Dual targeting of EZH2 and androgen receptor as a novel therapy for castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) emerges after androgen withdrawal therapy and remains incurable due to the lack of effective treatment protocols. Treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, offers an initial response followed by drug resistance and tumor relapse. Enhancer of zeste homolog 2 (EZH2), a member of PRC2 complex, is an important target that acts as a coactivator of AR-mediated gene suppression whose oncogenic activity increases during castration. We hypothesize that dual targeting of EZH2 and AR could be highly effective in CRPC treatment. The present study aimed to examine the effectiveness of combination using EZH2 inhibitor GSK126 with antiandrogen enzalutamide in the treatment of CRPC cells. Treatment of 22Rv1 and C42B CRPC cells with a combination of GSK126 and enzalutamide led to synergistic inhibition of cell proliferation, cell cycle arrest and marked increase in cell death. Mechanistically, this combination treatment significantly reduced expression of AR and AR-v7, decrease in PSA and Akt activity, diminution of EZH2 and other members of PCR2 complex including SUZ12 and EED, with simultaneous loss of H3K27 trimethylation and dissociation between AR and PRC2 complex members compared to individual treatment. This study provides preclinical proof-of-concept that combined treatment of EZH2 inhibitor with AR antagonist results in synergistic anticancer effects opening new possibilities for treatment of CRPC tumors.

Role of class I histone deacetylases in the regulation of maspin expression in prostate cancer.

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Oxidative Stress and Antioxidant Status in High-Risk Prostate Cancer Subjects.

The oxidant/antioxidant balance has been implicated in the pathophysiology of prostate cancer. We investigated oxidative damage and antioxidant status in high-risk prostate cancer subjects. Reduced glutathione (GSH) levels were measured in erythrocytes, 8-hydroxydeoxyguanosine (8-OHdG) in leukocytes and plasma levels of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-R), glutathione S-transferase (GST), superoxide dismutase (SOD), and lipid peroxide products were measured in high-risk and age-matched healthy subjects. Serum PSA levels were significantly higher (p < 0.0001) in high-risk subjects, whereas GST (p < 0.0001) and GSH (p < 0.002) were higher in healthy controls. Levels of 8-OHdG, an oxidized nucleoside of DNA, were significantly increased (p < 0.0001) in high-risk subjects. No marked difference in the levels of CAT (p = 0.237), GSH-Px (p = 0.74), GSH-R (p = 0.344), SOD (p = 0.109), and lipid peroxide products (p = 0129) were observed between two groups. Pearson's correlation between GST and PSA (r = -0.69 (p < 0.0001)), GST and 8-OHdG (r = -0.62 (p < 0.0004)), GSH and 8-OHdG (r= -0.39 (p = 0.038)), and CAT and GSH-Px (r= -0.33 (p = 0.04)) were found to be negatively correlated, whereas 8-OHdG and PSA were positively associated (r= 0.57 (p < 0.002). These results indicate a significant role of oxidative damage in prostate carcinogenesis, particularly during the early stages of development. In conclusion, our data support the importance of antioxidant defense as a valuable diagnostic and/or prognostic marker in prostate cancer.

Acquisition of tumorigenic potential and therapeutic resistance in CD133+ subpopulation of prostate cancer cells exhibiting stem-cell like characteristics.

The role of CD133 (Prominin-1) as a cancer stem cell marker may be useful for therapeutic approaches and prognostication in prostate cancer patients. We investigated the stem-cell-related function and biological features of a subpopulation of CD133+ cells isolated from established primary human prostate cancer cell lines. The CD133+ cells sorted from human prostate cancer 22Rv1 exhibited high clonogenic and tumorigenic capabilities, sphere forming capacity and serially reinitiated transplantable tumors in NOD-SCID mice. Gene profiling analysis of CD133+ cells showed upregulation of markers of stem cell differentiation (CD44, Oct4, SOX9 and Nanog), epithelial-to-mesenchymal transition (c-myc and BMI1), osteoblastic differentiation (Runx2), and skeletal morphogenesis (BMP2), compared to side population of CD133- cells. These cells are highly malignant and resistant to γ-radiation and chemotherapeutic drug, docetaxel. Importantly, a docetaxel-resistant subclone was more enriched in CD133+ cells with significant increase in Runx2 expression, compared to CD133- cells. Furthermore, knockdown of Runx2 in these cells resulted in differential response to chemotherapy, sensitizing them to increased cell death. These results demonstrate therapy-resistant population with stem-like features are distinct subpopulation of malignant cells that resides within parental cell lines. The molecular signature of CD133+ cells may lead to identification of novel therapeutic targets and prognostic markers in the treatment of prostate cancer.

MicroRNAs in prostate cancer: Functional role as biomarkers.

MicroRNAs (miRNAs) are small endogenous non-coding molecules that alters gene expression through post-transcriptional regulation of messenger RNA. Compelling evidence suggest the role of miRNA in cancer biology having potential as diagnostic, prognostic and predictive biomarkers. This review summarizes the current knowledge on miRNA deregulated in prostate cancer and their role as oncogene, tumor suppressor and metastasis regulators. The emerging information elucidating the biological function of miRNA is promising and may lead to their potential usefulness as diagnostic/prognostic markers and development as effective therapeutic tools for management of prostate cancer.

Correction: Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases.

[This corrects the article DOI: 10.1371/journal.pone.0162956.].

Dietary Flavones as Dual Inhibitors of DNA Methyltransferases and Histone Methyltransferases.

Methylation of DNA and histone proteins are mutually involved in the epigenetic regulation of gene expression mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). DNMTs methylate cytosine residues within gene promoters, whereas HMTs catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins, thus causing chromatin condensation and transcriptional repression, which play an important role in the pathogenesis of cancer. The potential reversibility of epigenetic alterations has encouraged the development of dual pharmacologic inhibitors of DNA and histone methylation as anticancer therapeutics. Dietary flavones can affect epigenetic modifications that accumulate over time and have shown anticancer properties, which are undefined. Through DNA binding and in silico protein-ligand docking studies with plant flavones viz. Apigenin, Chrysin and Luteolin, the effect of flavones on DNA and histone methylation was assessed. Spectroscopic analysis of flavones with calf-thymus DNA revealed intercalation as the dominant binding mode, with specific binding to a GC-rich sequence in the DNA duplex. A virtual screening approach using a model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones are tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Epigenetic studies performed with flavones exhibited a decrease in DNMT enzyme activity and a reversal of the hypermethylation of cytosine bases in the DNA and prevented cytosine methylation in the GC-rich promoter sequence incubated with the M.SssI enzyme. Furthermore, a marked decrease in HMT activity and a decrease in EZH2 protein expression and trimethylation of H3K27 were noted in histones isolated from cancer cells treated with plant flavones. Our results suggest that dietary flavones can alter DNMT and HMT activities and the methylation of DNA and histone proteins that regulate epigenetic modifications, thus providing a significant anticancer effect by altering epigenetic processes involved in the development of cancer.

Dietary phytochemicals as epigenetic modifiers in cancer: Promise and challenges.

The influence of diet and environment on human health has been known since ages. Plant-derived natural bioactive compounds (phytochemicals) have acquired an important role in human diet as potent antioxidants and cancer chemopreventive agents. In past few decades, the role of epigenetic alterations such as DNA methylation, histone modifications and non-coding RNAs in the regulation of mammalian genome have been comprehensively addressed. Although the effects of dietary phytochemicals on gene expression and signaling pathways have been widely studied in cancer, the impact of these dietary compounds on mammalian epigenome is rapidly emerging. The present review outlines the role of different epigenetic mechanisms in the regulation and maintenance of mammalian genome and focuses on the role of dietary phytochemicals as epigenetic modifiers in cancer. Above all, the review focuses on summarizing the progress made thus far in cancer chemoprevention with dietary phytochemicals, the heightened interest and challenges in the future.

Apigenin blocks IKKα activation and suppresses prostate cancer progression.

IKKα has been implicated as a key regulator of oncogenesis and driver of the metastatic process; therefore is regarded as a promising therapeutic target in anticancer drug development. In spite of the progress made in the development of IKK inhibitors, no potent IKKα inhibitor(s) have been identified. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone apigenin as a specific IKKα inhibitor. Here we report apigenin, in micro molar range, inhibits IKKα kinase activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays and exhibits anticancer efficacy in experimental tumor model. We found that apigenin directly binds with IKKα, attenuates IKKα kinase activity and suppresses NF-ĸB/p65 activation in human prostate cancer PC-3 and 22Rv1 cells much more effectively than IKK inhibitor, PS1145. We also showed that apigenin caused cell cycle arrest similar to knockdown of IKKα in prostate cancer cells. Studies in xenograft mouse model indicate that apigenin feeding suppresses tumor growth, lowers proliferation and enhances apoptosis. These effects correlated with inhibition of p-IKKα, NF-ĸB/p65, proliferating cell nuclear antigen and increase in cleaved caspase 3 expression in a dose-dependent manner. Overall, our results suggest that inhibition of cell proliferation, invasiveness and decrease in tumor growth by apigenin are mediated by its ability to suppress IKKα and downstream targets affecting NF-ĸB signaling pathways.

Cancer epigenetics: an introduction.

Epigenetic and genetic alterations contribute to cancer initiation and progression. Epigenetics refers to the study of heritable changes in gene expression without alterations in DNA sequences. Epigenetic changes are reversible and include key processes of DNA methylation, chromatin modifications, nucleosome positioning, and alterations in noncoding RNA profiles. Disruptions in epigenetic processes can lead to altered gene function and cellular neoplastic transformation. Epigenetic modifications precede genetic changes and usually occur at an early stage in neoplastic development. Recent technological advances offer a better understanding of the underlying epigenetic alterations during carcinogenesis and provide insight into the discovery of putative epigenetic biomarkers for detection, prognosis, risk assessment, and disease monitoring. In this chapter we provide information on various epigenetic mechanisms and their role in carcinogenesis, in particular, epigenetic modifications causing genetic changes and the potential clinical impact of epigenetic research in the future.

Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1.

The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P < 0.0001) levels of 8-oxo-2'-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r = 0.2) with loss of GSTP1 activity (34%; P < 0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2 O2 -mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2 O2 , these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused reexpression of GSTP1, which protected the cells from H2 O2 -mediated DNA damage through decreased ROS production compared to nonexposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer.

High temperature unfolding of a truncated hemoglobin by molecular dynamics simulation.

Heme containing proteins are associated with peroxidase activity. The proteins like hemoglobin, myoglobins, cytochrome c and micro-peroxidase other than peroxidases have been shown to exhibit weak peroxidase-like activity. This weak peroxidase-like activity in hemoglobin-like molecules is due to heme moiety. We conducted molecular dynamics (MD) studies to decipher the unfolding path of Ba-Glb (a truncated hemoglobin from Bacillus anthracis) and the role of heme moiety to its unfolding path. The similar unfolding path is also observed in vitro by UV/VIS spectroscopy. The data confirmed that the unfolding of Ba-Glb follows a three state process with a meta-stable (intermediate) state between the native and unfolded conformations. The present study is supported by several unfolding parameters like root-mean-square-deviation (RMSD), dictionary of protein secondary structure (DSSP), and free energy landscape. Understanding the structure of hemoglobin like proteins in unicellular dreaded pathogens like B. anthracis will pave way for newer drug discovery targets and in the disease management of anthrax.

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress.

AIMS: Protection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms. MAIN METHODS: The cytoprotective effect of chamomile was examined on H(2)O(2)-induced cellular stress in RAW 264.7 murine macrophages. KEY FINDINGS: RAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H(2)O(2). Treatment with 50µM H(2)O(2) for 6h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10-20µg/mL for 16h followed by H(2)O(2) treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus. SIGNIFICANCE: These molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.

Epigenetics and cancer.

Epigenetic modifications are central to many human diseases, including cancer. Traditionally, cancer has been viewed as a genetic disease, and it is now becoming apparent that the onset of cancer is preceded by epigenetic abnormalities. Investigators in the rapidly expanding field of epigenetics have documented extensive genomic reprogramming in cancer cells, including methylation of DNA, chemical modification of the histone proteins, and RNA-dependent regulation. Recognizing that carcinogenesis involves both genetic and epigenetic alterations has led to a better understanding of the molecular pathways that govern the development of cancer and to improvements in diagnosing and predicting the outcome of various types of cancer. Studies of the mechanism(s) of epigenetic regulation and its reversibility have resulted in the identification of novel targets that may be useful in developing new strategies for the prevention and treatment of cancer.