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The immune system represents the major primary defense line against carcinogenesis and acts by identifying and eradicating nascent transformed cells. A growing body of evidence is indicating that aberrant epigenetic reprogramming plays a key role in tumor immune escape through: 1) impaired efficient recognition of neoplastic cells by the immune system, resulting from a downregulation or loss of the expression of tumor-associated antigens, human leukocyte antigens, antigen processing and presenting machinery, and costimulatory molecule genes; 2) aberrant expression of immune checkpoint proteins and their ligands; and 3) modification of cytokine profiles and tumor-associated immune cell populations toward an immunosuppressive state in the tumor microenvironment. Consistent with the inherent reversibility of epigenetic alterations, epigenetic drugs, including DNA methyltransferase and histone deacetylase inhibitors, have the unique potential to favorably modify the tumor microenvironment, restore tumor recognition and stimulate an antitumor immune response. The objective of this review is to highlight selected, naturally occurring epigenetic modulators, namely, butyrate, curcumin, (-)-epigallocatechin-3-gallate, resveratrol, romidepsin, and trichostatin A, with a special focus on their antitumor immune properties.

Background Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi. Results We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski’s “rule of five,” in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib co-treatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib. Conclusions Collectively, the present findings show that combinations HDAC inhibitor-imatinib are likely to overcome drug resistance in CML pathology.

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Imatinib became the standard treatment for chronic myeloid leukemia (CML) about 20 years ago, which was a major breakthrough in stabilizing the pathology and improving the quality of life of patients. However, the emergence of resistance to imatinib and other tyrosine kinase inhibitors leads researchers to characterize new therapeutic targets. Several studies have highlighted the role of histone deacetylase 6 (HDAC6) in various pathologies, including cancer. This protein effectively intervenes in cellular activities by its primarily cytoplasmic localization. In this review, we will discuss the molecular characteristics of the HDAC6 protein, as well as its overexpression in CML leukemic stem cells, which make it a promising therapeutic target for the treatment of CML.

Imatinib became the standard treatment for chronic myeloid leukemia (CML) about 20 years ago, which was a major breakthrough in stabilizing the pathology and improving the quality of life of patients. However, the emergence of resistance to imatinib and other tyrosine kinase inhibitors leads researchers to characterize new therapeutic targets. Several studies have highlighted the role of histone deacetylase 6 (HDAC6) in various pathologies, including cancer. This protein effectively intervenes in cellular activities by its primarily cytoplasmic localization. In this review, we will discuss the molecular characteristics of the HDAC6 protein, as well as its overexpression in CML leukemic stem cells, which make it a promising therapeutic target for the treatment of CML.

Alterations of the epigenetic machinery, affecting multiple biological functions, represent a major hallmark enabling the development of tumors. Among epigenetic regulatory proteins, histone deacetylase (HDAC)6 has emerged as an interesting potential therapeutic target towards a variety of diseases including cancer. Accordingly, this isoenzyme regulates many vital cellular regulatory processes and pathways essential to physiological homeostasis, as well as tumor multistep transformation involving initiation, promotion, progression and metastasis. In this review, we will consequently discuss the critical implications of HDAC6 in distinct mechanisms relevant to physiological and cancerous conditions, as well as the anticancer properties of synthetic, natural and natural-derived compounds through the modulation of HDAC6-related pathways.

Simple Summary Ferroptosis and necroptosis are two non-apoptotic programmed cell death pathways with increasing therapeutic potential. The isothiocyanate sulforaphane (SFN) is a well-known naturally derived anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell death mechanisms remains poorly investigated. This work discovered that SFN activates apoptosis and ferroptosis dose-dependently in acute myeloid leukemia cells. At lower concentrations, SFN induces caspase-dependent apoptosis. At higher concentrations, ferroptosis is activated and accompanied by the depletion of intracellular glutathione (GSH) and decreased GSH peroxidase 4 protein expression levels. Necroptosis, instead, is not involved in SFN-induced cell death. Considering that cancer cells resist pro-apoptotic treatments, SFN’s ability to induce different types of cell death delineates it as a promising anticancer agent. Abstract In recent years, natural compounds have emerged as inducers of non-canonical cell death. The isothiocyanate sulforaphane (SFN) is a well-known natural anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell-death mechanisms remains poorly investigated. This work aimed to explore the capacity of SFN to induce non-apoptotic cell death modalities. SFN was tested on different acute myeloid leukemia cell lines. The mechanism of cell death was investigated using a multi-parametric approach including fluorescence microscopy, western blotting, and flow cytometry. SFN triggered different cell-death modalities in a dose-dependent manner. At 25 μM, SFN induced caspase-dependent apoptosis and at 50 μM ferroptosis was induced through depletion of glutathione (GSH), decreased GSH peroxidase 4 protein expression, and lipid peroxidation. In contrast, necroptosis was not involved in SFN-induced cell death, as demonstrated by the non-significant increase in phosphorylation of receptor-interacting protein kinase 3 and phosphorylation of the necroptotic effector mixed lineage kinase domain-like pseudokinase. Taken together, our results suggest that the antileukemic activity of SFN can be mediated via both ferroptotic and apoptotic cell death modalities.

Although tyrosine kinase inhibitors (TKIs) revolutionized the management of chronic myeloid leukemia (CML), resistance against TKIs and leukemia stem cell (LSC) persistence remain a clinical concern. Therefore, new therapeutic strategies combining conventional and novel therapies are urgently needed. Since telomerase is involved in oncogenesis and tumor progression but is silent in most human normal somatic cells, it may be an interesting target for CML therapy by selectively targeting cancer cells while minimizing effects on normal cells. Here, we report that hTERT expression is associated with CML disease progression. We also provide evidence that hTERT-deficient K-562 cells do not display telomere shortening and that telomere length is maintained through the ALT pathway. Furthermore, we show that hTERT depletion exerts a growth-inhibitory effect in K-562 cells and potentiates imatinib through alteration of cell cycle progression leading to a senescence-like phenotype. Finally, we demonstrate that hTERT depletion potentiates the imatinib-induced reduction of the ALDH+-LSC population. Altogether, our results suggest that the combination of telomerase and TKI should be considered as an attractive strategy to treat CML patients to eradicate cancer cells and prevent relapse by targeting LSCs.

Epigenetics refers to the study of heritable changes in gene function that are mediated bymechanisms other than nucleotide alterations in the primary DNA sequence [...]

Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1&rsquo, (R), 6&rsquo, (S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1&rsquo, (R), and 6&rsquo, (S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.

DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line. © 2014 American Chemical Society.

Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells.

Chronic myeloid leukemia (CML) is a hematological disorder caused by the oncogenic BCR-ABL fusion protein in more than 90% of patients. Despite the striking improvements in the management of CML patients since the introduction of tyrosine kinase inhibitors (TKis), the appearance of TKi resistance and side effects lead to treatment failure, justifying the need of novel therapeutic approaches. Histone deacetylase inhibitors (HDACis), able to modulate gene expression patterns and important cellular signaling pathways through the regulation of the acetylation status of both histone and non-histone protein targets, have been reported to display promising anti-leukemic properties alone or in combination with TKis. This review summarizes pre-clinical and clinical studies that investigated the mechanisms underlying the anticancer potential of HDACis and discusses the rationale for a combination of HDACis with TKis as a therapeutic option in CML.

NAD+-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor 7), as normal human primary CD34+ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics., SCOPUS: ar.j, info:eu-repo/semantics/published

Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals.

Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca2+-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca2+ levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca2+ accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca2+ overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and in vivo tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish.

A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.

© 2017 Bentham Science Publishers Background: Histone deacetylases (HDACs) emerged as important epigenetic regulators of gene expression. Method: In order to identify potential positron emission tomography (PET) tracers for imaging HDACs, we evaluated in vitro and in cellulo activities of some compounds that were reported as potent HDAC2-selective inhibitors. We observed marked differences between reported activity values and the values obtained in our assays for some of the compounds. To understand the structural basis of the activity of some of these inhibitors, we also performed molecular docking studies to understand their interaction patterns and binding modes with HDAC2. Results and Conclusion: We observed the low affinity compounds 4, 6 and 7 did not showed equal number of key ?-? interactions and hydrogen bonding when compared to high affinity compounds, and could be the possible reason for poor inhibition as reflected in in vitro assays. These preliminary experimental and computational results will help to interpret the HDAC affinity values of these key compounds with caution. ispartof: Letters in Drug Design & Discovery vol:14 issue:7 pages:787-797 status: published

Valproic acid (VPA) exhibits important pharmacological properties but has been reported to trigger side effects, notably on the hematological system. We previously reported that VPA affects hematopoietic homeostasis by inhibiting erythroid differentiation and promoting myeloid and megakaryocyte differentiation. Here, we analyzed the effect of VPA on regulatory factors involved in erythro-megakaryocytic differentiation pathways, including transcription factors and microRNAs (miRs). We demonstrate that VPA inhibited erythroid differentiation in erythropoietin (Epo)-stimulated TF1 leukemia cells and CD34(+)/hematopoietic stem cells (HSCs) and in aclacinomycin-(Acla)-treated K562 cells. Mir-144/451 gene expression was decreased in all erythroid and megakaryocyte models in correlation with GATA-1 inhibition. In Epo-stimulated CD34(+)/HSCs, VPA induced the expression of the ETS family transcription factors PU.1, ETS-1, GABP-α, Fli-1 and GATA-2, which are all known to be negative regulators of erythropoiesis, while it promoted the megakaryocytic pathway. PU.1 and ETS-1 expression were induced in correlation with miR-155 inhibition; however, the GATA-1/PU.1 interaction was promoted. Using megakaryoblastic Meg-01 cells, we demonstrated that VPA induced megakaryocyte morphological features and CD61 expression. GATA-2 and miR-27a expression were increased in correlation with a decrease in RUNX1 mRNA expression, suggesting megakaryocyte differentiation. Finally, by using valpromide and the Class I HDACi MS-275, we validated that the well-described HDACi activity of VPA is not required in the inhibitory effect on erythropoiesis. Overall, this report shows that VPA modulates the erythro-megakaryocytic differentiation program through regulatory micro-networks involving GATA and ETS transcription factors and miRNAs, notably the GATA-1/miR-144/451 axis.

Cancer remains a major public health problem in our society. The development of potent novel anti-cancer drugs selective for tumor cells is therefore still required. Deregulation of the epigenetic machinery including DNA methylation, histone modifications and non-coding RNAs is a hallmark of cancer, which provides potential new therapeutic targets. Natural products or their derivatives represent a major class of anti-cancer drugs in the arsenal available to the clinician. However, regarding epigenetically active anti-cancer agents for clinics, the oceans represent a largely untapped resource. This review focuses on marine natural compounds with epigenetic activities and their synthetic derivatives displaying anti-cancer properties including largazole, psammaplins, trichostatins and azumamides.

Increased proliferation rates as well as resistance to apoptosis are considered major obstacles for the treatment of patients with chronic myelogenous leukemia (CML), thus highlighting the need for novel therapeutic approaches. Since senescence has been recognized as a physiological barrier against tumorigenesis, senescence-based therapy could represent a new strategy against CML. DNA demethylating agent 5-aza-2′-deoxycytidine (DAC) was reported to induce cellular senescence but underlying mechanisms remain to be elucidated. Here, we report that exposure to DAC triggers senescence in chronic leukemia cell lines as evidenced by increased senescence-associated β-galactosidase activity and lysosomal mass, accompanied by an up-regulation of cell cycle-related genes. We provide evidence that DAC is able to decrease telomere length, to reduce telomerase activity and to decrease human telomerase reverse transcriptase (hTERT) expression through decreased binding of c-myc to the hTERT promoter. Altogether, our results reveal the role of c-myc in telomere-dependent DAC-induced senescence and therefore provide new clues for improving chronic human leukemia treatments.

Introduction/Objectifs Explorer les effets de l’alcaloide Iso-3, nouvel agent de demethylation d’acide desoxyribonucleique (ADN), sur deux lignes cellulaires issues des lymphomes humains. L’hypermethylation d’ADN induisant l’inactivation des genes suppresseurs de tumeurs est un mecanisme important de la cancerogenese [1] souvent realise par la surexpression des enzymes de la famille des ADN methyltransferases (DNMT). Par consequent, les inhibiteurs des DNMT representent une voie prometteuse dans le traitement de plusieurs cancers et notamment des lymphomes [2] . Materiels/Patients et methodes Les cellules RAJA et U-937 ont ete traitees par Iso-3. Les colorations Cyto-ID® Green (plus microscopie electronique), Hoechst (plus propidium iodide), trypan bleue (plus essai de clonogenicite) ont ete utilisees pour quantifier l’autophagocytose, l’apoptose et la viabilite des cellules. Les cellules ont ete xenogreffees dans des poissons Zebre et la taille des tumeurs mesuree. L’expression de plusieurs molecules impliquees le cycle cellulaire et l’apoptose a ete evaluee par Western Blot, RT-PCR en temps reel ou par cytometrie de flux. Resultats In vitro, Iso-3 induit la diminution de la methylation d’ADN et de l’activite des DNMT, de la survie des cellules et de leur clonogenicite et l’augmentation des taux d’apoptose en synergie avec tumornecrosis-factor related apoptosis inducing ligand (TRAIL) ; du stress du reticulum endoplasmique et provoque la formation de phagophores et d’autophagosomes et l’accumulation des cellules en phase G0/G1 accompagnee d’une augmentation de l’expression de p21 et p27 et d’une diminution de celle de PCNA, E1 et c-myc. In vivo, Iso-3 diminue de facon significative la taille des tumeurs se developpant dans les poissons Zebre a partir des cellules traitees. Conclusions L’alcaloide Iso-3 semble un candidat prometteur pour le developpement des inhibiteurs des DNMT pour le traitement des lymphomes.

In diseases such as cancer, cells need to degrade the extracellular matrix (ECM) and therefore require high protease levels. Thus, aberrant tissue degradation is associated to matrix metalloproteinases (MMPs) overexpression resulting from different mechanisms including epigenetic events. One of the most characterized epigenetic mechanisms is DNA methylation causing changes in chromatin conformation, thereby decreasing the accessibility to the transcriptional machinery and resulting in a robust gene silencing. Modulation of DNA methylation by DNA hypomethylating agents such as 5-aza-2'-deoxycytidine (5-azadC) is widely used in epigenetic anticancer treatments. Here, we focus on the effects of this drug on the expression level of MMP-1, -2, and -9 in human HT1080 fibrosarcoma cells. We demonstrate that 5-azadC increases MMP expression at both mRNA and protein levels, and promotes invasion potential of HT1080 cells. Using broad-spectrum and specific MMP inhibitors, we establish that MMP-1, but not MMP-2 and -9, plays a key role in 5-azadC-enhanced cell invasion. We show that 5-azadC induces MMP-1 expression through a transcriptional mechanism without affecting MMP-1 promoter methylation status. Finally, we demonstrate that 5-azadC treatment increases the nuclear levels of Sp1 and Sp3 transcription factors, and modulates their recruitment to the MMP-1 promoter, resulting in chromatin remodeling associated to 5-azadC-induced MMP-1 expression. All together, our data indicate that the hypomethylating agent 5-azadC modulates, mainly via Sp1 recruitment, MMP-1 expression resulting in an increased invasive potential of HT1080 cells.

International audience; Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency.

The remarkable complexity of cancer involving multiple mechanisms of action and specific organs led researchers Hanahan and Weinberg to distinguish biological capabilities acquired by cancer cells during the multistep development of human tumors to simplify its understanding. These characteristic hallmarks include the abilities to sustain proliferative signaling, evade growth suppressors, resist cell death, enable replicative immortality, induce angiogenesis, activate invasion and metastasis, avoid immune destruction, and deregulate cellular energetics. Furthermore, two important characteristics of tumor cells that facilitate the acquisition of emerging hallmarks are tumor-promoting inflammation and genome instability. To treat a multifactorial disease such as cancer, a combination treatment strategy seems to be the best approach. Here we focus on natural histone deacetylase inhibitors (HDACi), their clinical uses as well as synergies with modulators of the pro-inflammatory transcription factor signaling pathways.

Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.

MicroRNAs (miRNAs) represent a class of small (21–23 nucleotides) non-coding RNAs that emerged as key post-transcriptional gene regulators, implicated in numerous physiological and pathological processes. Currently, a main focus of miRNA research is related to the roles of miRNAs in cancer development. The biogenesis and modes of action of miRNAs have not been completely elucidated; however, miRNA-mediated translational repression is involved in the regulation of almost every cellular process. Thus, pathological alterations in miRNA expression signatures are commonly associated with disease development. This review specifically focuses on miRNAs in cancer, with an emphasis on their use as potential biomarkers for cancer diagnosis and prognosis. Then, we discuss the potential use of synthetic antisense or miRNA mimetic oligonucleotides and dietary agents to modulate miRNA expression for chemotherapy and chemoprevention of cancer, respectively.

Histone deacetylase enzymes (HDACs) are emerging as a promising biological target for cancer and inflammation. Using a fluorescence assay, we tested the in vitro HDAC inhibitory activity of twenty-one natural chalcones, a widespread group of natural products with well-known anti-inflammatory and antitumor effects. Since HDACs regulate the expression of the transcription factor NF-κB, we also evaluated the inhibitory potential of the compounds on NF-κB activation. Only four chalcones, isoliquiritigenin (no. 10), butein (no. 12), homobutein (no. 15) and the glycoside marein (no. 21) showed HDAC inhibitory activity with IC50 values of 60-190 µM, whereas a number of compounds inhibited TNFα-induced NF-κB activation with IC50 values in the range of 8-41 µM. Interestingly, three chalcones (nos. 10, 12 and 15) inhibited both TNFα-induced NF-κB activity and total HDAC activity of classes I, II and IV. Molecular modeling and docking studies were performed to shed light into dual activity and to draw structure-activity relationships among chalcones (nos. 1-21). To the best of our knowledge this is the first study that provides evidence for HDACs as potential drug targets for natural chalcones. The dual inhibitory potential of the selected chalcones on NF-κB and HDACs was investigated for the first time. This study demonstrates that chalcones can serve as lead compounds in the development of dual inhibitors against both targets in the treatment of inflammation and cancer.

Protein acetylation status results from a balance between histone acetyltransferase and histone deacetylase (HDAC) activities. Alteration of this balance leads to a disruption of cellular integrity and participates in the development of numerous diseases, including cancer. Therefore, modulation of these activities appears to be a promising approach for anticancer therapy. Histone deacetylase inhibitors (HDACi) are epigenetically active drugs that induce the hyperacetylation of lysine residues within histone and non-histone proteins, thus affecting gene expression and cellular processes such as protein-protein interactions, protein stability, DNA binding and protein sub-cellular localization. Therefore, HDACi are promising anti-tumor agents as they may affect the cell cycle, inhibit proliferation, stimulate differentiation and induce apoptotic cell death. Over the last 30 years, numerous synthetic and natural products, including a broad range of dietary compounds, have been identified as HDACi. This review focuses on molecules from natural origins modulating HDAC activities and presenting promising anticancer activities.

Enzymatic inhibitors of pro-inflammatory cyclooxygenase-2 (COX-2) possess multiple anti-cancer effects, including chemosensitization. These effects are not always linked to the inhibition of the COX-2 enzyme. Here we analyze the effects of three COX-2 enzyme inhibitors (nimesulide, NS-398 and celecoxib) on apoptosis in different hematopoietic cancer models. Surprisingly, COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We selected U937 cells as a model of sensitive cells for further studies. Here, we provide evidence that the protective effect is COX-independent. No suppression of the low basal prostaglandin (PG)E(2) production may be observed upon treatment by COX-2 inhibitors. Besides, the non-active celecoxib analog 2,5-dimethyl-celecoxib is able to protect from apoptosis as well. We demonstrate early prevention of the stress-induced apoptotic signaling, prior to Bax/Bak activation. This preventive effect fits with an impairment of the ability of chemotherapeutic agents to trigger apoptogenic stress. Accordingly, etoposide-induced DNA damage is strongly attenuated in the presence of COX-2 inhibitors. In contrast, COX-2 inhibitors do not exert any anti-apoptotic activity when cells are challenged with physiological stimuli (anti-Fas, TNFα or Trail) or with hydrogen peroxide, which do not require internalization and/or are not targeted by chemoresistance proteins. Altogether, our findings show a differential off-target anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at the very early steps of intracellular signaling, prior to commitment. The results imply that an exacerbation of the chemoresistance phenomena may be implicated.

Leukemogenesis is a multistep process in which successive transformational events enhance the ability of a clonal population arising from hematopoietic progenitor cells to proliferate, differentiate and survive. Clinically and pathologically, leukemia is subdivided into four main categories: chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic leukemia and acute myeloid leukemia. Leukemia has been previously considered only as a genetic disease. However, in recent years, significant advances have been made in the elucidation of the leukemogenesis-associated processes. Thus, we have come to understand that epigenetic alterations including DNA methylation, histone modifications and miRNA are involved in the permanent changes of gene expression controlling the leukemia phenotype. In this article, we will focus on the epigenetic defects associated with leukemia and their implications as biomarkers for diagnostic, prognostic and therapeutic applications.

International audience; Glutathione -transferase P1 (GSTP1) gene is commonly silenced by CpG island promoter hypermethylation in prostate, breast, and liver cancers. However, mechanisms leading to GSTP1 repression by promoter hypermethylation in leukemia and its relationship with pathological alterations of the chromatin structure remain poorly understood. A panel of leukemia cell lines was analyzed for their GSTP1 expression, revealing cell lines with high, moderate or no detectable GSTP1 expression. Bisulfite sequencing, methylation-specific PCR and combined bisulfite restriction analysis revealed that GSTP1 promoter was completely methylated in transcriptionally inactive RAJI and MEG-01 cell lines. In contrast, cell lines expressing GSTP1 exhibited an unmethylated and transcriptionally active promoter. Furthermore, histone marks and effector proteins associated with transcriptional activity were detected by chromatin immunoprecipitation in the GSTP1 expressing hypomethylated K-562 cell line. However, repressive chromatin marks and the recruitment of silencing protein complexes were found in the non-expressing hypermethylated RAJI and MEG-01 cell lines. Finally, we provide evidence that treatment of RAJI and MEG-01 cells with the DNA demethylating agent, 5-Aza-2′-deoxycytidine, resulted in GSTP1 promoter demethylation, drastic changes of histone modifications and promoter associated proteins and GSTP1 gene activation. In contrast, treatments with HDAC inhibitors failed to demethylate and reactivate the GSTP1 gene. Our study extends the knowledge on leukemia-specific epigenetic alterations of GSTP1 gene. Furthermore, we are showing the correlation of DNA methylation and histone modifications with the positive/negative GSTP1 transcriptional expression state. Finally, these data support the concept of the dominance of DNA methylation over HDAC inhibitor-sensitive histone deacetylation in gene silencing.

Next meetings Redox regulation—Natural compounds as regulators of inflammation signaling (A RedCat—satellite meeting to Natural Compounds 2012)—January 24–26, 2012. Natural compounds 2012—Regulators of cell signaling pathways and novel therapeutic tools—January 26–28, 2012. Under the patronage of Corena, a network aiming to create an internationally competitive cluster within the Greater Region (Saarland, Lorraine, Luxembourg, Wallonia, Rheinland-Pfalz). Corena is cofunded by European Regional development fund within the INTERREG IVA Greater Region program. The European Union invests in your future. Meeting information: http://www.transduction-meeting.lu.

Posttranslational histone modifications are a critical regulatory mechanism of gene transcription. Previous studies from our laboratory have shown that contingent on binding to its cognate promoter motifs in the Cyp1a1 gene, activation of the aryl hydrocarbon receptor (AHR) by benzo[a]pyrene (BaP) treatment induces histone modifications in the Cyp1a1 promoter that are required for activation of gene transcription. Here, we have studied different AHR ligands, including polychlorinated biphenyls (PCBs) of different toxic equivalency factors (TEF), to determine whether changes in histone modifications are linked to different levels of Cyp1a1 expression or dependent on AHR-ligand affinity. We find that all ligands lead to the same pattern of histone modifications in a relationship that parallels the strength of their AHR-ligand affinity. Thus, whereas PCB126 (TEF 0.1), 3-methylcholanthrene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) initiate a pattern of histone marks similar to those induced by BaP, PCB77 (TEF 0.0001) causes a lower level of change in the same marks and requires a longer activation time than PCB126, BaP, or TCDD. In contrast, the non-dioxin-like PCB153 recruits AHR to the Cyp1a1 enhancer causing a displacement of enhancer-associated histone H3 but does not cause the other observed histone mark changes nor does it induce transcription. These results indicate that AHR recruitment to the promoter is not sufficient to induce the histone modifications needed to activate gene expression and show that there is a good correlation between the regulatory chromatin changes associated with ligand-induced AHR target gene transcription and the resultant toxicity of the ligand.

International audience; In addition to its demethylating properties, 5-aza-2′-deoxycytidine (DAC) induces cell cycle arrest, differentiation, cell sensitization to chemotherapy, and cell death. However, the mechanisms by which DAC induces antiproliferation via these processes and how they are interconnected remain unclear. In this study, we found that a clinically relevant concentration of DAC triggered erythroid and megakaryocytic differentiation in the human chronic myeloid leukemia (CML) K-562 and MEG-01 cell lines, respectively. In addition, cells showed a marked increase in cell size in both cell lines and a more adhesive cell profile for MEG-01. Furthermore, DAC treatment induced cellular senescence and autophagy as shown by β-galactosidase staining and by autophagosome formation, respectively. After prolonged DAC treatment, phosphatidyl serine exposure, nuclear morphology analysis, and caspase cleavage revealed an activation of mitochondrial-dependent apoptosis in CML cells. This activation was accompanied by a decrease of anti-apoptotic proteins and an increase of calpain activity. Finally, we showed that combinatory treatment of relatively resistant CML with DAC and either conventional apoptotic inducers or with an histone deacetylase inhibitor increased synergistically apoptosis. We therefore conclude that induction of differentiation, senescence, and autophagy in CML are a key in cell sensitization and DAC-induced apoptosis.

As a histone deacetylase inhibitor, valproic acid (VPA) is a candidate for anticancer therapy. Besides, VPA exhibits various mechanisms of action and its effects on the molecular basis of hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we performed microarray analysis using K562 cells treated with 1mM VPA over a 72h time course. The association between gene ontology (GO) terms and the lists of differentially expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for cellular differentiation pathways was performed based on manually curated gene lists. Results from microarray analysis were confirmed by studying cell differentiation features at the molecular and cellular levels using other hematopoietic cell lines as well as hematopoietic stem/progenitor CD34(+) cells. Microarray analysis revealed 3440 modulated genes in the presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were up-regulated while genes of the erythroid pathway were down-regulated. This was confirmed by analyzing erythrocytic and myeloid membrane markers and lineage-related gene expression in HEL, MEG01, HL60 as well as CD34(+) cells. Moreover, GATA-1 and its co-factors (FOG1, SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement with an inhibition of erythropoiesis. Our functional profiling and cell phenotyping approach demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell population balance in the myeloid compartment. This may lead to a potential failure of erythropoiesis in patients with cancer or chronic inflammatory diseases having a well-described propensity to anemia.

Acetylation and deacetylation of lysine residues on histones, which are catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), are epigenetic modifications that play a very important role in the regulation of gene transcription. Perturbation of the balance between histone acetylation and deacetylation leads to a myriad of diseases, including cancer, AIDS, malaria, neurodegenerative diseases, and diabetes. HATs and HDACs have recently been recognized as key targets for chemoprevention and drug discovery, and numerous natural and synthetic compounds have been screened in order to identify promising regulators of subtle balance between histone acetylation and deacetylation. Here, we present dietary compounds and other natural products that have emerged as potent HAT or HDAC activity modulators, and we discuss their current and future applications as chemopreventive or therapeutic agents.

International audience; Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the biologic and toxic effects of its xenobiotic ligands. In recent years it has become evident that in the absence of ligand the AHR promotes cell cycle progression and that its activation by high-affinity ligands results in interactions with the retinoblastoma protein (RB) that lead to perturbation of the cell cycle, G0/G1 arrest, diminished capacity for DNA replication and inhibition of cell proliferation. Hence, the AHR has diametrically opposed pro-proliferative and anti-proliferative functions that have yet to be reconciled at the molecular level. Work from our own and from other laboratories suggests that the AHR may function as a tumor suppressor gene that becomes silenced in the process of tumor formation. To develop preliminary support for a more thorough examination of this hypothesis we characterized the expression levels of various tumor suppressor genes, transforming growth factor-beta (Tgfb) genes and the Ahr gene in liver tumor samples from mice with a liver-specific RB ablation and their wild-type littermates. In tumors arising in RB-positive livers, Cdkn2d and Tgfb1 were repressed and Cdkn2c, Tgfb2, Tgfb3 and Pai1 were induced, whereas in RB-negative tumors, only Cdkn2c and Tgfb3 were induced. Ahr was significantly repressed in tumors from both sets of mice, supporting the concept that Ahr silencing may be associated with cancer progression.