In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/ PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.
Acetylation ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; HL-60 Cells ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; chemistry ; Humans ; Hydroxamic Acids ; pharmacology

In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/ PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.
Acetylation ; Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; HL-60 Cells ; Histone Deacetylases/antagonists & inhibitors ; Histone Deacetylases/*chemistry ; Hydroxamic Acids/*pharmacology

OBJECTIVETo establish a rapid, relatively quantitative method of detecting acetylated proteins.

METHODSThe proteins of Jurkat cells were acetylated by Trichostatin A (TSA) at different concentrations, then enriched and purified by anti-acetylated lysine antibodies affinity chromatography colum. The components eluted by acid were fixed on the microplate, the levels of acetylated proteins were tested by ELISA, and their components were identified by MALDI-TOF-TOF mass spectrometry. Also the above-mentioned methods were applied to the other three agents (gallic acid, emodin and monoacetylated emodin A).

RESULTSThat 4 × 10(5) Jurkat cells treated with 1 µmol/L TSA produced the optimal acetylated effect, up to 22 acetylated proteins were identified by MALDI-TOF-TOF, of them 15 were acetylated histones. The other three agents also induced acetylation, the relative values of acetylated proteins of Jurkat cells treated with 35.09 µmol/L and 17.54 µmol/L gallic acid were 4.3% and 14.2% respectively; those as of 28.7% and 11.5% treated with 1.47 µmol/L and 2.94 µmol/L emodin; those as of 22.0% and 3.6% treated with 152.91 µmol/L and 30.58 µmol/L monoacetylated emodin A.

CONCLUSIONThe method based on affinity chromatography colum may be useful for the detection of acetylated proteins, and could be used to screen agents which target to histone deacetylase.

Acetylation ; Chromatography, Affinity ; Histones ; analysis ; Humans ; Hydroxamic Acids ; pharmacology ; Jurkat Cells ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the effect of trichostatin A(TSA) on SGC- 7901 cells.

METHODSCytotoxicity and cell viability of gastric cancer cell line SGC- 7901 were assayed by MTT method. Morphologic assessment of apoptosis was performed with fluorescence microscope. Cell cycle and apoptosis rate were analyzed by flow cytometry. Histone H3 acetylation was detected by Western blot.

RESULTSTSA showed apparently cytotoxicity in SGC- 7901 cells. The growth curve showed the growth ratio decreased with the increase of TSA concentration. Apoptosis rate were significantly different between TSA treated group(75 ng/ml for 72 h)and control group (P < 0.05). Morphologic changes of apoptosis including nuclear chromatin condensation and fluorescence strength were observed with fluorescence microscope.TSA treatment (75 ng/ml for 72 h) sensitively induced apoptosis in the cell,which was demonstrated by the migration of many cells to the sub- G1 phase,the reduction of G1- phase cells and the increment of apoptosis rate (29.54%) in flow cytometric analysis. The expression of acetylated histone H3 was increased in TSA group(75 ng/ml) for 48 h compared with control group by Western blot.

CONCLUSIONSTSA can induce SGC- 7901 cell apoptosis. The expression of acetylated histone H3 may contribute to the apoptosis.

Acetylation ; drug effects ; Apoptosis ; drug effects ; Cell Line, Tumor ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Stomach Neoplasms

To investigate the effect of monoamine oxidase inhibitor tranylcypromine (TCP) on the differentiation of human U251 glioma cells, we treated U251 cells with TCP and/or 100 nmol/L histone deacetylase inhibitor trychostatin A (TSA). The differentiation of U251 cells was observed with inverted microscopy. The cell proliferation and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Apoptosis was observed by Hoechst 33258 staining. The levels of differentiation-related genes were assessed by real-time PCR and Western blotting. TCP-induced differentiation was characterized by typical morphological changes, inhibition of cellular proliferation, accumulation of cells in the G1 phase of the cell cycle, decreased expression of the pluripotency transcription factors Oct4 and Sox2, and increased expression of glial fibrillary acid protein (GFAP). The combination of TCP and TSA treatment also triggered an over-expression of GFAP. These findings suggest that TCP may induce differentiation of U251 glioma cells, and the differentiation process may be promoted by histone deacetylase inhibitor TSA.
Brain Neoplasms ; pathology ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; drug effects ; Glioma ; pathology ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Hydroxamic Acids ; pharmacology ; Monoamine Oxidase Inhibitors ; pharmacology ; Tranylcypromine ; 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 explore the impact of a new generation of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on multiple myeloma (MM) cells proliferation and its mechanism.

METHODSMM cell line U266 and dexamethasone resistant cell line MM1R cells were treated with different concentrations of LBH589 alone or in combination with bortezomib, the inhibition of cells proliferation was detected by MTT, the cell cycle and apoptosis by flow cytometry. The expression level of histone H4 acetylation and PARP, Bcl-X protein was analyzed by western blot, expression level of caspase-3, APAF-1 and TOSO gene by real-time fluorescence quantitative PCR.

RESULTSU266 and MM1R cell proliferation were inhibited by different concentrations of LBH589 (0, 10, 20, 50 nmol/L) alone or 50 nmol/L of LBH589 in combination with bortezomib (10, 20 nmol/L) in a dose- and time-dependent manner. Inhibition effect was significantly higher in all combinative groups than in single agent groups (all P < 0.05). The percentage of G(0)/G(1) phase in MM1R cells were 36.60%, 46.50%, 51.40%, 57.10%, 75.48%, 79.73%, respectively, and the apoptosis rate were 5.27%, 31.41%, 39.78%, 44.07%, 73.60%, 83.27%, respectively. The effects appeared to occur in a dose-dependent manner, and being significantly higher in all combinative groups than in single agent groups (all P < 0.05). The expression of the caspase-3 and APAF-1 gene up-regulated gradually, while TOSO gene expression in MM1R cells down-regulated gradually in a dose- and time-dependent manner (all P < 0.05).

CONCLUSIONSLBH589 can inhibit the growth of MM cells, block the cell cycle and induce cell apoptosis, which has anti-resistant effect on multidrug resistant cell. At the same time LBH589 in combination with bortezomib on myeloma cell has a synergistic effect, its mechanism and reversal of drug resistance mechanism involves in multiple changes in gene expression.

Acetylation ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Hydroxamic Acids ; pharmacology ; Indoles ; pharmacology ; Multiple Myeloma ; pathology ; Pyrazines ; pharmacology

This study was purposed to explore the effect of a new generation of histone deacetylase inhibitor LBH589 alone or combined with bortezomib (Bor) on multiple myeloma cells (MM1R) in vitro. The effect of LBH589 (10, 20, 50 nmol/L) alone or combined with Bor (10, 20 nmol/L) on MM1R proliferation was detected by MTT method; the effect of LBH589 on cell cycle and apoptosis of MM1R cells were determined by flow cytometry; the histone H4 acetylation level of MM1R cells treated with LBH589 (10, 20, 50 nmol/L) for 24 h was analyzed by Western blot. The results showed that the LBH589 alone or combined with Bor all could inhibit the proliferation of MM1R cells in a concentration- and time-dependent manner. After MM1R cells were treated with drugs for 48 h, the cells in G(0)/G(1) phase increased, the cells in G(2)/M and S phase decreased, suggesting the arrest of cells in G(0)/G(1) phase, at the same time, the apoptosis rate of MM1R cells treated with drugs increased in a concentration-dependent manner, while the effect of LBH589 combined with Bor was more obvious than that of LBH589 alone (P < 0.001). Western blot analysis showed that the histone H4 acetylation level was enhanced in concentration-dependent manner after MM1R cells were treated with different concentrations of LBH589 for 24 h. It is concluded that the LBH589 can inhibit the proliferation of MM1R cells, block the cell cycle, induce cell apoptosis, moreover LBH589 combined with Bor has synergistic effect on MM1R cells.
Acetylation ; drug effects ; Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Hydroxamic Acids ; pharmacology ; Indoles ; pharmacology ; Multiple Myeloma ; pathology ; Pyrazines ; pharmacology

This study was aimed to investigate the methylation status of WT1 gene in leukemia cell lines and its relation with expression of WT1 gene. The WT1 gene was silenced by DNA methylation or histone deacetylation, and the expression of WT1 gene was induced by using HDAC inhibitor and/or demethylation agent of DNA. Some leukemia cell lines (U937, HL-60, K562, KG1) were detected by RT-PCR, MS-PCR, restriction analysis, and DNA sequencing. U937 leukemic cells without WT1 mRNA expression were incubated with HDAC inhibitor Trichostatin A (TSA) and/or demethylation agent decitabine. The results showed that the U937 cells did not express WT1 gene, but HL-60, K562 and KG1 cells highly expressed WT1 gene; WT1 gene was unmethylated in HL-60 cells, but methylated in K562 and U937 cells. WT1 expression could be reactivated by co-incubation with TSA and decitabine, but not was observed by using single drug. It is concluded that WT1 promoter is methylated in some leukemia cells, however, the methylation can not affect its expression. DNA methylation and deacetylation of histones are synergistic to inhibit the expression of WT1 in leukemic U937 cells, the combination of TSA with decitabine can induce expression of WT1 gene.
Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; Gene Silencing ; HL-60 Cells ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; Promoter Regions, Genetic ; U937 Cells ; WT1 Proteins ; genetics

OBJECTIVETo study the effect on promoter de-methylation, expression of ALDH1a2 gene and cell apoptosis by treated with 5-Aza-dC and TSA in five human bladder cancer cell lines.

METHODSHuman bladder cancer cell lines RT-4, 253J, 5637, BIU-87 and T24 were cultured and treated with 5-Aza-dC and(or) TSA. The expression of the ALDH1a2 gene was detected by RT-PCR and Western blot. The methylation status of gene promoter was determined by MSP, and the cell cycle profile was established by flow cytometry.

RESULTSALDH1a2 was silenced in five human bladder cancer cell lines. Re-expression of ALDH1a2 was detected after treated with 5-Aza-dC alone or TSA in combination. ALDH1a2 transcript was marked in each cell lines combined with 5-Aza-dC and TSA treatment which showed a synergistic effect on expression of ALDH1a2 transcript. Early apoptotic was the main mode of apoptosis and death of human bladder cancer cell lines induced by 5-Aza-dC and TSA. The percentage of early apoptotic cells was 1.4% in control group and 2.8% in TSA group, however, 20.2% in 5-Aza-dC group and 33.8% in 5-Aza-dC + TSA group, respectively. The groups of TSA, 5-Aza-dC and 5-Aza-dC + TSA were significantly different from control group (P < 0.05).

CONCLUSIONSAberrant methylation of ALDH1a2 gene is the main cause for gene transcriptional inactivation. Re-expression of ALDH1a2 gene and cell apoptosis are detected after either treatment with 5-Aza-dC alone or in combination with TSA.

Apoptosis ; drug effects ; Azacitidine ; pharmacology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Hydroxamic Acids ; pharmacology ; Retinal Dehydrogenase ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology