OBJECTIVETo investigate the mechanism of increasing chemosensitivity of gallbladder carcinoma stimulated by somatostatin.

METHODSGBC-SD cells were divided into four groups: SST-alone-treated group, Doxorubicin (DOX)-alone-treated group and co-treated group (co-treatment of SST and DOX). In the control group, the cells were cultivated by medium only. In SST-alone-treated group, the cells were cultivated by medium with SST in the concentration of 75 microg/ml. In DOX-alone-treated group, the cells were cultivated by medium with DOX in the gradient concentrations of 5, 10, 20 microg/ml. In the co-treated group, cells were first cultivated by medium with 75 microg/ml SST for 24 h, followed by the addition of DOX in the gradient concentrations mentioned above. Cell viability curve was measured by MTT assay at 24, 48, 72 and 96 h, respectively. Meanwhile, the alterations of protein expressions of ICBP90 and Topo IIalpha after treatment of SST were examined by Western blot.

RESULTSThe treatment of SST alone on GBC-SD cells did not exert significantly inhibitory effect compared to the control group (P > 0.05). However, 24 h after the treatment of SST, the protein expressions of ICBP90 and Topo IIalpha were both up-regulated (P < 0.05).

CONCLUSIONUp-regulated the expression of ICBP90 by somatostatin maybe the cause of overexpression of Topo IIalpha, which leads to the enhanced lethal effect of DOX.

Antigens, Neoplasm ; metabolism ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Topoisomerases, Type II ; metabolism ; DNA-Binding Proteins ; metabolism ; Doxorubicin ; pharmacology ; Drug Interactions ; Drug Resistance, Neoplasm ; drug effects ; Gallbladder Neoplasms ; drug therapy ; metabolism ; pathology ; Humans ; Somatostatin ; pharmacology

OBJECTIVETo clone differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene (BPDE).

METHODThe malignant transformation of human bronchial epithelial cell line 16HBE induced by BPDE in vitro was used as a model for comparing gene expression between the transformed cells and controls. cDNA representational difference analysis (cDNA-RDA) was performed to isolate differentially expressed cDNA fragment in transformed cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA were sequenced and compared with data in GenBank by BLASTN.

RESULTSFive cDNA sequences were found to be novel ones and were registered in dbest database, which assigned accession numbers in GenBank are BG354691, BG354692, BG354693, BG354694 and BG354695, respectively. Eight of the remaining cDNA sequences showed sequence homology to those previously reported such as ribosomal protein S23, MLN137, ACTN4, transforming growth factor and G protein gene.

CONCLUSIONSThese 13 genes may be involved in BPDE-induced malignant transformation, but their biological characteristics and functions are left to further studies.

Benzopyrenes ; metabolism ; pharmacology ; Carcinogens ; pharmacology ; Cell Transformation, Neoplastic ; chemically induced ; genetics ; Cells, Cultured ; Cloning, Molecular ; DNA, Complementary ; analysis ; drug effects ; DNA, Neoplasm ; analysis ; Gene Expression ; drug effects ; Humans

In order to explore the differences between the mechanisms of selenite-induced apoptosis and arsenic induced apoptosis in NB4 cells, growth inhibition was determined by MTT test, apoptosis determined by DNA electrophoresis and analysis of intracellular DNA contents, reactive oxygen species and reduced glutathione in the cell were measured by Lucigenin dependent chemoluminescent (CL) test and spectrophotometry, and mitochondrial transmembrane potential was measured by flow cytometry. The results showed that: 5 micro mol/L sodium selenite similar to 1 micro mol/L arsenic trioxide could induce the apoptosis of NB4 cells after treatment for 24 hours. Both could elevate the level of reactive oxygen species and intensify mitochondrial transmembrane potential collapse, accompanied with decrease of reduced glutathione centent. The effect of selenium selenite on these aspects was more significant than those of arsenic trioxide. Elevation of intracellular glutathione in N-acytlcysteine pretreated NB4 cells could enhance the selenite induced apoptosis and oxidative stress, but ameliorate the arsenic trioxide induced apoptosis and oxidative stress. It was concluded that sodium selenite and arsenic trioxide can induce the apoptosis of NB4 cells, but there are significant differences between the mechanisms of selenite-induced and arsenic-induced apoptosis in NB4 cells, particularly in the influence of intracellular glutathione content on the drug action.
Acetylcysteine ; pharmacology ; Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; Cell Division ; drug effects ; DNA, Neoplasm ; drug effects ; genetics ; metabolism ; Flow Cytometry ; Glutathione ; drug effects ; metabolism ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Oxides ; pharmacology ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; Tumor Cells, Cultured ; drug effects

Traditional theories suggest that tumor growth occurs when all tumor cells work together and result in proliferation, so treatment has been mainly directed against the majority of the cells in tumor tissue, which often relapse, metastasize, and lead to treatment failure. As cancer stem cells have been successfully isolated from different tumor tissues, in-depth study of their function in relation to traditional cancer treatment faces enormous challenges. At the same time, a new theoretical basis has been provided for the in-depth study of tumorigenesis and the evaluation of prognosis of cancer therapy. Also, new ideas have been introduced for cancer therapy. Therefore, radical treatment of cancer can be achieved through killing cancer stem cells. This article reviews the research progress on cancer stem cells and their drug resistance.
ATP-Binding Cassette Transporters ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Hypoxia ; Cell Transformation, Neoplastic ; DNA Repair ; DNA, Neoplasm ; genetics ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Humans ; Neoplasms ; genetics ; metabolism ; pathology ; Neoplastic Stem Cells ; drug effects ; pathology

Numerous types of cells have been shown to undergo apoptosis when exposed to oxidant agent such as hydrogen peroxide. In order to understand the functional relationship between the anti- and pro-apoptotic regulatory proteins in the cells under oxidant stress, we have studied the level of expression of apoptosis regulatory proteins, bcl-2 and bax, in human leukemia HL-60 cells. The exposure of HL-60 cells to different concentrations of H2O2 for 6 h resulted in a typical apoptosis of the cells as characterized by flow cytometry, cell cycle analysis, and DNA fragmantation. There was a block in G1 to S transition and apoptotic cells were mainly derived from S and G2 cells. Kinetic study demonstrated that the levels of both bcl-2-mRNA and -protein expression were decreased with the progression of cellular apoptosis whereas the level of bax-mRNA was unchanged but the expressed bax-protein was not detectable. Cycloheximide, a nonspecific translation inhibitor, did not prevent the hydrogen peroxide-mediated apoptosis in HL-60 cells. These results suggest that the regulation of bcl-2, but not of bax are important factor in the oxidative stress-induced apoptosis in HL-60 cells.
Apoptosis/drug effects* ; Blotting, Western ; Cycloheximide/pharmacology ; DNA Fragmentation ; DNA, Neoplasm/metabolism ; DNA, Neoplasm/genetics ; DNA, Neoplasm/drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/drug effects ; HL-60 Cells ; Human ; Hydrogen Peroxide/pharmacology* ; Oxidants/pharmacology* ; Protein Synthesis Inhibitors/pharmacology ; Proto-Oncogene Proteins/metabolism ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics* ; RNA, Messenger/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/drug effects

AIMTo investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells.

METHODSHL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit.

RESULTSDMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells.

CONCLUSIONDMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.

Apoptosis ; drug effects ; Carbocyanines ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; DNA Damage ; DNA Fragmentation ; drug effects ; DNA Primase ; antagonists & inhibitors ; Flow Cytometry ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Myeloid ; metabolism ; pathology ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism ; bcl-X Protein ; metabolism

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

OBJECTIVETo probe the possible mechanism of growth-inhibitory and apoptosis of oral squamous cell carcinoma by arsenic trioxide.

METHODSThe induction of apoptosis in two tongue squamous carcinoma cells treated by arsenic trioxide was investigated. The morphology changes of the cells was observed under electron microscope. The mitochondrial transmembrane potential was detected using rhodamine 123 and flow cytometry. The cell cycle was detected by flow cytometry, and p16, p53, BCL-2, Caspase-3, and PARP changes were examined by western blot.

RESULTS1. The antiproliferative effect on the oral squamous carcinoma cells by arsenic trioxide was carried out through two ways: induction of apoptosis and toxicity damage. 2. Activation of the caspase-3 and PARP, while no changes of p16, p53, BCL-2 occurred. 3. Mitochondrial transmembrane potential collapse and G(2)-M stagnation were correlated with apoptosis of oral squamous cell carcinoma.

CONCLUSIONS1. Tubulins and mitochondria may be the chief action position of arsenic trioxide, which is the start-up factors of mechanism. 2. Activation of the caspase-3 proteolytic pathway may be one of the pivotal ways of apoptosis procedure induced by arsenic trioxide.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell ; drug therapy ; metabolism ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; DNA, Neoplasm ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Intracellular Membranes ; drug effects ; physiology ; Membrane Potentials ; drug effects ; Mitochondria ; drug effects ; physiology ; Mouth Neoplasms ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Cells, Cultured ; drug effects ; ultrastructure ; Tumor Suppressor Protein p53 ; metabolism

BACKGROUNDOur previous study had cloned two glioma cell lines SWOZ1 and SWOZ2 isolated from parental glioma cell line SWO38. The 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance of SWOZ1 was higher than that of SWOZ2. Since O6-methylguanine-DNA methyltransferase (MGMT) was thought to be closely related to BCNU resistance in glioma, this study aimed to explore the function of MGMT in glioma resistant to BCNU.

METHODSA BCNU resistant glioma cell line SWOZ2-BCNU was established. The expression of MGMT was detected in SWOZ1, SWOZ2 and SWOZ2-BCNU. Small interferencing RNA targeting MGMT was used to silence the expression of MGMT in resistant cell lines SWOZ1 and SWOZ2-BCNU. The cytotoxicity of BCNU to these cells was measured using the cell counting kit-8 assay. Statistical analysis was carried out by one-way analysis of variance in statistical package SPSS 13.0.

RESULTSThe resistance of SWOZ1 and SWOZ2-BCNU against BCNU was 4.9-fold and 5.3-fold higher than that of SWOZ2. The results of quantitative RT-PCR and Western blotting confirmed that MGMT was both significantly increased in SWOZ1 and SWOZ2-BCNU compared to SOWZ2. After transfection with small interferencing RNA targeting MGMT, a decreased level of MGMT mRNA expression in SWOZ1 and SWOZ2-BCNU for more than 75% compared to negative control was found and confirmed by Western blotting. As a result, the resistance against BCNU was reversed for about 50% both in the BCNU-resistant cell lines SWOZ1 and SWOZ2-BCNU.

CONCLUSIONSSilencing MGMT with specific small interferencing RNA can reverse the BCNU resistant phenotype in these glioma cell lines. MGMT may play an important role both in intrinsic and acquired BCNU-resistance in glioma.

Blotting, Western ; Carmustine ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Glioma ; genetics ; metabolism ; Humans ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; metabolism ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Sincalide ; metabolism

OBJECTIVETo investigate the effects of hydroquinone (HQ) on expression of topoisomerase IIα (TOPOIIα) in human bone marrow mononuclear cells, and to explore the role and possible regulatory mechanism of TOPOIIα involved in toxicity of HQ to hematopoietic cells.

METHODSAfter human bone marrow mononuclear cells were exposed to 50 µmol/L HQ (used the cells which were exposed to sterile distilled water as control); the activity of TOPOII was measured by TOPOII assay kit; the expression levels of TOPOIIα mRNA and protein were detected by RT-PCR technique and Western blotting method respectively; the chromatin immunoprecipitation (ChIP) assay was carried out to study the possible mechanism of TOPOIIα expression changes.

RESULTS(1) The activity of TOPOII was inhibited obviously; the protein and mRNA expression of TOPOIIα were 0.017 ± 0.029 and 0.610 ± 0.128, significantly lower than that in the control with the significant difference (P < 0.01) after treated with HQ for 10 h; (2) The decreased content of TOPOIIα was associated with descended level of histone H4 acetylation than in the control, from 1.198 ± 0.056 to 0.324 ± 0.229, with the significant difference (P < 0.01), without accompanied descended level of histone H3 acetylation, from 1.253 ± 0.045 to 1.177 ± 0.025 (P > 0.05); (3) TOPOIIα mRNA expression decreased gradually after HQ processing, and the chemical modification (histone H4 acetylation) of TOPOIIα promoter happened prior to the mRNA expression.

CONCLUSIONHQ could repress the expression of TOPOIIα in human bone marrow mononuclear cells; the change of histone chemical modification plays an important role in the benzene's hematopoietic toxicity.

Acetylation ; Adult ; Antigens, Neoplasm ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; DNA Topoisomerases, Type II ; metabolism ; DNA-Binding Proteins ; metabolism ; Female ; Histones ; metabolism ; Humans ; Hydroquinones ; toxicity ; Male ; Young Adult