OBJECTIVETo study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.

METHODSAfter exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.

RESULTSIncubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.

CONCLUSIONSDiamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Arteriosclerosis ; pathology ; Cells, Cultured ; Chemokine CCL4 ; Diamide ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; Humans ; Macrophage Inflammatory Proteins ; metabolism ; RNA, Messenger ; drug effects ; metabolism ; Radiation-Sensitizing Agents ; pharmacology

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; physiology ; Diamide ; pharmacology ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Lipid Peroxidation ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Sulfhydryl Reagents ; pharmacology ; Umbilical Veins ; cytology

In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1 alpha (MIP-1 alpha), the expression of MIP-1 alpha protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1 alpha mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1 alpha was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1 alpha protein in endothelial cells exposed to 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 mumol/L, 5 mumol/L and 10 mumol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F = 188.93, P < 0.01). The mRNA expression in 5 mumol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t = 8.70, P < 0.05). Chemotactic response(99.50 +/- 4.31 microns) to the culture medium conditioned by 5 mumol/L diamide treated ECs, which was stronger than that(66.47 +/- 3.25 microns) conditioned by the ECs (F = 404.31, P < 0.05), was significantly decreased (F = 192.25, P < 0.05) after adding MIP-1 alpha antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1 alpha, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima.
Cells, Cultured ; Chemotaxis, Leukocyte/physiology ; Diamide/*pharmacology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Lipid Peroxidation ; Macrophage Inflammatory Protein-1/*biosynthesis ; Macrophage Inflammatory Protein-1/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Sulfhydryl Reagents/pharmacology ; Umbilical Veins/cytology

OBJECTIVETo investigate the effects of mitochondrial membrane permeability transition pore (MPT)-opened agent diamide and MPT-closed agent cyclosporin A on arsenic trioxide (As(2)O(3))-induced apoptosis in acute promyelocytic leukemia (APL) cell line NB4.

METHODSNB4 cells were treated with As(2)O(3) alone or in combination with diamide or cyclosporin A in different concentrations. Cell apoptosis was assessed by the morphological observation, Annexin-V assay, distribution of cellular DNA contents and genomic DNA electrophoresis. The mitochondrial transmembrane potentials (DeltaPsim) were detected by flow cytometry according to the intensity of rhodamine 123 uptake in cells.

RESULTSBoth diamide and cyclosporin A significantly enhanced As(2)O(3)-induced apoptosis in NB4 cells. The DeltaPsim collapse induced by As(2)O(3) was also enforced by combined treatment with diamide or cyclospo-rin A. 1 micromol/L As(2)O(3) alone treatment for 72 hours led to DeltaPsim disruption in 27.9% of cells, while combined treatment of As(2)O(3) and diamide or cyclosporin A increased DeltaPsim disruption cells to 59.7% and 42.2%, respectively.

CONCLUSIONSAs(2)O(3)-induced DeltaPsim disruption possibly involves with thiol oxidation or crosslink of important components especially ANT-related molecules.

Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; Arsenicals ; pharmacology ; therapeutic use ; Cyclosporine ; pharmacology ; Diamide ; pharmacology ; Drug Synergism ; Enzyme Inhibitors ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; pathology ; Membrane Potentials ; drug effects ; physiology ; Mitochondria ; physiology ; Oxides ; pharmacology ; therapeutic use ; Sulfhydryl Reagents ; pharmacology ; Tumor Cells, Cultured

Reactive oxygen species (ROS) has been implicated as an inducer of NF-kappaB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-kappaB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-kappaB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-kappaB activation by blocking IkappaB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-kappaB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-kappaB reporter gene was measured. NF-kappaB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-kappaB and inappropriate cytosolic IkappaB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-kappaB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-kappaB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-kappaB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-kappaB activity through an IKK-independent pathway.
Cell Nucleus/drug effects/metabolism ; Cytosol/drug effects/metabolism ; Diamide/pharmacology ; Hela Cells/drug effects/metabolism ; Human ; Hydrogen Peroxide/pharmacology ; I-kappa B/drug effects/metabolism ; NF-kappa B/drug effects/genetics/*metabolism ; Oxidants/pharmacology ; *Oxidative Stress ; Protein-Serine-Threonine Kinases/metabolism ; Signal Transduction/drug effects ; Time Factors ; Transcription, Genetic ; Tumor Necrosis Factor/pharmacology