The purpose of this investigation was to study the variation of p73 gene expression in the apoptotic process of acute myeloid leukemia (AML) cell line U937 induced by methotrexate (MTX). Morphological changes of apoptotic cells were observed with microscopy and Wright's + Giemsa staining. DNA ladder and cell cycle were examined by agarose gel electrophoresis and flow cytometry respectively. Using semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR), the expression of p73 mRNA was examined. Results showed that MTX could induce U937 cell apoptosis effectively. Condensed nuclei, fragmentation of chromosome and DNA ladder were seen after 6 hour following treatment of MTX 5 micro mol/L. Sub-G(1) peak and S + G(2)/M arrest were also determined by FCM, but the quantity of p73 expression was generally constant. In conclusion, U937 cell apoptosis induced by MTX did not change p73 mRNA level.
Acute Disease ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; genetics ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; DNA, Neoplasm ; drug effects ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, Tumor Suppressor ; Humans ; Leukemia, Myeloid ; drug therapy ; genetics ; pathology ; Methotrexate ; pharmacology ; Nuclear Proteins ; genetics ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Tumor Protein p73 ; Tumor Suppressor Proteins ; U937 Cells

Drug Evaluation, Preclinical ; methods ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Protein Binding ; drug effects ; Small Molecule Libraries ; chemistry ; pharmacology ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro.

METHODSU87 cells exposed to different concentrations of quercetin (50, 100, and 150 µmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions.

RESULTSQuercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40∓0.70)% at Q0, (22.53∓0.72)% at Q50, (29.06∓0.81)% at Q100, and (31.5∓0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells.

CONCLUSIONQuercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Glioma ; metabolism ; pathology ; Humans ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Quercetin ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism.

METHODSLuciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence.

RESULTSEndogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased.

CONCLUSIONHCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

Hepacivirus ; genetics ; Humans ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; drug effects ; genetics ; metabolism ; physiology ; Viral Core Proteins ; genetics ; Viral Nonstructural Proteins ; genetics ; pharmacology

OBJECTIVETo investigate the effect of demethylating agent 5-Aza-CdR (5-aza-2'- deoxycytidine) on demethylation and transcription-regulating of RASSF1A gene in gastric cancer cell SGC7901 in vitro, as well as on the growth inhibition of cells.

METHODSAfter SGC7901 cells were treated with 5-Aza-CdR, MTT assay, flow cytometry, and Annexin V-FITC staining were performed to analyze the cell proliferation, cell cycle and apoptotic rate respectively. Methylation- specific PCP (MSP), RT-PCR and Western blotting were used to detect methylation state, expression of mRNA and protein of RASSF1A gene.

RESULTSAfter SGC7901 cells were treated with different concentrations of 5-Aza-CdR, the cell growth was inhibited(P<0.05), the cell cycle was blocked at G(1) phase, and the apoptotic rate increased significantly(P<0.05). Hypermethylation was detected in the promoter region of RASSF1A gene in SGC7901 cells, and no expression of RASSF1A mRNA and protein was found. After treated with 5-Aza-CdR, demethylation occurred in RASSF1A gene,which subsequently induced re-expression of this gene at both mRNA and protein level.

CONCLUSIONDemethylating agent 5-Aza-CdR can regulate demethylation and re-expression of RASSF1A gene in gastric cancer cell SGC7901,and inhibit its growth.

Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Stomach Neoplasms ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo study the effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar and its mechanism.

METHODSFibroblasts from human hypertrophic scar were isolated and cultured with DMEM medium containing 10% FBS, and then they were divided into control (C, added with ethanol), low concentration (LC, added with 1 × 10(-5) mmol/L melatonin), middle concentration (MC, added with 1 × 10(-3) mmol/L melatonin), and high concentration (HC, added with 1 mmol/L melatonin) groups according to the random number table. After being cultured for 24 hours, cell morphologic change was observed under microscope; XTT-PMS assay was used to examine cell proliferative activity; cell cycle and apoptosis were assessed with flow cytometry after double staining of FITC and PI, and the levels of cyclin E, p53, and Fas mRNA were determined with fluorescence quantitative RT-PCR. Data were processed with analysis of variance and LSD test.

RESULTS(1) Fibroblasts in C group were spindle-shaped with growth in colonies. Along with the increase in melatonin concentration, fibroblasts in LC, MC, and HC groups gradually dispersed, deformed and atrophied, with shrunk cellular membrane, and decrease in ratio of nucleus and cytoplasm. (2) Proliferative activity of fibroblasts in LC, MC, and HC groups decreased along with an increase in melatonin concentration (1.49 ± 0.15, 1.24 ± 0.20, and 0.92 ± 0.09), which were lower that in C group (1.79 ± 0.10, F = 67.61, P < 0.05). Cell ratios of S and G2/M phases in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were all lower than those in C group [(10.6 ± 1.1)%, (6.1 ± 1.2)%, (3.2 ± 0.8)% vs.(16.9 ± 1.3)%, F = 286.10, P < 0.05; (13.5 ± 1.1)%, (9.8 ± 1.0)%, (6.0 ± 0.7)% vs. (16.7 ± 1.6)%, F = 162.69, P < 0.05]. Apoptotic rates in early and late stages of LC, MC, and HC groups increased along with an increase in melatonin concentration, all higher than those in C group (with F value respectively 424.05, 236.44, P values all below 0.05). The expressions of cyclin E mRNA in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were lower than that in C group (1.58 ± 0.21, 0.90 ± 0.20, and 0.24 ± 0.12 vs. 2.90 ± 0.30, F = 266.79, P < 0.05), while the expressions of p53 mRNA and Fas mRNA showed opposite tendency (with F value respectively 10.11, 12.03, P values all below 0.05).

CONCLUSIONSMelatonin can inhibit proliferation and induce apoptosis of fibroblasts in hypertrophic scar through regulating the gene expressions of cyclin E, p53, and Fas.

Adult ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Cyclin E ; metabolism ; Female ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Male ; Melatonin ; pharmacology ; Oncogene Proteins ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; fas Receptor ; metabolism

The tricarboxylic acid (TCA) cycle is a central route for oxidative phosphorylation in cells, and fulfills their bioenergetic, biosynthetic, and redox balance requirements. Despite early dogma that cancer cells bypass the TCA cycle and primarily utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated oncogene and tumor suppressor expression, rely heavily on the TCA cycle for energy production and macromolecule synthesis. As the field progresses, the importance of aberrant TCA cycle function in tumorigenesis and the potentials of applying small molecule inhibitors to perturb the enhanced cycle function for cancer treatment start to evolve. In this review, we summarize current knowledge about the fuels feeding the cycle, effects of oncogenes and tumor suppressors on fuel and cycle usage, common genetic alterations and deregulation of cycle enzymes, and potential therapeutic opportunities for targeting the TCA cycle in cancer cells. With the application of advanced technology and in vivo model organism studies, it is our hope that studies of this previously overlooked biochemical hub will provide fresh insights into cancer metabolism and tumorigenesis, subsequently revealing vulnerabilities for therapeutic interventions in various cancer types.
Animals ; Citric Acid Cycle ; drug effects ; Humans ; Molecular Targeted Therapy ; methods ; Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Oncogenes ; genetics ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo investigate the role and possible mechanism of JWA in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) inducing human bronchial epithelial (HBE) cells' neoplastic transformation.

METHODSJWA overexpression vector and its stable transfection HBE cells were established. The characteristics of transformed HBE cells were determined by methyl thiazolyl tetrazolium (MTT) assay and the soft agar colony formation assay. The expressions of JWA and P53 were detected by Western blot.

RESULTSThe growth rates of the HBE cells which were treated with MNNG were significantly accelerated than the JWA overexpression HBE cells and controlled HBE cells (P < 0.05). The soft agar colony formation of JWA overexpression HBE cells with and without MNNG treatment (8.06% and 10.14%) was significantly lower than that of the normal HBE cells with MNNG treatment (26.80%) (P < 0.01). After exposure of MNNG, the P53 expressions were gradually increased in HBE cells with the increased passages. However, the expression of P53 in JWA over expressed HBE cells showed a different manner. P53 reached an over expression peak at early stage (the first passage), and then with a gradually down-regulated expression spectrum with increased passages of the cells.

CONCLUSIONJWA might be a key molecule and play an important role in MNNG inducing neoplastic transformation in HBE cells through regulation of the expression of P53.

Cell Transformation, Neoplastic ; drug effects ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Heat-Shock Proteins ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Methylnitronitrosoguanidine ; toxicity ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate arsenic trioxide (As(2)O(3))-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML).

METHODSHepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 micro mol/L As(2)O(3) for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML.

RESULTSThe growth rates of HepG2 cells were slower in the As(2)O(3) treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As(2)O(3) treated groups. The expression of PML decreased in HepG2 cells with 2 micro mol/L As(2)O(3) treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 micro mol/L As(2)O(3), and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 micro mol/L As(2)O(3).

CONCLUSIONSOur results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As(2)O(3) induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As(2)O(3) may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As(2)O(3) treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As(2)O(3) therapy.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Humans ; Neoplasm Proteins ; metabolism ; Nuclear Matrix ; drug effects ; metabolism ; Nuclear Proteins ; Oxides ; pharmacology ; Promyelocytic Leukemia Protein ; Transcription Factors ; metabolism ; Tumor Cells, Cultured ; Tumor Suppressor Proteins

BACKGROUNDMethanesulfonic acid sodium salt (Dipyrone), an antipyretic and analgesic drug, has been demonstrated to improve cerebral ischemia through the inhibition of mitochondrial cell death cascades. The aim of this study was to evaluate the potential photoprotective activity of methanesulfonic acid sodium salt in a model of light-induced retinopathy.

METHODSOne hundred mice were assigned randomly into vehicle (V), methanesulfonic acid sodium salt (D), light damage model plus vehicle (MV) and light damage model plus methanesulfonic acid sodium salt (MD) groups (n = 25 each). In the MD group, methanesulfonic acid sodium salt (100 mg/kg) was administered by intraperitoneal injection 30 minutes before light exposure. Twenty-four hours after light exposure, hematoxylin and eosin staining and transmission electron microscopy (TEM) were used for histological evaluation. The thickness of the outer plus inner-segment and outer nuclear layer was measured on sections parallel to the vertical meridian of the eye at a distance of 1000 mm from the optic nerve. Electroretinography (ERG) test was performed to assess the functional change. The morphology of mitochondria was also revealed by TEM. Finally, the expression of cytochrome c (CytC) and the relative apoptotic proteins were detected by Western blotting, and the interaction between mitochondrial proteins was investigated by co-immunoprecipitation.

RESULTSThe photoreceptor inner and outer segments of the MV group were significantly disorganized than the MD group. The thicknesses of the outer plus inner-segment layers and the outer nuclear layer, and the amplitudes of the a and b waves of the scotopic ERG response markedly decreased in the MV group compared to those in the MD group (P < 0.05). TEM examination revealed that the mitochondria of the MV group were distinctly swollen and contained disrupted cristae. In contrast, the morphology of mitochondria in the MD group was unaffected. Western blotting analysis showed that CytC, apoptosis proteinase activating factor-1 (Apaf-1), caspase 3, p53, p53-upregulated modulator of apoptosis (PUMA), Bax, and Bad were increased, whereas the anti-apoptotic proteins Bcl-2 and Bcl-X(L) were significantly decreased in the MV group than the MD group. Co-immunoprecipitation detection revealed that PUMA immunoreactivity precipitated by Bcl-X(L) decreased, whereas Bax immunoreactivity precipitated by Bcl-X(L) increased in the MD group compared to those in the MV group.

CONCLUSIONMethanesulfonic acid sodium salt is an effective photoprotective agent against light-induced retinopathy through the inhibition of CytC-mediated mitochondrial impairment.

Animals ; Apoptosis ; drug effects ; radiation effects ; Blotting, Western ; Electroretinography ; Immunoprecipitation ; Light ; adverse effects ; Male ; Mesylates ; therapeutic use ; Mice ; Microscopy, Electron, Transmission ; Mitochondrial Proteins ; metabolism ; Random Allocation ; Retina ; drug effects ; radiation effects ; ultrastructure ; Tumor Suppressor Protein p53 ; metabolism