Sugar-terminated carbon-nanodots stimulate osmolyte accumulation and ROS detoxification for the alleviation of salinity stress in Vigna radiata.

In recent times, nanotechnology has emerged as an efficient tool to manage the adverse effect of environmental stresses on plants. In this connection, carbon-nanodots (CNDs) have been reported to ameliorate the negative impacts of salinity stress. Further, surface modification of CNDs is believed to augment their stress-alleviating potential, however, very little has been known about the potential of surface-functionalized CNDs. In this purview, two sugar (trehalose and glucose) terminated CNDs (CNPT and CNPG) have been synthesized and assessed for their stress-alleviating effects on Vigna radiata (a salt-sensitive legume) seedlings subjected to different concentrations of NaCl (0, 50, and 100 mM). The synthesized CNDs (CNPT and CNPG) exhibited a hydrodynamic size of 20-40 nm and zeta potential of up to - 22 mV with a 5-10 nm core. These water-soluble nanomaterials exhibited characteristic fluorescence emission properties viz. orange and greenish-yellow for CNPT and CNPG respectively. The successful functionalization of the sugar molecules on the CND cores was further confirmed using FTIR, XRD, and AFM. The results indicated that the application of both the CNDs improved seed germination, growth, pigment content, ionic and osmotic balance, and most importantly, the antioxidant defense which decreased ROS accumulation. At the same time, CNPT and CNPG exhibited no toxicity in the Allium cepa root tip bioassay. Therefore, it can be concluded that sugar-terminated CNDs improved the plant responses to salinity stress by facilitating sugar uptake to the aerial part of the seedlings.

Dissecting miRNA facilitated physiology and function in human breast cancer for therapeutic intervention.

MicroRNAs (miRNAs) are key epigenomic regulators of biological processes in animals and plants. These small non coding RNAs form a complex networks that regulate cellular function and development. MiRNAs prevent translation by either inactivation or inducing degradation of mRNA, a major concern in post-transcriptional gene regulation. Aberrant regulation of gene expression by miRNAs is frequently observed in cancer. Overexpression of various 'oncomiRs' and silencing of tumor suppressor miRNAs are associated with various types of human cancers, although overall downregulation of miRNA expression is reported as a hallmark of cancer. Modulations of the total pool of cellular miRNA by alteration in genetic and epigenetic factors associated with the biogenesis of miRNA machinery. It also depends on the availability of cellular miRNAs from its store in the organelles which affect tumor development and cancer progression. Here, we have dissected the roles and pathways of various miRNAs during normal cellular and molecular functions as well as during breast cancer progression. Recent research works and prevailing views implicate that there are two major types of miRNAs; (i) intracellular miRNAs and (ii) extracellular miRNAs. Concept, that the functions of intracellular miRNAs are driven by cellular organelles in mammalian cells. Extracellular miRNAs function in cell-cell communication in extracellular spaces and distance cells through circulation. A detailed understanding of organelle driven miRNA function and the precise role of extracellular miRNAs, pre- and post-therapeutic implications of miRNAs in this scenario would open several avenues for further understanding of miRNA function and can be better exploited for the treatment of breast cancers.

Paederia foetida induces anticancer activity by modulating chromatin modification enzymes and altering pro-inflammatory cytokine gene expression in human prostate cancer cells.

Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer.

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H2O2 induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.

miR-193a targets MLL1 mRNA and drastically decreases MLL1 protein production: Ectopic expression of the miRNA aberrantly lowers H3K4me3 content of the chromatin and hampers cell proliferation and viability.

Mixed-lineage leukaemia 1 (MLL1) enzyme plays major role in regulating genes associated with vertebrate development. Cell physiology and homeostasis is regulated by microRNAs in diverse microenvironment. In this investigation we have identified conserved miR-193a target sites within the 3'-UTR of MLL1 gene transcript. Utilizing wild type and mutated 3'-UTR constructs and luciferase reporter assays we have clearly demonstrated that miR-193a directly targets the 3'-UTR region of the MLL1 mRNA. Ectopic expression of miR-193a modulated global H3K4 mono-, di- and tri-methylation levels and affects the expression of CAV1, a gene which is specifically modulated by H3K4me3. To determine the implications of this in vitro finding in aberrant physiological conditions we analyzed prostate cancer tissue samples. In this context miR-193a RNA was undetectable and MLL1 was highly expressed with concomitantly high levels of H3K4me, H3K4me2, and H3K4me3 enrichment in the promoters of MLL1 responsive genes. Finally, we showed that prolonged ectopic expression of miR-193a inhibits growth and cell migration, and induces apoptosis. Thus, while our study unveils amplitude of the epigenome, including miRnome it establishes that; (i) miR-193a directly target MLL1 mRNA, (ii) miR-193a impair MLL1 protein production, (iii) miR-193a reduces the overall methylation marks of the genome.

DNA methylation regulates Microtubule-associated tumor suppressor 1 in human non-small cell lung carcinoma.

Microtubule associated tumor suppressor 1 (MTUS1) has been recognized as a tumor suppressor gene in multiple cancers. However, the molecular mechanisms underlying the regulation of MTUS1 are yet to be investigated. This study aimed to clarify the significance of DNA methylation in silencing MTUS1 expression. We report that MTUS1 acts as tumor suppressor in non-small cell lung carcinoma (NSCLC). Analysis of in silico database and subsequent knockdown of DNMT1 suggested an inverse correlation between DNMT1 and MTUS1 function. Interestingly, increased methylation at MTUS1 promoter is associated with low expression of MTUS1. Treatment with DNA methyltransferases (DNMTs) inhibitor, 5-aza-2'-deoxycytidine (AZA) leads to both reduced promoter methylation accompanied with enrichment of H3K9Ac and enhanced MTUS1 expression. Remarkably, knockdown of MTUS1 showed increased proliferation and migration of NSCLC cells in contrast to diminished proliferation and migration, upon treatment with AZA. We concluded that low expression of MTUS1 correlates to DNA methylation and histone deacetylation in human NSCLC.

Antiamyloidogenic Chemical/Biochemical-Based Designed Nanoparticle as Artificial Chaperone for Efficient Inhibition of Protein Aggregation.

Protein aggregation is linked to variety of neurodegenerative disorders and other diseases. Current research involves understanding the mechanism of protein aggregation, inhibiting protein aggregation under intra/extracellular space, lowering toxicity arising due to soluble oligomers, and augmenting the clearance of protein aggregates from the brain. Toward this direction, different types of antiamyloidogenic small molecules, macromolecules, and nanomaterials are identified that can inhibit protein aggregation, and extensive progress has been made for their effective utilization. Here, we summarize our effort in designing a nanoparticle form of antiamyloidogenic molecules with enhanced performance under in vitro and in vivo conditions. We found that the nanoparticle form of antiamyloidogenic molecules can perform up to 100,000-times better than the respective molecular form due to the combined effect of enhanced bioavailability at intra/extracellular space and multivalent binding property with aggregating protein. This work demonstrates that further research should be directed toward designing nanoparticle forms of antiamyloidogenic molecules for their effective performance.

Poly(trehalose) Nanoparticles Prevent Amyloid Aggregation and Suppress Polyglutamine Aggregation in a Huntington's Disease Model Mouse.

Prevention and therapeutic strategies for various neurodegenerative diseases focus on inhibiting protein fibrillation, clearing aggregated protein plaques from the brain, and lowering protein-aggregate-induced toxicity. We have designed poly(trehalose) nanoparticles that can inhibit amyloid/polyglutamine aggregation under extra-/intracellular conditions, reduce such aggregation-derived cytotoxicity, and prevent polyglutamine aggregation in a Huntington's disease (HD) model mouse brain. The nanoparticles have a hydrodynamic size of 20-30 nm and are composed of a 6 nm iron oxide core and a zwitterionic polymer shell containing ∼5-12 wt % covalently linked trehalose. The designed poly(trehalose) nanoparticles are 1000-10000 times more efficient than molecular trehalose in inhibiting protein fibrillation in extra-cellular space, in blocking aggregation of polyglutamine-containing mutant huntingtin protein in model neuronal cells, and in suppressing mutant huntingtin aggregates in HD mouse brain. We show that the nanoparticle form of trehalose with zwitterionic surface charge and a trehalose multivalency (i.e., number of trehalose molecules per nanoparticle) of ∼80-200 are crucial for efficient brain targeting, entry into neuronal cells, and suppression of mutant huntingtin aggregation. The present work shows that nanoscale trehalose can offer highly efficient antiamyloidogenic performance at micromolar concentration, compared with millimollar to molar concentrations for molecular trehalose. This approach can be extended to in vivo application to combat protein-aggregation-derived neurodegenerative diseases.

Epigenetics of reproductive infertility.

Infertility is a complex pathophysiological condition. It may caused by specific or multiple physical and physiological factors, including abnormalities in homeostasis, hormonal imbalances and genetic alterations. In recent times various studies implicated that, aberrant epigenetic mechanisms are associated with reproductive infertility. There might be transgenerational effects associated with epigenetic modifications of gametes and studies suggest the importance of alterations in epigenetic modification at early and late stages of gametogenesis. To determine the causes of infertility it is necessary to understand the altered epigenetic modifications of associated gene and mechanisms involved therein. This review is devoted to elucidate the recent mechanistic advances in regulation of genes by epigenetic modification and emphasizes their possible role related to reproductive infertility. It includes environmental, nutritional, hormonal and physiological factors and influence of internal structural architecture of chromatin nucleosomes affecting DNA and histone modifications in both male and female gametes, early embryogenesis and offspring. Finally, we would like to emphasize that research on human infertility by gene knock out of epigenetic modifiers genes must be relied upon animal models.

Sugar-Terminated Nanoparticle Chaperones Are 102-105 Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity.

Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 102-105 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 102-105 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.

Inhibition and Degradation of Amyloid Beta (Aß40) Fibrillation by Designed Small Peptide: A Combined Spectroscopy, Microscopy, and Cell Toxicity Study.

A designed nontoxic, nonhemolytic 11-residue peptide, NF11 (NAVRWSLMRPF), not only inhibits the aggregation of amyloid beta (Aß40) protein but also disaggregates the preformed oligomers and mature Aß fibrils, thereby reducing associated-toxicity. NMR experiments provide evidence of NF11's ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aß40. NF11 has micromolar binding affinity toward both monomeric and aggregated species for efficient clearance of toxic aggregates. From these in vitro results, the future development of a next generation peptidomimetic therapeutic agent for amyloid disease may be possible.

SOX2 function and Hedgehog signaling pathway are co-conspirators in promoting androgen independent prostate cancer.

Developmentally inclined hedgehog (HH) signaling pathway and pluripotency inducing transcription factor SOX2 have been known to work syngerstically during cellular reprogramming events to facilitate efficient differentiation. Hence, it is not surprising that both the factors are actively involved in arbitrating malignant growth, including prostate cancer progression. Here, we have described in details the potential mechanisms by which SOX2 effects neoplastic characteristics in prostate cancer and investigated the consequences of simultaneous down-regulation of SOX2 and HH pathway in androgen-independent human prostate cancer cells. Expression of SOX2 has been determined by qRT-PCR, western blot, immunohistochemistry and immunocytochemistry analyses; its functional role determined by gene knockdown using RNAi and over-expression via chemical activation in HaCaT, DU145 and PC-3 cells. Changes in level of cell proliferation, migration and apoptosis profiles were measured by MTT, FACS, chromatin condensation and scratch assays respectively. SOX2 was expressed in all the three cell lines and its inhibition reduced cell proliferation and induced apoptosis. Most importantly, when both SOX2 and HH pathway were targeted simultaneously, cell proliferation was greatly reduced, apoptotic cell population increased drastically and migration potential was reduced. Moreover, gene expression of EMT markers such as E-cadherin and apoptosis related Bcl-2 and Bax was also investigated wherein decrease in E-cadherin and Bcl-2 levels and increase in Bax expression further substantiating our claim. These findings could provide the basis for a novel therapeutic strategy targeting both the effector i.e. SOX2 and perpetuator i.e. HH pathway of aggressive tumorigenic properties in androgen independent prostate cancer.

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.

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.

Detection and Monitoring of Amyloid Fibrillation Using a Fluorescence "Switch-On" Probe.

Amyloid protein fibrillation is associated with a variety of neurodegenerative and other diseases, and their efficient detection and monitoring can greatly advance early diagnosis and therapy. Herein, we report a fluorescent "switch-on" probe for the reliable detection and monitoring of amyloid fibrils. The probe consists of a peptide component for binding with amyloid structure and a color component with an aggregation-induced green emission property. This probe is nonfluorescent in the presence of amyloid forming monomer protein/peptide, but fluorescence "switch-on" occurs after binding with amyloid fibrils. Compared to conventionally used thioflavin T, this probe offers a high signal-to-noise ratio, which is unaffected by the quencher ion/nanoparticle. The proposed new probe has been used for the detection and monitoring of amyloid fibrils produced by a wide variety of amyloid protein/peptides and can be extended for in vitro diagnostic applications.

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.