Nanoparticle-mediated diagnosis, treatment, and prevention of breast cancer.

By virtue of their advanced physicochemical properties, nanoparticles have attracted significant attention from researchers for application in diverse fields of medical science. Breast cancer, presenting a high risk of morbidity and mortality, frequently occurs in women and is considered a malignant tumor. Globally, breast cancer is considered the second leading cause of death. Accordingly, its poor prognosis, invasive metastasis, and relapse have motivated oncologists and nano-medical researchers to develop highly potent nanotherapies to cure this deadly disease. In this case, nanoparticles have emerged as responsive platforms for breast cancer management, providing new approaches to improve the diagnostic accuracy, deliver targeted therapies, and limit the progression of this disease. Recently, smart nano-carriers encapsulating drugs, ligands, and tracking probes have been developed for the specific therapy of breast cancers. Further, efforts have been devoted to developing various nano-systems with minimal toxicity. The aim of this review is to present a background on novel nanotheranostic methods that can be employed to diagnose and treat breast cancers and encourage readers to focus on the development of novel nanomedicine for breast cancers and other deadly diseases. In this context, we discuss different methods for the diagnosis, treatment, and prevention of breast cancers using different metal and metal oxide nanoparticles.

YB1 modulates the DNA damage response in medulloblastoma.

Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein-protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.

Photocatalytic activity of biosynthesized silver nanoparticle fosters oxidative stress at nanoparticle interface resulting in antimicrobial and cytotoxic activities.

Inside the biological milieu, nanoparticles with photocatalytic activity have potential to trigger cell death non-specifically due to production of reactive oxygen species (ROS) upon reacting with biological entities. Silver nanoparticle (AgNP) possessing narrow band gap energy can exhibit high light absorption property and significant photocatalytic activity. This study intends to explore the effects of ROS generated due to photocatalytic activity of AgNP on antimicrobial and cytotoxic propensities. To this end, AgNP was synthesized using the principle of green chemistry from the peel extract of Punica granatum L., and was characterized using UV-Vis spectroscope, transmission electron microscope and x-ray diffraction, and so forth. The antimicrobial activity of AgNP against studied bacteria indicated that, ROS generated at AgNP interface develop stress on bacterial membrane leading to bacterial cell death, whereas Alamar Blue dye reduction assay indicated that increased cytotoxic activity with increasing concentrations of AgNP. The γH2AX activity assay revealed that increasing the concentrations of AgNP increased DNA damaging activity. The results altogether demonstrated that both antimicrobial and cytotoxic propensities are triggered primarily due interfacial ROS generation by photocatalytic AgNP, which caused membrane deformation in bacteria and DNA damage in HT1080 cells resulting in cell death.

DNA methylation and not H3K4 trimethylation dictates the expression status of miR-152 gene which inhibits migration of breast cancer cells via DNMT1/CDH1 loop.

MicroRNAs (miRNA) are small non-coding RNAs which targets most protein-coding transcripts (mRNA) and destroy them. Thus miRNA controls the abundance of those specific proteins and impact on developmental, physiological and pathological processes. Dysregulation of miRNA function thus may lead to various clinicopathological complications, including breast cancer. Silencing of miR-152 gene due to promoter DNA methylation alter the expression pattern of several other genes. E-cadherin (CDH1) forms the core of adherent junctions between surrounding epithelial cells, link with actin cytoskeleton and affects cell signaling. CDH1 gene is down regulated by promoter DNA methylation during cancer progression. In this investigation, we attempt to elucidate the correlation of miR-152 and CDH1 function, as it is well known that the loss of CDH1 function is one of the major reasons for cancer metastasis and aggressiveness of spreading. For the first time we have shown that loss of CDH1 expression is directly proportional to the loss of miR-152 function in breast cancer cells. mRNA and protein expression profile of DNMT1 implicate that miR-152 targets DNMT1 mRNA and inhibits its protein expression. Tracing the molecular marks on DNA and histone 3 for understanding the mechanism of gene regulation by ChIP analyses leads to a paradoxical result that shows DNA methylation adjacent to active histone marking (enrichment of H3K4me3) silence miR-152 gene. Further experiments revealed that DNMT1 plays crucial role for regulation of miR-152 gene. When DNMT1 protein function is blocked miR-152 expression prevails and destroys the mRNA of DNMT1; this molecular regulatory mechanism is creating a cyclic feedback loop, which is now focused as DNMT1/miR-152 switch for on/off of DNMT1 target genes. We discovered modulation of CDH1 gene expression by DNMT1/miR-152 switches. We have demonstrated further that DNMT1 down regulation mediated upregulation of CDH1 (hereafter, DNMT1/CDH1 loop) in presence of ectopic-excess of miR-152 prevents migration of cancer cells. Our data provides novel insights into the regulation mechanism of miRNA and mRNA/protein coding genes and enhances the amplitude of cancer epigenome.

Silencing of ZRF1 impedes survival of estrogen receptor positive MCF-7 cells and potentiates the effect of curcumin.

The role and clinical implication of ZRF1 in breast cancer are poorly understood. So this study is aimed to explore the role of ZRF1 in breast cancer progression. With this context, we first assessed its expression pattern in FFPE primary and metastasis breast tissue samples as well as from publicly available databases. Moreover, we also explored the survival status of patients from the publicly available database and interestingly discover that high expression of ZRF1 decreases the survival of estrogen-positive breast cancer patients more than estrogen-negative status patients. In the perspective of this, we evaluated the role ZRF1 in MCF-7 breast cancer cells and found that it's silencing by knockdown results in decreased cell proliferation as well as cell viability. Results also show that expression of ZRF1 is down regulated in the presence of estrogen-depleted conditions but independent of RAS/MEK as well as AKT axes. Moreover, the decrease in viability of MCF-7 cells was accompanied by induction of apoptosis and DNA damage, well-marked with upregulation of cleaved PARP and downregulation of BCL2 and H2AUbK119 levels. Furthermore, we also explored that knockdown of ZRF1 sensitises the effect of curcumin, observed with decrease in cell viability and dropping of IC50 value from 25 to 15 µM. This investigation thus shed a new light on the role on ZRF1 in breast cancer cells and hence can be exploited to design better therapeutic intervention.

Epigenetic drift towards histone modifications regulates CAV1 gene expression in colon cancer.

BACKGROUND: Caveolin-1 (CAV1) is an important structural component of cellular caveolae involved in cell signaling. CAV1 gene on/off regulatory mechanism in multiple diseases, including cancer is not clearly understood. The tumor suppressor versus oncogene paradox of CAV1 during tumor development tempted us to investigate the role for the epigenetic drift of CAV1 gene regulation. METHODS: We have analyzed CAV1 gene expression and associated epigenetic marks (DNA methylation and histone 3 modifications) in the CAV1 promoter in two colon cancer cell lines, under treatment with well established epigenetic modulators, AZA, SAM, TSA and SFN at varying concentrations. CAV1 gene promoter DNA methylation and histone modifications were analyzed by DNA methylation specific PCR, bisulphite modification of DNA and ChIP analyses following PCR respectively. RESULTS: Ectopic expression of CAV1 by epigenetic modulators inhibits colon cancer cell growth. CAV1 promoter DNA remains unmethylated before and after treatment with epigenetic modulators, which confirmed that DNA methylation is not the regulator of CAV1 expression in colon cancer. There was enrichment of H3K4me3 and H3K9AcS10P and depletion of H3K9me3 modifications around the CAV1 promoter. CONCLUSIONS: Our data provides novel insight into the regulation of CAV1 gene by histone H3 modifications and enhance the amplitude of the cancer epigenome.

Insights into the molecular interactions of thymoquinone with histone deacetylase: evaluation of the therapeutic intervention potential against breast cancer.

Many HDAC inhibitors have passed through the gateway of clinical trials. However, they have limited therapeutic implications due to their pleiotropic pharmaceutical properties and off-target effects. In view of this, dietary active phytochemicals were evaluated. Based upon the chemical and structural insights of HDAC active pockets, thymoquinone (TQ) was investigated to uncover its active participation in HDAC inhibition. The synergistic analysis of docking and molecular dynamics simulation disclosed the elementary interaction and stability of TQ with human HDACs. The in silico findings were corroborated with an in vitro analysis, demonstrating the efficient role of TQ in the attenuation of global HDAC activity. Furthermore, TQ also elicited downstream effects of HDAC inhibition: reactivation of HDAC target genes (p21 and Maspin), induction of the pro-apoptotic gene Bax, down regulation of the anti-apoptotic gene Bcl-2 and arrest of the cell cycle at the G2/M phase. Finally, the result of a higher cytotoxicity of TQ towards MCF-7 breast cancer cells in comparison to normal cells indicates the potential of TQ to be an anticancer drug.

Clusterin gene is predominantly regulated by histone modifications in human colon cancer and ectopic expression of the nuclear isoform induces cell death.

Clusterin (CLU) is an important glycoprotein involved in various cellular functions. Different reports have mentioned that the two isoforms of CLU; secretary (sCLU) and nuclear (nCLU) have opposite (paradoxical) roles in cancer development. sCLU provides pro-survival signal, whereas nCLU is involved in pro-apoptotic signaling. However, the molecular mechanism of CLU gene regulation is not clear as of yet. We hypothesize that CLU gene is regulated by DNA methylation and histone modifications and clusterin plays an important role in colon cancer. To evaluate the hypothesis, we investigated CLU expression in colon cancer tissues and DNA methylation and histone modification status of CLU gene promoter. It is apparent from immonohistology data that both benign and cancerous (primary and metastasis) formalin fixed paraffin embedded (FFPE) tissue samples exhibit CLU expression. However and interestingly only noncancerous tissue samples show nCLU expression. Ectopic expression of nCLU either by epigenetic modulators or by nCLU transfection is responsible for colon cancer cell death. To clarify the molecular mechanisms for regulation of expression of CLU isoforms, we have analyzed DNA methylation and histone modifications, such as histone H3K9me3, H3K27me3, H3K4me3, and H3K9AcS10P patterns around the CLU promoter. There is no remarkable change in the DNA methylation status upon treatment of the cells by AZA, TSA and SAM. Our findings clearly show that promoter histone H3K9me3 and H3K27me3 marks are elevated in comparison to H3K4me3 and H3K9AcS10P marks in colon cancer cell lines.

Mechanisms of DNA methyltransferase-inhibitor interactions: Procyanidin B2 shows new promise for therapeutic intervention of cancer.

DNA methyltransferases (DNMTs) is a key epigenetic enzyme for pharmacological manipulation and is employed in cancer reprogramming. During past few years multiple strategies have been implemented to excavate epigenetic compounds targeting DNMTs. In light of the emerging concept of chemoinformatics, molecular docking and simulation studies have been employed to accelerate the development of DNMT inhibitors. Among the DNMT inhibitors known till date, epigallocathechin-3-gallate (EGCG) was identified to be effective in reducing DNMT activity. However, the broad spectrum of EGCG to other diseases and variable target enzymes offers some limitations. In view of this, 32 EGCG analogues were screened at S-Adnosyl-L-homocysteine (SAH) binding pocket of DNMTs and procyanidin B2-3, 3'-di-O-gallate (procyanidin B2) was obtained as potent inhibitor having medicinally relevant chemical space. Further, in vitro analysis demonstrates the efficiency of procyanidin B2 in attenuating DNMT activity at IC50 of 6.88±0.647 µM and subsequently enhancing the expression of DNMT target genes, E-cadherin, Maspin and BRCA1. Moreover, the toxic property of procyanidin B2 towards triple negative breast cancer cells to normal cells offers platform for pre-clinical trial and an insight to the treatment of cancer.

Expression profiling of DNA methylation-mediated epigenetic gene-silencing factors in breast cancer.

BACKGROUND: DNA methylation mediates gene silencing primarily by inducing repressive chromatin architecture via a common theme of interaction involving methyl-CpG binding (MBD) proteins, histone modifying enzymes and chromatin remodelling complexes. Hence, targeted inhibition of MBD protein function is now considered a potential therapeutic alternative for thwarting DNA hypermethylation prompted neoplastic progress. We have analyzed the gene and protein expression level of the principal factors responsible for gene silencing, that is, DNMT and MBD proteins in MCF-7 and MDA-MB-231 breast cancer cell lines after treatment with various epigenetic drugs. RESULTS: Our study reveals that the epigenetic modulators affect the expression levels at both transcript and protein levels as well as encourage growth arrest and apoptosis in MCF-7 and MDA-MB-231 cells. AZA, TSA, SFN, and SAM inhibit cell growth in MCF-7 and MDA-MB-231 cell lines in a dose-dependent manner, that is, with increasing concentrations of drugs the cell viability gradually decreases. All the epigenetic modulators promote apoptotic cell death, as is evident form increased chromatin condensation which is a distinct characteristic of apoptotic cells. From FACS analysis, it is also clear that these drugs induce G2-M arrest and apoptosis in breast cancer cells. Further, transcript and protein level expression of MBDs and DNMTs is also affected - after treatment with epigenetic drugs; the level of transcripts/mRNA of MBDs and DNMTs has consistently increased in general. The increase in level of gene expression is substantiated at the protein level also where treated cells show higher expression of DNMT1, DNMT3A, DNMT3B, and MBD proteins in comparison to untreated cells. In case of tissue samples, the expression of different DNMTs is tissue stage-specific. DNMT1 exhibits significantly higher expression in the metastatic stage, whereas, DNMT3A and DNMT3B have higher expression in the primary stage in comparison to the metastatic samples. CONCLUSION: The epigenetic modulators AZA, TSA, SFN, and SAM may provide opportunities for cancer prevention by regulating the components of epigenetic gene-silencing machinery especially DNMTs and MBDs.

Elucidation of caveolin 1 both as a tumor suppressor and metastasis promoter in light of epigenetic modulators.

Caveolin-1 (CAV1) is an integral part of plasma membrane protein playing a vital role in breast cancer initiation and progression. CAV1 acts both as a tumor suppressor as well as an oncogene, and its activity is thus highly dependent on cellular environment. Keeping this fact in mind, the recent work is designed to reveal the role of CAV1 in inhibiting cancer cell progression in presence of epigenetic modulators like 5-aza-2'-deoxycytidine (AZA), trichostatin A (TSA), S-adenosyl methionine (SAM) and sulforaphane (SFN). Forced expression of CAV1 by AZA, TSA, and SFN is correlated to induction of apoptosis and inhibition of cell migration in breast cancer. In breast cancer along with promoter DNA methylation, other epigenetic mechanisms are also involved in CAV1 expression. These observations clearly provide a new scenario regarding the role of CAV1 in cancer and as a possible therapeutic target in breast cancer.

Epigenetic choreography of stem cells: the DNA demethylation episode of development.

Reversible DNA methylation is a fundamental epigenetic manipulator of the genomic information in eukaryotes. DNA demethylation plays a very significant role during embryonic development and stands out for its contribution in molecular reconfiguration during cellular differentiation for determining stem cell fate. DNA demethylation arbitrated extensive make-over of the genome via reprogramming in the early embryo results in stem cell plasticity followed by commitment to the principal cell lineages. This article attempts to highlight the sequential phases and hierarchical mode of DNA demethylation events during enactment of the molecular strategy for developmental transition. A comprehensive knowledge regarding the pattern of DNA demethylation during embryogenesis and organogenesis and study of the related lacunae will offer exciting avenues for future biomedical research and stem cell-based regenerative therapy.

Histone deacetylases: a saga of perturbed acetylation homeostasis in cancer.

In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer.