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.
Epigenesis, Genetic ; Fetal Blood ; Hematopoietic Stem Cells ; Histone Deacetylase Inhibitors/pharmacology* ; Valproic Acid/pharmacology*

OBJECTIVETo study the effect of valproate acid sodium(VPA) on apoptosis of human gastric cancer cell BGC-823 and to explore its possible mechanism.

METHODSCell growth inhibition was examined by MTT assay. Apoptosis rate was detected by FCM with Annexin V/PI staining. The activities and protein expression levels of caspase 3, caspase 8 and caspase 9 were examined by spectrophotometry and indirect immunofluorescence technique respectively.

RESULTSThe growth inhibition rate and apoptosis rate of human gastric cancer cells, treated with 0.75-4.00 mmol/L VPA for 24 h and 48 h, elevated in time- and dose-dependent manner. Apoptosis rates of VPA 0.75 mmol/L 24 h and 48 h were (7.2 +/- 0.5)% and (9.2 +/- 1.0)%, of VPA 4.00 mmol/L 24 h and 48 h were (16.7 +/- 2.2)% and (20.4 +/- 1.6)% respectively, which were significantly different as compared to the control [24 h, (4.9 +/- 0.2)%, 48 h, (5.1 +/- 0.8)%] (P< 0.001). The activities and protein expression levels of caspase 3 and caspase 9 were up-regulated compared with the control group (P< 0.001), meanwhile the activity and protein expression of caspase 8 enhanced slightly after VPA treatment for 48 h.

CONCLUSIONVPA can inhibit the growth and induce the apoptosis of BGC-823 cells mainly through the activation of caspase 9 pathway.

Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Line, Tumor ; Humans ; Stomach Neoplasms ; pathology ; Valproic Acid ; pharmacology

OBJECTIVETo investigate the effects of two histone deacetylase (HDAC) inhibitors, valproic acid (VPA) and TSA, on the expression of vascular endothelial growth factor (VEGF) and its receptor KDR of the leukemia cell line Kasumi-1 cells, and to explore their potential mechanism in leukemia angiogenesis.

METHODKasumi-1 cells were treated with VPA and TSA at different concentrations for 3 days. The mRNA and protein expression levels of VEGF and KDR were determined by semi-quantitative RT-PCR and Western blot, and the bFGF mRNA by semi-quantitative RT-PCR.

RESULTSAs compared with that of control groups, VPA at 3 mmol/L downregulated the VEGF mRNA expression level for VEGF(121) from 0.632 ± 0.014 to 0.034 ± 0.004 and for VEGF(165) from 0.526 ± 0.021 to 0.015 ± 0.001, for KDR mRNA from 0.258 ± 0.034 to 0.038 ± 0.000, and for bFGF mRNA from 0.228 ± 0.017 to 0.086 ± 0.015. TSA downregulated the VEGF mRNA and KDR mRNA at concentration of 100 nmol/L, but its effect on bFGF mRNA only at higher concentration.

CONCLUSIONHDAC inhibitors might inhibit the leukemia angiogenesis by regulating the expression of VEGF and its recptor.

Angiogenesis Inducing Agents ; Cell Line ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology ; Vascular Endothelial Growth Factor A

Animals ; Anticonvulsants ; pharmacology ; Drug Evaluation, Preclinical ; methods ; GABA-A Receptor Antagonists ; pharmacology ; Motor Activity ; drug effects ; Pentylenetetrazole ; pharmacology ; Valproic Acid ; pharmacology ; Zebrafish

OBJECTIVETo study the antitumour effects of sodium valproate (VPA) on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms.

METHODSAfter Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 (DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR.

RESULTSVPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G(0)/G(1) phase was (66.87 ± 3.31)% and in S phase was (8.47 ± 2.56)%, while in control group, the cells in G(0)/G(1) phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration.

CONCLUSIONVPA has a demethylation effect on p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G(0)/G(1) phase.

Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA Methylation ; drug effects ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology

To study the effects of sodium valproate (VPA) on human myelodysplastic syndrome cell line MUTZ-1. The cell proliferation was determined by MTT assay, apoptotic morphological features were observed by light microscopy and transmission electronmicroscopy, cell apoptosis and cell cycle shift were analyzed by flow cytometry (FCM). The results showed that VPA could inhibit the growth of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in MUTZ-1 cells treated with 4 mmol/L VPA for 72 hours. Pyknosis of cells and nuclei, disintegration of nuclear chromatin and apoptotic body could be observed by light microscopy. Aggregation and margination of nuclear chromatin, concentration of plasm, increment of density and chromatin mass of irregular size could be observed by transmission electronmicroscope. The flow cytometric analysis indicated that the VPA could induce cell apoptosis, apoptosis rate increased in dose-dependent manner, ratio of cells at G(0)/G(1) phase increased and ratio of cells at S phase decreased in dose-dependent manner, the cells were arrested at G(0)/G(1) phase. It is concluded that the VPA can induce apotosis and inhibite proliferation of MUTZ-1 cells via arresting cells at G(0)/G(1) phase.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Humans ; Myelodysplastic Syndromes ; pathology ; Valproic Acid ; pharmacology

OBJECTIVETo study the effects of topiramate (TPM) and valproate acid (VPA) on serum insulin and leptin levels in young and adult rats.

METHODSThirty healthy female young rats (21 days old) and thirty healthy female adult rats (2 months old) were randomly administered with TPM (50 mg/kg daily), VPA (200 mg/kg daily) or normal saline (control group) by intragastric administration for 5 weeks. After 5 weeks, serum leptin and insulin levels were detected by radioimmunoassay (RIA).

RESULTSSerum leptin and insulin levels in both the young and adult TPM groups were remarkably lower than those of the corresponding control group (P < 0.05). The adult TPM group had significantly lower serum leptin and insulin levels than the young TPM group (P < 0.05). In contrast, serum leptin and insulin levels in both the young and adult VPA groups were remarkably higher than those of the corresponding control group (P < 0.05). The young TPM group had significantly higher serum leptin and insulin levels than the adult TPM group (P < 0.05).

CONCLUSIONSTPM decreases serum leptin and insulin levels in young and adult rats, especially in adult rats. VPA increases serum levels of both in young and adult rats, especially in young rats.

Age Factors ; Animals ; Anticonvulsants ; pharmacology ; Body Weight ; drug effects ; Female ; Fructose ; analogs & derivatives ; pharmacology ; Insulin ; blood ; Leptin ; blood ; Rats ; Rats, Sprague-Dawley ; Valproic Acid ; pharmacology

The objective of this study was to investigate antineoplastic effects of valproic acid (VPA) and trichostatin (TSA) on HL-60 and K562 cells in vitro, and the synergic effects of VPA or TSA in combination with ATRA. The inhibitory effects of VPA, TSA and ATRA in various concentrations and different combinations on proliferation of HL-60 and K562 cells were observed by cell growth curves, 50% inhibitory concentration (IC(50)), as well as inhibition of leukemia colony growth at different time points. The characteristics of cell differentiation or apoptosis were analyzed by cytochemical staining, differentiation antigen detection, cell cycle assay and A(NBT)/A(MMT) value determination. The results showed that HL-60 cell had a lower IC(50) of VPA and TSA compared with K562 cells. ATRA could significantly enhance the inhibition of VPA, TSA on clonegenicity of HL-60 cells and inhibition of VPA on clonegenicity of K562 cells. HL-60 cells treated with VPA displayed the phenotype of neutrophilic like cells, and showed the increases of NBT reduction rate and CD11b expression. No evidence for K562 differentiation was found. It is concluded that both VPA and TSA inhibit HL-60 cells growth in vitro. VPA induces differentiation of HL-60 cells to granulocyte. VPA and TSA have a moderate anti-proliferative effect on K562 cells. None of these agents induces K562 cell differentiation.
Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Drug Synergism ; HL-60 Cells ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Inhibitory Concentration 50 ; K562 Cells ; Valproic Acid ; pharmacology

OBJECTIVETo investigate in vivo inhibitory effect of histone deacetylase (HDAC) inhibitor valproic acid (VPA) on xenografted Kasumi-1 tumor in nude mice and its mechanism.

METHODSXenografted Kasumi-1 tumor mouse model was established by subcutaneous inoculation of Kasumi-1 cells. Xenotransplanted nude mice were assigned into control or VPA treatment groups. Volume of the xenografted tumors was measured and compared between the two groups. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was applied to detection of tumor cell apoptosis. The gene expression of GM-CSF, HDAC1, Ac-H3 and survivin was studied with semi-quantitative RT-PCR and Western blotting. ChIP method was used to assay the effects of VPA on acetylation of histone H3 within GM-CSF promoter region.

RESULTS(1) VAP significantly inhibited xenografted Kasumi-1 tumor growth. The calculated inhibition rate was 57.25%. (2) Morphologic study showed that VPA induced differentiation and apoptosis of Kasumi-1 tumor cells. The apoptosis index of VAP treatment group [(3.661 +/- 0.768)%] was significantly higher than that of control group [(0.267 +/- 0.110)%]. (3) Comparing to those in control group, the level of nuclear HDAC1 protein was significantly decreased, the Ac-H3 protein expression level was increased, the mRNA and protein expression levels of GM-CSF and acetylation of histone H3 were remarkably increased, and the gene expression level of survivin significantly decreased in VPA treatment group.

CONCLUSIONVAP significantly inhibits xenografted Kasumi-1 tumor growth and induces tumor cell differentiation and apoptosis. The mechanism may be decrease of survivin gene expression, inhibition of nuclear expression of HDAC, promotion of histone protein acetylation level and acetylation of histone H3 within GM-CSF promoter region, and increase of GM-CSF transcription.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Mice ; Mice, Nude ; Valproic Acid ; pharmacology ; Xenograft Model Antitumor Assays

The aim of this study was to investigate the tumor suppression efficacy of a histone deacetylase inhibitor, sodium valproate combined with adriamycin in the treatment of myelodysplastic syndrome cell line MUTZ-1. After treated with different concentrations of sodium valproate alone, adriamycin alone or combination of them, growth curve of MUTZ-1 cell line were detected; growth of the tumor cells were analyzed by flow cytometry and morphology method. The results indicated that when the myelodysplastic syndrome cell line MUTZ-1 was treated with adriamycin (0.039 microg/ml, 0.078 microg/ml, 0.156 microg/ml, 0.312 microg/ml, 0.4 microg/ml), the tumor growth inhibition rates were 5.08 +/- 0.79%, 12.32 +/- 2.39%, 23.65 +/- 1.34%, 43.33 +/- 2.38% and 47.85 +/- 1.46% (p < 0.05), 0.25 mmol/L sodium valproate did not show apoptosis effect, but could synergize adriamycin to promote apoptosis. When the myelodysplastic syndrome cell line MUTZ-1 treated with two drug combination, the tumor growth inhibition rates were 23.46 +/- 1.12%, 49.87 +/- 0.84%, 52.37 +/- 1.05%, 78.43 +/- 4.34% and 82.47 +/- 1.04% (p < 0.05), and displayed concentration-dependent manner. All the data above were significantly different from those in control (p < 0.05). Sodium valproate showed obvious effect at concentration of 0.078 microg/ml adriamycin. After treated with 0.25 mmol/L sodium valproate and 0.078 microg/ml adriamycin for 72 hours, MUTZ-1 cell line showed typical apoptosis morphological character. It is concluded that sodium valproate may enhance the efficacy of adriamycin on MUTZ-1 cell line.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Doxorubicin ; pharmacology ; Drug Synergism ; Humans ; Myelodysplastic Syndromes ; pathology ; Valproic Acid ; pharmacology