Paternal Combined Botanicals Contribute to the Prevention of Estrogen Receptor-Negative Mammary Cancer in Transgenic Mice.

BACKGROUND: Parental nutritional interventions have considerably affected gametogenesis and embryogenesis, leading to the differential susceptibility of offspring to chronic diseases such as cancer. Moreover, combinatorial bioactive diets are more efficacious in ameliorating epigenetic aberrations in tumorigenesis. OBJECTIVES: We sought to investigate the transgenerational influence and epigenetic regulation of paternal sulforaphane (SFN)-rich broccoli sprouts (BSp) and epigallocatechin-3-gallate (EGCG)-rich green tea polyphenols (GTPs) consumption in the prevention of estrogen receptor-negative [ER(-)] mammary cancer in transgenic mice. METHODS: Human breast cancer cells were used to detect cell viability and epigenetic-related gene expression after treatment with EGCG and/or SFN. Twenty-four C3 or HER2/neu males were randomly assigned into 4 groups and treated with control, 26% BSp (w/w) in food, 0.5% GTPs (w/v) in drinking water or combined BSp and GTPs for 7 wk before mating. Tumor growth of nontreated female pups was monitored weekly for 19 wk (C3) and 25 wk (HER2/neu). Tumor- and epigenetic-related protein expression and enzyme activities in mammary tumors were measured. Sperms were isolated from treated males for RNA sequencing and reduced-representation bisulfite sequencing analysis. Data were analyzed with a 2-factor or 3-factor analysis of variance. RESULTS: EGCG and SFN inhibited breast cancer cell growth via epigenetic regulation. Combined BSp and GTPs synergistically (combination index < 1) suppressed tumor growth over time (P < 0.001) in 2 mouse models. Key tumor-related proteins were found differentially expressed (P < 0.05) along with epigenetic regulations in offspring mammary tumors. The transcriptome profile of sperm derived from dietary-treated males revealed differentially expressed genes correlated with spermatogenesis and breast cancer progression. DNA methylomes of the sperm and further integrated analysis with transcriptomes indicate that DNA methylation alone may not contribute to sufficient regulation in dietary-treated sperm pronucleus, leading to offspring tumor suppression. CONCLUSIONS: Collectively, paternal consumption of combined BSp and GTPs shows potential for preventing ER(-) mammary cancer through transgenerational effects. J Nutr 2023;xx:xx-xx.

Combinatorial epigenetic mechanisms of sulforaphane, genistein and sodium butyrate in breast cancer inhibition.

Dietary phytochemicals are currently being studied with great interest due to their ability to regulate the epigenome resulting in prevention of cancer. Some natural botanicals have been reported to have enhanced and synergistic impact on cancer suppression when administered at optimum concentrations and in-conjunction. Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables and sodium butyrate (NaB) is a short-chain fatty acid produced by gut microbiota. They have been intensively explored due to numerous anti-cancerous properties and ability to modulate epigenetic machinery by inhibition of histone deacetylase (HDAC). Genistein (GE), present in soy, is a known DNA methyltransferase (DNMT) inhibitor. While combined chemoprotective epigenetic effects induced by SFN and GE have been investigated, the key impact of combinatorial SFN-NaB, GE-NaB, and SFN-GE-NaB bioactive components in regulation of various mechanisms are poorly defined. In the present study, we found that combinations of dietary compounds had synergistic effects in decreasing cellular viability at lower dosages than their single dosages in breast cancer cell lines. The respective combinations limited growth and increased apoptosis and necrosis in cancerous cells among which the tri-combination displayed the most significant impact. Additionally, the respective combinations of compounds arrested MDA-MB-231 and MCF-7 breast cancer cells at G2/M phase. Our further mechanistic evaluation revealed that respective di-combinations and tri-combination had higher impact in down-regulation of DNMTs (DNMT3A and DNMT3B), HDACs (HDAC1, HDAC6 and HDAC11), histone methyltransferases (EZH2 and SUV39H1) and histone acetyltransferases (GCN5, PCAF, P300 and CBP) levels as compared to singly administered compounds. We also found that these combinations exhibited global epigenetic changes by inhibition of DNMT and HDAC activity, histone H3 at lysine 27 methylation (H3K27me) and histone H3 at lysine 9 methylation (H3K9me) levels, and by induction of histone acetyltransferases activity. Collectively, our investigation indicates that combined SFN, GE and NaB is highly effective in inhibiting breast cancer genesis by, at least in part, regulating epigenetic modifications, which may have implications in breast cancer therapy.

Dietary Supplementation of Inulin Contributes to the Prevention of Estrogen Receptor-Negative Mammary Cancer by Alteration of Gut Microbial Communities and Epigenetic Regulations.

Breast cancer (BC) is among the most frequently diagnosed malignant cancers in women in the United States. Diet and nutrition supplementation are closely related to BC onset and progression, and inulin is commercially available as a health supplement to improve gut health. However, little is known with respect to inulin intake for BC prevention. We investigated the effect of an inulin-supplemented diet on the prevention of estrogen receptor-negative mammary carcinoma in a transgenic mouse model. Plasma short-chain fatty acids were measured, the gut microbial composition was analyzed, and the expression of proteins related to cell cycle and epigenetics-related genes was measured. Inulin supplementation greatly inhibited tumor growth and significantly delayed tumor latency. The mice that consumed inulin had a distinct microbiome and higher diversity of gut microbial composition compared to the control. The concentration of propionic acid in plasma was significantly higher in the inulin-supplemented group. The protein expression of epigenetic-modulating histone deacetylase 2 (Hdac2), Hdac8, and DNA methyltransferase 3b decreased. The protein expression of factors related to tumor cell proliferation and survival, such as Akt, phospho-PI3K, and NF-kB, also decreased with inulin administration. Furthermore, sodium propionate showed BC prevention effect in vivo through epigenetic regulations. These studies suggest that modulating microbial composition through inulin consumption may be a promising strategy for BC prevention.

Maternal soybean genistein on prevention of later-life breast cancer through inherited epigenetic regulations.

Breast cancer has strong developmental origins and maternal nutrition composition may influence later-life breast cancer risk in the offspring. Our study focused on a bioactive dietary component, genistein (GE) enriched in soybean products, to investigate specific timing of maternal GE exposure that may influence preventive efficacy of GE on offspring breast cancer later in life, and to explore the potential epigenetic mechanisms. Our results indicate a time-dependent effect of maternal GE exposure on early-life breast cancer development in offspring mice. Through integrated transcriptome and methylome analyses, we identified several candidate genes showing significantly differential gene expression and DNA methylation changes. We further found maternal long-term GE treatment can induce inherited epigenetic landmark changes in a candidate tumor suppressor gene, Trp63, resulting in transcriptional activation of Trp63 and induction of the downstream target genes. Our results suggest that maternal long-term exposure to soybean GE may influence early-life epigenetic reprogramming processes, which may contribute to its temporal preventive effects on breast cancer in the offspring. This study provides important mechanistic insights into an appropriate maternal administration of soybean products on prevention of breast cancer later in offspring life.

Computational methods and next-generation sequencing approaches to analyze epigenetics data: Profiling of methods and applications.

Epigenetics is mainly comprised of features that regulate genomic interactions thereby playing a crucial role in a vast array of biological processes. Epigenetic mechanisms such as DNA methylation and histone modifications influence gene expression by modulating the packaging of DNA in the nucleus. A plethora of studies have emphasized the importance of analyzing epigenetics data through genome-wide studies and high-throughput approaches, thereby providing key insights towards epigenetics-based diseases such as cancer. Recent advancements have been made towards translating epigenetics research into a high throughput approach such as genome-scale profiling. Amongst all, bioinformatics plays a pivotal role in achieving epigenetics-related computational studies. Despite significant advancements towards epigenomic profiling, it is challenging to understand how various epigenetic modifications such as chromatin modifications and DNA methylation regulate gene expression. Next-generation sequencing (NGS) provides accurate and parallel sequencing thereby allowing researchers to comprehend epigenomic profiling. In this review, we summarize different computational methods such as machine learning and other bioinformatics tools, publicly available databases and resources to identify key modifications associated with epigenetic machinery. Additionally, the review also focuses on understanding recent methodologies related to epigenome profiling using NGS methods ranging from library preparation, different sequencing platforms and analytical techniques to evaluate various epigenetic modifications such as DNA methylation and histone modifications. We also provide detailed information on bioinformatics tools and computational strategies responsible for analyzing large scale data in epigenetics.

DNA methylation methods: Global DNA methylation and methylomic analyses.

DNA methylation provides a pivotal layer of epigenetic regulation in eukaryotes that has significant involvement for numerous biological processes in health and disease. The function of methylation of cytosine bases in DNA was originally proposed as a "silencing" epigenetic marker and focused on promoter regions of genes for decades. Improved technologies and accumulating studies have been extending our understanding of the roles of DNA methylation to various genomic contexts including gene bodies, repeat sequences and transcriptional start sites. The demand for comprehensively describing DNA methylation patterns spawns a diversity of DNA methylation profiling technologies that target its genomic distribution. These approaches have enabled the measurement of cytosine methylation from specific loci at restricted regions to single-base-pair resolution on a genome-scale level. In this review, we discuss the different DNA methylation analysis technologies primarily based on the initial treatments of DNA samples: bisulfite conversion, endonuclease digestion and affinity enrichment, involving methodology evolution, principles, applications, and their relative merits. This review may offer referable information for the selection of various platforms for genome-wide analysis of DNA methylation.

Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects.

Cancer therapeutics is an ever-evolving field due to incessant demands for effective and precise treatment options. Over the last few decades, cancer treatment strategies have shifted somewhat from surgery to targeted precision medicine. CRISPR-dCas9 is an emerging version of precision cancer therapy that has been adapted from the prokaryotic CRISPR-Cas system. Once ligated to epigenetic effectors (EE), CRISPR-dCas9 can function as an epigenetic editing tool and CRISPR-dCas9-EE complexes could be exploited to alter cancerous epigenetic features associated with different cancer hallmarks. In this article, we discuss the rationale of epigenetic editing as a therapeutic strategy against cancer. We also outline how sgRNA-dCas9 was derived from the CRISPR-Cas system. In addition, the current status of sgRNA-dCas9 use (in vivo and in vitro) in cancer is updated with a molecular illustration of CRISPR-dCas9-mediated epigenetic and transcriptional modulation. As sgRNA-dCas9 is still at the developmental phase, challenges are inherent to its use. We evaluate major challenges in targeting cancer with sgRNA-dCas9 such as off-target effects, lack of sgRNA designing rubrics, target site selection dilemmas and deficient sgRNA-dCas9 delivery systems. Finally, we appraise the sgRNA-dCas9 as a prospective cancer therapeutic by summarizing ongoing improvements of sgRNA-dCas9 methodology.

Combined Broccoli Sprouts and Green Tea Polyphenols Contribute to the Prevention of Estrogen Receptor-Negative Mammary Cancer via Cell Cycle Arrest and Inducing Apoptosis in HER2/neu Mice.

BACKGROUND: Aberrations in the regulation of cell proliferation perturb cellular homeostasis and lead to malignancies in which dysregulation of the cell cycle and suppressed apoptosis are 2 common phenomena. Combinatorial nutritional approaches could be efficacious in ameliorating these aberrations. OBJECTIVES: We sought to investigate the effect of dietary broccoli sprouts (BSp) and green tea polyphenol (GTP) administration on cell cycle progression and apoptosis in mammary tumors. METHODS: Forty female HER2/neu transgenic mice were randomly divided into 4 groups and treated with control, 26% BSp (wt:wt) in food, 0.5% GTPs (wt:vol) in drinking water, or combined BSp and GTPs from dams' conception until their pups were killed at 29 wk of age. Pups' tumor growth was monitored weekly for 27 wk. Tumor cell cycle- and apoptosis-related protein expression was measured. Data were analyzed with 2-factor or 3-factor (repeated-measures) ANOVA. RESULTS: Compared with the control group, BSp and/or GTPs decreased tumor incidence (P < 0.05) and combined BSp and GTPs synergistically [combination index (CIn) < 1] reduced tumor volume over time (P-time < 0.01). BSp and/or GTPs upregulated the expression of phosphatase and tension homolog, P16, and P53 (P < 0.05) and downregulated myelocytomatosis oncogene, Bmi1 polycomb ring finger oncogene, and telomerase reverse transcriptase (P < 0.05) compared with the control group. Combined BSp and GTPs synergistically (CIn < 1) downregulated the expression of cyclin B1, D1, and E1 and cyclin-dependent kinase 1, 2, and 4 (P < 0.05) compared with the control group. Moreover, combined BSp and GTPs induced apoptosis by regulating Bcl-2-associated X protein and B-cell lymphoma 2 (P < 0.05). BSp and/or GTPs also reduced the expression of DNA methyltransferase 1, 3A, and 3B and histone deacetylase 1 compared with the control group (P < 0.05). CONCLUSIONS: Collectively, lifelong BSp and GTP administration can prevent estrogen receptor-negative mammary tumorigenesis through cell cycle arrest and inducing apoptosis in HER2/neu mice.

Association of body composition with odds of breast cancer by molecular subtype: analysis of the Mechanisms for Established and Novel Risk Factors for Breast Cancer in Nigerian Women (MEND) study.

BACKGROUND: The association between obesity and breast cancer (BC) has been extensively studied among US, European and Asian study populations, with often conflicting evidence. However, despite the increasing prevalence of obesity and associated conditions in Africa, the continent with the highest age-standardized BC mortality rate globally, few studies have evaluated this association, and none has examined in relation to molecular subtypes among African women. The current analysis examines the association between body composition, defined by body mass index (BMI), height, and weight, and BC by molecular subtype among African women. METHODS: We estimated odds ratios (ORs) and 95% confidence intervals (95% CI) for the association between measures of body composition and BC and molecular subtypes among 419 histologically confirmed cases of BC and 286 healthy controls from the Mechanisms for Established and Novel Risk Factors for Breast Cancer in Women of Nigerian Descent (MEND) case-control study. RESULTS: Higher BMI (aOR: 0.79; 95% CI: 0.67, 0.95) and weight (aOR: 0.83; 95% CI: 0.69, 0.98) were associated with reduced odds of BC in adjusted models, while height was associated with non-statistically significant increased odds of BC (aOR: 1.07, 95% CI: 0.90, 1.28). In pre/peri-menopausal, but not post-menopausal women, both higher BMI and weight were significantly associated with reduced odds of BC. Further, higher BMI was associated with reduced odds of Luminal A, Luminal B, and HER2-enriched BC among pre/peri-menopausal women, and reduced odds of triple-negative BC among post-menopausal women. CONCLUSIONS: Higher BMI and weight were associated with reduced odds of BC overall and by molecular subtype among West African women. Larger studies of women of African descent are needed to definitively characterize these associations and inform cancer prevention strategies.

Impact of Stilbenes as Epigenetic Modulators of Breast Cancer Risk and Associated Biomarkers.

With the recent advancement of genetic screening for testing susceptibility to mammary oncogenesis in women, the relevance of the gene-environment interaction has become progressively apparent in the context of aberrant gene expressions. Fetal exposure to external stressors, hormones, and nutrients, along with the inherited genome, impact its traits, including cancer susceptibility. Currently, there is increasing interest in the role of epigenetic biomarkers such as genomic methylation signatures, plasma microRNAs, and alterations in cell-signaling pathways in the diagnosis and primary prevention of breast cancer, as well as its prognosis. Polyphenols like natural stilbenes have been shown to be effective in chemoprevention by exerting cytotoxic effects that can stall cell proliferation. Besides possessing antioxidant properties against the DNA-damaging effects of reactive oxygen species, stilbenes have also been observed to modulate cell-signaling pathways. With the increasing trend of early-life screening for hereditary breast cancer risks, the potency of different phytochemicals in harnessing the epigenetic biomarkers of breast cancer risk demand more investigation. This review will explore means of exploiting the abilities of stilbenes in altering the underlying factors that influence breast cancer risk, as well as the appearance of associated biomarkers.

Identification of DNA methylation associated enrichment pathways in adults with non-specific chronic low back pain.

Chronic low back pain (cLBP) that cannot be attributable to a specific pathoanatomical change is associated with high personal and societal costs. Still, the underlying mechanism that causes and sustains such a phenotype is largely unknown. Emerging evidence suggests that epigenetic changes play a role in chronic pain conditions. Using reduced representation bisulfite sequencing (RRBS), we evaluated DNA methylation profiles of adults with non-specific cLBP (n = 50) and pain-free controls (n = 48). We identified 28,325 hypermethylated and 36,936 hypomethylated CpG sites (p < 0.05). After correcting for multiple testing, we identified 159 DMRs (q < 0.01and methylation difference > 10%), the majority of which were located in CpG island (50%) and promoter regions (48%) on the associated genes. The genes associated with the differentially methylated regions were highly enriched in biological processes that have previously been implicated in immune signaling, endochondral ossification, and G-protein coupled transmissions. Our findings support inflammatory alterations and the role of bone maturation in cLBP. This study suggests that epigenetic regulation has an important role in the pathophysiology of non-specific cLBP and a basis for future studies in biomarker development and targeted interventions.

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms.

Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

Combinatorial Epigenetics Impact of Polyphenols and Phytochemicals in Cancer Prevention and Therapy.

Polyphenols are potent micronutrients that can be found in large quantities in various food sources and spices. These compounds, also known as phenolics due to their phenolic structure, play a vital nutrient-based role in the prevention of various diseases such as diabetes, cardiovascular diseases, neurodegenerative diseases, liver disease, and cancers. However, the function of polyphenols in disease prevention and therapy depends on their dietary consumption and biological properties. According to American Cancer Society statistics, there will be an expected rise of 23.6 million new cancer cases by 2030. Due to the severity of the increased risk, it is important to evaluate various preventive measures associated with cancer. Relatively recently, numerous studies have indicated that various dietary polyphenols and phytochemicals possess properties of modifying epigenetic mechanisms that modulate gene expression resulting in regulation of cancer. These polyphenols and phytochemicals, when administrated in a dose-dependent and combinatorial-based manner, can have an enhanced effect on epigenetic changes, which play a crucial role in cancer prevention and therapy. Hence, this review will focus on the mechanisms of combined polyphenols and phytochemicals that can impact various epigenetic modifications such as DNA methylation and histone modifications as well as regulation of non-coding miRNAs expression for treatment and prevention of various types of cancer.

Pterostilbene down-regulates hTERT at physiological concentrations in breast cancer cells: Potentially through the inhibition of cMyc.

Human telomerase reverse transcriptase (hTERT) encodes the catalytic subunit of telomerase, which has been shown to be upregulated in many cancers. Pterostilbene is a naturally occurring stilbenoid and phytoalexin found primarily in blueberries that exhibits antioxidant activity and inhibits the growth of various cancer cell types. Therefore, the aim of this study was to determine whether treatment with pterostilbene, at physiologically achievable concentrations, can inhibit the proliferation of breast cancer cells and down-regulate the expression of hTERT. We found that pterostilbene inhibits the cellular proliferation of MCF-7 and MDA-MB-231 breast cancer cells in both a time- and dose-dependent manner, without significant toxicity to the MCF10A control cells. Pterostilbene was also shown to increase apoptosis in both breast cancer cell lines. Dose-dependent cell cycle arrest in G1 and G2/M phase was observed after treatment with pterostilbene in MCF-7 and MDA-231 cells, respectively. hTERT expression was down-regulated after treatment in both a time- and dose-dependent manner. Pterostilbene also reduced telomerase levels in both cell lines in a dose-dependent manner. Moreover, cMyc, a proposed target of the pterostilbene-mediated inhibition of hTERT, was down-regulated both transcriptionally and posttranscriptionally after treatment. Collectively, these findings highlight a promising use of pterostilbene as a natural, preventive, and therapeutic agent against the development and progression of breast cancer.

Combinational Proanthocyanidins and Resveratrol Synergistically Inhibit Human Breast Cancer Cells and Impact Epigenetic⁻Mediating Machinery.

Breast cancer is the second most common cancer and the second leading cause of death from cancer among women in the United States (US). Cancer prevention and therapy through the use of phytochemicals that have epigenetic properties has gained considerable interest during the past few decades. Such dietary components include, but are not limited to, grape seed proanthocyanidins (GSPs) and resveratrol (Res), both of which are present in red wine. In this study, we report for the first time the synergistic effects of GSPs and Res on inhibiting MDA-MB-231 and MCF-7 human breast cancer cells. Our results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and clonogenic assays indicate that treatments with the combinations of GSPs and Res synergistically decreased cell viability and posttreatment cell proliferation in both cell lines. Additional analyses show that treatments with GSPs and Res in combination synergistically induced apoptosis in MDA-MB-231 cells by upregulating Bax expression and down-regulating Bcl-2 expression. DNA methyltransferase (DNMT) activity and histone deacetylase (HDAC) activity were greatly reduced in MDA-MB-231 and MCF-7 cells after treatments with GSPs and Res in combination. Collectively, our findings suggest that GSPs and Res synergistically inhibit human breast cancer cells through inducing apoptosis, as well as modulating DNA methylation and histone modifications.

The Effects of Combinatorial Genistein and Sulforaphane in Breast Tumor Inhibition: Role in Epigenetic Regulation.

Dietary compounds that possess the properties of altering epigenetic processes are gaining popularity as targets for cancer prevention studies. These compounds when administered at optimal concentrations and especially in combination can have enhanced effects in cancer prevention or therapy. It is important to study the interaction of two or more compounds in order to assess their role in enhancing prevention. Genistein (GEN), found in soy, has been extensively studied for its role as an epigenetic modifier especially as a DNA methyltransferase (DNMT) inhibitor and sulforaphane (SFN), found in cruciferous vegetables, is known as a histone deacetylase (HDAC) inhibitor. However, very little is known about the effects of these two compounds in conjunction in breast cancer prevention or therapy. In our current study, we determined that, at certain doses, the compounds have synergistic effects in decreasing cellular viability of breast cancer cell lines. Our results indicate that the combination of GEN and SFN is much more effective than their single doses in increasing the rate of apoptosis and lowering the colony forming potential of these cells. We determined that these compounds inhibit cell cycle progression to G2 phase in MDA-MB-231 and G1 phase in MCF-7 breast cancer cell lines. Additionally, we determined that the combination is effective as an HDAC and histone methyltransferase (HMT) inhibitor. Furthermore, we demonstrated that this combination downregulates the levels of HDAC2 and HDAC3 both at the mRNA and protein levels. We also found that these compounds have the potential to downregulate KLF4 levels, which plays an important role in stem cell formation. The combination of GEN and SFN is also effective in downregulating hTERT levels, which is known to be activated when KLF4 binds to its promoter region. Our hypothesis is further strengthened by in vivo studies, where the combination is administered to transgenic mice in the form of genistein and SFN-enriched broccoli sprouts. We have demonstrated that the combination is more effective in preventing or treating mammary cancer via extending tumor latency and reducing tumor volumes/sizes than either of these dietary components administered alone. These results are consistent with our in vitro study suggesting potential preventive and therapeutic effects of this novel dietary combinatorial approach against breast cancer.

Epigenetic biomarkers: Current strategies and future challenges for their use in the clinical laboratory.

Epigenetic modifications and regulators represent potential molecular elements which control relevant physiological and pathological features, thereby contributing to the natural history of human disease. These epigenetic modulators can be employed as disease biomarkers, since they show several advantages and provide information about gene function, thus explaining differences among patient endophenotypes. In addition, epigenetic biomarkers can incorporate information regarding the effects of the environment and lifestyle on health and disease, and monitor the effect of applied therapies. Technologies used to analyze these epigenetic biomarkers are constantly improving, becoming much easier to use. Laboratory professionals can easily acquire experience and techniques are becoming more affordable. A high number of epigenetic biomarker candidates are being continuously proposed, making now the moment to adopt epigenetics in the clinical laboratory and convert epigenetic marks into reliable biomarkers. In this review, we describe some current promising epigenetic biomarkers and technologies being applied in clinical practice. Furthermore, we will discuss some laboratory strategies and kits to accelerate the adoption of epigenetic biomarkers into clinical routine. The likelihood is that over time, better markers will be identified and will likely be incorporated into future multi-target assays that might help to optimize its application in a clinical laboratory. This will improve cost-effectiveness, and consequently encourage the development of theragnosis and the application of precision medicine.

Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-ß pathways in MCF-7 Cancer cells with PX-866 and Raloxifene.

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-ß pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-ß pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.

A Novel Combination of Withaferin A and Sulforaphane Inhibits Epigenetic Machinery, Cellular Viability and Induces Apoptosis of Breast Cancer Cells.

With cancer often classified as a disease that has an important epigenetic component, natural compounds that have the ability to regulate the epigenome become ideal candidates for study. Humans have a complex diet, which illustrates the need to elucidate the mechanisms of interaction between these bioactive compounds in combination. The natural compounds withaferin A (WA), from the Indian winter cherry, and sulforaphane (SFN), from cruciferous vegetables, have numerous anti-cancer effects and some report their ability to regulate epigenetic processes. Our study is the first to investigate the combinatorial effects of low physiologically achievable concentrations of WA and SFN on breast cancer cell proliferation, histone deacetylase1 (HDAC1) and DNA methyltransferases (DNMTs). No adverse effects were observed on control cells at optimal concentrations. There was synergistic inhibition of cellular viability in MCF-7 cells and a greater induction of apoptosis with the combinatorial approach than with either compound administered alone in both MDA-MB-231 and MCF-7 cells. HDAC expression was down-regulated at multiple levels. Lastly, we determined the combined effects of these bioactive compounds on the pro-apoptotic BAX and anti-apoptotic BCL-2 and found decreases in BCL-2 and increases in BAX. Taken together, our findings demonstrate the ability of low concentrations of combinatorial WA and SFN to promote cancer cell death and regulate key epigenetic modifiers in human breast cancer cells.

Combinatorial PX-866 and Raloxifene Decrease Rb Phosphorylation, Cyclin E2 Transcription, and Proliferation of MCF-7 Breast Cancer Cells.

As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-ß pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc.