• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.261-264
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• 2002

CHO (Chinese hamster ovary) cells were transfected with plasmids containing both cis-acting HRE (hypoxia response element) and CMV-promoter that controls tissue-type plasminogen activator (t-PA). CHO cells with HRE produced 16.2 fold higher t-PA concentration than CHO cells without HRE. It was noted that hypoxia strongly induced CHO cell apoptosis. which resulted in decrease of cell viability and protein production. In this study. by introducing Bcl-2, anti-apoptotic gene, we tried to recover cell viability and increase the protein production. When batch culture of both control cells without transfection of Bcl-2 and cells transfected with Bcl-2 were performed in the absence of CoCl ι hypoxia mimic condition. the cells with Bcl-2 were effected specific cell growth rates, maximum cell density. Immunoblotting assay showed Bcl-2 was recombinant with HRE dependent t- P A expression cassette, and their expression level was depended on hypoxia. By introducing Bcl-2, both cell viability and maximum cell density could be increased.

• Journal of Sasang Constitutional Medicine
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• v.15 no.1
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• pp.72-89
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• 2003

To elucidate the neuroprotective effect of Yeoldahanso-tang(YHT) on nerve cells damaged by hypoxia, the cytotoxic effects of exposure to hypoxia were determined by XTT(SODIUM3,3'-{I-[(PHENYLAMINO) CARBONYL]-3,4-TETRAZOLIUM}- BIS (4-METHOXY-6-NITRO) BENZENE SULFONIC ACID HYDRATE), NR(Neutral red), MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and SRB(Sulforhodamin B) asssay. The activity of catalase and SOD(Superoxide dismutase) was measured by spectrophometry, and $TNF-{\alpha}$(Tumor cell necrosis $fector-{\alpha}$) and PKC(Protein kinase C) activity was measured after exposure to hypoxia and treatment of YHTWE. Also the neuroprotective effect of YHTWE was researched for the elucidatioion of neuroprotective mechanism. The results were as follows; 1. Hypoxia decreased cell viability measured by XTT, NR assay when cultured cerebral neurons were exposed to 95% N2/5% CO2 for $2{\sim}26$ minutes in these cultures and YHTWE inhibited the decrease of cell viability. 2. H2O2 treatment decreased cell viability measured by MTT, and SRB assay when cultured cerebral neurons were exposed to 1-80 ${\mu}M$ for 6 hours, but YHTWE inhibited the decrease of cell viability. 3. Hypoxia decreased catalase and SOD activity, and also $TNF-{\alpha}$ and PKC activity in these cultured cerebral neurons, but YHTWE inhibited the decrease of the catalase and SOD activity in these cultures. 4. Hypoxia triggered the apoptosis via caspase activation and internucleosomal DNA fragmentation. Also hypoxia stimulate the release of cytochrome c forom mitochondria. YHTWE inhibited the apoptosis via caspase activation induced by hypoxia. From these results, it can be suggested that brain ischemia model induced hypoxia showed neurotoxicity on cultured mouse cerebral neurons, and the YHTWE has the neuroprotective effect in blocking the neurotoxicity induced by hypoxia in cultured mouse cerebral neurons.

• YAKHAK HOEJI
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• v.49 no.1
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• pp.97-102
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• 2005

The protective effect of Yangguksanwha-tang (YST) against hypoxia-reperfusion insult was investigated in PC12 cells. To elucidate the mechanism of the protective effect of YST, cell viability, the changes in activities of superoxide dismutase, glutathione peroxidase, catalase, caspase 3 and the production of malondialdehyde were observed after treating PC12 cells with YST which was metabolized by rat liver homogenate. Pretreatment of YST with liver homogenate appeared to increase its protective effect against hypoxia-reperfusion insult. The result showed that YST had the highest protective effect against hypoxia/reperfusion at the dose of $2\;{\mu}g/ml$ in PC12 cells, probably by recovering the redox enzyme activities and MDA to control level.

• BMB Reports
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• v.32 no.2
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• pp.112-118
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• 1999

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment such as in radiation therapy or treatment with some anticancer drugs. It has been suggested that hypoxic cells are involved in the development of a more aggressive phenotype and contribute to metastasis. In this study, as an attempt to understand how tumor cells adapt to hypoxic stress, we investigated the regulation of the hypoxia-induced expression of proteins that control essential processes of tumor cell survival and angiogenesis. We first examined whether hypoxia induces stress protein gene expression of murine solid tumor RIF cells. We also examined hypoxia-induced changes in angiogenic gene expression in these cells. Finally, we investigated the association of the elevated levels of stress proteins with the regulation of hypoxia-induced angiogenic gene expression. Results demonstrated that hypoxia induced the expression of the erythropoietin (EPO) gene and at least two major members of stress proteins, heat shock protein 70 (HSP70) and 25 (HSP25) in RIF tumor cells. Evidence that the expression of EPO gene was greatly potentiated in TR cells suggested that the elevated levels of HSPs may play an important role in the regulation of the hypoxia-induced EPO gene expression. One of the RIF variant cell lines, TR, displays elevated levels of HSPs constitutively. Taken together, our results suggest that a hypoxic tumor microenvironment may promote the survival and malignant progression of the tumor cells by temporarily increasing the level of stress proteins and expressing angiogenic genes. We suspect that stress proteins may be associated with the increase of the angiogenic potential of tumor cells under hypoxia.

• Biomedical Science Letters
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• v.19 no.3
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• pp.180-187
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• 2013

Hypoxia is an integral part of the environment during luteolysis. In this study we examined whether hypoxia could directly stimulate endothelial cells to produce nitric oxide (NO). Endothelial cells were cultured in hypoxic (5% $O_2$) or normoxic (20% $O_2$) conditions and the levels of total NO, inducible NO and endothelial NO was measured. We found that hypoxia but not normoxia upregulated NO production. The increased NO levels correlated with increased inducible NO synthase (iNOS) expression whereas expression of endothelial NOS (eNOS) expression remained constant. Addition of the iNOS specific inhibitor 1400W to hypoxic cultures prevented NO production suggesting that hypoxia-induced NO production in endothelial cells was due mainly to upregulation of iNOS. We also found that prostaglandin $F_{2{\alpha}}$ (PGF) production was unaffected by hypoxia suggesting that upregulation of NO was not due to increased synthesis of PGF. In summary, we report that endothelial cells cultured under hypoxic conditions produce NO via the iNOS pathway. This study provides the importance of the relation between the hypoxic environment and the induction of NO by endothelial cells during regression of the corpus luteum in the ovary.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.4
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• pp.1082-1091
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• 2003

To elucidate the neuroprotective effect of Kamijihwang-hwan(KSH) on nerve cells damaged by hypoxia, the cytotoxic effects of exposure to hypoxia were determined by XTT, NR, MTT and SRB asssay. The activity of catalase and SOD was measured by spectrophometry, and TNF-α and PKC activity was measured after exposure to hypoxia and treatment of Kamijihwang-hwan(KSH) water extract(KJHWE). Also the neuroprotective effect of KJHWE was researched for the elucidation of neuroprotective mechanism. The results were as follows ; Hypoxia decreased cell viability measured by XTT, NR assay when cultured cerebral neurons were exposed to 95% N2/5% CO₂ for 2～26 minutes in these cultures and KJHWE inhibited the decrease of cell viability. H₂O₂ treatment decreased cell viability measured by MTT, and SRB assay when cultured cerebral neurons were exposed to 1-80 uM for 6 hours, but KJHWE inhibited the decrease of cell viability. Hypoxia decreased catalase and SOD activity, and also TNF-α and PKC activity in these cultured cerebral neurons, but KJHWE inhibited the decrease of the catalase and SOD activity in these cultures. Hypoxia triggered the apoptosis via caspase activation and internucleosomal DNA fragmentation. Also hypoxia stimulate the release of cytochrome c form mitochondria. KJHWE inhibited the apoptosis via caspase activation induced by hypoxia. From these results, it can be suggested that brain ischemia model induced hypoxia showed neurotoxity on cultured mouse cerebral neurons, and the KJHWE has the neuroprotective effect in blocking the neurotoxity induced by hypoxia in cultured mouse cerebral neurons.

• Genomics & Informatics
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• v.9 no.4
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• pp.161-172
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• 2011

Angiogenesis is regulated by a large number of molecules and complex signaling mechanisms. The G protein $G{\alpha}_{13}$ is a part of this signaling mechanism as an endothelial cell movement regulator. Gene expression analysis of $G{\alpha}_{13}$ knockout mouse embryos was carried out to identify the role of $G{\alpha}_{13}$ in angiogenesis signaling during embryonic development. Hypoxia-inducible response factors including those acting as regulators of angiogenesis were over expressed, while genes related to the cell cycle, DNA replication, protein modification and cell-cell dissociation were under expressed. Functional annotation and network analysis indicate that $G{\alpha}_{13}{^{-/-}}$ embryonic mice were exposed to hypoxic conditions. The present analysis of the time course highlighted the significantly high levels of disorder in the development of the cardiovascular system. The data suggested that hypoxia-inducible factors including those associated with angiogenesis and abnormalities related to endothelial cell division contributed to the developmental failure of $G{\alpha}_{13}$ knockout mouse embryos.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.5
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• pp.2031-2035
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• 2015

Intra-tumoral hypoxia is an environment that promotes tumor cell migration, angiogenesis and epithelial-mesenchymal transition that accounts for a major mechanism of metastasis. Chloroquine potentially offers a new therapeutic approach with an 'old' drug for effective and safe cancer therapies, as it exerts anti-metastatic activity. We investigated the inhibitory effect of chloroquine on cholangiocarcinoma (CCA) cell migration under cobalt chloride ($CoCl_2$)-stimulated hypoxia. We showed that chloroquine suppressed CCA cell migration under hypoxic-mimicking conditions on exposure to $100{\mu}M$ $CoCl_2$. Moreover, chloroquine stabilized the protein level of prolyl hydroxylase domain proteins (PHD-2) but reduced the levels of hypoxic responsive proteins such as hypoxia-inducible factor (HIF-$1{\alpha}$) and vascular endothelial growth factor (VEGF). It also suppressed epithelial mesenchymal transition (EMT) by increasing the ratio of E-cadherin to N-cadherin under hypoxic conditions. In conclusion, chloroquine can inhibit hypoxia-stimulated metastasis via HIF-$1{\alpha}$/VEGF/EMT which may serve as a useful additional strategy for CCA therapy.

• Radiation Oncology Journal
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• v.22 no.3
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• pp.217-224
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• 2004

Purpose: It is well known that the radiosensitivity of tumor cells can be significantly reduced under hypoxic conditions. Hypoxia-inducible factor-1 $\alpha$ (HIF-1 $\alpha$) plays a pivotal role in the essential adaptive responses to hypoxia. Therefore this study investigated the relationship between HIF-1 $\alpha$ expression and radiosensitivity. M Mouse hepatoma cell line hepafcic7 and HIF-1 $\beta$-deficient mutant cell line hepa1C4 were used to analyze the role of HIF-1 a. on radiosensitivity. These cells were exposed for 6 h to desferrioxamine (DFX) before radiation. HIF-1$\alpha$. expression was examined by Western blot. Apoptosis was assessed by DNA fragmentation, propidium iodide staining, and apoptotic cell death detection ELISA kit. Radiation sensitivity was determined using MTT assay. The radiobioiogical parameters, surviving fractions at 2 Gy and 8 Gy, and mean inactivation dose (MID) from the linear-quadratic model were used to assess radiation sensitivity in the statistical analyses. Results: The expression of HIF-1 $\alpha$. was Increased, whereas apoptosis was decreased, by radiation In the presence of DFX In hepal cl c7, but not In hepal C4. The radlosensitivity of hepal C4 cells was not significantly affected by DFX treatment. The radiosensitivlty of hepal cl c7 cells was significantly decreased in the presence of DFX Conclusion: The expression of HIF-1 w by hypoxia-mimic agent DFX reduced apoptosls and radiosensitlvity in mouse hepatoma cell line hepafclc7. These results suggested that HIF-1 u could be Induced by irradiation in hypoxic ceils of tumor masses, and that this mlght Increase radioresistance in hypoxic cells.

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