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Keywords:

  • prostate cancer;
  • resveratrol;
  • chromatin remodelling;
  • MTA1;
  • NuRD;
  • acetylated p53;
  • apoptosis;
  • SAHA

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Dietary compounds and epigenetic influences are well recognized factors in cancer progression. Resveratrol (Res), a dietary compound from grapes, has anticancer properties; however, its epigenetic effects are understudied. Metastasis-associated protein 1 (MTA1) is a part of the nucleosome remodeling deacetylation (NuRD) corepressor complex that mediates posttranslational modifications of histones and nonhistone proteins resulting in transcriptional repression. MTA1 overexpression in prostate cancer (PCa) correlates with tumor aggressiveness and metastasis. In this study, we have identified a novel MTA1-mediated mechanism, by which Res restores p53-signaling pathways in PCa cells. We show, for the first time, that Res causes down-regulation of MTA1 protein, leading to destabilization of MTA1/NuRD thus allowing acetylation/activation of p53. We demonstrated that MTA1 decrease by Res was concomitant with accumulation of Ac-p53. MTA1 knockdown further sensitized PCa cells to Res-dependent p53 acetylation and recruitment to the p21 and Bax promoters. Furthermore, MTA1 silencing maximized the levels of Res-induced apoptosis and pro-apoptotic Bax accumulation. HDAC inhibitor SAHA, like MTA1 silencing, increased Res-dependent p53 acetylation and showed cooperative effect on apoptosis. Our results indicate a novel epigenetic mechanism that contributes to Res anticancer activities: the inhibition of MTA1/NuRD complexes due to MTA1 decrease, which suppresses its deacetylation function and allows p53 acetylation and subsequent activation of pro-apoptotic genes. Our study identifies MTA1 as a new molecular target of Res that may have important clinical applications for PCa chemoprevention and therapy, and points to the combination of Res with HDAC inhibitors as an innovative therapeutic strategy for the treatment of PCa.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers and the second leading cause of cancer death in American men. As PCa is typically diagnosed in the elderly population with a relatively slower rate of growth and progression, and a “Western” diet is implicated in the development of PCa,1, 2 the use of dietary compounds for chemoprevention and therapy has big potential.3

Resveratrol (Res), a naturally occurring phytochemical in grapes, peanuts and berries, has exceptional potential as a treatment modality due to its cardioprotective, anti-inflammatory, chemopreventive and anti-angiogenic properties.2In vitro studies and preclinical research strongly support the wide-ranging anticancer effects of Res, which include the induction of apoptosis and inhibition of cell growth and angiogenesis. Indeed, Res inhibits the proliferation of a variety of cancer cell lines, including PCa cells4 and inhibits angiogenesis-dependent processes, such as wound healing, tumor growth, invasion and metastasis.1, 3, 5–8 Resveratrol inhibits angiogenesis by blocking a common pathway for VEGF and FGF-2 in endothelial cells6 and by decreasing the binding of VEGF to endothelial cells.1 Resveratrol inhibits the expression of MMP2 and MMP9,9, 10 which are involved in the degradation of the vascular basement membrane and associated with aggressive tumor growth and metastasis.11 Notably, Res also inhibits the function of androgen receptor (AR).12 These studies support the use of Res as potential chemotherapeutic agent.2, 9, 13 The molecular mechanisms of cancer prevention by Res occur by blocking cell cycle progression and inducing apoptosis14–17 and/or by inhibiting signal transduction through the PI3/Akt, MAPK and NF-κB pathways.9, 18–22 Preclinical studies show that very low doses of Res (200 μg–2 mg/kg/day) that give peak plasma concentrations between 20 nM and 2 μM are sufficient to exert potent cancer chemopreventive efficacy in rat carcinogenesis model.23, 24 These studies made Res one of the most potent diet-derived chemopreventive agents.

It is known that, under physiological conditions, Res contributes to the maintenance of genome stability.25 However, there is growing evidence that Res may also contribute to chromatin remodeling encompassing events associated with epigenetic alterations that are important in cancer progression.26, 27 Chromatin remodeling is determined by DNA methylation and multiple histone modifications including methylation, ubiquitination, sumoylation, phosphorylation and acetylation.26 Deacetylation of histones is mediated by a histone deacetylase (HDAC) family of enzymes and ensures a locked, inactive chromatin conformation, while histone acetylation catalyzed by histone acetyltransferases (HATs), such as p300, allows their disengagement from DNA, DNA association with transcription factors and gene transcription.28, 29 Increasing evidence imply that epigenetic silencing mechanisms (HDAC activities) play a major role in human carcinogenesis. Recent studies demonstrate that HDAC inhibitors (HDACi) have strong anti-cancer activity, via mechanisms, that are not completely understood.30–32 Several HDACi are now in various stages of clinical trials,33 and Vorinostat (suberoylanilide hydroxamic acid, SAHA) is approved by the Food and Drug Administration (FDA) for treatment of T-cell lymphoma. Unfortunately, the doses required to achieve in vivo activity are very high and frequently produce nonspecific toxic effects. Thus, there is growing interest in identifying pharmacologically safe dietary compounds with HDAC inhibitory activities.34 Resveratrol is one such compound, which alters chromatin conformation by acting directly on sirtuin 1 (silent information regulator 1, Sirt1), a member of the HDAC family35 and indirectly through modulation of p300, which promotes Sirt2 acetylation thus reducing its deacetylation activity.36 Furthermore, Res promotes acetylation of p53 and thus increases the expression of p53 target genes that participate in cell death and growth arrest.37, 38 However, while different posttranslational modifications of p53 induced by Res have been documented,38, 39 the upstream epigenetic events remain unknown.

As mentioned earlier, epigenetic silencing mechanisms play a major role in the development of human cancer, including PCa.40 Metastasis-associated protein 1 (MTA1) is part of the nucleosome remodeling deacetylation (NuRD) complex involved in global and gene-specific histone deacetylation, alteration of chromatin structure and transcriptional repression.41, 42 The complex also contains HDAC1 and HDAC2, CpG-methyl binding protein MBD3 and the ATP-dependent chromatin-remodeling protein Mi2. In the NuRD co-repressor complex, MTA1/HDAC1 subunit plays an essential role in governing deacetylation of histones and nonhistone proteins, such as p53.42, 43 MTA1 is expressed in numerous human cancers, including breast, gastrointestinal, pancreatic and lung, and its expression correlates with tumor aggressiveness and metastasis.44–49 In PCa, MTA1 protein expression is higher in hormone-refractory metastatic PCa compared to clinically localized disease and benign prostatic tissues.50

This study was designed to test the hypothesis that Res exerts its anticancer effects in PCa, in part, through chromatin remodeling. We discovered that Res, at 50–100 μM concentrations, dramatically down-regulates MTA1 protein, a key component of repressive chromatin implicated in cancer progression and metastasis. Our study is the first to identify MTA1 as a new molecular target of Res. We report here, for the first time, a novel pathway of chromatin remodeling by Res through function blockade of the MTA1/NuRD complex: Res initiated MTA1 decrease thus deregulating MTA1/HDAC1 complexes leading to increased p53 acetylation (Ac-p53) and enhanced binding to the p21 and Bax promoters in PCa cells. Furthermore, Res treatment on MTA1-null background caused a dramatic increase in Res's apoptotic function suggesting MTA1 as an anti-apoptotic protein. Finally, combined treatment of Res and SAHA cooperatively increased Ac-p53 levels and apoptosis, suggesting novel therapeutic strategies with the use of HDACi that are already clinically approved.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cells and reagents

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) was purchased from Sigma. Resveratrol in 100% ethanol (EtOH) was stored in the dark at −20°C. LNCaP and DU145 cells were grown in RPMI 1640 (Gibco BRL), 10% FBS and antibiotics. For Res treatment, phenol red-free media containing 10% charcoal-stripped serum was used to provide steroid-free background.

Western blotting

Western blot analysis was performed as described previously.51 Briefly, the cells were maintained in regular RPMI 1640 media, and then media was changed to phenol red-free for treatments. Cells were harvested and lysed in lysis buffer [50 nM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.5% NP40 and 10 mM β-glycerphosphate, pH 8, containing a 1:100 dilution of a protease inhibitor cocktail (Sigma)]. Equal amounts of protein (50–75 μg) were size-fractionated by 7–10% Tris-HCl Ready gels and transferred to nitrocellulose membrane by Mini Trans-Blot Electrophoresis Transfer System (Bio-Rad Laboratories). The membranes were then blocked with TBS-Tween and 5% dry milk for 2 hr at room temperature and subsequently probed with the following antibodies: anti-MTA1 (A-11), anti-p53 (DO-1), anti-HDAC1 (10E2), anti-Bax (P-19) and anti-Bcl-2 (N-19) (Santa Cruz Biotechnology, Inc); anti-Ac-p53 (Lys 382) (Cell Signaling); anti-AR and anti-β-actin (NeoMarkers), which was used as a loading control. Signals were visualized using enhanced chemiluminescence. Images were quantified using Image J software.

Real-time RT-PCR

Cells were cultured in steroid-free medium before treatment with Res. Total RNA was isolated using RNeasy kit (Qiagen). RT-PCR was performed on the MiniOpticon Real-Time PCR system (Bio-Rad Laboratories) using the protocol of the SuperScript III platinum two-step qRT-PCR kit with SYBR green (Invitrogen). Following primers were used: MTA1 sense: 5′-AGC TAC GAG CAG CAC AAC GGG GT-3′, MTA1 antisense 5′-CAC GCT TGG TTT CCG AGG AT-3′; β-actin sense 5′-CGT GGG CCG CCC TAG GCA CCA-3′, β-actin antisense 5′-TTG GCT TAG GGT TCA GGG GGG-3′. The quantitation and analysis was done using Opticon Monitor software v.3.1 and calculations are performed using double-delta Ct method.52

Immunoprecipitation (IP)

Protein A Immunoprecipitation Kit (Roche) was used as recommended. Briefly, cells were harvested after treatments with Res and then lysed in lysis buffer provided. After centrifugation, antibodies (MTA1, AR or IgG) and beads were added to the supernatant and incubated for 1 hr at 4°C. After a series of washes, proteins were analyzed by Western blot with antibodies for MTA1, AR, HDAC1 and p53.

RNA interference

We established stable MTA1 knockdowns in DU145 and LNCaP cells using the Expression Arrest™GIPZ lentiviral shRNAmir vectors expressing shRNA for MTA1 (Open Biosystems). Vectors contained green fluorescence protein (GFP) and puromycin resistance gene for selection of stable transfectants. Packaging psPAX2 and envelope pMD2.G plasmids were purchased from Addgene. Transfection of packaging 293T cells to generate viral particles was done using LipofectAmine2000. To infect cells, transduction cocktail (media, virus, HEPES buffer and polybrene) was added to the cells for 3 days, after which GFP-positive cells were selected by fluorescence-activated cell sorting (FACS). Additional selection for puromycin resistance was applied. For some experiments, cells were transiently transfected with ON-TARGETplus SMARTpool siRNA against MTA1 and NON-Targeting control siRNA (siGENOME) per manufacturers' protocol (Thermo Scientific).

Apoptosis

Cells were plated at 2 × 105 cells/well of a 24-well plate containing round cover slips in complete media. The next day media was changed to phenol red-free media with 2% FBS for 5 hr after which cells were treated with vehicle or Res. After 24 hr treatment, the cells were processed using the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (Millipore). Slides were examined using the fluorescence microscope. Apoptosis was quantified with the MetaMorph software.

Chromatin immunoprecipitation (ChIP)

Resveratrol-treated cells were processed using EZ-ChIP kit (Upstate). Briefly, cells were cross-linked with 1% formaldehyde, washed, sonicated and lysed. IP step was performed using anti-Ac-p53Lys373/382 antibody. DNA amplification was accomplished using primers for p21 and Bax promoter fragments encompassing p53 binding sites.53 p21 sense 5′-GTG GCT CTG ATT GGC TTT CTG-3′, p21 antisense 5′-CTG AAA ACA GGC AGC CCA AG-3′; Bax sense 5′-TAA TCC CAG CGC TTT GGA A-3′; Bax antisense 5′-TGC AGA GAC CTG GAT CTA GCA A-3′. PCR was performed using MJ Mini Thermal Cycler and analyzed by Opticon Monitor software. Calculations were done using ΔΔCt method.52

Statistical analyses

Statistical significance (p values) in mean values of two-sample comparison was determined with unpaired two-tailed Student's t-test. Statistical significance was defined as a p value < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Resveratrol down-regulates MTA1 protein in a dose-dependent manner

Because Res dosage in vitro is typically 1–150 μM in most studies,14–17, 22, 51 we used 1–100 μM range to study the effect of Res on MTA1 expression in PCa cell lines, DU145 and LNCaP. Resveratrol had only modest effects on MTA1 mRNA levels but showed a dose-dependent decrease of MTA1 protein levels (Fig. 1). MTA1 down-regulation was independent of AR status, as it was detected in AR-null DU145 and PC3 (Supporting Information Fig. S1) cells as well as in AR-positive LNCaP cells.

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Figure 1. Res down-regulates MTA1 protein expression in PCa cells. DU145 and LNCaP cells were maintained in regular media then transferred to phenol red-free media with stripped serum. Next day cells were treated for 24 hr with indicated concentrations of Res and vehicle control (EtOH). Western blot was performed, and 80 kDA protein was detected. (a) representative of three independent experiments is shown for each cell line. (b) Effect of Res on the MTA1 mRNA expression. Total RNA was isolated from the cells following treatment with different doses of Res. Real-time PCR was performed as described in Material and Methods. ΔCt for MTA1 was calculated by subtracting the Ct of β-actin mRNA from the Ct of MTA1 mRNA. ΔΔCt was then calculated by subtracting the ΔCt of the EtOH-treated Ctl from the ΔCt of the Res-treated samples. Fold changes for the expression of MTA1 mRNA were calculated by the equation 2−ΔΔCt. Expression levels of MTA1 mRNA are displayed as relative expression over β-actin mRNA with EtOH-treated cells setting at 1.0.

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Resveratrol destabilizes MTA1-containing NuRD complexes

Considering the central role of AR in PCa progression, we examined MTA1/AR interactions using LNCaP cells. Immunoprecipitation followed by Western blot analysis (IP-Western) of MTA1 or AR immunoprecipitates showed the lack of physical association between MTA1 and AR (Fig. 2a). Consistent with previous reports, we detected MTA1 in complexes with HDAC1 and p53.43, 54 Interestingly, these data showed higher association of HDAC1, p53 and MTA1 in control compared to Res-treated cells (Fig. 2a, IP:MTA1). Further IP-Western analysis showed that between 24 and 72 hr the amount of MTA1/HDAC1 complexes dropped by more than 50% (Fig. 2b, IP:MTA1), suggesting that when MTA1 levels are low, the stability of the complexes is decreased. In contrast, total MTA1 levels (Fig. 2b, Cell lysate) declined by 80% at 24 hr and remained relatively stable up to 72 hr whereas only a slight decrease in total HDAC1 levels was detected in Res-treated cells. Thus, Res treatment resulted in decrease of the MTA1/HDAC1 in the NuRD complexes, likely leading to deregulation of their function.

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Figure 2. Destabilization of the MTA1/HDAC1/p53 complexes by Res. (a) MTA1 is not in direct contact with AR, and Res decreases MTA1-associated proteins' levels. LNCaP cells were treated with EtOH (Ctl) and Res (50, 100 μM) for 24 hr. Cell lysates were precipitated with antibody for MTA1 and AR followed by Western blot for the presence of MTA1, HDAC1, AR and p53 (left). Cell lysates were precipitated with the same antibodies (right). (b) Graphic representation of MTA1 and HDAC1 levels at 24 and 72 hr, normalized against loading control (IgG). The Ctl was set to 100% and Res-induced changes in protein levels are expressed as a percentage of Ctl. Note the decrease in MTA1 and HDAC1 levels at 72 hr in immunocomplexes but not in total cell lysates.

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Destabilization of MTA1/HDAC1 complexes by Res is accompanied by increased p53 acetylation

As MTA1 and HDAC1 cooperate in maintaining p53 deacetylated state and repressing p53-dependent gene transcription,43, 55 we hypothesized that MTA1 degradation and subsequent inhibition of MTA1/HDAC1 complexes by Res may reverse p53 deacetylation and repression of target genes in PCa cells. To test this hypothesis, we measured p53 acetylation (Lys 382) in Res-treated DU145 and LNCaP cells, which express mutant (mt) p53 and wild type (wt) p53, respectively. Resveratrol treatment for 24 hr reduced MTA1 levels and elevated both total and Ac-p53 levels; HDAC1 levels were unaffected (Fig. 3a). Basal levels of mt p53 in untreated DU145 cells were higher than those of wt p53 in LNCaP, due to its shorter half-life.55 Upon Res treatment, p53 became acetylated, which resulted in stabilization and accumulation of the protein. Notably, in Res-treated LNCaP cells, the ratio of Ac-p53/total p53 was 14-fold higher than the baseline, whereas in Res-treated DU145 (mt p53), this ratio was increased only 2-fold (Fig. 3b). Nevertheless, it was apparent that Res, simultaneously with MTA1 down-regulation, increased acetylation of preferably wt but also mt p53 thus activating p53-mediated pathways in PCa cells.

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Figure 3. (a) Res -induced MTA1 down-regulation is concomitant with increased p53 acetylation. DU145 and LNCaP cells were treated with EtOH and Res (50 μM) for 24 hr. Western blot was performed and quantified as described in Material and Methods. (b) The levels of Ac-p53 and total p53 were normalized against β-actin and the ratios calculated; mean values and standard deviations are calculated from three independent. *p < 0.05.

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MTA1 knockdown enhances p53 acetylation and transcriptional activation by Res

To further investigate how MTA1 affects Res-dependent increase in p53 acetylation, we silenced MTA1 using lentiviral shRNAmir in DU145 and LNCaP cells (Supporting Information Fig. S2) and measured levels of p53 and Ac-p53 in response to Res (Fig. 4a). MTA1 silencing alone produced only slight increase in Ac-p53 (compare Lanes 3 to 1 for each cell line). Remarkably, Ac-p53 increase by Res in MTA1-null background was much more dramatic, especially for wt p53 (LNCaP cells) (Fig. 4a, lane 4). These data indicate that MTA1 silencing sensitized the cells to Res-dependent p53 acetylation, and support the involvement of MTA1 in Res-mediated p53 acetylation.

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Figure 4. A. Knockdown of MTA1 sensitized PCa cells to Res-induced p53 acetylation. DU145 and LNCaP cells infected with lentiviral vectors expressing shRNAmir (MTA1 shRNA) or control (Ctl shRNA) (Supporting Information Fig. S2) were treated with 50 μM Res for 18 hr. Cell lysates were analyzed by Western blot for MTA1, Ac-p53, total p53 and β-actin. Lanes 1 and 2 for each cell line are cells expressing MTA1, and Lanes 3 and 4 are MTA1 knockdowns. (a) Representative experiment of three independent ones is shown. (b) Knockdown of MTA1 increased promoter recruitment of Ac-p53 in response to Res. Cells were harvested for ChIP, and real- time PCR was done with primers specific to promoters of p21 and Bax (Material and Methods). Fold enrichment over input and EtOH-treated cells was calculated using ΔΔCt method.52 Data from 3 independent experiments were pooled together, mean values and standard errors calculated. (c) MTA1 silencing potentiates Res-induced apoptosis in PCa cells. Left, DU145 cells were transiently tranfected with Ctl siRNA or MTA1 siRNA. The cells were treated with 50 μM of Res or EtOH for 24 hr and apoptosis was measured as described in Material and Methods. Digital images were taken using the Kodak Diaphot 2000 fluorescence microscope. Representative images are shown. Red, DAPI nuclear staining, healthy cells; Green, FITC signal, damaged cells; Yellow, overlay, apoptotic cells. (d) Apoptotic cells were counted using MetaMorph software in 10 random 20× fields and percent was calculated. Mean values and standard errors were calculated using data from three independent experiments. Comparison between groups was performed using unpaired two-tailed Student's t test. *p < 0.001. (e) MTA1 silencing potentiates Res-induced increase in pr-oapoptotic Bax protein. LNCaP cells expressing shRNAmir (MTA1 shRNA) or control (Ctl shRNA) were treated with 50 μM Res for 18 hr. Cell lysates were analyzed by Western blot for Bax (23 kDa), Bcl-2 (26 kDa) and β-actin. Lanes 1 and 2 are cells expressing MTA1, and Lanes 3 and 4 are MTA1 knockdowns. A representative experiment is shown.

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As a key transcriptional regulator of genes involved in growth arrest and apoptosis, the Ac-p53 has increased transcriptional activity, promotes co-activator recruitment and enhances site-specific DNA binding.56 We examined the effect of Res and MTA1 on p53 occupancy of the promoters of two p53-responsive pro-apoptotic genes, p21 and Bax using ChIP assay. We found maximal Ac-p53 recruitment at 18 hr of Res-treatment (data not shown) and used this time point in our subsequent experiments. We found that MTA1 silencing combined with Res treatment (MTA1 shRNA) increased p21 and Bax promoter contents compared with Res-treated cells that express MTA1 (Ctl shRNA): for mt p53 the increase was 6.2 fold for p21 and 4.3-fold for Bax whereas for wt p53 fold increases were less pronounced (1.7-fold for p21 and 2.9-fold for Bax). These results indicate that MTA1 knockdown allows maximal acetylation activity for both mt and wt p53 due to Res (Fig. 4b).

MTA1 silencing sensitizes PCa cells to Res-induced apoptosis

To demonstrate the functional involvement of MTA1 in Res-mediated effects, we examined Res-induced apoptosis in DU145 cells expressing MTA1 and silenced for MTA1 (Fig. 4c). Cells transfected with control, nontarget siRNA and treated with Res displayed about the same 5–7% of apoptotic cells as untreated MTA1 siRNA cells. Importantly, in MTA1 knockdown cells Res induced over 40% apoptosis (Fig. 4d). These results indicate that MTA1 silencing induced cell death by itself and significantly increased the sensitivity to Res. We next assessed whether pro-apoptotic Bax protein and anti-apoptotic Bcl-2 were modulated by MTA1 silencing and Res. LNCaP cells expressing MTA1 (Ctl shRNA) and silenced for MTA1 (MTA1 shRNA) were treated with EtOH or 50 μM Res, and the levels of Bax and Bcl-2 was evaluated by Western blot (Fig. 4e). Res increased pro-apoptotic Bax protein levels (2.6-fold) and decreased anti-apoptotic Bcl-2 levels (1.5-fold) in control cells. Notably, Bax was significantly increased in MTA1 knockdown cells (2.56-fold) (compare Lanes 3 to 1) and achieved further increase when cells were treated with Res (2.9-fold) (compare Lanes 4 and 1). Accordingly, Bcl-2 levels were decreased by Res with slight additional effect from MTA1 silencing (1.6-fold, Lanes 4 and 1). These data one more time point out that MTA1 is anti-apoptotic protein and that MTA1 silencing alone can induce apoptosis. More importantly, MTA1 silencing sensitizes PCa cells to Res-induced apoptosis.

Res and SAHA cooperate in inducing p53 acetylation and apoptosis

Our findings suggested that the inhibition of MTA1/HDAC1 complexes facilitates the acetylation and apoptotic action of Res. We therefore asked if SAHA, a clinically approved HDACi,57 would have similar effects. We examined effects of these 2 compounds alone and in combination on the levels of p53, Ac-p53 and HDAC1 in DU145 and LNCaP cells (Fig. 5a). Res alone up-regulated levels of total and Ac-p53 in both DU145 and LNCaP cells but to different extent: up-regulation of Ac-p53/ p53 ratio was not significant with mt p53 while it was statistically significant with wt p53 (Fig. 5a). Contrary to its expected affect as HDACi, SAHA alone down-regulated mt p53 (total and acetylated) and wt p53 (total). Ac-wt p53 levels remained unaltered compared to control untreated cells. However, SAHA treatment amplified Res-dependent increase of the Ac-p53/p53 ratio in DU145 cells and had a profound synergistic effect on accumulation of wt Ac-p53 in LNCaP cells (Fig. 5a and 5b). Neither Res nor SAHA altered HDAC1 levels, the observation with SAHA consistent with other reports.58 We next examined the effect of Res and SAHA alone and in combination on cell death and found that the combined treatment significantly increased apoptosis in DU145 cells compared to SAHA alone (Fig. 5c and 5d).

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Figure 5. (a) SAHA potentiates Res-induced p53 acetylation. DU145 and LNCaP cells were treated with Res (50 μM), SAHA (10 μM) and combinations (R + S). Cell lysates were collected for Western blot. (b) The levels of Ac-p53 and total p53 were normalized to β-actin and the ratios were calculated; mean values and standard errors are calculated from 3 independent experiments. *p < 0.05; **p < 0.01. (c) Combined treatment by SAHA and Res increased apoptosis. Apoptosis and imaging were performed as described in Figure 4c. Apoptotic cells appear yellow on overlayed images. (d) Apoptotic cells were counted using MetaMorph software in 10 random 20× fields and percent was calculated. Mean values and standard errors were calculated using data from 3 independent experiments. Comparison between groups was performed using unpaired two-tailed Student's t test. *p < 0.01; **p < 0.001.

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In summary, we discovered a novel epigenetic mechanism of the anticancer action of Res where MTA1 down-regulation leads to increased p53 acetylation and pro-apoptotic gene activation. Our results suggest MTA1 as a new molecular target of Res and point to the Res and HDACi combination as a possible effective therapeutic strategy against PCa.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

To our knowledge, this is the first report that describes a mechanism of p53 acetylation by Res, which involves the MTA1/NuRD complex and underscores the epigenetic, chromatin remodeling capacity of Res as an anticancer agent. In this study, we found that Res potently increased p53 acetylation and its transcriptional activation of pro-apoptotic genes if MTA1 was eliminated from NuRD repressor complex. We showed that Res down-regulated MTA1 protein levels in a dose-dependent manner in three PCa cell lines (Fig. 1a and Supporting Information Fig. S1). Because in these studies we found that the maximal MTA1 inhibition in three PCa cell lines occurred at higher than 50 μM, we used 50 or 100 μM Res in the subsequent experiments. Although concentrations of 10 μM or less is considered to be physiologically relevant, available data are insufficient to predict levels in most tissues due to low bioavailability and extensive metabolism of Res.59 In addition, in vitro and in vivo responses to the same dose did not necessarily correlate. The fact that Res can inhibit MTA1 protein levels may have clinical significance since MTA1 overexpression in several types of human cancer correlates with clinicopathological parameters typical for advanced, aggressive tumors such as frequent lymph node metastasis and increased tumor grade and angiogenesis.46–49, 60

In our study, Res had no effect on MTA1 mRNA level; however, MTA1 protein levels were dramatically decreased by Res. We have ruled out the possibility of caspase-dependent cleavage of MTA1 and our accumulated data (not shown) suggesting involvement of ubiquitin-proteasome system. Res facilitated MTA1 degradation with fast kinetics: ubiquitinated MTA1 could be detected after 1 hr of treatment and was completely cleared after 24 hr (data not shown). Because ubiquitin is not only a modifier of proteolysis, but also a general post-translational signaling molecule that can participate in protein relocation,61 one might presume that Res-stimulated MTA1 ubiquitilation also may affect its accumulation in cytoplasm and prevent MTA1 translocation to the nucleus. A detailed analysis of these potential mechanisms deserves further investigation and is outside the scope of this manuscript.

MTA1 is an integral part of the NuRD multiprotein complex, which functions to maintain locked chromatin conformation by deacetylating histone and non-histone proteins.41, 42, 45 In breast cancer cells, MTA1 directly interacts with estrogen receptor α (ERα) and represses its transactivation function through HDAC1 and HDAC2.62 We therefore asked whether MTA1 could also bind AR in PCa cells. We found, however, that neither MTA1 was in contact with AR (Fig. 2) nor its expression was AR-dependent, as the treatment of AR-positive LNCaP cells with DHTα and/or antiandrogen flutamide had no effect on MTA1 (or HDAC1) protein levels while regulating PSA levels (Supporting Information Fig. S3a). This finding is consistent with our original observation where MTA1 down-regulation by Res in different PCa cells was not affected by their AR status. Because Res is a phytoestrogen, which binds to both ERα and ERβ with high affinity,63, 64 we examined whether MTA1 down-regulation by Res was ER-mediated. We treated PC3 cells that are shown to express both ERs65 with Res, 17-β estradiol (E2), 4-hydroxytamoxifen (4OHT) and pure antiestrogen Fulvestrant (Fulv) to manipulate ER activity and found that E2, 4OHT had no effects on MTA1 and Fulvestrant failed to alter Res effect on MTA1 levels, suggesting ER-independent mechanism of action (Supporting Information Fig. S3b and S3c).

Given the role of MTA1 role in NuRD complex, its down-regulation by Res was likely to affect NuRD components. Indeed, Res treatment decreased amount of HDAC1 in MTA1 immunoprecipitates. This can be attributed to a decreased MTA1 level due to degradation; however, it is also possible that Res treatment dissociated or decreased association between MTA1 and HDAC1 as the amount of HDAC1 went down in IP experiments dramatically compared to effects in cell lysates. Therefore, it is reasonable to suggest that Res also lowers the stability of MTA1/NuRD complex thus deregulating their function on deacetylation of histones and nonhistone proteins. Indeed, we were able to show that down-regulation of MTA1 by Res was concomitant with up-regulation of both total and Ac-p53 in cells expressing wt p53 and mt p53. Unexpectedly, MTA1 knockdown alone failed to show increased p53 acetylation; however, p53 acetylation by Res was significantly higher upon MTA1 silencing. Predictably, increased p53 acetylation dictated increase occupancy of the p21 and Bax promoters although with differences between cells with mt p53 and wt p53, possibly due to elevated basal occupancy of both promoters by Ac-wt p53. This is consistent with known distinct promoter selectivity of the mt p53 and wt p53 or their ability to recruit different protein complexes to the same promoters.66, 67 MTA1 silencing also dramatically enhanced apoptosis by Res while MTA1 silencing alone caused apoptosis at a level similar to Res alone in MTA1-positive cells. The results of apoptosis detected by in situ method were consistent with the increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 proteins detected by western blot. Our results suggest that MTA1 acts as a brake on Res-dependent p53 protein acetylation, which is thought to be mediated by HAT, namely p300.38 Therefore, eradication of deacetylation provided by MTA1 knockdown and boost of acetylation provided by Res-induced activation of p300 results in the synergistic effect of combined MTA1 silencing and Res treatment.

Because Res acted as an indirect HDACi by down-regulating MTA1, we investigated the potential use of Res in combination with clinically approved HDACi SAHA. SAHA blocks HDACs' activity by displacing zinc from the site in the catalytic pocket of HDACs68 ultimately leading to accumulation of acetylated histones and nonhistone proteins and chromatin alterations.69 We hypothesized that because these two compounds act through different mechanisms, combined treatment might result in enhanced anticancer activity. Indeed, in our study, SAHA alone had no effect on wt p53 levels and acetylation and down-regulated both total and acetylated mt p53 levels. Similar results on mt p53 and wt p53 regulation by SAHA were recently reported for breast cancer cells.70 Importantly, when combined with Res, SAHA synergistically increased wt p53 acetylation (the effect on mt p53 was insignificant) pointing to a selective activation of wt p53 signaling pathways, the hallmark of successful anticancer therapy. Although the exact mechanisms of Res and SAHA are unknown, this is the first demonstration of combined SAHA and Res treatment resulting in substantial increase in wt p53 acetylation. These results open new avenues for combination therapies with Res and its potent analogues/derivatives and chemotherapeutic HDAC inhibitors.

It has been shown that Res can cause apoptosis through p53-dependent and -independent pathways.71, 72 SAHA, on the other hand, causes p21 induction and apoptosis mainly in a p53-independent manner.70, 73–77 Our apoptosis experiments underscore the role of MTA1 in apoptosis, regardless of p53 status of PCa cells. Indeed, MTA1 cooperates with Res even in the presence of defective mt p53 (223Leu and 274Phe mutations in both alleles cooperate to gain oncogenic functions).78 It is possible that by inhibiting MTA1 and promoting mt p53 acetylation, Res may secure the conformation of the mt p53-associated protein complexes favorable for the activation of pro-apoptotic genes. Once more, in the combination experiments, we found that Res significantly (p < 0.01) enhanced apoptosis caused by SAHA alone.

In conclusion, we propose a novel mechanism of Res chromatin remodeling anticancer activity mediated through MTA1/NuRD co-repressor complexes (Fig. 6). Our data demonstrate that Res destabilizes MTA1/NuRD suppressor complex by inhibiting MTA1 protein, which results in p53 acetylation, increased stability and transcriptional activity. We also demonstrated that although MTA1 silencing does not directly increase acetylation it renders cells more sensitive to the effects of Res by maximizing its acetylation function, which augments p53 acetylation and promoter binding. In addition, we showed that MTA1 is an anti-apoptotic protein as its silencing not only caused apoptosis but strongly facilitates induction of apoptosis by Res. Moreover, we showed that adding HDACi SAHA to Res treatment mimics, at least in part, MTA1 silencing and may evolve as a new anticancer strategy since SAHA is a FDA approved anticancer agent57 and Res is a pharmacologically safe dietary compound.3 Finally, defining a novel epigenetic pathway of Res anticancer activity opens new opportunities for the development of preventive and treatment strategies based on Res and its potent analogues. In addition, as a result of our research, Res may be considered as chemotherapeutic agent of potent synergistic activity with HDAC inhibitors.

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Figure 6. Proposed mechanism of chromatin remodeling by Res: the blockade of MTA1/NuRD-mediated p53 deacetylation. (a) In the absence of Res, MTA1/HDAC1/NuRD complexes are intact and repress transcriptional activation of the genes implicated in cell cycle and apoptosis (p21 and Bax) through deacetylation, which results in cell cycle progression and survival. (b) Resveratrol causes MTA1 down-regulation; decreased MTA1 levels destabilize MTA1/HDAC1 interactions in NuRD complexes, decreasing NuRD activity, which results in accumulation of p53 accessible for acetylation. In parallel, Res switches on acetylation mechanisms by HATs increasing global protein acetylation, including p53. Increased acetylation of chromatin elements by Res favors transcriptional activation of genes implicated in cell cycle arrest and apoptosis.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Drs. Volpert and Mirochnik for providing SAHA and valuable discussions. They especially thank Dr. S. Rosen, the director of Robert H Lurie Comprehensive Cancer Center of Northwestern University, for his support (Director's Fund).

References

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  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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