• BMB Reports
• /
• v.43 no.8
• /
• pp.554-560
• /
• 2010

Smad4 is involved in cancer progression and metastasis. Using a pair of human syngeneic epithelial ovarian cancer cells with low (HO-8910) and high (HO-8910PM) metastatic abilities, we aimed to reveal the role of Smad4 in ovarian cancer metastasis in vitro. Smad4 was down-regulated in HO-8910PM cell line relative to HO-8910 by implicating Smad4 was probably a potential tumor suppressor gene for ovarian cancer. Re-expression of Smad4 decreased the migration ability and inhibited the invasion capacity of HO-8910PM, while promoted the cell adhesion capacity for HO-8910PM. The stable expression of Smad4 increased the expression of E-cadherin, reduced the expression of plasminogen activator inhibitor-1 (PAI-1) and slightly down-regulated the expression of VEGF. Smad4 suppresses human ovarian cancer cell metastasis potential through its effect on the expressions of PAI-1, E-cadherin and VEGF. Results from current work implicate Smad4 might suppress the invasion and metastasis of human ovarian tumor cells through a TGF-$\beta$/Smad-mediated pathway.

• The Korean Journal of Physiology and Pharmacology
• /
• v.22 no.6
• /
• pp.679-688
• /
• 2018

Autism spectrum disorders (ASDs) are neurodevelopmental disorders that share behavioral features, the results of numerous studies have suggested that the underlying causes of ASDs are multifactorial. Behavioral and/or neurobiological analyses of ASDs have been performed extensively using a valid model of prenatal exposure to valproic acid (VPA). Abnormal synapse formation resulting from altered neurite outgrowth in neural progenitor cells (NPCs) during embryonic brain development has been observed in both the VPA model and ASD subjects. Although several mechanisms have been suggested, the actual mechanism underlying enhanced neurite outgrowth remains unclear. In this study, we found that VPA enhanced the expression of brain-derived neurotrophic factor (BDNF), particularly mature BDNF (mBDNF), through dual mechanisms. VPA increased the mRNA and protein expression of BDNF by suppressing the nuclear expression of methyl-CpG-binding protein 2 (MeCP2), which is a transcriptional repressor of BDNF. In addition, VPA promoted the expression and activity of the tissue plasminogen activator (tPA), which induces BDNF maturation through proteolytic cleavage. Trichostatin A and sodium butyrate also enhanced tPA activity, but tPA activity was not induced by valpromide, which is a VPA analog that does not induce histone acetylation, indicating that histone acetylation activity was required for tPA regulation. VPA-mediated regulation of BDNF, MeCP2, and tPA was not observed in astrocytes or neurons. Therefore, these results suggested that VPA-induced mBDNF upregulation was associated with the dysregulation of MeCP2 and tPA in developing cortical NPCs.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.5
• /
• pp.695-703
• /
• 2006

The dissolved oxygen level of any cell culture environment has a critical effect on cellular metabolism. Specifically, hypoxia condition decreases cell viability and recombinant protein productivity. In this work, to develop CHO cells producing recombinant protein with high productivity, mammalian expression vectors containing a human tissue-type plasminogen activator (t-PA) gene with hypoxia response element (HRE) were constructed and stably transfected into CHO cells. CHO/2HRE-t-PA cells produced 2-folds higher recombinant t-PA production than CHO/t-PA cells in a $Ba^{2+}-alginate$ immobilized culture, and 16.8-folds in a repeated batch culture. In a non-aerated batch culture of suspension-adapted cells, t-PA productivity of CHO/2HRE/t-PA cells was 4.2-folds higher than that of CHO/t-PA cells. Our results indicate that HRE is a useful tool for the enhancement of protein productivity in mammalian cell cultures.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.15
• /
• pp.6327-6330
• /
• 2015

Background: Glioblastoma is a highly aggressive and malignant brain tumor. Risk factors are largely unknown however, although several biomarkers have been identified which may support development, angiogenesis and invasion of tumor cells. One of these biomarkers is PAI-1.4G/5G and A-844G are two common polymorphisms in the gene promotor of PAI 1 that may be related to high transcription and expression of this gene. Studies have shown that the prevalence of the 4G and 844G allele is significantly higher in patients with some cancers and genetic disorders. Materials and Methods: We here assessed the association of 4G/5G and A-844G polymorphisms with glioblastoma cancer risk in Iranians in a case-control study. All 71 patients with clinically confirmed and 140 volunteers with no history and symptoms of glioblastoma as control group were screened for 4G/5G and A-844G polymorphisms of PAI-1, using ARMS-PCR. Genotype and allele frequencies of case and control groups were analyzed using the DeFinetti program. Results: Our results showed significant associations between 4G/5G (p=0.01824) and A-844G (p = 0.02012) polymorphisms of the PAI-1 gene with glioblastoma cancer risk in our Iranian population. Conclusions: The results of this study supporting an association of the PAI-1 4G/5G (p=0.01824) and A-844G (p = 0.02012) polymorphisms with increasing glioblastoma cancer risk in Iranian patients.

• The Journal of Korean Physical Therapy
• /
• v.25 no.4
• /
• pp.224-231
• /
• 2013

Purpose: The purpose of this case study was to investigate three poor fibrinolytic responders with chronic ischemic stroke to acute exercise intensity and time. Methods: Three ischemic stroke patients (male) from the stroke center located at Busan metropolitan area in Republic of Korea volunteered at this study. They performed two single session exercises that were a VO2peak test and a single bout treadmill walking (70-75%HRpeak, 30 min, 50min). Fasting blood samples for determination of tissue Plasminogen Activator (tPA) and Plasminogen Activator Inhibitor-1 (PAI-1) were obtained before, immediately after, 30min after acute exercise. SPSS 12.0 was used for analyzing of data and computing mean and standard deviation, and change rate was conducted between times. Results: In fibrinolytic activity according to the intensity and time of acute exercise, tPA change increased steadily during the recovery stage after the VO2peak in the cases, but PAI-1 activity showed different patterns among the cases. In a single bout treadmill walking (70-75%HRpeak, 30 min, 50min), tPA change increased between 30min and 50min. Conclusion: In conclusion, these results suggest that the exercise prescription for poor fibrinolytic responder with three male chronic ischemic stroke patients without motor disability recommend at 70-75%HRpeak, over 30min.

• 한국생물공학회:학술대회논문집
• /
• 2002.04a
• /
• pp.257-260
• /
• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• Journal of Life Science
• /
• v.19 no.6
• /
• pp.836-838
• /
• 2009

Tissue plasminogen activator (tPA) is very useful for dissolving the clots of blood, however, the use of tPA is limited to only 3-5% of ischemic stroke patients because of the narrow therapeutic time windows and negative side effects. Previous evidences suggest that limitation of tPA in thrombolytic therapy may be related to the upregulation of MMPs. However, little is known about the regulatory mechanism. In this study, we examined the role of tPA on MMP upregulation in rat neuronal cells. tPA (5 ${\mu}g$/ml) increased MMP-9 levels of neuronal cells in a time dependent manner. Hypoxia/reoxygenation amplified tPA-induced MMP-9 levels significantly. Pretreatment with JNK inhibitor SP600125 reduced the MMP-9 response. These results suggest that tPA can upregulate MMPs in neuronal cells and that JNK kinase may be involved.

• Biomolecules & Therapeutics
• /
• v.14 no.1
• /
• pp.30-35
• /
• 2006

Tissue-type plasminogen activator (t-PA) is a multidomain serine protease containing two kringle domains, TK1-2. Previously, Pichia-derived recombinant human TK1-2 has been reported as an angiogenesis inhibitor although t-PA plays an important role in endothelial and tumor cell invasion. In this work, in order to improve in vivo efficacy of TK1-2 through elimination of immune reactivity, we mutated wild type TK1-2 into non-glycosylated form (NE-TK1-2) and examined whether it retains anti-angiogenic activity. The plasmid expressing NE-TK1-2 was constructed by replacing $Asn^{l17}\;and\;Asn^{184}$ with glutamic acid residues. After expression in Pichia pastoris, the secreted protein was purified from the culture broth using S-sepharose and UNO S1-FPLC column. The mass spectrum of NE-TK1-2 showed closely neighboring two peaks, 19631.87 and 19,835.44 Da, and it migrated as one band in SDS-PAGE. The patterns of CD-spectra of these two proteins were almost identical. Functionally, purified NE-TK1-2 was shown to inhibit endothelial cell migration in response to bFGF stimulation at the almost same level as wild type TK1-2. Therefore, the results suggest that non-glycosylated NETK1-2 can be developed as an effective anti-angiogenic and anti-tumor agent devoid of immune reactivity.

• KSBB Journal
• /
• v.9 no.5
• /
• pp.518-524
• /
• 1994

Serum free medium was developed to cultivate Human Embryonic Kidney cells with Cytodex III microcarriers. The cells normally attached and spreaded on the microcarriers in serum free medium, and grew well by bead to bead processes. 85% of attachment yield was obtained on Cytodex III in this medium, compared to about 93% in 1% serum containing medium. About 90% of the attached HEK cells spreaded after 6 hours of post-attachment periods on the surface of microcarrier. Maximum cell density and scu-PA concentration in a serum free medium were $9.1{\times}10^5$ cells/ml and 1790 IU/ml, respectively, with fed-batch cultivation. Maximum cell density and scu-PA concentration in this medium with perfusion cultivation were $2.5{\times}10^6$ cells/ml and 1820 IU/ml, respectively. The conversion of single chain urokinase type plasminogen activator (scu-PA) into two chain type plasminogen activator (tc-PA) was less than 5% in a serum free medium compared to about 10% in 0.5% serum containing medium.

• The Korean Journal of Zoology
• /
• v.32 no.1
• /
• pp.48-54
• /
• 1989

Several proteolytic activities and the level of and-trypsin in neoplastic tissues of human cervix and liver were compared to those in normal tissues to examine if any correlation exists between malignant behavior of the tumors and the changes in overall proteolytic capacity. Proteolysis against casein and insulin in cervix tumor was increased to 2-to 3-fold while that in liver tumor was reduced to one-tenth to one-half. By contrast, the level of anti-trypsin in cervix tumor was lowered to nearly one-tenth of that in normal tissues while the level rose to about 2-fold in malignant tissues of liver. On the other hand, the activities of plasmin-like protease and plasminogen activator were enhanced 10-20% over the activities in normals. These results suggest that the changes in proteolytic capacity are at least in part due to outbalance in either of proteolytic or its inhibitory activity over the other and occur distinctively to each tumor systems for their malignant behavior.