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Aim: To investigate the effect of diallyl disulfide (DADS), a component of garlic, on apoptosis in human mammary cancer cell line (MCF-7) and its mechanisms. Methods: Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Morphology of apoptotic cells was detected by acridine orange and ethidium bromide staining. Apoptotic cells stained with propidium iodide were examined using flow cytometry. Protein levels were detected by Western blot analysis. Results: DADS inhibited the proliferation of MCF-7 cells and induced the apoptotic ratio to increase rapidly. Cleavage of the caspase-3 and caspase-3 substrate poly(ADP-ribose) polymerase was observed in MCF-7 cells after 24 h of treatment with DADS. When the MCF-7 cells were treated with 200 μmol·DL−1 DADS, the stress-activated protein kinase extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase, was inhibited after 6 h; c-Jun N-terminal kinase (JNK), that is stress-activated protein kinase (SAPK), and p38 mitogen-activated protein kinase were activated after 6 h. Conclusion: These results suggest that DADS both inhibits the proliferation of MCF-7 cells and induces apoptosis of MCF-7 cells. The mechanisms may include the inhibition of ERK and the activation of the SAPK/JNK and p38 pathways. [ABSTRACT FROM AUTHOR]

Aim: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as a molecular chaperone on the activated extracellular signal-regulated kinase 1/2 (ERK1/2) and cell proliferation stimulated by reactive oxygen species (ROS) in rat vascular smooth muscle cells (VSMC). Methods: VSMC were exposed to 1 μmol/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the presence or absence of 5 μmol/L geldanamycin, a specific inhibitor of HSP90. Then the total, soluble, and insoluble proteins of the cells were collected. HSP90, ERK1/2, and phosphor-ERK1/2 in the cell lysate were measured by Western blotting. The interaction of HSP90 and phosphor-ERK1/2 was analyzed by immunoprecipi-tation assay, and the nuclear phosphor-ERK1/2 was measured by Western blotting and immunofluorescence. Cell proliferation was tested by cell counting and 3-(4,5-dimethylthiazol-2-y1)-3,5-di-phenyltetrazolium bromide (MTT). Results: The cytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent manner with the peak at 120 min, which is consistent with the late peak of phosphor-ERK1/2. Immunoprecipitation and Western blotting analyses showed that LY83583 increased the interaction of HSP90 with phosphor-ERK1/2, the phosphor-ERK1/2 level, and the soluble phosphor-ERK1/2 level by 1.8-, 2.5-, and 2.9-fold, respectively. In contrast, the insoluble phosphor-ERK1/2 of VSMC was decreased. Interestingly, LY83583 treatment promoted the nuclear phosphor-ERK1/2 by 7.6-fold as confirmed by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC proliferation with the increased expression of HSP90 and levels of soluble and nuclear phosphor-ERK1/2. Pretreatment of geldanamycin antagonized the effect of LY83583. Conclusion: HSP90 could mediate the oxidative stress-stimulated, late-phase activation of ERK1/2 and VSMC proliferation by promoting the ERK1/2 phosphorylation, the association of itself with phosphor-ERK1/2, and the solubility and nuclear translocation of phosphor-ERK1/2. [ABSTRACT FROM AUTHOR]

Abstract Aim  To study the relationship between Daxx expression and the antiapoptotic effects of probucol in THP-1 macrophage. Materials and methods  Apoptosis of THP-1 derived macrophages was induced by exposure to oxidized low density lipoprotein (oxLDL). The development of apoptosis was determined by flow cytometry analysis and nucleic acid-binding dye acridin orange. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and indirect immunofluorescence were used to evaluate the expression of Daxx and caspase-3 at both mRNA and protein level. Results  As expected, THP-1 macrophages exposed to 100 mg/l oxLDL for 48 h exhibited typical morphologic changes of apoptosis, including condensed chromatin and shrunken nucleus. oxLDL treatment markedly increased Daxx expression in a time- and dose-dependent manner, and facilitated Daxx translocation from cytoplasm to nucleus. The percentage of cells with Daxx in nuclei was significantly increased from 8 to 59%. Treatment with probucol (50 μmol/l) for 4 h prior to exposure to oxLDL significantly inhibited Daxx expression and THP-1 macrophage apoptosis by 61.3%. Furthermore, oxLDL enhanced caspase-3 expression with increased mRNA and protein levels, but without obvious change in translocation of caspase-3 (the cells with nuclear Daxx: 14 vs 8%). In contrast, probucol attenuated oxLDL-stimulated caspase-3 expression in THP-1 macrophages. Conclusion  OxLDL-induced apoptosis of THP-1 macrophage is associated with Daxx up-regulation; while inhibition of apoptosis by probucol is related to decreased Daxx expression and nuclear translocation. [ABSTRACT FROM AUTHOR]

Aim: To investigate the protective effect of high density lipoprotein 3 (HDL3) on oxidized low density lipoprotein (ox-LDL)-induced apoptosis in RAW264.7 cells. Methods: RAW264.7 cells were exposed to 50 mg/L ox-LDL for various durations up to 48 h, and apoptosis was detected using Hoechst 33258 staining and flow cytometric analysis. Total cholesterol levels were detected by high performance liquid chromatography, cholesterol efflux was determined by Tritium labeling, and the cellular lipid droplets were assayed by oil red O staining. Results: Treatment with 50 mg/L ox-LDL for 12, 24, and 48 h increased the apoptotic rate of RAW264. 7 cells in a time-dependent manner. The peak apoptotic rate (47.7%) was observed after 48 h incubation. HDL3 at various concentrations (50 mg/L, 100 mg/L, and 200 mg/L) inhibited the ox-LDL (50 mg/L for 48 h)-mediated apoptosis that was accompanied by an increased rate of intracellular cholesterol efflux, and decreased total cholesterol levels in cells in a concentration-dependent manner. Blockage of cholesterol efflux by brefeldin decreased the protective effect of HDL3 on ox-LDL induced apoptosis. Increase of the cholesterol efflux effected by another cholesterol acceptor, β-cyclodextrin, led to a dramatic decrease in the apoptotic rate of cells. Conclusion: HDL3 antagonizes ox-LDL-induced apoptosis in RAW264.7 cells, through reducing the accumulation of toxic cholesterol. [ABSTRACT FROM AUTHOR]

To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS).Rat VSMCs were incubated with onychin 1–50μmol/L or genistein 10μmol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies.The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2%vs70.0%,P<0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3%vs16.4%,P<0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS.Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation. [ABSTRACT FROM AUTHOR]