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Ai Zheng ; 26(9): 957-62, 2007 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-17927852


BACKGROUND & OBJECTIVE: Phospholipase C-gamma 1 (PLC-gamma1) is a vital signal transducer in transmembrane signaling, which regulates cell proliferation and apoptosis. It is overexpressed in many cancers, such as colorectal cancer, which indicates that it is closely related to the genesis and development of tumors. This study was to explore the effects of blocking PLC-gamma1 signaling pathway on the proliferation and apoptosis of human colorectal cancer cell line LoVo, and investigate the signaling mechanisms. METHODS: LoVo cells were treated with PLC-gamma1-specific chemical blocking agent U73122. Cell proliferation was examined by cell counting, MTT assay, and flow cytometry (FCM). Cell apoptosis was observed under a microscope, and measured by agarose gel electrophoresis and FCM with PI simple staining. The expression of hot shock protein 70(HSP70) and Caspase-3 in LoVo cells were detected by Western blot. RESULTS: The proliferation of LoVo cells was inhibited after blocking PLC-gamma1 signaling pathway and the effect was enhanced along with the increasing concentration of U73122. The inhibition rate reached 35% and 45% when treated with 10 micromol/L U73122 for 24 h and 48 h respectively. After blocking PLC-gamma1 signaling pathway, the G1 phase proportion of LoVo cells was increased while the S phase proportion was decreasedû no apoptosis-specific cell shrinkage was found under a light microscope, and no apoptosis-specific DNA ladder was found by agarose gel electrophoresisû no activated Caspase-3 was detected by Western blot, while increased expression of HSP70 was detected. CONCLUSIONS: Blocking PLC-gamma1 signaling pathway can inhibit the proliferation and cell cycle progress of LoVo cells, which may be due to the up-regulated expression of HSP70. PLC-gamma1 is not a vital signal molecule regulating the apoptosis of LoVo cells.

Apoptose , Proliferação de Células , Neoplasias Colorretais/patologia , Estrenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Pirrolidinonas/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Fosfolipase C gama/metabolismo , Transdução de Sinais/efeitos dos fármacos
J Biol Chem ; 280(17): 17497-506, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15731115


It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Reabsorção Óssea , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Humanos , Peróxido de Hidrogênio/farmacologia , Ligantes , Camundongos , Osteoclastos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Ligante RANK , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Regulação para Cima
Br J Haematol ; 125(4): 512-20, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15142122


Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.

Antineoplásicos/uso terapêutico , Arsenicais/uso terapêutico , Linfoma de Burkitt/enzimologia , Peróxido de Hidrogênio/metabolismo , Óxidos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Androstadienos/farmacologia , Apoptose , Trióxido de Arsênio , Linfoma de Burkitt/tratamento farmacológico , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Wortmanina , Proteína bcl-X
Di Yi Jun Yi Da Xue Xue Bao ; 24(4): 375-8, 2004 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-15090301


OBJECTIVE: To examine the effects of hydrogen peroxide (H2O2) on arsenic trioxide (As2O3)-induced apoptosis of human Burkitt lymphoma cells and evaluate the value of fluorescence microscope in analyzing cellular apoptosis. METHODS: As2O3 was used to induce apoptosis in human Burkitt lymphoma cells BJAB, and the cellular changes were analyzed quantitatively using transmission electron microscope and fluorescence microscope after staining with Hoechst 33342/ propidium iodide (PI). RESULTS: Under fluorescence microscope and electron microscope, BJAB cells displayed chromatin aggregation, nuclear margination and fragmentation in response to As2O3 treatment. In the presence of relatively low levels of H2O2 (200 micromol/L), the cell death resulting from apoptosis was inhibited with pyknosis and necrosis becoming the major pathway, while As2O3 failed to induce cell apoptosis. The cells showed none of the typical markers of apoptosis nor any characteristic necrotic changes, and the nuclei exhibited condensation instead of swelling. In addition, the rate of cell death induced by As2O3 was decreased in the presence of H2O2. CONCLUSIONS: Low levels of H2O2 (200 micromol/L) inhibits As2O3-induced (at clinically achievable concentrations) apoptosis of the human malignant lymphoma cells. Fluorescence microscopy makes possible quantitative analysis of apoptosis, capable of distinguishing not only cell apoptosis from necrosis, but also membrane-intact from membrane-permeable apoptotic cells.

Apoptose/efeitos dos fármacos , Arsenicais/antagonistas & inibidores , Linfoma de Burkitt/tratamento farmacológico , Peróxido de Hidrogênio/farmacologia , Óxidos/antagonistas & inibidores , Trióxido de Arsênio , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência
Di Yi Jun Yi Da Xue Xue Bao ; 23(8): 820-2, 2003 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-12919908


OBJECTIVE: To find a new method for obtaining abundant, well-purified and functionally active rat islet cells cultured in vitro. METHOD: The pancreatic tissues of suckling rats underwent repeated digestion for short durations with collagenase, and the cell growth was observed under inverted microscope 18 h after cell inoculation. The cultured cells were then transferred to a new culture plate and the supernate was harvested regularly to determine the concentration of insulin, amylase and glucose-stimulated insulin release. RESULTS: The fibroblast cells in the primary cultured suckling rat cells were obviously reduced, and the ratio of dithizone-stained cells were 85%-90% with the viability exceeding 90% as assessed by trypan blue staining. The secretion function of the cultured cells remained normal after a 7- to 11-day culture. CONCLUSION: Repeated digestion of the pancreatic tissues for short durations with collagenase and timely cell transfer to new plate may help achieve highly purified viable monolayer islet cells that are well applicable for experimental studies.

Ilhotas Pancreáticas/citologia , Animais , Animais Lactentes , Separação Celular , Células Cultivadas , Imuno-Histoquímica , Insulina/análise , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Ratos , Ratos Sprague-Dawley