Melatonin ameliorates histone modification disorders in mammalian aged oocytes by neutralizing the alkylation of HDAC1.

Aging-associated histone modification changes in oocytes have been sporadically reported, but the underlying mechanisms remain elusive. Here, we systematically characterize multiple histone modifications in oocytes during aging. We find that maternal and postovulatory aging markedly alter the status of histone modifications, specifically H4K12ac and H3K4me3, in both mouse and porcine oocytes. Meanwhile, we identify a substantial reduction in HDAC1 (histone deacetylase 1) protein in aged oocytes, which contributes to the changes in H4K12ac and H3K4me3. Moreover, by employing methylglyoxal (MG) and site-directed mutagenesis, we demonstrate that the elevated reactive carbonyl species (RCS) level induces HDAC1 degradation, likely through attacking the cysteine residues, thereby influences histone modification state. Importantly, supplementation of melatonin not only prevents the loss of HDAC1 protein, but also partially corrects the H4K12ac and H3K4me3 status in aged oocytes. To sum up, this study established the link between redox disequilibrium and histone modification alterations during mammalian oocyte aging.

Oroxylin A inhibits the migration of hepatocellular carcinoma cells by inducing NAG-1 expression.

Hepatocellular carcinoma (HCC), the most prevalent liver cancer, is considered one of the most lethal malignancies with a dismal outcome mainly due to frequent intrahepatic and distant metastasis. In the present study, we demonstrated that oroxylin A, a natural product extracted from Scutellaria radix, significantly inhibits transforming growth factor-beta1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) and metastasis in HCC. Oroxylin A blocked the TGF-ß1/Smad signaling via upregulating the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression. Oroxylin A promoted NAG-1 transcription by regulating the acetylation of CCAAT/enhancer binding protein ß (C/EBPß), a transcription factor that binds to the NAG-1 promoter. In terms of the underlying mechanism, oroxylin A may interact with histone deacetylase 1 (HDAC1) by forming hydrogen bonds with GLY149 residue and induce proteasome-mediated degradation of HDAC1 subsequently impairing HDAC1-mediated deacetylation of C/EBPß and promoting the expression of NAG-1. Taken together, our findings revealed a previously unknown tumor-suppressive mechanism of oroxylin A. Oroxylin A should be further investigated as a potential clinical candidate for inhibiting HCC metastasis.

Entinostat, a histone deacetylase inhibitor, increases the population of IL-10+ regulatory B cells to suppress contact hypersensitivity.

IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].

Snail/HDAC1/2 mediate skeletal growth retardation in fetuses caused by prenatal nicotine exposure.

Previous findings have confirmed that prenatal nicotine exposure (PNE) leads to retarded cartilage development in the fetal growth plate. It is characterized by insufficient matrix synthesis and decreased expression of matrix phenotype genes aggrecan (ACAN) and Col2A1 in the fetal growth plate chondrocytes; however, the specific molecular mechanism is yet unclear. This study intends to clarify the specific molecular mechanism of fetal osteochondral retardation caused by PNE through animal and cellular experiments. The present study demonstrated that in male offspring of the PNE group (the pregnant rats were subcutaneously administered nicotine 1.0 mg/kg twice per day (2.0 mg/kg.d) at GD11-20), the cartilage matrix of the fetal growth plate was lightly stained, the collagen was reduced, and expression of the matrix phenotype genes, ACAN and Col2A1, was significantly decreased. It was further found that PNE decreased histone acetylation (H3K9/H3K14) levels in the ACAN and Col2A1 promoter regions. Moreover, the expression of Snail and HDAC1/2 was increased in the PNE group. in vitro, the nicotine treatment at different concentrations elevated the expression of Snail/HDAC1/2 while decreasing the H3K9/H3K14 levels in the ACAN and Col2A1 promoter regions. Snail-siRNA transfection partially abolished the nicotine-induced increase in HDAC1/2 expression and decreased the histone acetylation levels in the ACAN and Col2A1 promoter regions. Trichostatin A (TSA) treatment partially reversed the nicotine-induced changes in downstream parameters. In summary, PNE-induced decreased cartilage matrix synthesis in the fetal growth plate of male offspring is effectuated by Snail/HDAC1/2-mediated decreased H3K9/H3K14 levels in the ACAN and Col2A1 promoter regions.

Comparison of the sensitizing effects of cocaine and ethanol on histone deacetylase isoforms in the rat brain.

Behavioral sensitization, an animal model of drug addiction, persists for a prolonged period after repeated exposure to drugs of abuse. The persistence of an addiction behavioral phenotype suggests long-lasting changes in gene regulation at the epigenetic level. We measured the expression of histone deacetylases (HDACs) isoforms in the prefrontal cortex and dorsal striatum following the development of sensitization to cocaine (15 mg/kg, administered five times) and ethanol (0.5 g/kg, administered 15 times) to investigate the epigenetic changes that mediate sensitization. Animals sensitized to ethanol exhibited augmented locomotor activity in response to the cocaine challenge. Similarly, those sensitized to cocaine exhibited increased locomotor activity in response to an ethanol challenge. These findings indicate cross-sensitization between ethanol and cocaine and suggest that a common molecular mechanism underlying the cross-sensitization. In animals sensitized to cocaine or ethanol, mRNA levels of class II HDACs (HDAC4 and HDAC5) were decreased in the prefrontal cortex and dorsal striatum, whereas acute treatments with either drug had no effect on the expression of class II HDACs. By contrast, class I HDACs (HDAC1 and HDAC2) responded to the acute cocaine challenge, whereas sensitization itself did not have a consistent effect on class I HDAC levels. These findings support the hypothesis of a common epigenetic mechanism underlying persistent behavioral sensitization induced by different drugs, which may be mediated by the altered expression of class II HDACs.

Discovery of Novel c-Mesenchymal-Epithelia transition factor and histone deacetylase dual inhibitors.

Clinically, a single agent that simultaneously inhibits multiple targets has been widely used in cancer treatment to overcome complicated dose design and anti-cancer resistance. Inspired by the synergistic effects between c-Met and HDAC in tumor development, a novel series of c-Met/HDAC bifunctional inhibitors was designed and synthesized by merging the pharmacophores of HDAC inhibitor into a c-Met inhibitor. All the target compounds were evaluated for their biological activity, the most potent compound, 14x, exhibited strong inhibition against HDAC1 with an IC50 of 18.49 nM and remarkable inhibitory activity against c-Met with an IC50 of 5.40 nM, respectively. In addition, 14x efficiently inhibited the proliferation of HCT-116, MCF-7 and A549 cell lines with IC50 values of 0.22 µM, 1.59 µM and 0.22 µM, respectively, which were superior to the reference compounds Cabozantinib and SAHA. Futhermore, 14x induced apoptosis and cause cell cycle arrest in G2/M phase. Docking experiments on c-Met and HDAC enzymes revealed the key interactions between 14x with the target protein. These results indicated that 14x was a potent dual c-Met/HDAC inhibitor and deserved for further investigation.

Histone deacetylase inhibitor pre-treatment enhances the efficacy of DNA-interacting chemotherapeutic drugs in gastric cancer.

BACKGROUND: The prognosis of gastric cancer continues to remain poor, and epigenetic drugs like histone deacetylase inhibitors (HDACi) have been envisaged as potential therapeutic agents. Nevertheless, clinical trials are facing issues with toxicity and efficacy against solid tumors, which may be partly due to the lack of patient stratification for effective treatments. AIM: To study the need of patient stratification before HDACi treatment, and the efficacy of pre-treatment of HDACi as a chemotherapeutic drug sensitizer. METHODS: The expression activity of class 1 HDACs and histone acetylation was examined in human gastric cancer cells and tissues. The potential combinatorial regime of HDACi and chemotherapy drugs was defined on the basis of observed drug binding assays, chromatin remodeling and cell death. RESULTS: In the present study, the data suggest that the differential increase in HDAC activity and the expression of class 1 HDACs are associated with hypo-acetylation of histone proteins in tumors compared to normal adjacent mucosa tissue samples of gastric cancer. The data highlights for the first time that pre-treatment of HDACi results in an increased amount of DNA-bound drugs associated with enhanced histone acetylation, chromatin relaxation and cell cycle arrest. Fraction-affected plots and combination index-based analysis show that pre-HDACi chemo drug combinatorial regimes, including valproic acid with cisplatin or oxaliplatin and trichostatin A with epirubicin, exhibit synergism with maximum cytotoxic potential due to higher cell death at low combined doses in gastric cancer cell lines. CONCLUSION: Expression or activity of class 1 HDACs among gastric cancer patients present an effective approach for patient stratification. Furthermore, HDACi therapy in pre-treatment regimes is more effective with chemotherapy drugs, and may aid in predicting individual patient prognosis.

HDAC7 regulates histone 3 lysine 27 acetylation and transcriptional activity at super-enhancer-associated genes in breast cancer stem cells.

Chromatin regulation through histone modifications plays an essential role in coordinated expression of multiple genes. Alterations in chromatin induced by histone modifiers and readers regulate critical transcriptional programs involved in both normal development and tumor differentiation. Recently, we identified that histone deacetylases HDAC1 and HDAC7 are necessary to maintain cancer stem cells (CSCs) in both breast and ovarian tumors. Here, we sought to investigate the CSC-specific function of HDAC1 and HDAC7 mechanistically by using a stem-like breast cancer (BrCa) cell model BPLER and matched nonstem tumor cell (nsTC)-like HMLER, along with conventional BrCa cell lines with different CSC enrichment levels. We found that HDAC1 and HDAC3 inhibition or knockdown results in HDAC7 downregulation, which is associated with a decrease in histone 3 lysine 27 acetylation (H3K27ac) at transcription start sites (TSS) and super-enhancers (SEs) prominently in stem-like BrCa cells. Importantly, these changes in chromatin landscape also correlate with the repression of many SE-associated oncogenes, including c-MYC, CD44, CDKN1B, SLUG, VDR, SMAD3, VEGFA, and XBP1. In stem-like BrCa cells, HDAC7 binds near TSS and to SEs of these oncogenes where it appears to contribute to both H3K27ac and transcriptional regulation. These results suggest that HDAC7 inactivation, directly or through inhibition of HDAC1 and HDAC3, can result in the inhibition of the CSC phenotype by downregulating multiple SE-associated oncogenes. The CSC selective nature of this mechanism and the prospect of inhibiting multiple oncogenes simultaneously makes development of HDAC7 specific inhibitors a compelling objective.

Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment.

Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Ɩ/ʎ (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.

The Novel HDAC8 Inhibitor WK2-16 Attenuates Lipopolysaccharide-Activated Matrix Metalloproteinase-9 Expression in Human Monocytic Cells and Improves Hypercytokinemia In Vivo.

Dysregulated human monocytes/macrophages can synthesize and secrete matrix metalloproteinases (MMPs), which play important roles in the progression of sepsis. In this study, we investigated the effects and mechanism of a novel histone deacetylase (HDAC8) inhibitor, (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16), on MMP-9 production and activation in stimulated human monocytic THP-1 cells. Our results demonstrated that the acetylation level of structural maintenance of chromosomes 3 (SMC3) was up-regulated by WK2-16 in THP-1 cells. Consistently, an in vitro enzyme study demonstrated that WK2-16 selectively inhibited HDAC8 activity. Moreover, the WK2-16 concentration dependently suppressed MMP-9-mediated gelatinolysis induced by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). Additionally, WK2-16 significantly inhibited both MMP-9 protein and mRNA expression without cellular toxicity. Nevertheless, WK2-16 suppressed the extracellular levels of interleukin (IL)-6 from LPS-stimulated THP-1 cells. For the signaling studies, WK2-16 had no effect on LPS/TLR4 downstream signaling pathways, such as the NF-κB and ERK/JNK/P38 MAPK pathways. On the other hand, WK2-16 enhanced the recruitment of acetylated Yin Yang 1 (YY1) with HDAC1. Finally, in vivo studies indicated that WK2-16 could reduce the serum levels of TNF-α and IL-6 in endotoxemic mice. These results suggested that HDAC8 inhibition might provide a novel therapeutic strategy of hypercytokinemia in sepsis.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Anti-CSC effects in human esophageal squamous cell carcinomas and Eca109/9706 cells induced by nanoliposomal quercetin alone or combined with CD 133 antiserum.

CD133 was recently reported to be a cancer stem cell and prognostic marker. Quercetin is considered as a potential chemopreventive agent due to its involvement in suppression of oxidative stress, proliferation and metastasis. In this study, the expression of CD133/CD44 in esophageal carcinomas and Eca109/9706 cells was explored. In immunoflurorescence the locations of CD133+ and multidrug resistance 1 (MDR 1)+ in the same E-cancer cells were coincident, mainly in cytomembranes. In esophageal squamous cell carcinomas detected by double/single immunocytochemistry, small CD133+ cells were located in the basal layer of stratified squamous epithelium, determined as CSLC (cancer stem like cells); CD44+ surrounding the cells appeared in diffuse pattern, and the larger CD44+ (hi) cells were mainly located in the prickle cell layer of the epithelium, as progenitor cells. In E-cancer cells exposed to nanoliposomal quercetin (nLQ with cytomembrane permeability), down-regulation of NF-κBp65, histone deacetylase 1 (HDAC1) and cyclin D1 and up-regulation of caspase-3 were shown by immunoblotting, and attenuated HDAC1 with nuclear translocation and promoted E-cadherin expression were demonstrated by immunocytochemistry. In particular, enhanced E-cadherin expression reflected the reversed epithelial mesenchymal transition (EMT) capacity of nLQ, acting as cancer attenuator/preventive agent. nLQ acting as an HDAC inhibitor induced apoptotic cells detected by TUNEL assay mediated via HDAC-NF-κB signaling. Apoptotic effects of liposomal quercetin (LQ, with cytomembrane-philia) combined with CD133 antiserum were also detected by CD133 immunocytochemistry combined with TUNEL assay. The combination could induce greater apoptotic effects than nLQ induced alone, suggesting a novel anti-CSC treatment strategy.

Apigenin reactivates Nrf2 anti-oxidative stress signaling in mouse skin epidermal JB6 P + cells through epigenetics modifications.

Nrf2 is a crucial transcription factor that controls a critical anti-oxidative stress defense system and is implicated in skin homeostasis. Apigenin (API), a potent cancer chemopreventive agent, protects against skin carcinogenesis and elicits multiple molecular signaling pathways. However, the potential epigenetic effect of API in skin cancer chemoprotection is not known. In this study, bisulfite genomic DNA sequencing and methylated DNA immunoprecipitation were utilized to investigate the demethylation effect of API at 15 CpG sites in the Nrf2 promoter in mouse skin epidermal JB6 P + cells. In addition, qPCR and Western blot analyses were performed to evaluate the mRNA and protein expression of Nrf2 and the Nrf2 ARE downstream gene, NQO1. Finally, the protein expression levels of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) were evaluated using API and the DNMT/HDAC inhibitor 5-aza/ trichostatin A. Our results showed that API effectively reversed the hypermethylated status of the 15 CpG sites in the Nrf2 promoter in a dose-dependent manner. API enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein expression of Nrf2 and the Nrf2 downstream target gene, NQO1. Furthermore, API reduced the expression of the DNMT1, DNMT3a, and DNMT3b epigenetic proteins as well as the expression of some HDACs (1-8). Taken together, our results showed that API can restore the silenced status of Nrf2 in skin epidermal JB6 P + cells by CpG demethylation coupled with attenuated DNMT and HDAC activity. These results may provide new therapeutic insights into the prevention of skin cancer by dietary phytochemicals.

Inhibitors selective for HDAC6 in enzymes and cells.

Histone deacetylase inhibitors with anticancer or anti-inflammatory activity bind to Class I or Class I and II HDAC enzymes. Here we compare selectivity of inhibitors of a Class II HDAC enzyme (HDAC6) and find one that retains high selectivity in macrophages.