Energy metabolism in H460 lung cancer cells: effects of histone deacetylase inhibitors

Background

Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.

Methodology/Principal Findings

Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed that NaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O₂ consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged.

Conclusion

NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.     

Benzothiazole-containing hydroxamic acids as histone deacetylase inhibitors and antitumor agents

Data from clinical studies indicate that inhibitors of Class I and Class II histone deacetylase (HDAC) enzymes show great promise for the treatment of cancer. Zolinza (SAHA, Zolinza) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma. As a part of our ongoing effort to identify novel small molecules to target these important enzymes, we have prepared two series of benzothiazole-containing analogues of SAHA. It was found that several compounds with 6C-bridge linking benzothiazole moiety and hydroxamic functional groups showed good inhibition against HDAC3 and 4 at as low as 1 μg/ml and exhibited potent cytotoxicity against five cancer cell lines with average IC₅₀ values of as low as 0.81 μg/ml, almost equipotent to SAHA.     

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.     

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC₅₀ values 10–200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.     

Discovery of a series of small molecules as potent histone deacetylase inhibitors

Abstract

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC₅₀ of 0.14?μM and tumor cell growth inhibition IC₅₀ of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.     

Design of novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.     

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

Background

Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

Methods

HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity^® Pathway Analysis.

Results

Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

Conclusion

This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.     

Targeting inflammation in heart failure with histone deacetylase inhibitors

Cardiovascular insults such as myocardial infarction and chronic hypertension can trigger the heart to undergo a remodeling process characterized by myocyte hypertrophy, myocyte death and fibrosis, often resulting in impaired cardiac function and heart failure. Pathological cardiac remodeling is associated with inflammation, and therapeutic approaches targeting inflammatory cascades have shown promise in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors block adverse cardiac remodeling in animal models, suggesting unforeseen potential for this class of compounds for the treatment of heart failure. In addition to their beneficial effects on myocardial cells, HDAC inhibitors have potent antiinflammatory actions. This review highlights the roles of HDACs in the heart and the potential for using HDAC inhibitors as broad-based immunomodulators for the treatment of human heart failure.     

Synthesis and biological evaluation of histone deacetylase and DNA topoisomerase II-Targeted inhibitors

HDAC inhibitors enable histones to maintain a high degree of acetylation. The resulting looser state of chromatin DNA may increase the accessibility of DNA drug targets and consequently improve the efficiency of anticancer drugs targeting DNA, such as Topo II inhibitors. A novel class of nucleoside-SAHA derivatives has been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities toward histone deacetylases and Topo II, and their cytotoxicities in cancer cell lines, were evaluated. Among the synthesized hybrid compounds, compound 16b showed the potent HDAC inhibitory activity at a low nanomolar level and exhibited antiproliferative activity toward cancer cell lines including MCF-7 (breast), HCT-116 (colon), and DU-145 (prostate) cancer cells at a low micromolar level. Moreover, compound 16a showed HDAC6-selectivity 20-fold over HDAC1.     

Discovery of histone deacetylase 8 selective inhibitors

We have developed an efficient method for synthesizing candidate histone deacetylase (HDAC) inhibitors in 96-well plates, which are used directly in high-throughput screening. We selected building blocks having hydrazide, aldehyde and hydroxamic acid functionalities. The hydrazides were coupled with different aldehydes in DMSO. The resulting products have the previously identified ‘cap/linker/biasing element’ structure known to favor inhibition of HDACs. These compounds were assayed without further purification. HDAC8-selective inhibitors were discovered from this novel collection of compounds.     

Heterocyclic ketones as inhibitors of histone deacetylase

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.     

Fluoroalkene modification of mercaptoacetamide-based histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.     

Novel alkoxyamide-based histone deacetylase inhibitors reverse cisplatin resistance in chemoresistant cancer cells

Although histone deacetylase inhibitors (HDACi) have shown promising antitumor effects in specific types of blood cancer, their effects on solid tumors are limited. Previously, we developed LMK235 (5), a class I and class IIb preferential HDACi with chemosensitizing effects on breast cancer, ovarian cancer and HNSCC. Based on its promising effects on solid tumor cells, we modified the cap group of 5 to improve its anticancer activity. The tri- and dimethoxy-phenyl substituted compounds 13a and 13d turned out to be the most potent HDAC inhibitors of this study. The isoform profiling revealed a dual HDAC2/HDAC6 inhibition profile, which was confirmed by the acetylation of α-tubulin and histone H3 in Cal27 and Cal27CisR. In combination with cisplatin, both compounds enhanced the cisplatin-induced cytotoxicity via caspase-3/7 activation. The effect was more pronounced in the cisplatin resistant subline Cal27CisR. The pretreatment with 13d resulted in a complete resensitisation of Cal27CisR with IC₅₀ values in the range of the parental cell line. Therefore, 13d may serve as an epigenetic tool to analyze and modulate the cisplatin resistance of solid tumors.     

Trifluoromethyl ketones as inhibitors of histone deacetylase

Trifluoromethyl ketones were found to be inhibitors of histone deacetylases (HDACs). Optimization of this series led to the identification of submicromolar inhibitors such as 20 that demonstrated antiproliferative effects against the HT1080 and MDA 435 cell lines.     

Alpha-keto amides as inhibitors of histone deacetylase

Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.     

Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity

A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a-i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a-l). Compounds 3a-i and 4a-l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5 microM against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5 microM, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and alpha-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with alpha-tubulin as a substrate.