The class I-specific HDAC inhibitor MS-275 modulates the differentiation potential of mouse embryonic stem cells.
ABSTRACT: Exploitation of embryonic stem cells (ESC) for therapeutic use and biomedical applications is severely hampered by the risk of teratocarcinoma formation. Here, we performed a screen of selected epi-modulating compounds and demonstrate that a transient exposure of mouse ESC to MS-275 (Entinostat), a class I histone deacetylase inhibitor (HDAC), modulates differentiation and prevents teratocarcinoma formation. Morphological and molecular data indicate that MS-275-primed ESCs are committed towards neural differentiation, which is supported by transcriptome analyses. Interestingly, in vitro withdrawal of MS-275 reverses the primed cells to the pluripotent state. In vivo, MS275-primed ES cells injected into recipient mice give only rise to benign teratomas but not teratocarcinomas with prevalence of neural-derived structures. In agreement, MS-275-primed ESC are unable to colonize blastocysts. These findings provide evidence that a transient alteration of acetylation alters the ESC fate.
Project description:MS-275 (Entinostat) is a histone deacetylase (HDAC) inhibitor currently in clinical trials for the treatment of several types of cancer. Recent reports have noted that MS-275 can cross the blood brain barrier (BBB) and cause region specific changes in rodent brain histone acetylation. To characterize the pharmacokinetics and distribution of MS-275 in the brain using positron emission tomography (PET), we labeled the carbamate carbon of MS-275 with carbon-11. Using PET, we determined that [(11)C]MS-275 has low uptake in brain tissue when administered intravenously to non-human primates. In rodent studies, we observed that pharmacokinetics and brain accumulation of [(11)C]MS-275 were not changed by the co-administration of large doses of unlabeled MS-275. These results, which both highlight the poor brain penetration of MS-275, clearly suggest its limitation as a therapeutic agent for the central nervous system (CNS). Moreover, our study demonstrates the effectiveness of PET at providing brain pharmacokinetic data for HDAC inhibitors. These data are important not only for the development of new compounds for peripheral cancer treatment (where CNS exclusion is often advantageous), but also for the treatment of neurological disorders (where CNS penetration is critical).
Project description:Transcriptional profiling of U937 miR-194-5p (UmiR-194-5p) vs U937 miR-194-5p (UmiR-194-5p) treated with MS275 (SNDX 275;Entinostat) for 24 h at 5uM concetration Overall design: Different Experimental Conditions: U937 miR-194-5p (UmiR-194-5p) (U937 transfected with miR-194-5p vector) vs UmiR-194-5p (U937 transfected with miR-194-5p vector) treated with MS275 for 24 h at 5uM concetration. Two replicates.
Project description:The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.
Project description:OBJECTIVE:Non-selective histone deacetylase (HDAC) inhibitors are known to improve hypertension. Here, we investigated the therapeutic effect and regulatory mechanism of the class I HDAC selective inhibitors, MS-275 and RGFP966, in angiotensin (Ang) II-induced hypertensive mice. METHODS AND RESULTS:MS-275 inhibited the activity of HDAC1, HDAC2, and HDAC3, while RGFP966 weakly inhibited that of HDAC3 in a cell-free system. MS-275 and RGFP966 treatment reduced systolic blood pressure and thickness of the aorta wall in Ang II-induced hypertensive mice. MS-275 treatment reduced aorta collagen deposition, as determined by Masson's trichrome staining. MS-275 decreased the components of the renin angiotensin system and increased vascular relaxation of rat aortic rings via the nitric oxide (NO) pathway. NO levels reduced by Ang II were restored by MS-275 treatment in vascular smooth muscle cells (VSMCs). However, MS-275 dose (3 mg·kg-1·day-1) was not enough to induce NO production in vivo. In addition, MS-275 did not prevent endothelial nitric oxide synthase (eNOS) uncoupling in the aorta of Ang II-induced mice. Treatment with MS-275 failed to inhibit Ang II-induced expression of NADPH oxidase (Nox)1, Nox2, and p47phox. MS-275 treatment reduced proinflammatory cytokines such as tumor necrosis factor (TNF)-?, interleukin (IL)-1?, and monocyte chemoattractant protein (MCP)-1, as well as adhesion molecules. Histological analysis showed that Ang II-induced macrophage infiltration was reduced by MS-275 and RGFP966 administration. CONCLUSIONS:Our results indicate that class I HDAC selective inhibitors may be good therapeutic agents for the treatment of hypertension through the regulation of vascular remodeling and vasoconstriction, as well as inflammation.
Project description:This study describes transcriptome profiling of mouse ES cells treated with MS-275. We included RNA-Seq of ds_cDNA synthesized from double poly(A) selected mRNA RNA-Seq profiling on mouse ES cells treated with MS-275
Project description:Transplantation of induced pluripotent stem cell-derived cardiac tissue constructs is a promising regenerative treatment for cardiac failure: however, its tumourigenic potential is concerning. We hypothesised that the tumourigenic potential may be eliminated by the host immune response after allogeneic cell transplantation. Scaffold-free iPSC-derived cardaic tissue sheets of C57BL/6 mouse origin were transplanted into the cardiac surface of syngeneic C57BL/6 mice and allogeneic BALB/c mice with or without tacrolimus injection. Syngeneic mice and tacrolimus-injected immunosuppressed allogeneic mice formed teratocarcinomas with identical phenotypes, characteristic, and time courses, as assessed by imaging tools including (18)F-fluorodeoxyglucose-positron emission tomography. In contrast, temporarily immunosuppressed allogeneic mice, following cessation of tacrolimus injection displayed diminished progression of the teratocarcinoma, accompanied by an accumulation of CD4/CD8-positive T cells, and finally achieved complete elimination of the teratocarcinoma. Our results indicated that malignant teratocarcinomas arising from induced pluripotent stem cell-derived cardiac tissue constructs provoked T cell-related host immune rejection to arrest tumour growth in murine allogeneic transplantation models.
Project description:OBJECTIVE:To assess class I-histone deacetylase (HDAC) inhibition on formation of lipid-accumulating, disease-promoting phagocytes upon myelin load in vitro, relevant for neuroinflammatory disorders like multiple sclerosis (MS) and cerebral X-linked adrenoleukodystrophy (X-ALD). METHODS:Immunohistochemistry on postmortem brain tissue of acute MS (n = 6) and cerebral ALD (n = 4) cases to analyze activation and foam cell state of phagocytes. RNA-Seq of in vitro differentiated healthy macrophages (n = 8) after sustained myelin-loading to assess the metabolic shift associated with foam cell formation. RNA-Seq analysis of genes linked to lipid degradation and export in MS-275-treated human HAP1 cells and RT-qPCR analysis of HAP1 cells knocked out for individual members of class I HDACs or the corresponding enzymatically inactive knock-in mutants. Investigation of intracellular lipid/myelin content after MS-275 treatment of myelin-laden human foam cells. Analysis of disease characteristic very long-chain fatty acid (VLCFA) metabolism and inflammatory state in MS-275-treated X-ALD macrophages. RESULTS:Enlarged foam cells coincided with a pro-inflammatory, lesion-promoting phenotype in postmortem tissue of MS and cerebral ALD patients. Healthy in vitro myelin laden foam cells upregulated genes linked to LXR?/PPAR? pathways and mimicked a program associated with tissue repair. Treating these cells with MS-275, amplified this gene transcription program and significantly reduced lipid and cholesterol accumulation and, thus, foam cell formation. In macrophages derived from X-ALD patients, MS-275 improved the disease-associated alterations of VLCFA metabolism and reduced the pro-inflammatory status of these cells. INTERPRETATION:These findings identify class I-HDAC inhibition as a potential novel strategy to prevent disease promoting foam cell formation in CNS inflammation.
Project description:Insulin-like growth factor-binding protein-3 (IGFBP-3) expression is frequently suppressed in liver cancers and can be reactivated by histone deacetylase (HDAC) inhibition. This study examined the role of IGFBP-3 in mediating the effects of the HDAC inhibitor MS-275 in liver cancer cells and identified IGFBP-3-dependent proteins that regulate proliferation and migration. In HepG2 cells, MS-275 inhibited DNA synthesis, cell cycle activity, and cell viability concomitantly with increased binding of acetylated histone H3 to IGFBP-3 promoter sequences and induction of IGFBP-3 expression. IGFBP-3 down-regulation by siRNA significantly reversed the inhibition of cell viability and DNA synthesis by MS-275, indicating an intermediary role for IGFBP-3. Induction of the cyclin-dependent kinase inhibitor p21 by MS-275 was attenuated by IGFBP-3 down-regulation, providing an explanation for IGFBP-3-dependent effects of MS-275 on cell cycle activity. In contrast, MS-275 stimulated HepG2 cell migration, an effect also inhibited by IGFBP-3 down-regulation. Among genes whose induction by MS-275 was attenuated by IGFBP-3 down-regulation, LYVE1 and THBS2 (thrombospondin-2) were identified as mediators of IGFBP-3-dependent effects of MS-275. Silencing of either protein had no effect on the inhibition of HepG2 viability by MS-275 but reversed its stimulatory effect on cell migration. We conclude that among genes up-regulated by MS-275, IGFBP-3 is a key mediator of effects on hepatoma cell growth and migration, involving IGFBP-3-dependent proteins p21 (proliferation) and LYVE1 and THBS2 (migration). The enhanced cell motility that accompanies reactivation of IGFBP-3 expression in liver cancer by HDAC inhibition suggests the possibility of increased metastatic spread despite inhibited cell proliferation.
Project description:New strategies for the treatment of alcohol dependence are a pressing need, and recent evidence suggests that targeting enzymes involved in epigenetic mechanisms seems to have great potential. Among these mechanisms, alteration of histone acetylation by histone deacetylases is of great importance for gene expression and has also been implicated in addiction. Here, we examined whether intra-cerebroventricular administration of MS-275, a class I-specific histone deacetylase inhibitor, could alter ethanol self-administration, motivation to consume ethanol, and relapse in heavy drinking rats.Male Long Evans rats trained to self-administer high levels of ethanol received intra-cerebroventricular micro-infusions of MS-275 (250 µM, 500 µM, and 1000 µM) 3 hours prior to the self-administration sessions.First, we demonstrated that intra-cerebroventricular infusion of MS-275 increases acetylation of Histone 4 within the nucleus accumbens nucleus accumbens and the dorsolateral striatum. Second, we observed that MS-275 decreases ethanol self-administration by about 75%. We found that 2 consecutive daily injections are necessary to decrease ethanol self-administration. Additionally, the dose-response curve test indicated that MS-275 has a U-shape effect on ethanol self-administration with the dose of 500 µM as the most efficient dose. Furthermore, we showed that MS-275 also diminished the motivation to consume ethanol (25% decrease), and finally, we demonstrated that MS-275 reduced relapse (by about 50%) and postponed reacquisition even when the treatment was stopped.Our study confirms the potential therapeutic interest of targeting epigenetic mechanisms in excessive alcohol drinking and strengthens the interest of focusing on specific isoforms of histone deacetylases.
Project description:Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes.