Anti-Cancer Effects of Epigenetics Drugs Scriptaid and Zebularine in Human Breast Adenocarcinoma Cells.

BACKGROUND: High relapse and metastasis progression in breast cancer patients have prompted the need to explore alternative treatments. Epigenetic therapy has emerged as an attractive therapeutic strategy due to the reversibility of epigenome structures. OBJECTIVE: This study investigated the anti-cancer effects of epigenetic drugs scriptaid and zebularine in human breast adenocarcinoma MDA-MB-231 and MCF-7 cells. METHODS: First, the half maximal Inhibitory Concentration (IC50) of scriptaid and zebularine, and the combination of both drugs on human breast adenocarcinoma MDA-MB-231 cells were determined. Next, MDA-MB-231 and MCF-7 cells were treated with IC50 of scriptaid, zebularine and the combination of both. After IC50 treatments, the anti-cancer effects were evaluated via cell migration assay, cell cycle analysis and apoptotic studies which included histochemical staining and reverse-transcriptase polymerase chain reaction (RT-PCR) of the apoptotic genes. RESULTS: Both epigenetic drugs inhibited cell viability in a dose-dependent manner with IC50 of 2 nM scriptaid, 8 µM zebularine and a combination of 2 nM scriptaid and 2 µM zebularine. Both MDA-MB-231 and MCF-7 cells exhibited a reduction in cell migration after the treatments. In particular, MDA-MB-231 cells exhibited a significant reduction in cell migration (p < 0.05) after the treatments of zebularine and the combination of scriptaid and zebularine. Besides, cell cycle analysis demonstrated that scriptaid and the combination of both drugs could induce cell cycle arrest at the G0/G1 phase in both MDA-MB-231 and MCF-7 cells. Furthermore, histochemical staining allowed the observation of apoptotic features, such as nuclear chromatin condensation, cell shrinkage, membrane blebbing, nuclear chromatin fragmentation and cytoplasmic extension, in both MDA-MB-231 and MCF-7 cells after the treatments. Further, apoptotic studies revealed the upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2 and elevation of Bax/Bcl-2 ratio in MDA-MB-231 cells treated with zebularine and MCF-7 cells treated with all drug regimens. CONCLUSION: Collectively, these findings suggest that scriptaid and zebularine are potential anti-cancer drugs, either single or in combination, for the therapy of breast cancer. Further investigations of the gene regulatory pathways directed by scriptaid and zebularine are definitely warranted in the future.

Direct Amidation to Access 3-Amido-1,8-Naphthalimides Including Fluorescent Scriptaid Analogues as HDAC Inhibitors.

Methodology to access fluorescent 3-amido-1,8-naphthalimides using direct Buchwald-Hartwig amidation is described. The protocol was successfully used to couple a number of substrates (including an alkylamide, an arylamide, a lactam and a carbamate) to 3-bromo-1,8-naphthalimide in good yield. To further exemplify the approach, a set of scriptaid analogues with amide substituents at the 3-position were prepared. The new compounds were more potent than scriptaid at a number of histone deacetylase (HDAC) isoforms including HDAC6. Activity was further confirmed in a whole cell tubulin deacetylation assay where the inhibitors were more active than the established HDAC6 selective inhibitor Tubastatin. The optical properties of these new, highly active, compounds make them amenable to cellular imaging studies and theranostic applications.

The Effect of Vicinal Difluorination on the Conformation and Potency of Histone Deacetylase Inhibitors.

Histone deacetylase enzymes (HDACs) are potential targets for the treatment of cancer and other diseases, but it is challenging to design isoform-selective agents. In this work, we created new analogs of two established but non-selective HDAC inhibitors. We decorated the central linker chains of the molecules with specifically positioned fluorine atoms in order to control the molecular conformations. The fluorinated analogs were screened against a panel of 11 HDAC isoforms, and minor differences in isoform selectivity patterns were observed.

Intracellular fluorescence competition assay for inhibitor engagement of histone deacetylase.

An intracellular fluorescence competition assay was developed to assess the capability of inhibitor candidates to engage histone deacetylase (HDAC) inside living cells and thus diminish cell uptake and staining by the HDAC-targeted fluorescent probe APS. Fluorescence cell microscopy and flow cytometry showed that pre-incubation of living cells with candidate inhibitors led to diminished cell uptake of the fluorescent probe. The assay was effective because the fluorescent probe (APS) possessed the required performance properties, including bright fluorescence, ready membrane diffusion, selective intracellular HDAC affinity, and negligible acute cytotoxicity. The concept of an intracellular fluorescence competition assay is generalizable and has broad applicability since it obviates the requirement to use the isolated biomacromolecule target for screening of molecular candidates with target affinity.

Differential Transcriptomes and Methylomes of Trophoblast Stem Cells From Naturally-Fertilized and Somatic Cell Nuclear-Transferred Embryos.

Trophoblast stem cells (TSCs) are critical to mammalian embryogenesis by providing the cell source of the placenta. TSCs can be derived from trophoblast cells. However, the efficiency of TSC derivation from somatic cell nuclear transfer (NT) blastocysts is low. The regulatory mechanisms underlying transcription dynamics and epigenetic landscape remodeling during TSC derivation remain elusive. Here, we derived TSCs from the blastocysts by natural fertilization (NF), NT, and a histone deacetylase inhibitor Scriptaid-treated NT (SNT). Profiling of the transcriptomes across the stages of TSC derivation revealed that fibroblast growth factor 4 (FGF4) treatment resulted in many differentially expressed genes (DEGs) at outgrowth and initiated transcription program for TSC formation. We identified 75 transcription factors (TFs) that are continuously upregulated during NF TSC derivation, whose transcription profiles can infer the time course of NF not NT TSC derivation. Most DEGs in NT outgrowth are rescued in SNT outgrowth. The correct time course of SNT TSC derivation is inferred accordingly. Moreover, these TFs comprise an interaction network important to TSC stemness. Profiling of DNA methylation dynamics showed an extremely low level before FGF4 treatment and gradual increases afterward. FGF4 treatment results in a distinct DNA methylation remodeling process committed to TSC formation. We further identified 1,293 CpG islands (CGIs) whose DNA methylation difference is more than 0.25 during NF TSC derivation. The majority of these CGIs become highly methylated upon FGF4 treatment and remain in high levels. This may create a barrier for lineage commitment to restrict embryonic development, and ensure TSC formation. There exist hundreds of aberrantly methylated CGIs during NT TSC derivation, most of which are corrected during SNT TSC derivation. More than half of the aberrantly methylated CGIs before NT TSC formation are inherited from the donor genome. In contrast, the aberrantly methylated CGIs upon TSC formation are mainly from the highly methylated CGIs induced by FGF4 treatment. Functional annotation indicates that the aberrantly highly methylated CGIs play a role in repressing placenta development genes, etc., related to post-implantation development and maintaining TSC pluripotency. Collectively, our findings provide novel insights into the transcription dynamics, DNA methylation remodeling, and the role of FGF4 during TSC derivation.

Zinc finger protein-440 promotes cartilage degenerative mechanisms in human facet and knee osteoarthritis chondrocytes.

OBJECTIVES: To investigate the role of zinc finger protein 440 (ZNF440) in the pathophysiology of cartilage degeneration during facet joint (FJ) and knee osteoarthritis (OA). METHODS: Expression of ZNF440 in FJ and knee cartilage was determined by immunohistochemistry, quantitative (q)PCR, and Western blotting (WB). Human chondrocytes isolated from FJ and knee OA cartilage were cultured and transduced with ZNF440 or control plasmid, or transfected with ZNF440 or control small interfering RNA (siRNA), with/without interleukin (IL)-1ß. Gene and protein levels of catabolic, anabolic and apoptosis markers were determined by qPCR or WB, respectively. In silico analyses were performed to determine compounds with potential to inhibit expression of ZNF440. RESULTS: ZNF440 expression was increased in both FJ and knee OA cartilage compared to control cartilage. In vitro, overexpression of ZNF440 significantly increased expression of MMP13 and PARP p85, and decreased expression of COL2A1. Knockdown of ZNF440 with siRNA partially reversed the catabolic and cell death phenotype of human knee and FJ OA chondrocytes stimulated with IL-1ß. In silico analysis followed by validation assays identified scriptaid as a compound with potential to downregulate the expression of ZNF440. Validation experiments showed that scriptaid reduced the expression of ZNF440 in OA chondrocytes and concomitantly reduced the expression of MMP13 and PARP p85 in human knee OA chondrocytes overexpressing ZNF440. CONCLUSIONS: The expression of ZNF440 is significantly increased in human FJ and knee OA cartilage and may regulate cartilage degenerative mechanisms. Furthermore, scriptaid reduces the expression of ZNF440 and inhibits its destructive effects in OA chondrocytes.

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Effect of supplemetation of Zebularine and Scriptaid on efficiency of in vitro developmental competence of ovine somatic cell nuclear transferred embryos.

Somatic cell nuclear transfer (SCNT) technology has been applied in the construction of disease model, production of transgenic animals, therapeutic cloning, and other fields. However, the cloning efficiency remains limited. In our study, to improve SCNT efficiency, brilliant cresyl blue (BCB) staining were chosen to select recipient oocytes. In addition, DNA methyltransferase inhibitor Zebularine (5 nmol/L) and histone deacetylase inhibitor Scriptaid (0.2 µmol/L) were jointly used to treat sheep donor cumulus cells and reconstructed embryo. Moreover, the expression levels of embryonic development-related genes (OCT4, SOX2, H19, IGF2 and Dnmt1) of reconstructed embryo were also detected. Using BCB + oocytes as recipient cell, donor cumulus cells and reconstructed embryos were treated with 5 nmol/L Zebularine and 0.2 µmol/L Scriptaid, the blastocyst rate in Zeb + SCR-SCNT group (28.25%) was significantly higher than SCNT (21.16%) (p < 0.05). Furthermore, results showed that expression levels of OCT4, SOX2, H19, IGF2 and Dnmt1 genes in Zeb + SCR-SCNT embryos were more similar to IVF embryos. Our study proved that 5 nmol/L Zebularine and 0.2 µmol/L Scriptaid treating with sheep donor cumulus cells and reconstructed embryos improved SCNT blastocyst rate and relieve the abnormal expression of embryonic developmental related genes.

Pharmacokinetics and Acute Toxicity of a Histone Deacetylase Inhibitor, Scriptaid, and its Neuroprotective Effects in Mice After Intracranial Hemorrhage.

BACKGROUND & OBJECTIVE: The pharmacokinetics and acute toxicity of a histone deacetylase inhibitor, Scriptaid, was unknown in the mouse. The aim of this study was to determine the pharmacokinetics, acute toxicity, and tissue distribution of Scriptaid, a new histone deacetylase inhibitor, in mice, and its neuroprotective efficacy in a mouse intracranial hemorrhage (ICH) model. METHODS: The pharmacokinetics, acute toxicity, and tissue distribution were determined in C57BL/6 male and female mice after the intraperitoneal administration of a single dose. Behavioral tests, as well as investigations of brain atrophy and white matter injury, were used to evaluate the neuroprotective effect of Scriptaid after ICH. Western blotting was used to investigate if Scriptaid could offer antiinflammatory benefits after ICH. RESULTS: No significant differences were observed in body weight or brain histopathology between the group that received Scriptaid at 50 mg/kg and the group that received dimethyl sulfoxide (control). The pharmacokinetics of Scriptaid in mice was nonlinear, and it was cleared rapidly at low doses and slowly at higher doses. Consistent with the pharmacokinetic data, Scriptaid was found to distribute in several tissues, including the spleen and kidneys. In the ICH model, we found that Scriptaid could reduce neurological deficits, brain atrophy, and white matter injury in a dose-dependent manner. Western blotting results demonstrated that Scriptaid could decrease the expression of pro-inflammatory cytokines IL1ß and TNFα, as well as iNOS, after ICH. CONCLUSION: These findings indicate that Scriptaid is safe and can alleviate brain injury after ICH, thereby providing a foundation for the pharmacological action of Scriptaid in the treatment of brain injury after ICH.

Scriptaid/exercise-induced lysine acetylation is another type of posttranslational modification occurring in titin.

Titin serves important functions in skeletal muscle during exercise, and posttranslational modifications of titin participate in the regulation of titin-based sarcomeric functions. Scriptaid has exercise-like effects through the inhibition of HDAC and regulatory acetylation of proteins. However, it remains mostly unclear if exercise could result in titin's acetylation and whether Scriptaid could regulate acetylation of titin. We treated C57BL/6 mice with 6-wk treadmill exercise and 6-wk Scriptaid administration to explore Scriptaid's effects on mice exercise capacity and whether Scriptaid administration/exercise could induce titin's acetylation modification. An exercise endurance test was conducted to explore their effects on mice exercise capacity, and proteomic studies were conducted with gastrocnemius muscle tissue of mice from different groups to explore titin's acetylation modification. We found that Scriptaid and exercise did not change titin's protein expression, but they did induce acetylation modification changes of titin. In total, 333 acetylated lysine sites were identified. Exercise changed the acetylation levels of 33 lysine sites of titin, whereas Scriptaid changed acetylation levels of 31 titin lysine sites. Exercise treatment and Scriptaid administration shared 11 lysine sites. In conclusion, Scriptaid increased exercise endurance of mice by increasing the time mice spent running to fatigue. Acetylation is a common type of posttranslational modification of titin, and exercise/Scriptaid changed the acetylation levels of titin and titin-interacting proteins. Most importantly, titin may be a mediator through which Scriptaid and exercise modulate the properties and functions of exercise-induced skeletal muscle at the molecular level.NEW & NOTEWORTHY Scriptaid administration increased mouse exercise endurance. Acetylation is another type of posttranslational modification of titin. Scriptaid/exercise changed acetylation levels of titin and titin-interacting proteins. Titin may mediate exercise-induced skeletal muscle properties and functions.

Effects of histone deacetylase inhibitor Scriptaid and parathyroid hormone on osteocyte functions and metabolism.

Bone is a highly metabolic organ that undergoes continuous remodeling to maintain its structural integrity. During development, bones, in particular osteoblasts, rely on glucose uptake. However, the role of glucose metabolism in osteocytes is unknown. Osteocytes are terminally differentiated osteoblasts orchestrating bone modeling and remodeling. In these cells, parathyroid hormone (PTH) suppresses Sost/sclerostin expression (a potent inhibitor of bone formation) by promoting nuclear translocation of class IIa histone deacetylase (HDAC) 4 and 5 and the repression of myocyte enhancer factor 2 (MEF2) type C. Recently, Scriptaid, an HDAC complex co-repressor inhibitor, has been shown to induce MEF2 activation and exercise-like adaptation in mice. In muscles, Scriptaid disrupts the HDAC4/5 co-repressor complex, increases MEF2C function, and promotes cell respiration. We hypothesized that Scriptaid, by affecting HDAC4/5 localization and MEF2C activation, might affect osteocyte functions. Treatment of the osteocytic Ocy454-12H cells with Scriptaid increased metabolic gene expression, cell respiration, and glucose uptake. Similar effects were also seen upon treatment with PTH, suggesting that both Scriptaid and PTH can promote osteocyte metabolism. Similar to PTH, Scriptaid potently suppressed Sost expression. Silencing of HDAC5 in Ocy454-12H cells abolished Sost suppression but not glucose transporter type 4 (Glut4) up-regulation induced by Scriptaid. These results demonstrate that Scriptaid increases osteocyte respiration and glucose uptake by mechanisms independent of HDAC complex inhibition. In osteocytes, Scriptaid, similar to PTH, increases binding of HDAC5 to Mef2c with suppression of Sost but only partially increases receptor activator of NF-κB ligand (Rankl) expression, suggesting a potential bone anabolic effect.

Highly fluorescent and HDAC6 selective scriptaid analogues.

Fluorescent scriptaid analogues with excellent HDAC6 selectivity (HDAC1/6 > 500) and potency (HDAC6 IC50 < 5 nM) have been synthesised and evaluated. The highly fluorescent nature of the compounds (up to ΦF = 0.83 in DMSO and 0.38 in aqueous buffer) makes them ideally suited for cellular imaging and visualisation of their cytoplasmic localisation was readily accomplished. Whole organism imaging in zebrafish confirmed both the vascular localisation of the new inhibitors and the impact of HDAC6 inhibition on in vivo development.

HDAC3 Inhibition Promotes Alternative Activation of Macrophages but Does Not Affect Functional Recovery after Spinal Cord Injury.

After spinal cord injury (SCI), monocyte derived macrophages play a detrimental role. Histone deacetylases (HDACs) are central epigenetic regulators of macrophage-polarization. We hypothesized that HDAC3 inhibition suppresses the pro-inflammatory macrophage phenotype (M1), promotes the anti-inflammatory phenotype (M2) and improves functional recovery after SCI. Therefore, two inhibitors of HDAC3 were selected, namely scriptaid and RGFP966. The impact on macrophage polarization was studied by investigating the effect on gene and protein expression of selected M1 and M2 markers. We show that scriptaid differentially influences M1 and M2 markers. It increases CD86 and iNOS gene expression and decreases GPR18, CD38, FPR2 and Arg-1 gene expression as well as the production of IL-6 and NO. RGFP966 primarily increased the expression of the M2 markers Arg-1 and Ym1 and reduced the production of IL-6 (M1). RGFP966 and scriptaid reduced the formation of foamy macrophages. Finally, to investigate the impact of HDAC3 inhibition on functional recovery after SCI, we studied the effects of RGFP966 and scriptaid in an in vivo T-cut hemisection SCI model. Histological analyses were performed on spinal cord sections to determine lesion size and astrogliosis, demyelinated area and selected infiltrating immune cells. RGFP966 and scriptaid did not affect functional recovery or histopathological outcome after SCI. In conclusion, these results indicate that specific HDAC3 inhibition with RGFP966 promotes alternative activation of macrophages and reduces the formation of foamy macrophages, but does not lead to a better functional recovery after SCI.

Anticancer effect of histone deacetylase inhibitor scriptaid as a single agent for hepatocellular carcinoma.

Recurrence is one of the major causes of poor prognosis for patients with hepatocellular carcinoma (HCC), and drug resistance is closely associated with disease recurrence. Histone deacetylase (HDAC) inhibitor scriptaid functions as an anticancer agent in many different types of tumors, but its possible roles in HCC progression have not been explored to date. Herein, we show that HDAC inhibitor scriptaid decreases HCC cell proliferation and induces cell cycle G2/M-phase arrest in a dose-dependent manner. Furthermore, scriptaid triggered HCC cell death via transcriptional activation of p21 and subsequent elevated global H3Ac levels. Importantly, we found that scriptaid showed robust antitumor activity against HCC. Thus, our findings indicate that HDAC inhibitor scriptaid could be an important potential candidate for treatment of HCC patients.

Effects of the HDAC inhibitor scriptaid on the in vitro development of bovine embryos and on imprinting gene expression levels.

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis -including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting (GRB10, GNAS, XIST) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified.

Pharmacological targeting of exercise adaptations in skeletal muscle: Benefits and pitfalls.

Exercise exerts significant effects on the prevention and treatment of many diseases. However, even though some of the key regulators of training adaptation in skeletal muscle have been identified, this biological program is still poorly understood. Accordingly, exercise-based pharmacological interventions for many muscle wasting diseases and also for pathologies that are triggered by a sedentary lifestyle remain scarce. The most efficacious compounds that induce muscle hypertrophy or endurance are hampered by severe side effects and are classified as doping. In contrast, dietary supplements with a higher safety margin exert milder outcomes. In recent years, the design of pharmacological agents that activate the training program, so-called "exercise mimetics", has been proposed, although the feasibility of such an approach is highly debated. In this review, the most recent insights into key regulatory factors and therapeutic approaches aimed at leveraging exercise adaptations are discussed.

Scriptaid improves the reprogramming of donor cells and enhances canine-porcine interspecies embryo development.

Histone methylation, histone acetylation, and DNA methylation are the important factors for somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have been used to improve cloning efficiency. In particular, scriptaid, an HDACi, has been shown to improve SCNT efficiency. However, no studies have been performed on canines. Here, we evaluated the effects of scriptaid on histone modification in canine ear fibroblasts (cEFs) and cloned canine embryos derived from cEFs. The early development of cloned canine-porcine interspecies SCNT (iSCNT) embryos was also examined. cEFs were treated with scriptaid (0, 100, 250, 500, 750, and 1000nM) in a medium for 24h. Scriptaid treatment (all concentrations) did not significantly affect cell apoptosis. Treatment with 500nM scriptaid caused a significant increase in the acetylation of H3K9, H3K14, and H4K5. cEFs treated with 500nM scriptaid showed significantly decreased Gcn5, Hat1, Hdac6, and Bcl2 and increased Oct4 and Sox2 expression levels. After SCNT with canine oocytes, H3K14 acetylation was significantly increased in the one- and two-cell cloned embryos from scriptaid-treated cEFs. In iSCNT, the percentage of embryos in the 16-cell stage was significantly higher in the scriptaid-treated group (21.6±2.44%) than in the control (7.5±2.09%). The expression levels of Oct4, Sox2, and Bcl2 were significantly increased in 16-cell iSCNT embryos, whereas that of Hdac6 was decreased. These results demonstrated that scriptaid affected the reprogramming of canine donor and cloned embryos, as well as early embryo development in canine-porcine iSCNT, by regulating reprogramming and apoptotic genes.

Scriptaid cause histone deacetylase inhibition and cell cycle arrest in HeLa cancer cells: A study on structural and functional aspects.

Scriptaid (SCR), a well-known histone deacetylase inhibitor, cause various cellular effects such as cell growth inhibition and apoptosis. In this study, we have evaluated the anti-cancer effects of Scriptaid in HeLa cells, IMR-32 and HepG2 cells. Scriptaid inhibited the growth of HeLa cells with IC50 of 2µM at 48h in a dose-dependent manner. Flow-cytometric analysis indicated that SCR induced apoptosis. Scriptaid was found to inhibit HDAC-8 effectively than other HDAC inhibitor such as TSA as observed by HDAC-8 assay, Western blotting and modelling study. This observation was further strengthened by an artificial neuronal network (ANN) model.

Scriptaid Upregulates Expression of Development-Related Genes, Inhibits Apoptosis, and Improves the Development of Somatic Cell Nuclear Transfer Mini-Pig Embryos.

The present study was undertaken to investigate the mechanisms by which Scriptaid treatment improves the developmental competence of somatic cell nuclear transfer (SCNT) mini-pig embryos in vitro. We found that treatment with 500 nmol/L Scriptaid for 15 hours significantly improved the development of mini-pig SCNT embryos. Compared with the control group, the blastocyst rate was higher (18.3% vs. 10.7%; p < 0.05). The acetylation level on H3K14 of the Scriptaid-treated group was higher compared with the control group in SCNT embryos at two-cell, four-cell, and blastocyst stages (p < 0.05). After Scriptaid treatment, histone deacetylase gene HDAC5 expression level was significantly decreased in four-cell embryos and blastocysts, while the expression levels of the embryos' development-related genes AKT, Oct4, and apoptosis inhibited gene PGC-1α were significantly increased in blastocysts (p < 0.05). The number of apoptotic cells per blastocyst in the Scriptaid-treated group was lower compared with the control group (p < 0.05). These results indicate that Scriptaid repressed HDCA5 gene expression, increased the acetylation level of H3K14, upregulated the expression of AKT, Oct4, and PGC-1α genes, improved embryos' development, and reduced apoptosis, which favors development of the SCNT mini-pig embryos to blastocysts.

Isoliquiritigenin inhibits TNF-α-induced release of high-mobility group box 1 through activation of HDAC in human intestinal epithelial HT-29 cells.

The suppression of pro-inflammatory cytokine-induced inflammation responses is an attractive pharmacological target for the development of therapeutic strategies for inflammatory bowel disease (IBD). In the present study, we evaluated the anti-inflammatory properties of flavonoid isoliquiritigenin (ISL) in intestinal epithelial cells and determined its mechanism of action. ISL suppressed the expression of inflammatory molecules, including IL-8, IL-1ß and COX-2, in TNF-α-stimulated HT-29 cells. Moreover, ISL induced activation of Nrf2 and expression of its target genes, such as HO-1 and NQO1. ISL also inhibited the TNF-α-induced NF-κB activation in HT-29 cells. High-mobility group box 1 (HMGB1), which is one of the critical mediators of inflammation, is actively secreted from inflammatory cytokine-stimulated immune or non-immune cells. ISL inhibited HMGB1 secretion by preventing TNF-α-stimulated HMGB1 relocation, whereas the RNA and protein expression levels of cellular HMGB1 did not change in response to TNF-α or ISL. Moreover, we found that HMGB1 acetylation was associated with HMGB1 translocation to the cytoplasm and the extracellular release in TNF-α-stimulated HT-29 cells; however, ISL significantly decreased the amount of acetylated HMGB1 in both the cytoplasm and extracellular space of HT-29 cells. Histone deacetylase (HDAC) inhibition by Scriptaid abrogated ISL-induced HDAC activity and reversed the ISL-mediated decrease in acetylated HMGB1 release in TNF-α-stimulated HT-29 cells, suggesting that, at least in TNF-α-stimulated HT-29 cells, ISL suppresses acetylated HMGB1 release via the induction of HDAC activity. Together, the current results suggest that inhibition of HMGB1 release via the induction of HDAC activity using ISL may be a promising therapeutic intervention for IBD.