[Show abstract][Hide abstract] ABSTRACT: Recent studies show that up-regulation of cyclooxygenase-2 (COX-2) in human cancer cells induces activation of matrix metalloproteinases (MMPs) and increase of metastatic potential. In this study, we investigate the effect of a COX-2 selective inhibitor, NS398, on the expression and enzymatic activity of MMPs in human lung cancer cells. We found that NS398 inhibited MMP-2, not MMP-9, mRNA expression. NS398 also reduced the amount of MMP-2, not MMP-9, released into the medium. Additionally, this COX-2 inhibitor attenuated the degrading activity of MMP-2 as demonstrated by gelatin zymography. Investigation of cellular MMP-2 by Western blotting indicated that synthesis and processing of MMP-2 was significantly suppressed by NS398. We performed promoter activity assay to address whether NS398 might affect MMP-2 gene transcription. Our results indicated that NS398 directly inhibited MMP-2 promoter activity. However, the inhibitory effect of NS398 is not fully dependent on inhibition of COX-2 because a high concentration of NS398 was needed to suppress MMP-2 expression and addition of prostaglandin E2 only partially reversed the action of NS398. Moreover, a non-selective COX inhibitor indomethacin also suppressed the expression of MMP-2. Taken together, these results indicate that non-steroidal anti-inflammatory drugs suppress MMP-2 expression via repression of transcription and support the notion that COX inhibitors may be useful in inhibition and/or prevention of metastasis.
[Show abstract][Hide abstract] ABSTRACT: Advanced glycation end products (AGEs) are important in the pathogenesis of diabetic nephropathy, which leads to renal fibrosis. Previously, we found that the janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is necessary for AGE-induced cellular proliferation in normal rat kidney interstitial fibroblast (NRK-49F) cells. However, a direct link between JAK/STAT and cell-cycle progression has not been well established. In this regard, STAT5 has been found to induce cyclin D1 and proliferation in hematopoietic cells. Therefore, we examined effects of AGE on STAT5 and cell-cycle-dependent mitogenesis in NRK-49F cells. We found that AGE increased cyclin D1 expression and cyclin-dependent kinase (cdk)4 activity while decreasing p21(WAF1/CIP1) expression. We also found that AGE (100 microg/mL) induced STAT5 tyrosine phosphorylation. Meanwhile, AGE induced STAT5 protein-DNA binding activity, which was reversed by AG-490 (a specific JAK2 inhibitor) and STAT5 decoy oligodeoxynucleotide (ODN). In addition, STAT5 decoy ODN reversed AGE-induced cell-cycle-dependent cellular proliferation and cyclin D1 protein expression. We concluded that AGE induced cell-cycle-dependent cellular proliferation by inducing the JAK2-STAT5-cyclin D1 and cdk4 pathways in NRK-49F cells.
American Journal of Kidney Diseases 12/2001; 38(5):1096-104. · 5.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our previous work has shown that a number of sphingolipid metabolites including sphingosine, sphinganine, and other long-chain bases potently induced apoptosis in human hepatoma cells. In this study, we examined the possibility that sphingosine may trigger apoptosis in human hepatoma cells via inhibition of anti-apoptotic pathways. We investigated the effect of sphingosine on AKT kinase, a serine/threonine kinase which was found to protect cells from apoptosis induced by a variety of extracellular stresses. Our results indicated that sphingosine inhibited basal and serum-stimulated AKT kinase activity in a dose-dependent manner in hepatoma cells. Additionally, sphingosine-induced inhibition of AKT kinase was correlated with induction of apoptosis in these cells. Pretreatment of insulin, a potent stimulator of AKT kinase, partially reversed the inhibition of AKT kinase by sphingosine and counteracted the apoptotic action of this sphingolipid. Expression of activated AKT kinase partially protected cells from sphingosine-induced apoptosis, whereas expression of kinase-dead AKT kinase had no effect. The molecular mechanism by which AKT kinase suppressed the apoptotic action of sphingosine was investigated. Our results showed that increased release of cytochrome C from mitochondria and subsequent activation of caspase-3 were detected in sphingosine-treated hepatoma cells. On the contrary, expression of activated AKT kinase in Hep3B cells attenuated cytochrome C release and caspase-3 activation induced by sphingosine. Taken together, these findings suggest that suppression of AKT kinase is one of the mechanisms by which sphingosine induces apoptosis in hepatoma cells and activation of AKT kinase may inhibit sphingosine-induced apoptosis by blocking a step upstream of cytochrome C release and caspase-3 activation.
Journal of Cellular Physiology 09/2001; 188(2):188-93. · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy (DN), and captopril (an angiotensin converting enzyme inhibitor) is effective in treating this disorder. We have shown that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cascade is responsible for AGE-induced mitogenesis in NRK-49F (normal rat kidney fibroblast) cells, but its role in renal fibrosis in DN remains unknown. Therefore, we have sought to determine whether JAK/STAT is involved in AGE-regulated collagen production in NRK-49F cells. We found that AGE time (1-7 days) and dose (10-200 microg/ml)-dependently increased collagen production in these cells. Additionally, AGE increased RAGE (receptor for AGE) protein expression. AGE-induced RAGE expression was dose-dependently inhibited by antisense RAGE oligodeoxynucleotide (ODN) and captopril. AGE-induced type I collagen production and JAK2-STAT1/STAT3 activation were decreased by AG-490 (a specific JAK2 inhibitor), antisense RAGE ODN and captopril. Meanwhile, STAT1 and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. We concluded that RAGE and the JAK2-STAT1/STAT3 pathway were involved in AGE-induced collagen production in NRK-49F cells. Furthermore, captopril was found to reverse AGE-induced collagen production, probably by attenuating RAGE expression and JAK2-STAT1/STAT3 activities.
Journal of Cellular Biochemistry 02/2001; 81(1):102-13. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased expression of cyclooxygenase-2 (COX-2) causes enhanced production of prostaglandins, which are emerging as important mediators of growth stimulation of cancer cells. Overexpression of COX-2 has been found in human non-small cell lung cancer tissues and cell lines. In vitro and in vivo studies showed that nonselective cyclooxygenase inhibitors (like aspirin and indomethacin) may suppress growth of lung cancer cells and may prevent lung tumorigenesis induced by the tobacco-specific carcinogens. However, the molecular mechanisms that mediated the anticancer action of these inhibitors are not well defined. In this study, we examined the effect of a specific COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398), on high COX-2-expressing A549 lung cancer cells. Our results indicated that NS398 inhibited prostaglandin E(2) synthesis and induced G(1) growth arrest in these cells. NS398 specifically up-regulated cyclin-dependent kinase inhibitor p27(KIP1), whereas the expressions of G(1)-acting cyclins and cyclin-dependent kinases were not changed. Additionally, NS398 effectively suppressed cyclin E-associated kinase activity in A549 cells. The molecular mechanism responsible for the induction of p27(KIP1) by NS398 was characterized. We found that NS398 did not induce p27(KIP1) through transcriptional activation because this drug could not stimulate the p27(KIP1) promoter. Metabolic labeling experiments showed that the synthesis rate of p27(KIP1) protein was not altered by NS398. Conversely, pulse-chase assays demonstrated that degradation of p27(KIP1) protein was obviously reduced in NS398-treated cells. We conclude that NS398 enhances p27(KIP1) expression via post-translational regulation, and our results provide a new mechanism by which specific COX-2 inhibitors suppress proliferation of cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Our previous results demonstrated that the plant amino acid mimosine blocked cell cycle progression and suppressed proliferation of human lung cancer cells in vitro by multiple mechanisms. Inhibition of cyclin D1 expression or induction of cyclin-dependent kinase inhibitor p21WAF1 expression was found in mimosine-treated lung cancer cells. However, whether mimosine may modulate the expression of these cell cycle regulatory proteins and suppress tumor growth in vivo is unknown. In this study, we examined the anti-cancer effect of mimosine on human H226 lung cancer cells grown in nude mice. Our results demonstrated that mimosine inhibits cyclin D1 and induces p21WAF1 expression in vivo. Furthermore, results of TUNEL analysis indicated that mimosine may induce apoptosis to suppress tumor growth in nude mice. Collectively, these results suggest that mimosine exerts anti-cancer effect in vivo and might be useful in the therapy of lung cancer.
International Journal of Oncology 11/2000; 17(4):659-65. · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although originally synthesized as an anti-estrogen, tamoxifen (Tam) was found to be able to inhibit proliferation of estrogen receptor (ER)-negative cancer cells in vitro. However, the molecular basis of such ER-independent growth inhibition is largely unknown. We have previously demonstrated that Tam induces p21WAF1 and p27KIP1 expression in human lung cancer cells which lack ER-alpha and -beta. We found that Tam induced p21WAF1 expression via transcriptional activation. In order to determine the molecular mechanism responsible for p21WAF1 induction by Tam, we performed a deletion analysis on the p21WAF1 promoter. The minimal region in the p21WAF1 promoter required for Tam-activated induction was mapped to a contiguous stretch of 10 bp located 83 bases upstream of the transcription initiation site. Our results showed that transcription factor Sp1 and Sp3 bound to this GC-rich region and mutation of Sp1-binding sites dramatically attenuated Tam-induced p21WAF1 promoter activity. We also tried to elucidate the signaling pathway that mediated the activation of p21WAF1 by Tam. Inhibition of mitogen-activated protein kinase pathways did not block Tam-induced p21WAF1. Similarly, protein kinase C inhibitor calphostin C could not suppress Tam-induced p21WAF1. Conversely, pretreatment of a specific protein kinase A inhibitor H89 significantly attenuated the induction of p21WAF1 by Tam. Furthermore, PKA activators forskolin and dibutyryl-cAMP activated p21WAFI promoter activity and increased p21wAF1 protein level in lung cancer cells. Taken together, these results demonstrate that Tam activates the p21WAF1 promoter via Sp1-binding sites and suggest that PKA may be involved in the induction of p21wAF1 by Tam in ER-negative lung cancer cells.
[Show abstract][Hide abstract] ABSTRACT: In this study, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Rutaecarpine (200 microg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules when it was intravenously injected. Rutaecarpine (200 microg/g) prolonged occlusion time by approximately 1.5-fold (control 127 +/- 29 vs. taecarpine 188 +/- 23 s). Furthermore, aspirin (250 microg/g) also showed a similar prolongation of the occlusion time in this experiment. On a molar basis, rutaecarpine was approximately twofold more potent than aspirin at prolonging the occlusion time. Furthermore, rutaecarpine was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 25 and 50 microg/g. Intravenous injection of rutaecarpine (50 microg/g) significantly prolonged the bleeding time by approximately 1.5-fold compared with normal saline in the severed mesenteric arteries of rats. Continuous infusion of rutaecarpine (5 microg/g/min) also significantly increased the bleeding time 1. 5-fold, and the bleeding time returned to baseline within 60 min after cessation of rutaecarpine infusion. These results suggest that rutaecarpine has an effective anti-platelet effect in vivo and that it may be a potential therapeutic agent for arterial thrombosis, but it must be assessed further for toxicity.
British Journal of Haematology 08/2000; 110(1):110-5. · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetramethylpyrazine (TMPZ) is an active ingredient of a Chinese herbal medicine (Ligusticum wallichii Franchat). In this study, TMPZ (50-200 microM) significantly increased production of nitrate and cyclic GMP in human platelets within a 15-min incubation period. TMPZ concentration-dependently inhibited intracellular Ca2+ mobilization in human platelets stimulated by collagen (5 microg/ml). Furthermore, TMPZ concentration (50 and 200 microM)- and time (15 and 30 min)-dependently triggered endothelial-type constitutive nitric oxide synthase (ecNOS) protein expression in human platelets. These results indicated that TMPZ at micromolar concentrations stimulated nitric oxide production in human platelets via a novel mechanism that activated ecNOS protein expression.
Life Sciences 08/2000; 67(8):937-47. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21-/- cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine specifically inhibited cyclin D1 expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21CIP1 expression in H226 and H358 cells, while it activated p27KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because significant changes in cyclin D1 or CDKI expression were observed at 12-24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control.
Cancer Letters 11/1999; 145(1-2):1-8. · 5.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advanced glycation end product (AGE) is important in the pathogenesis of diabetic nephropathy, which is characterized by cellular hypertrophy/hyperplasia leading to renal fibrosis. However, the signal transduction pathways of AGE remain poorly understood. The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway has been associated with cellular proliferation in some extra-renal cells. Because interstitial fibroblast proliferation might be important in renal fibrosis, we studied the role of the JAK/STAT pathway in NRK-49F (normal rat kidney fibroblast) cells cultured in AGE/BSA and non-glycated BSA. We showed that AGE dose-dependently (10-200 microgram/ml) increased cellular mitogenesis in NRK-49F cells at 5 and 7 days. However, cellular mitogenesis was unaffected by the simultaneous presence of BSA. Regarding the JAK/STAT pathway, AGE (100 microgram/ml) induced tyrosine phosphorylation of JAK2 (but not JAK1, JAK3 or TYK2) at 15-60 min; it also induced the tyrosine phosphorylation of STAT1 and STAT3 at 1-2 h and 0.5-4 h respectively. Being a transcription factor, AGE also increased the DNA-binding activities of STAT1 and STAT3 AG-490 (a specific JAK2 inhibitor) (5 microM) inhibited tyrosine phosphorylation of JAK2 and the DNA-binding activities of STAT1 and STAT3. The same results were obtained by using specific 'decoy' oligodeoxynucleotides (ODNs) that prevented STAT1 and STAT3 from binding to DNA. Meanwhile, the STAT1 or STAT3 decoy ODN and AG-490 were effective in reversing AGE-induced cellular mitogenesis. We concluded that the JAK2-STAT1/STAT3 signal transduction pathway is necessary for AGE-induced cellular mitogenesis in NRK-49F cells.
[Show abstract][Hide abstract] ABSTRACT: Tamoxifen (Tam), besides its action as an anti-estrogen, also inhibits cell proliferation of estrogen receptor (ER)-negative cancer cells by an unknown mechanism. In this study, we used ER-negative lung cancer cells to clarify such ER-independent inhibitory effect of Tam. We found that Tam induced G1 growth arrest in these cells. However, our results indicated that the expression of G1 cyclins (including D1, 2, 3 and E) was not regulated by Tam in these lung cancer cells. Additionally, the protein levels of G1 acting cyclin-dependent kinases (CDKs), CDK2, 4 and 6, was unaltered in Tam-treated lung cancer cells with the exception of CDK2 expression in H322 cells which was attenuated by Tam in a cell line-specific manner. We next examined the effect of Tam on the expression of cyclin-dependent kinase inhibitors (CDKIs) and our results demonstrated that the expression of p21WAF1 and p27KIP1, but not p57KIP2, was strongly activated by Tam in these cells. The amounts of p21WAF1 and p27KIP1 co-immunoprecipitated with cyclin E were obviously increased after Tam treatment and reduced activity of cyclin E-associated kinases and accumulation of hypo-phosphorylated retinoblastoma (Rb) protein were clearly detected in Tam-incubated cells. No consentaneous induction of CDKIs was found when ER-negative lung cancer cells were incubated with cytotoxic drugs, cisplatin and etoposide, this indicates that enhancement of CDKI expression is not a non-specific effect of Tam. We also found that Tam may up-regulate p21WAF1 expression via transcription activation. Considered together, these results suggest that Tam-induced growth inhibition in ER-negative lung cancer cells is associated with induction of p21WAF1 and p27KIP1.
[Show abstract][Hide abstract] ABSTRACT: PMC, a potent alpha-tocopherol derivative, dose-dependently (5-25 microM) inhibited the ATP-release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose-dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25 microM) significantly inhibited collagen-stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 microM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 microM) did not affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. PMC (10 and 25 microM) markedly inhibited the exogenous arachidonic acid (100 microM)-induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 microM) in washed human platelets, indicating that PMC inhibits cyclo-oxygenase activity. We conclude that PMC may exert its anti-platelet aggregation activity by inhibiting cyclooxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP-release.
British Journal of Pharmacology 08/1999; 127(5):1206-12. · 4.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thrombocytopenia frequently occurs early in the course of Gram-negative bacterial infections. Triflavin, an Arg-Gly-Asp-containing disintegrin, has been suggested to interfere with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. The present study was undertaken to determine whether triflavin could prevent thrombocytopenia in lipopolysaccharide (LPS)-treated rats.
In this study, 51Cr-labeled platelets were used to assess blood and tissue platelet accumulation after LPS challenge. The administration of LPS (4 mg/kg IV bolus) for 4 hours induced a reduction in radiolabeled platelets in blood and an obvious accumulation of platelets in liver. Triflavin (500 microg/kg) but not GRGDS (20 mg/kg) significantly prevented the alteration of radiolabeled platelet distribution in blood and liver when induced by LPS. Furthermore, triflavin but not GRGDS markedly suppressed the elevation in plasma thromboxane B2 concentration within the 4-hour period of LPS administration. In LPS-treated rats, the 5-hydroxytryptamine level was lower in the blood and higher in the liver compared with levels in normal saline-treated rats. Pretreatment with triflavin (500 microg/kg) significantly reversed the 5-hydroxytryptamine concentration in blood and liver of LPS-treated rats. In histological examinations and platelet adhesion assay, triflavin markedly inhibited the adhesion of platelets to subendothelial matrixes in vivo and in vitro.
The results indicate that triflavin effectively prevents thrombocytopenia, possibly through the following 2 mechanisms: (1) Triflavin markedly inhibits platelet aggregation, resulting in decreased thromboxane A2 formation. (2) It inhibits the adhesion of platelets to subendothelial matrixes, thereby leading to a reversal in the distribution of platelets in blood and liver in LPS-treated rats.
[Show abstract][Hide abstract] ABSTRACT: Although interferon-alpha (IFN-alpha) has shown great promise in the treatment of chronic viral hepatitis, the anti-tumour effect of this agent in the therapy of liver cancer is unclear. Recent studies have demonstrated that differentiation-inducing agents could modulate the responsiveness of cancer cells to IFN-alpha by regulating the expression of signal transducers and activators of transcription (STAT) proteins, a group of transcription factors which play important roles in the IFN signalling pathway. We have reported that sodium butyrate is a potent differentiation inducer for human hepatoma cells. In this study, we investigated whether this drug could regulate the expression of STAT proteins and enhance the anti-tumour effect of IFN-alpha in hepatoma cells. We found that sodium butyrate specifically activated STAT1 gene expression and enhanced IFN-alpha-induced phosphorylation and activation of STAT1 proteins. Co-treatment with these two drugs led to G1 growth arrest, accompanied by down-regulation of cyclin D1 and up-regulation of p21WAF-1, and accumulation of hypophosphorylated retinoblastoma protein in hepatoma cells. Additionally, internucleosomal DNA fragmentation, a biological hallmark of apoptosis, was detected in hepatoma cells after continuous incubation with a combination of these two drugs for 72 h. Our results show that sodium butyrate potently enhances the anti-tumour effect of IFN-alpha in vitro and suggest that a rational combination of these two drugs may be useful for the treatment of liver cancer.
British Journal of Cancer 06/1999; 80(5-6):705-10. · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, Escherichia coli LPS dose-dependently (100-500 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human and rabbit platelets stimulated by agonists. LPS also dose-dependently inhibited the intracellular Ca2+ mobilization in human platelets stimulated by collagen. In addition, LPS (200 and 500 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) significantly increased the production of nitrate within a 10-min incubation period. Furthermore, LPS also dose-dependently inhibited platelet aggregation induced by PDBu (30 nmol/l), a protein kinase C activator. These results indicate that the antiplatelet activity of E. coli LPS may be involved in the activation of a nitric oxide/cyclic GMP pathway in platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.
European Journal Of Haematology 06/1999; 62(5):317-26. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sphingosine and other long-chain bases (including sphinganine, dimethylsphingosine and stearylamine), but not octylamine (a short-chain analogue of sphinganine), induced apoptosis in Hep3B hepatoma cells. Because both D- and L-erythrosphingosine and stearylamine exert potent apoptotic effects on Hep3B cells, it is possible that these long-chain bases may activate apoptosis by inhibiting protein kinase C (PKC) activity. However, pretreatment with the PKC activator PMA could not rescue cells from apoptosis triggered by long-chain bases. Therefore the involvement of PKC in this apoptotic process requires further characterization. We also investigated whether these long-chain bases might be metabolized into ceramide in order to elicit their apoptotic action. We found that long-chain bases acted independently of ceramide in the induction of apoptosis, since addition of fumonisin B1, a fungal agent which effectively inhibits ceramide synthesis from sphingosine, did not protect against apoptosis. Additionally, we found that sphingosine-induced apoptosis was accompanied by activation of caspases. The functional role of caspases in this apoptotic process was examined by using specific caspase inhibitors. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, which exhibits a broad specificity for caspase-family proteases, effectively blocked sphingosine-induced apoptosis. Furthermore, our results indicate that caspase-3-like proteases, but not caspase-1, are activated during apoptosis triggered by sphingosine. Enhancement of caspase-3-like activity and cleavage of poly(ADP-ribose) polymerase, an in vivo substrate for caspase-3, was clearly demonstrated in sphingosine-treated Hep3B cells. Considered together, these results suggest that caspase-3-like proteases participate in apoptotic cell death induced by sphingosine.
[Show abstract][Hide abstract] ABSTRACT: In this study, PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), a potent antioxidant derived from alpha-tocopherol, dose-dependently inhibited agonist-induced platelet aggregation in human platelet-rich plasma. PMC is over 5-10 times more potent than alpha-tocopherol in inhibiting human platelet aggregation. Moreover, PMC (25-350 microM) dose-dependently reduced the relative fluorescence intensity of platelet membrane tagged with diphenylhexatriene (DPH). PMC is about 6-times more potent than alpha-tocopherol on this effect. Furthermore, antioxidative activity of PMC was investigated using two in vitro models. PMC inhibited non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 0.21+/-0.05 microM. It was more potent than alpha-tocopherol or other classical antioxidants. PMC also scavenged the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The concentration of PMC resulting in a decrease of 0.20 in the absorbance of DPPH was about 12.1+/-3.6 microM, was comparable in potency to alpha-tocopherol, butylated hydroxytoluence and Trolox. The antiplatelet activity of PMC may possibly be due initially to an increase in fluidity of the platelet membrane followed by inhibition of platelet aggregation. Our results indicate that PMC is a potentially effective antioxidant and antiaggregating agent, and could be helpful the design of compounds with more clinical effectiveness.
Life Sciences 02/1999; 65(2):197-206. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, Escherichia coli lipopolysaccharide (LPS) dose-dependently (100-300 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LPS also dose-dependently inhibited the phosphoinositide breakdown and the intracellular Ca+2 mobilization in human platelets stimulated by collagen. LPS (300 microg/ml) also significantly inhibited the thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LPS (100-300 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. In addition, LPS (200 and 300 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) also significantly increased the production of nitrate within a 30 min incubation period. Rapid phosphorylation of a platelet protein of Mr 47,000, a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by LPS (200 microg/ml) within a 30 min incubation period. These results indicate that the antiplatelet activity of LPS may be involved in two important pathways. (1) LPS may induce conformational changes in the platelet membrane, leading to change in the activity of phospholipase C. (2) LPS also activated the formation of nitric oxide (NO)/cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicaemic and endotoxaemic patients.
British Journal of Haematology 11/1998; 103(1):29-38. · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of apoptosis in Hep3B hepatoma cells by transforming growth factor beta 1 (TGF-beta 1) was accompanied by the activation of interleukin-1-beta-converting-enzyme-like proteases, which have recently been renamed as caspases. The caspase inhibitor ZVAD-FMK, which has a broader specificity for caspase family proteases, blocked TGF-beta 1-induced apoptosis in a concentration-dependent manner. The caspases in this apoptotic process were further characterized by using a more specific caspase inhibitor, DEVD-FMK, for CPP32-like (caspase-3-like) proteases. Our results demonstrated that CPP32-like proteases were activated during apoptosis triggered by TGF-beta 1. Enhancement of CPP32-like activity was clearly detected in TGF-beta 1-treated Hep3B cells. Furthermore, cleavage of poly(ADP-ribose) polymerase, an in vivo substrate for CPP32, in these cells was confirmed by immunoblotting. Thus, we suggest that CPP32-like proteases participate in apoptotic cell death induced by TGF-beta 1.