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1.
Keio J Med ; 59(1): 10-8, 2010.
Article in English | MEDLINE | ID: mdl-20375653

ABSTRACT

We explored the possibility of the cysteinyl leukotriene receptor antagonists, pranlukast and montelukast, preventing tumor cell migration through both cerebral and peripheral capillaries. To study tumor cell migration through brain capillaries, male Fisher rats were cannulated via the cisterna magna under pentobarbital anesthesia. RCN9 cells labeled with a fluorescent marker PKH67 were intravenously administered following arachidonic acid administration into the subarachnoid space, and specimens of the central nervous system were collected every 30 min for 8 h. Arachidonic acid increased the fluid volume with elevated white blood cell and RCN9 cell counts. When given 2 h before arachidonic acid administration, pranlukast, but not montelukast, reduced the fluid volume and inhibited white blood cell and RCN9 cell extravasation through the brain capillary. In addition, a Lewis lung carcinoma metastasis model in mice was used to study the inhibitory effect of pranlu kast and montelukast against cancer cell extravasation through general capillaries. When mice were given food containing either pranlukast or montelukast, immediately after paw amputation, tumor metastasis was prevented by both drugs, and their survival was prolonged. These results show that pranlukast can inhibit tumor cell migration through both the brain and peripheral capillaries, whereas montelukast inhibits tumor cell migration only in the peripheral capillaries.


Subject(s)
Capillary Permeability/drug effects , Leukotriene Antagonists/pharmacology , Neoplasm Metastasis/prevention & control , Acetates/pharmacology , Animals , Carcinoma, Lewis Lung/blood supply , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Lewis Lung/secondary , Cell Line, Tumor , Chromones/pharmacology , Colonic Neoplasms/blood supply , Colonic Neoplasms/drug therapy , Colonic Neoplasms/secondary , Cyclopropanes , Female , Male , Mice , Neoplastic Cells, Circulating/drug effects , Quinolines/pharmacology , Rats , Rats, Inbred F344 , Sulfides
2.
J Biol Chem ; 279(6): 4075-83, 2004 Feb 06.
Article in English | MEDLINE | ID: mdl-14610081

ABSTRACT

Carbonyl stress is one of the important mechanisms of tissue damage in vascular complications of diabetes. In the present study, we observed that the plasminogen activator inhibitor-1 (PAI-1) levels in serum and its gene expression in adipose tissue were up-regulated in aged OLETF rats, model animals of obese type 2 diabetes. To study the mechanism of PAI-1 up-regulation, we examined the effect of advanced glycation end products (AGEs) and the product of lipid peroxidation (4-hydroxy-2-nonenal (HNE)), both of which are endogenously generated under carbonyl stress. Stimulation of primary white adipocytes by either AGE or HNE resulted in the elevation of PAI-1 in culture medium and at mRNA levels. The up-regulation of PAI-1 was also observed by incubating the cells in high glucose medium (30 mm, 48 h). The stimulatory effects by AGE or high glucose were inhibited by antioxidant, pyrrolidine dithiocarbamate, and reactive oxygen scavenger, probucol, suggesting a pivotal role of oxidative stress in white adipocytes. We also found that the effect by HNE was inhibited by antioxidant, N-acetylcysteine and that a specific inhibitor of glutathione biosynthesis, l-buthionine-S,R-sulfoximine, augmented the effect of subthreshold effect of HNE. Bioimaging of reactive oxygen species (ROS) by a fluorescent indicator, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, revealed ROS production in white adipocytes treated with AGE or HNE. These results suggest that cellular carbonyl stress induced by AGEs or HNE may stimulate PAI-1 synthesis in and release from adipose tissues through ROS formation.


Subject(s)
Adipocytes/metabolism , Deoxyguanosine/analogs & derivatives , Plasminogen Activator Inhibitor 1/blood , Plasminogen Activator Inhibitor 1/genetics , Reactive Oxygen Species/metabolism , 8-Hydroxy-2'-Deoxyguanosine , Acetylcysteine/pharmacology , Adipocytes/drug effects , Aldehydes/metabolism , Aldehydes/pharmacology , Animals , Antioxidants/pharmacology , Buthionine Sulfoximine/pharmacology , Cells, Cultured , Deoxyguanosine/blood , Diabetes Mellitus, Type 2/metabolism , Diabetic Angiopathies/etiology , Diabetic Angiopathies/metabolism , Free Radical Scavengers/pharmacology , Glycation End Products, Advanced/metabolism , Glycation End Products, Advanced/pharmacology , Lipid Peroxidation , Male , NF-kappa B/metabolism , Oxidative Stress , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred OLETF , Up-Regulation
3.
Kidney Int ; 63(5): 1671-80, 2003 May.
Article in English | MEDLINE | ID: mdl-12675842

ABSTRACT

BACKGROUND: Uremic toxins have been suggested to promote progression of chronic renal failure. We have shown that organic anion transporter-mediated uptake of uremic toxins induces oxidative stress in opossum kidney renal tubular cells overexpressing the transporter. Plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-kappa B (NF-kappaB) are major factors known to promote tubulointerstitial fibrosis. The present study examined the signaling pathway that is activated by uremic toxins to induce PAI-1 and activate NF-kappaB in human renal proximal tubular cells (HK-2). METHODS: Uremic toxins in the form of organic anion were examined their ability to induce oxidative stress, PAI-1 gene expression, and NF-kappaB activation in HK-2. PAI-1 expression was measured by enzyme-linked immunosorbent assay (ELISA) and the Northern blotting. Human PAI-1 promoter activity was estimated by luciferase reporter gene (NKkappaB-luc) assay. NF-kappaB activation was measured by the pNFkappaB-luc reporter gene and electrophretic gel mobility shift assay. RESULTS: Among organic anion species tested, indoxyl sulfate and indoleacetic acid induced free radical production in HK-2. A nonspecific transporter inhibitor (probenecid) suppressed the IS-stimulated radical production. Indoxyl sulfate and indoleacetic acid dose dependently increased the expressions of PAI-1 mRNA and protein in these cells. The luciferase reporter gene assay revealed that indoxyl sulfate and indoleacetic acid dose dependently activated NF-kappaB and PAI-1 promoter. Activation of NF-kappaB was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-kappaB inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate. CONCLUSION: Uremic toxins induce free radical production by renal tubular cells and activate NF-kappaB which, in turn, up-regulates PAI-1 expression. Thus, progression of chronic renal failure may be promoted by PAI-1 up-regulation induced by uremic toxins.


Subject(s)
Indican/pharmacokinetics , Indoleacetic Acids/pharmacokinetics , Kidney Tubules, Proximal/cytology , NF-kappa B/metabolism , Plasminogen Activator Inhibitor 1/genetics , Anions/pharmacokinetics , Cells, Cultured , Free Radicals/metabolism , Gene Expression/drug effects , Humans , Kidney Failure, Chronic/physiopathology , Kidney Tubules, Proximal/metabolism , Oxidative Stress/drug effects , Promoter Regions, Genetic/physiology , Signal Transduction/drug effects , Toxins, Biological/pharmacology , Up-Regulation/drug effects , Uremia/physiopathology
4.
J Pharmacol Exp Ther ; 303(2): 656-63, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12388648

ABSTRACT

The effect of heat shock protein (hsp) induction on lipopolysaccharide (LPS)-induced increase in vascular permeability was studied in mice as a model of inflammatory mediator-induced inflammatory response. Mice were exposed to an ambient temperature of 43 degrees C for 1 h and then returned to 23 degrees C to recover up to 24 h. Dermal contents of hsp70 and hsp90 but not heat shock cognate protein (hsc)70 increased at 6 h after heat exposure and returned to the basal level at 24 h. LPS was injected subcutaneously at 0, 2, 4, 6, or 24 h after heat exposure. Two hours after LPS injection, vascular permeability was assessed by dermal accumulation of intravenously injected dye. LPS-induced dye leakage was reduced by 42 and 49% in heat-exposed mice after recovery for 4 and 6 h, respectively. Increases in dermal tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) contents induced by LPS were significantly reduced in the heat-stressed mice recovered for 6 h. LPS-induced increase in cyclooxygenase-2 but not TNF-alpha mRNA was attenuated in heat-stressed mice. Deoxyspergualin, an inhibitor of hsc70 and hsp90, and geldanamycin, a specific hsp90 inhibitor, dose dependently reversed the inhibitory effect of heat stress on LPS-induced dye leakage and dermal TNF-alpha content but not PGE(2) content. These results suggest that heat stress attenuated LPS-induced vascular permeability change by inducing hsp90, leading to inhibition of TNF-alpha production.


Subject(s)
Capillary Permeability/drug effects , Heat Stress Disorders/physiopathology , Lipopolysaccharides/pharmacology , Animals , Benzoquinones , Blotting, Western , Cyclooxygenase 2 , Cysteine Proteinase Inhibitors , Dinoprostone/biosynthesis , Fever/physiopathology , Guanidines , Heat-Shock Proteins/metabolism , Isoenzymes/biosynthesis , Isoenzymes/genetics , Lactams, Macrocyclic , Male , Mice , Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandin-Endoperoxide Synthases/genetics , Quinones , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Salmonella typhimurium/chemistry , Skin/drug effects , Skin/metabolism , Tumor Necrosis Factor-alpha/biosynthesis
5.
Br J Pharmacol ; 135(2): 555-63, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11815391

ABSTRACT

A direct effect of uraemic toxins in promoting progression of chronic renal disease has not been established. In this study, we investigated the toxic effects of organic anions which characteristically appeared in the patients with progressive renal disease on renal proximal tubular cells expressing human organic anion transporter (hOAT) 1. A renal proximal tubular cell line, opossum kidney (OK) cells, was transformed with hOAT1. Among the organic anions examined, hippuric acid, para-hydroxyhippuric acid, ortho-hydroxyhippuric acid, indoxyl sulphate and indoleacetic acid showed a high affinity for hOAT1 expressed in the OK cells. Indoxyl sulphate and indoleacetic acid concentration-dependently inhibited proliferation of the hOAT1-transformed cells. The h.p.l.c. analysis demonstrated that cellular uptake of these organic anions was significantly elevated in hOAT1-transformed cells. These organic anions also concentration-dependently stimulated cellular free radical production. The degrees of inhibition of cell proliferation and the stimulation of free radical production induced by the organic anions were significantly higher in the hOAT1-transformed cells than vector-transformed cells. The stimulatory effect of indoxyl sulphate on free radical production was abolished by anti-oxidants and probenecid. Less free radical production was observed in the hOAT1-transformed cells treated with p-hydroxyhippuric acid, o-hydroxyhippuric acid compared with indoxyl sulphate and indoleacetic acid. Hippuric acid had little effect on free radical production. Organic anions present in the serum of patients with progressive renal disease may cause proximal tubular injury via hOAT1-mediated uptake. The mechanism of cellular toxicity by these uraemic toxins involves free radical production. Thus, some uraemic toxins may directly promote progression of chronic renal disease.


Subject(s)
Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Organic Anion Transport Protein 1/physiology , Toxins, Biological/pharmacology , Uremia/metabolism , Animals , Binding Sites , Cell Line , Cell Line, Transformed , Dose-Response Relationship, Drug , Free Radicals/metabolism , Growth Inhibitors/metabolism , Growth Inhibitors/pharmacology , Growth Inhibitors/physiology , Humans , Kidney Tubules, Proximal/metabolism , Opossums , Organic Anion Transport Protein 1/biosynthesis , Organic Anion Transport Protein 1/genetics , Organic Anion Transport Protein 1/metabolism , Rats , Toxins, Biological/metabolism , p-Aminohippuric Acid/pharmacology
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