Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies

Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC(50) potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of alpha-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer.     

CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo

CRA-024781 is a novel, broad spectrum hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor activity in vitro and in vivo preclinically and is under evaluation in phase I clinical trials for cancer. CRA-024781 inhibited pure recombinant HDAC1 with a K(i) of 0.007 mumol/L, and also inhibited the other HDAC isozymes HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-024781 resulted in the accumulation of acetylated histone and acetylated tubulin, resulting in an inhibition of tumor cell growth and the induction of apoptosis. CRA-024781 parenterally administered to mice harboring HCT116 or DLD-1 colon tumor xenografts resulted in a statistically significant reduction in tumor growth at doses that were well tolerated as measured by body weight. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells, and an alteration in the expression of many genes in the tumors, including several involved in apoptosis and cell growth. These results reveal CRA-024781 to be a novel HDAC inhibitor with potent antitumor activity.     

CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo

CRA-026440 is a novel, broad-spectrum, hydroxamic acid-based inhibitor of histone deacetylase (HDAC) that shows antitumor and antiangiogenic activities in vitro and in vivo preclinically. CRA-026440 inhibited pure recombinant isozymes HDAC1, HDAC2, HDAC3/SMRT, HDAC6, HDAC8, and HDAC10 in the nanomolar range. Treatment of cultured tumor cell lines grown in vitro with CRA-026440 resulted in the accumulation of acetylated histone and acetylated tubulin, leading to an inhibition of tumor cell growth and the induction of apoptosis. CRA-026440 inhibited ex vivo angiogenesis in a dose-dependent manner. CRA-026440 parenterally given to mice harboring HCT116 or U937 human tumor xenografts resulted in a statistically significant reduction in tumor growth. CRA-026440, when used in combination with Avastin, achieved greater preclinical efficacy in HCT 116 colorectal tumor model. Inhibition of tumor growth was accompanied by an increase in the acetylation of alpha-tubulin in peripheral blood mononuclear cells and an alteration in the expression of many genes in the tumors, including several involved in angiogenesis, apoptosis, and cell growth. These results reveal CRA-026440 to be a novel HDAC inhibitor with potent antitumor activity.     

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity in vitro and in vivo

Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.     

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor, exerts antimyeloma effects by DNA damage and mitochondrial signaling

Histone deacetylase inhibitors have emerged as promising anticancer drugs. Using an unbiased ultrahigh throughput screening system, a novel mercaptoketone-based histone deacetylase inhibitor series was identified that was optimized to the lead compound, KD5170. KD5170 inhibited the proliferation of myeloma cell lines and the viability of CD138(+) primary myeloma cells by induction of apoptosis, accompanied by an increase of acetylation of histones and activation of caspase-3, caspase-8, and caspase-9. Treatment with KD5170 caused a loss of mitochondrial membrane potential resulting in release of apoptogenic factors such as cytochrome c, Smac, and apoptosis-inducing factor. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells as evidenced by the up-regulation of heme oxygenase-1 and H2A.X phosphorylation. Combination of KD5170 with proteasome inhibitor bortezomib or tumor necrosis factor-related apoptosis-inducing ligand synergistically enhanced the antimyeloma activity. We further found that resistance of myeloma cells to KD5170 was associated with activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway under treatment with KD5170. Pretreatment with the mitogen-activated protein kinase inhibitor U0126 restored sensitivity to KD5170, suggesting that the combination of KD5170 with U0126 could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited and survival was prolonged. Histone acetylation was increased in spleen and tumor tissues of animals treated with KD5170. Our data indicate that KD5170 has potent antimyeloma activity in vitro and in vivo, which is mediated by DNA damage and mitochondrial signaling and subsequent induction of apoptosis.     

Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.     

Induction of CYP1A1 in tumor cells by the antitumor agent 2-[4-amino-3-methylphenyl]-5-fluoro-benzothiazole: a potential surrogate marker for patient sensitivity

A candidate antitumor agent, 2-(4-amino-3-methylphenyl)-5-fluoro-benzothiazole (5F-203), like its non-fluorinated parent compound (DF-203), has a unique cytotoxicity pattern in the National Cancer Institute in vitro anticancer drug screen. These compounds show selective toxicity for a subset of cell types including estrogen receptor positive breast cancer and certain renal and ovarian cancer cell lines. Metabolic activation of these benzothiazoles seems to be mediated through the CYP1 family of cytochrome P450s. In an effort to characterize the involvement of CYP1A1 and CYP1B1 in the unique toxicity response of 5F-203, constitutive and 5F-203-induced gene expression patterns were measured in 60 cell lines of the National Cancer Institute drug screen using TaqMan real-time PCR. The patterns of CYP1A1 and CYP1B1 gene expression in the 60 cell lines were correlated with the toxicity pattern of 5F-203 and DF-203. There was significant correlation between drug sensitivity and induced CYP1A1 (R = 0.752, P < 0.001), but not constitutive CYP1A1 mRNA expression. CYP1A1 protein expression was found to mirror the corresponding gene expression, indicating that gene expression changes were concordant with function. Treatment of sensitive cell lines with 10 micro M resveratrol, an inhibitor of CYP1A1 induction, in combination with either 1 or 10 micro M 5F-203 showed an ablation of the observed CYP1A1, but not CYP1B1 mRNA induction in parallel with a decreased sensitivity to 5F-203. Fine needle aspirates were obtained from a variety of human tumor xenografts, and treated ex vivo with 1 micro M 5F-203 for 24 h. In these samples, induction of CYP1A1 by 5F-203 correlated with in vitro sensitivity (R = 0.711, P < 0.05), and corresponded to in vivo sensitivity in human tumor xenografts. These data are concordant with the idea that toxicity of 5F-203 requires activation by CYP1A1, and therefore induction of CYP1A1 mRNA in response to 5F-203 treatments ex vivo may provide a possible surrogate marker for determination of drug-sensitive tumors in patients.     

Induction of apoptosis in renal tubular cells by histone deacetylase inhibitors, a family of anticancer agents

Inhibitors of histone deacetylases, including suberoylanilide hydroxamic acid (SAHA) and Trichostatin A, are a new class of anticancer agents. With potent chemotherapy effects in cancers, these agents are not obviously toxic in normal nonmalignant cells or tissues. However, their toxicity in kidney cells has not been carefully evaluated. Here, we demonstrate a potent apoptosis-inducing activity of SAHA in cultured renal proximal tubular cells. SAHA induces apoptosis at low micromolar concentrations. At 5 muM, SAHA induces 30 to approximately 40% apoptosis in 18 h. The apoptosis is accompanied by notable caspase activation; however, the general caspase inhibitor VAD can only partially suppress SAHA-induced apoptosis, suggesting the involvement of both caspase-dependent and -independent mechanisms. SAHA treatment leads to cytochrome c release from mitochondria, which is suppressed by Bcl-2 but not by VAD. Bcl-2 consistently blocks SAHA-induced apoptosis. During SAHA treatment, Bcl-2 and Bcl-XL decrease, and Bid is proteolytically cleaved, whereas Bax and Bak expression remains constant. Bid cleavage, but not Bcl-2/Bcl-XL decrease, is completely suppressed by VAD. SAHA does not activate p53, and pifithrin-alpha (a pharmacological p53 inhibitor) does not attenuate SAHA-induced apoptosis, negating a role of p53 in SAHA-induced apoptosis. SAHA induces histone acetylation, which is not affected by VAD, Bcl-2, or pifithrin-alpha. Trichostatin A can also induce apoptosis and histone acetylation in renal tubular cells. Together, the results have shown evidence for renal toxicity of histone deacetylase inhibitors. The toxicity may be related to protein acetylation and decrease of antiapoptotic proteins including Bcl-2 and Bcl-XL.     

In vivo synergy between topoisomerase II and histone deacetylase inhibitors: predictive correlates

Histone deacetylase inhibitors (HDACi) are a promising class of anticancer agents, yet the specific biological effects resulting in cell death are still poorly understood and clinically relevant markers of response are not adequately defined. The anticonvulsant valproic acid has recently emerged as an HDACi, and in vitro studies suggested that valproic acid may potentiate cytotoxic agents. We evaluated the pharmacologic and biological effects of valproic acid on histone acetylation, chromatin structure, and DNA damage induced by topoisomerase II inhibitors in mice bearing breast cancer tumors and developed an ex vivo methodology for response prediction using comet assays. The exposure of mice to valproic acid before exposure to epirubicin led to tumor regression when valproic acid was given for 48 hours at concentrations sufficient for histone hyperacetylation, down-regulation of heterochromatin maintenance proteins, and chromatin decondensation. Tumor response was accurately predicted by ex vivo comet moments. Valproic acid did not exacerbate epirubicin-related toxicity. Antitumor effects were not observed with valproic acid alone despite biologically active valproic acid concentrations. These findings suggest that exposure of tumor-bearing mice to valproic acid potentiated the antitumor effects of topoisomerase II inhibitors without enhancing toxicity. The HDACi-induced histone acetylation and modulation of heterochromatin correlated with potentiation of epirubicin-mediated DNA damage. However, these effects did not result in antitumor activity when using a HDACi alone and hence should not be considered a surrogate marker. Ex vivo comet assays may be useful as a predictive tool when tumor cells are limited and serial biopsies are difficult to obtain.     

西达本胺对人B淋巴瘤细胞株的作用及其机制研究

本研究旨在探讨组蛋白去乙酰化酶抑制剂(HDACI)西达本胺对3种B淋巴瘤细胞株Raji(Burkitt淋巴瘤)、Maver及Z-138(套细胞淋巴瘤)的抑制增殖和诱导凋亡的作用及其机制。以不同浓度的西达本胺及不同时间作用于体外培养的3种B淋巴瘤细胞,采用CCK-8法检测细胞的增殖;流式细胞术检测细胞的凋亡及线粒体膜电位;Western blot方法检测细胞内组蛋白H3、H4乙酰化水平及caspase-3蛋白的表达。结果表明,西达本胺可抑制这3种B淋巴瘤细胞的增殖,其抑制作用具有一定的时间和浓度依赖性,尤其能较早较快地抑制Z-138细胞的增殖;另外,西达本胺还可诱导3种B淋巴瘤细胞发生凋亡并引起细胞线粒体膜电位下降,Maver及Z-138细胞对西达本胺较Raji细胞更为敏感;西达本胺可以提高细胞内组蛋白H3、H4乙酰化水平及Maver和Z-138细胞的caspase-3活性。结论:西达本胺能够抑制B淋巴瘤细胞株的增殖,诱导细胞凋亡,其机制可能与西达本胺上调组蛋白H3、H4乙酰化水平,触发线粒体凋亡途径及活化caspase-3有关。     

MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo

Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotype-selective HDAC inhibitor. We have developed an isotype-selective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted.     

Angiostatic activity of DNA methyltransferase inhibitors

Inhibitors of DNA methyltransferases (DNMT) and histone deacetylases can reactivate epigenetically silenced tumor suppressor genes and thereby decrease tumor cell growth. Little, however, is known on the effects of these compounds in endothelial cell biology and tumor angiogenesis. Here, we show that the DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine markedly decrease vessel formation in different tumor models. We show that DNMT inhibitors are antiproliferative for tumor-conditioned endothelial cells, without affecting endothelial cell apoptosis and migration. Furthermore, these compounds inhibit angiogenesis in vitro and in vivo as shown by inhibition of endothelial cells sprouting in a three-dimensional gel and inhibition of microvessel formation in the chorioallantoic membrane, respectively. 5-Aza-2'-deoxycytidine, as well as the histone deacetylase inhibitor trichostatin A, reactivates the growth-inhibiting genes TSP1, JUNB, and IGFBP3, which are suppressed in tumor-conditioned endothelial cells. Despite enhanced DNMT activity and increased overall genomic methylation levels in tumor-conditioned endothelial cells, silencing of these genes seemed not to be regulated by direct promoter hypermethylation. For IGFBP3, gene expression in endothelial cells correlated with histone H3 acetylation patterns. In conclusion, our data show that DNMT inhibitors have angiostatic activity in addition to their inhibitory effects on tumor cells. This dual action of these compounds makes them promising anticancer therapeutics.     

Histone deacetylase inhibitors induced caspase-independent apoptosis in human pancreatic adenocarcinoma cell lines

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor-induced apoptosis is due to a serine protease-dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.     

姜黄素调节NB4细胞P53和组蛋白H3乙酰化抑制细胞增殖的研究

目的 研究姜黄素对NB4细胞组蛋白H3和非组蛋白P53的乙酰化和细胞增殖的作用，探讨姜黄素抗白血病机制。方法 应用小同浓度（50，25，12．5，6．25，3．125μmol／L）的姜黄素作用NB4细胞不同时间（0，4，8，12，24h），MTT法测定姜黄素对NB4细胞增殖的影响，Western blot法检测乙酰化组蛋白H3和乙酰化P53的水平。结果 姜黄素以时间和剂量依赖方式抑制NB4细胞增殖，在24h和36h的IC值分别为40μmol／L和25μmol／L；能明显上调组蛋白H3的乙酰化水平，促进P53的表达和P53的乙酰化。结论 姜黄素具有去乙酰化酶抑制剂作用，能上调组蛋白H3乙酰化水平，促进肿瘤抑制凶子P53表达和活化，抑制白血病细胞增殖。     

The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines

The differentiating agent and histone deacetylase inhibitor, sodium butyrate (NaB), was shown previously to cause a transient, 3-17-fold induction of human DNA topoisomerase II alpha (topo II alpha) gene promoter activity and a 2-fold increase in topo II alpha protein early in monocytic differentiation of HL-60 cells. This observation has now been extended to other short chain fatty acids and aromatic butyrate analogues, and evidence is presented that human topo II alpha promoter induction correlates closely with histone H4 acetylation status. Because increased topo II alpha expression is associated with enhanced efficacy of topo II-poisoning antitumor drugs such as etoposide, the hypothesis tested in this report was whether NaB pretreatment could sensitize HL-60 myeloid leukemia and K562 erythroleukemia cells to etoposide-triggered DNA damage and cell death. A 24-72 h NaB treatment (0.4-0.5 mM) induced topo II alpha 2-2.5-fold in both HL-60 and K562 cells and caused a dose-dependent enhancement of etoposidestimulated, protein-linked DNA complexes in both cell lines. At concentrations with minimal effects on cell cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB pretreatment also modestly enhanced etoposidetriggered apoptosis in HL-60 cells, as determined morphologically after acridine orange/ethidium bromide staining, and substantially increased K562 growth inhibition and poly(ADP-ribose)polymerase cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a cytotoxic antitumor agent. These results indicate that histone deacetylase inhibitors should be investigated for etoposide sensitization of other butyrate-responsive hematopoietic and nonhematopoietic tumor lines in vitro and in vivo.