Treatment of canine haemangiosarcoma with suberoylanilide hydroxamic acid, a histone deacetylase inhibitor

A case report is presented by describing the treatment of a 12‐year‐old dog – diagnosed with haemangiosarcoma (HSA) – with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor. The drug was administered orally, on a daily basis, approximately 2 weeks post‐splenectomy at a dose of 3 mg kg^?1. HSA is a lethal malignancy of the endothelium, which is usually disseminated by the time it is diagnosed. Median survival time, usually, is no longer than 80 days. Following treatment with SAHA, no sign of malignant growth could be discerned by means of diagnostic abdominal ultrasound, chest X‐ray or with the help of clinical symptoms, over a period of >1000 days. The precise mechanism by which HDAC inhibitors exert their anti‐cancer effects is uncertain, but evidence suggests that exposure to SAHA generates hyperacetylated chromosomal histones, which, in turn, facilitates the expression of tumour suppressor genes turned off by epigenetic mechanisms during neoplastic transformation of the endothelium.     

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre‐implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.     

Effects of melatonin on maturation,histone acetylation,autophagy of porcine oocytes and subsequent embryonic development

Melatonin (MLT) is an endogenous hormone with roles in animal germ cell development. However, the effect of MLT on porcine oocyte maturation and its underlying mechanisms remain largely unknown. Here, we investigated the effects of exogenous MLT on oocyte maturation, histone acetylation, autophagy and subsequent embryonic development. We found that 1 nmol/L MLT supplemented in maturation medium was the optimal concentration to promote porcine oocyte maturation and subsequent developmental competence and quality of parthenogenetic embryos. Interestingly, the beneficial effects of 1 nmol/L MLT treatment on porcine oocyte maturation and embryo development were mainly attributed to the first half period of in vitro maturation. Simultaneously, MLT treatment could also improve maturation of small follicle‐derived oocytes, morphologically poor (cumulus cell layer ≤1) and even artificially denuded oocytes and their subsequent embryo development. Furthermore, MLT treatment not only could decrease the levels of H3K27ac and H4K16ac in metaphase II (MII) oocytes, but also could increase the expression abundances of genes associated with cumulus cell expansion, meiotic maturation, histone acetylation and autophagy in cumulus cells or MII oocytes. These results indicate that MLT treatment can facilitate porcine oocyte maturation and subsequent embryonic development probably, through improvements in histone acetylation and autophagy in oocytes.     

Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC‐AF), adipose tissue (MSC‐AT) and fibroblasts (FIB‐control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT‐PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC‐AF were less hydroxymethylated for both animals. MSC‐AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC‐AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC‐AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.     

Effect of histone acetylation modification with sodium butyrate, a histone deacetylase inhibitor, on cell cycle, apoptosis, ploidy and gene expression in porcine fetal fibroblasts

The present study evaluated the effective dose of sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, for determination of the level of enhancement of histone acetylation in porcine fetal fibroblasts (PFFs) based on their morphology, growth, apoptosis and cell cycle status. Cells were analyzed for their histone acetylation levels at H3, H4 and H2A and expression of genes related to histone deacetylation (HDAC1, HDAC2 and HDAC3), pro-apoptosis (Bax and Bak) and anti-apoptosis (Bcl-2). PFFs at passage 3-4 were cultured with 0, 0.5, 1.0, 2.0 and 3.0 mM NaB for 96 h. NaB inhibited cell proliferation at all tested concentrations in a dose-dependent manner. However, there was slow cell growth for PFFs treated with 2.0 and 3.0 mM NaB compared with those of untreated PFFs and those treated with other lower concentrations (0.5 and 1.0 mM). More than 85% of the cells that were untreated or treated with 0.5 or 1.0 mM NaB had intact membranes, whereas, approximately 30% of the cells treated with 2.0 or 3.0 mM NaB had increased cell sizes and a more flattened and elongated appearance. NaB induced apoptosis in a dose-dependent manner; the rates of apoptosis were 2.5 +/- 0.4% for 1.0 mM NaB, 7.6 +/- 1.1% for 2.0 mM NaB and 11.2 +/- 1.4% for 3.0 mM NaB. The chromosomal sets of PFFs treated with 0.5 and 1.0 mM NaB were normal, whereas a lower proportion of PFFs treated with 2.0 and 3.0 mM were classified as normal. NaB at 0.5 and 1.0 mM showed little effect on cell cycle. However, 2.0 and 3.0 mM resulted in an increased cell population at the G(0)/G(1) phase. Increased NaB concentrations led to elevated acetylation of H3, H4 and H2A. NaB altered the expression of histone deacetylation and apoptosis-related genes. In conclusion, 1.0 mM NaB induced histone hyperacetylation in the PFFs and produced less deleterious effects than other concentrations; these PFFs might serve as suitable donors for porcine somatic cell nuclear transfer (SCNT).     

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation‐treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation‐treated embryos.