MSRE-PCR for analysis of gene-specific DNA methylation

Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/μl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes

Stilbenes Inhibit Androgen Receptor Expression in 22Rv1 Castrate-resistant Prostate Cancer Cells

Androgen receptor (AR) signaling plays an important role in the development and progression of prostate cancer (PCa). Importantly, AR continues to be expressed in advanced castrate-resistant PCa (CRPC), where the AR can have ligand-independent activity. Identification of naturally occurring substances that can inhibit AR expression holds promise for PCa chemoprevention and therapy. Earlier research demonstrated that resveratrol (Res) inhibited androgenpromoted growth, AR expression, and transactivation in androgen-responsive non-metastatic LNCaP PCa cells. In the current study, the effects of Res and three natural analogs [trimethoxyresveratrol (3M-Res), pterostilbene (Pter), and piceatannol (Pic)] were investigated for effects on the growth of 22Rv1 castrate-resistant cells that express full-length (AR114/110) and truncated form (AR80) of AR. Although all the stilbenes tested inhibited the proliferation of 22Rv1 cells in a dose-dependent manner, 3M-Res was the most potent inhibitor. While AR114/110 responded to the syn-thetic androgen agonist methyltrienolone (R1881) as well as to antiandrogen flutamide, AR80, which lacks a ligand-binding domain, did not respond to R1881, but was inhibited by flutamide. Interestingly, Res, Pter, and Pic, but not 3M-Res, similar to flutamide, inhibited both AR114/110 and AR80 with the effect on AR80 being more prominent with the use of high concentrations of stilbenes. Collectively, the data indicate that both AR-independent (3M-Res) and possibly AR-dependent (Res, Pter, and Pic) mechanisms of cell growth inhibition occurs via these stilbenes. These findings provide evidence for plant-derived stilbenes as attractive and promising pharmacologically safe compounds to be used for diminishing progression and curb worsening of CRPC

In Vitro Anticancer Properties of Table Grape Powder Extract (GPE) in Prostate Cancer

Although the link between diet and cancer is complex, epidemiological data confirm that diet is a risk factor for prostate cancer and indicate a reduced prostate cancer incidence associated with a diet rich in vegetables and fruits. Because of the known protective effect of grape seed extract (GSE) against prostate cancer, we evaluated the effects of grape powder extract (GPE) on cell viability, proliferation, and metastatic capability. Importantly, we explored the possible novel mechanism of GPE through metastasis-associated protein 1 (MTA1) downregulation in prostate cancer, since our previous studies indicated resveratrol (Res)- and pterostilbene (Pter)-induced MTA1-mediated anticancer activities in prostate cancer. We found that GPE inhibited the cell viability and growth of prostate cancer cells only at high 100 &#956;g/mL concentrations. However, at low 1.5&#8315;15 &#956;g/mL concentrations, GPE significantly reduced the colony formation and wound healing capabilities of both DU145 and PC3M cells. Moreover, we found that GPE inhibited MTA1 in a dose-dependent manner in these cells, albeit with considerably less potency than Res and Pter. These results indicate that stilbenes such as Res and Pter specifically and potently inhibit MTA1 and MTA1-associated proteins compared to GPE, which contains low concentrations of Res and mainly consists of other flavonoids and anthocyanidins. Our findings support continued interest in GPE as a chemopreventive and anti-cancer agent against prostate cancer but also emphasize the unique and specific properties of stilbenes on MTA1-mediated anticancer effects on prostate cancer

Gnetin C Intercepts MTA1-Associated Neoplastic Progression in Prostate Cancer

Nutritional chemoprevention is particularly suitable for prostate cancer. Gnetin C, a resveratrol dimer found abundantly in the melinjo plant (Gnetum gnemon), may possess more potent biological properties compared to other stilbenes. We examined the effects of gnetin C in a high-risk premalignant transgenic mouse model overexpressing tumor-promoting metastasis-associated protein 1 (MTA1) on the background of Pten heterozygosity (R26MTA1; Pten+/f; Pb-Cre+). Mice were fed diets supplemented with the following compounds: pterostilbene (70 mg/kg diet); gnetin C, high dose (70 mg/kg diet); and gnetin C, low dose (35 mg/kg diet). Prostate tissues were isolated after 17 weeks and examined for histopathology and molecular markers. Serum was analyzed for cytokine expression. Gnetin C-supplemented diets substantially delayed the progression of preneoplastic lesions compared to other groups. Prostate tissues from gnetin C-fed mice showed favorable histopathology, with decreased severity and number of prostatic intraepithelial neoplasia (PIN) foci, reduced proliferation, and angiogenesis. A decreased level of MTA1, concurrent with the trend of increasing phosphatase and tensin homolog expression and reduced interleukin 2 (IL-2) levels in sera, were also detected in gnetin C-fed mice. Importantly, gnetin C did not exert any visible toxicity in mice. Our findings demonstrate that a gnetin C-supplemented diet effectively blocks MTA1-promoted tumor progression activity in high-risk premalignant prostate cancer, which indicates its potential as a novel form of nutritional interception for prostate cancer chemoprevention

MTA1 drives malignant progression and bone metastasis in prostate cancer

Prostate cancer often metastasizes to the bone, leading to morbidity and mortality. While metastasis‐associated protein 1 (MTA1) is highly overexpressed in metastatic tumors and bone metastatic lesions, its exact role in the development of metastasis is unknown. Here, we report the role of MTA1 in prostate cancer progression and bone metastasis in vitro and in vivo. We found that MTA1 silencing diminished formation of bone metastases and impaired tumor growth in intracardiac and subcutaneous prostate cancer xenografts, respectively. This was attributed to reduced colony formation, invasion, and migration capabilities of MTA1 knockdown cells. Mechanistic studies revealed that MTA1 silencing led to a significant decrease in the expression of cathepsin B (CTSB), a cysteine protease critical for bone metastasis, with an expected increase in the levels of E‐cadherin in both cells and xenograft tumors. Moreover, meta‐analysis of clinical samples indicated a positive correlation between MTA1 and CTSB. Together, these results demonstrate the critical role of MTA1 as an upstream regulator of CTSB‐mediated events associated with cell invasiveness and raise the possibility that targeting MTA1/CTSB signaling in the tumor may prevent the development of bone metastasis in prostate cancer

Pterostilbene Acts through Metastasis-Associated Protein 1 to Inhibit Tumor Growth, Progression and Metastasis in Prostate Cancer

<div><p>The development of natural product agents with targeted strategies holds promise for enhanced anticancer therapy with reduced drug-associated side effects. Resveratrol found in red wine, has anticancer activity in various tumor types. We reported earlier on a new molecular target of resveratrol, the metastasis-associated protein 1 (MTA1), which is a part of nucleosome remodeling and deacetylation (NuRD) co-repressor complex that mediates gene silencing. We identified resveratrol as a regulator of MTA1/NuRD complex and re-activator of p53 acetylation in prostate cancer (PCa). In the current study, we addressed whether resveratrol analogues also possess the ability to inhibit MTA1 and to reverse p53 deacetylation. We demonstrated that pterostilbene (PTER), found in blueberries, had greater increase in MTA1-mediated p53 acetylation, confirming superior potency over resveratrol as dietary epigenetic agent. In orthotopic PCa xenografts, resveratrol and PTER significantly inhibited tumor growth, progression, local invasion and spontaneous metastasis. Furthermore, MTA1-knockdown sensitized cells to these agents resulting in additional reduction of tumor progression and metastasis. The reduction was dependent on MTA1 signaling showing increased p53 acetylation, higher apoptotic index and less angiogenesis <i>in vivo</i> in all xenografts treated with the compounds, and particularly with PTER. Altogether, our results indicate MTA1 as a major contributor in prostate tumor malignant progression, and support the use of strategies targeting MTA1. Our strong pre-clinical data indicate PTER as a potent, selective and pharmacologically safe natural product that may be tested in advanced PCa.</p> </div

Chemical structures of stilbenes.

<p>Resveratrol (Res), <i>trans</i>-3,5,4′-trihydroxystilbene; Pterostilbene (PTER), <i>trans</i>-3,5-dimethoxystilbene; Trimethoxy-Resveratrol (3M-Res), <i>trans</i>-3,5,4′-trimethoxystilbene; Piceatannol (PIC), <i>trans</i>-3,5,3′4′-tetrahydroxystilbene; Dimethoxystyrylaniline (DMSA), <i>trans</i>-4-(3,5-dimethoxystyryl)aniline; Diacetyl-Resveratrol (2Ac-Res), <i>trans</i>-3,5-diacetylstilbene; Triacetyl-Resveratrol (3Ac-Res), <i>trans</i>-3,5,4′-triacetylstilbene.</p