts of methanol in the control wells. After 24 and 96 h of treatment, the cells were incubated with a solution of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) at a concentration of 0.5 g/L for 3 h at 37°C. The cells were lysed in 100 µL of solubilizing solution (10% SDS, 0.01 mol/L HCl). Colored formazan converted from MTT by viable cells was measured at 570 nm using a microplate reader. Experiments were performed in triplicate.

    Determination of the induction of apoptosis. Apoptotic events were analyzed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay using the In Situ Cell Death detection kit (Roche) after treating the cells with 2 µmol/L APE. We used an equal volume of methanol in the control wells. Apoptotic cells were visualized under an AxioSkop2 multichannel epifluorescence microscope and processed by AxioVision software (Carl Zeiss). We repeated this experiment 3 independent times.

    Cell cycle analysis. The effects of APE on cell cycle profiles were evaluated by flow cytometry. Cell cycle distribution was based on an evaluation of the amount of the DNA stained with propidium iodide. Cells were plated at a density of 5 x 105 cells per plate in 100-mm dishes, synchronized by serum deprivation for 48 h, and finally treated with 2 µmol/L of APE for a total duration of 96 h. DNA content was evaluated by a FACSCalibur Flow Cytometer (BD Biosciences). Cell cycle distribution was determined using the ModFit DNA Analysis Software (Verity Software House). We repeated this experiment 3 separate times

    Western blotting analysis. We assessed protein expressions by Western blot. Protein extraction was performed using radioimmunoprecipitation Buffer (Santa Cruz Biotechnology) combined with 10 µL/mL of phenylmethylsulfonyl fluoride solution, 10 µL/mL sodium orthovanadate solution and 10 µL/mL protease inhibitor cocktail. Forty micrograms of proteins was separated on 10% SDS-PAGE gels. The separated proteins were transferred onto polyvinylidene fluoride membrane (Amersham Pharmacia Biotech). After blocking, membranes were probed with the specific primary antibody followed by incubation with a peroxidase-conjugated anti-mouse secondary antibody (80 µg/L; Santa Cruz) for 30 min. The protein bands were visualized using the ECL Plus Chemiluminescence system and the membranes were scanned with a STORM 840 Phosphoimager (Amersham Biosciences). Quantification of the bands was performed using IMAGEQUANT 5.2 spot densitometric software (Molecular Dynamics). The expression levels of the proteins were corrected by normalization to the expression of the housekeeping protein ß-actin. Cell cycle protein primary antibodies, including anti-cyclin D1 (clone A-12), anti-cyclin E (clone HE 12), anti-cyclin B1 (clone D11), anti-p53 (clone DO-1), and anti-p21 (clone F5), were obtained from Santa Cruz Biotechnology and incubated for 3 h at room temperature. Anti-hMLH1 and anti-DNMT-1 antibodies were purchased from BD Biosciences Pharmingen and the anti-DNMT-3b antibody was purchased from Imgenex. These antibodies were incubated overnight at 4°C. All antibodies were used at a working concentration of 2 mg/L. We evaluated each protein in at least 2 independent experiments.

    Bisulfite modification, methylation-specific PCR, and combined bisulfite restriction assay. After treatment with APE or 5-aza-2dC, DNA extraction was performed using the QIAamp DNA Mini kit (Qiagen) and 500 ng of DNA was subjected to bisulfite modification with the Epitect Bisulfite kit (Qiagen) as recommended by the manufacturer's protocol. Modified DNA was used as a template. The status of hMLH1 promoter methylation was assessed by methylation-specific PCR and p14ARF and p16INK4a promoter methylation were assessed by combined bisulfite restriction analysis (COBRA), as previously described (10,22,23).

    Conventional RT-PCR. After treatment with APE or 5-aza-2dC, we extracted total RNA using Trizol reagents (Invitrogen) according to the manufacturer's instructions. Conventional RT-PCR was also performed to assess the messenger RNA (mRNA) expression of p14ARF (24), p16INK4a (20), hMLH1 (25), DNMT-1 (26), and DNMT-3b (27). cDNA was generated with random hexamers using 2 µg of RNA. The PCR was performed as previously reported (28). ß-Actin was used as an endogenous control.

    Statistical analysis. Multifactorial ANOVA with interactions between the factors was used to evaluate the relationships between cell viability and the included factors and Tukey-Kramer honestly significant difference test for pair-wise comparisons was utilized when the main effect was significant. In addition, a 2-way ANOVA including the effects for cell line and treatment was used to evaluate the treatment effects on cell cycle dynamics. We used a Student's 2 sample t test to compare protein expressions between the 2 groups. Results are expressed as means ± SE. Differences were considered significant at P < 0.05.


   Results
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    APE inhibit cell viability and induce apoptosis in RKO and SW480 cell lines. We tested the anticancer effects of APE on cell lines displaying different genetic and epigenetic profiles. RKO and SW48 are in vitro models of CIMP and both cell lines have undergone silencing of the DNA mismatch repair gene hMLH1 and subsequently developed MSI. The tumor suppressor genes p14ARF and p16INK4a are also methylated in both lines (1,29–31). SW480 cells, on the other hand, are a model of CIN, do not have CIMP features (although they show a certain degree of methylation in the p16INK4a promoter), and the DNA mismatch repair system is fully functional in these cells (29,31).

We determined the effect of APE treatment on cell viability by the MTT assay after treating the cells with different concentrations of APE (0–20 µmol/L) for 24 and 96 h (Fig. 1A). The treatment significantly reduced the viability of RKO and SW480 cell lines in a dose- and time-dependent manner. No significant changes in the cell viability were detected after 24 h at any concentration, whereas cell viability decreased after 96 h of treatment at concentrations of 2 µmol/L for RKO and 200 nmol/L for SW480 (P < 0.05) (Fig. 1). At 2 µmol/L, cell viability was decreased by 49 ± 3.0% for SW480 and by 47 ± 2.7% for RKO. We used this schedule of treatment for subsequent evaluations. The levels of phenolic compounds in 100 g of apple flesh and the percentage of each phenolic compound in 2 µmol APE/L (i.e. the concentration of APE we used in our experiments) are in Table 1. The APE concentrations were arbitrarily expressed in catechin equivalents. As previously reported, catechin, chlorogenic acid, and epicatechin were the main phenolic components (6,7).


Figure 1
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  		FIGURE 1  Cell viability and apoptosis in RKO and SW480 cells after treatment with APE. Cell viability (A) was assessed after 24 and 96 h of incubation with APE. Values are means ± SEM, n = 3. Asterisks indicate a difference from untreated counterparts, P < 0.05:*RKO, ** SW480. APE treatment induced apoptosis (B) in both RKO and SW480 cells compared with the untreated control cells. Pretreatment with DNaseI was used as a positive control and incubation without terminal deoxynucleotidyltransferase was performed as the negative control (C).

 
We evaluated apoptosis by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, as a measure of DNA fragmentation, after 4 d of treatment. Apoptotic cells were visible after treatment of either SW480 or RKO, whereas no apoptosis was detectable in control cells (Fig. 1B).

    Effects of APE in cell cycle dynamics. We synchronized the cells by serum deprivation and subsequently treated with APE for 4 d. The cell cycle profile showed a nonsignificant S phase arrest in both RKO (P = 0.493; Fig. 2A) and SW480 (P = 0.052; Fig. 2B). To confirm these results, we assessed the levels of expression of the principal cell cycle regulatory proteins by western blot (Fig. 2C). We observed significant increases in p53 expression in RKO (P < 0.05; Fig. 2D).


Figure 2
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  		FIGURE 2  Cell cycle analysis and cell cycle regulatory protein expressions in SW480 and RKO cells after APE treatment. An example of cell cycle distribution is shown (A). The graph in B shows the distribution within the cell cycle in control (C) and APE-treated (A) RKO and SW480 cells. Values are means ± SEM, n = 3. Expression of cell cycle regulatory proteins was evaluated by western blot before and after APE treatment in RKO and SW480 cells (C). Quantitative data are presented in D. Values are means ± SEM, n = 3. *Different from RKO-C, P < 0.05.

 
    APE induce reversal of methylation and reactivate hMLH1, p14ARF, and p16INK4a. Based on reported data of demethylating activity on esophageal squamous cell carcinoma after treatment with epigallocatechin-3 gallate (EGCG) extracted from green tea (19) and soy isoflavones (20), we investigated the effects of APE on the DNA methylation status of CRC cells. Following treatment with APE, methylation-specific PCR of the hMLH1 promoter revealed the appearance of the specific unmethylated band in RKO cells beginning on d 2 after the end of treatment until d 8 (Fig. 3A). The demethylating effect resulted in the reexpression of hMLH1 mRNA detected by conventional RT-PCR (Fig. 3B). Nevertheless, the methylated promoter band was still visible during the appearance of the unmethylated band, confirming a prior report that total demethylation of the promoter is not necessary for reexpression of the transcripts or proteins (32). In fact, western blot analysis performed for hMLH1 protein showed increasing expression of the protein from d 3 after the termination of treatment, peaking at d 5. Although resulting in a weaker and less lasting effect, the reversal of hMLH1 DNA hypermethylation and reexpression of the mRNA and protein were qualitatively comparable to the results obtained after treating the cells with 5-aza-2dc (Fig. 3C). There were no changes in the hMLH1 expression profiles after treating SW480 cells with demethylating agents.


Figure 3
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  		FIGURE 3  Reversal of methylation and reactivation of hMLH1 after APE treatment in RKO cells. Evaluation of the methylation status of the hMLH1 promoter (A). A specific unmethylated band was present beginning on d 2 after treatment. Untreated RKO DNA was used as a methylated positive control (PC; M). Untreated SW480 DNA was used as an unmethylated positive control (PC; U). BL, Blank; MW, size standard. The expression of hMLH1 mRNA in RKO cells was obtained after treatment with either 5-aza-2dC or APE (B). SW480 cells were used as a positive control for hMLH1 expression. Increased hMLH1 protein expression (C) was obtained in RKO cells after APE treatment.

 
We investigated the DNA methylation status of the promoters of p14ARF and p16INK4a, both methylated in RKO (28,33), by COBRA. Treatment with APE resulted in the appearance of an unmethylated band for both promoters (Fig. 4A), followed by an increase in RNA transcripts. This was qualitatively comparable to the results obtained after treatment the cells with 5-aza-2-dC (Fig. 4B).


Figure 4
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  		FIGURE 4  Evaluation of p16INK4a and p14ARF methylation and RNA expression in RKO cells by COBRA and RT-PCR, respectively. Appearance of the unmethylated band (indicated by arrows) occurred after APE treatment (A). Reexpression of p16INK4a and p14ARF mRNA in RKO and SW48 cells after either APE or 5-aza-2dC treatment (B). SW480 was employed as a positive control for p14ARF gene expression. Also in SW480 cells, the mRNA expression of p16INK4a, which is partially methylated, increased after APE treatment.

 
Furthermore, we evaluated the transcript levels of p14ARF and p16INK4a in SW48 cells and RNA transcripts appeared for both genes after treatment with either APE or 5-aza-2dC (Fig. 4B).

Untreated SW480 cells were used as controls. As previously reported (31), SW480 has a complete unmethylated p14ARF and a partially methylated p16INK4a pattern at COBRA (Fig. 4A). As expected, increase in p16INK4a mRNA expression was detected in SW480 after APE treatment (Fig. 4B).

    APE inhibit DNMT-1 and -3b in colon cancer cells. To clarify the mechanism by which APE induces DNA demethylation, we evaluated changes in DNMT levels induced by APE. For this purpose, DNMT-1 and DNMT-3b mRNA and protein expression patterns were assessed. We found no significant differences in the transcript levels between treated and control cells (Fig. 5A), whereas we observed a significant reduction in protein expression (Fig. 5B) 48 h after the end of the treatment for both DNMT-1 (P < 0.001; Fig. 5C) and DNMT-3b (P < 0.005; Fig. 5D). The time course of the inhibition of DNMT was consistent with the timing of promoter demethylation, as well as reexpression of both mRNA and protein of the tumor suppressor genes tested. Taken together, these results suggest that APE induce demethylation through post-translational inhibition of both DNMT-1 and DNMT-3b.


Figure 5
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  		FIGURE 5  Effects of APE treatment on DNMT expression in RKO and SW480 cells. No changes in DNMTs mRNA expression was found in RKO and SW480 after 48 h of treatment with APE (A) compared with controls (C). An example of a western blot for the expression of DNMT-1 and DNMT-3b proteins in RKO cells before (C) and 24 and 72 h after APE treatment is in B. Quantitative data for DNMT-1 are in C and for DNMT-3b, in D. Values are means ± SEM, n = 3. Asterisks indicate different from control: *P < 0.001, **P < 0.005.

 

   Discussion
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LITERATURE CITED
 
In this study, we demonstrated that polyphenols extracted from APE, containing chlorogenic acid, catechin, and epicatechin as major components, are active in regulating apoptosis and cell viability without exerting significant effects on cell cycle dynamics in 2 different models of CRC: RKO (MSI and CIMP) and SW480 (CIN and no CIMP). More importantly, our results show that APE lead to the reactivation of silenced tumor suppression genes by inhibiting DNMT-1 and DNMT-3b protein expression. Interestingly, the effects of APE were similar to what we achieved with the clinically used compound, 5-aza-2dC, a potent but toxic synthetic DNMT inhibitor. Moreover, these findings are compatible to what has been observed with other nutriceuticals such as tea catechins and soy bioflavonoids, each of which can modulate DNMT-1 protein levels (34). This suggests that it is possible to achieve a potent biological response with well-tolerated dietary substances, including at least 1 variety of apple.

Polyphenols are present in virtually all plant-derived foods and they represent key components of the Mediterranean diet, which is rich in vegetables, fruits, nuts, seeds, olive oil, grains, wine, and honey (4,35–37). The Mediterranean diet is universally associated with lower incidences of cancer, including CRC (2,4).

Of particular importance, our results were obtained with doses of APE reflecting the dietary consumption. In fact, the concentration of APE we used in this study (i.e. 2 µmol/L) is, in all likelihood, comparable to that obtained in the lower gut following the consumption of 1 apple, which contains ~20 mg/100 g polyphenols (i.e. 1 mmol/L). Even though some of the polyphenols ingested with an apple are either absorbed or degraded during transit through the gut, a significant proportion is recoverable in the feces (38,39). Therefore, even considering a dilution effect along the intestinal tract, a residual APE concentration between 1 and 10 µmol/L is likely to reach the colon.

Our data show that APE inhibit cell viability in a time- and concentration-dependent manner in the CRC models analyzed. However, no important changes in cell cycle dynamics were found, whereas increased apoptosis was evident in both RKO and SW480. Because SW480 cells lack in a fully functional p53 system (29), the increased apoptosis seems to occur in a p53-independent manner. Nevertheless, p53 protein expression significantly increased in RKO, where the p53 cascade is intact (29,40).

In the second part of the study, we tested the hypothesis that there might be effects of APE on the DNA methylation status of CRC cells. Tea EGCG (19) and soy isoflavones (20) have been demonstrated to reverse the methylation of p16INK4a, RARß, and other genes, which is thought to be mediated through the inhibition of DNMT-1. In our study, we used the naturally occurring combination of polyphenols present in Annurca extracts. The main phenolic compounds of APE are catechins, chlorogenic acid, and epicatechin. These extracts have been demonstrated to prevent exogenous gastric damage in vitro and in vivo and protective antioxidant effects have been obtained at concentrations of 2.5 mmol/L and 10 mmol/L, respectively (6). In our study, low concentrations of catechins were sufficient to obtain a potent demethylating effect with subsequent reexpression of previously silenced tumor suppressor genes. The different experimental models used might explain this apparent discrepancy with the previous study.

Our observations indicate that the demethylating activity could be a key protective mechanism of these compounds. In silico molecular modeling studies have demonstrated that structural analogs of EGCG may interact with the catalytic domain of DNMT-1 (19).

Using synthetic compounds, it has been demonstrated that catechin and epicatechin are ideal candidate DNMT inhibitors. Although EGCG seems to be more potent as a direct inhibitor, catechin and epicatechin are better substrates for catechol-O-methyltransferase methylation with stronger negative feedback on the DNMT and better intracellular bioavailability (34,41). Nevertheless, our data on the expression of DNMT show suppression at the protein level, whereas no differences were evident in the transcripts of either DNMT-1 or DNMT-3b, implying that translational inhibition might be the main mechanism of APE in this pathway. In fact, recent findings demonstrate that Glu1265 on the human DNMT-1 protein is the key catalytic site that interacts with EGCG by forming H-bonds, suggesting a direct inhibition of the protein by catechins (19,34). Strong DNMT protein inhibition occurred by d 2 after APE treatment and the time course of inhibition coincided with the timing of promoter demethylation, mRNA appearance, and protein reexpression of the previously silenced genes. Moreover, our data demonstrated that these effects were robust. In RKO, the unmethylated DNA band and reexpression of the hMLH1 mRNA were evident until d 8 after the end of the treatment and protein expression was evident for at least 6 d after the cessation of treatment. Our data were reproducible after testing a broader panel of tumor suppressor genes (p14ARF and p16INK4a) and confirmed on a second model of colon cancer with CIMP (SW48). As expected, increase in p16INK4a mRNA levels was also demonstrated in SW480 after treatment.

Additionally, we wanted to compare the demethylating effects of APE with the clinically used DNMT inhibitor 5-aza-2dC. Our study indicates that APE can inhibit DNMT expression and reactivate silenced genes (hMLH1, p14ARF, and p16INK4a) at very low concentrations. Although our results show weaker demethylating activity for APE compared with 5-aza-2'dC in vitro, which is consistent with previous reports on EGCG (42), excessive inhibition of DNMT might be dangerous, as it has been associated with the induction of CIN in vitro and sarcomas and T-cell lymphoma in vivo (43,44). There has never been a suggestion of toxicity due to excessive hypomethylation in individuals who regularly consume apples. Although issues of dose and tissue distribution make speculation difficult here, it would be hard to imagine that a commonly consumed variety of apples is toxic.

To date, no demethylating drugs are available for the treatment of colon cancer patients or, perhaps more importantly, for its chemoprevention. Currently, 5-aza-cytidine and its metabolite 5-aza-2-dC have been approved for the treatment of myelodysplasia (17) and several clinical trials are currently being conducted. Although promising results have been achieved with hematological malignancies, the limited clinical responses (45–47) and the spectrum of side effects have discouraged the use of these agents in other tumors (18,45,46,48). Nevertheless, the reactivation of hypermethylated tumor suppressor genes remains an attractive strategy for cancer therapy.

In some geographical areas, Annurca apples have long been consumed as a dietary staple and their long-term effects, in addition to other protective nutrients in the Mediterranean diet, may be reflected in the lower incidences of cancer, including CRC. Our data permit a reasonable speculation that APE could be adapted to our preventive or therapeutic armamentaria against CRC. Future clinical studies are necessary to determine whether these compounds will be as interesting as preventive or therapeutic agents as they are in this preclinical study.


   FOOTNOTES
 
1 Supported by a grant from the National Cancer Institute of the NIH: R01 CA72851 (to C.R.B.) and by the Baylor Research Institute. Back

2 Author disclosures: L. Fini, M. Selgrad, G. Graziani, Y. A. Daoud, E. B. De Vol, E. Hotchkiss, no conflicts of interest; V. Fogliano, M. Romano, C. R. Boland, and L. Ricciardiello hold patent rights for APE. Back

8 Abbreviations used: APE, Annurca polyphenol extract; 5-aza-2dC, 5-aza-2'deoxycytidine; CIMP, CpG island methylator phenotype; CIN, chromosomal instability; COBRA, combined bisulfite restriction analysis; CRC, colorectal cancer; DNMT, DNA methyltransferase; EGCG, epigallocatechin-3 gallate; MSI, microsatellite instability; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back

Manuscript received 24 August 2007. Initial review completed 29 August 2007. Revision accepted 26 September 2007.


   LITERATURE CITED
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 Introduction
 Methods
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 Discussion
 LITERATURE CITED
 

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