HDAC Activity Research Articles

Comparison of a histone deacetylase inhibitor plus exemestane with exemestane alone in hormone receptor-positive advanced breast cancer that progressed on prior endocrine therapy: A meta-analysis.

Currently, endocrine therapy is the standard treatment for hormone receptor-positive advanced breast cancer (ABC). Despite the high sensitivity of anti-estrogen therapy, many breast cancer patients still experience disease progression, relapse, and reduced overall survival (OS) because of endocrine resistance. Several underlying mechanisms of this phenomenon include a change in hormone receptor expression, mutations in ESR1 and modification of important signaling pathways, but thus far none of these can be defined as the complete explanation. Additionally, it has been shown that in some breast cancers, expression of the estrogen receptor (ER) can be repressed by epigenetic modifications such as DNA methylation and histone deacetylation, and this could be a mechanism for endocrine resistance. Interestingly, although the efficacy of the combination of histone deacetylase (HADC) inhibitors and exemestane in hormone receptor-positive ABC that progressed on prior endocrine therapy has been investigated in several studies, whether pharmacologic blocking of HDAC activity acts as a therapeutic strategy remains highly controversial. Herein, we conducted a meta-analysis to evaluate the efficacy and safety of an HDAC inhibitor plus exemestane vs. exemestane alone in this setting. Our meta-analysis demonstrated that the combination group exhibited significantly prolonged progression-free survival (PFS) [hazard ratio (HR)=0.776, 95% confidence interval (CI)=0.675-0.892, P=0.000] and an improved objective response rate (ORR) (RR=1.612, 95% CI=1.085-2.396, P=0.018) compared to those treated with exemestane alone. Additionally, in terms of OS, the combination group failed to achieve a significant clinical OS benefit (HR=0.811, 95% CI=0.596-1.104, P=0.183). Although grade 3/4 toxicities were more common in the combination group, those toxicities were mostly asymptomatic and manageable. In conclusion, the addition of an HDAC inhibitor to exemestane significantly improves PFS over exemestane alone in hormone receptor-positive ABC patients who progressed on previous endocrine therapy. Identification of novel biomarkers to select patients who will benefit from this combination strategy is a high priority.

H19 inhibition increases HDAC6 and regulates IRS1 levels and insulin signaling in the skeletal muscle during diabetes

BackgroundHistone deacetylases (HDACs) that catalyze removal of acetyl groups from histone proteins, are strongly associated with several diseases including diabetes, yet the precise regulatory events that control the levels and activity of the HDACs are not yet well elucidated.MethodsLevels of H19 and HDACs were evaluated in skeletal muscles of normal and diabetic db/db mice by Western Blot analysis. C2C12 cells were differentiated and transfected with either the scramble or H19 siRNA and the levels of HDACs and Prkab2, Pfkfb3, Srebf1, Socs2, Irs1 and Ppp2r5b were assessed by Western Blot analysis and qRT-PCR, respectively. Levels of H9, HDAC6 and IRS1 were evaluated in skeletal muscles of scramble/ H19 siRNA injected mice and chow/HFD-fed mice.ResultsOur data show that the lncRNA H19 and HDAC6 exhibit inverse patterns of expression in the skeletal muscle of diabetic db/db mice and in C2C12 cells, H19 inhibition led to significant increase in HDAC activity and in the levels of HDAC6, both at the transcript and protein levels. This was associated with downregulation of IRS1 levels that were prevented in the presence of the HDAC inhibitor, SAHA, and HDAC6 siRNA suggesting the lncRNA H19-HDAC6 axis possibly regulates cellular IRS1 levels. Such patterns of H19, HDAC6 and IRS1 expression were also validated and confirmed in high fat diet-fed mice where as compared to normal chow-fed mice, H19 levels were significantly inhibited in the skeletal muscle of these mice and this was accompanied with elevated HDAC6 levels and decreased IRS1 levels. In-vivo inhibition of H19 led to significant increase in HDAC6 levels and this was associated with a decrease in IRS1 levels in the skeletal muscle.ConclusionsOur results suggest a critical role for the lncRNA H19-HDAC6 axis in regulating IRS1 levels in the skeletal muscle during diabetes and therefore restoring normal H19 levels might hold a therapeutic potential for the management of aberrant skeletal muscle physiology during insulin resistance and type 2 diabetes.

Design of a Fluorogenic Probe Based on Intramolecular Condensation for Specific Detection of HDAC3.

It is crucial to develop fluorogenic probes for selective targeting of HDACs to explore the roles of HDACs in the tumor onset and progression as well as HDAC-related drug development. However, considerable non-specific signals were produced by spontaneous hydrolysis and undesirable intermolecular attack of the unstable caging moiety in the detection of HDACs with previous probes. To improve the detection specificity, we proposed an intramolecular condensation strategy by the replacement of the traditional acetamide moiety with a trans-enamide unit. Upon deacetylation by HDACs, rapid intramolecular condensation reaction between newly formed terminal aldehyde and hydrazine moiety would occur to afford highly fluorescent hydrazone product. Systematic studies demonstrated that the probe exhibited an extraordinary selectivity for HDAC3 over other HDAC isoforms and interfering substances. The stability and specificity of the indicator make it a powerful tool for HDAC3 activity detection and HDAC3-related drug development.

HDAC Inhibitory and Anti-Cancer Activities of Curcumin and Curcumin Derivative CU17 against Human Lung Cancer A549 Cells.

Previous research reported that the curcumin derivative (CU17) inhibited several cancer cell growths in vitro. However, its anticancer potential against human lung cancer cells (A549 cell lines) has not yet been evaluated. The purpose of this research was to examine the HDAC inhibitory and anti-cancer activities of CU17 compared to curcumin (CU) in A549 cells. An in vitro study showed that CU17 had greater HDAC inhibitory activity than CU. CU17 inhibited HDAC activity in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 0.30 ± 0.086 µg/mL against HDAC enzymes from HeLa nuclear extract. In addition, CU17 could bind at the active pockets of both human class I HDACs (HDAC1, 2, 3, and 8) and class II HDACs (HDAC4, 6, and 7) demonstrated by molecular docking studies, and caused hyperacetylation of histone H3 (Ac-H3) in A549 cells shown by Western blot analysis. MTT assay indicated that both CU and CU17 suppressed A549 cell growth in a dose- and time-dependent manner. Besides, CU and CU17 induced G2/M phase cell cycle arrest and p53-independent apoptosis in A549 cells. Both CU and CU17 down-regulated the expression of p53, p21, Bcl-2, and pERK1/2, but up-regulated Bax expression in this cell line. Although CU17 inhibited the growth of lung cancer cells less effectively than CU, it showed less toxicity than CU for non-cancer cells. Accordingly, CU17 is a promising agent for lung cancer treatment. Additionally, CU17 synergized the antiproliferative activity of Gem in A549 cells, indicating the possibility of employing CU17 as an adjuvant treatment to enhance the chemotherapeutic effect of Gem in lung cancer.

Design, Synthesis and Cytotoxic Activity Evaluation of Newly Synthesized Amides-Based TMP Moiety as Potential Anticancer Agents over HepG2 Cells.

A novel series of amides based TMP moiety was designed, synthesized and evaluated for their antiproliferative as well as enzyme inhibition activity. Compounds 6a and 6b showed remarkable cytotoxic activity against HepG2 cells with IC50 values 0.65 and 0.92 μM, respectively compared with SAHA and CA-4 as reference compounds. In addition, compound 6a demonstrated good HDAC-tubulin dual inhibition activity as it showed better HDAC activity as well as anti-tubulin activity. Moreover, compound 6a exhibited G2/M phase arrest and pre-G1 apoptosis as demonstrated by cell cycle analysis and Annexin V assays. Further apoptosis studies demonstrated that compound 6a boosted the level of caspase 3/7. Caspase 3/7 activation and apoptosis induction were evidenced by decrease in mitochondrial permeability suggesting that activation of caspase 3/7 may occur via mitochondrial apoptotic pathway.

Givinostat-Liposomes: Anti-Tumor Effect on 2D and 3D Glioblastoma Models and Pharmacokinetics.

Simple SummaryGlioblastoma is the most common malignant brain tumor with a high grade of recurrence, invasiveness, and aggressiveness. Currently, there are no curative treatments; therefore, the discovery of novel molecules with anti-tumor activity or suitable drug delivery systems are important research topics. The aim of the present study was to investigate the anti-tumor activity of Givinostat, a pan-HDAC inhibitor, and to design an appropriate liposomal formulation to improve its pharmacokinetics profile and brain delivery. The present work demonstrates that the incorporation of Givinostat in liposomes composed of cholesterol and sphingomyelin improves its in vivo half-life and increases the amount of drug reaching the brain in a mouse model. Furthermore, this formulation preserves the anti-tumor activity of glioblastoma in 2D and 3D in vitro models. These features make liposome-Givinostat formulations potential candidates for glioblastoma therapy.Glioblastoma is the most common and aggressive brain tumor, associated with poor prognosis and survival, representing a challenging medical issue for neurooncologists. Dysregulation of histone-modifying enzymes (HDACs) is commonly identified in many tumors and has been linked to cancer proliferation, changes in metabolism, and drug resistance. These findings led to the development of HDAC inhibitors, which are limited by their narrow therapeutic index. In this work, we provide the proof of concept for a delivery system that can improve the in vivo half-life and increase the brain delivery of Givinostat, a pan-HDAC inhibitor. Here, 150-nm-sized liposomes composed of cholesterol and sphingomyelin with or without surface decoration with mApoE peptide, inhibited human glioblastoma cell growth in 2D and 3D models by inducing a time- and dose-dependent reduction in cell viability, reduction in the receptors involved in cholesterol metabolism (from −25% to −75% of protein levels), and reduction in HDAC activity (−25% within 30 min). In addition, liposome-Givinostat formulations showed a 2.5-fold increase in the drug half-life in the bloodstream and a 6-fold increase in the amount of drug entering the brain in healthy mice, without any signs of overt toxicity. These features make liposomes loaded with Givinostat valuable as potential candidates for glioblastoma therapy.

HDAC6-specific inhibitor alleviates hashimoto's thyroiditis through inhibition of Th17 cell differentiation

ObjectiveHashimoto's thyroiditis (HT) is one of the commonest autoimmune disorders. This study was performed to investigate the potential effect of histone deacetylase 6-specific inhibitor (HDAC6i) on Th17 cell differentiation in animal model and the underlying mechanism. MethodsExperimental autoimmune thyroiditis (EAT) mouse model was established by subcutaneously immunization of porcine thyroglobulin (pTg) and adjuvant, and the HDACi Tubastatin A (TSA) or HDAC6i (ACY-1215) was intraperitoneally injected into mice in the following. The histological examination and immune analysis in EAT mice were carried out. Next, the CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of EAT mice followed by Th17 cell differentiation assay. The associated factor levels, and the protein interaction between HDAC6 and PKM2 were examined. Subsequently, the effect of STAT3 activation on Th17 cell differentiation was explored. ResultsACY-1215 or TSA treatment reduced lymphocytic infiltration and alleviated thyroid tissue injury in EAT mice. Correspondingly, either ACY-1215 or TSA treatment reduced the levels of anti-thyroglobulin (Tg), anti-thyroid peroxidase (TPO), IL-17A, and IFN-γ in the serum, decreased Th17 cell differentiation, but enhanced TGF-β level and promoted Treg cell differentiation. In vitro, after induction of Th17 cell differentiation from CD4+ T cells, HDAC6 activity and Th17 cell differentiation were significantly decreased when treated with ACY-1215 or TSA. HDAC6 could interact with PKM2, and HDAC6 overexpression promoted the phosphorylation of STAT3 and PKM2 nuclear translocation. Furthermore, the STAT3 activator treatment reversed the effects of ACY-1215 or TSA treatment. ConclusionHDAC6i suppresses Th17 cell differentiation and attenuates HT via PKM2/STAT3 axis.

Identification of novel HDAC8 selective inhibitors through ligand and structure based studies: Exploiting the acetate release channel differences among class I isoforms

Histone deacetylases (HDACs) are key regulators of gene expression and have emerged as crucial therapeutic targets for cancer. Among the HDACs, inhibition of HDAC8 enzyme has been reported to be a novel strategy in the treatment of female-specific cancers. Most of the HDAC inhibitors discovered so far inhibit multiple HDAC isoforms causing toxicities in the clinic thus limiting their potential. Therefore, the discovery of isoform-selective HDAC8 inhibitors is highly desirable. In the present study, a combination of ligand and structure based drug design tools were utilized to build a statistically significant pharmacophore based 3D QSAR model with statistical parameters R 2 : 0.9964, and Q 2 : 0.7154, from a series of 31 known HDAC8 inhibitors. Top 1000 hits obtained from Virtual screening of Phase database were subjected to docking studies against HDAC8. Top 100 hits obtained were redocked into HDAC Class I (HDAC 1,2,3) and Class II isoforms (HDAC 4, 6) and rescored with XP Glide Score. Based on fitness score, XP glide score and interacting amino acid residues, five HDAC8 inhibitors ( 1 – 5 ) were selected for in vitro studies. The HDAC8 activity assay followed by enzyme kinetics clearly indicated Compounds 1 , 2 and 3 to be potent HDAC8 selective inhibitors with IC 50 of 126 pM, 112 nM, and 442 nM respectively. These compounds were cytotoxic to HeLa cells where HDAC8 is overexpressed but not to normal cells, HEK293. Also, they were able to induce apoptosis by modulating Bax/Bcl2, cleavage of PARP and release of Cytochrome C . Molecular Dynamics simulations observed most favorable interaction patterns and presented a rationale for the activities of the identified compounds. Selectivity against HDAC8 was due to exploitation of the architectural difference in the acetate release channel among class I HDAC isoforms.

Intervening Effects and Molecular Mechanism of Quercitrin on PCV2-Induced Histone Acetylation, Oxidative Stress and Inflammatory Response in 3D4/2 Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing porcine circovirus-associated diseases (PCVD/PCVADs), and infection of the host induces immunosuppression. Since quercitrin (QUE) has anti-inflammatory and antiviral activity, it is worth exploiting in animal diseases. In this study, the interventional effects and the molecular mechanism of QUE on PCV2-induced oxidative stress and inflammatory responses in 3D4/2 cells and the modulation of histone acetylation modifications were investigated. The ROS production was measured by DCFH-DA fluorescent probes. HAT and HDAC enzyme activity were determined by ELISA. Histone acetylation, oxidative stress and inflammation-related gene expression levels were measured by q-PCR. Histone H3 and H4 (AcH3 and AcH4) acetylation, oxidative stress and inflammation-related protein expression levels were measured by Western blot. The results showed that QUE treatment at different concentrations on PCV2-infected 3D4/2 cells was able to attenuate the production of ROS. Moreover, QUE treatment could also intervene in oxidative stress and decrease the enzyme activity of HAT and the mRNA expression level of HAT1, while it increased the enzyme activity of HDAC and HDAC1 mRNA expression levels and downregulated histone H3 and H4 (AcH3 and AcH4) acetylation modification levels. In addition, QUE treatment even downregulated the mRNA expression levels of IL-6, IL-8, IκB, AKT and p38, but upregulated the mRNA expression levels of IL-10, SOD, GPx1, p65, Keap1, Nrf2, HO-1 and NQO1. As to protein expression, QUE treatment downregulated the levels of iNOS, p-p65 and IL-8 as well as the phosphorylation expression of IκB and p38, while it upregulated the levels of HO-1 and NQO1. It was shown that QUE at 25, 50 or 100 μmol/L regulated p38MAPK and PI3K/AKT signaling pathways by downregulating cellular histone acetylation modification levels while inhibiting the NF-κB inflammatory signaling pathway and activating the Nrf2/HO-1 antioxidant signaling pathway, thus regulating the production of inflammatory and antioxidant factors and exerting both anti-inflammatory and antioxidant effects.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

A novel aromatic amide derivative SY-65 co-targeted tubulin and histone deacetylase 1 with potent anticancer activity in vitro and in vivo

Given the essential role of Epigenetic regulation in many biological processes, targeted epigenetic drugs have been gradually applied to the treatment of tumors. Histone deacetylases (HDACs) are a class of epigenetic enzymes, which play key roles in chromosome structural modification and gene expression regulation. Targeted microtubules drugs have achieved great success in clinical application for decades. Development of novel agents with multitargeting capabilities specially dual-target has become a popular research field for the treatment of human cancers, which may provide synergistic anticancer effects. Here, we reported a novel aromatic amide derivative SY-65 co-targeted tubulin and histone deacetylase 1 with potent anticancer activity in vitro and in vivo. Compound SY-65 was identified as a dual inhibitor of tubulin/HDAC1 (IC50 = 3.64 and 0.529 μM, respectively) with excellent antiproliferative activity against MGC-803, HCT-116, KYSE-450, HGC-27, SGC-7901 and MKN-45 cells. Especially, compound SY-65 exhibited potent antiproliferative activity against MGC-803, HGC-27 and SGC-7901 cells with IC50 values <55 nM, which was better than that of Colchicine, MS-275 and SAHA. Compound SY-65 effectively inhibited tubulin polymerization and bound to the colchicine binding site of tubulin, as well as inhibited HDAC1 activity both intra/extracellularly. Molecular docking results suggested there were the well-defined binding modes of compound SY-65 in HDAC1 and tubulin. In addition, compound SY-65 inhibited colony formation, interfered with the cell cycle distribution, induced cell cycle arrest at the G2/M phase and apoptosis in MGC-803 and HGC-27 cells. Compound SY-65 also exhibited a good tumor inhibitory effect in vivo without obvious toxicity. Therefore, compound SY-65 could be developed as a novel tubulin/HDAC1 candidate inhibitor for future cancer therapeutics.

Characterization of Acetylation of Histone H3 at Lysine 9 in the Trigeminal Ganglion of a Rat Trigeminal Neuralgia Model.

Trigeminal neuralgia (TN) is a chronic neuropathic pain disorder characterized by spontaneous and elicited paroxysms of electric-shock-like or stabbing pain in a region of the face. The epigenetic regulation of TN is still obscure. In current study, a rat TN model subject to carbamazepine (CBZ) treatment was established, and transcriptome- and genome-scale profiling of H3K9ac and HDAC3 was performed by RNA-seq and ChIP-seq. We observed that H3K9ac levels in the trigeminal ganglion were lower in the TN rats compared with those in the control, and CBZ treatment led to recovery of H3K9ac levels. Further, we found that HDAC3 was overactivated, which interfered with H3K9 acetylation due to higher phosphorylation in TN compared with that in the control. Finally, the phosphokinase leucine-rich repeat kinase 2 (LRRK2) was demonstrated to contribute to HDAC3 activity via the MAPK signaling pathway. Taken together, we identified a regulatory mechanism in which the phosphate groups transferred from activated ERK and LRRK2 to HDAC3 caused genome-scale deacetylation at H3K9 and resulted in the silencing of a large number of genes in TN. The kinases or important enzymes within this regulatory axis may represent important targets for TN therapy and prevention.

Epigenetic regulation of genes involved in fluid‐electrolyte balance in the kidney

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional regulators via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid‐electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase‐Accessible Chromatin‐sequencing (snATAC‐seq) and snRNA‐seq to profile kidney nuclei from adult male and female, control and littermate inducible kidney epithelial‐specific Hdac1/Hdac2 knockdown mice (iKSHdac1/2KO). Compared to littermate control mice iKSHdac1/2KO mice had significant elevations in plasma sodium (male control n=17, PNa= 143.6 ± 1.5 vs KO n=8, 147.6 ± 3.5, P=0.002, female control n=6, 141.8 ± 2.5 vs KO n=8 144.6 ± 1.8, P=0.03). iKSHdac1/2KO mice also had a significant increase in the kidney to body mass ratio (males 11.5 ±1.4 mg/g vs 13.1 ±1.5 P=0.1, females 9.7 ± 1.0 vs 12.3 ± 1.4, P=0.001) but no differences in liver to body mass ratio. Kidney histological analyses determined significant interstitial fibrosis, tubular atrophy and necrosis, dilated tubules, and protein casts in the iKSHdac1/2KO regardless of sex. Kidney structure was normal in all control mice. Survival was also significantly impacted in the iKSHdac1/2KO as all of these mice died (male mice died 27‐28 days after induction of knockdown). At 2 weeks after induction of KO, kidney nuclei from control and iKSHdac1/2KO mice were isolated and snATAC‐seq and snRNA‐seq analyses completed. We identified 26 unique clusters in both datasets that represented epithelial, endothelial, immune, stromal, and podocyte cells. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid‐electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Aqp1 expressed in the proximal tubules was reduced 60% and principal cell Aqp2 was reduced 50% in the iKSHdac1/2KO mice compared to controls. However, not all aquaporins were affected as for example, principal cell Aqp4 was not significantly different between the mice (P=0.98). Moreover, sex‐specific transcriptomic and chromatin structure were apparent in the proximal tubule clusters. Hdac1/Hdac2 knockdown resulted in different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24‐30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function and survival.

Effects of cinnamon extract on sirtuin activity: potential for metabolic regulation blood glucose control

Sirtuins (SIRTs) are a family of enzymes that are involved in several metabolic and inflammatory pathways in diseases. Sirtuins are histone‐deacetylase enzymes which remove an acetyl group from lysine residues in histone proteins. Acetylation of histones plays a role in epigenetic regulation of gene expression. Recent studies demonstrate that SIRT enzymes can regulate fasting glucose, concentration of circulating insulin, insulin resistance, pancreatic activity, and inflammation. Metabolic parameters, like glucose homeostasis and chronic inflammation, contribute to the development of diseases like type 2 diabetes mellitus (T2DM). Increasing expression of SIRT can aid in the regulation of these metabolic parameters and may be involved in diseases like T2DM. Our research aims to analyze the activity of HDACs and SIRT in 3T3L‐1 cells. 3T3L‐1 cells were grown in bovine calf serum and nuclear extracts were collected using a nuclear extraction kit. Nuclear extracts were then analyzed for protein concentration and added to the Epigenase™ Universal SIRT Activity/Inhibition Assay Kit. The data collected suggests that cinnamon does alter the activity of SIRT and further research will be explored to determine the extent to which this effect proves useful in the treatment of T2DM.

Intestinal Zip14 ablation dysregulates tight junction protein expression, inflammatory genes and lncRNAs in intestinal epithelial cells and enteroids

The mechanisms by which Zip14 ablation mediates mild intestinal inflammation and decreased barrier function are not completely understood. We used RNA sequencing to profile the transcriptome of intestinal epithelial cells (IEC) isolated from Zip14 Cre/flox and control flox/flox mice. RNA‐seq analysis identified 1,345 differentially expressed genes in Zip14 Cre/flox mice compared with control mice. In‐silico analyses revealed that Zip14ablation promoted dysregulation of specific proinflammatory genes (e.g. IL‐6 and Ifn‐γ) and genes influencing intestinal barrier homeostasis (specifically, Claudins 1‐and‐2). Highly dysregulated genes were selected for confirmation by qPCR, and proteins by western analysis, using extracts from isolated IECs from mice of the two genotypes. Highly upregulated genes in the Zip14 Cre/flox mice included Fabp6, Cldn2and Cldn8, while downregulated genes included Cldn1. To explore the possibility that Zip14ablation may alter transcriptional activity, ChIP assays of selected promoters were conducted. Increased binding of Stat3, FXR and NF‐kB was observed in Cldn2, FABP6, and Cldn8promoters, respectively, with chromatin from the Zip14 Cre/floxmice compared to control mice. By contrast, Cdx2 binding was decreased for the Cldn1promoter site of the knockout mice. Zip14 ablation also altered expression levels of long noncoding RNAs (lncRNAs). Specifically, the lncRNAs H19, U90926, and Meg3 were highly upregulated, and Gata4 was downregulated as validated by qPCR. Using organoids derived from intestines of mice of both genotypes, it was demonstrated that growth was not impaired with Zip14 ablation. Other biochemical markers of Zip14ablation were recapitulated in organoid cultures. This included indices of enhanced Zn2+ trafficking and differential expression of specific tight junction genes. Decreased HDAC3 activity in IECs from the Zip14 knockout mice suggest that epigenetic modifications account in part for the genomic changes observed. We conclude zinc transported by Zip14 controls intestinal permeability through epigenetic modifications, thus representing a novel health‐promoting role for this micronutrient.

Naphthoquinone derivatives as P-glycoprotein inducers in inflammatory bowel disease: 2D monolayers, 3D spheroids, and in vivo models

Inflammatory bowel disease (IBD) represents a chronic inflammation of the gastrointestinal tract characterized by an overreaction of immune responses and damage at the intestinal mucosal barrier. P-glycoprotein (P-gp) plays a key role to protect the intestinal barrier from xenobiotic accumulation and suppressing excessive immune responses. Therefore, induction/activation of P-gp function could serve as a novel therapeutic target to treat IBD. This study aimed to evaluate the potential therapeutic values of naphthoquinone derivatives (NQ-1 - NQ-8) as P-gp modulators to counterbalance intestinal inflammation. The data indicate that NQ-2, NQ-3, and NQ-4 act as P-gp inducers/activators and are recognized as substrates for P-gp. The three derivatives possess anti-inflammatory effects mediated by suppression of NF-κB and HDAC6 activity in Caco2 monolayer cells. Besides, they reversed LPS-induced intestinal barrier dysfunction by enhancing the expression of P-gp and ZO-1 tight junction proteins in a Caco-2 spheroid model. NQ-2, NQ-3, and NQ-4 showed a robust inhibitory effect on IL-1β maturation in LPS-primed THP-1 cells. This effect may contribute to alleviate the inflammatory cascades associated with IBD. Distinctively, NQ-2 and NQ-3 exerted anti-NLRP3 inflammasome activity evidenced by the inhibition of CASP-1 activity and the promotion of autophagy. Both compounds induced disruptions of the microtubule network in transfected U2OS-GFP-α-tubulin cells. Treatment with NQ-2 remarkably attenuated dextran sulfate sodium (DSS)-induced colitis in rats by suppressing changes in colon length, colon mass index, and intestinal histopathology scores. Thus, 1,4-naphthoquinone derivatives such as NQ-2 may provide potential therapeutic anti-inflammatory effects for IBD patients and for other NLRP3-associated inflammatory diseases.

Determinants of epigenetic resistance to HDAC inhibitors in dystrophic fibro‐adipogenic progenitors

Pharmacological treatment of Duchenne muscular dystrophy (DMD) with histone deacetylase inhibitors (HDACi) is currently being tested in clinical trials; however, pre‐clinical studies indicated that the beneficial effects of HDACi are restricted to early stages of disease. We show that FAPs from late‐stage mdx mice exhibit aberrant HDAC activity and genome‐wide alterations of histone acetylation that are not fully reversed by HDACi. In particular, combinatorial H3K27 and/or H3K9/14 hypo‐acetylation at promoters of genes required for cell cycle activation and progression, as well as glycolysis, are associated with their downregulation in late‐stage mdx FAPs. These alterations could not be reversed by HDACi, due to a general resistance to HDACi‐induced H3K9/14 hyperacetylation. Conversely, H3K9/14 hyper‐acetylation at promoters of Senescence Associated Secretory Phenotype (SASP) genes is associated with their upregulation in late‐stage mdx FAPs; however, HDACi could reduce promoter acetylation and blunt SASP gene activation. These data reveal that during DMD progression FAPs develop disease‐associated features reminiscent of cellular senescence, through epigenetically distinct and pharmacologically dissociable events. They also indicate that HDACi might retain anti‐fibrotic effects at late stages of DMD.

Minocycline suppresses lipogenesis via inhibition of p300 histone acetyltransferase activity in human SZ95 sebocytes.

Minocycline is a second-generation tetracycline drug, which is widely used to treat diverse infectious and inflammatory diseases such as acne vulgaris. The effects of minocycline on acne vulgaris have been mainly attributed to its anti-inflammatory effect; however, its sebum-regulating effect and the relevance to epigenetic regulation in human sebaceous glands remain uninvestigated. To identify the potential underlying epigenetic mechanism of sebum-inhibitory effects of minocycline in human SZ95 sebocytes. The quantity of lipid droplets and the expression of key lipogenic genes were analysed in minocycline-treated SZ95 sebocytes. To examine whether the sebum-inhibitory effects of minocycline are relevant to histone acetylation, we analysed the effects of minocycline on p300 HAT and total HDAC activity. To elucidate the functional implication of p300 HAT inhibition by minocycline in sebocytes, we assessed the effect of p300 knockdown, inhibition and overexpression on lipid accumulation in SZ95 sebocytes. Minocycline suppressed the insulin and liver X receptor agonist-induced lipid accumulation and the expression of the key lipogenic transcription factor sterol regulatory element-binding protein 1 (SREBP1) and its downstream genes, fatty acid synthase (FAS) and acetyl-CoA carboxylase α (ACCα). Minocycline inhibited p300 HAT activity in a concentration-dependent manner, but demonstrated no effect on global HDAC activity, resulting in a significant decrease in histone acetylation. Downregulation of p300 by knockdown or inhibition significantly suppressed SREBP1 expression, histone acetylation and lipid accumulation, whereas p300 overexpression enhanced these effects. Moreover, p300 overexpression rescued minocycline-inhibited SREBP1 expression and lipid synthesis. Our findings revealed a novel sebum-regulating effect of minocycline. Moreover, as p300 HAT is a key epigenetic regulator of sebaceous lipogenesis, its inhibitors could be used for the treatment of acne vulgaris.

Synthesis, in vitro and structural aspects of cap substituted Suberoylanilide hydroxamic acid analogs as potential inducers of apoptosis in Glioblastoma cancer cells via HDAC /microRNA regulation

Glioblastoma multiforme (GBM) is a heterogeneous, aggressive brain cancer characterized by chemo-resistance and cancer stemness. Histone deacetylases (HDACs) are a group of enzymes that regulate chromatin epigenetics which were in turn found to be controlled by microRNAs (miRs). The drug employed in chemotherapy for the treatment of GBM is Temozolomide (TMZ). Unfortunately, many GBM patients exhibit chemo-resistance to this drug. Here we have synthesized various Suberoyl anilide hydroxamic acid (SAHA) analogs with many substitutions at the cap site majority of which not yet studied. These SAHA analogs have exhibited profound cytotoxicity at 2 μM, and 4 μM concentrations in GBM cancer cell line U87MG, and 1 μM, and 2 μM concentrations in breast cancer cell line MCF-7. Surprisingly, these analogs have exhibited cytotoxic effects in chronic lymphoid leukemia cells (Raji) at 64 μM, and 128 μM concentrations due to mutated p53. Among all the synthesized analogs 3-Chloro-SAHA, 3-Chloro-4-fluoro SAHA have exhibited effective cytotoxicity in all cancer cells. These potent analogs inhibited HDAC-8 enzyme activity by 2-folds in U87MG, and MCF-7 cell lines and 7-folds decrease in HDAC-8 activity was observed in Raji cell line. These analogs decreased the expression of HDAC-2, HDAC-3 genes and enhanced the expression of p53 tumor suppressor. Interestingly, these compounds decreased the expression of Rictor, the main component of the mTORC2 complex involved cancer cell metabolism. Furthermore, these molecules have decreased oncogenic microRNA expression such as miR-21 and enhanced the expression of tumor suppressor microRNAs such as miR-143. The HDAC binding ability of these molecules was highly significant and have exhibited the ability to cross blood-brain barrier (BBB), and followed the Lipinski rule of five. Thus, these molecules need to be taken up further to clinics for better therapy against GBM either singly or combination therapy.

Rutin increases alpha-tubulin acetylation via histone deacetylase 6 inhibition.

Microtubules are dynamic cytoskeletal filaments composed of alpha- (α) and beta (β)-tubulin proteins. α-tubulin proteins are posttranslationally acetylated, and loss of acetylation is associated with axonal transport defects, a common alteration contributing to the pathomechanisms of several neurodegenerative diseases. Restoring α-tubulin acetylation by pharmacological inhibition of HDAC6, a primary α-tubulin deacetylase, can rescue impaired transport. Therefore, HDAC6 is considered a promising therapeutic target for neurodegenerative diseases, but currently, there is no clinically approved inhibitor for this purpose. In this study, using drug repurposing strategy, we aimed to identify compounds possessing HDAC6 inhibition activity and inducing α-tubulin acetylation. We systematically analyzed the FDA-approved library by utilizing virtual screening and consensus scoring approaches. Inhibition activities of promising compounds were tested using in vitro assays. Motor neuron-like NSC34 cells were treated with the candidate compounds, and α-tubulin acetylation levels were determined by Western blot. Our results demonstrated that rutin, a natural flavonoid, inhibits cellular HDAC6 activity without inducing any toxicity, and it significantly increases α-tubulin acetylation level in motor neuron-like cells.