Novel inhibitors of human histone deacetylases: Design,synthesis and bioactivity of 3-alkenoylcoumarines

Histone deacetylases (HDACs) are well-established, promising targets for anticancer therapy due to their critical role in cancer development. Accordingly, an increasing number of HDAC inhibitors displaying cytotoxic effects against cancer cells have been reported. Among them, a large panel of chemical structures was described including coumarin-containing molecules. In this study, we described synthesis and biological activity of new coumarin-based derivatives as HDAC inhibitors. Among eight derivatives, three compounds showed HDAC inhibitory activities and antitumor activities against leukemia cell lines without affecting the viability of peripheral blood mononuclear cells from healthy donors.     

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.     

Aza-PBHA,a potent histone deacetylase inhibitor,inhibits human gastric-cancer cell migration via PKCα-mediated AHR-HDAC interactions

Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.     

Design, synthesis, and activity of HDAC inhibitors with a N-formyl hydroxylamine head group

Histone deacetylases (HDAC) are promising targets for cancer chemotherapy. HDAC inhibitors are thought to act in part by disrupting normal cell cycle regulation, resulting in apoptosis and/or differentiation of transformed cells. Several HDAC inhibitors, which contain hydrophobic tails and the Zn(2+) chelator hydroxyamic acid as a head group, are potent inhibitors of HDACs both in vitro and in vivo. In this study, a related class of compounds with a N-formyl hydroxylamino head group has been synthesized and their ability to inhibit HDACs have been assayed in biochemical and cellular assays. These compounds were found to have comparable activities to suberoylanilide hydroxyamic acid (SAHA) in HDAC enzymatic assays and histone hyperacetylation cellular assays.     

Methyl-CpG-DNA binding proteins in human prostate cancer: expression of CXXC sequence containing MBD1 and repression of MBD2 and MeCP2

We analyzed gene expression of MBD1, MBD2, MBD3, MBD4, and MeCP2 and protein expression of MBD1, MBD2, and MeCP2 in prostate cancer cell lines, benign prostate epithelium (BPH-1) cell line, 49 BPH tissues, and 46 prostate cancer tissues. The results of this study demonstrate that MBD2 gene is expressed in all samples and MeCP2 gene is expressed in all cancer cell lines but not in BPH-1 cell line. However, there was no protein expression for MBD2 and MeCP2 in cancer cell lines and cancer tissues. For CXXC sequence containing MBD1, both protein and mRNA were expressed in cancer cell lines, cancer tissues, BPH-1 cell line, and BPH tissues. We observed that, in BPH tissues and low-grade cancer tissues, MBD1 protein expression was very high and gradually decreased with increase of cancer grade. Treatment of cancer cell lines with proteasome inhibitor (MG-132) did not restore expression of MBD2 and MeCP2 proteins. When prostate cancer cell lines were treated with hypomethylating agent, 5-aza-2(')-deoxycytidine (DNMT inhibitor), HDAC1 and HDAC2 expression was decreased. This is the first report demonstrating that CXXC sequence containing MBD1 is overexpressed and can be the major factor of hypermethylated chromatin segments through HDAC1/2 translocation and histone deacetylation in human prostate cancer.     

Histone deacetylase inhibitors induce mitochondrial elongation

Although various stimuli-inducing cell demise are known to alter mitochondrial morphology, it is currently debated whether alteration of mitochondrial morphology is per se responsible for apoptosis execution or prevention. This study was undertaken to examine the effect of histone deacetylase (HDAC) inhibitors on mitochondrial fusion-fission equilibrium. The mechanism underlying HDAC inhibitor-induced alteration of mitochondrial morphology was examined in various cells including primary cultured cells and untransformed and cancer cell lines treated with seven different HDAC inhibitors. Suberoylanilide hydroxamic acid (SAHA)-induced mitochondrial elongation in both Hep3B and Bcl-2-overexpressing Hep3B cells, apart from its apoptosis induction function. SAHA significantly decreased the expression of mitochondrial fission protein Fis1 and reduced the translocation of Drp1 to the mitochondria. Fis1 overexpression attenuated SAHA-induced mitochondrial elongation. In addition, depletion of mitochondrial fusion proteins, Mfn1 or Opa1, by RNA interference also attenuated SAHA-induced mitochondrial elongation. All of the HDAC inhibitors we examined induced mitochondrial elongation in all the cell types tested at both subtoxic and toxic concentrations. These results indicate that HDAC inhibitors induce mitochondrial elongation, irrespective of the induction of apoptosis, which may be linked to alterations of mitochondrial dynamics regulated by mitochondrial morphology-regulating proteins. Since mitochondria have recently emerged as attractive targets for cancer therapy, our findings that HDAC inhibitors altered mitochondrial morphology may support the rationale for these agents as novel therapeutic approaches against cancer. Further, the present study may provide insight into a valuable experimental strategy for simple manipulation of mitochondrial morphology.     

miR-335 Targets SIAH2 and Confers Sensitivity to Anti-Cancer Drugs by Increasing the Expression of HDAC3

We previously reported the role of histone deacetylase 3 (HDAC3) in response to anti-cancer drugs. The decreased expression of HDAC3 in anti-cancer drug-resistant cancer cell line is responsible for the resistance to anti-cancer drugs. In this study, we investigated molecular mechanisms associated with regulation of HDAC3 expression. MG132, an inhibitor of proteasomal degradation, induced the expression of HDAC3 in various anti-cancer drug-resistant cancer cell lines. Ubiquitination of HDAC3 was observed in various anti-cancer drug-resistant cancer cell lines. HDAC3 showed an interaction with SIAH2, an ubiquitin E3 ligase, that has increased expression in various anti-cancer drug-resistant cancer cell lines. miRNA array analysis showed the decreased expression of miR-335 in these cells. Targetscan analysis predicted the binding of miR-335 to the 3′-UTR of SIAH2. miR-335-mediated increased sensitivity to anti-cancer drugs was associated with its effect on HDAC3 and SIAH2 expression. miR-335 exerted apoptotic effects and inhibited ubiquitination of HDAC3 in anti-cancer drug-resistant cancer cell lines. miR-335 negatively regulated the invasion, migration, and growth rate of cancer cells. The mouse xenograft model showed that miR-335 negatively regulated the tumorigenic potential of cancer cells. The down-regulation of SIAH2 conferred sensitivity to anti-cancer drugs. The results of the study indicated that the miR-335/SIAH2/HDAC3 axis regulates the response to anti-cancer drugs.     

TGF-β1 Reduces miR-29a Expression to Promote Tumorigenicity and Metastasis of Cholangiocarcinoma by Targeting HDAC4

Transforming growth factor β1 (TGF-β1) and miRNAs play important roles in cholangiocarcinoma progression. In this study, miR-29a level was found significantly decreased in both cholangiocarcinoma tissues and tumor cell lines. TGF-β1 reduced miR-29a expression in tumor cell lines. Furthermore, anti-miR-29a reduced the proliferation and metastasis capacity of cholangiocarcinoma cell lines in vitro, overexpression of miR-29a counteracted TGF-β1-mediated cell growth and metastasis. Subsequent investigation identified HDAC4 is a direct target of miR-29a. In addition, restoration of HDAC4 attenuated miR-29a-mediated inhibition of cell proliferation and metastasis. Conclusions: TGF-β1/miR-29a/HDAC4 pathway contributes to the pathogenesis of cholangiocarcinoma and our data provide new therapeutic targets for cholangiocarcinoma.     

CUDC‐907, a novel dual PI3K and HDAC inhibitor,in prostate cancer: Antitumour activity and molecular mechanism of action

Targeting the androgen receptor (AR) signalling pathway remains the main therapeutic option for advanced prostate cancer. However, resistance to AR‐targeting inhibitors represents a great challenge, highlighting the need for new therapies. Activation of the PI3K/AKT pathway and increased expression of histone deacetylases (HDACs) are common aberrations in prostate cancer, suggesting that inhibition of such targets may be a viable therapeutic strategy for this patient population. Previous reports demonstrated that combination of PI3K inhibitors (PI3KIs) with histone deacetylase inhibitors (HDACIs) resulted in synergistic antitumour activities against preclinical models of prostate cancer. In this study, we demonstrate that the novel dual PI3K and HDAC inhibitor CUDC‐907 has promising antitumour activity against prostate cancer cell lines in vitro and castration‐resistant LuCaP 35CR patient‐derived xenograft (PDX) mouse model in vivo. CUDC‐907‐induced apoptosis was partially dependent on Mcl‐1, Bcl‐xL, Bim and c‐Myc. Further, down‐regulation of Wee1, CHK1, RRM1 and RRM2 contributed to CUDC‐907‐induced DNA damage and apoptosis. In the LuCaP 35CR PDX model, treatment with CUDC‐907 resulted in significant inhibition of tumour growth. These findings support the clinical development of CUDC‐907 for the treatment of prostate cancer.     

Synthesis,evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents

Histone deacetylase inhibitors (HDACIs) are a promising class of anticancer agents. To examine whether a slight change in the recognition domain could alter their inhibitory activity, we synthesized a series of cyclo(?l ‐Am7(S2Py)‐Aib‐l ‐Phe(n‐Me)‐d ‐Pro)derivatives and evaluated their HDAC inhibitory and anticancer activities. The peptides exhibited potent HDAC inhibitory activity and inhibited three human cancer cell lines with IC₅₀ in the micromolar range. Docking and molecular dynamics simulation were conducted to explore the interaction mechanisms of class I and II HDACs with these inhibitors. It revealed that the zinc ion in the active site coordinated five atoms of HDACs and the sulfur atom of the inhibitor. The metal binding domains of these compounds interacted with HDAC2, and the surface recognition domains of these compounds interacted with HDAC4 through hydrogen bonding. The hydrophobic interactions also provided favorable contributions to stabilize the complexes. The results obtained from this study would be helpful for us to design some novel cyclic tetrapeptides that may act as potent HDACIs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synergistic effect of trichostatin A and 5‐aza‐2′‐deoxycytidine on growth inhibition of pancreatic endocrine tumour cell lines: A proteomic study

Our research group recently reported that pancreatic endocrine cancer cell lines are sensitive to the HDAC inhibitor trichostatin A (TSA). In the present paper, we show that the combined treatment of pancreatic endocrine tumour cell lines with TSA and the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (DAC) determines a strong synergistic inhibition of proliferation mainly due to apoptotic cell death. Proteomic analysis demonstrates that the modulation of specific proteins correlates with the antiproliferative effect of the drugs. A schematic network clarifies the most important targets or pathways involved in pancreatic endocrine cancer growth inhibition by single or combined drug treatments, which include proteasome, mitochondrial apoptotic pathway and caspase related proteins, p53 and Ras related proteins. A comparison between the patterns of proteins regulated by TSA or DAC in endocrine and ductal pancreatic cancer cell lines is also presented.     

Selective repression of YKL-40 by NF-kappaB in glioma cell lines involves recruitment of histone deacetylase-1 and -2

Here we show that in contrast to other cancer types, tumor necrosis factor (TNF)-alpha suppresses YKL-40 expression in glioma cell lines in a nuclear factor kappaB (NF-kappaB) dependent manner. Even though TNF-alpha causes recruitment of p65 and p50 subunits of NF-kappaB to the YKL-40 promoter in all cell types, recruitment of histone deacetylases (HDAC)-1 and -2, and a consequent deacetylation of histone H3 at the YKL-40 promoter occurs only in glioma cells. Importantly, using chromatin immunoprecipitation assays in frozen glioblastoma multiforme tissues, we show that YKL-40 levels decrease consistent with HDAC1 recruitment despite high levels of nuclear p-p65. This study presents a paradigm for NF-kappaB regulation of one of its targets in a strict cell type specific manner.     

Identification of potential isoform-selective histone deacetylase inhibitors for cancer therapy: a combined approach of structure-based virtual screening,ADMET prediction and molecular dynamics simulation assay

Histone deacetylases (HDACs) have gained increased attention as targets for anticancer drug design and development. HDAC inhibitors have proven to be effective for reversing the malignant phenotype in HDAC-dependent cancer cases. However, lack of selectivity of the many HDAC inhibitors in clinical use and trials contributes to toxicities to healthy cells. It is believed that, the continued identification of isoform-selective inhibitors will eliminate these undesirable adverse effects – a task that remains a major challenge to HDAC inhibitor designs. Here, in an attempt to identify isoform-selective inhibitors, a large compound library containing 2,703,000 compounds retrieved from Otava database was screened against class I HDACs by exhaustive approach of structure-based virtual screening using rDOCK and Autodock Vina. A total of 41 compounds were found to show high-isoform selectivity and were further redocked into their respective targets using Autodock4. Thirty-six compounds showed remarkable isoform selectivity and passed drug-likeness and absorption, distribution, metabolism, elimination and toxicity prediction tests using ADMET Predictor? and admetSAR. Furthermore, to study the stability of ligand binding modes, 10 ns-molecular dynamics (MD) simulations of the free HDAC isoforms and their complexes with respective best-ranked ligands were performed using nanoscale MD software. The inhibitors remained bound to their respective targets over time of the simulation and the overall potential energy, root-mean-square deviation, root-mean-square fluctuation profiles suggested that the detected compounds may be potential isoform-selective HDAC inhibitors or serve as promising scaffolds for further optimization towards the design of selective inhibitors for cancer therapy.     

Rational design,synthesis and preliminary antitumor activity evaluation of a chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory activity

Histone deacetylases (HDACs) play a pivotal role not only in gene expression but also in DNA repair. Herein, we report the successful design, synthesis and evaluation of a chlorambucil derivative named vorambucil with a hydroxamic acid tail as a DNA/HDAC dual-targeting inhibitor. Vorambucil obtained both potent DNA and HDACs inhibitory activities. Molecular docking results supported the initial pharmacophoric hypothesis and rationalized the potent inhibitory activity of vorambucil against HDAC1, HDAC2 and HDAC6. Vorambucil showed potent antiproliferative activity against all the test four cancer cell lines with IC₅₀ values of as low as 3.2–6.2 μM and exhibited 5.0–18.3-fold enhanced antiproliferative activity than chlorambucil. Vorambucil also significantly inhibits colony formation of A375 cancer cells. Further investigation showed that vorambucil remarkably induced apoptosis and arrested the cell cycle of A375 cells at G2/M phase. Vorambucil could be a promising candidate and a useful tool to elucidate the role of those DNA/HDAC dual-targeting inhibitors for cancer therapy.