ABSTRACT
Belinostat is a hydroxamate-type histone deactylase inhibitor (HDACi), which has recently entered phase I and II clinical trials. Microarray-based analysis of belinostat-treated cell lines showed an impact on genes associated with the G2/M phase of the cell cycle and downregulation of the aurora kinase pathway. Expression of 25 dysregulated genes was measured in eight differentially sensitive cell lines using a novel high-throughput assay that combines multiplex reverse transcriptase-PCR and fluorescence capillary electrophoresis. Sensitivity to belinostat and the magnitude of changes in overall gene modulation were significantly correlated. A belinostat-gene profile was specific for HDACi in three cell lines when compared with equipotent concentrations of four mechanistically different chemotherapeutic agents: 5-fluorouracil, cisplatin, paclitaxel, and thiotepa. Belinostat- and trichostatin A (HDACi)-induced gene responses were highly correlated with each other, but not with the limited changes in response to the other non-HDACi agents. Moreover, belinostat treatment of mice bearing human xenografts showed that the preponderance of selected genes were also modulated in vivo, more extensively in a drug-sensitive tumor than a more resistant model. We have demonstrated a gene signature that is selectively regulated by HDACi when compared with other clinical agents allowing us to distinguish HDACi responses from those related to other mechanisms.
Subject(s)
Enzyme Inhibitors/pharmacology , Gene Expression Profiling , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Xenograft Model Antitumor Assays , Animals , Antineoplastic Agents/pharmacology , Aurora Kinases , Cell Cycle/drug effects , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Chromosomal Proteins, Non-Histone/genetics , Down-Regulation/genetics , Enzyme Inhibitors/therapeutic use , Female , Gene Expression/drug effects , Gene Expression/genetics , Guanine Nucleotide Exchange Factors/genetics , HCT116 Cells , Humans , Hydroxamic Acids/therapeutic use , Male , Mice , Mice, Inbred Strains , Neoplasms/drug therapy , Neoplasms/metabolism , Protein Serine-Threonine Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Sulfonamides , Up-Regulation/genetics , ran GTP-Binding Protein/geneticsABSTRACT
Enzymatic inhibition of histone deacetylase (HDAC) activity is emerging as an innovative and effective approach for the treatment of cancer. A series of novel amide derivatives have been synthesized and evaluated for their ability to inhibit human HDACs. Multiple compounds were identified as potent HDAC inhibitors (HDACi), with IC(50) values in the low nanomolar (nM) range against enzyme activity in HeLa cell extracts and sub-microM for their in vitro anti-proliferative effect on cell lines. The introduction of an unsaturated linking group between the terminal aryl ring and the amide moiety was the key to obtain good potency. This approach yielded compounds such as (E)-N-[6-(hydroxyamino)-6-oxohexyl]-3-(7-quinolinyl)-2-propenamide (27) (HDAC IC(50) 8 nM) which showed potent in vivo activity in the P388 mouse leukemia syngeneic model (an increased lifespan (ILS) of 111% was obtained).
Subject(s)
Amides/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Amides/chemistry , Enzyme Inhibitors/chemistry , Humans , Magnetic Resonance Spectroscopy , Structure-Activity RelationshipABSTRACT
Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors.
Subject(s)
Antineoplastic Agents/therapeutic use , Histones/metabolism , Hydroxamic Acids/therapeutic use , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Acetylation/drug effects , Animals , Antibodies, Monoclonal , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Cell Line, Tumor , Female , HCT116 Cells , Histone Deacetylase Inhibitors , Histone Deacetylases/immunology , Histones/immunology , Humans , Hydroxamic Acids/blood , Hydroxamic Acids/pharmacokinetics , Leukemia P388/drug therapy , Leukemia P388/enzymology , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/enzymology , Male , Mice , Mice, Nude , Neoplasms, Experimental/enzymology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Sulfonamides , Transplantation, HeterologousABSTRACT
PURPOSE: Histone deacetylase inhibitors (HDACi) inhibit the growth of cancer cells, and combinations of HDACi with established chemotherapeutics can lead to synergistic effects. We have investigated effects of PXD101 (HDACi in phase II clinical trials) in combination with 5-fluorouracil, on tumour cell proliferation and apoptosis both in vitro and in vivo. EXPERIMENTAL DESIGN: HCT116 cells were studied using proliferation and clonogenic assays. Synergistic inhibition of proliferation and clonogenicity was determined by incubation with PXD101 and 5-fluorouracil, and analysis using CalcuSyn software. The effect of combining PXD101 and 5-fluorouracil on apoptosis was examined in vitro using PARP-cleavage and TUNEL. Finally, the effectiveness of combining PXD101 and 5-fluorouracil in vivo was tested using both HT-29 and HCT116 xenograft models. RESULTS: Synergistic inhibition of proliferation and clonogenicity was obtained when HCT116 cells were incubated with PXD101 and 5-fluorouracil. 5-fluorouracil combined with PXD101 also increased DNA fragmentation and PARP cleavage in HCT116 cells. Incubation with PXD101 down regulated thymidylate synthase expression in HCT116 cells. In vivo studies, using mouse HT29 and HCT116 xenograft models, showed improved reductions in tumour volume compared to single compound, when PXD101 and 5-fluorouracil were combined. CONCLUSIONS: PXD101 and 5-fluorouracil synergistically combine in their anti-tumour effects against colon cancer cells in vitro and show enhanced activity when combined in vivo. Based on the results presented herein, a rationale for the use of PXD101 and 5-fluorouracil in combination in the clinic has been demonstrated.
Subject(s)
Colorectal Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Fluorouracil/pharmacology , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Neoplasms, Experimental/drug therapy , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , Female , Humans , Mice , Neoplasms, Experimental/pathology , Sulfonamides , Tumor Stem Cell Assay/methods , Xenograft Model Antitumor AssaysABSTRACT
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.
