Soy-derived isoflavones as chemo-preventive agents targeting multiple signalling pathways for cancer prevention and therapy.

The chemopreventive and chemotherapeutic properties of soy and soy-derived compounds, especially isoflavones, have been extensively studied in recent years. However, in contrast to their anticancer effects, such as cell growth inhibition, cell cycle arrest and apoptosis induction, isoflavones have also been found to promote the growth of cancer cells. Therefore, the aim of this comprehensive review article is to present the current state of knowledge regarding the molecular mechanisms by which soy-derived isoflavones target multiple cellular signalling pathways in cancer cells. Our findings indicate that soy-derived isoflavones act as, among other things, potent modulators of HOX transcript antisense RNA (HOTAIR)/SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), vascular endothelial growth factor (VEGF)/C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor type 4 (CXCR4), 17-ß-oestradiol (E2)/oestrogen receptor-α (ERα)/neuroglobin (NGB) and sonic hedgehog signalling pathways, epigenetic modulatory agents (i.a. miR-155, miR-34a and miR-10a-5p) and cancer stem cells and epithelial-to-mesenchymal transition inhibitors. The paper also discusses the latest epidemiological studies and clinical trials and provides an insight into recent extensive research on the chemo-preventive and therapeutic potential of soy-derived isoflavones.

Lunasin and Its Epigenetic Impact in Cancer Chemoprevention.

Cancer diseases are a leading cause of death worldwide. Therefore, it is pivotal to search for bioactive dietary compounds that can avert tumor development. A diet rich in vegetables, including legumes, provides chemopreventive substances, which have the potential to prevent many diseases, including cancer. Lunasin is a soy-derived peptide whose anti-cancer activity has been studied for over 20 years. The results of the previous research have shown that lunasin inhibits histone acetylation, regulates the cell cycle, suppresses proliferation and induces apoptosis of cancer cells. Thus, lunasin seems to be a promising bioactive anti-cancer agent and a potent epigenetic modulator. The present review discusses studies of the underlying molecular mechanisms and new perspectives on lunasin application in epigenetic prevention and anti-cancer therapy.

An unusual non­metastatic, mismatch repair­deficient primary gastric squamous cell carcinoma presenting as a large, exophytic, bleeding tumor: A case report.

Primary gastric squamous cell carcinoma (GSCC) is an extremely rare malignancy with a poor prognosis. Despite the improved knowledge regarding its pathogenesis and biology, the treatment options remain limited. The present study reported on the unique case of a mismatch repair-deficient (dMMR) primary GSCC in a 79-year-old woman reporting fatigue and symptoms of upper gastrointestinal tract bleeding. Physical examination revealed abdominal pain at palpation. Gastroscopy revealed a large, exophytic, bleeding tumor. Medical imaging confirmed a mushroom-like polyp in the lumen of the stomach, with no signs of disease spread. Total gastrectomy and D2 lymphadenectomy were performed. Pathological examination of the post-operational material confirmed a well-differentiated SCC invading the mucosa, submucosa and muscle layer. There were no signs of dissemination observed in any of the 32 excised lymph nodes. Notably, according to the last follow-up, the patient remains well, supporting the 5-year GSCC survival rate statistics. To the best of our knowledge, this is the first such GSCC case reported in the Surgical Oncology Outpatient Clinic (Copernicus Memorial Hospital, Lodz, Poland) and these findings add to the limited data on GSCC. Although this is a very rare condition, it should always be considered during the process of diagnosis of gastric tumors.

Curcumin from Turmeric Rhizome: A Potential Modulator of DNA Methylation Machinery in Breast Cancer Inhibition.

One of the most systematically studied bioactive nutraceuticals for its benefits in the management of various diseases is the turmeric-derived compounds: curcumin. Turmeric obtained from the rhizome of a perennial herb Curcuma longa L. is a condiment commonly used in our diet. Curcumin is well known for its potential role in inhibiting cancer by targeting epigenetic machinery, with DNA methylation at the forefront. The dynamic DNA methylation processes serve as an adaptive mechanism to a wide variety of environmental factors, including diet. Every healthy tissue has a precise DNA methylation pattern that changes during cancer development, forming a cancer-specific design. Hypermethylation of tumor suppressor genes, global DNA demethylation, and promoter hypomethylation of oncogenes and prometastatic genes are hallmarks of nearly all types of cancer, including breast cancer. Curcumin has been shown to modulate epigenetic events that are dysregulated in cancer cells and possess the potential to prevent cancer or enhance the effects of conventional anti-cancer therapy. Although mechanisms underlying curcumin-mediated changes in the epigenome remain to be fully elucidated, the mode of action targeting both hypermethylated and hypomethylated genes in cancer is promising for cancer chemoprevention. This review provides a comprehensive discussion of potential epigenetic mechanisms of curcumin in reversing altered patterns of DNA methylation in breast cancer that is the most commonly diagnosed cancer and the leading cause of cancer death among females worldwide. Insight into the other bioactive components of turmeric rhizome as potential epigenetic modifiers has been indicated as well.

The effects of clofarabine in ALL inhibition through DNA methylation regulation.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analog, possesses manifold anti-cancer activities. Our previous reports and some of others demonstrate the potential capacity of ClF to regulate the epigenetic machinery. The study presented here is the first to investigate the influence of ClF on modulators of the DNA methylation machinery, including DNMT1 and CDKN1A, in acute lymphoblastic leukemia (ALL) cells. ClF effects on promoter methylation and transcriptional activity of hypermethylated and silenced tumor suppressor genes (TSGs), including APC, CDKN2A, PTEN, and RARB, have been tested as well. Methylation level of the proximal promoter region of APC, CDKN2A, PTEN, and RARB, as well as expression of those TSGs, DNMT1 and CDKN1A, were estimated by using a methylation-sensitive restriction analysis and qPCR, respectively. The Nalm-6 cell line was used as an experimental in vitro model of ALL cells. We observed ClF-mediated inhibition of cellular viability and apoptosis induction of Nalm-6 cells with an increased percentage of cells positive for active Caspase-3. Interestingly, exposure of Nalm-6 cells to CIF at 20 nM concentration for three days has led to a significant DNMT1 downregulation, accompanied by robust CDKN1A upregulation. ClF caused hypomethylation of APC, CDKN2A, and PTEN, with a concomitant increase in their transcript levels. Taken together, our results demonstrate the ability of ClF to reactivate DNA methylation-silenced TSGs in ALL cells. This may implicate translational significance of our findings and support ClF application as a new epigenetic modulator in the anti-leukemia therapy.

Clofarabine­phytochemical combination exposures in CML cells inhibit DNA methylation machinery, upregulate tumor suppressor genes and promote caspase­dependent apoptosis.

Clofarabine (2­chloro­2'­fluoro­2'­deoxyarabinosyladenine, CIF), a second­generation 2'­deoxyadenosine analog, possesses a variety of anti­cancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and all­trans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylation­silenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase­3­dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylation­mediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in anti­leukemic epigenetic therapy.

Novel Clofarabine-Based Combinations with Polyphenols Epigenetically Reactivate Retinoic Acid Receptor Beta, Inhibit Cell Growth, and Induce Apoptosis of Breast Cancer Cells.

An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential.

Relationship of muscle function to circulating myostatin, follistatin and GDF11 in older women and men.

BACKGROUND: Myostatin, its inhibitor follistatin, and growth/differentiation factor 11 (GDF11) have been proposed as factors that could potentially modify biological aging. The study aimed to test whether there is a relationship between these plasma circulating proteins and muscle strength, power and optimal shortening velocity (υopt) of older adults. METHODS: The cross-sectional study included 56 women and 45 men aged 60 years and older. Every participant underwent examination which included anthropometric and bioimpedance analysis measurements, functional and cognitive performance tests, muscle strength of upper and lower extremities, muscle power testing with two different methods and blood analyses. RESULTS: Women had higher plasma levels of myostatin and GDF11 than men. Men had higher plasma level of follistatin than women. In women, plasma level of myostatin was negatively correlated with left handgrip strength and υopt. Follistatin was negatively correlated with maximum power output (Pmax), power relative to kg of body mass (Pmax∙kg- 1) (friction-loaded cycle ergometer) and power at 70% of the 1-repetition maximum (1RM) strength value (P70%) of leg press (Keiser pneumatic resistance training equipment), and positively correlated with the Timed Up & Go (TUG) test. GDF11 was negatively correlated with body mass, body mass index, waist circumference, fat mass and the percentage of body fat. In men, there were no significant correlations observed between circulating plasma proteins and muscle function measures. CONCLUSIONS: The circulating plasma myostatin and follistatin are negatively associated with muscle function in older women. There is stronger relationship between these proteins and muscle power than muscle strength. GDF11 has a higher association with the body mass and composition than muscle function in older women.

Inhibition of breast cancer cell growth by the combination of clofarabine and sulforaphane involves epigenetically mediated CDKN2A upregulation.

Many antineoplastic nucleoside analogue-based combinatorial strategies focused on remodelling aberrant DNA methylation patterns have been developed. The number of studies demonstrate high efficacy of bioactive phytochemicals in support of conventional chemotherapy. Our recent discoveries of the epigenetic effects of clofarabine (2'-deoxyadenosine analogue, antileukaemic drug) and clofarabine-based combinations with dietary bioactive compounds in breast cancer cells led us to look for more DNA methylation targets of these cancer-preventive agents. In the present study, using methylation-sensitive restriction analysis (MSRA) and qPCR, we showed that clofarabine in combination with sulforaphane, a phytochemical from cruciferous vegetables, significantly reactivates DNA methylation-silenced CDKN2A tumour suppressor and inhibits cancer cell growth at a non-invasive breast cancer stage.

Epigenetic changes in cancer by Raman imaging, fluorescence imaging, AFM and scanning near-field optical microscopy (SNOM). Acetylation in normal and human cancer breast cells MCF10A, MCF7 and MDA-MB-231.

This paper examines epigenetic changes in breast cancer by Raman imaging, fluorescence imaging, AFM and SNOM and discusses how they contribute to different aspects of tumourigenesis in malignant human breast epithelial cell lines MCF7 and MDA-MB-231 compared with non-malignant MCF10A cell lines. The paper focuses on information that can be extracted from Raman microscopy and Raman imaging for the biological material of nucleoli contained within the cell nucleus and lipid droplets within the cell cytoplasm. The biochemical composition of the nuclei and lipid droplets in the non-malignant and malignant human breast epithelial cell lines has been monitored. The potential of Raman microspectroscopy to monitor acetylation processes and a prognostic value of Raman biomarkers in breast cancer have been discussed.

The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation.

Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells.

BACKGROUND/AIM: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1 , p53 , and p21 , in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. METHODS: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. RESULTS: In both MCF-7 and MDA-MB-231 cells, SFN at IC 50 (22 and 46 µ M , respectively) and a physiologically relevant 10 µ M concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene upregulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. CONCLUSIONS: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

Clofarabine, a novel adenosine analogue, reactivates DNA methylation-silenced tumour suppressor genes and inhibits cell growth in breast cancer cells.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF) is a second-generation 2'-deoxyadenosine analogue that is structurally related to cladribine (2-chloro-2'-deoxyadenosine, 2CdA) and fludarabine (9-beta-d-arabinosyl-2-fluoroadenine, F-ara-A). It demonstrates potent antitumour activity at much lower doses than parent compounds with high therapeutic efficacy in paediatric blood cancers. Our previous studies in breast cancer cells indicate that 2CdA and F-ara-A are involved in epigenetic regulation of gene transcription. We therefore investigated whether ClF influences methylation and expression of selected tumour suppressor genes, such as adenomatous polyposis coli (APC), phosphatase and tensin homologue (PTEN), and retinoic acid receptor beta 2 (RARbeta2), as well as expression of p53, p21 and DNA methyltransferase 1 (DNMT1) in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive potential. Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. ClF demonstrated potent growth inhibitory activity in MCF-7 and MDA-MB-231 cells after 96h treatment with IC50 determined as equal to 640nM and 50nM, respectively. In both breast cancer cell lines, ClF led to hypomethylation and up-regulation of APC, PTEN and RARbeta2 as well as increase in p21 expression. Only in non-invasive MCF-7 cells, these changes were associated with down-regulation of DNMT1. Our results provide first evidence of ClF implications in epigenetic regulation of transcriptional activity of selected tumour suppressor genes in breast cancer. It seems to be a new important element of ClF anticancer activity and may indicate its potential efficacy in epigenetic therapy of solid tumours, especially at early stages of carcinogenesis.

Folic acid enforces DNA methylation-mediated transcriptional silencing of PTEN, APC and RARbeta2 tumour suppressor genes in breast cancer.

Folate, one of the most studied dietary compounds, has recently become the main topic of debates on food fortification. Although low folate levels may be associated with increased risk of cancer development, simultaneously several reports indicate a detrimental effects mediated by high folate concentrations. Using the methylation sensitive restriction analysis (MSRA) and real-time RT-PCR we tested the effect of folic acid on DNA promoter methylation and expression of PTEN, APC and RARbeta2 tumour suppressor genes in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive capacity. The tested genes encode proteins involved in regulation of oncogenic intracellular signaling pathways. The results show that the increasing concentrations of folic acid lead to a dose-dependent down-regulation of tumour suppressor genes which may be linked to the increased DNA methylation detected within their promoter regions. The effects were more remarkable in non-invasive MCF-7 cells where we also observed 30% up-regulation of DNMT1 expression at the highest folate concentration used. Our findings show that caution need to be used when introducing folic acid supplementation since it may lead to cancer progression.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine)--biochemical aspects of anticancer activity.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine) is a second generation analogue of 2'-deoxyadenosine connecting biochemical activities of its prototypes: cladribine (2-chloro-2'-deoxyadenosine) and fludarabine (2-fluoro-arabinosyladenine). This new anticancer drug is more effective (in low doses) and indicates higher oral bioavailability in comparison to its congeners. The studies indicated that the molecular mechanism of clofarabine cytotoxic action includes cell apoptosis, which results from inhibition (by the drug triphosphate nucleotides) of ribonucleotide reductase and DNA polymerases. The most recent research demonstrated also that action of the drug may cause up-expression of some genes on mRNA and protein levels. Clofarabine was synthesized in 1992 and in 2004 was approved for treatment of pediatric patients with refractory or relapsed acute lymphoblastic leukemia (ALL). Encouraging results of clinical trials with clofarabine in acute leukemias inclined to present background knowledge about multidirectional biomolecular mechanism of its cytotoxicity.

Influence of clofarabine on transcriptional activity of PTEN, APC, RARB2, ZAP70 genes in K562 cells.

BACKGROUND: In this study, the effect of clofarabine, a new generation 2'-deoxyadenosine analogue, on promoter methylation and transcriptional activity of selected genes (PTEN, APC, RARB2, ZAP70) in K562 cells was assessed. MATERIALS AND METHODS: Promoter methylation was estimated using methylation-sensitive restriction analysis. The mRNA level of the genes was measured with real-time PCR. RESULTS: The inhibitory cytostatic index (IG(50)) for clofarabine in K562 cells cultured for 72 (or 96) h was 8 nM. The drug (20 nM) caused: (i) potent diminution in methylation of PTEN promoter, moderate methylation reduction of APC and RARB2 promoters, and complete methylation of ZAP70 promoter; (ii) significant stimulation of PTEN, APC, RARB, and p21 mRNA expression and (iii) decline in mRNA level of ZAP70 and DNMT1 genes. CONCLUSION: The results indicated that clofarabine is involved in epigenetic regulation of transcriptional activity of the tested tumour suppressor genes and genes encoding proteins involved in DNA methylation process.

Identification of the csp gene and molecular modelling of the CspA-like protein from Antarctic soil-dwelling psychrotrophic bacterium Psychrobacter sp. B6.

We cloned and sequenced the cspA-like gene from a psychrotrophic Antarctic soil-dwelling bacterial strain Psychrobacter sp. B6. The gene is 213 bp long and shows 99% and 98% sequence identity with the Psychrobacter cryohalolentis K5 gene encoding a cold-shock DNA-binding domain protein and the Psychrobacter arcticus transcriptional regulator-CspA gene, respectively. The protein encoded by the Psychrobacter sp. B6 cspA-like gene shows 100% identity with the two proteins mentioned above, and also 61% sequence identity with CspB from Bacillus subtilis and Csp from Bacillus caldolyticus, and 56% - with Escherichia coli CspA protein. A three-dimensional model of the CspA-like protein from Psychrobacter sp. B6 was generated based on three known structures of cold shock proteins: the crystal structure of the major cold shock protein from Escherichia coli (CspA), the NMR structure of the latter protein, and the NMR structure of Csp from Thermotoga maritima. The deduced structure of the CspA-like protein from Psychrobacter sp. B6 was found to be very similar to these known structures of Csp-like proteins.