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Importance: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express N pro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its N pro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving N pro -mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms., Competing Interests: The authors declare no conflict of interest.

Breast cancer resistance protein (BCRP/ABCG2) is a critical drug efflux transporters by limiting drugs' transplacental transfer rates. More investigations on the regulation of placental BCRP offer great promise for enabling pronounced progress in individualized and safe pharmacotherapy during pregnancy. Histone deacetylases (HDACs) play an important role in epigenetic regulation of placental genes. It was reported recently by us that HDAC1 was involved in placental BCRP regulation in vitro. The aim of this study was to further explore the effect of HDAC1 on placental BCRP expression and functionality in animals. Randomly assigned C57BL pregnant dams received intraperitoneal injections of a negative control siRNA or Hdac1 siRNA from embryonic day 7.5 (E7.5) to E15.5, respectively. At E16.5, glyburide (GLB), a probe for evaluating placental BCRP efflux functionality, was injected via the tail vein. Animals were sacrificed through cervical dislocation at various times (5-180 min) after drug administration. The maternal blood, placentas, and fetal-units were collected. GLB concentrations were determined by a validated high-performance liquid chromatography/mass spectrometry (HPLC-MS) assay. Real-time quantitative PCR (qRT-PCR), Western blot, and immunohistochemical (IHC) analysis were employed to identify mRNA/protein levels and localization of gene expressions, respectively. It was noted that Hdac1 inhibition significantly decreased placental Bcrp expression, with markedly increases of GLB concentrations and area under the concentration-time curve (AUC) in fetal-units. Particularly, the ratios of fetal-unit/maternal plasma GLB concentrations were also significantly elevated following Hdac1 repression. Taken together, these findings suggested that HDAC1 was involved in positive regulation of placental BCRP expression and functionality in vivo., (© 2021. The Author(s).)

Colorectal cancer (CRC) has ranked first in terms of incidence in Taiwan. Surgical resection combined with chemo-, radio-, or targeted-therapies are the main treatments for CRC patients in current clinical practice. However, many CRC patients still respond poorly to these treatments, leading to tumor recurrence and an unacceptably high incidence of metastasis and death. Therefore, appropriate diagnosis, treatment, and drug selection are pressing issues in clinical practice. The Mi-2/nucleosome remodeling and deacetylase complex is an important epigenetic regulator of chromatin structure and gene expression. An important component of this complex is chromodomain-helicase-DNA-binding protein 4 (CHD4), which is involved in DNA repair after injury. Recent studies have indicated that CHD4 has oncogenic functions that inhibit multiple tumor suppressor genes through epigenetic regulation. However, the role of CHD4 in CRC has not yet been well investigated. In this study, we compared CHD4 expression in CRC patients from The Cancer Genome Atlas database. We found higher levels of CHD4 expression in CRC patients. In a series of in vitro experiments, we found that CHD4 affected cell motility and drug sensitivity in CRC cells. In animal models, the depletion of CHD4 affected CRC tumor growth, and the combination of a histone deacetylase 1 (HDAC1) inhibitor and platinum drugs inhibited CHD4 expression and increased the cytotoxicity of platinum drugs. Moreover, CHD4 expression was also a prognostic biomarker in CRC patients. Based on the above results, we believe that CHD4 expression is a viable biomarker for predicting metastasis CRC patients, and it has the potential to become a target for drug development., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Selective serotonin reuptaker inhibitors, including fluoxetine, are widely studied and prescribed antidepressants, while their exact molecular and cellular mechanism are yet to be defined. We investigated the involvement of HDAC1 and eEF2 in the antidepressant mechanisms of fluoxetine using a lipopolysaccharide (LPS)-induced depression-like behavior model., Methods: For in vivo analysis, mice were treated with LPS (2 mg/kg BW), fluoxetine (20 mg/kg BW), HDAC1 activator (Exifone: 54 mg/kg BW) and NH125 (1 mg/kg BW). Depressive-like behaviors were confirmed via behavior tests including OFT, FST, SPT, and TST. Cytokines were measured by ELISA while Iba-1 and GFAP expression were determined by immunofluorescence. Further, the desired gene expression was measured by immunoblotting. For in vitro analysis, BV2 cell lines were cultured; treated with LPS, exifone, and fluoxetine; collected; and analyzed., Results: Mice treated with LPS displayed depression-like behaviors, pronounced neuroinflammation, increased HDAC1 expression, and reduced eEF2 activity, as accompanied by altered synaptogenic factors including BDNF, SNAP25, and PSD95. Fluoxetine treatment exhibited antidepressant effects and ameliorated the molecular changes induced by LPS. Exifone, a selective HDAC1 activator, reversed the antidepressant and anti-inflammatory effects of fluoxetine both in vivo and in vitro, supporting a causing role of HDAC1 in neuroinflammation allied depression. Further molecular mechanisms underlying HDAC1 were explored with NH125, an eEF2K inhibitor, whose treatment reduced immobility time, altered pro-inflammatory cytokines, and NLRP3 expression. Moreover, NH125 treatment enhanced eEF2 and GSK3β activities, BDNF, SNAP25, and PSD95 expression, but had no effects on HDAC1., Conclusions: Our results showed that the antidepressant effects of fluoxetine may involve HDAC1-eEF2 related neuroinflammation and synaptogenesis.

TP53 mutation is an indicator of poor prognostic in chronic lymphocytic leukemia (CLL). Worse still, CLL patients with TP53 mutation are associated with poor efficacy to current chemotherapeutic, such as Fludarabine. Here, we confirmed that high expression of HDAC1 in CLL patients with TP53 mutation, which is closely related to poor prognosis and drug-resistance. Subsequently, we demonstrated Entinostat (HDAC1 inhibitor) combination with Fludarabine significantly induced apoptosis in TP53 mutations CLL cells. Its mechanism was associated with up-regulation of the pro-apoptotic protein Bax and the down-regulation of HDAC1, HO-1 and BCL-2 proteins. More importantly, we also confirmed that upregulation of HDAC1 could resistant Entinostat-induced apoptosis in TP53 mutations CLL cells by activating the HDAC1/P38/HO-1 pathway. In vivo, we found that Entinostat combination with Fludarabine significantly induced tumor cells apoptosis and prolong survival time in xenograft mouse model. Finally, combining vitro and vivo experiments, we presented the first demonstration that Entinostat combination with Fludarabine had a synergistic effect on the induction of apoptosis in TP53 mutations CLL cells. In conclusion, we provide valuable pre-clinical experimental evidence for the treatment of CLL patients with poor prognosis, especially for TP53 mutations., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: This study aimed to investigate the function and mechanism of neddylation of HDAC1 underlying drug resistance of AML cells., Methods: Evaluation experiments of effects of HDAC1 on drug resistance of AML cells were performed with AML cell transfected with constructs overexpressing HDAC1 or multi-drug resistance AML cells transfected with siRNA for HDAC1 through observing cell viability, percentage of apoptotic cell, doxorubicin-releasing index and multidrug resistance associated protein 1 (MRP1) expression. Neddylation or ubiquitination of HDAC1 was determined by immunoprecipitation or Ni2 + pull down assay followed by western blot. The role of HDAC1 was in vivo confirmed by xenograft in mice., Results: HDAC1 was significantly upregulated in refractory AML patients, and in drug-resistant AML cells (HL-60/ADM and K562/A02). Intracellular HDAC1 expression promoted doxorubicin resistance of HL-60, K562, and primary bone marrow cells (BMCs) of remission AML patients as shown by increasing cell viability and doxorubicin-releasing index, inhibiting cell apoptosis. Moreover, HDAC1 protein level in AML cells was regulated by the Nedd8-mediated neddylation and ubiquitination, which further promoted HDAC1 degradation. In vivo, HDAC1 overexpression significantly increased doxorubicin resistance; while HDACs inhibitor Panobinostat markedly improved the inhibitory effect of doxorubicin on tumor growth. Furthermore, HDAC1 silencing by Panobinostat and/or lentivirus mediated RNA interference against HDAC1 effectively reduced doxorubicin resistance, resulting in the inhibition of tumor growth in AML bearing mice., Conclusion: Our findings suggested that HDAC1 contributed to the multidrug resistance of AML and its function turnover was regulated, at least in part, by post-translational modifications, including neddylation and ubiquitination.

Colorectal cancer (CRC) is one of the most common cancer diagnoses. Histone deacetylase (HDAC) overactivity in CRC could promote cancer progression. HDAC1, a member of the HDAC family, is found aberrantly expressed in CRC, but it remains unclear whether the expression of HDAC1 can be regulated by microRNA. In the present study, we confirmed the overexpression status of HDAC1 in CRC tissues and cell lines, and its overexpression could promote CRC cell proliferation and invasion in vitro . We saw that HDAC1 was a direct target gene of miR-761 in CRC by bioinformatic and luciferase reporter analyses. HDAC1 expression could be regulated and was negatively correlated with miR-761 in CRC. We also indicated that the expression of miR-761 was abnormally downregulated in CRC. Transfection with a miR-761 mimic impeded the growth and invasion of CRC cells. In addition, we showed that ectopic expression of miR-761 mitigated HDAC1 stimulation of CRC cell proliferation and invasion. Our results demonstrate that miR-761 represents a potential strategy against CRC.

The homeobox transcription factor Nkx6.1 is sufficient to increase functional β-cell mass, where functional β-cell mass refers to the combination of β-cell proliferation, glucose-stimulated insulin secretion (GSIS) and β-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional β-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase β-cell proliferation in primary rat islets and the INS-1 832/13 β-cell line. The increase in HDAC1-mediated proliferation occurs while maintaining GSIS and increasing β-cell survival in response to apoptotic stimuli. We demonstrate that HDAC1 overexpression results in decreased expression of the cell cycle inhibitor Cdkn1b/p27 which is essential for inhibiting the G1 to S phase transition of the cell cycle. This corresponds with increased expression of key cell cycle activators, such as Cyclin A2, Cyclin B1 and E2F1, which are activated by activation of the Cdk4/Cdk6/Cyclin D holoenzymes due to down-regulation of Cdkn1b/p27. Finally, we demonstrate that overexpression of Cdkn1b/p27 inhibits HDAC1-mediated β-cell proliferation. Our data suggest that HDAC1 is critical for the Nkx6.1-mediated pathway that enhances functional β-cell mass., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

This study aimed to explore the roles of miRNA-34a (miR-34a) in ovarian cancer (OC) cells and uncover possible mechanisms. The proliferation of OC cells was measured with an MTT assay and soft agar colony formation assay. TargetScan analysis, real-time PCR, and a luciferase reporter assay were used to demonstrate the downstream target of miR-34a in OC cells. HDAC1 expression levels were detected by immunoblot analysis. miR-34a inhibited the proliferation of SKOV3 and OVCA433 cells and enhanced cisplatin sensitivity in cisplatin-resistant SKOV3cp cells. The results of TargetScan analysis, real-time PCR, and luciferase reporter assay confirmed that miR-34a downregulated HDAC1 expression by directly targeting the 3'-UTR of HDAC1 mRNA. The overexpression of HDAC1 decreased cisplatin sensitivity and promoted proliferation in OC cells. MTT assay and soft agar colony formation assay showed that HDAC1 overexpression blocked the suppressive effects of miR-34a on SKOV3 cell proliferation. In addition, treatment with the miR-34a mimic partially recovered the cisplatin sensitivity of SKOV3cp cells, whereas HDAC1 overexpression blocked the above phenomena caused by treatment with the miR-34a mimic. miR-34a exhibited suppressive effects on OC cells via directly binding and downregulating HDAC1 expression, which subsequently decreased the resistance to cisplatin and suppressed proliferation in OC cells.

Histone deacetylases (HDACs) are involved in multiple physical and pathological processes in classical Hodgkin lymphoma (cHL). The prognostic value of HDACs in cHL patients has not been discussed. The aim of the current study is to investigate the HDAC1, HDAC2, HDAC3, and HDAC11 expressions, and to evaluate the correlation of HDAC1, HDAC2, HDAC3, and HDAC11 expressions with the survival rate in cHL patients. We retrospectively analyzed clinicopathological data of 28 patients who were diagnosed with cHL between August 2002 and March 2010. Immunohistochemistry was used to detect the expression of HDAC1, HDAC2, HDAC3, and HDAC11 in these patients. The results showed that HDAC1, HDAC3, and HDAC11 were expressed at a higher level in Hodgkin Reed-Sternberg cells, whereas HDAC2 was expressed at a lower level in Hodgkin Reed-Sternberg cells. The expression of HDAC2 had a relationship with pathological type (P=0.012). There was also a correlation between the expression of HDAC11 and the erythrocyte sedimentation rate (P=0.054). Other clinicopathological parameters had no significant correlation with the expression of HDAC1, HDAC2, HDAC3, and HDAC11 in terms of survival (P>0.05). The 10-year total survival rate by Cox multivariate analysis, after taking into account all clinical and pathologic factors, showed that bulky disease retained significance (P=0.028). Higher expression of HDAC1 predicted shorter progression-free survival and overall survival (OS) in cHL patients (P<0.05, in both cases), and higher expression of HDAC11 might be correlated with lower OS (P=0.05). The study showed that the expressions of HDAC2 and HDAC11 have a particular relationship with the pathologic subtype. Increased expression of HDAC1 was correlated negatively with progression-free survival and OS, and increased expression of HDAC11 had a borderline relationship with the OS rate in patients with cHL.

Silibinin, extracted from milk thistle ( Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin's pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.

Unlabelled: Cutaneous T-cell lymphoma (CTCL) is the most common type of primary cutaneous lymphoma. Here, we report that patients with CTCL show increased IL15 in a clinical stage-dependent manner. Mechanistically, we show that ZEB1 is a transcriptional repressor of IL15 in T cells and that hypermethylation of the ZEB1 binding region within the IL15 promoter, as seen in patients with CTCL, prevents ZEB1 binding and causes increased transcription of IL15 Using a transgenic mouse model of IL15, we provide evidence that overexpression of IL15 induces a spontaneous CTCL that mimics the human neoplasm. Excessive autocrine production of IL15 in T cells inhibits an HDAC1-mediated negative autoregulatory loop, resulting in the upregulation of HDAC1 and HDAC6 and transcriptional induction of the onco-miR-21. Interruption of IL15 downstream signaling with isotype-specific HDAC inhibitors halts (HDAC1) or significantly delays (HDAC6) the progression of CTCL in vivo and provides preclinical evidence supporting a hierarchical model of oncogenic signaling in CTCL., Significance: To date, CTCL pathogenesis remains unknown, and there are no curative therapies. Our findings not only demonstrate a critical role for IL15-mediated inflammation in cutaneous T-cell lymphomagenesis, but also uncover a new oncogenic regulatory loop in CTCL involving IL15, HDAC1, HDAC6, and miR-21 that shows differential sensitivity to isotype-specific HDAC inhibitors. Cancer Discov; 6(9); 986-1005. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932., (©2016 American Association for Cancer Research.)

Conclusion: HDAC1 may be a prognostic biomarker for LSCC malignant potency and a potent factor resulting in decreased sensitivity of LSCC in radiotherapy., Objective: The aim of this study was to evaluate the correlation between histone deacetylase 1 (HDAC1) over-expression and clinical features in laryngeal squamous cell carcinoma (LSCC)., Methods: This study assessed the HDAC1 expressions in 90 formalin-fixed paraffin-embedded LSCC samples, 30 adjacent non-neoplastic tissues, and 30 laryngeal polyp tissues by immunohistochemistry (IHC). In addition, another 40 LSCC samples were collected that were divided into two groups after 3-month radiotherapy: the high radio-sensitive group (HRS) and low radio-sensitive group (LRS). Overall survival curves of all the LSCC patients were constructed by Kaplan-Meier method and long-rank test., Results: All ninety samples were positively immunostained for HDAC1. The expression of HDAC1 was up-regulated and significantly associated with T classification, lymph node metastases, tumor location and clinical stage. HDAC1 was mainly labeled in the epithelial cells of laryngeal polyp tissues and adjacent non-neoplastic tissues. In addition, the expression of HDAC1 was significantly higher in LRS than that in HRS. The positive rates for stage III-IV tumor were significantly higher than those for stage II. LSCC patients with HDAC1 over-expression and LRS presented a shorter 5-year overall survival rate.

HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ulcerative colitis (UC) is an important risk factor for colorectal cancer (CRC). Histone modifications are one of the epigenetic mechanisms that may have key roles in the carcinogenesis of CRC. At present, there are no studies comparing histone modification patterns of UC and CRC in the literature. Therefore the aim of the present study was to investigate whether genes, particularly those involved in histone modification, have value in patient monitoring with regards to CRC development in UC. Key gene expressions of the histone modification enzyme were assessed and compared in CRC, UC and control groups using the RT-PCR array technique. Patients were divided into subgroups based on the extent and duration of the disease and inflammatory burden, which are considered risk factors for CRC development in UC patients. In UC and CRC groups, a significantly higher overexpression of the NEK6 and AURKA genes compared to the control group was identified. In addition, there was a significantly higher overexpression of HDAC1 and PAK1 genes in the UC group, and of HDAC1, HDAC7, PAK1 and AURKB genes in the CRC group. NEK6, AURKA, HDAC1 and PAK1 were significantly overexpressed in patients with a longer UC duration. Overexpression of AURKA and NEK6 genes was significantly more pronounced in UC patients with more extensive colon involvement. HDAC1, HDAC7, PAK1, NEK6, AURKA and AURKB are important diagnostic and prognostic markers involved in the carcinogenesis of CRC. HDAC1, PAK1, NEK6 and AURKA may be considered as diagnostic markers to be used in CRC screening for UC patients.

Downregulation of Rpd3, a homologue of mammalian Histone Deacetylase 1 (HDAC1), extends lifespan in Drosophila melanogaster. Once revealed that long-lived fruit flies exhibit limited cardiac decline, we investigated whether Rpd3 downregulation would improve stress resistance and/or lifespan when targeted in the heart. Contested against three different stressors (oxidation, starvation and heat), heart-specific Rpd3 downregulation significantly enhanced stress resistance in flies. However, these higher levels of resistance were not observed when Rpd3 downregulation was targeted in other tissues or when other long-lived flies were tested in the heart-specific manner. Interestingly, the expressions of anti-aging genes such as sod2, foxo and Thor, were systemically increased as a consequence of heart-specific Rpd3 downregulation. Showing higher resistance to oxidative stress, the heart-specific Rpd3 downregulation concurrently exhibited improved cardiac functions, demonstrating an increased heart rate, decreased heart failure and accelerated heart recovery. Conversely, Rpd3 upregulation in cardiac tissue reduced systemic resistance against heat stress with decreased heart function, also specifying phosphorylated Rpd3 levels as a significant modulator. Continual downregulation of Rpd3 throughout aging increased lifespan, implicating that Rpd3 deacetylase in the heart plays a significant role in cardiac function and longevity to systemically modulate the fly's response to the environment.

Chronic cerebral hypoperfusion (CCH) has been commonly associated with Alzheimer's disease and other types of dementia, but therapies that can improve cerebral blood flow displayed little effect on impaired cognition. Epigenetic intervention with histone deacetylase inhibitors, such as sodium butyrate (SB), on the other hand has been shown to improve cognition in several animal models of dementia. To investigate the effect of SB on cognitive impairment induced by CCH in rats, adult male SD rats were given intraperitoneal injections of SB at a daily dose of 840mg/kg for 4weeks, from the 29th day after permanent occlusion of bilateral common carotid arteries (2VO). Learning and memory were assessed by Morris water maze and novel object recognition. Following behavioral tests, western blotting of histone acetylation, of transcription factors, of neuronal/synaptic proteins, were performed using rat hippocampus and cortex. The data showed that SB treatment alleviated hippocampal dependent spatial learning disability in 2VO rats, and altered HDAC1/2 mRNA level, histone H4 acetylation and Nrf2 transcriptional activation in rat hippocampus. Accordingly, cognition-protective effect of SB appeared to be partially mediated by enhancing histone acetylation and hence by facilitating the transcription of Nrf2 downstream genes in the hippocampus. Thus, SB might be considered for putative treatment for CCH-related cognitive impairment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective and Design: Cigarette smoke (CS)-induced inflammation is critical in chronic obstructive pulmonary disease (COPD). However, the role of acetylation at histone 3 lysine 9 (H3K9) in COPD inflammation remains unclear. The present study assessed the effect of acetylation of H3K9 on transcription both in rat lungs and in macrophages., Methods: Sprague-Dawley rats were exposed to CS for either 6 or 12 weeks and rat lungs were collected. Rat macrophages were subjected to 20 % cigarette smoke extract (CSE) for 48 h., Results: CS increased MCP-1 and IL-8 expressions at both mRNA and protein levels in rat lungs after 6 and 12 weeks; increased TNF-α and MMP9 expressions at both levels were noted only after 12 weeks. CSE increased these genes expression in macrophages after 48 h exposure. Increased abundance of acetylated H3K9 protein in rat lungs and in macrophages were associated with decreased expression of histone deacetylase-1(HDAC1). Chromatin immunoprecipitation demonstrated increased level of acetylated H3K9 on promoter regions of these genes both in vivo and in vitro. Knockdown of HDAC1 increased these genes mRNA expression., Conclusions: CS increased H3K9 acetylation and subsequently altered the expression of pro-inflammatory mediators and protease genes through HDAC1 depression in CS-induced rat lungs and in macrophages.

The aim of the present study was to investigate the synergistic effects of valproic acid (VPA) and arsenic trioxide (ATO) on the proliferation of RPMI8226 cells and the possible underlying mechanisms. Cell apoptosis was assessed by flow cytometry. The mRNA expression levels were analyzed by semi-quantitative polymerase chain reaction, and the protein expression levels were analyzed by western blotting. The histone acetylation and methylation states of the gene promoters were detected using a chromatin immunoprecipitation technique. The apoptotic rates of the RPMI8226 cells in the combined drug groups were significantly increased compared with those of the single drug groups (P<0.05). The mRNA and protein expression levels of Bcl-2 and the expression levels of HDAC1 mRNA and H3K9me2 protein decreased significantly in the combined groups compared with the single drug groups. The mRNA and protein expression levels of Bax, Caspase 8, Caspase 9 and LSD1, and the protein expression of acetylated H3 increased significantly in the combination groups compared with the single drug groups. Histone methylation and acetylation of the Bcl-2 and bax gene promoters were increased in the combination groups compared with the single drug groups. VPA and ATO had synergistic effects on the proliferation of RPMI8226 cells, which may have been associated with the decreased expression of Bcl-2 and the increased expression levels of Bcl-2-associated X protein, Caspase 8 and Caspase 9. Therefore, the expression levels of the Bcl-2 gene family may have been regulated by the levels of gene promoter methylation and acetylation.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.

Background/aim: Gastric cancer is one of the most common types of cancer. Cancer stem cells (CSCs) have been reported to play important roles in multiple cancer types. This study investigated the correlation between cluster of differentiation 133 (CD133), histone deacetylase 1 (HDAC1) and thrombospondin-1 (THBS1) expression in advanced gastric cancer., Materials and Methods: The study included 65 patients with gastric cancer with recurrence after surgery. Expression of CD133, HDAC1 and THBS1 was examined by immunohistochemistry. Prognostic factors were investigated by multivariate analysis using Cox's proportional hazard model., Results: Clinicopathological variables, including survival, of patients positive for CD133 expression (n=6, 23%), were compared with those without CD133 expression (n=20, 77%). Positive HDAC1 expression and THBS1 expression were observed in 34 (52%) and 17 (26%) patients, respectively. Using univariate analysis, positive expression of CD133 and negative expression of THBS1 predicted significantly worse prognosis. Multivariate analysis revealed CD133-positive and THBS1-negative expression were independent prognostic indicators., Conclusion: CD133 expression and THBS1 expression were prognostic factors, and a negative relationship between HDAC and THBS1 was observed in advanced gastric cancer. These biomarkers may help determine postoperative treatment in patients with gastric cancer., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

There has been increasing evidence that numerous bioactive dietary agents can hamper the process of carcinogenesis by targeting epigenetic alterations including DNA methylation. This therapeutic approach is considered as a significant goal for cancer therapy due to the reversible nature of epigenetic-mediated gene silencing and warrants further attention. One such dietary agent, green tea catechin, (-)-epigallocatechin-3-gallate (EGCG) has been shown to modulate many cancer-related pathways. Thus, the present study was designed to investigate the role of EGCG as an epigenetic modifier in HeLa cells. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition assays were conducted, and the transcription levels of DNMT3B and HDAC1 were assessed by enzymatic activity assay and RT-PCR, respectively. Furthermore, we studied the binding interaction of EGCG with DNMT3B and HDAC1 by molecular modeling as well as promoter DNA methylation and expression of retinoic acid receptor-β (RARβ), cadherin 1 (CDH1) and death-associated protein kinase-1 (DAPK1) in EGCG-treated HeLa cells by RT-PCR and MS-PCR. In the present study, time-dependent EGCG-treated HeLa cells were found to have a significant reduction in the enzymatic activity of DNMT and HDAC. However, the expression of DNMT3B was significantly decreased in a time-dependent manner whereas there was no significant change in HDAC1 expression. Molecular modeling data also supported the EGCG-mediated DNMT3B and HDAC1 activity inhibition. Furthermore, time-dependent exposure to EGCG resulted in reactivation of known tumor-suppressor genes (TSGs) in HeLa cells due to marked changes in the methylation of the promoter regions of these genes. Overall, the present study suggests that EGCG may have a significant impact on the development of novel epigenetic-based therapy.

Hypoxia-inducible factor (HIF)-1 is the key transcriptional activator mediating both adaptive and pathological responses to hypoxia. The purpose of this study was to find the role of HIF-1 in regulating neprilysin (NEP) at the early stage of hypoxia and explore the underlying mechanism. In this study, we demonstrated that both NEP mRNA and protein levels in neuroblastoma cells were elevated in early stages of hypoxia. Over-expression of HIF-1α gene increased NEP mRNA/protein levels, as well as enzyme activity while knockdown of HIF-1α decreased them. Meanwhile, HIF-1α was shown to bind to histone deacetylase (HDAC)-1 and reduced the association of HDAC-1 with NEP promoter, thus activating NEP gene transcription in a de-repression way. In summary, our results indicated that hypoxia in the early stages would up-regulate NEP expression, in which interaction of HIF-1α and HDAC-1 may play a role. This study suggested that NEP up-regulation might be an adaptive response to hypoxia, which was mediated by HIF-1α binding to HDAC-1 at the early stage of hypoxia., (© 2014 International Society for Neurochemistry.)

Cigarette smoking (CS) is considered one of the major risk factors to cause neurodegenerative disorders. Nicotine is the main chemical in CS which is responsible for dysfunction of the brain as a neuroteratogen. Also, nicotine dependency is a real mental illness and disease. Recently, chronic nicotine exposure has been shown to cause oxidative/nitrosative stress leading to a deleterious condition to cellular death in different brain regions. However, little is known about the effects of nicotine on mouse neural stem cells (mNSCs). The aim of this study is to investigate the effects of nicotine on mNSCs and elucidate underlying mechanisms involved in expression of a diversity of genes regulated by nicotine. When mNSCs were isolated from the whole brain of embryonic day 16 mice treated with nicotine at vehicle, 100, 400, and 800 μM for 5 d, nicotine significantly decreased the number and size of neurospheres. In immunocytochemistry, nicotine-exposed mNSCs expressing nestin showed the shortened filaments and condensed nuclei. In RT-PCR, messenger RNA (mRNA) levels of proliferating cell nuclear antigen (PCNA) and sirtuin1 (SIRT1) were significantly decreased, while the production of nitric oxide and mRNA levels of cyclooxygenase2 (COX-2), tumor necrosis factor-alpha TNF-α, and histone deacetylase 1 (HDAC1) were increased in a dose-dependent manner. In addition, sodium butyrate and valproic acid, HDAC inhibitors, partially rescue proliferation of mNSCs via inhibition of HDAC1 expression and NO production. Taken together, these data demonstrate that prolonged exposure of nicotine decreased proliferation of mNSCs by increased NO and inflammatory cytokine through increased HDAC1. Furthermore, this study could help in the development of a therapy for nicotine-induced neurodegenerative disorder and drug abuse.

Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro.

Activation of the endothelium by alkyl-glycerophosphate (AGP) has been implicated in the development of atherosclerosis. Our previous study suggested that cyclic phosphatidic acid (cPA) inhibits arterial wall remodeling in a rat model in vivo. However, the mechanisms through which specific target genes are regulated during this process remain unclear. Here, we examined whether cPA inhibited AGP-induced expression of class I histone deacetylases (HDACs, namely HDAC1, HDAC2, HDAC3, and HDAC8), which may affect subsequent transcriptional activity of target genes. Our experimental results showed that human coronary artery endothelial cells (HCAECs) expressed high levels of HDAC2 and low levels HDAC1, HDAC3, and HDAC8. Moreover, AGP treatment induced downregulation of HDAC2 expression in HCAECs. However, cotreatment with cPA inhibited this downregulation of HDAC2 expression. Interestingly, treatment with AGP increased the expression and secretion of endogenous interleukin (IL)-6 and IL-8; however, this effect was inhibited when HCAECs were cotreated with cPA or the synthetic peroxisome proliferator-activator receptor gamma (PPARγ) antagonist T0070907. Thus, our data suggested that cPA may have beneficial effects in inflammation-related cardiovascular disease by controlling HDAC2 regulation.

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.

Gastric cancer is still the second leading cause of cancer-related death worldwide, even though its incidence and mortality have declined over the recent few decades. Epigenetic control using histone deacetylase inhibitors, such as trichostatin A (TSA), is a promising cancer therapy. This study aimed to assess the messenger RNA (mRNA) levels of three histone deacetylases (HDAC1, HDAC2, and HDAC3), two histone acetyltransferases (GCN5 and PCAF), and two possible targets of these histone modifiers (MYC and CDKN1A) in 50 matched pairs of gastric tumors and corresponding adjacent nontumors samples from patients with gastric adenocarcinoma, as well as their correlations and their possible associations with clinicopathological features. Additionally, we evaluated whether these genes are sensitive to TSA in gastric cancer cell lines. Our results demonstrated downregulation of HDAC1, PCAF, and CDKN1A in gastric tumors compared with adjacent nontumors (P < 0.05). On the other hand, upregulation of HDAC2, GCN5, and MYC was observed in gastric tumors compared with adjacent nontumors (P < 0.05). The mRNA level of MYC was correlated to HDAC3 and GCN5 (P < 0.05), whereas CDKN1A was correlated to HDAC1 and GCN5 (P < 0.05 and P < 0.01, respectively). In addition, the reduced expression of PCAF was associated with intestinal-type gastric cancer (P = 0.03) and TNM stages I/II (P = 0.01). The increased expression of GCN5 was associated with advanced stage gastric cancer (P = 0.02) and tumor invasion (P = 0.03). The gastric cell lines treated with TSA showed different patterns of histone deacetylase and acetyltransferase mRNA expression, downregulation of MYC, and upregulation of CDKN1A. Our findings suggest that alteration of histone modifier genes play an important role in gastric carcinogenesis, contributing to MYC and CDKN1A deregulation. In addition, all genes studied here are modulated by TSA, although this modulation appears to be dependent of the genetic background of the cell line.

The recent evidences indicate that autophagy is associated with a number of pathological processes including cancer, muscular disorder and neurodegeneration in addition to longevity. The efficacy of spermine was investigated on induction of autophagy through histone deacetylation and p53 activation in human fibrosarcoma cell line, HT1080. In this study, it was discovered that spermine increases the activity of HAT and autophagy. It was also identified that the transcriptional activation of p53 and the activation of p21 promoter by spermine are related to the induction of autophagy in reporter gene assay. Furthermore, western blot analyses demonstrated that spermine modulates the expression of proteins related to autophagy and apoptosis. The expression levels of Ac-histone H3, HDAC1, HAT1, p300 and SIRT1 were increased in HT1080 cells treated with spermine. In addition, the expression levels of protein such as acetyl-p53, p-p53, Bcl-2 and caspase-9 inducing apoptosis were increased in the presence of spermine. Moreover, the levels of Mdm2 and caspase-3 expression were reduced in the cells exposed to spermine compared to blank group. These results suggest that activation of HAT in the presence of spermine promotes the induction of autophagy in HT1080 cells through the enhanced activity of p-p53 and acetyl p53., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Epigenetic regulators like histone deacetylases (1 and 2), and viral onco-proteins (E6/E7) are known to be overexpressed in cervical cancer cells. The present study was designed to investigate the effect of curcumin on HDACs (1 and 2) and HPV E6/E7 in the cervical cancer cell line SiHa and a drug resistant clone SiHaR (derived from SiHa). It was further intended to investigate whether curcumin could sensitize the cells towards cisplatin induced cell killing by modulation of multi drug resistant proteins like MRP1 and Pgp1. Curcumin inhibited HDACs, HPV expression and differentially increased acetylation and up-regulation of p53 in SiHa and SiHaR, leading to cell cycle arrest at G1-S phase. Up-regulation of pRb, p21, p27 and corresponding inhibition of cyclin D1 and CDK4 were observed. Cisplatin resistance in SiHaR due to over-expression of MRP1 and Pgp1 was overcome by curcumin. Curcumin also sensitized both the cervical cancer cells towards cisplatin induced cell killing. Inhibition of HDACs and HPVs led to cell cycle arrest at G1/S phase by alteration of cell cycle regulatory proteins. Suppression of MRP1 and Pgp1 by curcumin resulted in sensitization of cervical cancer cells, lowering the chemotherapeutic dose of the drug cisplatin.

Background: Postmortem brain studies have shown that HDAC1-a lysine deacetylase with broad activity against histones and nonhistone proteins-is frequently expressed at increased levels in prefrontal cortex (PFC) of subjects diagnosed with schizophrenia and related disease. However, it remains unclear whether upregulated expression of Hdac1 in the PFC could affect cognition and behavior., Methods: Using adeno-associated virus, an Hdac1 transgene was expressed in young adult mouse PFC, followed by behavioral assays for working and long-term memory, repetitive activity, and response to novelty. Prefrontal cortex transcriptomes were profiled by microarray. Antipsychotic drug effects were explored in mice treated for 21 days with haloperidol or clozapine., Results: Hdac1 overexpression in PFC neurons and astrocytes resulted in robust impairments in working memory, increased repetitive behaviors, and abnormal locomotor response profiles in novel environments. Long-term memory remained intact. Over 300 transcripts showed subtle but significant changes in Hdac1-overexpressing PFC. Major histocompatibility complex class II (MHC II)-related transcripts, including HLA-DQA1/H2-Aa, HLA-DQB1/H2-Ab1, and HLA-DRB1/H2-Eb1, located in the chromosome 6p21.3-22.1 schizophrenia and bipolar disorder risk locus, were among the subset of genes with a more robust (>1.5-fold) downregulation in expression. Hdac1 levels declined during the course of normal PFC development. Antipsychotic drug treatment, including the atypical clozapine, did not affect Hdac1 levels in PFC but induced expression of multiple MHC II transcripts., Conclusions: Excessive HDAC1 activity, due to developmental defects or other factors, is associated with behavioral alterations and dysregulated expression of MHC II and other gene transcripts in the PFC., (© 2013 Society of Biological Psychiatry.)

Histone deacetylases (HDACs) are epigenetic regulatory proteins that repress gene transcription by changing DNA conformation. The regulation of gene expression through histone deacetylation is an important mechanism for the development of the central nervous system. Although the disruption of the balance in epigenetic gene regulation has been implicated in many CNS developmental abnormalities and diseases, the expression pattern of HDACs in various cell types in the brain during its maturation process has had limited exploration. Therefore, in this study, we investigate the cell type-specific and developmental stage-specific expression pattern of HDAC1 and HDAC2 in the mouse cerebellum. Our experimental results show that the cerebellar progenitors and glial cells express high levels of HDAC1 and low levels of HDAC2. On the other hand, the post-mitotic migrating neuronal cells of the cerebellar cortex show strong HDAC2 and weak HDAC1 expressions. In more differentiated neuronal cells, including Purkinje cells, granule cells, unipolar brush cells, and GABAergic interneurons, we found a consistent expression pattern, high levels of HDAC2 and low levels of HDAC1. Therefore, our data provide support for the potential important roles of HDAC1 in cell proliferation and HDAC2 in migration and differentiation.

Objective: To investigate the effect of vitrification on Dnmt1o, Hat1, and Hdac1 promoter CpG island methylation patterns and messenger RNA (mRNA) expression levels in mouse metaphase II (MII) oocytes., Design: In vitro study., Setting: Academic institution., Animal(s): Kunming white mice., Intervention(s): After vitrification, surviving mouse MII oocytes subjected to methylation and expression analysis with fresh oocytes used as a control., Main Outcome Measure(s): Expression levels of mRNA as measured by real-time reverse-transcriptase polymerase chain reaction of methylation patterns of the CpG islands in the Dnmt1o, Hat1, and Hdac1 promoters analyzed by bisulfite mutagenesis and sequencing., Result(s): The methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, and Hdac1 were not statistically significantly when comparing vitrified oocytes and fresh oocytes. The expression levels of Hat1 and Hdac1 mRNA were not statistically significantly different in comparing in vitrified oocytes and fresh oocytes. The expression of Dnmt1o mRNA was statistically significantly lower in vitrified oocytes compared with fresh oocytes., Conclusion(s): Vitrification did not statistically significantly alter the methylation patterns of the promoter CpG islands in Dnmt1o, Hat1, or Hdac1, but did statistically significantly decrease the expression of Dnmt1o mRNA in mouse MII oocytes., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Our recent studies of microRNA (miRNA) expression signature demonstrated that microRNA-874 (miR-874) was significantly downregulated in maxillary sinus squamous cell carcinoma (MSSCC), and a putative tumour-suppressive miRNA in human cancers. Our aim of this study was to investigate the functional significance of miR-874 in cancer cells and to identify novel miR-874-mediated cancer pathways and responsible genes in head and neck squamous cell carcinoma (HNSCC)., Methods: Gain-of-function studies using mature miR-874 were performed to investigate cell proliferation and cell cycle distribution in HNSCC cell lines (SAS and FaDu). To identify miR-874-mediated molecular pathways and targets, we utilised gene expression analysis and in silico database analysis. Loss-of-function assays were performed to investigate the functional significance of miR-874 target genes., Results: Expression levels of miR-874 were significantly downregulated in HNSCC tissues (including oral, pharyngeal and laryngeal SCCs) compared with normal counterpart epithelia. Restoration of miR-874 in SAS and FaDu cell lines revealed significant inhibition of cell proliferation and induction of G2/M arrest and cell apoptosis. Our expression data and in silico analysis demonstrated that miR-874 modulated the cell cycle pathway. Moreover, histone deacetylase 1 (HDAC1) was a candidate target of miR-874 regulation. Luciferase reporter assays showed that miR-874 directly regulated HDAC1. Silencing of the HDAC1 gene significantly inhibited cell proliferation and induced G2/M arrest and cell apoptosis in SAS cells., Conclusions: Downregulation of miR-874 was a frequent event in HNSCC. miR-874 acted as a tumour suppressor and directly targeted HDAC1. Recognition of tumour-suppressive miRNA-mediated cancer pathways provides new insights into the potential mechanisms of HNSCC oncogenesis and suggests novel therapeutic strategies for the disease.

Background: Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac))., Methods: We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry., Results: Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression., Conclusions: Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma, Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.

Histone deacetylases (HDACs) have a critical role in epigenetic gene silencing, rendering a compact chromatin structure by removing acetyl groups from lysine residues within the tails of core histones, thereby repressing gene expression. Epigenetic transcriptional dysregulation is an important oncogenic mechanism in some sarcomas associated with translocations, for which antitumor activity by HDAC inhibitors has been shown in preclinical studies. Nevertheless, the expression of the protein targets of these drugs has not yet been broadly surveyed in this neoplasia. In this study, we assess the expression of HDAC1 and 2 by immunohistochemistry in a tissue microarray series of 1332 cases, representing 44 categories of malignant and borderline mesenchymal tumors. HDAC2 was the more highly expressed isoform, and was more strongly expressed in translocation-associated sarcomas than in other mesenchymal tumors or normal tissues. HDAC1, in contrast, displayed lower expression in translocation-associated sarcomas than in other mesenchymal tumors or in normal tissues. These results indicate that HDAC1 and HDAC2 are differentially expressed in mesenchymal neoplasms, and suggest that HDAC2 is the isoform more likely contributing to the pathogenesis of many translocation-associated sarcomas and to their response to HDAC inhibitors.

Histone deacetylase (HDAC) is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors induce the differentiation or apoptosis of cancer cells. Valproic acid (VPA) is one of the clinically available HDAC inhibitors. We investigated the anticancer effects of VPA in combination with gemcitabine (GEM) in the human cholangiocarcinoma cell line HuCCT1, and explored the mechanisms of the anticancer effects using microarray analysis. The anticancer effects of VPA or gemcitabine (GEM), and the effects of VPA combined with GEM, were studied by a cell proliferation assay. A microarray analysis was performed and the genes were picked up using GeneSpring GX11.5, followed by Ingenuity Pathways Analysis (IPA) and determination of gene expression by RT-PCR. GEM (5 nM) and VPA (0.5 mM) reduced proliferation by 23%, which significantly augmented the anticancer effect of GEM alone or VPA alone (P<0.01). Using microarray analysis, 43 genes were identified with the comparison between the GEM group and the GEM plus VPA combination group. Interactions were identified between genes of the 'Cellular Development' network relevant to the differentiation of cancer cells using IPA. Furthermore, GEM combined with VPA up-regulated the HLA-DRA expression compared to the single agents (P<0.01). VPA augmented the effects of GEM by enhancing the gene network mainly including HLA-DRA, possibly through the modification of cancer cell differentiation.

The aim of this study was to identify and evaluate novel prognostic markers for gastric cancer. Differential mRNA displays comparing paired tumor/normal stomach samples were assessed. Several differentially expressed cDNA fragments of candidate genes were identified, and one of these was further studied using quantitative reverse transcription-PCR in 140 human gastric carcinomas. To evaluate protein expression, immunohistochemical staining was performed in selected cases. One of the genes abundantly expressed in tumor tissue on the differential mRNA displays was identified as histone deacetylase 1 (HDAC). HDAC was overexpressed in the tumor tissue in 77% of the cases as determined by quantitative reverse transcription-PCR. Immunohistochemical staining revealed analogous results, showing strong expression in cancer cells. Patients were then classified into high (n=78) and low (n=62) expression groups according to the mean value of HDAC expression. High frequencies of lymph vessel and vascular vessel permeations, and advanced stage of the disease were recognized in the high expression group compared to the low expression group (p<0.05). Prognosis was significantly worse for the high expression group than for the low expression group (p<0.05), and multi-variate analysis demonstrated that HDAC expression was an independent prognostic factor. Although not significantly different, lymph node metastasis was recognized more frequently in the high expression group (p=0.07). In conclusion, the findings show that HDAC expression is associated with aggressive behavior of primary gastric cancer, and imply that determination of the HDAC expression status is useful for predicting prognosis in patients with gastric cancer.

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 (Tis7/IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o(-) cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o(-) cells compared to normal bronchial epithelial cells 16HBE14o(-). Surprisingly, messenger RNA level of IFRD1 in CFBE41o(-) cells was found elevated. Treating CFBE41o(-) cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Histone deacetylase 1 (HDAC1) and metastasis-associated protein 1 (MTA1) form the nucleosome remodeling and histone deacetylation (NuRD) complex and may possibly play a central role in cancer development. However, limited data has been reported regarding the expression of both HDAC1 and MTA1. The aim of the present study was to clarify the clinical role of HDAC1 and MTA1 expression in colon cancer. Seventy-four patients with colon cancer, who underwent colectomy at our institution, were enrolled in this study. Expression of HDAC1 and MTA1 was examined immunohistochemically. The patients were divided into four groups: HDAC1-positive group (n=58), HDAC1-negative group (n=16), MTA1-positive group (n=38) and MTA1-negative group (n=36). Clinicopathological factors and survival rates were compared between the groups. Regarding the clinicopathological factors, the depth of tumor invasion and stage correlated significantly with HDAC1 expression (p<0.05). Age, depth of tumor invasion and vascular invasion tended to correlate with MTA1 expression. The 5-year survival rate in the HDAC1-positive group (55.1%) was significantly worse compared to the HDAC1-negative group (86.5%) (p<0.05), and the 5-year survival rate of the MTA1-positive group (50.5%) was significantly worse than that of the MTA1-negative group (73.1%) (p=0.05). In patients with stages II-IV and curability A, B, the survival rate in those with HDAC1-positive expression was significantly worse than those with HDAC1-negative expression (p<0.05), and the survival rate of the MTA1-positive group tended to be worse than that of the MTA1-negative group (p=0.07). Overall survival in both the HDAC1 and MTA1-positive groups was significantly worse than overall survival of the other groups (p<0.05). Disease-free survival in both the HDAC1- and MTA1-positive groups, among patients with stages II-IV and curability A, B, was also significantly worse than that of the other groups (p<0.05). HDAC1 and MTA1 expression levels were significantly related to poorer prognosis. Therefore, HDAC1 and MTA1 expression levels are potential prognostic indicators for colon cancer.

Histone deacetylases (HDACs) are associated with the development and progression of cancer, but it is not known which of the HDAC isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMP-9) compared to MCF-7 cells. We compared the expression of HDAC isoforms between MCF-7 and MDA-MB-231 cells and found greater expression of HDAC4, 6 and 8 in MDA-MB-231 cells by RT-PCR and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific siRNAs directed against HDAC1, 4, 6 and 8, we show that inhibition of HDAC1, 6 and 8, but not HDAC4, are responsible for invasion and MMP-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of MCF-7 cells overexpressing HDAC1, 4, 6 or 8 and found that overexpression of HDAC1, 6 or 8 increased invasion and MMP-9 expression. By developing HDAC isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Valproic acid (VPA) is a wide spread anticonvulsant and mood-stabilizing agent, the use of which is associated with hepatotoxicity, bone marrow suppression and osteomalacia. In the current paper we propose a possible mechanism of VPA-induced osteomalacia involving accelerated catabolism of 1α,25(OH)(2)-vitamin D3 (VD3) due to increased expression of CYP24. We demonstrate that VPA strongly potentiates CYP24 mRNA expression by VD3 in human hepatocytes (HH) and in human embryonic kidney cells (HEK293). By the method of gene reporter assay we found that VPA increases basal and VD3-inducible activity of CYP24 promoter (pCYP24-luc) in human liver adenocarcinoma (HepG2) and in HEK293 cells in dose-dependent manner. In order to delineate the role of inhibitory effects of VPA on histone deacetylase 1 (HDAC1), we compared the effects of VPA with trichostatin A (TSA) on basal and inducible levels of CYP24 mRNA and pCYP24-luc transactivation. Transactivation of CYP24 promoter by VD3 was enhanced in the presence of both TSA and VPA. In contrast, VD3-inducible expression of CYP24 mRNA was enhanced by VPA but not by TSA, implying that HDAC1 inhibition is not the major reason for VPA effects on CYP24. We examined the effects of VPA on mitogen-activated protein kinases as the important transcriptional regulators of VDR. VPA activated extracellular signal-regulated kinase (ERK) but not c-Jun-N-terminal kinase (JNK) and p38 MAPKs. In conclusion, VPA enhances transcriptional activity of VDR and increases expression of CYP24 mRNA in the presence of VD3 in physiological concentrations. The mechanism involves activation of ERK and partly the inhibition of HDAC1., (© 2010 Elsevier Ireland Ltd. All rights reserved.)

Transcriptional repressors such as nuclear receptor corepressors (NCORs) and class I histone deacetylases (HDACs) are considered potential therapeutic targets in various human malignancies. In astrocytic gliomas, however, there is still a need to understand the role of these transcriptional repressors in tumor proliferation, tumor differentiation, and patient survival. We immunohistochemically analyzed the expression of NCOR1 and 2 as well as HDAC1, 2, and 3 on a tissue microarray comprising tumor samples from 283 astrocytic gliomas and correlated the expression levels with tumor differentiation, tumor proliferation, and patient survival. Strong nuclear expression was found in glioma cells for HDAC1, HDAC2, and NCOR2. In contrast, weak expression of NCOR1 and HDAC3 was detected in the cytoplasm and nuclei of tumor cells. HDAC3 expression was inversely associated with tumor grade. Consequently, increased HDAC3 expression was associated with better patient survival in univariate regression. Expression of HDAC1 and HDAC2 increased during tumor recurrence and malignant tumor progression, respectively, whereas expression of the remaining antigens did not seem to depend on tumor grade and was comparable to expression levels found in non-neoplastic brain tissues. Finally, we detected a positive association between HDAC2 expression and tumor proliferation as well as between NCOR1 and expression of the stem cell-associated intermediate filament protein nestin. Our findings suggest that "classical" transcriptional repressors are expressed in astrocytic tumors and that the roles of HDAC2 and HDAC3 in these tumors deserve further investigation., (© 2010 Japanese Cancer Association.)

Overexpression of histone deacetylases has been reported in various human malignancies; however, the expression of histone deacetylases in endometrial tissue is not fully understood. In the present study, the expression of histone deacetylase 1, histone deacetylase 2, and Ki-67 was examined immunohistochemically in 30 normal and 66 malignant endometrial tissue samples. The results were expressed as a positivity index and compared with the positivity index for Ki-67 and rates of patient survival. The effect of 2 histone deacetylase inhibitors, trichostatin A and apicidine, on cell proliferation and the expression of cell cycle regulators such as cyclins (D1, E, and A), p21, p27, and p16 were investigated using 6 endometrial carcinoma cell lines. The positivity index for histone deacetylase 1 (79.8 +/- 33.0, mean +/- SD) and histone deacetylase 2 (106.3 +/- 41.9) was higher in endometrial carcinoma than the normal endometrium, with a significant difference for histone deacetylase 2. The positivity index for histone deacetylase 2 was significantly increased in higher-grade carcinomas (positivity index for grade 3, 124.9 +/- 28.4) compared with grade 1 tumors (86.0 +/- 41.0) and was positively correlated with that for Ki-67. In addition, patients with histone deacetylase 2-positive carcinomas had a poor prognosis compared with those with histone deacetylase 2-negative carcinoma (P = .048). Treatment with trichostatin A or apicidine suppressed the proliferation in all cell lines examined, in association with increased expression of p21 and down-regulation of cyclin D1 and cyclin A expression. These results indicated that increased histone deacetylase 2 expression is involved in the acquisition of aggressive behavior by endometrial carcinoma and suggest histone deacetylase inhibitor to be a promising anticancer drug for this carcinoma., (Copyright 2010 Elsevier Inc. All rights reserved.)

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.