CUDC-101 as a dual-target inhibitor of EGFR and HDAC enhances the anti-myeloma effects of bortezomib by regulating G2/M cell cycle arrest / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

CUDC-101, an effective and multi-target inhibitor of epidermal growth factor receptor (EGFR), histone deacetylase (HDAC), and human epidermal growth factor receptor 2 (HER2), has been reported to inhibit many kinds of cancers, such as acute promyelocytic leukemia and non-Hodgkin's lymphoma. However, no studies have yet investigated whether CUDC-101 is effective against myeloma. Herein, we proved that CUDC-101 effectively inhibits the proliferation of multiple myeloma (MM) cell lines and induces cell apoptosis in a time- and dose-dependent manner. Moreover, CUDC-101 markedly blocked the signaling pathway of EGFR/phosphoinositide-3-kinase (PI3K) and HDAC, and regulated the cell cycle G2/M arrest. Moreover, we revealed through in vivo experiment that CUDC-101 is a potent anti-myeloma drug. Bortezomib is one of the important drugs in MM treatment, and we investigated whether CUDC-101 has a synergistic or additive effect with bortezomib. The results showed that this drug combination had a synergistic anti-myeloma effect by inducing G2/M phase blockade. Collectively, our findings revealed that CUDC-101 could act on its own or in conjunction with bortezomib, which provides insights into exploring new strategies for MM treatment.

Romidepsin (FK228) improves the survival of allogeneic skin grafts through downregulating the production of donor-specific antibody via suppressing the IRE1α-XBP1 pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Antibody-mediated rejection (AMR) is one of the major causes of graft loss after transplantation. Recently, the regulation of B cell differentiation and the prevention of donor-specific antibody (DSA) production have gained increased attention in transplant research. Herein, we established a secondary allogeneic in vivo skin transplant model to study the effects of romidepsin (FK228) on DSA. The survival of grafted skins was monitored daily. The serum levels of DSA and the number of relevant immunocytes in the recipient spleens were evaluated by flow cytometry. Then, we isolated and purified B cells from B6 mouse spleens in vitro by magnetic bead sorting. The B cells were cultured with interleukin-4 (IL-4) and anti-clusters of differentiation 40 (CD40) antibody with or without FK228 treatment. The immunoglobulin G1 (IgG1) and IgM levels in the supernatant were evaluated by enzyme-linked immunosorbent assay (ELISA). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were conducted to determine the corresponding levels of messenger RNA (mRNA) and protein expression in cultured cells and the recipient spleens. The results showed that FK228 significantly improved the survival of allogeneic skin grafts. Moreover, FK228 inhibited DSA production in the serum along with the suppression of histone deacetylase 1 (HADC1) and HDAC2 and the upregulation of the acetylation of histones H2A and H3. It also inhibited the differentiation of B cells to plasma cells, decreased the transcription of positive regulatory domain-containing 1 (Prdm1) and X-box-binding protein 1 (Xbp1), and decreased the expression of phosphorylated inositol-requiring enzyme 1 α (p-IRE1α), XBP1, and B lymphocyte-induced maturation protein-1 (Blimp-1). In conclusion, FK228 could decrease the production of antibodies by B cells via inhibition of the IRE1α-XBP1 signaling pathway. Thus, FK228 is considered as a promising therapeutic agent for the clinical treatment of AMR.

Histone Deacetylase Inhibitors in the in Vitro Expansion of Hematopoietic Stem Cells / 中国医学科学院学报

The self-renewal and differentiation of hematopoietic stem cells(HSCs)are highly regulated by epigenetic modification,in which histone acetylation can activate or silence gene transcription.Histone deacetylase inhibitors(HDACIs)can inhibit the activity of histone deacetylase in HSCs to increase histone acetylation.A variety of HDACIs,such as trichostatin A and valproic acid,are used to expand HSCs in vitro,especially cord blood HSCs,combined with cytokines in serum-free culture to obtain more long-term repopulating cells.HDACIs promote the transcription of pluripotent genes related to stem cell self-renewal and inhibit the expression of genes related to differentiation,so as to promote the expansion and inhibit differentiation of HSCs.The expansion of cord blood HSCs by small molecular HDACIs in vitro is expected to improve the quantity of cord blood HSCs.The further research will focus on high-throughput screening for the most powerful HDACIs and the highly selective HDACIs,exploring the combination of epigenetic modifiers of different pathways.

The Role of Histone Deacetylase Inhibitors in Regulation of Akt/GSK-3ß Signaling Pathway in Mice Following Transient Focal Cerebral Ischemia

Abstract Purpose: To investigate whether the neuroprotective effect of TSA on cerebral ischemia reperfusion injury is mediated by the activation of Akt/GSK-3β signaling pathway. Methods: Mice were randomly divided into four groups (n=15): sham group (S); ischemia reperfusion group (IR); ischemia reperfusion and pretreated with TSA group (IR+T); ischemia reperfusion and pretreated with TSA and LY294002 group (IR+T+L). The model of cerebral ischemia reperfusion was established by 1h of MCAO following 24h of reperfusion. TSA (5mg/kg) was intraperitoneally given for 3 days before MCAO, Akt inhibitor, LY294002 (15 nmol/kg) was injected by tail vein 30 min before the MCAO. Results: TSA significantly increased the expression of p-Akt, p-GSK-3β proteins and the levels of SOD, Bcl-2, reduced the infarct volume and the levels of MDA, ROS, TNF-α, IL-1β, Bax, Caspase-3, TUNEL and attenuated neurological deficit in mice with transient MCAO, LY294002 weakened such effect of TSA dramatically. Conclusions: TSA could significantly decrease the neurological deficit and reduce the cerebral infarct volume, oxidative stress, inflammation, as well as apoptosis during cerebral ischemia reperfusion injury, which was achieved by activation of the Akt/GSK-3β signaling pathway.

Measurements of body fat distribution: assessment of collinearity with body mass, adiposity and height in female adolescents / Medidas de localização da gordura corporal: uma avaliação da colinearidade com massa corporal, adiposidade e estatura em adolescentes do sexo feminino

OBJECTIVE : To verify the correlation between body fat location measurements with the body mass index (BMI), body fat percentage (BF%) and height, according to the nutritional status in female adolescents. METHODS : A controlled cross-sectional study was carried out with 113 adolescents (G1: 38 with normal weight, but with high body fat level, G2: 40 with normal weight and G3: 35 overweight) from public schools in Viçosa-MG, Brazil. The following measures were assessed: weight, height, waist circumference (WC), umbilical circumference (UC), hip circumference (HC), thigh circumference, waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), waist-to-thigh ratio (WTR), conicity index (CI), sagittal abdominal diameter (SAD), coronal diameter (CD), central (CS) and peripheral skinfolds (PS). The BF% was assessed by tetrapolar electric bioimpedance. RESULTS : The increase in central fat, represented by WC, UC, WHtR, SAD, CD and CS, and the increase in peripheral fat indicated by HC and thigh circumference were proportional to the increase in BMI and BF%. WC and especially the UC showed the strongest correlations with adiposity. Weak correlation between WHR, WTR, CI and CS/PS with adiposity were observed. The height showed correlation with almost all the fat location measures, being fair or weak with waist measurements. CONCLUSIONS : The results indicate colinearity between body mass and total adiposity with central and peripheral adipose tissue. We recommend the use of UC for assessing nutritional status of adolescents, as it showed the highest capacity to predict adiposity in each group, and also showed fair or weak correlation with height. .

OBJETIVO: Verificar a correlação entre medidas de localização da gordura corporal com índice de massa corporal (IMC), percentual de gordura corporal (%GC) e estatura, de acordo com o estado nutricional em adolescentes do sexo feminino. MÉTODOS: Realizou-se estudo transversal controlado, com 113 adolescentes (G1: 38 eutróficas mas com gordura corporal elevada; G2: 40 eutróficas e G3: 35 com excesso de peso), de 14 a 19 anos, de escolas públicas de Viçosa-MG. Aferiu-se peso, estatura, circunferência da cintura (CC), circunferência umbilical (CUm), circunferência do quadril (CQ), circunferência da coxa, relação cintura/quadril (RCQ), relação cintura/estatura (RCE), relação cintura/coxa (RCC), índice de conicidade (IC), diâmetro abdominal sagital (DAS), diâmetro coronal (DC), pregas cutâneas centrais (PCC) e periféricas (PCP). Avaliou-se o %GC por bioimpedância elétrica tetrapolar. RESULTADOS: O aumento da gordura central, representada pela CC, CUm, RCE, DAS, DC e PCC, e o aumento da gordura periférica indicado pela CQ e da coxa foram proporcionais ao aumento do IMC e %GC. A CC e principalmente CUm apresentaram as correlações mais fortes com a adiposidade, enquanto RCQ, RCC, IC e PCC/PCP as mais fracas. A estatura apresentou correlação com praticamente todas as medidas de localização de gordura, sendo de fraca a regular com as medidas da cintura. CONCLUSÕES: Os resultados indicam colinearidade entre massa corporal e adiposidade total com tecido adiposo central e periférico. Recomenda-se o emprego da CUm na avaliação do estado nutricional de adolescentes, pois ela apresentou maior capacidade para predizer adiposidade em cada grupo, além de correlação fraca a regular com a estatura. .

A comparative 2D QSAR study on a series of hydroxamic acid-based histone deacetylase inhibitors vis-à-vis comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA).

A quantitative structure-activity relationship (QSAR) study was performed on a series of indole amide analogues reported by Dai et al. [Bioorg Med Chem Lett (2003), 13, 1897-1901] to act as histone deacetylase (HDAC) inhibitors. The multiple regression analysis (MRA) revealed a model showing the significant dependence of the activity on molar refractivity (MR) and global topological charge index (GTCI) of the compounds, suggesting that inhibition of the HDAC by this series of compounds might involve the dispersion interaction with the receptor, where charge transfer between pairs of atoms might greatly help to polarize the molecule. The MRA results were then compared with those obtained by Guo et al. [Bioorg Med Chem (2005), 13, 5424-5434] by comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). It was found that MRA gave as good results and had as good predictive ability as CoMFA and CoMSIA. Besides, MRA was also able to throw the light on the physicochemical properties of the molecules that were involved in drug-receptor interactions, while CoMFA and CoMSIA could not. The dispersion interaction between the molecule and the active site of the receptor is suggested to be the main interaction.

Role of histone deacetylase activity in the developing lateral line neuromast of zebrafish larvae

Histone deacetylases are involved in many biological processes and have roles in regulating cell behaviors such as cell cycle entry, cell proliferation and apoptosis. However, the effect of histone deacetylases on the development of hair cells (HCs) has not been fully elucidated. In this study, we examined the influence of histone deacetylases on the early development of neuromasts in the lateral line of zebrafish. Hair cell development was evaluated by fluorescent immunostaining in the absence or presence of histone deacetylase inhibitors. Our results suggested that pharmacological inhibition of histone deacetylases with inhibitors, including trichostatin A, valproic acid and MS-275, reduced the numbers of both HCs and supporting cells in neuromasts. We also found that the treatment of zebrafish larvae with inhibitors caused accumulation of histone acetylation and suppressed proliferation of neuromast cells. Real-time PCR results showed that the expression of both p21 and p27 mRNA was increased following trichostatin A treatment and the increase in p53 mRNA was modest under the same conditions. However, the expression of p53 mRNA was significantly increased by treatment with a high concentration of trichostatin A. A high concentration of trichostatin A also led to increased cell death in neuromasts as detected in a TUNEL assay. Moreover, the nuclei of most of these pyknotic cells were immunohistochemically positive for cleaved caspase-3. These results suggest that histone deacetylase activity is involved in lateral line development in the zebrafish and might have a role in neuromast formation by altering cell proliferation through the expression of cell cycle regulatory proteins.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

ATM modulates transcription in response to histone deacetylase inhibition as part of its DNA damage response

Chromatin structure has a crucial role in a diversity of physiological processes, including development, differentiation and stress responses, via regulation of transcription, DNA replication and DNA damage repair. Histone deacetylase (HDAC) inhibitors regulate chromatin structure and activate the DNA damage checkpoint pathway involving Ataxia-telangiectasia mutated (ATM). Herein, we investigated the impact of histone acetylation/deacetylation modification on the ATM-mediated transcriptional modulation to provide a better understanding of the transcriptional function of ATM. The prototype HDAC inhibitor trichostain A (TSA) reprograms expression of the myeloid cell leukemia-1 (MCL1) and Gadd45alpha genes via the ATM-mediated signal pathway. Transcription of MCL1 and Gadd45alpha is enhanced following TSA treatment in ATM+ cells, but not in isogenic ATM- or kinase-dead ATM expressing cells, in the ATM-activated E2F1 or BRCA1-dependent manner, respectively. These findings suggest that ATM and its kinase activity are essential for the TSA-induced regulation of gene expression. In summary, ATM controls the transcriptional upregulation of MCL1 and Gadd45alpha through the activation of the ATM-mediated signal pathway in response to HDAC inhibition. These findings are important in helping to design combinatory treatment schedules for anticancer radio- or chemo-therapy with HDAC inhibitors.

Curcumin-induced histone acetylation in malignant hematologic cells / 华中科技大学学报（医学）（英德文版）

This study investigated the inhibitory effects of curcumin on proliferation of hematological malignant cells in vitro and the anti-tumor mechanism at histone acetylation/histone deacetylation levels. The effects of curcumin and histone deacetylase inhibitor trichostatin A (TSA) on the growth of Raji cells were tested by MTT assay. The expression of acetylated histone-3 (H(3)) in Raji, HL60 and K562 cells, and peripheral blood mononuclear cells (PBMCs) treated with curcumin or TSA was detected by immunohistochemistry and FACS. The results showed curcumin inhibited proliferation of Raji cells significantly in a time- and dose-dependent fashion, while exhibited low toxicity in PBMCs. Curcumin induced up-regulation of the expression of acetylated H(3) dose-dependently in all malignant cell lines tested. In conclusion, curcumin inhibited proliferation of Raji cells selectively, enhanced the level of acetylated (H(3)) in Raji, HL60, and K562 cells, which acted as a histone deacetylase inhibitor like TSA. Furthermore, up-regulation of H(3) acetylation may play an important role in regulating the proliferation of Raji cells.