Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Effects of moxibustion with seed-sized moxa cone on apoptosis of myocardial cells after sport fatigue in mice / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of moxibustion on factors related with apoptosis of myocardial cells after sports fatigue in mice as well as the relationship among histone acetyltransferases p300 (p300), CREB binding protein (CBP) and cell apoptosis to discuss the role of p300 and CBP in moxibustion against apoptosis of myocardial cells.</p><p><b>METHODS</b>Sixty clean-grade male Kunming mice were randomly divided into a control group, a sport group and a moxibustion group, 20 cases in each one. Mice in all group received identical feeding environment. Mice in the control group did not received sport nor moxibustion; mice in the sport group and moxibustion group received non-weight swimming training which lasted from 30 min per day to 90 min per day gradually for 21 days; 1 h after swimming training, mice in the moxibustion group received moxibustion with seed-sized moxa cone at "Zusanli" (ST 36) and "Guanyuan" (CV 4), 5 cones at each acupoint, once a day for 21 days. 24 h after the final swimming training, cardiac muscle tissue was collected to test factor associated suicide (Fas), B cell lymphoma/lewkmia-2 (Bcl-2) by immunohistochemical method and expression of p300 and CBP.</p><p><b>RESULTS</b>Compared with the control group, the apoptosis rate of myocardial cells in the sport group was significantly increased (P<0.01), and apoptosis body with dense distribution and deep coloring can be seen in the field of microscope; the expression of Fas protein was significantly increased (P<0.01), and expression of Bcl-2, p300 and CBP was reduced (all P<0.01). The equally distributed apoptosis body with slight coloring was seen in the moxibustion group. Compared with the sport group, the apoptosis rate of myocardial cells in the moxibustion group was significantly reduced (P<0.05); the expression of Fas protein was significantly reduced (P<0.05), and expression of Bcl-2, p300 and CBP was increased (all P<0.05).</p><p><b>CONCLUSION</b>Moxibustion could promote the expression of p300 and CBP in myocardial cells after sports fatigue in mice to inhibit the starting of apoptotic process, therefore reducing the apoptosis of myocardial cells after heavy exercise and protecting heart function.</p>

Structure-based identification of drug-like inhibitors of p300 histone acetyltransferase / 药学学报

A growing body of evidence suggests that p300 histone acetyltransferase plays important roles in cancer cell differentiation and proliferation. Here, we employed structure-based hierarchical virtual screening method to identify novel lead compounds of p300 histone acetyltransferase. From a screening library containing approximate 100 000 diverse druglike compounds, 33 compounds were chosen for experimental testing and one compound, 4-acetyl-2-methyl-N-morpholino-3,4-dihydro-2H-benzo[b][1, 4]thiazine-7-sulfonamide (17), showed as micromolar inhibitor. Based on its predicted binding pose, we investigated its binding characteristics by designing two series of structural modifications. The obtained structure-activity relationship results are consistent with the predicted binding model. We expect that the identified novel p300 histone acetyltransferase inhibitors will serve as starting points for further development of more potent and specific histone acetyltransferase inhibitors.

Mechanism of E1A-mediated escape from ras-induced senescence in human fibraoblasts / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of binding activities of the NH(2) terminus of E1A to the proteins regulating cell growth on ras-induced cell senescence and explore the mechanism of E1A-mediated escape from ras-induced senescence by E1A in human fibroblast.</p><p><b>METHODS</b>In primary human fibroblasts, the proteins regulating cell growth in association with E1A NH(2) terminus, including the Rb family proteins, p300/CBP, and p400, were inactivated or interfered. The effect of alterations in the binding activities of these proteins on cell senescence bypass mediated by E1A was evaluated by cell growth curve.</p><p><b>RESULTS</b>The Inactivation of Rb family proteins alone was not sufficient to rescue ras-induced cell senescence, whereas inactivation of both the Rb proteins and p300/CBP blocked ras-induced senescence of human fibroblasts.</p><p><b>CONCLUSION</b>Rb and p300/CBP binding activities are both required for E1A to bypass ras-induced senescence in human fibroblasts.</p>

Effect of artemisinin combined with glucocorticoid on the expressions of glucocorticoid receptor α mRNA, glucocorticoid receptor β mRNA and P300/CBP protein in lupus nephritis mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic effects and mechanisms of using artemisinin (Art) combined with glucocorticoid (GC) to treat lupus nephritis (LN) mice.</p><p><b>METHODS</b>Forty hybrid female mice were randomly and equally divided into four groups with the method of random number table: control group, model group, prednisone group administrated with 6.45 mg/(kg·d) prednisone suspension, and Art+prednisone group administrated with 150 mg/(kg·d) Art suspension and 3.225 mg/(kg·d) prednisone suspension. A mice model of LN was established by injection with living lymph cell suspension. The changes of urine protein/24h, the expressions of GC receptor α (GRα) mRNA, GC receptor β (GRβ) mRNA in peripheral blood mononuclear cells (PBMCs), and transcriptional coactivator P300/CBP protein in renal tissue were measured.</p><p><b>RESULTS</b>Compared with the model group, the treatment groups had significant decrease in urine protein/24 h, and renal pathological lesion (P<0.01). In the same groups, the expression of transcriptional coactivator P300/CBP protein in renal tissue and GRα mRNA were significantly increased, and GRβ mRNA expression was significantly decreased (P<0.01). And the Art+prednisone group has a better therapeutic effect than the prednisone group (P<0.01).</p><p><b>CONCLUSIONS</b>Art has therapeutic sensitization effects on GC in the LN mice. The underlying mechanism could be correlated with the effect of Art on the increase of the expressions of GRα mRNA and transcriptional coactivator P300 300/CBP protein in renal tissue and on the decrease of the expression of GRβ mRNA in PBMC.</p>

Exploring the binding affinities of p300 enzyme activators CTPB and CTB using docking method.

CREB binding protein (CBP) and E1A binding protein p300, also known as p300 are functionally related transcriptional co-activators (CoAs) and histone acetyltransferases (HATs). Some small molecules, which target HATs can activate or inhibit the p300 enzyme potently. Here, we report the binding affinities of two small molecules CTPB [N-(4-chloro- 3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide] and CTB [N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide] with p300 using docking method to obtain the insight of their interaction with p300. These small molecules bind to the enzyme, subsequently causing a structural change in the enzyme, which is responsible for the HAT activation. CTB exhibits higher binding affinity than CTPB, and their lowest docked energies are -7.72, -1.18 kcal/mol, respectively. In CTPB molecule, phenolic hydroxyl of Tyr1397 interacts with the non-polar atoms C(5E) and C(5F), and forms polar-non polar interactions. Similar interactions have also been observed in CTB. The residues Tyr1446 and Cys1438 interact with the non-pentadecyl atoms. Further, the docking study predicts a N-HO hydrogen bonding interaction between CTB and Leu1398, in which the HO contact distance is 2.06 Å. The long pentadecyl chain of CTPB reduces the formation of hydrogen bond with the p300. The H-bond interaction could be the key factor for the better activation of CTB.

The effect of p21 on transcription of survivin in hepatocellular carcinoma HepG2 cells and its regulation mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms.</p><p><b>METHODS</b>Doxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300.</p><p><b>RESULTS</b>After treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01).</p><p><b>CONCLUSION</b>p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.</p>

Transcriptional induction of DLC-1 gene through Sp1 sites by histone deacetylase inhibitors in gastric cancer cells

We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.

Constructions of Gcn5 shRNAs interfere the histone acetylation modification with stem cell differentiation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct the Gcn5 shRNA plasmids and to explore the Gcn5 shRNA role in histone acetylation modification with the differentiation of stem cells.</p><p><b>METHODS</b>Seven shRNA fragments were recombined into pGenesil-1 vector to form 7. Gcn5 shRNA constructions. The mesenchymal stem cells (MSCs) induced for two weeks with 5-aza were transfected by the plasmids with lipofectamine2000. Polyclonal antibodies labeled with TRITC were used to identify the acetylation in MSCs with or without Gcn5 shRNA constructions. The efficiencies of transfection and RNAi were calculated based on the ratio of GFP (green fluorescence)/DAPI (blue fluorescence) and TRITC (red fluorescence)/DAPI, respectively.</p><p><b>RESULTS</b>Seven Gcn5 shRNA plasmids or constructions were identified by restriction endonucleases Pst I/Sal I and DNA sequencing. Acetylation block was observed after Gcn5 shRNA plasmids transfected into cells. Fluorescent intensity of TRITC in nucleuses were decreased remarkably, or even disappeared in MSCs. The efficiencies of transfection and RNAi were 93.7% and 46.6%, respectively.</p><p><b>CONCLUSION</b>The Gcn5 shRNA plasmids constructed in the present study can decrease the histone acetylation during cell differentiation. It sets the basis for further exploring the role of acetylation in the regulation of cell differentiation.</p>