Inhibition Of Gene Transcription Research Articles

Role of programmed cell death 4 in diseases: a double-edged sword.

The molecule of programmed cell death 4 (PDCD4) is named based on its upregulation during apoptosis. It is now known that PDCD4 is highly conserved during evolution and widely expressed in the immune cells and non-immune cells of humans and other animals. PDCD4, as an inhibitor of gene transcription and translation, is very important in maintaining the normal function of cells.1, 2 PDCD4 protein contains an N-terminal domain and two MA-3 domains in the central and C-terminal regions. The MA-3 domains can competitively bind to the translation initiation factor eIF4A with eIF4G to inhibit eIF4A activity and to impact the translation of specific mRNAs containing structured 5′-untranslated regions (5′UTRs).2 In this eIF4A-dependent manner, PDCD4 is able to inhibit the translational initiation of multiple genes, including tumor suppressor gene p53, apoptosis-related gene pro-caspase 3, autophagy-related gene Atg5 and metabolism-related gene LXR-α.3, 4, 5, 6 With its N-terminal domain, PDCD4 interacts with specific RNA secondary structures, such as c-myb and A-myb (proto-oncogene) mRNAs, and thereby suppresses translation elongation.7 The N-terminal domain is also responsible for PDCD4 functional interference by binding to certain proteins, such as poly (A)-binding protein (PABP), Daxx (a scaffold protein with roles in diverse processes, including transcriptional regulation and DNA-damage signaling), transcription factor Twist1 and the p65 subunit of NF-κB.8, 9, 10, 11 In addition, PDCD4 can also inhibit the activation of the ERK/P38 MAPK pathway and thereby suppress the expression of cytokines, such as Interleukin-10 (IL-10) (Figure 1a).12

Inhibitors of the Histone Methyltransferases EZH2/1 Induce a Potent Antiviral State and Suppress Infection by Diverse Viral Pathogens.

ABSTRACTEpigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo. Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses.

Abstract 4777: Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway

Abstract Stemness-high cancer cells, or cancer stem cells (CSC) represent a subpopulation of cancer cells with enhanced tumorigenic capacity, metastasis-forming potential, and resistance to conventional chemotherapy and radiation. One such key CSC pathway is regulated by STAT3, a transcription factor that is downstream of several cytokines and growth factor receptors which controls the expression of a broad range of target genes and plays essential roles in CSC biology in many tumor types. Cancer stemness inhibitor Napabucasin (BBI-608), a small molecule that inhibits gene transcription driven by STAT3, can inhibit stemness gene expression, block spherogenesis, and kill CSCs. In vivo, BBI-608 effectively blocks cancer relapse and metastasis in xenograft models while sparing normal hematopoietic stem cells, suggesting that targeting stemness-high cancer cells is a feasible approach for developing next-generation cancer therapeutics to combat cancer recurrence. SOX2 (sex-determining region Y-box protein 2), a transcription factor that is essential for self-renewal and pluripotency, is amplified in various cancer types and has been shown to be required for CSC self-renewal and maintenance. Mucin 1 (MUC1), a glycoprotein normally expressed at the apical surface of epithelial cells, is overexpressed in most human epithelial cancers. MUC1 has been implicated in regulating tumor proliferation, metabolism, invasion, angiogenesis, chemoresistance, and inflammation. Here, we generated a stemness-high culture system based on the reporter activity of a SOX2-regulatory region construct in MKN28 gastric cancer cells (MKN28 SOX2-reporter GFP+ cells). BBI-608 treatment inhibited the STAT3-MUC1 pathway in stemness-high cells. High MUC1 status in stemness-high cells was associated with Paclitaxel resistance. Down-regulation of MUC1 sensitized stemness-high cells to Paclitaxel. Moreover, BBI-608 synergized with Paclitaxel in inhibiting spherogenesis of stemness-high cells. This study provides a new mechanism for the association of cancer stemness with drug resistance. Our findings support this combination therapy, which pairs a conventional chemotherapy (Paclitaxel) with a cancer stemness inhibitor (BBI-608), as a promising strategy to combat cancer. Citation Format: Harry A. Rogoff, Juying Li, Chiang Li. Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2017-4777

Identification of a novel metastasis inducing lncRNA which suppresses the KAI1/CD82 metastasis suppressor gene and is upregulated in triple-negative breast cancer.

Inactivation of tumor/metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. KAI1/CD82 is a metastasis suppressor gene whose normal protecting activity is deficient in twelve different solid malignancies. Here we have identified and characterized a primarily nuclear non-polyadenylated, antisense (as)-lncRNA, initiating upstream of the KAI1 human metastasis suppressor gene transcription start site; and elongating in the opposite direction to KAI1 mRNA. We show that the KAI1 promoter is bi-directional giving rise to KAI1 mRNA and its as-lncRNA. Moreover, expression of this lncRNA transcript emerges to be inversely related to the KAI1 mRNA expression, and in direct relationship to the invasiveness level of human breast cancer derived cell lines. Importantly, knockdown of the KAI1 as-lncRNA in the triple-negative breast cancer cell line MDA-MB-231 have led to increased KAI1 mRNA and protein expression, manifested in stronger adhesion to fibronectin, retardation of cell migration and reduced cell invasion in vitro. Accordingly we have named this lncRNA, SKAI1BC, standing for “Suppressor of KAI1 in Breast Cancer”. These results uncover a potential way to harness tumor metastasis via targeting SKAI1BC in human breast cancer, and perhaps also in other KAI1-deficient human malignancies.

NF-κB upregulation through epigenetic silencing of LDOC1 drives tumor biology and specific immunophenotype in Group A ependymoma.

Inflammation has been identified as a hallmark of high-risk Group A (GpA) ependymoma (EPN). Chronic interleukin (IL)-6 secretion from GpA tumors drives an immune suppressive phenotype by polarizing infiltrating monocytes. This study determines the mechanism by which IL-6 is dysregulated in GpA EPN. Twenty pediatric GpA and 21 pediatric Group B (GpB) EPN had gene set enrichment analysis for MSigDB Hallmark gene sets performed. Protein and RNA from patients and cell lines were used to validate transcriptomic findings. GpA cell lines 811 and 928 were used for in vitro experiments performed in this study. The nuclear factor-kappaB (NF-κB) pathway is a master regulator of IL-6 and a signaling pathway enriched in GpA compared with GpB EPN. Knockdown of NF-κB led to significant downregulation of IL-6 in 811 and 928. NF-κB activation was independent of tumor necrosis factor alpha (TNF-α) stimulation in both cell lines, suggesting that NF-κB hyperactivation is mediated through an alternative mechanism. Leucine zipper downregulated in cancer 1 (LDOC1) is a known transcriptional repressor of NF-κB. In many cancers, LDOC1 promoter is methylated, which inhibits gene transcription. We found decreased LDOC1 gene expression in GpA compared with GpB EPN, and in other pediatric brain tumors. EPN cells treated with 5AZA-DC, demethylated LDOC1 regulatory regions, upregulated LDOC1 expression, and concomitantly decreased IL-6 secretion. Stable knockdown of LDOC1 in EPN cell lines resulted in a significant increase in gene transcription of v-rel avian reticuloendotheliosis viral oncogene homolog A, which correlated to an increase in NF-κB target genes. These results suggest that epigenetic silencing of LDOC1 in GpA EPN regulates tumor biology and drives inflammatory immune phenotype.

Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference

Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhfr and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhfr and foreign gene. CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to be exploited in CHO cells. Here we constructed vectors expressing the functional CRISPRi system and proved effective CRISPRi-mediated suppression of dhfr transcription in CHO cells. We next generated stable CHO cell clones coexpressing DHFR, the model protein (EGFP), dCas9 and gRNA targeting dhfr. Combined with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to coamplify more copies of dhfr and egfp genes. Compared with the traditional method relying on MTX selection (up to 250 nM), the CRISPRi approach increased the dhfr copy number ∼3-fold, egfp copy number ∼3.6-fold and enhanced the EGFP expression ∼3.8-fold, without impeding the cell growth. Furthermore, we exploited the CRISPRi approach to enhance the productivity of granulocyte colony stimulating factor (G-CSF) ∼2.3-fold. Our data demonstrate, for the first time, the application of CRISPRi in CHO cells to enhance recombinant protein production and may pave a new avenue to CHO cell engineering.

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

SET oncoprotein accumulation regulates transcription through DNA demethylation and histone hypoacetylation.

Epigenetic modifications are essential in the control of normal cellular processes and cancer development. DNA methylation and histone acetylation are major epigenetic modifications involved in gene transcription and abnormal events driving the oncogenic process. SET protein accumulates in many cancer types, including head and neck squamous cell carcinoma (HNSCC); SET is a member of the INHAT complex that inhibits gene transcription associating with histones and preventing their acetylation. We explored how SET protein accumulation impacts on the regulation of gene expression, focusing on DNA methylation and histone acetylation. DNA methylation profile of 24 tumour suppressors evidenced that SET accumulation decreased DNA methylation in association with loss of 5-methylcytidine, formation of 5-hydroxymethylcytosine and increased TET1 levels, indicating an active DNA demethylation mechanism. However, the expression of some suppressor genes was lowered in cells with high SET levels, suggesting that loss of methylation is not the main mechanism modulating gene expression. SET accumulation also downregulated the expression of 32 genes of a panel of 84 transcription factors, and SET directly interacted with chromatin at the promoter of the downregulated genes, decreasing histone acetylation. Gene expression analysis after cell treatment with 5-aza-2′-deoxycytidine (5-AZA) and Trichostatin A (TSA) revealed that histone acetylation reversed transcription repression promoted by SET. These results suggest a new function for SET in the regulation of chromatin dynamics. In addition, TSA diminished both SET protein levels and SET capability to bind to gene promoter, suggesting that administration of epigenetic modifier agents could be efficient to reverse SET phenotype in cancer.

Lipid-soluble Cigarette Smoke Particles Induced Vascular Endothelin Type A Receptor Up-Regulation through Activation of ERK1/2 Signal Pathways.

Abnormal contraction of vessels termed 'vasospasm' is associated with various cardiovascular diseases. Smoking is a well-known risk factor that increases vasospasm. However, the molecular mechanisms by which smoking leads to vasospasm and cardiovascular disease are not fully understood. This study was designed to examine whether DMSO-extracted cigarette smoke particles (DSP) could induce up-regulation of vascular endothelin type A (ETA ) receptors, and whether ETA receptor is up-regulated through activation of extracellular regulated protein kinases 1 and 2 (ERK1/2) signal pathways. Mesenteric arterial segments from rats were cultured in the presence of DSP, water-extracted cigarette smoke particles (WSP) or equivalent concentration of nicotine for up to 24 hr. The results showed that DSP, but not WSP or nicotine, induced ETA receptor up-regulation with increased ETA receptor-mediated contraction (myograph, p < 0.001). Simultaneously, the expression of ETA receptor mRNA (real-time PCR, p < 0.001) and protein (immunohistochemistry) were enhanced in the smooth muscle cells, suggesting that the lipid-soluble substances contained in cigarette smoke were responsible for the effects of DSP. Actinomycin D (a general transcriptional inhibitor) decreased ETA receptor mRNA expression and attenuated receptor-mediated contraction (p < 0.001), while DSP accelerated ETA receptor mRNA degradation (p < 0.01) and promoted the translation of ETA receptor mRNA into protein. Furthermore, the up-regulation of ETA receptors was significantly attenuated by inhibition of ERK1/2 signal pathways (p < 0.001). In conclusion, DSP most likely activate ERK1/2 signal pathway-mediated transcriptional and post-transcriptional (translational) mechanisms that lead to vascular ETA receptor up-regulation, which might contribute to vasospasm and the development of smoking-associated cardiovascular diseases.

Triptolide Promotes Senescence of Prostate Cancer Cells Through Histone Methylation and Heterochromatin Formation

Background:Triptolide is a medicinal herb-derived diterpene triepoxide with potent anti-tumor activity, mainly, correlated with its ability to inhibit and inactivate subunits of RNA polymerase II, thereby suppressing global gene transcription. Epigenetic imbalance including histone methylation are well known to play important roles in prostate cancer (PCa) onset and progression. The goal of this study was to investigate whether triptolide might exert anti-PCa influence by reshaping the histone methylation landscape.Methods:Triptolide-treated PCa cell lines were analyzed by RT-qPCR and western blotting for expression of histone demethylases and associated markers. Detection of senescence was achieved using senescence associated β-galactosidase staining and analyses of apoptosis and cell cycle were performed by flow cytometry. Senescence–associated heterochromatin foci were detected by immunofluorescence while chromatin immunoprecipitation associated with qPCR (CHIP-qPCR) was applied to assess accumulation of histone markers on promoters of target genes. Cell viability was determined using the CCK-8 assay.Results:We found triptolide to enhance H3K27me3 levels by down-regulating JMJD3 and UTX and also H3K9me3 through up-regulation of SUV39H1. Furthermore, it up-regulated expression of HP1α. Thereby, heterochromatin formation and deposition on promoters of E2F1-target genes was promoted, correlating with suppression of gene transcription, decreased cell viability and induction of a senescence-like phenotype in PCa cells.Conclusions:Our results indicate that triptolide exerts anti-tumor effects including PCa cell senescence at least partially through increasing the levels of repressive histone H3 methylation and formation of a repressive chromatin state in PCa cells. Further studies of its potential as an epigenetic anti-PCa drug appear warranted.

Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model.

Histone acetyl transferases (HATs) and histone deacetylases (HDAC) control transcription during myogenesis. HDACs promote chromatin condensation, inhibiting gene transcription in muscle progenitor cells until myoblast differentiation is triggered and HDACs are released. HATs, namely CBP/p300, activate myogenic regulatory and elongation factors promoting myogenesis. HDAC inhibitors are known to improve regeneration in dystrophic muscles through follistatin upregulation. However, the potential of directly modulating HATs remains unexplored. We tested this possibility in a well-known zebrafish model of Duchenne muscular dystrophy. Interestingly, CBP/p300 transcripts were found downregulated in the absence of Dystrophin. While investigating CBP rescuing potential we observed that dystrophin-null embryos overexpressing CBP actually never show significant muscle damage, even before a first regeneration cycle could occur. We found that the pan-HDAC inhibitor trichostatin A (TSA) also prevents early muscle damage, however the single HAT CBP is as efficient even in low doses. The HAT domain of CBP is required for its full rescuing ability. Importantly, both CBP and TSA prevent early muscle damage without restoring endogenous CBP/p300 neither increasing follistatin transcripts. This suggests a new mechanism of action of epigenetic regulators protecting dystrophin-null muscle fibres from detaching, independent from the known improvement of regeneration upon damage of HDACs inhibitors. This study builds supporting evidence that epigenetic modulators may play a role in determining the severity of muscle dystrophy, controlling the ability to resist muscle damage. Determining the mode of action leading to muscle protection can potentially lead to new treatment options for muscular dystrophies in the future.

Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models.

Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large-scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell-based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two-dimensional and three-dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery. © 2016 International Society for Advancement of Cytometry.

Using Histone Deacetylase Inhibitors to Analyze the Relevance of HDACs for Translation.

Gene expression is regulated in part through the reversible acetylation of histones, by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). HAT activity results in the addition of acetyl groups on the lysine residues of histone tails leading to decondensation of the chromatin, and increased gene transcription in general, whereas HDACs remove these acetyl groups, thus leading to an overall suppression of gene transcription. Recent evidence has elucidated that histones are not the only components of the proteome that are targeted by HATs and HDACs. A large number of nonhistone proteins undergo posttranslational acetylation. They include proteins involved in mRNA stability, protein localization and degradation, as well as protein-protein and protein-DNA interactions. In recent years, numerous studies have discovered increased HDAC expression and/or activity in numerous disease states, including cancer, where the upregulation of HDAC family members leads to dysregulation of genes and proteins involved in cell proliferation, cell cycle regulation, and apoptosis. These observations have pushed HDAC inhibitors (HDACi) to the forefront of therapeutic development of oncological conditions. HDACi, such as Vorinostat (Suberoylanilide hydroxamic acid (SAHA)), affect cancer cells in part by suppressing the translation of key proteins linked to tumorigenesis, such as cyclin D1 and hypoxia inducible factor 1 alpha (HIF-1α). Herein we describe methodologies to analyze the impact of the HDACi Vorinostat on HIF-1α translational regulation and downstream effectors.

Regulation of protein and mRNA expression of the mTORC1 repressor REDD1 in response to leucine and serum

Expression of the mTORC1 repressor, Regulated in DNA Damage and Development 1 (REDD1), is elevated in skeletal muscle during various catabolic conditions including fasting, hindlimb immobilization, and sepsis. Conversely, REDD1 expression is suppressed by anabolic stimuli such as resistance exercise or nutrient consumption following a fast. Though it is known that nutrient consumption reduces REDD1 expression, it is largely unknown how nutrients and hormones individually contribute to the reduction in REDD1 expression. Therefore, the purpose of the present study was to determine how nutrients and hormones individually regulate REDD1 expression. HeLa cells were deprived of leucine or serum for 10h, after which either leucine or serum was reintroduced to cell culture medium for 60min. Re-supplementation of either leucine or serum resulted in a reduction in REDD1 protein levels by 34.8±5.8% and 54.1±3.4%, respectively, compared to the deprived conditions. Re-supplementation of leucine or serum to deprived cells also led to a reduction in REDD1 mRNA content by 49.1±2.7% and 65.0±1.4%, respectively, compared to the deprived conditions. Interestingly, rates of REDD1 protein degradation were unaffected by either leucine or serum re-supplementation, as assessed in cells treated with cycloheximide to block protein synthesis. Likewise, addition of leucine- or serum to cells treated with Actinomycin D to inhibit gene transcription failed to alter the rate of REDD1 mRNA degradation. The data indicate that the leucine or serum-induced suppression of REDD1 expression occurs independent of changes in the rate of degradation of either the REDD1 protein or mRNA. Thus, the leucine- or serum-induced suppression likely occurs through alternative mechanism(s) such as reduced REDD1 gene transcription and/or mRNA translation.

Large tumor suppressor homologs 1 and 2 regulate mouse liver progenitor cell proliferation and maturation through antagonism of the coactivators YAP and TAZ.

Hippo signaling, mediated by the LATS1 and LATS2 kinases, is required to restrict YAP and TAZ activation during both biliary and hepatocyte differentiation. These findings suggest that dynamic regulation of the Hippo signaling pathway plays an important role in differentiation and functional maturation of the liver. (Hepatology 2016;64:1757-1772).

YIA 03-04 EPIGENETIC CHANGES AFTER ACUTE TREATMENT WITH ACUTE ANGIOTENSIN CONVERTING ENZYME INHIBITORS (ACEi)

Objective: The ‘legacy effect’ of hypertension treatment is where short term treatment with blood pressure (BP) lowering drugs such as angiotensin converting enzyme inhibitors (ACEi) is followed by a persistent reduction in BP, reduced cardiovascular complications and increased lifespan. However, the involvement of epigenetics mechanisms remains unclear. DNA methylation is the binding of a methyl group to DNA which inhibits gene transcription. The aim of this study is to investigate DNA methylation changes after short term treatment with ACEi. Design and Method: Spontaneously hypertensive rats were treated with the ACEi perindopril for 48 hours at 6 weeks old. Average global DNA methylation was quantified in renal cortices from perindopril (n = 6) and vehicle-treated animals (n = 6) using the 5-mC ELISA Kit (Zymo Research, USA) which features a unique anti-5-methylcytosine monoclonal antibody that is both sensitive and specific for 5-mC. We also measured the expression of epigenetic regulators histone deacetylase 1 (Hdac1), solute carrier family 16 member 3 (monocarboxylate transporter, Mct3) and DNA (cytosine-5-)-methyltransferase 1 (Dnmt1) using quantitative PCR. Results: Global DNA methylation was reduced in the renal cortices of animals treated with ACEi (P < 0.05, Figure 1). Acute treatment with perindopril did not significantly change the renal expression of Hdac1, Dnmt1 and Mct3 (P > 0.05). Conclusions: Acute treatment by ACEi results in a reduction in global DNA methylation indicating that epigenetics may play an important role in the ‘legacy effect’. Further investigation into which specific genes contribute to these changes and if longer course of treatment have a higher impact in the expression of such genes is important.

Baicalein Attenuates Neurological Deficits and Preserves Blood–Brain Barrier Integrity in a Rat Model of Intracerebral Hemorrhage

Previous studies have demonstrated that baicalein has protective effects against several diseases, which including ischemic stroke. The effect of baicalein on the blood-brain barrier (BBB) in intracerebral hemorrhage (ICH) and its related mechanisms are not well understood. We aimed to investigate the mechanisms by which baicalein may influence the BBB in a rat model of ICH. The rat model of ICH was induced by intravenous injection of collagenase IV into the brain. Animals were randomly divided into three groups: sham operation, vehicle, and baicalein group. Each group was then divided into subgroups, in which the rats were sacrificed at 24 and 72h after ICH. We assessed brain edema, behavioral changes, BBB leakage, apoptosis, inducible nitric oxide synthase (iNOS), zonula occludens (ZO)-1, Mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). Treatment with baicalein reduced brain water content, BBB leakage, apoptosis, and neurologic deficits, compared with vehicle. Baicalein also decreased ICH-induced changes in the levels of iNOS but increased the levels of ZO-1. The protective effect of baicalein on the BBB in ICH rats was possibly invoked by attenuated p-38 MAPK and JNK phosphorylation, and decreased activation of the NF-κB signaling pathway, which may have suppressed gene transcription, including iNOS, and eventually decreased formation of peroxynitrite (ONOO-). Our results suggest that baicalein exerts a protective effect on BBB disruption in the rat model of ICH. The likely mechanism is via inhibition of MAPKs and NF-κB signaling pathways, leading to decreased formation of iNOS and ONOO-, thereby improving neurological function.

Acute Stress Suppresses Synaptic Inhibition and Increases Anxiety via Endocannabinoid Release in the Basolateral Amygdala

Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoid-induced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoid-induced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis. We provide a cellular mechanism in the basolateral amygdala (BLA) for the rapid stress regulation of anxiogenesis in rats. We demonstrate a nongenomic glucocorticoid induction of long-lasting suppression of synaptic inhibition that is mediated by retrograde endocannabinoid release at GABA synapses. The rapid glucocorticoid-induced endocannabinoid suppression of synaptic inhibition is initiated by a membrane-associated glucocorticoid receptor in BLA principal neurons. We show that acute stress increases anxiety-like behavior via an endocannabinoid-dependent mechanism centered in the BLA. The stress-induced endocannabinoid modulation of synaptic transmission in the BLA contributes, therefore, to the stress regulation of anxiety, and may play a role in anxiety disorders of the amygdala.

Abstract 3090: Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules

Abstract Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3’ capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3’ capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3’ capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs. Citation Format: Clement Lin, Prashansa Agrawal, Yuwei Chen, Salil Kalarn, Nanjie Deng, Laurence Hurley, Danzhou Yang. Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3090.

Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs

Abstract Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3766.