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O H Lee

Publications and source records attributed to O H Lee.

16 recordsLinked to original sources
PubMed2000

Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase.

Here we report the inhibition of cellular invasion by a recombinant mouse endostatin and the possible mechanism of the inhibition. Endostatin significantly reduced endothelial as well as tumor cellular invasion into the reconstituted basement membrane in vitro. Gelatin zymographic analysis revealed that the activation of promatrix metalloproteinase-2 (proMMP-2) that was secreted from endothelial cells was blocked upon endostatin treatment. Studies with recombinant MMPs confirmed that endostatin inhibited proMMP-2 activation, mediated by both membrane-type 1 MMP and 4-aminophenylmercuric acetate. Furthermore, enzymatic assays using a peptide substrate demonstrated that endostatin inhibited the catalytic activities of both MMP-2 and membrane-type 1 MMP. Finally, coimmunoprecipitation experiments revealed that endostatin formed a stable complex with proMMP-2. These novel findings would, at least in part, explain the mechanism of the potent antiangiogenic and antitumor activities of endostatin.

Angiogenesis Inhibitors↗
PubMed2000

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway.

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.

Cell Adhesion Molecules↗
PubMed2000

Identification of angiogenic properties of insulin-like growth factor II in in vitro angiogenesis models.

Insulin-like growth factor II (IGF-II), highly expressed in a number of human tumours, has been recently known to promote neovascularization in vivo. Yet, the detailed mechanism by which IGF-II induces angiogenesis has not been well defined. In the present study, we explored an angiogenic activity of IGF-II in in vitro angiogenesis model. Human umbilical vein endothelial cells (HUVECs) treated with IGF-II rapidly aligned and formed a capillary-like network on Matrigel. In chemotaxis assay, IGF-II remarkably increased migration of HUVECs. A rapid and transient activation of p38 mitogen-activated protein kinase (p38 MAPK) and p125 focal adhesion kinase (p125FAK) phosphorylation was detected in HUVECs exposed to IGF-II. IGF-II also stimulated invasion of HUVECs through a polycarbonate filter coated with Matrigel. Quantitative gelatin-based zymography identified that matrix metalloproteinase-2 (MMP-2) activity generated from HUVECs was increased by IGF-II. This induction of MMP-2 activity was correlated with Northern blot analysis, showing in HUVECs that IGF-II increased the expression of MMP-2 mRNA, while it did not affect that of TIMP-2, a tissue inhibitor of MMP-2. These results provide the evidence that IGF-II directly induces angiogenesis by stimulating migration and morphological differentiation of endothelial cells, and suggest that IGF-II may play a crucial role in the progression of tumorigenesis by promoting the deleterious neovascularization.

Cell Movement↗
PubMed1999

Egr-1 mediates transcriptional activation of IGF-II gene in response to hypoxia.

We have previously reported that the exposure of human HepG2 cells to hypoxic conditions results in the overexpression of human insulin-like growth factor II (IGF-II) mRNA whose size is 6.0 kb. This particular size of IGF-II mRNA is transcribed under the control of the IGF-II P3 promoter. In the present study, to delineate the molecular mechanism for the activation of the IGF-II gene, we examined the induction of P3 promoter activity in HepG2 cells by hypoxia in the transient expression system. In this system, hypoxia induced a linear increase within 24 h in the expression of luciferase that was driven by the IGF-II P3 promoter. To further delineate which factors mediate this response, the expression pattern of regulators of the P3 promoter, Egr-1, Sp1, and WT1, were analyzed by reverse transcription-PCR and Northern blot analysis. We found that hypoxia increased the expression of Egr-1 but not of Sp1. In contrast, the level of WT1, a repressor of IGF-II expression, was markedly decreased during hypoxia. The mRNA stability assay revealed that the induction of transcription is the mechanism of underlying Egr-1 mRNA elevation. We then investigated the effects of hypoxia on the DNA binding activity of Egr-1. Both electrophoretic mobility shift assay and supershift assay demonstrated that the DNA binding activity of the Egr-1 protein was increased by hypoxia. In addition, the level of Egr-1 protein was also increased under the hypoxia as determined by Western blot analysis. Cotransfection of HepG2 cells with an Egr-1 expression vector and an IGF-II P3 promoter-luciferase reporter plasmid showed that the transcription of IGF-II was activated by Egr-1 in a dose-dependent manner. Moreover, the elevation of IGF-II P3 promoter activity was induced synergistically by the cotreatment of hypoxia with Egr-1 overexpression. Deletion of sequences in the IGF-II P3 promoter containing Egr-1 binding sites did not respond to hypoxic stress. Taken together, these data strongly indicate that hypoxia-induced IGF-II expression in HepG2 cells is due to the enhanced activity of Egr-1 on the IGF-II P3 promoter and that the Egr-1 binding site in the IGF-II P3 promoter is essential for the transcriptional regulation of IGF-II under hypoxic conditions.

Carcinoma, Hepatocellular↗
PubMed1999

Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite abundantly stored in platelets and released upon platelet activation. Recently, S1P has been postulated for its potential roles in angiogenesis. In this study, we provided several lines of evidence showing that S1P has angiogenic activity. In vitro, S1P stimulated DNA synthesis and chemotactic motility of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, reaching a near maximum at 1 microM. S1P also significantly induced tube formation of HUVECs on Matrigel. Matrigel plug assay in mice revealed that S1P promotes angiogenesis in vivo. In addition, exposure of HUVECs to S1P led to rapid activation of extracellular signal-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in a pertussis toxin (PTX)-sensitive manner. Notably, HUVEC migration and tube formation in response to S1P were completely blocked by pretreatment with PTX. Further, the MEK inhibitor U0126 markedly inhibited S1P-induced tube formation but S1P-induced migration was not affected by inhibition of ERK and p38 MAPK. Taken together, these results indicate that S1P induces angiogenesis predominantly via G(i) protein-coupled receptors in endothelial cells and suggest that S1P may act as an important modulator of platelet-induced angiogenesis.

Animals↗
PubMed1999

Glucocorticoids prevent NF-kappaB activation by inhibiting the early release of platelet-activating factor in response to lipopolysaccharide.

Lipopolysaccharide (LPS) is a known inducer of numerous pro-inflammatory events including the production of platelet-activating factor (PAF). PAF released in response to LPS is a major contributor to the pathological events associated with endotoxemia. The present study demonstrates that dexmethasone (DEX) inhibited the LPS-induced early plasma PAF raise in a dose- and time-dependent manner. In addition, DEX prevented the subsequent PAF-mediated pathological phenomena such as anaphylactic shock-like symptoms, symptoms of disseminated intravascular coagulation and hemorrhage in renal medullae. DEX or the PAF antagonist BN 50739 significantly inhibited LPS-induced NF-kappaB activation. The inhibition of NF-kappaB activation by DEX was overcome by the injection of exogenous PAF. Administration of PAF or LPS resulted in a rapid loss of IkappaBalpha protein. The LPS-induced degradation of IkappaBalpha was prevented by pretreatment with BN 50739, suggesting that PAF is a critical intermediate in the LPS-triggered degradation of IkappaBalpha protein. DEX prevented the LPS-induced IkappaBalpha degradation, which was also reversed by exogenous PAF. Administration of DEX or BN 50739 caused an increase in cytoplasmic IkappaBalpha level. Our results indicate that DEX inhibits IkappaBalpha degradation and subsequent NF-kappaB activation through blocking the initial release of PAF.

Animals↗
PubMed1999

A novel angiogenic factor derived from Aloe vera gel: beta-sitosterol, a plant sterol.

Aloe vera gel has a beneficial effect on wound healing. Because angiogenesis is an essential process in wound healing, we hypothesized that Aloe vera gel might contain potent angiogenic compounds. Here we demonstrate that Aloe vera gel and its extracts are angiogenic on the chorioallantoic membrane (CAM) of chick embryo. Out of the three compounds purified from the final fraction of Aloe vera gel, beta-sitosterol showed a potent angiogenic activity in the CAM assay. In the presence of heparin, beta-sitosterol stimulated neovascularization in the mouse Matrigel plug assay and the motility of human umbilical vein endothelial cells in an in vitro wound migration assay. Thus beta-sitosterol is a novel plant-derived angiogenic factor which may have potential pharmaceutical applications for the management of chronic wounds.

Journal Article↗
PubMed1998

Insulin-like growth factor II (IGF-II) secreted from HepG2 human hepatocellular carcinoma cells shows angiogenic activity.

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. Since insulin-like growth factor II (IGF-II) has been reported to play a significant role in liver regeneration and hepatocarcinogenesis, we initially examined its angiogenic effect on the chorioallantoic membrane (CAM) of 9-day-old chick embryos. We also investigated whether IGF-II secreted from HepG2 human hepatocellular carcinoma cells induces vascularization using the chick embryo CAM. We found that the concentrated conditioned media (CCM) of HepG2 cell culture induced angiogenesis on the CAM. We also identified IGF-II protein in the CCM from HepG2 cells by Western blot analysis. However, CCM from Chang liver cells, which are normal human liver cells and were free of IGF-II, did not induce angiogenesis in the CAM. These results suggest that IGF-II secreted from hepatocellular carcinoma cells may act as an angiogenic factor for the hypervascularization of HCC.

Allantois↗
PubMed1998

Hypoxia-induced apoptosis in human hepatocellular carcinoma cells: a possible involvement of the 6-TG-sensitive protein kinase(s)-dependent signaling pathway.

Apoptosis is a morphologically and biochemically distinct form of cell death which can be triggered by a variety of extracellular agents both during normal developments and in adult pathological states. However, the molecular mechanism of apoptotic cell death due to hypoxia has not been clearly elucidated. In this study, we investigated critical factors involved in hypoxia-induced apoptosis using HepG2, a human hepatocellular carcinoma cell line, as an experimental model. We found that 24 h of exposure of HepG2 cells to hypoxia induced apoptosis, for which de novo protein synthesis was required. Apoptosis was demonstrated by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Hypoxia-induced apoptosis was associated with a marked induction of c-jun and c-fos messenger RNAs. Electromobility shift assay showed the increased DNA binding activity of AP-1 during hypoxia, suggesting that AP-1 may be involved in the induction of cell death by acting as a transcriptional regulator. A purine analogue, 6-thioguanine (6-TG), significantly blocked the induction of apoptosis by hypoxia. Moreover, the inductive effect of hypoxia on c-jun expression was also inhibited by 6-TG, whereas the levels of c-fos mRNA and its protein were rather strongly increased. Iodoacetamide (IAA), a non-specific inhibitor of ICE family proteases, also has an inhibitory effect on hypoxia-induced apoptosis. These results suggest that the 6-TG-sensitive protein kinase(s)-dependent signaling pathway may be involved in the apoptotic response of HepG2 cells exposed to hypoxia by increasing the level of c-jun and c-fos and the activity of AP-1 and/or by activating ICE family protease(s).

Apoptosis↗
PubMed1998

Insulin-like growth factor II induced by hypoxia may contribute to angiogenesis of human hepatocellular carcinoma.

Insulin-like growth factor II (IGF-II) is highly expressed during hepatocarcinogenesis (P. Schirmacher et al., Cancer Res., 52: 2549-2556, 1992; B. C. Park et al., J. Hepatol., 22: 286-294, 1995). However, the mechanism of its enhanced expression is largely unknown. In this study, we show that IGF-II mRNA levels are increased within six h of exposing human hepatoma cell cultures to hypoxia, suggesting that hypoxia may be a strong stimulus for the induction of IGF-II expression in the process of hepatocarcinogenesis. This finding and the fact that hepatocellular carcinoma (HCC) is a typical hypervascular tumor (M. Mise et al., Hepatology, 23: 455-464, 1996) imply that IGF-II may play an important role in the development of neovascularization of HCC. Here we demonstrate that IGF-II substantially increases vascular endothelial growth factor (VEGF) mRNA and protein levels in a time-dependent manner in human hepatoma cells. The induction of VEGF by IGF-II was additively increased by hypoxia. Moreover, the direct angiogenic activity of IGF-II was observed in the quantitative chick chorioallantoic membrane assay (M. Nguyen et al., Microvasc. Res., 47: 31-40, 1994). These data suggest that IGF-II may be a hypoxia-inducible angiogenic factor in HCC.

Animals↗
PubMed1998

In vitro angiogenic activity of Aloe vera gel on calf pulmonary artery endothelial (CPAE) cells.

Angiogenic activity of Aloe vera gel was investigated by in vitro assay. We obtained the most active fraction from dichloromethane extract of Aloe vera gel by partitioning between hexane and 90% aqueous methanol. The most active fraction (F3) increased the proliferation of calf pulmonary artery endothelial (CPAE) cells. In addition, F3 fraction induced CPAE cells to invade type 1 collagen gel and form capillary-like tube through in vitro angiogenesis assay, and increased the invasion of CPAE cells into matrigel through in vitro invasion assay. Furthermore, the effect on the mRNA expression of proteolytic enzymes which are key participants in the regulation of extracellular matrix degradation was investigated by northern blot analysis. F3 fraction enhanced mRNA expression of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase-2 (MMP-2), and membrane-type MMP (MT-MMP) in CPAE cells whereas the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA was not changed.

Aloe↗
PubMed1998

Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control.

The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression required spo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducing srf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of srf-lacZ at neutral pH. The results suggest that srf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.

Bacillus subtilis↗
PubMed1996

Anti-invasive activity of ursolic acid correlates with the reduced expression of matrix metalloproteinase-9 (MMP-9) in HT1080 human fibrosarcoma cells.

We examined the anti-invasive activity of ursolic acid (UA) on the highly metastatic HT1080 human fibrosarcoma cell line. UA reduced tumor cell invasion through a reconstituted basement membrane in a transwell chamber. A significant down-regulation of matrix metalloproteinase-9 [MMP-9; Mr 92,000 gelatinase/type IV collagenase (gelatinase B)] by UA was detected by Northern blot analysis. However, MMP-2 [Mr 72,000 gelatinase/type IV collagenase (gelatinase A)] and membrane-type MMP were constantly expressed, and the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 also was not changed after 3 and 6 days of treatment with UA. Quantitative gelatin-based zymography confirmed a markedly reduced expression of MMP-9 but not MMP-2 after treatment with UA. To confirm the UA-induced down-regulation of MMP-9 expression, we constructed a secreted alkaline phosphatase (SEAP) reporter vector including MMP-9 promoter. After transfection of MMP-9/SEAP reporter vector into HT1080 cells, reduced SEAP activity was detected after treatment with UA. These results suggest that down-regulation of MMP-9 contributes to the anti-invasive activity of UA in HT1080 cells.

Basement Membrane↗
PubMed1996

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Bacillus subtilis↗