组蛋白脱乙酰化酶抑制剂在治疗白血病中的研究进展

组蛋白的乙酰化和脱乙酰化在调节基因表达方面起着重要作用,与肿瘤的发生密切相关。具有组蛋乙酰基转移酶(HATs)活性的蛋白均为转录因子活性相关蛋白;组蛋白脱乙酰化酶(HDACs)是转录因子辅助抑制复合物的关键成分。目前发现的HDAc抑制剂结合HDAC催化袋状结构的锌离子,其他部位阻断活性位点的通道而发挥作用;体外培养多种白血病细胞系细胞导致不同程度的分化、凋亡和细胞周期阻滞。体内能抑制荷瘤动物肿瘤生长。     

组蛋白去乙酰化酶9在肿瘤中的作用

组蛋白去乙酰化酶9（histone deacetylase 9,HDAC9）属于组蛋白去乙酰化酶（histone deacetylases,HDACs）Ⅱa亚型,在体内通过催化组蛋白3（H3）、组蛋白4（H4）和非组蛋白的去乙酰化,改变染色体的结构,调节基因的转录。研究表明HDAC9表达异常与肿瘤关系密切,但不同肿瘤中HDAC9的表达和功能不同,最终造成促癌或抑癌的相反结果,具体机制尚不明确。当前利用组蛋白去乙酰化酶抑制剂（histone deacetylases inhibitors,HDACIs）的表观遗传治疗已成为热点,研制特异性HDACIs并与化疗、放疗以及免疫治疗相结合已成为未来的方向,但靶向针对HDAC9的治疗研究非常有限。本文就HDAC9在肿瘤中的作用进行综述。      

组蛋白去乙酰化酶及其抑制剂在骨肉瘤分子机制中的作用研究进展

骨肉瘤 ( osteosarcoma,OS ) 起源于间叶细胞,是一种最常见于 10～30 岁青少年的原发恶性骨肿瘤,多见于长骨干骺端,具有侵袭性强且易转移等特点 [1-4].目前主要的治疗包括手术结合新辅助化疗,但是存在耐药、转移、复发等问题 [5-6].已发生肺部等远处转移的骨肉瘤患者几乎不可治愈,5 年总体生存率...     

组蛋白去乙酰化酶抑制剂在卵巢癌中的研究进展

摘 要：研究发现在基因的核苷酸序列不发生改变的情况下,基因表达发生的可遗传变化即表观遗传改变与卵巢癌的发生,发展密切相关。近年来,组蛋白去乙酰化酶抑制剂因主要抑制组蛋白去乙酰化酶功能,影响表观遗传改变,在卵巢癌细胞中具有抑制肿瘤生长、分化、促进癌细胞凋亡等作用备受关注。全文就组蛋白去乙酰化酶抑制剂在卵巢癌中的研究进展作一综述。     

急性白血病组蛋白乙酰化修饰及其对错配修复基因表达的调控作用

 目的　探讨急性白血病患者组蛋白乙酰化修饰规律,并探索组蛋白乙酰化对错配修复基因hMSH2 和hMLH1差异表达的调控作用。方法　用反转录-聚合酶链反应（RT-PCR）方法检测56例急性白血病患者和30名健康志愿者单个核细胞（MNC）的错配修复基因hMSH2 和hMLH1 mRNA的表达,用Western blot法检测组蛋白H3、H4、去乙酰化酶（HDAC1）、hMSH2 和hMLH1基因的蛋白表达情况。用组蛋白去乙酰转移酶抑制剂（TSA）诱导30例白血病患者MNC乙酰化,并检测处理后MNC的组蛋白H3、H4、HDAC1、hMSH2 和hMLH1的表达状态变化。结果　急性白血病组的hMSH2 和hMLH1、组蛋白H3、H4的蛋白表达量分别为0.4610±0.1211、0.4013±0.1143、0.4103±0.1241和0.4251±0.1081,均明显低于健康志愿者组的蛋白表达量（0.9461±0.1841、0.9960±0.2021、0.8971±0.1194、0.9513±0.1953）,差异均有统计学意义（t值分别为3.341、3.935、2.843、3.575,P＜0.05）;而急性白血病患者组的HDAC1表达（0.8841±0.2018）高于健康志愿者组的表达量（0.5142±0.1340）,差异有统计学意义（t＝2.634,P＜0.05）;TSA作用于白血病单个核细胞后,组蛋白H3、H4、hMSH2 和hMLH1的表达上调,分别比阴性对照组表达上调2.9倍、3.4倍、1.5倍和1.6倍,而HDAC1的蛋白表达出现明显的抑制,表达下调为阴性对照组的40 %。结论　急性白血病患者的组蛋白乙酰化呈低表达现象,组蛋白乙酰化在急性白血病患者中对错配修复基因差异表达具有调控作用。     

组蛋白去乙酰化酶抑制剂在非霍奇金淋巴瘤中的研究进展

组蛋白去乙酰化酶（HDAC）是一种可以调控基因表达、修饰染色体结构的蛋白酶。在维持细胞的正常功能和调控基因表达的过程中,组蛋白乙酰化和去乙酰化水平的平衡起着重要作用。 HDAC过度表达并被转录因子募集,抑制某些基因的表达,导致肿瘤和其他疾病的发生和发展。近年来,随着对肿瘤表观遗传学研究的深入,HDAC 抑制剂在肿瘤发生、发展中的作用越来越引起研究者的重视。 HDAC抑制剂具有广泛的抗肿瘤活性,尤其在恶性血液病的治疗中,临床应用日益广泛。目前已有几种 HDAC抑制剂被用于非霍奇金淋巴瘤的治疗和临床研究,如伏立诺他、贝利司他、西达本胺、CUDC-907等。文章就以上 HDAC 抑制剂针对 NHL 的临床研究进展进行阐述。     

组蛋白去乙酰化酶2在肝细胞癌组织中的表达及其临床意义

目的 探讨组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)在肝细胞癌组织中的表达及其与预后的关系.方法 采用Western blot法和免疫组化检测肝细胞癌组织及对应癌旁组织中HDAC2蛋白的表达情况,并分析其与患者临床病理学参数及预后的关系.结果 HDAC2蛋白在肝细胞癌组织中的表达明显高...     

组蛋白去乙酰化酶抑制剂对胶质瘤细胞增殖及MKK7表达的影响

目的 探讨组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor, HDACI）对胶质瘤细胞增殖及MKK7表达的影响。方法 体外培养神经胶质瘤细胞株U251,用脂质体转染MKK7-siRNA1、MKK7-siRNA2和对照siRNA,48 h后Western blot检测MKK7表达及JNK/c-Jun活性,BrdU掺入实验检测细胞增殖情况;用0.5 μM组蛋白去乙酰化酶抑制剂曲古菌素A（trichostatin A, TSA）处理细胞8 h,DMSO作对照,Western blot检测MKK7、p-JNK、JNK、p-c-Jun、c-Jun表达或磷酸化水平;用TSA分别处理细胞8、12、24 h,检测各时间点细胞增殖率;用其他组蛋白去乙酰化酶抑制剂包括伏立诺他（suberoylanilide hydroxamic acid, SAHA）,丙戊酸（valproic acid, VPA）,M344处理细胞检测MKK7表达及细胞增殖。结果 同对照组相比,沉默MKK7表达抑制了JNK/c-Jun活性和细胞增殖（P=0.008）;TSA处理细胞8 h显著抑制MKK7表达及JNK/c-Jun活性,SAHA、M344、VPA均显著抑制MKK7表达。TSA处理细胞8 h没有抑制细胞增殖,处理12 h显著抑制增殖（P=0.006）, 24 h抑制效应更明显（P=0.002）;SAHA（P=0.001）, M344（P=0.008）或VPA（P=0.002）处理细胞12 h均抑制了细胞增殖。结论 组蛋白去乙酰化酶抑制剂可通过抑制MKK7表达抑制神经胶质瘤细胞增殖。     

组蛋白去乙酰化酶4在人肝癌细胞Bel7402中的表达及其意义

目的：研究组蛋白去乙酰化酶4（histone deacetyase4,HDAC4）在人肝癌细胞株Bel-7402中的表达,及其对Bel-7402细胞增殖和分化的调控作用。方法：培养Bel-7402细胞,以组蛋白去乙酰化酶抑制剂苯丁酸钠（sodium phenylbutyrate,SPB）作用于Bel-7402,RT—PCR检测用药前后HDAC4 mRNA的表达水平,倒置相差显微镜观察细胞形态改变,MTT比色法观察SPB对Bel-7402生长的抑制作用,流式细胞术分析细胞周期,免疫组化法观察Bel-7402细胞P27蛋白的表达水平。结果：SPB处理后Bel-7402中HDAC4mRNA的表达水平降低（0．88±0．13,0．12±0．04,P〈0．05）;SPB处理后Bel-7402细胞生长受到明显抑制,且与SPB剂量和作用时间相关;同时细胞形态出现成纤维细胞样改变;细胞周期阻滞于G。期[（50．6±4．0）％,（78．8±3．6）％,P〈0．05）];P27蛋白表达水平增强（23±11,61±7,P〈0．05）。结论：SPB降低HDAC4在人肝癌细胞中的表达,从而诱导部分人肝癌细胞分化,该作用与P27蛋白水平变化有关。     

组蛋白去乙酰化酶6在非小细胞肺癌组织中的表达及意义

目的 探讨组蛋白去乙酰化酶6 (HDAC6)在非小细胞肺癌(NSCLC)组织中的表达及其临床意义.方法 应用免疫组织化学SP法检测HDAC6在70例NSCLC组织和18例正常肺组织中的表达.结果 在NSCLC组织中HDAC6阳性率为58.57％,显著高于正常肺组织的16.67％ (P ＜0.01);NSCLC组织中HD...     

Expression of p53 in human leukemic cell lines

The cell-encoded p53 antigen seems to be tightly associated with various human malignancies. We have analyzed biochemical properties of p53 in two different cell lines derived from patients with ALL or ANLL. p53 was found in elevated levels in both leukemic cell lines compared to unstimulated or stimulated normal lymphocytes. High levels of p53 in these cell lines are due to an extended stability of p53 protein rather than to different rates of synthesis. p53 from both cell lines formed low- and high-molecular weight oligomers which revealed that p53 exists in a heterogenous population in these tumor cells. The presence of immunologically different subsets of p53 was demonstrated by sequential immunoprecipitation experiments with different p53 specific monoclonal antibodies. Our results showed structural and immunological variabilities of p53 in cell lines derived from human tumors and may thus provide an insight into the role p53 may play in human malignancies.     

Microarray profiling of the effects of histone deacetylase inhibitors on gene expression in cancer cell lines

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.     

The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines

We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, ?9, ?3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.     

去甲基化和组蛋白去乙酰化转移酶抑制剂对Ishikawa和AN3细胞增殖和凋亡的影响

目的：探讨去甲基化制剂5-氮-2′-脱氧胞苷（5-Aza-CdR or DAC）和组蛋白去乙酰化酶抑制剂曲古抑菌素A（trichostatin A,TSA）对人子宫内膜腺癌Ishikawa和AN3细胞增殖和凋亡的影响.方法：台盼蓝拒染法观察药物对细胞生长曲线的影响;Annexin Ⅴ染色法检测细胞凋亡;流式细胞仪分析细胞周期; Western blot检测凋亡信号通路中Caspase-8 、Caspase-9活化及多聚ADP核糖聚合酶（PARP）裂解情况.结果：DAC和 TSA对Ishikawa和AN3细胞均有时间依赖性的抑制作用,联用后抑制作用更强.单用DAC或TSA均可诱导细胞凋亡.单用DAC或TSA对细胞G1期无影响,单用TSA使G2/M期细胞比例增高,联合用药发生明显的G1期阻滞.Western blot检测表明,DAC和 TSA诱导了Caspase-8、Caspase-9裂解活化及PARP裂解.结论：DAC与TSA协同可抑制细胞生长,并使细胞阻滞在G1期,且细胞凋亡发生的时相不同于单独用药组.     

In vitro study of CI-994, a histone deacetylase inhibitor, in non-small cell lung cancer cell lines

CI-994 (N-acetyldinaline) is a novel oral compound with a wide spectrum of antitumor activity in preclinical models, in vitro and in vivo. The mechanism of action may involve inhibition of histone deacetylation and cell cycle arrest. We studied the action of CI-994 on two non-small cell lung cancer (NSCLC) cell lines: A-549 (adenocarcinoma) and LX-1 (squamous cell carcinoma). Different drug concentrations were tested, ranging from 0.01 to 160 microM at 24, 48, and 72 h of treatment, with MTT assay. A concentration-dependent cell survival inhibition was observed, with an IC50 at 80 microM. The effect of CI-994, as demonstrated by recovery experiments, was cytostatic and seemed to be superimposable in both cell lines. Cytofluorimetric analysis to assess cell cycle perturbation and apoptosis was performed after 24 h of treatment, indicating a cell block with concomitant increase at G0/G1 phase, a reduction at S phase level at 20, 40, 80, and 160 microM, and apoptosis at the higher concentration (160 microM). When CI-994 was combined with antineoplastic agents commonly used in NSCLC management, a marked synergism of action (R = 1.8, R = 1.5) was observed between CI-994 (40 microM) and gemcitabine (0.01 microM) at 48 and 72 h of treatment. The same result was obtained with docetaxel (0.001 microM) combination (R = 1.4, R = 1.2), but no synergism of action was noted with paclitaxel. CI-994 showed no radiopotentiating effects, when combined with 100, 200, or 400 cGy irradiation. In conclusion, our experiments indicate that CI-994 is a promising novel cytostatic for the treatment of NSCLC. Its use in combination with standard anticancer agents, such as gemcitabine and docetaxel, is warranted.     

Induction of differentiation and apoptosis in leukaemic cell lines by the novel benzamide family histone deacetylase 2 and 3 inhibitor MI-192

Histone deacetylase inhibitors (HDACIs) are in advanced clinical development as cancer therapeutic agents. However, first generation HDACIs such as butyrate and valproate are simple short chain aliphatic compounds with moieties resembling acetyl groups, and have a broad spectrum of activity against HDACs. More complex second generation HDACIs undergoing clinical trials, such as the benzamide group compounds MS-275 and MGCD0103, are specific primarily for HDAC1 and HDAC2. To expand the repertoire of available HDACIs and HDAC specificities we created a novel benzamide-based compound named MI-192. When tested against purified recombinant HDACs, MI-192 had marked selectivity for the class I enzymes, HDAC2 and HDAC3. Screening in the NCI60 screen demonstrated that MI-192 had greatly enhanced efficacy against cells of leukaemic origin. When tested in culture against the acute myeloid leukaemic cell lines U937, HL60 and Kasumi-1, MI-192 induced differentiation and was cytotoxic through promotion of apoptosis. MI-192 therefore justifies further investigation and development as a potential therapeutic agent for use in leukaemia.