Plasminogen activator(PA) system such as urokinase plasminogen activator(uPA), urokinase PA Receptor(uPAR), tissue, tissue PA, and PA inhibitor-1&2(PAI-1&2) play a role in tumor invasion, metastasis, and proliferation. It is interested that these factors in patients with primary oral squamous cell carcinoma(Oral SCC) will be evaluated and correlated with clinicopathologic variables. Recently, these expression of primary oral SCC has been restricted to clinical or immunohistochemical study such in vivo study. The purpose of this study were to investigate the mRNA expression and cytologic concentration of uPA, uPAr, tPA, and PAI-1,2 in oral SCC cell lines compared to NHOK and to apply these results to evaluate early detection biomarkers of oral SCC in future. All the cell lines(NHOK, HN 4 and SCC 25) were cultured under KBM bullet kit at 37℃ in a 5% CO2 incubator. We studied a possible association between mRNA expression and cytosolic concentrations of uPA, uPAR, tPA, and PAI-1,2 in oral SCC cell lines compared to NHOK using RT-PCR and an enzyme-linked immunoassay(ELISA) method. uPA mRNA expression was about 5-6 folds, while uPAR was a bout 3 f olds, and PAI-1 was about 1 .5-1.6 f olds. PAI-2 was a bout1.2 -1.3 f olds t han that o f NHOK, w hile t PA w as l ower t han that of NHOK. uPA cytosolic concentrations was about 15-19 folds, while uPAR was about 8 folds, and PAI-1 was about 3-4.5 folds. PAI-2 was about 2 folds than that of NHOK, while tPA was lower than that of NHOK. Both uPA, uPAR, and PAI-1,2 cytologic concentrations were correlated with mRNA expression of oral SCC cell lines. From the aboving results, high cytosolic concentrations of uPA, uPAR, and PAI-1 & 2 were correlated with mRNA expression. It suggested that these might be specific markers for oral SCC cell lines and these results would be contributed to evaluate early detection biomarkers for human oral squamous cell carcinoma.

Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

The present study was performed to identify the role of plasminogen activator (PA) and the location of PA expression in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from ovary in pre-ovulatory (Pre-Ov), post-ovulatory stage (Post-Ov) and early to mid-luteal stage (Early-mid L). The uterus tissue was immediately fixed by PBS with 10% formalin. There were fixed porcine uterus tissue for 24 hours at room temperature and porcine uterus tissue dehydrate for 12 hour in sucrose solution. For immunohistochemical staining, porcine uterus tissues were cut to 4 μm by micro frozen section microtome. The nucleus and cytoplasm of porcine uterus tissues were stained by Hematoxin and Eosin. Porcine uterus tissues were evaluated by Immunofluorescence using anti-tissue type PA (tPA) and urokinase type PA (uPA). The location of PA expression was identified by observing the PA fluorescence using fluorescent microscope and optical telescopes. As a results, when Pre-Ov and Post-Ov were identified endometrial blood vessel in an inner layer that were observed tPA and uPA. Especially, expression of PA was observed around secretory gland. But the expression of PA were not confirm in Early-mid L. Also, The expression of PA were higher in Post-Ov than Early-mid L. In conclusion, during the estrous cycle, the expression of PA were increased from Pre-Ov to Post-Ov and was decreased from Post-Ov to Early-mid L.

The present study was performed to identify the role of plasminogen activator (PA) and the location of PA expression in porcine uterus tissues during the estrous cycle. Porcine uterus tissues were obtained from ovary in pre-ovulatory (Pre-Ov), post-ovulatory stage (Post-Ov) and early to mid-luteal stage (Early-mid L). The uterus tissue was immediately fixed by PBS with 10% formalin. There were fixed porcine uterus tissue for 24 hours at room temperature and porcine uterus tissue dehydrate for 12 hour in sucrose solution. For immunohistochemical staining, porcine uterus tissues were cut to 4 μm by micro frozen section microtome. The nucleus and cytoplasm of porcine uterus tissues were stained by Hematoxin and Eosin. Porcine uterus tissues were evaluated by Immunofluorescence using anti-tissue type PA (tPA) and urokinase type PA (uPA). The location of PA expression was identified by observing the PA fluorescence using fluorescent microscope and optical telescopes. As a results, when Pre-Ov and Post-Ov were identified endometrial blood vessel in an inner layer that were observed tPA and uPA. Especially, expression of PA was observed around secretory gland. But the expression of PA were not confirm in Early-mid L. Also, The expression of PA were higher in Post-Ov than Early-mid L. In conclusion, during the estrous cycle, the expression of PA were increased from Pre-Ov to Post-Ov and was decreased from Post-Ov to Early-mid L.

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide (H2O2), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decreased with addition of H2O2, and were significantly (p<0.05) lower in medium with 0.1 mM H2O2 than control group. Also, the rate of degenerated oocytes was increased in as H2O2 concentration increased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When H2O2 concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM H2O2 and (3) control medium with 1.0 mM H2O2 along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. H2O2 decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity against oxidative stress caused by H2O2. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM H2O2 alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.

Tumor cell biological factors, such as urokinase plasminogen activator(uPA) and its inhibitor plasminogen activator inhibitor- 1(PAI-1) play a role in tumor invasion, metastasis, and proliferation. These factors in patients with primary oral squamous cell carcinoma(Oral SCC) will be evaluated and correlated with clinicopathologic variables. However, relatively rarely has been known in oral squamous cell carcinoma in vivo and in vitro study . The purpose of this study were to investigate the protein expression of uPA and PAI-1 in oral SCC cell lines cell line compared to NHOK and to study migration and adhesion assay. All the cell lines were cultured under KBM bullet kit at 37℃ in a 5% CO2 incubator. We studied a possible association between cytosolic uPA and PA-1 concentrations in oral SCC cell line compared to NHOK using an enzyme-linked immunoassay(ELISA). Cell adhesion and migration assay were done in all the cell l ines. In migration assay oral SCC cell lines were about 70 folds higher than NHOK. In adhesion assay oral SCC cell line were about 7-12 folds higher than NHOK. uPA cy tosolic concentrations was about 15-19 folds and PAI-1 was 3 to 4.5 folds than that of NHOK. Both uPA and PAI-1 concentrations were correlated with migration and adhesion assay. High cytosolic concentrations o f uPA and PAI-1 were correlated with migration and adhesion assay . It suggested that these markers might be specific for oral SCC cell line and these results would be contributed to treatment and prognosis of human oral squamous cell carcinoma.

Plasminogen activators (PAs) are serine protease that cleave plasminogen to form the active protease plasmin and may participate in mammalian fertilization. Although correlations have been reported between reactive oxygen species (ROS) and sperm function, the relationship between PA activity and ROS is unknown. We determined the effects of ROS on sperm function and PA activities in boar spermatozoa preincubated under the X-XO system. When spermatozoa were treated with the X+XO group, a significant increase (p<0.05) was observed in the percentage of acrosome reacted spermatozoa compared with that of the control group. However, when antioxidants were added to the medium with X+XO, the rate of acrosome reaction tended to decrease. Also, a significantly lower percentage of acrosome reacted spermatozoa was observed in the X+XO+catalase group at 6 hr of incubation compared with that of X+XO group. The density of malondialdehyde (MDA) was higher in the X+XO group than in different treatment groups. In another experiment, incubation of spermatozoa in medium with X+XO was associated with a significant (p<0.05) increase in activity of tPA-PAI and tPA compared with the control group. Antioxidants decreased the increased activity of tPA-PAI and tPA by preincubation in the X-XO system. Also, a significantly lower (p<0.05) activities of tPA-PAI and tPA were observed in the X+XO+catalase group compared with the X+XO group. No significant differences, however, were observed in the activity of uPA. These results suggest that the increase of acrosome reaction by the X-XO system resulted in increase of PAs activity in the sperm incubation medium.

Plasminogen activators (PAs) are serine proteases that convert plasminogen to plasmin. The PA/plasmin system has been associated with a number of physiological processes such as fibrinolysis, ovulation and fertilization. Although correlations have been reported between reactive oxygen species (ROS) and oocyte maturation, the relationship between PA activity and ROS is unknown. The present study was undertaken to determine the effects of cumulus cells on PA activity in matured porcine oocytes under xanthine (X)-xanthine oxidase (XO) system. When oocytes were matured under the X-XO system, the proportion of oocytes remaining GV stage was higher (p<0.05) in oocytes without cumulus cells. The incidence of degenerated oocytes was higher (p<0.05) in the X+XO ( and ) than in the control group ( and ). The proportion of TUNEL-positive oocytes and activity of caspase-3 were higher (p<0.05) in cumulus-free oocytes and oocytes exposed to ROS. Tissue-type plasminogen activator-plasminogen activator inhibitor (tPA-PAI) and tissue-type plasminogen activator (tPA) activity were detected in oocytes that were separated from cumulus-oocytes complexs (COCs) at 44 h of maturation culture, and only tPA was produced in oocytes that were denuded before the onset of maturation culture. On the other hand, the activities of PA were increased (p<0.05) when oocytes were cultured under the X-XO system. The higher activity of tPA was observed in denuded oocytes (DOs) underwent apoptotic changes by oxidative stress. In COCs, however, tPA-PAI as well as tPA activity was detected and apoptotic changes such as DNA cleavage or caspase-3 activation were not observed. These results suggest that tP A may be relevant to apoptotic cell death in porcine oocytes by oxidative stress.

Urokinase-type plasminogen activator (uPA) and plasminogen activator type 1 (PAI-1) inhibitor contribute to the invasiveness of many carcinomas. It will be helpful to study clinical behavior of patients with malignant tumor by analysis of their expression. Expression of uPA and PAI-1 in human salivary gland tumors has been rarely reported in vitro. The purpose of this study were to investigate the protein expression of uPA and PAI-1 in SGT cell line compared to oral SCC and HeLa cell lines and to study migration and adhesion assay. All the cell lines were cultured under DMEM with 10% FBS at at 37oC in a 5% CO2 incubator. We studied a possible association between cytosolic uPA and PA-1 concentrations in SGT cell line compared to any other cell lines through cell migration and adhesion assay, and enzyme-linked immunoassay(ELISA). In migration assay SGT cell line was about 2 .5-4 folds higher than another cell lines. In adhesion assay SGT cell line was about 1.1-2 folds higher than another cell lines. uPA cytosolic concentrations of SGT cell line was about 3-10 folds, while PAI-1 was about 2.5-10 folds. Oral SCC cell lines were the lowest value. Both uPA and PAI-1 concentrations were correlated with migration and adhesion assay. High cytosolic concentrations of uPA and PAI-1 was correlated with migration and adhesion assay. It suggested that these markers might be specific marker for SGT cell line and would be contributed to treatment and prognosis of human salivary gland adenocarcinoma

Nerve gro때h factor-induced B (NGFI-B, Nur77) is an orphan nuclear receptor with no known endogenous Iigands , however‘ recent stuclies on a series of methylene -substituted diindolylmethanes (C-DIMs) have identified 1,l-bis(3’ - In dolyl) -l-(phenyl)methane (DIM-C-Ph) and l , l -bis(3’ indolyl)-l-(p-anisyl)methane (DIM-C-pPhOCHa) as Nur77 agonist Nur77 is expressed in several colon cancer cell lines (RKO, SW480, HCT-116, HT-29 and HCT-15) and we a lso observed by irnmunostaining that Nur77 was overexpressed in colon tumors compared to normal colon tIssue DIM-C-Ph and DlM-C-pPhOCH3 decreased survival and induced apoptosis in RKO colon cancer cells and this was accompanied by in ductdion of tumor nec rosis factor-related apoptosis-incluced ligand (TRAlL) protein, The induct ion of a poptosis and TRAlL by DIM-C-pPhOCH3 was significantly inhibited by a small inhibitory RNA for Nur77 (iNur77); however, it was evide nt from RNA in terference studies that DIM-C-pPhOCH3 a1so induced Nur77-independent apoptosis. Analysis o( DIM-C-pPhOCH3-induced gene expression using microarrays idontifiod sovoral proapoptotic genos and analysis by ro verse t ranscriptase PCR in the presence 0 1' absence of iNru77 showed that incluction of prograrnmed cell death gene 1 (PDcm) was Nur77-dependent‘ whereas induction of cystathionase (CSE) and activating transcription factor 3 (ATF3) was Nur77-independent, DIM-C-pPhOCHa (25 mg/kg/day) also inhi bited tumor growth in athymic nude mice bearing RKO cell xenograft, These results demonstrate that Nur77-active C-DIM compounds represent a new class of anti-colon cancer drugs that act through receptor- dependent and - independent pathway

1,1- Bis(3’-indolyl)-l-(p-methoxyphenyl)methane (DIM- C- pPhOCH.3) is a methylenc - substituted diindolylmethanes (C-DIM) ana log that acti vates the orphan receptOl‘ nerve growth factor-induced-B (NGFI-B, Nur77) , RNA inteference studies with small inhibitory RNA for Nur77 demonstrate that DIM-C-pPhOCH:J induces Nur77-dependent and - independent apoptosis, and this study has focused on delineating the Nur77-independent proapoptotic pathways induced by the C-DIM analog DIM-C-pPhOCH3 induced caspase-dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl-2 ratios and release of cytochrome c into the cytosol DlM-C一pPhOCH.3 also induced phosphatidylinositol-3-kinase-dependent activation of early growth response gene-l whi ch, in turn, induced expression of the proapoptotic nonsteroidal anti-inflammatory drug- activated gene-l (NAG- l) in colon tumors in athyrnic nude mice bearing RKO cells as xenografts, DIM-C-pPhOCH.3 also activated the extrinsic apoptosis pathway through increased phosphorylation of c- jun N-terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5) , Thus, the effectiveness of DIM-C-pPhOCH.3 as a tumor growth inhibitor is through activation of Nur77-dependent and -independent pathways