Hyperacetylation Of Histone H4 Research Articles

Structural impotence of PDX-1 protein in the epigenetic control of insulin expression

This study investigates the role of pancreatic and duodenal homeobox 1 (PDX-1) as an important transcription factor in the epigenetic regulation of insulin expression. The research focused on PDX-1 as a disordered protein, and how the flexibility of its intrinsically disordered regions (IDRs) allows PDX-1 to interact with various transcription factors and epigenetic enzymes, facilitating histone modifications and insulin transcription. Particularly, the role of PDX-1 in processes like acetylation, methylation, and demethylation was discussed by detailing its interactions with key enzymes such as p300, Set7, and JMJD3. For instance, PDX-1’s interaction with p300 leads to histone H4 hyperacetylation, enhancing insulin gene expression, while Set7-mediated methylation of H3K4 further promotes transcription. In addition, the demethylation of H3K27 by JMJD3 is also facilitated by the PDX-1 protein. Finally, structural predictions of PDX-1 using AlphaFold III and EMS Fold also highlight the importance of its IDRs in these interactions. The findings highlight the significance of PDX-1 in insulin regulation and offer insights into potential therapeutic targets for treating diabetes from an epigenetic approach.

Sodium valproate affects the expression of p16INK4a and p21WAFI/Cip1 cyclin‑dependent kinase inhibitors in HeLa cells.

p16INK4a and p21WAF1/Cip1 are cyclin-dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus-induced cervical cancer, p16 INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21 WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16 INK4a and p21 WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription-quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16 INK4a and a tendency for p16INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16 INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16 INK4a and p21 WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Generation and Characterization of a Human Neuronal In Vitro Model for Rett Syndrome Using a Direct Reprogramming Method.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Hyperacetylated histone H4 is a source of carbon contributing to lipid synthesis.

Histone modifications commonly integrate environmental cues with cellular metabolic outputs by affecting gene expression. However, chromatin modifications such as acetylation do not always correlate with transcription, pointing towards an alternative role of histone modifications in cellular metabolism. Using an approach that integrates mass spectrometry-based histone modification mapping and metabolomics with stable isotope tracers, we demonstrate that elevated lipids in acetyltransferase-depleted hepatocytes result from carbon atoms derived from deacetylation of hyperacetylated histone H4 flowing towards fatty acids. Consistently, enhanced lipid synthesis in acetyltransferase-depleted hepatocytes is dependent on histone deacetylases and acetyl-CoA synthetase ACSS2, but not on the substrate specificity of the acetyltransferases. Furthermore, we show that during diet-induced lipid synthesis the levels of hyperacetylated histone H4 decrease in hepatocytes and in mouse liver. In addition, overexpression of acetyltransferases can reverse diet-induced lipogenesis by blocking lipid droplet accumulation and maintaining the levels of hyperacetylated histone H4. Overall, these findings highlight hyperacetylated histones as a metabolite reservoir that can directly contribute carbon to lipid synthesis, constituting a novel function of chromatin in cellular metabolism.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( > 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

Reduction of SIRT1-Mediated Epigenetic Upregulation of Nav1.7 Contributes to Oxaliplatin-Induced Neuropathic Pain

BACKGROUND: Clinically, neuropathic pain is a severe side effect of oxaliplatin chemotherapy, which usually leads to dose reduction or cessation of treatment. Due to the unawareness of detailed mechanisms of oxaliplatin-induced neuropathic pain, it is difficult to develop an effective therapy and limits its clinical use. OBJECTIVES: The aim of the present study was to identify the role of sirtuin 1 (SIRT1) reduction in epigenetic regulation of the expression of voltage-gated sodium channels 1.7 (Nav1.7) in the dorsal root ganglion (DRG) during oxaliplatin-induced neuropathic pain. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: The von Frey test was performed to evaluate pain behavior in rats. Real-time quantitative polymerase chain reaction, western blotting, electrophysiological recording, chromatin immunoprecipitation, and small interfering RNA (siRNA) were used to illustrate the mechanisms. RESULTS: In the present study, we found that both the activity and expression of SIRT1 were significantly decreased in rat DRG following oxaliplatin treatment. The activator of SIRT1, resveratrol, not only increased the activity and expression of SIRT1, but also attenuated the mechanical allodynia following oxaliplatin treatment. In addition, local knockdown of SIRT1 by intrathecal injection of SIRT1 siRNA caused mechanical allodynia in naive rats. Besides, oxaliplatin treatment enhanced the action potential firing frequency of DRG neurons and the expression of Nav1.7 in DRG and activation of SIRT1 by resveratrol reversed this effect. Furthermore, blocking Nav1.7 by ProTx II (a selective Nav1.7 channel blocker) reversed oxaliplatin-induced mechanical allodynia. In addition, histone H3 hyperacetylation at the Nav1.7 promoter in DRG of rats following oxaliplatin treatment was significantly suppressed by activation of SIRT1 with resveratrol. Moreover, both the expression of Nav1.7 and histone H3 acetylation at the Nav1.7 promoter were upregulated in the DRG by local knockdown of SIRT1 with SIRT1 siRNA in naive rats. LIMITATIONS: More underlying mechanism(s) of SIRT1 reduction after oxaliplatin treatment needs to be explored in future research. CONCLUSIONS: These findings suggest that reduction of SIRT1-mediated epigenetic upregulation of Nav1.7 in the DRG contributes to the development of oxaliplatin-induced neuropathic pain in rats. The intrathecal drug delivery treatment of activating SIRT1 might be a novel therapeutic option for oxaliplatin-induced neuropathic pain. KEY WORDS: SIRT1, neuropathic pain, dorsal root ganglion, NAV1.7 voltage-gated sodium channel, oxaliplatin

Histone H4 hyperacetylation but not DNA methylation regulates the expression of decidualization-associated genes during induced human endometrial stromal cells decidualization.

The efficiency of endometrial stromal cells (ESC) decidualization is a critical player in successful embryo implantation and further pregnancy development. Epigenetic mechanisms strictly regulate massive changes that affect endometrium in each cycle, so investigating epigenetic patterns could help identify endometrial targets for infertility treatment solutions. The aim of our study was to analyze the changes in epigenetic modulators, histone modifications, and DNA methylation during induced human ESC in vitro decidualization. Decidualization markers and epigenetic factors' gene and protein expression levels were assessed during ESC cells in vitro decidualization, performing RT-qPCR and immunoblot tests. Furthermore, chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP) analysis by the following qPCR were conducted to evaluate the level of H4hyperAc and 5-methylcytosine in the decidualization-associated gene promoter and exon regions accordingly. Our results revealed that ESC decidualization caused the down-regulation of HDAC2 and subunits of PRC2. We observed the increased global level of H4hyperAc and H3K27me3. We also demonstrated that H4hyperAc was specifically enriched in the decidualization-associated genes (WNT4, HAND2, STAT5A) promoter regions during ESC decidualization. In contrast, the DNA methylation level in these promoter regions was relatively low before ESC induction and did not vary through ESC decidualization. Our findings demonstrate that specific gene promoters' histone acetylation increases during the induced ESC decidualization, which indicates the importance of epigenetic regulation in endometrial decidualization.

Orthodontic Compression Enhances Macrophage M2 Polarization via Histone H3 Hyperacetylation

Orthodontic tooth movement is a complex periodontal remodeling process triggered by compression that involves sterile inflammation and immune responses. Macrophages are mechanically sensitive immune cells, but their role in orthodontic tooth movement is unclear. Here, we hypothesize that orthodontic force can activate macrophages, and their activation may be associated with orthodontic root resorption. After force-loading and/or adiponectin application, the migration function of macrophages was tested via scratch assay, and Nos2, Il1b, Arg1, Il10, ApoE, and Saa3 expression levels were detected using qRT-PCR. Furthermore, H3 histone acetylation was measured using an acetylation detection kit. The specific inhibitor of H3 histone, I-BET762, was deployed to observe its effect on macrophages. In addition, cementoblasts were treated with macrophage-conditioned medium or compression force, and OPG production and cellular migration were measured. We further detected Piezo1 expression in cementoblasts via qRT-PCR and Western-blot, and its effect on the force-induced impairment of cementoblastic functions was also analyzed. Compressive force significantly inhibited macrophage migration. Nos2 was up-regulated 6 h after force-loading. Il1b, Arg1, Il10, Saa3, and ApoE increased after 24 h. Meanwhile, higher H3 histone acetylation was detected in the macrophages subjected to compression, and I-BET762 dampened the expression of M2 polarization markers (Arg1 and Il10). Lastly, even though the activated macrophage-conditioned medium showed no effect on cementoblasts, compressive force directly impaired cementoblastic function by enhancing mechanoreceptor Piezo1. Compressive force activates macrophages; specifically, it causes M2 polarization via H3 histone acetylation in the late stage. Compression-induced orthodontic root resorption is macrophage-independent, but it involves the activation of mechanoreceptor Piezo1.

Distinct histone H3 modification profiles correlate with aggressive characteristics of salivary gland neoplasms

Post-translational modification of histones is the crucial event that affect many tumor-specific traits. A diverse type of histone modifications had been reported in different cancers with prognostic implications. This study aimed to examine the degree of histone H3 modifications in salivary gland neoplasms and their associations with tumor pathologic characteristics and proliferative activity. The expression of H3K9Ac, H3K18Ac, H3K9Me3 and Ki-67 in 70 specimens of salivary gland neoplasms, consisting of 30 mucoepidermoid carcinoma (MEC), 20 adenoid cystic carcinoma (ACC) and 20 pleomorphic adenoma (PA), were investigated immunohistochemically. The immunohistochemical scoring of 3 histone modification types and Ki-67 labeling index were determined. Overall, MEC demonstrated elevated H3K9Ac level compared with benign PA. Increased H3K9Me3 in MEC was positively correlated with small nest invasion at tumor front, advanced pathologic grade, and elevated proliferative index. In addition, the significant upregulation of all 3 types of histone H3 modification was noted in solid subtype of ACC and associated with increased cell proliferation. This study indicates that salivary gland neoplasms differentially acquire distinct patterns of histone H3 modification, which impact prognostically relevant cancer phenotypes. The hyperacetylation and methylation of histone H3 could be underpinning the prognostically worsen solid type of ACC, and the trimethylation of H3K9 may be involved in aggressive characteristics of MEC.

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.

Cyclic Tetrapeptide HDAC Inhibitors with Improved Plasmodium falciparum Selectivity and Killing Profile.

Cyclic tetrapeptide histone deacetylase inhibitors represent a promising class of antiplasmodial agents that epigenetically disrupt a wide range of cellular processes in Plasmodium falciparum. Unfortunately, certain limitations, including reversible killing effects and host cell toxicity, prevented these inhibitors from further development and clinical use as antimalarials. In this study, we present a series of cyclic tetrapeptide analogues derived primarily from the fungus Wardomyces dimerus that inhibit P. falciparum with low nanomolar potency and high selectivity. This cyclic tetrapeptide scaffold was diversified further via semisynthesis, leading to the identification of several key structural changes that positively impacted the selectivity, potency, and in vitro killing profiles of these compounds. We confirmed their effectiveness as HDAC inhibitors through the inhibition of PfHDAC1 catalytic activity, in silico modeling, and the hyperacetylation of histone H4. Additional analysis revealed the in vitro inhibition of the most active epoxide-containing analogue was plasmodistatic, exhibiting reversible inhibitory effects upon compound withdrawal after 24 or 48 h. In contrast, one of the new diacetyloxy semisynthetic analogues, CTP-NPDG 19, displayed a rapid and irreversible action against the parasite following compound exposure for 24 h.

Histone deacetylase inhibitors improve MeJA-induced ginsenoside production in ginseng adventitious roots

Post-translational histone modifications such as histone acetylation play essential roles in plant growth, development, and stress responses by modulating gene expression. However, little is known about epigenetic regulation in plant secondary metabolism. To analyze the involvement of histone acetylation in elicitor-induced metabolite production, ginseng adventitious roots were treated with methyl jasmonate (MeJA) together with histone deacetylase (HDAC) inhibitors. Western blot results revealed that HDAC inhibitors cause hyperacetylation of histone H3, especially acetylation of histone H3 lysine 18, under MeJA treatment. In addition, the HDAC inhibitor-induced increase in the transcript levels of MeJA-induced ginsenoside biosynthetic genes resulted in enhancement of MeJA-induced ginsenoside production, indicating that HDAC activity serves as a negative factor in MeJA-induced transcriptional induction of ginsenoside biosynthesis pathways. In addition, we identified 25 HDAC genes from the ginseng genome, which suggested that MeJA-induced PgHDACs (PgHDA05, PgHDA10, PgHDA16, PgHDA17, PgHDA18, PgHDA19, and PgHDA20) might be key factors controlling MeJA-induced ginsenoside production in ginseng adventitious roots. This unique information regarding histone acetylation and ginsenoside production would be helpful in understanding epigenetic regulation in plant secondary metabolism.

HSP70-mediated neuroprotection by combined treatment of valproic acid with hypothermia in a rat asphyxial cardiac arrest model.

It has been reported that valproic acid (VPA) combined with therapeutic hypothermia can improve survival and neurologic outcomes in a rat asphyxial cardiac arrest model. However, neuroprotective mechanisms of such combined treatment of valproic acid with hypothermia remains unclear. We hypothesized that epigenetic regulation of HSP70 by histone acetylation could increase HSP70-mediated neuroprotection suppressed under hypothermia. Male Sprague-Dawley rats that achieved return of spontaneous circulation (ROSC) from asphyxial cardiac arrest were randomized to four groups: normothermia (37°C ± 1°C), hypothermia (33°C ± 1°C), normothermia + VPA (300 mg/kg IV initiated 5 minutes post-ROSC and infused over 20 min), and hypothermia + VPA. Three hours after ROSC, acetyl-histone H3 was highly expressed in VPA-administered groups (normothermia + VPA, hypothermia + VPA). Four hours after ROSC, HSP70 mRNA expression levels were significantly higher in normothermic groups (normothermia, normothermia + VPA) than in hypothermic groups (hypothermia, hypothermia + VPA). The hypothermia + VPA group showed significantly higher HSP70 mRNA expression than the hypothermia group. Similarly, at five hours after ROSC, HSP70 protein levels were significantly higher in normothermic groups than in hypothermic groups. HSP70 levels were significantly higher in the hypothermia + VPA group than in the hypothermia group. Only the hypothermia + VPA group showed significantly attenuated cleaved caspase-9 levels than the normothermia group. Hypothermia can attenuate the expression of HSP70 at transcriptional level. However, VPA administration can induce hyperacetylation of histone H3, leading to epigenetic transcriptional activation of HSP70 even in a hypothermic status. Combining VPA treatment with hypothermia may compensate for reduced activation of HSP70-mediated anti-apoptotic pathway.

Effects of histone H4 hyperacetylation on inhibiting MMP2 and MMP9 in human amniotic epithelial cells and in premature rupture of fetal membranes.

Histone modification is closely associated with several diseases. The aim of the current study was to investigate the associations among histone acetylation, matrix metalloproteinases (MMPs) and premature rupture of membranes (PROM) during pregnancy. A total of 180 puerperants were divided into three groups: i) Preterm-PROM (PPROM), ii) term-PROM (TPROM) and iii) full-term labor (FTL). Enzyme-linked immunosorbent assay (ELISA) kits and western blotting were used to determine the protein concentrations of MMP2, MMP9, histone deacetylase (HDAC)1, HDAC2 and HDAC6, and the protein levels of histone H4 lysine (H4K)5 and H4K8 acetylation, respectively, in three types of fetal membranes. Additionally, human amniotic epithelial cells were used to determine the effects of the HDAC inhibitors droxinostat and chidamide on cell viability, histone acetylation and the levels of MMP2, MMP9, HDAC1, HDAC2 and HDAC6 in vitro, using the Cell Counting Kit-8 assay, western blotting and ELISA, respectively. Furthermore, the effects of droxinostat and chidamide on the invasion and migration abilities of human amniotic epithelial cells were investigated using transwell assays. In fetal membranes, the activities of MMP2 and MMP9 increased in PPROM, but decreased in TPROM. Further, the expression of HDAC1 was decreased and histone hyperacetylation was increased in both PPROM and TRPOM. In vitro experiments revealed that 5 µM droxinostat and 0.5 µM chidamide selectively decreased the level of HDAC and induced acetylation of H4K5 and H4K8. Additionally, the aforementioned HDAC inhibitors reduced human amniotic epithelial cell viability, invasion and migration, and decreased the expression levels of MMP2 and MMP9. The current study revealed a high expression level of MMP2 and MMP9 in PPROM compared with TPROM and FL tissue, which was in accordance with previously published studies. Furthermore, the in vitro tests performed in the current study revealed the effect of histone H4 hyperacetylation on inhibiting MMP2 and MMP9 levels in vitro was similar to that observed in TPROM. The results obtained in the current study may be used as a theoretical guide for clinical treatment of premature rupture of membranes.

Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines.

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI-H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFRT790M resistance mutation harboring NCI-H1975cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI-H1975cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring tumor cell lines (NCI-H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Immunoregulation induced by autologous serum collected after acute exercise in obese men: a randomized cross-over trial

In this study, we evaluated the effects of autologous serum collected after two types of exercise on the in vitro inflammatory profile and T cell phenotype of resting peripheral blood mononuclear cells (PBMCs) in obese men. Serum samples and PBMCs were obtained from eight obese men who performed two exercise bouts—high intensity interval exercise (HIIE) and exhaustive exercise session to voluntary fatigue—in a randomized cross-over trial. Pre-exercise PBMCs were incubated with 50% autologous serum (collected before and after each exercise bout) for 4 h. In vitro experiments revealed that post-HIIE serum reduced the histone H4 acetylation status and NF-κB content of PBMCs and suppressed the production of both TNF-α and IL-6 by PBMCs, while increasing IL-10 production. Post-exhaustive exercise serum induced histone H4 hyperacetylation and mitochondrial depolarization in lymphocytes and increased TNF-α production. In vitro post-HIIE serum incubation resulted in an increase in the frequencies of CD4 + CTLA-4 + and CD4 + CD25+ T cells expressing CD39 and CD73. Post-exhaustive exercise serum decreased the frequency of CD4 + CD25 + CD73+ T cells but increased CD4 + CD25-CD39 + T cell frequency. Both post-exercise serums increased the proportions of CD4 + PD-1 + and CD8 + PD-1+ T cells. Blood serum factors released during exercise altered the immune response and T cell phenotype. The type of exercise impacted the immunomodulatory activity of the post-exercise serum on PBMCs.

An ELISA method to assess HDAC inhibitor-induced alterations to P. falciparum histone lysine acetylation

The prevention and treatment of malaria requires a multi-pronged approach, including the development of drugs that have novel modes of action. Histone deacetylases (HDACs), enzymes involved in post-translational protein modification, are potential new drug targets for malaria. However, the lack of recombinant P. falciparum HDACs and suitable activity assays, has made the investigation of compounds designed to target these enzymes challenging. Current approaches are indirect and include assessing total deacetylase activity and protein hyperacetylation via Western blot. These approaches either do not allow differential compound effects to be determined or suffer from low throughput. Here we investigated dot blot and ELISA methods as new, higher throughput assays to detect histone lysine acetylation changes in P. falciparum parasites. As the ELISA method was found to be superior to the dot blot assay using the control HDAC inhibitor vorinostat, it was used to evaluate the histone H3 and H4 lysine acetylation changes mediated by a panel of six HDAC inhibitors that were shown to inhibit P. falciparum deacetylase activity. Vorinostat, panobinostat, trichostatin A, romidepsin and entinostat all caused an ~3-fold increase in histone H4 acetylation using a tetra-acetyl lysine antibody. Tubastatin A, the only human HDAC6-specific inhibitor tested, also caused H4 hyperacetylation, but to a lesser extent than the other compounds. Further investigation revealed that all compounds, except tubastatin A, caused hyperacetylation of the individual N-terminal H4 lysines 5, 8, 12 and 16. These data indicate that tubastatin A impacts P. falciparum H4 acetylation differently to the other HDAC inhibitors tested. In contrast, all compounds caused hyperacetylation of histone H3. In summary, the ELISA developed in this study provides a higher throughput approach to assessing differential effects of antiplasmodial compounds on histone acetylation levels and is therefore a useful new tool in the investigation of HDAC inhibitors for malaria.

Liver-Targeting Class I Selective Histone Deacetylase Inhibitors Potently Suppress Hepatocellular Tumor Growth as Standalone Agents.

Simple SummaryLiver cancers are among the leading causes of global cancer deaths. The current therapy options for liver cancers, including hepatocellular carcinoma (HCC), which accounts for over 80% of all cases, have afforded limited benefit to patients with an advanced disease state. HCC results from genetic and epigenetic alterations, including gene-silencing chromatin histone hypoacetylation. The aim of this study was to investigate the potential utility of liver tissue-targeting HDAC inhibitors (HDACi) as a new class of anti-HCC agents. We showed that a class of macrolide-based HDACi, which are selective for sub-class I HDACs, preferentially accumulated in the liver tissue and robustly suppressed tumor growths in an orthotopic model of HCC. The liver tissue-selective accumulation property of these compounds gives them a unique advantage over most of the current HDACi, including those currently in clinical use.Dysfunctions in epigenetic regulation play critical roles in tumor development and progression. Histone deacetylases (HDACs) and histone acetyl transferase (HAT) are functionally opposing epigenetic regulators, which control the expression status of tumor suppressor genes. Upregulation of HDAC activities, which results in silencing of tumor suppressor genes and uncontrolled proliferation, predominates in malignant tumors. Inhibition of the deacetylase activity of HDACs is a clinically validated cancer therapy strategy. However, current HDAC inhibitors (HDACi) have elicited limited therapeutic benefit against solid tumors. Here, we disclosed a class of HDACi that are selective for sub-class I HDACs and preferentially accumulate within the normal liver tissue and orthotopically implanted liver tumors. We observed that these compounds possess exquisite on-target effects evidenced by their induction of dose-dependent histone H4 hyperacetylation without perturbation of tubulin acetylation status and G0/G1 cell cycle arrest. Representative compounds 2 and 3a are relatively non-toxic to mice and robustly suppressed tumor growths in an orthotopic model of HCC as standalone agents. Collectively, our results suggest that these compounds may have therapeutic advantage against HCC relative to the current systemic HDACi. This prospect merits further comprehensive preclinical investigations.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

The epigenetic treatment remodel genome-wide histone H4 hyper-acetylation patterns and affect signaling pathways in acute promyelocytic leukemia cells

Although majority of acute promyelocytic leukemia (APL) patients achieve complete remission after the standard treatment, 5–10% of patients are shown to relapse or develop resistance to treatment. In such cases, medications that target epigenetic processes could become an appealing supplementary approach. In this study, we tested the anti-leukemic activity of histone deacetylase inhibitor Belinostat (PXD101) and histone methyltransferase inhibitor 3-Deazaneplanocin A combined with all-trans retinoic acid in APL cells NB4, promyelocytes resembling HL-60 cells and APL patients' cells. After HL-60 and NB4 cell treatment, ChIP-sequencing was performed using antibodies against hyper-acetylated histone H4. Hyper-acetylated histone H4 distribution peaks were compared in treated vs untreated HL-60 and NB4 cells. Results demonstrated that in treated HL-60 cells, the majority of peaks were distributed within the regions of proximal promoters, whereas in treated NB4 cells, hyper-acetylated histone H4 peaks were mainly localized in gene body regions. Further ChIP-seq data analysis revealed the changes in histone H4 hyper-acetylation in promoter/gene body regions of genes involved in cancer signaling pathways. In addition, quantitative gene expression analysis proved changes in various cellular pathways important for carcinogenesis. Epigenetic treatment down-regulated the expression of MTOR, LAMTOR1, WNT2B, VEGFR3, FGF2, FGFR1, TGFA, TGFB1, TGFBR1, PDGFA, PDGFRA and PDGFRB genes in NB4, HL-60 and APL patients’ cells. In addition, effect of epigenetic treatment on protein expression of aforementioned signaling pathways was confirmed with mass spectrometry analysis. Taken together, these results provide supplementary insights into molecular changes that occur during epigenetic therapy application in in vitro promyelocytic leukemia cell model.