Development of a histone deacetylase 6 inhibitor and its biological effects

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.Histone deacetylase 6 (HDAC6) is unique among the eleven zinc-dependent HDACs in humans. HDAC6 is located in the cytoplasm, and it has two catalytic domains and an ubiquitin-binding domain at the C-terminal region (1–3). This study focused on the development of a HDAC6-selective inhibitor and its biological effects. The substrates of HDAC6 include nonhistone proteins such as α-tubulin, peroxiredoxin (PRX), cortactin, and heat shock protein 90 (Hsp90) but not histones (4–7). HDAC6 plays a key role in the regulation of microtubule dynamics including cell migration and cell–cell interactions. The reversible acetylation of Hsp90, a substrate of HDAC6, modulates its chaperone activity and, accordingly, the stability of survival and antiapoptotic factors, including epidermal growth factor receptor (EGFR), protein kinase AKT, proto-oncogene C-RAF, survivin, and other factors. HDAC6, through its ubiquitin-binding activity and interaction with other partner proteins, plays a role in the degradation of misfolded proteins by binding polyubiquitinated proteins and delivering them to the dynein and motor proteins for transport into aggresomes which are degraded by lysosomes (8–10). Thus, HDAC6 has multiple biological functions through deacetylase-dependent and -independent mechanisms modulating many cellular pathways relevant to normal and tumor cell growth, migration, and death. HDAC6 is an attractive target for potential cancer treatment.There are several previous reports on the development of HDAC6-selective inhibitors (11–15). The most extensively studied is tubacin (16, 17). Tubacin has non–drug-like qualities, high lipophilicity, and difficult synthesis and has proved to be more useful as a research tool rather than as a potential drug (18). We and others (12–15, 19) have developed HDAC6-selective inhibitors whose pharmacokinetics, toxicity, and efficacy make them potentially more useful than tubacin as therapeutic agents. ACY-1215, 2-(Diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide, a HDAC6-selective inhibitor, is currently being evaluated in clinical trials (http://clinicaltrials.gov).HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), consist of three structural domains: a metal-binding domain, a linker domain, and a surface domain (20). The catalytic pocket of HDAC1 is deeper and narrower than the catalytic pocket of HDAC6 (14). To develop HDAC6-selective inhibitors, we synthesized small molecules with bulkier and shorter linker domains than the pan-HDAC inhibitor SAHA (20, 21). A hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) was synthesized that selectively inhibits HDAC6. We report the effects of this HDAC6-selective inhibitor on normal and transformed cells. Further, we found that selective inhibition of HDAC6 increases the effectiveness of anticancer agents, etoposide, doxorubicin, and SAHA in inducing cell death of transformed cells but not normal cells.     

The anti-histaminic cyproheptadine synergizes the antineoplastic activity of bortezomib in mantle cell lymphoma through its effects as a histone deacetylase inhibitor

Cyproheptadine, an inhibitor of the H1 histamine receptors, has recently shown activity in models of leukaemia and myeloma, presumably through inhibition of cyclin-D expression. Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphoma characterized by overexpression of cyclin-D1. We investigated the effect of cyproheptadine alone and in combination with the proteasome inhibitor bortezomib in models of MCL. The combination of these drugs was mathematically synergistic, producing significant reductions in the mitochondrial membrane potential leading to apoptosis. In a severe combined immunodeficient beige mouse model, cyproheptadine plus bortezomib demonstrated a statistically significant advantage compared to either agent alone.     

Genetic dissection of histone deacetylase requirement in tumor cells

Histone deacetylase inhibitors (HDACi) represent a new group of drugs currently being tested in a wide variety of clinical applications. They are especially effective in preclinical models of cancer where they show antiproliferative action in many different types of cancer cells. Recently, the first HDACi was approved for the treatment of cutaneous T cell lymphomas. Most HDACi currently in clinical development act by unspecifically interfering with the enzymatic activity of all class I HDACs (HDAC1, 2, 3, and 8), and it is widely believed that the development of isoform-specific HDACi could lead to better therapeutic efficacy. The contribution of the individual class I HDACs to different disease states, however, has so far not been fully elucidated. Here, we use a genetic approach to dissect the involvement of the different class I HDACs in tumor cells. We show that deletion of a single HDAC is not sufficient to induce cell death, but that HDAC1 and 2 play redundant and essential roles in tumor cell survival. Their deletion leads to nuclear bridging, nuclear fragmentation, and mitotic catastrophe, mirroring the effects of HDACi on cancer cells. These findings suggest that pharmacological inhibition of HDAC1 and 2 may be sufficient for anticancer activity, providing an experimental framework for the development of isoform-specific HDAC inhibitors.     

Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation

Protein acetylation, especially histone acetylation, is the subject of both research and clinical investigation. At least four small-molecule histone deacetylase inhibitors are currently in clinical trials for the treatment of cancer. These and other inhibitors also affect microtubule acetylation. A multidimensional, chemical genetic screen of 7,392 small molecules was used to discover "tubacin," which inhibits alpha-tubulin deacetylation in mammalian cells. Tubacin does not affect the level of histone acetylation, gene-expression patterns, or cell-cycle progression. We provide evidence that class II histone deacetylase 6 (HDAC6) is the intracellular target of tubacin. Only one of the two catalytic domains of HDAC6 possesses tubulin deacetylase activity, and only this domain is bound by tubacin. Tubacin treatment did not affect the stability of microtubules but did decrease cell motility. HDAC6 overexpression disrupted the localization of p58, a protein that mediates binding of Golgi elements to microtubules. Our results highlight the role of alpha-tubulin acetylation in mediating the localization of microtubule-associated proteins. They also suggest that small molecules that selectively inhibit HDAC6-mediated alpha-tubulin deacetylation, a first example of which is tubacin, might have therapeutic applications as antimetastatic and antiangiogenic agents.     

Combined effects of histone deacetylase inhibitor and rituximab on non-Hodgkin's B-lymphoma cells apoptosis

OBJECTIVE: The anti-CD20 monoclonal antibody rituximab has shown promising results in the clinical treatment of patients with B-cell non-Hodgkin's lymphoma (B-NHL). However, its therapeutic effect could still be improved. METHODS: This study examined the anti-tumor activity of rituximab combined with histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in CD20-positive B-NHL cell lines, as well as in primary B-NHL cells and a murine B-NHL model. RESULTS: The combination treatment sensitized B-NHL cells to apoptosis in a synergistic manner, concomitant with mitochondrial instability and Bcl-2/Bcl-XL downregulation. Particularly in Daudi cells relatively resistant to rituximab, these events were associated with nuclear factor-kappaB (NF-kappaB) inactivation and c-Myc degradation. SAHA presented functional complementation with rituximab, through decreasing IKKalpha/beta and IkappaBalpha phosphorylation, thus preventing NF-kappaB nuclear translocation. In addition, SAHA induced IkappaBalpha cleavage to a stable inhibitory form and caused NF-kappaB degradation in response to caspase-3 activation. More importantly, rituximab-SAHA combination significantly promoted primary B-NHL cells apoptosis and improved survival time of a severe combined immunodeficient mouse lymphoma model established with intravenous injection of Daudi cells. CONCLUSION: These findings emphasized the value of targeting apoptosis signaling pathway in lymphoma therapy. Rituximab in conjunction with histone deacetylase inhibitor may represent a novel strategy in treating patients with B-NHL.     

组蛋白去乙酰化酶6的研究进展

本文介绍了组蛋白去乙酰化酶6的发现和发展,并详细叙述了它的分子结构及其与肿瘤发生、细胞迁移、蛋白质降解等相关的生物学功能。     

Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

AIM： To explore the effect of histone deacetylase inhibitor, trichostatin A （TSA） on the growth of biliary tract cancer cell lines （gallbladder carcinoma cell line and cholangiocarcinoma cell line） in vivo and in vitro, and to investigate the perspective of histone deacetylase inhibitor in its clinical application. METHODS： The survival rates of gallbladder carcinoma cell line （Mz-ChA-l cell line） and cholangiocarcinoma cell lines （QBC939, KMBC and OZ cell lines） treated with various doses of TSA were detected by methylthiazoy tetrazolium （MTT） assay. A nude mouse model of transplanted gallbladder carcinoma （Mz-ChA-l cell line） was successfully established, and changes in the growth of transplanted tumor after treated with TSA were measured. RESULTS： TSA could inhibit the proliferation of gallbladder carcinoma cell line （Mz-ChA-l cell line） and cholangiocarcinoma cell lines （QBC939, KMBC and OZ cell lines） in a dose-dependent manner. After the nude mouse model of transplanted gallbladder carcinoma （Mz- ChA-l cell line） was successfully established, the growth of cancer was inhibited in the model after treated with TSA. CONCLUSION： TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.     

组蛋白去乙酰酶抑制剂对结肠癌细胞增殖和ID4基因表达的影响

目的:研究组蛋白去乙酰酶抑制剂曲古菌素A (TSA)对结肠癌细胞Caco2增殖的影响和机制.方法:5-500μg/L TSA处理结肠癌Caco2细胞0-5d,在药物处理前及36h后用MTT法检测细胞增殖状况.流式细胞仪检测细胞周期的改变.RT-PCR检测ID4 mRNA的表达.结果:TSA在100μg/L浓度以上可以明显抑制结肠癌Caco2细胞的增殖(P<0.05),但在5和20μg/L浓度时抑制作用不明显.100μg/L TSA可导致细胞G_1期阻滞,但没有诱导明显的细胞凋亡.RT-PCR显示20μg/L TSA作用36 h后ID4 mRNA表达增强(P<0.05),在100μg/L其表达显著增强(P<0.01).结论:TSA可以通过阻滞Caco2细胞的G_1期和重新表达ID4来发挥抑癌作用.     

Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7

VEGF has been shown to regulate endothelial cell (EC) proliferation and migration. However, the nuclear mediators of the actions of VEGF in ECs have not been fully defined. We show that VEGF induces the phosphorylation of three conserved serine residues in histone deacetylase 7 (HDAC7) via protein kinase D, which promotes nuclear export of HDAC7 and activation of VEGF-responsive genes in ECs. Expression of a signal-resistant HDAC7 mutant protein in ECs inhibits proliferation and migration in response to VEGF. These results demonstrate that phosphorylation of HDAC7 serves as a molecular switch to mediate VEGF signaling and endothelial function.     

组蛋白脱乙酰基酶抑制剂曲古霉素对心肌细胞肥大的影响

目的利用体外培养的大鼠乳鼠心肌细胞,研究曲古霉素(TSA)对血管紧张素II(Ang II)诱导的心肌细胞肥大的影响,并探讨相关机制。方法分离乳鼠心肌细胞进行原代培养,应用血管紧张素Ⅱ制备心肌细胞肥大模型。分别给予不同浓度的TSA预处理心肌细胞,观察TSA对心肌细胞面积、3H亮氨酸掺入率、心房利钠肽(ANP)、脑利钠肽(BNP)的mRNA表达水平的影响。同时通过检测乙酰化组蛋白3的蛋白表达水平,观察AngⅡ和TSA对组蛋白脱乙酰基酶(HDAC)活性的影响。应用Western blot检测AngⅡ和TSA处理后磷酸化的c-Jun氨基末端激酶(JNK)表达水平的变化。结果10-6mmol/L AngⅡ作用48 h后,心肌细胞面积增加至对照组的(1.63±0.46)倍(P<0.01),而10-7mmol/L和3×10-7mmol/L TSA预处理可以剂量依赖性的抑制AngⅡ引起的心肌细胞面积增大。TSA干预也阻断了AngⅡ引起的蛋白合成速率增加以及ANP和BNP的蛋白表达水平的增加(均为P<0.05)。AngⅡ刺激心肌细胞使乙酰化组蛋白3的蛋白表达水平降低,TSA可逆转这一效应。同时TSA抑制了AngⅡ介导的磷酸化JNK表达水平的升高。结论 TSA可显著抑制AngⅡ诱导的心肌细胞肥大和HDAC活性增加,可能通过抑制JNK的激活而发挥抑制心室肥厚的作用。     

Therapeutic effects of histone deacetylase inhibitor givinostat on air inflammation and high airway resistance in a murine asthma model

正Objective To investigate the therapeutic effects of givinostat,a histone deacetylase inhibitor(HDACI),on the development of chronic asthma with airway inflammation,airway remodeling and airway hyperresponsiveness(AHR).Methods BALB/C mice were randomly divided into control group,asthma group,dexamethasone group     

Combination therapy of established cancer using a histone deacetylase inhibitor and a TRAIL receptor agonist

Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.     

Efficacy of a novel histone deacetylase inhibitor in murine models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. This study was aimed at assessing the antitumor effect of a novel phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, OSU-HDAC42, vis-à-vis suberoylanilide hydroxamic acid (SAHA), in in vitro and in vivo models of human HCC. OSU-HDAC42 was several times more potent than SAHA in suppressing the viability of PLC5, Huh7, and Hep3B cells with submicromolar median inhibitory concentration (IC(50)) values. With respect to SAHA, OSU-HDAC42 exhibited greater apoptogenic potency, which was associated with reduced levels of the apoptotic regulators phosphorylated Akt B-cell lymphoma-xL, survivin, cellular inhibitor of apoptosis protein 1, and cellular inhibitor of apoptosis protein 2. The in vivo efficacy of OSU-HDAC42 versus SAHA was assessed in orthotopic and subcutaneous xenograft tumor models in athymic nude mice. Daily oral treatments with OSU-HDAC42 and SAHA, both at 25 mg/kg, suppressed the growth of orthotopic PLC5 tumor xenografts by 91% and 66%, respectively, and of established subcutaneous PLC5 tumor xenografts by 85% and 56%, respectively. This differential tumor suppression correlated with the modulation of intratumoral biomarkers associated with HDAC inhibition and apoptosis regulation. Moreover, the oral administration of OSU-HDAC42 at 50 mg/kg every other day markedly suppressed ectopic tumor growth in mice bearing large tumor burdens (500 mm(3)) at the start of treatment. CONCLUSION: OSU-HDAC42 is a potent, orally bioavailable inhibitor of HDAC with a broad spectrum of antitumor activity that includes targets regulating multiple aspects of cancer cell survival. These results suggest that OSU-HDAC42 has clinical value in therapeutic strategies for HCC.     

Antileukemia activity of the combination of an anthracycline with a histone deacetylase inhibitor

We studied the cellular and molecular effects of the combination of an anthracycline with 2 different histone deacetylase inhibitors (HDACIs): vorinostat (suberoylanilide hydroxamic acid) and valproic acid (VPA). The 10% inhibitory concentration (IC(10)) of idarubicin was 0.5 nM in MOLT4 and 1.5 nM in HL60 cells. Concentrations above 0.675 microM of vorinostat resulted in at least 80% loss of cell viability in both cell lines. Concentrations of 1.5 to 3 mM of VPA induced 50% to 60% loss in viability in HL60 and 80% in MOLT4 cells. The combination of idarubicin with vorinostat at 0.075 microM or VPA at 0.25 mM resulted in at least an additive loss of cell viability in both lines. Vorinostat (0.35 microM) and VPA (0.25 mM) in combination with idarubicin (0.5 nM) resulted in a significant increase in apoptotic cells in MOLT4 cells. The combination resulted in an increase in histone H3 and H4 acetylation at 24 hours, phosphorylated H2AX, as well as in the induction of p21(CIP1) mRNA. No effect on cell cycle transition was observed. Of importance, the cellular and molecular effects observed were independent of the sequence used. In summary, the combination of an anthracycline with an HDACI should have significant clinical activity in patients with leukemia.     

Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against Bcr-Abl-expressing human leukemia cells

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) has potent in vitro and in vivo activity against the unmutated and most common mutant forms of Bcr-Abl. Treatment with the histone deacetylase inhibitor LBH589 (Novartis) depletes Bcr-Abl levels. We determined the effects of AMN107 and/or LBH589 in Bcr-Abl-expressing human K562 and LAMA-84 cells, as well as in primary chronic myelogenous leukemia (CML) cells. AMN107 was more potent than imatinib mesylate (IM) in inhibiting Bcr-Abl tyrosine kinase (TK) activity and attenuating p-STAT5, p-AKT, Bcl-x(L), and c-Myc levels in K562 and LAMA-84 cells. Cotreatment with LBH589 and AMN107 exerted synergistic apoptotic effects with more attenuation of p-STAT5, p-ERK1/2, c-Myc, and Bcl-x(L) and increases in p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of Bcr-Abl or the IM-resistant, point-mutant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis of IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared with either agent alone, cotreatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Thus, cotreatment with LBH589 and AMN107 is active against cultured or primary IM-resistant CML cells, including those with expression of Bcr-AblT315I.