Purpose: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (−)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation.
Experimental Design: Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor β (RARβ), p16INK4a, and O6-methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2′-deoxy-5-aza-cytidine (5-aza-dCyd).
Results: Genistein (2-20 μmol/L) reversed DNA hypermethylation and reactivated RARβ, p16INK4a, and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARβ by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 μmol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate- and methyl donor–dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARβ, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth.
Conclusions: These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.
Methylation of CpG islands in the promoter region is a key epigenetic mechanism for the silencing of many genes including tumor suppressor genes, as well as genes encoding hormone receptors, DNA repair enzymes, mediators of the apoptosis pathway, and detoxification enzymes (1, 2). Unlike many other genomic alterations that occur during carcinogenesis, DNA methylation is potentially reversible via the use of preventive or therapeutic agents (3, 4). 5-Aza-cytidine and 2′-deoxy-5-aza-cytidine (5-aza-dCyd), nucleoside analogue inhibitors of DNA methytransferases, have been widely used in attempts to reverse abnormal DNA hypermethylation in cancer cells and restore “silenced” gene expression. However, clinical utility of these nucleoside analogue DNA methyltransferase inhibitors has been somewhat limited by myelosuppression and other side effects (5). We have previously shown the reactivation of methylation-silenced retinoic acid receptor β (RARβ), p16INK4a (p16), O6-methylguanine methyltransferase (MGMT), and human mutL homologue 1 (hMLH1) genes by (−)-epigallocatechin-3-gallate (EGCG), the major polyphenol from green tea, and this activity has been attributed to the inhibition of DNA methyltransferase by EGCG (6). To determine whether other polyphenolic compounds have similar activities, we tested the ability of soy isoflavones to inhibit DNA methylation.
Genistein (4′,5,7-trihydroxyisoflavone), the major isoflavone present in soy bean, has been shown to prevent carcinogenesis in animal models for tumor development at different organ sites (7). Many mechanisms have been proposed for this activity. Some are believed to be closely related to the estrogenic and antiestrogenic activities of genistein (7). Prepubertal exposure to soy or genistein reduced mammary carcinogenesis in rats treated with carcinogens, possibly by modulating the development of the mammary end buds (8, 9). Various soy products containing genistein have been found to inhibit the growth of transplanted human prostate carcinoma, reduce the incidence of poorly differentiated prostate adenocarcinoma in a transgenic mouse model, and inhibit 2-amino-1-methyl-6-phenylimidazo[4,5]pyridine induced rat prostate carcinogenesis (10–13). Carcinogenesis or metastasis in stomach, colon, bladder, and lung is also inhibited by genistein and related isoflavones (14–20).
The effects of genistein on various cancer cell lines have been extensively studied. It has been reported that genistein can inhibit cancer cell growth (21–23), induce apoptotic cell death accompanied by cell cycle arrest at G2-M phase, and inhibit angiogenesis (17, 24–28). The precise molecular mechanisms responsible for these activities, however, are not clearly known. One potential mechanism that has recently received considerable attention is that genistein may be involved in regulation of gene activity by modulating epigenetic events such as DNA methylation and/or histone acetylation directly or through an estrogen receptor dependent process (29–31). This hypothesis is supported by a report indicating that dietary genistein causes epigenetic changes in mouse prostate (32). Genistein can also up-regulate mRNA expression of the BRCA1 gene during mammary tumorigenesis, which is frequently inactivated by epigenetic events in breast cancer (8).
In this preclinical study, we examined the effect of genistein and its combination with other agents on the reactivation of methylation-silenced RARβ, p16, and MGMT genes, as well as its effect on the activity of DNA methyltransferase and histone deacetylase (HDAC) in the esophageal squamous cell carcinoma cell line KYSE 510. These genes were selected for study because they have been shown to be progressively inactivated by DNA hypermethylation in human esophageal squamous carcinogenesis and to be reactivated in KYSE 510 cells by 5-aza-dCyd and EGCG (33–35). Our results show that genistein inhibits DNA methyltransferase and reverses the methylation status, with accompanying reexpression of RARβ, p16, and MGMT genes. These activities may contribute to the chemopreventive activity of genistein.
Materials and Methods
Cell lines and cell culture
The human esophageal squamous cell carcinoma cell lines KYSE 510 and KYSE 150 were a gift from Dr. Yutaka Shimada (Kyoto University, Kyoto, Japan). The cells were maintained in 5% CO2 atmosphere at 37°C in RPMI 1640/Ham's F-12 mixed (1:1) medium containing 5% fetal bovine serum. To determine the dose-dependent changes, KYSE 510 cells were treated with 2, 5, 10, or 20 μmol/L of genistein (Sigma, St. Louis, MO) or 8.7 μmol/L of 5-aza-dCyd (Sigma) administered in fresh culture medium every other day, when the medium was changed, for 6 days. For the time-course study, cells were treated with 5 μmol/L of genistein in fresh culture medium on days 0, 2, 4, and 5. Prostate cancer cell lines LNCaP and PC3 were obtained from American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 containing 10% fetal bovine serum. KYSE 150, LNCaP, and PC3 cells were treated with 10 or 20 μmol/L of genistein for 6 days as described above. For the combination study, KYSE 510 cells were treated with either 5 μmol/L of genistein or 2 μmol/L 5-aza-dCyd for 5 days and then cultured for 1 additional day in fresh medium with 0.5 μmol/L trichostatin or 15 μmol/L sulforaphane; or treated with 5 μmol/L of genistein or 2 μmol/L 5-aza-dCyd alone or together for 5 days and 1 additional day in fresh medium.
Bisulfite modification and methylation-specific PCR
DNA was extracted from the cells using the DNeasy tissue kit (Qiagen, Valencia, CA) following the procedure of the manufacturer and modified by a bisulfite reaction as described by Herman et al. (36). Primers specific for the methylated and unmethylated DNA were the same as we previously reported (6). To verify the specificity of the PCR, we used normal human placental DNA (Sigma) as a negative control and CpGenome universal methylated DNA (Chemicon, Temecula, CA) as a positive control for methylation-specific PCR. Methylation-specific PCR was carried out using a nested two-stage PCR approach (37) with similar PCR conditions as previously described (6). In brief, stage I PCR was done on bisulfite-modified DNA to amplify the CpG-rich promoter regions of the p16, RARβ, or MGMT gene with the primers that recognize the bisulfite-modified template, but do not discriminate between methylated and unmethylated alleles. DNA fragments from the first PCR were used for methylation-specific PCR with primers specific for methylated and unmethylated DNA. Amplification was carried out using a 9700 Perkin-Elmer thermal cycler (Applied Biosystems, Foster City, CA).
Total RNA was isolated from cells using Tri reagent (Sigma). Reverse transcription of RNA was done using the Advantage RT-for-PCR kit (Clontech, Palo Alto, CA). The primers and conditions used in the PCR for the genes analyzed were previously summarized (6). The PCR was carried out in an approximate linear range (for example, 25 to 28 cycles for p16, 30 to 35 cycles for RARβ, and 30 to 35 cycles for MGMT). The PCR products were separated on 2% agarose gel containing ethidium bromide and then photographed. Negative controls for PCR were run under the same conditions without RNA or reverse transcriptase. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was used as an endogenous control.
Total RNA was extracted using ABI Prism 6100 Nucleic Acid PrepStation. Analysis of relative gene expression for RARβ, p16, and MGMT was done using real-time quantitative PCR and the comparative threshold cycle method (Ct). All reagents, kits, and instruments used in RNA extraction, reverse transcription, and real-time PCR were purchased from Applied Biosystems. In the reverse transcription step, 12.5 ng of RNA were reversely transcribed to cDNA using the High Capacity cDNA Archive Kit following the instruction manual. In the real-time PCR step, cDNA was amplified with Assays-on-Demand Products containing two gene-specific primers for RARβ, p16, and MGMT, respectively, and one TaqMan MGB probe (6-FAM dye–labeled) using the TaqMan Universal PCR Master Mix in ABI Prism 7000 Sequencing Detector. Thermal cycling conditions included 2 minutes at 50°C, 10 minutes at 95°C, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute according to the TaqMan Universal PCR Protocol. No-template control was included in each assay. β-Actin was used as an endogenous control and vehicle control was used as a calibrator. Each sample was run in triplicate. The comparative Ct method was used to calculate the relative changes in gene expression in the ABI Prism sequence detection system and Microsoft Excel as previously described by Livak and Schmittgen (38) and Ariani et al. (39). The relative changes of gene expression were calculated using the following formula: Fold change in gene expression, 2 –ΔΔCt = 2−[ΔCt (genistein treated samples) − ΔCt (untreated control)], where ΔCt = Ct (detected gene) − Ct (β-actin) and Ct represents threshold cycle number.
DNA methyltransferase assays
Assays with nuclear extracts as the source of DNA methyltransferase. Cultured KYSE 510 cells were harvested, and nuclear extracts were prepared with a nuclear extraction kit (Pierce, Rockford, IL). The DNA methyltransferase assay was modified in our lab according to the published methods (40, 41). In brief, the nuclear extracts (4.5 μg of protein) were incubated for 1.5 hours at 37°C with 20 nmol/L (0.75 μg) of poly(deoxyinosine-deoxycytosine) [poly(dI-dC)·poly(dI-dC)] and 10 μmol/L of S-adenosyl-l-[methyl-3H]methionine (2 μCi; Amersham, Piscataway, NJ) in a total volume of 40 μL of reaction buffer (pH 7.4), containing 20 mmol/L Tris-HCl, 10% glycerol (v/v), 10 mmol/L EDTA, 0.2 mmol/L phenylmethylsulfonylfluoride, and 2 mmol/L 2-mercaptoethanol. Genistein was dissolved in DMSO, and all incubations were adjusted to contain an equivalent concentration of DMSO (10%), which had no significant effect on DNA methyltransferase activity. Reactions were initiated by the addition of nuclear extracts and stopped by mixing with 200 μL of a solution containing 1% SDS, 3% 4-aminosalicylate, 5% butanol, 2.0 mmol/L EDTA, 125 mmol/L NaCl, 0.25 mg/mL carrier salmon testes DNA (Life Technologies, Inc., Gaithersburg, MD), and 1 mg/mL protease K. DNA was extracted using the phenol/chloroform method, purified with ethanol precipitation, and then washed thrice with 70% ethanol. The radioactivity in the pellets was counted in a scintillation counter. Each assay was done in duplicate. Background levels were determined in incubations without the template poly(dI-dC)·poly(dI-dC). For kinetics, the results were analyzed using GraphPad Prism 4 (GraphPad Software, San Diego, CA).
Assays with purified recombinant DNA methyltransferase 1. Recombinant murine DNA methyltransferase 1 (DNMT1) with an NH2-terminal hexahistidine tag added for purification was expressed in Spodoptera frugiperda 9 cells and purified as previously described (42). Standard reaction mixtures (25 μL) contained 60 nmol/L recombinant DNMT1, 15 μmol/L S-adenosyl-l-[methyl-3H]methionine (specific activity, 275 GBq/mmol), and 10 ng poly(dI-dC)·poly(dI-dC) (average chain length: 3,000) in M2 buffer [100 mmol/L Tris (pH 8), 10 mmol/L EDTA, 10 mmol/L DTT, 200 μg/mL bovine serum albumin] and were incubated for 1 hour at 37°C. Reactions were terminated by the addition of NaOH to a final concentration of 0.3 mol/L. Salmon sperm DNA (100 μg/mL) was added as a carrier, and the solution brought to 860 mmol/L with perchloric acid to precipitate DNA. Precipitated DNA was collected and washed on glass fiber filters before analysis for 3H-methyl by liquid scintillation counting in aqueous fluor. To measure the effect of genistein on the rate of substrate methylation, sets of duplicate reactions were initiated by adding poly(dI-dC)·poly(dI-dC) in the presence of increasing concentrations of EGCG or genistein dissolved in DMSO as described above.
Cell growth inhibition and other studies
KYSE 510 cells were treated with 1, 2, 5, 10, and 20 μmol/L genistein, biochenin A, or daidzein added in fresh culture medium every other day for 6 days or with 5 μmol/L genistein for 1, 2, 4, or 6 days. Living cells were counted after staining with 0.04% trypan blue. After treatment for 2 or 6 days, the cells were photographed under an inverted microscope for morphologic analysis. In other experiments, KYSE 510 cells were treated with 2, 5, 10, and 20 μmol/L genistein for 3 days; cell proliferation was determined with the BrdUrd ELISA method according to the instruction manual (Roche Diagnostics Co., Indianapolis, IN) and apoptosis was determined with fluorescence 4′,6-diamidino-2-phenylindole staining or caspase-3 immunocytochemistry staining. For colony formation efficiency assay, KYSE 510 cells were treated with 2, 5, 10, or 20 μmol/L genistein for 2 days and then cultured for 10 additional days in fresh medium, and then colony numbers were analyzed.
The effect of genistein on HDAC activity was carried out according to the instruction manual of the HDAC Assay Kit (Fluorometric Detection, Upstate Biotechnology, Lake Placid, NY). In brief, the reaction mixture contained nuclear extract from KYSE 510 cells (4.0 μg protein), substrate (CH3-CO-NH-CH2-(CH2)3-Lys-X2-, 48 μmol/L), and genistein (5, 10, 20, 50, or 100 μmol/L) in 40 μL total volume. Trichostatin (1 μmol/L) was used as a positive control. After incubation for 30 minutes at 25°C, activator solution was added to release a fluorophore from the deacetylated substrate. The product was determined by a fluorescence plate reader (excitation, 360 nm; emission, 465 nm). Each assay was done in triplicate.
Statistical significance between treatment and control groups was evaluated using the Student's t test. One-way ANOVA and Tukey's honest significant difference test were used for comparing the effects of different treatments. Pearson's correlation coefficient (r) with P value was also determined to examine the association between concentration and efficacy. All statistical analyses were done using GraphPad Instat.
Reversal of hypermethylation and reactivation of RARβ, p16, and MGMT by genistein
KYSE 510 cells, with hypermethylated RARβ, p16, and MGMT genes, showed only the methylation-specific bands of these genes in methylation-specific PCR, with the loss of the respective mRNA expression, as we previously reported (6, 34). After treating the cells with 2, 5, 10, or 20 μmol/L genistein for 6 days, the unmethylation-specific bands of these three genes were detectable at 2 μmol/L, with higher intensity at 5 μmol/L and even higher intensity at 10 and 20 μmol/L (Fig. 1A). The expression of mRNA from all three genes increased approximately proportional to the appearance of unmethylated DNA, whereas PCR products from methylated DNA decreased as genistein concentration increased. The reversal of hypermethylation and reexpression of these three genes by genistein were similar to that produced by the classic DNA methyltransferase inhibitor 5-aza-dCyd. On treating the cells with 5 μmol/L genistein, PCR products from unmethylated RARβ and MGMT genes began to appear after 2 days, whereas that from methylated p16 was only observed after 6 days (Fig. 1A). The mRNA expression of RARβ, p16, and MGMT roughly correlated with the appearance of the unmethylated PCR products. Under these conditions, the loss of methylation was not apparent. Similar results were obtained in a repeated experiment.
To determine whether the reactivation of methylation-silenced genes by genistein is a general phenomenon and occurs in other cell lines, we studied the effects of genistein treatment on the methylation status and mRNA levels of RARβ in three other human cancer cell lines (Fig. 1B). The appearance of an unmethylation-specific band and mRNA band of RARβ in KYSE 150 was observed after treating the cells with 10 or 20 μmol/L genistein for 6 days. In the LNCaP and PC3 cells, unmethylation-specific band and mRNA band of RARβ only appeared after treating the cells with 20 μmol/L genistein for 6 days. Methylation-specific bands in all three cell lines were slightly decreased after treatment with 20 μmol/L of genistein. The results show that the reactivation of methylation-silenced genes by genistein does occur in different cell lines as a general phenomenon, although the effective concentrations are somewhat different in the different cell lines.
To confirm the effect of genistein on the reactivation of these genes, real-time quantitative PCR with TaqMan MGB detector was employed to determine the mRNA expression levels of RARβ, p16, and MGMT genes in the KYSE 510 cells after treatment with different concentrations of genistein. The results are summarized in Fig. 2. The results show that the relative amount of mRNA expression from these three genes was increased by treatment with 5 μmol/L of genistein and increased further with higher concentrations of genistein (10 or 20 μmol/L). However, genistein was not as effective as 5-aza-dCyd (8.7 μmol/L). This result was generally consistent with those from general reverse transcription-PCR (RT-PCR) shown in Fig. 1A.
To compare the effects of different isoflavones from soy on the reactivation of RARβ gene, KYSE 510 cells were treated with 5, 10, or 20 μmol/L of genistein, biochanin A, or daidzein for 6 days and mRNA levels of RARβ were determined by RT-PCR (Fig. 3). All three compounds caused the reexpression of RARβ mRNA, but biochanin A and daidzein were significantly less effective than genistein (P < 0.05).
Inhibition of DNA methyltransferase and histone deacetylase activities
Genistein exhibited a dose-dependent inhibition of DNA methyltransferase activity with nuclear extracts from KYSE 510 cells as the enzyme source and poly(dI-dC)·poly(dI-dC) as the substrate. The IC50 of genistein was ∼67 μmol/L. The structural analogues of genistein, biochanin A, and daidzein had weaker inhibitory activities than genistein (Fig. 4A). Genistein also showed a dose-dependent inhibitory effect on recombinant DNMT1 activity with an IC50 of 30 μmol/L (Fig. 4B). In kinetic studies with varying concentrations of poly(dI-dC)·poly(dI-dC), genistein decreased Vmax and increased Km, showing a mixed inhibition with a Kic of 189.3 μmol/L for competitive and a Kin of 80.2 μmol/L for noncompetitive actions (Fig. 4C). With different concentrations of S-adenosyl-l-[methyl-3H]methionine, genistein decreased Vmax without a significant change of Km, suggesting a noncompetitive inhibition with a Kin of 140.5 μmol/L (Fig. 4D). The same conclusion was reached when this experiment was repeated thrice.
HDAC activity was assayed using an artificial fluorescence substrate, and the activity was completely inhibited by 1 μmol/L trichostatin. Significant inhibition was observed at 5 μmol/L genistein (13.2%), but the extent of inhibition was low even at 100 μmol/L genistein (33%; Fig. 5).
Enhanced reactivation of RARβ, p16, and MGMT and growth inhibition by combination of genistein with trichostatin, sulforaphane, or 2′-deoxy-5-aza-cytidine
Treatment of KYSE 510 cells with trichostatin (0.5 μmol/L) alone for 1 day or with 5-aza-dCyd (2 μmol/L) alone for 5 days reactivated the RARβ gene, but had little or no effect on p16 and MGMT (Fig. 6A). However, treatment of the cells with 5 μmol/L genistein for 5 days and then with 0.5 μmol/L trichostatin for 1 additional day significantly enhanced the reactivation of these three genes. In combination with 2 μmol/L 5-aza-dCyd, genistein also significantly enhanced the reexpression of RARβ and p16, but not MGMT. The reactivation caused by the combination of genistein with trichostatin or 5-aza-dCyd was higher than the sum of the effect of individual agents, suggesting a synergistic action. The combination of 2 μmol/L 5-aza-dCyd and 0.5 μmol/L trichostatin seemed to produce an additive effect on RARβ and a synergistic effect on p16 and MGMT. Under these experimental conditions, trichostatin (0.5 μmol/L) was more effective than genistein (5 μmol/L) in inhibiting cell growth, and the combination of these two agents caused a more pronounced inhibition (48%, P < 0.05). Similarly, 15 μmol/L sulforaphane alone can reactivate the RARβ gene, but cannot induce reexpression of p16 and MGMT genes; however, treatment of the cells with 5 μmol/L genistein for 5 days and then with 15 μmol/L sulforaphane for 1 additional day significantly enhanced the reactivation of these three genes, especially p16 and MGMT, whereas the combination of genistein and sulforaphane significantly enhanced the inhibitory effect on cell growth (P < 0.05) compared with genistein or sulforaphane alone (Fig. 6B).
Effects on inhibition of cell growth and other factors
After treatment with genistein, biochanin A, or daidzein for 6 days, a dose-dependent inhibition of cell growth was observed (Fig. 7A). At concentrations as low as 2 μmol/L, genistein inhibited cell growth by ∼30%, and at 20 μmol/L, by more than 90%. The inhibitory effects of biochanin A and daidzein were weaker, with biochanin A the weakest. In a time-dependent study with 5 μmol/L genistein, slight growth inhibition was seen after 4 days and significant inhibition was observed after 6 days (Fig. 7A). Signs of toxicity and growth inhibition were observed in cells treated for 2 or 6 days with 10 or 20 μmol/L genistein; the greatest effect was observed in cells treated with 20 μmol/L genistein for 6 days (Fig. 7B). In cells treated with different concentrations of genistein for 3 days, cell proliferation was significantly inhibited after treatment with 10 or 20 μmol/L genistein (Fig. 7C), but induction of apoptosis was not observed even after treatment with 20 μmol/L genistein (data not shown). In colony formation assays of KYSE 510 cells, treatment with genistein, especially at 10 or 20 μmol/L, for 2 days, significantly decreased the number of colonies formed (Fig. 7D).
RT-PCR results with total RNA of KYSE 510 cells treated with 1, 2, 5, 10, and 20 μmol/L genistein for 6 days indicated that genistein treatment did not affect the mRNA expression levels of DNMT1, DNMT3a, DNMT3b, and methyl-CpG binding domain 2. The protein level of 5-methylcytosine DNA glycosylase, an enzyme that may be involved in the removal of methylated DNA (43), also did not change after treatment with 10 or 20 μmol/L of genistein for 24 hours (data not shown).
The present study clearly shows that genistein reverses DNA hypermethylation and reactivates the methylation-silenced genes RARβ, p16, and MGMT. This activity is similar to our previous observations with EGCG. Genistein is also an inhibitor of DNA methyltransferase activity in nuclear extracts from KYSE 510 cells, with 14% inhibition at 20 μmol/L (IC50, ∼67 μmol/L), and this activity is weaker than that of EGCG (IC50, ∼20 μmol/L). Kinetic studies indicate that genistein inhibits DNA methyltransferase activity in a substrate- and methyl donor–dependent manner, which differs from our previous results with EGCG as a competitive inhibitor of DNA methyltransferase. The inhibition activity of genistein was also observed using recombinant DNMT1 as the enzyme source, showing an IC50 of 30 μmol/L. In comparison with genistein, biochanin A and daidzein are weaker inhibitors of DNA methyltransferase, and they are also less effective in the reactivation of RARβ gene, suggesting a correlation between the inhibition of DNA methyltransferase activity and reactivation of methylation-silenced genes by these dietary isoflavonoids. However, the fact that the effective concentrations of genistein in the inhibition of DNA methyltransferase are higher than those for the reactivation of methylation-silenced genes suggests that other factors are involved in the action of genistein. Our results, however, suggest genistein did not exert its effect by modulating the expression levels of DNMT1, 3a, and 3b, methyl-CpG binding domain 2, and 5-methylcytosine DNA glycosylase.
For the DNA hypermethylation-related silencing of RARβ, p16, and MGMT genes, histone deacetylation is also known to be involved (44, 45). In addition to inhibition of DNA methyltransferase, the presently observed weak inhibitory effect of genistein on HDAC activity (13-17% inhibition at 5-20 μmol/L) may also contribute to the reactivation of these genes. The weak inhibitory effects of genistein on DNA methyltransferase and HDAC may have an intrinsic synergistic effect on the reactivation of methylation-silenced genes. Genistein may also be expected to have a synergistic or an additive effect with other HDAC or DNA methyltransferase inhibitors. Indeed, our results (Fig. 6) indicate that genistein, at 5 μmol/L, enhanced the activity of lower concentrations of 5-aza-dCyd (2 μmol/L) or trichostatin (0.5 μmol/L) in the reactivation of RARβ, p16, and MGMT genes. In addition, combination of genistein and sulforaphane, a HDAC inhibitor found in broccoli (46), also enhanced the reactivation of these genes, especially p16 and MGMT genes. The possible synergistic actions in the combination of these agents warrant detailed investigation.
In theory, the demethylation and reactivation of p16 and RARβ by genistein should result in cell growth inhibition. Genistein (5-20 μmol/L) significantly inhibited cell proliferation without induction of apoptosis and also affected long-term cell survival after only 2 days of treatment, and this could be related to the reexpression of p16. More studies, however, are needed to determine the relationship between the reactivation of these genes and the growth inhibition by genistein.
Oral administration of soy products containing genistein and other isoflavones has been shown to inhibit tumorigenesis in different organs, and different mechanisms have been proposed (7). In our present study, the effective concentration of genistein for DNA demethylation, transcriptional reactivation of these three genes, and cell growth inhibition was 2 to 10 μmol/L. These concentrations are lower than those used in many other studies (26, 47, 48). A concentration of 10 μmol/L is higher than the plasma level of genistein in women consuming soy products (0.74-6.0 μmol/L; http://www.aminoup.co.jp; refs. 49, 50), but lower micromolar concentrations of genistein are still achievable. Because genistein is a weak inhibitor of DNA methyltransferase, genomic global hypomethylation is not expected to occur due to dietary intake of soy isoflavones. Even if hypomethylation occurred, it would not be to the extent as to induce genomic instability as has been reported with some DNA methyltransferase inhibitors (51, 52).
In this study, we showed that genistein and related soy isoflavones can inhibit DNA methyltransferase activity, reverse DNA hypermethylation, and reactivate methylation-silenced genes. Genistein in combination with other DNA methyltransferase or HDAC inhibitors can enhance the reactivation of methylation-silenced genes. It remains to be determined whether dietary administration of genistein or soy products could cause the reactivation of methylation-silenced genes in vivo. An attractive approach that needs to be explored in vivo is the combination of genistein with sulforaphane, and low concentrations of these two dietary factors may produce a synergistic effect in the prevention of hypermethylation-induced inactivation of tumor suppressor genes. The combination of genistein with low doses of trichostatin or 5-aza-dCyd for the prevention or reversal of hypermethylation also warrants further investigations in vitro and in vivo.
Grant support: NIH grants CA105331, CA88961, and CA65871 (C.S. Yang).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Jungil Hong, Shengmin Sang, and Charles desBordes for helpful discussions.