Histone deacetylase inhibitors as a novel therapeutic approach for pheochromocytomas and paragangliomas

Epigenetic mechanisms,such as DNA methylation and histone modifications(e.g.,acetylation and deacetylation),are strongly implicated in the carcinogenesis of various malignancies.During transcription,the expression and functionality of coding gene products are altered following the histone acetylation and deacetylation.These processes are regulated by histone acetyltransferases(HATs)and histone deacetylases(HDACs),respectively.HDAC inhibitors(HDACis)have been developed as promising therapeutic agents,to limit exposure to traditional and toxic chemotherapies and offer more alternatives for some specific malignant diseases with limited options.Mechanistically,these agents affect many intracellular pathways,including cell cycle arrest,apoptosis and differentiation,and their mechanism of action mainly depends on the type of cancer.Currently,five HDACis have been approved for the treatment of several hematological malignancies(e.g.,T-cell lymphoma subtypes and multiple myeloma);while,many of them are tested for further therapeutic indications in solid tumors(e.g.,colorectal,thyroid,breast,lung and pancreatic cancer).Herein,we review the literature and gather all available evidence,from in vitro and in vivo data to clinical trial results,that recognizes the antitumor activity of HDACis on pheochromocytomas and paragangliomas;and supports their clinical implementation in the treatment of these rare neuroendocrine tumors at metastatic setting.

Curcumin inhibits hepatitis B virus infection by downregulating ccc DNA-bound histone acetylation

AIM To investigate the potential effect of curcumin on hepatitis B virus(HBV) covalently closed circular DNA(ccc DNA) and the underlying mechanism.METHODS A Hep G2.2.15 cell line stably transfected with HBV was treated with curcumin, and HBV surface antigen(HBs Ag) and e antigen(HBe Ag) expression levels were assessed by ELISA. Intracellular HBV DNA replication intermediates and ccc DNA were detected by Southern blot and real-time PCR, respectively. The acetylation levels of histones H3 and H4 were measured by Western blot. H3/H4-bound ccc DNA was detected by chromatin immunoprecipitation(Ch IP) assays. The deacetylase inhibitors trichostatin A and sodium butyrate were used to study the mechanism of action for curcumin. Additionally, short interfering RNAs(si RNAs) targeting HBV were tested along with curcumin.RESULTS Curcumin treatment led to time-and dose-dependent reductions in HBs Ag and HBe Ag expression and significant reductions in intracellular HBV DNA replication intermediates and HBV ccc DNA. After treatment with 20 μmol/L curcumin for 2 d, HBs Ag and ccc DNA levels in Hep G2.2.15 cells were reduced by up to 57.7%(P < 0.01) and 75.5%(P < 0.01), respectively, compared with levels in non-treated cells. Meanwhile, time-and dose-dependent reductions in the histone H3 acetylation levels were also detected upon treatment with curcumin, accompanied by reductions in H3-and H4-bound ccc DNA. Furthermore, the deacetylase inhibitors trichostatin A and sodium butyrate could block the effects of curcumin. Additionally, transfection of si RNAs targeting HBV enhanced the inhibitory effects of curcumin.CONCLUSION Curcumin inhibits HBV gene replication via downregulation of ccc DNA-bound histone acetylation and has the potential to be developed as a ccc DNA-targeting antiviral agent for hepatitis B.

Histone deacetylases:Regulation of vascular homeostasis via endothelial cells and vascular smooth muscle cells and the role in vascular pathogenesis

tHistone deacetylases(HDACs)are proteases that play a key role in chromosome structural modification and gene expression regulation,and the involvement of HDACs in can-cer,the nervous system,and the metabolic and immune system has been well reviewed.Our understanding of the function of HDACs in the vascular system has recently progressed,and a significant variety of HDAC inhibitors have been shown to be effective in the treatment of vascular diseases.However,few reviews have focused on the role of HDACs in the vascular sys-tem.In this study,the role of HDACs in the regulation of the vascular system mainly involving endothelial cells and vascular smooth muscle cells was discussed based on recent updates,and the role of HDACs in different vascular pathogenesis was summarized as well.Furthermore,the therapeutic effects and prospects of HDAC inhibitors were also addressed in this review.

Curcumin-induced Histone Acetylation in Malignant Hematologic Cells

This study investigated the inhibitory effects of curcumin on proliferation of hematological malignant cells in vitro and the anti-tumor mechanism at histone acetylation/histone deacetylation levels. The effects of curcumin and histone deacetylase inhibitor trichostatin A （TSA） on the growth of Raji cells were tested by MTT assay. The expression of acetylated histone-3 （H3） in Raji, HL60 and K562 cells, and peripheral blood mononuclear cells （PBMCs） treated with curcumin or TSA was detected by immunohistochemistry and FACS. The results showed curcumin inhibited pro- liferation of Raji cells significantly in a time- and dose-dependent fashion, while exhibited low toxicity in PBMCs. Curcumin induced up-regulation of the expression of acetylated H3 dose-dependently in all malignant cell lines tested. In conclusion, curcumin inhibited proliferation of Raji cells selectively, enhanced the level of acetylated H3 in Raji, HL60, and K562 cells, which acted as a histone deacetylase inhibitor like TSA. Furthermore, up-regulation of H3 acetylation may play an important role in regulating the proliferation of Raji cells.

组蛋白乙酰化/甲基化在口腔疾病中的研究进展

组蛋白乙酰化和甲基化能影响染色质构象,进而调控多种生物学活动。异常的组蛋白乙酰化和甲基化修饰与多种口腔疾病的发生发展有关。在牙的发育过程中,组蛋白乙酰化和甲基化修饰有序地升高或降低,调控牙的发育,氟离子能够破坏组蛋白乙酰化和甲基化修饰的平衡,这可能与氟牙症的发生有关。此外,组蛋白乙酰化和甲基化修饰也参与调控了口腔的炎症性疾病,炎症微环境下,组蛋白乙酰转移酶GCN5表达下降,使Dickkopf 1(DKK1)表达下降,从而激活Wnt/β⁃catenin通路,最终抑制牙周膜干细胞的成骨分化。Zeste增强子同源物2(enhancer of zeste homolog 2,EZH2)与H3K27me3在炎症牙髓组织和牙髓细胞中下降,抑制EZH2可抑制炎症刺激导致的人牙髓细胞中白细胞介素⁃1b、白细胞介素⁃6和白细胞介素⁃8的表达。组蛋白乙酰化/甲基化修饰能够与多条信号通路相互作用,促进口腔肿瘤的发生发展,并与唾液腺肿瘤的高侵袭性有关。靶向组蛋白乙酰化和甲基化相关酶的小分子药物能调控组蛋白甲基化/乙酰化修饰水平,在口腔颌面部疾病治疗中展现出应用潜能,例如组蛋白去乙酰化酶抑制剂——伏立诺他,其既能够抑制炎症的相关细胞因子的分泌,还能促进成牙本质细胞分化并形成牙本质相关基质,展现出了在保髓治疗中的潜力。了解组蛋白乙酰化/甲基化修饰在口腔疾病发生发展中的作用,有助于推进表观遗传修饰在口腔疾病的研究深入,提供新的疾病诊疗视角。

Targeting histone deacetylase suppresses tumor growth through eliciting METTL14-modified m^(6)A RNA methylation in ocular melanoma

Background Diversified histone deacetylation inhibitors(HDACis)have demonstrated encouraging outcomes in multiple malignancies.N6-methyladenine(m6A)is the most prevalent messenger RNA modification that plays an essential role in the regulation of tumorigenesis.Howbeit,an in-depth understanding of the crosstalk between histone acetylation and m6A RNA modifications remains enigmatic.This study aimed to explore the role of histone acetylation and m6A modifications in the regulation of tumorigenesis of ocular melanoma.Methods Histone modification inhibitor screening was used to explore the effects of HDACis on ocular melanoma cells.Dot blot assay was used to detect the global m6A RNA modification level.Multi-omics assays,including RNA-sequencing,cleavage under targets and tagmentation,single-cell sequencing,methylated RNA immunoprecipitation-sequencing(meRIP-seq),and m6A individual nucleotide resolution cross-linking and immunoprecipitation-sequencing(miCLIP-seq),were performed to reveal the mechanisms of HDACis on methyltransferase-like 14(METTL14)and FAT tumor suppressor homolog 4(FAT4)in ocular melanoma.Quantitative real-time polymerase chain reaction(qPCR),western blotting,and immunofluorescent staining were applied to detect the expression of METTL14 and FAT4 in ocular melanoma cells and tissues.Cell models and orthotopic xenograft models were established to determine the roles of METTL14 and FAT4 in the growth of ocular melanoma.RNA-binding protein immunoprecipitation-qPCR,meRIP-seq,miCLIP-seq,and RNA stability assay were adopted to investigate the mechanism by which m6A levels of FAT4 were affected.Results First,we found that ocular melanoma cells presented vulnerability towards HDACis.HDACis triggered the elevation of m6A RNA modification in ocular melanoma.Further studies revealed that METTL14 served as a downstream candidate for HDACis.METTL14 was silenced by the hypo-histone acetylation status,whereas HDACi restored the normal histone acetylation level of METTL14,thereby inducing its expression.Subsequently,METTL14 served as a tumor suppressor by promoting the expression of FAT4,a tumor suppressor,in a m6A-YTH N6-methyladenosine RNA-binding protein 1-dependent manner.Taken together,we found that HDACi restored the histone acetylation level of METTL14 and subsequently elicited METTL14-mediated m6A modification in tumorigenesis.Conclusions These results demonstrate that HDACis exert anti-cancer effects by orchestrating m6A modification,which unveiling a“histone-RNA crosstalk”of the HDAC/METTL14/FAT4 epigenetic cascade in ocular melanoma.

Reversible histone acetylation/ deacetylation modification by p300 and HDAC3 is involved in the regulation of IL-18 promoter activity

Interleukin-18 (IL-18) is a pleiotropic cytokine involved in the development of T helper type 1 (Thl) cells, and it plays important roles in regulation of both the innate and acquired immune responses. The aim of this study was to elucidate whether the reversible histone acetylation/ deacetylation modification participates in the regulation of IL-18 transcription expression. The transcription coactivator p300 containing the histone acetyltransferase (HAT) activity, and the histone deacetylase 3 (HDAC3) were used in this study to analyze the effect of this modification in the regula-tion of mouse IL-18 gene. The results demonstrate that transfection of p300-expression plasmid promotes the en-dogenous IL-18 mRNA synthesis in J774 cells, and stimulates the activation of IL-18 promoter. It has been found that this stimulating effect of p300 was reversed by HDAC3, indicat-ing the involvement of the reversible histone acetyla-tion/deacetylation modification in IL-18 regulation. Fur-thermore, the data show that the HAT activity of p300 was essential to its function in activating IL-18 promoter. In ad-dition, p300 was shown to be able to work synergistically with the transcription factor c-Fos on activation of IL-18 promoter and this effect could also be impaired by HDAC3. Results presented in this paper indicate that the reversible histone acetylation/deacetylation modification plays an im-portant role in the transcriptional regulation of IL-18.

A domesticated Harbinger transposase forms a complex with HDA6 and promotes histone H3 deacetylation at genes but not TEs in Arabidopsis

In eukaryotes,histone acetylation is a major modification on histone N-terminal tails that is tightly connected to transcriptional activation.HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements(TEs)in Arabidopsis thaliana.HDA6 has been shown to participate in several complexes in plants,including a conserved SIN3 complex.Here,we uncover a novel protein complex containing HDA6,several Harbinger transposon-derived proteins(HHP1,SANT1,SANT2,SANT3,and SANT4),and MBD domain-containing proteins(MBD1,MBD2,and MBD4).We show that mutations of all four SANT genes in the sant-null mutant cause increased expression of the flowering repressors FLC,MAF4,and MAF5,resulting in a late flowering phenotype.Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes,TE silencing is unaffected in sant-null mutants.Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation.Collectively,our findings suggest that Harbinger transposon-derived SANT domain-containing proteins are required for histone deacetylation and flowering time control in plants.

Relationship between histone acetylation/deacetylation and gene transcription

In eukaryotes, nucleosome is the basic unit of chromatin. Nucleosome is composed of an octamer of histone proteins (two molecules each of histones H 2A , H 2B , H 3 and H 4) and DNA strand wound around the octamer. Some data show that core histone octamer can affect gene transcription both \%in vitro\% and \%in vivo.\% Recent results indicate that histone acetylation/deacetylation is a key step to regulate activity of genes. This article summarizes some coactivators, such as GCN5p, P300/CBP and TAF Ⅱ 250, which are recently found to have histone acetyltransferase activity. The relationship between these coactivators and gene activation is also described. Besides, this article concerns some corepressors which have histone deacetylase activity, such as Rpd3p, HDAC2. These corepressors combine with other protein complex and then repress transcription. Finally, some problems to be solved and the future direction in this active field are discussed.

组蛋白去乙酰化酶6对畜禽热应激和脂代谢的调节作用

组蛋白去乙酰化酶6(HDAC6)是位于细胞质中的一种具有特殊结构和功能的去乙酰化酶,通过蛋白质泛素化以及蛋白质赖氨酸乙酰化共同调控细胞内的异常蛋白降解,影响细胞的周期、增殖、迁移和凋亡等过程。文章综述了HDAC6作为细胞内管理错误折叠蛋白质的关键者,间接调控了畜禽热应激以及脂代谢过程,为动物养殖生产过程中防治热应激以及脂肪代谢研究提供参考。

The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis

Plasticity in root system architecture(RSA)allows plants to adapt to changing nutritional status in the soil.Phosphorus availability is a major determinant of crop yield,and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition.Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants,whether and how epigenetic regulatory mechanisms contribute is poorly understood.Here,we report that the Switch defective/sucrose non-fermentable(SWI/SNF)ATPase BRAHMA(BRM)is involved in the local response to phosphate(Pi)starvation.The loss of BRM function induces iron(Fe)accumulation through increased LOW PHOSPHATE ROOT1(LPR1)and LPR2 expression,reducing primary root length under Pi deficiency.We also demonstrate that BRM recruits the histone deacetylase(HDA)complex HDA6-HDC1 to facilitate histone H3 deacetylation at LPR loci,thereby negatively regulating local Pi deficiency responses.BRM is degraded under Pi deficiency conditions through the 26 S proteasome pathway,leading to increased histone H3 acetylation at the LPR loci.Collectively,our data suggest that the chromatin remodeler BRM,in concert with HDA6,negatively regulates Fe-dependent local Pi starvation responses by transcriptionally repressing the RSA-related genes LPR1 and LPR2 in Arabidopsis thaliana.

姜黄素诱导Raji、HL-60和K562组蛋白乙酰化的研究

目的研究姜黄素对淋巴瘤细胞系Raji细胞的抑制增殖作用,并在组蛋白乙酰化/去乙酰化水平对其抗肿瘤机制进行探讨。方法MTT法检测不同浓度姜黄素、TSA作用于Raji细胞的抑制增殖率。以TSA处理后的细胞为阳性对照,免疫组化法定量分析及免疫荧光流式细胞化学定量检测姜黄素作用后Raji、HL-60、K562细胞的组蛋白乙酰化H3水平。结果姜黄素抑制Raji细胞增殖,并呈时间剂量依赖性;25μmol.L-1姜黄素可致Raji、HL-60、K562细胞的乙酰化H3水平增加(P<0.05),50μmol.L-1姜黄素的诱导作用增强(P<0.01)。结论姜黄素可以选择性地抑制Raji细胞增殖;且类似于TSA诱导Raji,HL-60,K562细胞组蛋白乙酰化增加。

丙戊酸钠抑制人多发性骨髓瘤U266细胞增殖及组蛋白乙酰化调控的研究

本研究探讨丙戊酸钠(valproic acid sodium,VPA)对U266细胞增殖的抑制作用及对组蛋白乙酰化修饰调控的影响。采用CCK-8法检测VPA对U266细胞增殖的抑制作用,流式细胞术检测VPA作用前后U266细胞周期的变化,半定量RT-PCR检测VPA作用后U266细胞中组蛋白去乙酰化酶1(HDAC1)mRNA的表达变化,Western blot方法检测VPA作用后HDAC1及组蛋白H3、H4乙酰化蛋白水平的变化。结果表明,VPA对U266细胞增殖的抑制作用呈时间-浓度依赖性;2.5mmol/L的VPA处理细胞48小时后,细胞周期检测显示G0/G1期细胞比例增高,S期细胞比例下降,细胞被阻滞在G0/G1期(p<0.05);RT-PCR证实VPA能够抑制HDAC1 mRNA的表达水平;Western blot方法证实不同浓度VPA作用细胞48小时后HDAC1蛋白水平降低,组蛋白H3、H4乙酰化表达水平提高。结论:VPA能够抑制U266细胞增殖,使细胞阻滞于G0/G1期,这可能与VPA抑制HDAC1表达,上调组蛋白H3、H4乙酰化水平有关。

组蛋白乙酰化/去乙酰化与基因表达调控研究进展

在真核生物中,组蛋白是构成核小体的重要成分,其乙酰化与去乙酰化修饰在真核生物基因表达调控中起重要作用。组蛋白乙酰化异常(低乙酰化或高乙酰化)常会引起基因表达紊乱,进而引起疾病的发生。对组蛋白乙酰转移酶(Histone acetyltransferase,HAT)及组蛋白去乙酰化酶(Histone deacetylase,HDAC)的种类,组蛋白乙酰化与去乙酰化与基因表达调控、疾病发生的关系进行了综述。

狼疮性肾炎患者血浆组蛋白乙酰化酶和组蛋白去乙酰化酶表达及其与IL-6、INF-γ的关系

目的研究狼疮性肾炎(LN)患者血浆组蛋白乙酰化酶(HAT)、组蛋白去乙酰化酶(HDAC)表达水平,分析其与LN活动性、炎性细胞因子等的关系,探究组蛋白乙酰化酶在LN发病机制中的作用。方法共收入正常对照组(N组,8例),狼疮性肾炎患者组(18例)。狼疮性肾炎组分为非活动期(I组,8例),活动期(A组,10例)。采用ELISA检测血浆HAT、HDAC、IL-6、INF-γ水平;采用直线相关性分析HAT、HDAC表达与IL-6、INF-γ相关性。结果 I组患者血浆HAT含量低于N组,但差异无统计学意义,A组患者HAT含量明显低于N组,差异有统计学意义[(9.74±3.67)vs(15.32±3.32),P<0.05],但I组与A组患者比较,差异无统计学意义;与N组比较,I组和A组患者中血浆HDAC含量明显降低[(6.75±2.05)vs(17.08±6.46),P<0.05;(4.53±3.62)vs(17.08±6.46),P<0.05],但两组之间比较差异无统计学意义。I组和A组患者血浆IL-6、INF-γ均明显高于N组,A组患者血浆中IL-6、INF-γ均明显高于I组。相关性分析,发现HAT、HDAC与IL-6呈负相关(r分别为-0.532和-0.665);而HAT、HDAC与INF-γ呈负相关(r分别为-0.621和-0.720)。结论LN患者血浆HDAC含量、活动期患者HAT低于正常人群,与IL-6、INF-γ呈负相关,HAT、HDAC可能影响IL-6、INF-γ等细胞因子水平参与LN的发病。

番茄HDACs家族基因在胁迫条件下的表达分析

组蛋白去乙酰化酶(Histone deacetylases,HDACs)家族基因在植物的生长发育、器官构建及逆境胁迫和激素信号应答中发挥重要作用。利用生物信息学方法对番茄的HDACs家族成员、分布及结构和功能等进行分析。结果表明,番茄HDACs家族包含15个成员,分为3个亚家族。遗传进化分析表明,番茄HDACs家族成员与拟南芥HDACs家族具有相似分类。利用实时荧光定量PCR对番茄HDACs家族基因的组织表达分析表明,HDACs具有组织特异性表达差异,Sl HDT1、Sl HDT2和Sl HDT3在根中表达较高,而Sl HDA1、Sl HDA3、Sl HDA5、Sl HDA6在果实发育过程中表达较高;利用RT-PCR对番茄HDACs的胁迫响应分析表明,在盐、SA、ABA、高温和低温胁迫条件下,15个番茄HDACs成员的表达模式不同,其中部分基因的表达水平被显著地诱导增加或者降低,推测这些基因很可能参与了调控番茄逆境胁迫条件下的防御应答反应。结果将为进一步解析番茄HDACs家族基因的功能奠定基础。

曲古霉素A增强抗肿瘤药物对膀胱癌T24细胞的杀伤作用

目的观察HDAC抑制剂曲古霉素A(TSA)和针对DNA的抗癌药物联用对膀胱癌T24细胞的杀伤作用。方法用MTT法测定TSA单用及分别与ADM、MMC和DDP联用对T24细胞的抑制率,用金氏公式法判断联合用药的效果。结果TSA分别与ADM、MMC、DDP联合用药对T24细胞的抑制率均随着浓度的增加而明显增加,TSA与MMC联用的协同作用最明显,而TSA与ADM和DDP在中低剂量下联用亦表现为协同作用。结论HDAC抑制剂TSA可明显增强针对DNA的抗癌药物对膀胱癌细胞的杀伤作用,有希望应用于晚期膀胱癌的化疗方案中。

牦牛SIRT3基因的克隆及其在各组织和睾丸不同发育阶段的表达

旨在克隆牦牛沉默蛋白调节因子3(sirtuin 3,SIRT 3)基因,并检测其在牦牛不同组织及不同年龄睾丸中的表达水平,从而为研究SIRT 3在牦牛睾丸发育过程中的作用机制提供试验依据。本研究选取胎牛期(5~6个月)、幼年期(1~2岁)、性成熟期(3~5岁)及老年期(7~9岁)健康雄性牦牛共12头(每组各3头),其中来源于同一年龄阶段的3个组织样本视为生物学重复。采集性成熟期牦牛肝、心、肺、脾、脑、肾、肌肉、小肠、睾丸和大肠以及4个时期的睾丸组织。分别提取各组织的总RNA,并利用反转录PCR(reverse transcription PCR,RT-PCR)技术克隆获得牦牛SIRT 3基因序列,使用相关生物信息学软件预测其编码蛋白质的结构和功能;通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测SIRT 3 mRNA在牦牛各组织及睾丸4个发育阶段的表达水平。结果表明,SIRT 3基因的ORF(open reading frame,开放阅读框)为1002 bp,编码333个氨基酸,与黄牛和绵羊的同源性分别达到99.9%和96.7%,该基因的mRNA在牦牛的各个组织中广泛表达,在脾、睾丸、肺和肌肉中的表达量较高。随年龄增长SIRT 3 mRNA在牦牛睾丸中表达呈现先上升后下降的趋势,其中在性成熟期的表达量最高。本研究为揭示SIRT 3在牦牛生长发育,尤其是睾丸发育过程中的调控提供了一定的基础数据。

组蛋白乙酰化/去乙酰化与白血病的研究进展

组蛋白乙酰化/去乙酰化与基因调控密切相关。组蛋白乙酰化水平的异常在白血病的发展、增殖和分化中起着很重要的作用。随着对组蛋白乙酰化的深入研究,组蛋白去乙酰化酶抑制剂在白血病中的作用机制越来越受到人们的关注。