Induction Of Histone Acetylation Research Articles

Abstract LB-342: HDAC inhibition promotes tubulin-independent cancer cell migration

Abstract Breast cancer remains the most common cancer and second-leading cause of cancer-related death in women, with metastatic disease carrying a particularly poor prognosis. Despite advances with chemotherapeutic drugs aimed at tumor growth, we do not fully understand the effects of these drugs on the metastatic process. Histone deacetylase inhibitors (HDACi) are novel epigenetic therapies that induce cancer cell death through various mechanisms, including apoptosis, cell-cycle arrest and antiangiogenesis. Clinical trials investigating HDACi use in solid tumors are ongoing but have yielded mixed results. To date, very few studies have examined how HDACi influence the metastatic potential of breast tumor cells. Since HDAC enzymes act on both histone and non-histone targets, HDACi drugs cause dramatic increases in non-histone protein acetylation, including acetylation of alpha-tubulin. A well-known marker of stable microtubules, tubulin acetylation increases in metastatic breast tumors and predicts poor patient survival. However, the effects of HDACi on tubulin-related cytoskeletal dynamics of tumor cells have not been previously examined. The current study investigates whether the use of HDACi in breast cancer cells could promote metastatic phenotypes. Clinically-relevant HDAC pan-inhibitors that target multiple Class I HDAC enzymes (Entinostat) or both Class I and Class II (FDA-approved Vorinostat) yielded strong and rapid increases in the migration of both MCF-7 and Bt-549 breast cancer cells. In addition, Vorinostat and Entinostat were each able to strongly induce histone acetylation, while only Vorinostat induced tubulin acetylation. To determine if the increased tubulin acetylation induced by HDACi drugs was sufficient to induce tumor cell migration, we used an isotype-specific HDACi (Tubastatin) that selectively targets the Class II HDAC6 and therefore can increase tubulin acetylation. At doses which strongly induce tubulin acetylation, Tubastatin did not increase tumor cell migration or other tubulin-dependent cancer cell processes, including reattachment and microtentacle formation. Vorinostat and Entinostat therefore induce tumor cell migration through a tubulin-independent mechanism, indicating that more precise targeting of HDAC enzymes could help avoid therapeutic approaches that induce metastatic phenotypes. Citation Format: Lindsay K. Hessler, Amanda E. Boggs, Rebecca A. Whipple, Michele I. Vitolo, Kristi R. Chakrabarti, Lekhana Bhandary, Keyata N. Thompson, Stuart S. Martin. HDAC inhibition promotes tubulin-independent cancer cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-342.

BRD4 regulates fructose-inducible lipid accumulation-related genes in the mouse liver

ObjectiveFructose intake induces hepatic steatosis by activating fat synthesis. In this study, we searched for genes that showed acute induction in the livers of mice force-fed with fructose, and examined how this induction is regulated. Materials/MethodsWe identified genes induced at 6h after the fructose force-feeding using a microarray and quantitative real-time RT-PCR. Histone acetylation and an acetylated histone binding protein bromodomain containing (BRD)4 binding around the fructose-inducible genes were examined using a chromatin immunoprecipitation assay. We examined whether (+)-JQ1, an inhibitor of the binding between the BRD4 and acetylated histones, inhibited the expressions of fructose-inducible genes, histone acetylation and BRD4 binding around the genes. ResultsWe identified upregulated genes related to lipid accumulation, such as Cyp8b1, Dak and Plin5, in mice force-fed with fructose compared with those force-fed with glucose. Acetylation of histones H3 and H4, and BRD4 binding around the transcribed region of those fructose-inducible genes, were enhanced by fructose force-feeding. Meanwhile, (+)-JQ1 treatment reduced expressions of fructose-inducible genes, histone acetylation and BRD4 binding around these genes. ConclusionsAcute induction of genes related to lipid accumulation in the livers of mice force-fed with fructose is associated with the induction of histone acetylation and BRD4 binding around these genes.

In vivo analysis of the effect of panobinostat on cell-associated HIV RNA and DNA levels and latent HIV infection.

BackgroundThe latent reservoir in resting CD4+ T cells presents a major barrier to HIV cure. Latency-reversing agents are therefore being developed with the ultimate goal of disrupting the latent state, resulting in induction of HIV expression and clearance of infected cells. Histone deacetylase inhibitors (HDACi) have received a significant amount of attention for their potential as latency-reversing agents.ResultsHere, we have investigated the in vitro and systemic in vivo effect of panobinostat, a clinically relevant HDACi, on HIV latency. We showed that panobinostat induces histone acetylation in human PBMCs. Further, we showed that panobinostat induced HIV RNA expression and allowed the outgrowth of replication-competent virus ex vivo from resting CD4+ T cells of HIV-infected patients on suppressive antiretroviral therapy (ART). Next, we demonstrated that panobinostat induced systemic histone acetylation in vivo in the tissues of BLT humanized mice. Finally, in HIV-infected, ART-suppressed BLT mice, we evaluated the effect of panobinostat on systemic cell-associated HIV RNA and DNA levels and the total frequency of latently infected resting CD4+ T cells. Our data indicate that panobinostat treatment resulted in systemic increases in cellular levels of histone acetylation, a key biomarker for in vivo activity. However, panobinostat did not affect the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells.ConclusionWe have demonstrated robust levels of systemic histone acetylation after panobinostat treatment of BLT humanized mice; and we did not observe a detectable change in the levels of cell-associated HIV RNA, HIV DNA, or latently infected resting CD4+ T cells in HIV-infected, ART-suppressed BLT mice. These results are consistent with the modest effects noted in vitro and suggest that combination therapies may be necessary to reverse latency and enable clearance. Animal models will contribute to the progress towards an HIV cure.

Distinct Activities of Myf5 and MyoD Indicate Separate Roles in Skeletal Muscle Lineage Specification and Differentiation

Most transcription factor families contain highly related paralogs generated by gene duplication, and functional divergence is generally accomplished by activation of distinct sets of genes by each member. Here we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find that MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits Pol II, and robustly activates gene transcription. Therefore, the initial specification of the muscle lineage by Myf5 occurs without significant induction of gene transcription. Transcription of the skeletal muscle program is then achieved by the subsequent expression of MyoD, which binds to the same sites as Myf5, indicating that each factor regulates distinct steps in gene initiation and transcription at a shared set of binding sites.

Droxinostat, a Histone Deacetylase Inhibitor, Induces Apoptosis in Hepatocellular Carcinoma Cell Lines via Activation of the Mitochondrial Pathway and Downregulation of FLIP

Background: The current chemotherapeutic outcomes for hepatocellular carcinoma (HCC) are not encouraging, and long-term survival of this patient group remains poor. Recent studies have demonstrated the utility of histone deacetylase inhibitors that can disrupt cell proliferation and survival in HCC management. However, the effects of droxinostat, a type of histone deacetylase inhibitor, on HCC remain to be established. Methods: The effects of droxinostat on HCC cell lines SMMC-7721 and HepG2 were investigated. Histone acetylation and apoptosis-modulating proteins were assessed via Western blot. Proliferation was examined with 3-(4, 5 dimetyl-2-thiazolyl)-2, 5-diphenyl 2H-tetrazolium bromide, cell proliferation, and real-time cell viability assays, and apoptosis with flow cytometry. Results: Droxinostat inhibited proliferation and colony formation of the HCC cell lines examined. Hepatoma cell death was induced through activation of the mitochondrial apoptotic pathway and downregulation of FLIP expression. Droxinostat suppressed histone deacetylase (HDAC) 3 expression and promoted acetylation of histones H3 and H4. Knockdown of HDAC3 induced hepatoma cell apoptosis and histone H3 and H4 acetylation. Conclusions: Droxinostat suppresses HDAC3 expression and induces histone acetylation and HCC cell death through activation of the mitochondrial apoptotic pathway and downregulation of FLIP, supporting its potential application in the treatment of HCC.

Thymoquinone Promotes Pancreatic Cancer Cell Death and Reduction of Tumor Size through Combined Inhibition of Histone Deacetylation and Induction of Histone Acetylation.

Pancreatic ductal adenocarcinoma (PDAC) is virtually therapy-resistant. As noninvasive lesions progress to malignancy, the precursor period provides a window for cancer therapies that can interfere with neoplastic progression. Thymoquinone (Tq), a major bioactive component of essential oil from Nigella sativa's seeds, has demonstrated antineoplastic activities in multiple cancers. In this study, we investigated antineoplastic potential of Tq in human PDAC cell lines, AsPC-1 and MiaPaCa-2. Tq (10–50 μM) inhibited cell viability and proliferation and caused partial G2 cycle arrest in dose-dependent manner in both cell lines. Cells accumulated in subG0/G1 phase, indicating apoptosis. This was associated with upregulation of p53 and downregulation of Bcl-2. Independently of p53, Tq increased p21 mRNA expression 12-fold. Tq also induced H4 acetylation (lysine 12) and downregulated HDACs activity, reducing expression of HDACs 1, 2, and 3 by 40–60%. In vivo, Tq significantly reduced tumor size in 67% of established tumor xenografts (P < 0.05), along with increased H4 acetylation and reduced HDACs expression. Our results showed that Tq mediated posttranslational modification of histone acetylation, inhibited HDACs expression, and induced proapoptotic signaling pathways. These molecular targets demonstrate rationale for using Tq as a promising antineoplastic agent to prevent postoperative cancer recurrence and to prolong survival of PDAC patients after surgical resection.

Acute Ethanol Administration Increases Mir-124 Expression via Histone Acetylation in the Brain

Acute ethanol administration causes an enhancement of mesolimbic dopaminergic activity in the brain. The microRNAs miR-124 and miR-132 are highly expressed in the central nervous system and contribute to the regulation of mesolimbic dopaminergic activity. However, the nature of a potential mechanistic association between ethanol and these microRNAs has remained unclear. Recent studies reported that the expression of some microRNAs was regulated by histone acetylation. In this study, we investigated whether ethanol administration can change the expression of miR-124 and miR-132 via histone acetylation in mouse brain. We administered 2 g/kg ethanol (s.c.) to male C57BL / 6J mice. Total RNA was extracted using TRIzol in the whole brain 1 to 12 hr after administration. MicroRNAs were detected by northern blotting. Acetylated histone H3 and histone deacetylases (HDACs) were detected by western blotting analysis and immunohistochemistry. Trichostatin A (TSA) was administered intracerebroventricularly. Acute ethanol administration increased expression of miR-124 and miR-132 in the mouse whole brain 12 hr after ethanol administration. Acute ethanol administration significantly increased acetylated histone H3 in the nuclear fraction of mouse whole brain, especially the nucleus accumbens and ventral tegmental area. HDAC4 expression was significantly decreased in the nuclear fraction after acute ethanol administration. These findings suggest that acute administration induces histone acetylation by reducing nuclear HDAC4 expression. TSA treatment increased the expression of miR-124 in mouse whole brain. These findings suggest that HDAC4 may directly regulate the expression of miR-124 in the brain. In conclusion, acute ethanol administration induces histone acetylation via reducing nuclear HDAC4 protein, resulting in increased microRNA expression in the brain.

Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Background:Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism.Methods:We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1).Results:Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells.Conclusion:These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic stability in the adult mouse cortex under conditions of pharmacologically induced histone acetylation.

Histone acetylation is considered a major epigenetic process that affects brain development and synaptic plasticity, as well as learning and memory. The transcriptional effectors and morphological changes responsible for plasticity as a result of long-term modifications to histone acetylation are not fully understood. To this end, we pharmacologically inhibited histone deacetylation using Trichostatin A in adult (6-month-old) mice and found significant increases in the levels of the acetylated histone marks H3Lys9, H3Lys14 and H4Lys12. High-resolution transcriptome analysis of diverse brain regions uncovered few differences in gene expression between treated and control animals, none of which were plasticity related. Instead, after increased histone acetylation, we detected a large number of novel transcriptionally active regions, which correspond to long non-coding RNAs (lncRNAs). We also surprisingly found no significant changes in dendritic spine plasticity in layers 1 and 2/3 of the visual cortex using long-term in vivo two-photon imaging. Our results indicate that chronic pharmacologically induced histone acetylation can be decoupled from gene expression and instead, may potentially exert a post-transcriptional effect through the differential production of lncRNAs.

Epigenetic Modifications of Histones in Periodontal Disease

Periodontitis is a chronic infectious disease driven by dysbiosis, an imbalance between commensal bacteria and the host organism. Periodontitis is a leading cause of tooth loss in adults and occurs in about 50% of the US population. In addition to the clinical challenges associated with treating periodontitis, the progression and chronic nature of this disease seriously affect human health. Emerging evidence suggests that periodontitis is associated with mechanisms beyond bacteria-induced protein and tissue degradation. Here, we hypothesize that bacteria are able to induce epigenetic modifications in oral epithelial cells mediated by histone modifications. In this study, we found that dysbiosis in vivo led to epigenetic modifications, including acetylation of histones and downregulation of DNA methyltransferase 1. In addition, in vitro exposure of oral epithelial cells to lipopolysaccharides resulted in histone modifications, activation of transcriptional coactivators, such as p300/CBP, and accumulation of nuclear factor–κB (NF-κB). Given that oral epithelial cells are the first line of defense for the periodontium against bacteria, we also evaluated whether activation of pathogen recognition receptors induced histone modifications. We found that activation of the Toll-like receptors 1, 2, and 4 and the nucleotide-binding oligomerization domain protein 1 induced histone acetylation in oral epithelial cells. Our findings corroborate the emerging concept that epigenetic modifications play a role in the development of periodontitis.

Abstract 1771: Metabolic reprogramming in KRAS mutant cancer cells may cause sensitivity to the histone deacetylase (HDAC) inhibitor romidepsin

Abstract Ras pathway mutations are very common in cancer, with the highest prevalence for RAS itself, found in about 30% of tumors. Despite multiple efforts, targeting the RAS pathway has not yet been amenable to therapeutic intervention. Taking a different approach, our laboratory has focused on combination treatment with romidepsin, an HDAC inhibitor approved by the FDA to treat T-cell lymphoma. Our group indeed demonstrated perturbation of RAS downstream pathways after romidepsin treatment, by analyzing gene expression in samples obtained from patients treated with romidepsin for T-cell lymphoma (Chakraborty et al, Blood 2013, 121: 4115). The NCI-60 panel was screened with romidepsin plus or minus RAS pathway downstream inhibitors (MEK and/or Akt inhibitors). We found that mutations in RTK/RAS/BRAF correlated with romidepsin sensitivity (R = 0.472), with cell death ranging from 40% to 95% after 48h (assayed by annexin V labeling). Three KRAS mutant and three KRAS WT cell lines were then chosen to investigate the mechanism of this selectivity. Romidepsin induced histone acetylation and DNA damage from 30min treatment, and an increase in reactive oxygen species (ROS) after 10h exposure, independent of KRAS status. Only KRAS mutant cells showed mitochondrial membrane depolarization, cytochrome c release, and apoptosis, all starting about 24h after the beginning of treatment. Interestingly, cytoplasmic - but not mitochondrial - ROS scavengers rescued KRAS mutant cells from death, suggesting that ROS is required but not sufficient for romidepsin-induced apoptosis. Since Son et al (Nature 2013, 496: 101) reported that glutamine metabolism was reprogrammed by oncogenic KRAS to maintain redox balance, we explored this pathway using the Seahorse XF96 analyzer. We observed that, while all cell lines utilized glutamine for oxidative phosphorylation (OXPHOS) before drug exposure, romidepsin impaired the ability of only KRAS mutant cells to use glutamine for OXPHOS. Metabolomic analysis confirmed reduced glutamine use in KRAS mutant cells after drug treatment, as well as an inability to replenish their reduced glutathione stock. Moreover, this analysis also highlighted a romidepsin-induced drop in nucleotide synthesis in KRAS mutant cells, potentially leading to impaired DNA repair. Accordingly, adding back nucleosides to media blunted the effect of romidepsin on cell growth in KRAS mutant, but not in KRAS WT cells. From these results, we conclude that romidepsin causes cell damage through DNA damage and subsequent ROS release. And while KRAS WT cells are able to scavenge ROS and repair DNA breaks, KRAS mutant cells cannot. We hypothesize that the reprogramming of glutamine metabolism, which supports redox maintenance and may support nucleoside synthesis, makes KRAS mutant cells particularly sensitive to romidepsin treatment. Citation Format: Agnes Basseville, Carole Sourbier, Robert W. Robey, Dan L. Sackett, W. Marston Linehan, Susan E. Bates. Metabolic reprogramming in KRAS mutant cancer cells may cause sensitivity to the histone deacetylase (HDAC) inhibitor romidepsin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2015-1771

Abstract B40: Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma

Abstract Deregulation of cellular epigenetics is essential for malignant transformation. However, the mechanisms that cause epigenetic alterations in cancer cells are incompletely understood. Recent evidence has shown that cellular metabolism has a direct impact on the epigenome, since many chromatin-modifying enzymes rely on intracellular metabolites as cofactors or donor substrates. We have previously shown that the metabolic enzyme ATP-citrate lyase (ACLY), which generates nuclear-cytoplasmic acetyl-CoA from glucose, is required for maintaining histone acetylation levels in multiple mammalian cell types, suggesting that alterations in acetyl-CoA metabolism in cancer cells might also impact histone acetylation levels. Cellular metabolism is massively rewired in multiple cancer types, including pancreatic cancer, although the impact of metabolic alterations on the tumor epigenome is poorly understood. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. In this study, we demonstrate that acetyl-CoA abundance in cancer cells is dynamically regulated by glucose availability and that histone acetylation levels are responsive to the ratio of acetyl-CoA:coenzyme A within the nucleus. To test whether oncogene activation could mediate changes in histone acetylation in vivo, we performed immunohistochemical analysis comparing pancreata from mice expressing KrasG12D with those from mice with WT Kras. Whereas the acinar cells of WT mice exhibited very low levels of histone H4 (K5/8/12/16) acetylation, in KPC (LSL-KrasG12D; p53L/+; Pdx1-Cre) mice, acinar H4 acetylation was markedly increased, prior to the appearance of histological abnormalities or aberrant cell proliferation. High H4 acetylation persisted in pancreatic intraepithelial neoplasia (PanIN) and PDA. Histone acetylation in PanIN-derived primary cells was selectively impaired by PI3K and Akt inhibitors, correlating with suppression of glucose consumption and cellular acetyl-CoA levels. Moreover, addition of supraphysiological doses of acetate, a source of acetyl-CoA alternative to glucose, restored histone acetylation levels. These data suggest that oncogenic Kras promotes elevated histone acetylation preceding tumor development through Akt-dependent regulation of cellular acetyl-CoA levels. Further mechanistic analysis suggests that Akt promotes elevated histone acetylation through combined effects on promoting glucose uptake and phosphorylation and activation of ATP-citrate lyase, a metabolic enzyme that produces nuclear-cytoplasmic acetyl-CoA. The aberrant activation of the PI3K/Akt pathway occurs in broad variety of human malignancies. pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors, suggesting that PI3K-Akt-dependent promotion of acetyl-CoA metabolism may contribute to histone acetylation levels in multiple cancer types. Our data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in tumors and offer novel insights on Kras-induced pancreatic carcinogenesis. Citation Format: Alessandro Carrer, Joyce V. Lee, Supriya Shah, Nathaniel W. Snyder, Ellen Jackson, Nicole M. Aiello, Benjamin A. Garcia, Lewis A. Chodosh, Ben Z. Stanger, Ian A. Blair, Kathryn E. Wellen. Oncogenic Kras induces histone acetylation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B40.

Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

Osteogenic Gene Transcription Is Regulated via Gap Junction-Mediated Cell–Cell Communication

An analytical study of cell-cell communications between murine osteoblast-like MLO-A5 cells and bone marrow mesenchymal stem cell (BMSC)-like C3H10T1/2 cells was performed. C3H10T1/2 cells expressing green fluorescent protein (10T-GFP cells) were generated to enable the isolation of the BMSC-like cells from co-cultures with MLO-A5 cells. The mRNA expression levels of several osteogenic transcription factors (Runx2, Osterix, Dlx5, and Msx2) did not differ between the co-cultured and mono-cultured 10T-GFP cells, but those of alkaline phosphatase (ALP) and bone sialoprotein (BSP) were 300- to 400-fold higher in the co-cultured cells. Patch clamp and biocytin transfer assays revealed gap junction-mediated communication between co-cultured 10T-GFP and MLO-A5 cells. The addition of a gap junction inhibitor suppressed the increases in the expression levels of the ALP and BSP mRNAs in co-cultured 10T-GFP cells. Furthermore, the histone acetylation levels were higher in co-cultured 10T-GFP cells than in mono-cultured 10T-GFP cells. These results suggest that osteoblasts and BMSCs associate via gap junctions, and that gap junction-mediated signaling induces histone acetylation that leads to elevated transcription of the genes encoding ALP and BSP in BMSCs.

Romidepsin Synergizes with Lenalidomide in T Cell Lymphoma Cell Lines By Increasing Reactive Oxygen Species and Modulating PI3K/AKT and MAPK/ERK Signaling Pathways

Abstract Background and purpose.T-cell lymphoma (TCL) is an heterogeneous and aggressive group of lymphoproliferative disorders which commonly relapse with current chemotherapy regimens and then difficult to treat. Therefore, novel treatment approach are urgently needed. Romidepsin is a potent and specific inhibitor of class I and II HDAC inhibitor that has shown remarkable activity in the treatment of TCL in preclinical studies and early-phase clinical trials. Lenalidomide (IMiD), is an immunomodulatory agent highly effective for the treatment of a wide range of hematological malignancies with a low toxicity profile. In this study, we sought to define the potential anticancer effect of romidepsin (Celgene) in combination with lenalidomide (Celgene) in TCL preclinical model. Methods . The TCL cell lines Hut-78 and Karpas-299 were treated with increasing concentrations of romidepsin (0,5-25 nM) and lenalidomide (1-100 µM) as single agents from 24-72h to identify the IC50 of each drug. The interaction between romidepsin (0,5nM, 1nM, 2,5nM) and lenalidomide (2µM, 4µM, 10µM) was evaluated using the Chou-Talalay method. The cell proliferation was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. The cell cytotoxicity was quantified by MTT. Clonogenic survival was studied with the methylcellulose clonogenic assay. Apoptosis, reactive oxygen species (ROS), cell cycle analysis and Bcl-2 protein expression were measured by flow cytometry, whereas caspase activation, AKT/PI3K and MAPK/ERK pathways were analyzed by Western blot. Results. Treatment with romidepsin alone resulted in time- and dose-dependent increase in cytotoxicity in Hut-78 and Karpas-299 cell lines with an IC50 at 24h of 5.87 nM and 6.36 nM for Hut-78 and Karpas-299, respectively. Lenalidomide alone did not inhibit cells viability up to72 h of treatment in the two TCL cell lines, as we previously described in other cell lines probably due to a more pleitropic (or cytostatic) effects. However, the combination of lenalidomide (10µM) with a low dose of romidepsin (2.5 nM) showed a strong synergistic interaction with combination index (CI) of 0.14 in Hut-78 cells and an additive effect in Karpas-299 cells, (CI of 1.08) after 24h. In Hut-78 cells sequential treatment with romidespin for 6h followed by washout and the next addition of lenalidomide for 24h enhanced the cytotoxic effect of romidepsin and confirmed its irreversible effect. Treatment with romidepsin and lenalidomide for 24 h reduced clonogenic survival to approximately 15 % in HUT-78 and to 28 % in Karpas-299. The combination did not trigger relevant decreased in the viability of normal peripheral blood mononuclear cells and suppressed cell proliferation of TCL cell lines when co-cultured with bone marrow mesenchymal stromal cells in a system that mimics the bone marrow microenvironment. Romidepsin and lenalidomide in combination induced apoptosis with little effect on cell cycle. Interestingly, the treatment with the caspase inhibitor z-VAD-fmk allowed detection of cell cycle arrest in G0/G1 phase and a decrease of S phase. The induction of apoptosis by drugs combination was associated with an increase in ROS production. The co-administration of the antioxidant N-acetyl cysteine (NAC) blocked the increased ROS generation and apoptosis observed in the drugs combination compared with control or single-agent treatments. Cell death was associated with the activation of caspases -3, -9 -8 and PARP and was mediated by Bim, Bax, Bad, Bcl-xL and Mcl-1 but did not modify Bcl-2 expression. The drugs combination induced up-regulation of cell cycle protein p21, p27 and a slight decrease of cyclin E and cyclin D. These events were associated with the dephosphorylation of PI3K/Akt and MAPK/ERK pathways. Finally, the induction of histone acetylation (H3) and acetylated alpha-tubulin was confirmed. Conclusion . The combination of romidepsin with lenalidomide triggered cell death through interacting mechanisms including ROS generation, caspase activation and down-regulation of PI3K/AKT and MAPK/ERK pathways. These results provided a rational basis for the clinical development of the combination of romidepsin and lenalidomide for the treatment of TCL Disclosures No relevant conflicts of interest to declare.

Epigenetic activation of the Foxa2 gene is required for maintaining the potential of neural precursor cells to differentiate into dopaminergic neurons after expansion.

Dysregulation of forkhead box protein A2 (Foxa2) expression in fetal ventral mesencephalon (VM)-derived neural precursor cells (NPCs) appears to be associated with the loss of their potential to differentiate into dopaminergic (DA) neurons after mitogenic expansion in vitro, hindering their efficient use as a transplantable cell source. Here, we report that epigenetic activation of Foxa2 in VM-derived NPCs by inducing histone hyperacetylation rescues the mitogenic-expansion-dependent decrease of differentiation potential to DA neurons. The silencing of Foxa2 gene expression after expansion is accompanied by repressive histone modifications, including hypoacetylation of histone H3 and H4 and trimethylation of H3K27 on the Foxa2 promoter, as well as on the global level. In addition, histone deacetylase 7 (HDAC7) is highly expressed during differentiation and recruited to the Foxa2 promoter. Induction of histone acetylation in VM-derived NPCs by either knockdown of HDAC7 or treatment with the HDAC inhibitor apicidin upregulates Foxa2 expression via hyperacetylation of H3 and a decrease in H3K27 trimethylation on the promoter regions, leading to the expression of DA neuron developmental genes and enhanced differentiation of DA neurons. These effects are antagonized by the expression of shRNAs specific for Foxa2 but enhanced by shRNA for HDAC7. Collectively, these findings indicate that loss of differentiation potential of expanded VM-derived NPCs is attributed to a decrease in Foxa2 expression and suggest that activation of the endogenous Foxa2 gene by epigenetic regulation might be an approach to enhance the generation of DA neurons.

The histone deacetylase inhibitor trichostatin A induces neurite outgrowth in PC12 cells via the epigenetically regulated expression of the nur77 gene

Histone deacetylase (HDAC) inhibitors induce histone acetylation and gene expression by changing local chromatin structures. They can thereby influence various cells to proliferate or differentiate. It has been reported that trichostatin A (TSA) or valproic acid (VPA) can induce the neuronal differentiation of mouse embryonic neural stem cells and rat cerebellar granule cells. It is unclear however which gene is responsible for the neuronal differentiation induced by HDAC inhibitors. In this study, we investigated the contribution of immediate early gene (IEG) nur77 to the neuronal differentiation induced by TSA. We report that TSA induces neurite outgrowth in PC12 cells, and C646, an inhibitor of HAT (histone acetyl transferase) (p300), prevents TSA-induced neurite formation. The acetylation of the Lys14 residue of histone H3, and mRNA and protein expression of nur77 gene were found to be stimulated after treatment with TSA, but not in the presence of C646. A knock-down of nur77 inhibits the neurite outgrowth induced by TSA. Furthermore, the ectopic expression of nur77 significantly elicits neurite formation in PC12 cells. These results suggest that the expression of nur77, which is up-regulated via the TSA-induced acetylation of Lys14 on histone H3, is essential for the neuronal differentiation in TSA-induced PC12 cells.

A novel class I HDAC inhibitor, MPT0G030, induces cell apoptosis and differentiation in human colorectal cancer cells via HDAC1/PKCδ and E-cadherin.

Accumulation of genetic and epigenetic changes contributes to cancer development and progression. Compared with gene mutations or deletions, epigenetic changes are reversible, which alter the chromatin structure remodeling instead of changes in DNA sequence, and therefore become a promising strategy for chemotherapy. Histone deacetylases (HDACs) are a class of enzymes that responsible for the epigenetic regulation of gene expression. MPT0G030 is a potent and selective class I HDAC inhibitor which showed broad-spectrum cytotoxicity against various human cancer cell lines. in vitro fluorometric HDAC activity assay showed that MPT0G030 effectively inhibited Class I HDACs (HDAC1~3), which were overexpressed in many malignant neoplasms. Interestingly, MPT0G030 not only induced histone acetylation and tumor suppressor p21 transcription, but also redistributed E-cadherin and activated Protein Kinase C δ (PKCδ), which was linked to cell apoptosis and differentiation. Further, activation of PKCδ was demonstrated to be modulated through HDAC1. The in vivo anticancer activity of MPT0G030 and the importance of PKCδ were confirmed in the HT-29 tumor xenograft models. Taken together, those results indicate that MPT0G030, a class I HDAC inhibitor, has great potential as a new drug candidate for cancer therapy.

Acetate treatment increases fatty acid content in LPS-stimulated BV2 microglia.

Acetate supplementation increases plasma acetate, brain acetyl-CoA, histone acetylation, phosphocreatine levels, and is anti-inflammatory in models of neuroinflammation and neuroborreliosis. Although radiolabeled acetate is incorporated into the cellular lipid pools, the effect that acetate supplementation has on lipid deposition has not been quantified. To determine the impact acetate-treatment has on cellular lipid content, we investigated the effect of acetate in the presence of bacterial lipopolysaccharide (LPS) on fatty acid, phospholipid, and cholesterol content in BV2 microglia. We found that 1, 5, and 10 mM of acetate in the presence of LPS increased the total fatty acid content in BV2 cells by 23, 34, and 14 % at 2 h, respectively. Significant increases in individual fatty acids were also observed with all acetate concentrations tested with the greatest increases occurring with 5 mM acetate in the presence of LPS. Treatment with 5 mM acetate in the absence of LPS increased total cholesterol levels by 11 %. However, neither treatment in the absence of LPS significantly altered the content of individual phospholipids or total phospholipid content. To determine the minimum effective concentration of acetate we measured the time- and concentration-dependent changes in histone acetylation using western blot analysis. These studies showed that 5 mM acetate was necessary to induce histone acetylation and at 10 mM acetate, the histone acetylation-state increased as early as 0.5 h following the start of treatment. These data suggest that acetate increases fatty acid content in LPS-stimulated BV2 microglia that is reflected by an increase in fatty acids esterified into membrane phospholipids.

Thalidomide dithiocarbamate and dithioate derivatives induce apoptosis through inhibition of histone deacetylases and induction of caspases

Anti-cancer effect and mechanism of cell death were investigated in a battery of five thalidomide analogs containing one sulfur atom 2 or two sulfur atoms 3–6 and were compared with thalidomide 1 activity. The cytotoxic effect of thalidomide analogs 2–6 against Hep-G2, 1301, and HCT-116 cells was estimated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis and necrosis cell percentage was stained by ethidium bromide and acridine orange, DNA fragmentation, inhibition of histone deacetylase (HDAC), and total caspases were assayed by universal procedures and kits. We report here for the anti-cancer activity of thalidomide dithiocarbamate analog 3 and thalidomide dithioate analog 5 against Hep-G2 and HCT-116 cells, which was more cytotoxic than thalidomide itself, and that the cytotoxicity was associated with DNA fragmentation and was due to apoptosis and not necrosis. Moreover, we suggest that the cell death pathway is evoked by thalidomide dithiocarbamate analog 3 and thalidomide dithioate analog 5 in human hepatocellular carcinoma cells through multiple consequences that trigger apoptotic cell death; involving the enhancement of DNA fragmentation, the activation of caspases, and the induction of histone acetylation. In conclusion, thalidomide dithiocarbamate analog 3 and thalidomide dithioate analog 5 are promising anti-cancer agents more than thalidomide.