• Nuclear Medicine and Molecular Imaging
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• v.43 no.5
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• pp.468-477
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• 2009

Purpose: Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyD-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. Materials and Methods: HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynaminc injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. Results: In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. Conclusion: In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.117-122
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• 2012

Aim: To elucidate the effects of hyperthermic $CO_2$ pneumoperitoneum on human gastric AGS cells. Methods: Based on a newly devised in vitro study model, we evaluated the anti-cancer effects of HT-$CO_2$ ($42-44^{\circ}C$ for 2-4h) on human gastric cancer cells, and also the corresponding mechanisms. Results: HT-$CO_2$ ($42-44^{\circ}C$ for 2-4h) severely inhibited cell proliferation as assessed by Cell Counting Kit-8 assay, while inducing apoptosis in a temperature- and time-dependent manner demonstrated by annexin-V/PI flow cytometry and morphological analysis (Hoechst/PI fluorescence). In addition, it was found that HT-$CO_2$ ($42-44^{\circ}C$ for 2-4h) promoted the up-regulation of Bax by western blotting. Significantly, it could also suppress gastric cancer cell invasion and metastasis by in vitro invasion and motility assay. Conclusion: In conclusion, HT-$CO_2$ had an efficacious cytotoxic effect on gastric cancer cells through Bax-induced mitochondrial apoptotic signaling. Our studies indicate that it may serve as a potential therapy for peritoneal carcinomatosis of gastric cancer. Further investigations in vivo using animal models are now urgently needed.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.17
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• pp.7129-7135
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• 2014

Background: In this study, we aimed to evaluate the growth inhibitory effect of the combination of ethaselen (BBSKE) and low fixed dose of selenite against A549 human non-small cell lung cancer cells in vitro. Materials and Methods: Growth inhibitory effects against A549 cells were determined by SRB assay. Combination index (CI) values were calculated based on Chou-Talalay median-effect analyses. Dose reduction index (DRI) values were applied to calculate dose reduction of selenite. Contents of free thiols and GSH were determined by DTNB assay and intracellular ROS levels by DCFH-DA fluorescence labeling. Results: Compared with BBSKE or selenite single treatment, the combined application of ethaselen and a low fixed dose of selenite shortened the onset time of sodium selenite, reduced $IC_{50}$ values, and increased the maximum inhibition rates, suggesting a possible molecular mechanism of the synergism. Obvious synergistic effects were observed after different times of combination treatment, especially after 24 h. Compared with selenite single treatment, dosage of selenite could be remarkably reduced in combination therapy to gain the same inhibitory effect on cell proliferation. Compared with BBSKE single treatment, the content of free thiols and GSH were significantly reduced and ROS levels greatly elevated in the combination group. For the combination treatment, cell viability increased as greater concentrations of GSH were added. Conclusions: All these results indicate that the combination treatment of BBSKE and selenite showed synergism to inhibit A549 cell proliferation in vitro, and also reduced the selenite dosage to mitigate its toxicity which is very meaningful for combination chemotherapy of lung cancer. The synergism was probably caused by the accelerated exhaustion of intracellular reductive substances, such as free thiols and GSH, which ultimately leads to enhanced oxidative stress and apoptosis.

• YAKHAK HOEJI
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• v.48 no.2
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• pp.147-152
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• 2004

A wide variety of cancer chemotherapeutic agents have been shown to induce programmed cell death (PCD, APOPTOSIS) in various tumor cell lines in vitro. cis-Malonato [(4R,5R)-4,5-bis(aminomethyl)-2-isoprpopyl-1,3-dioxolane] platinum(II) (heptaplatin), which is a new drug approved by KFDA in 1999, in a novel platinum-based antitumor agent with clinical potential against stomach cancer and the 3rd generation of the cisplatin. This study was performed to know how heptaplatin and cisplatin and sunpla (mixture of heptaplatin and mannitol) affect on SK-MEL-28 cell line, and how they induce the apoptosis. At EM analysis, the morphology of the cell was changed by treatment of the cisplatin, heptaplatin and sunpla. Apoptotic body formed around plasma membrane, and chromatin condensation represented in nucleus. This phenomenon is one of the characteristic of the apoptosis. The DNA of SK-MEL-28 cell line truncated by cisplatin and sunpla treatment was identified on 2% agarose gel electrophoresis. TUNEL assay was performed to know whether SK-MEL-28 cell die as apoptosis or necrosis by cisplatin, heptaplatin and sunpla. At this result, fluorescence intensity increased according to increase of time and concentration. Therefore, it was identified that cislatin, heptaplatin and sunpla induced apoptosis. Fas expressed on SK-MEL-28 cell membrane by cisplatin, heptaplatin and sunpla was identified by using flow cytometer and the expression of bcl-2(anti-apoptotic gene) decreased according to increase of concentration of the cisplatin, heptaplatin and sunpla. Cisplatin, heptaplatin and sunpla induced apoptosis against SK-MEL-28 cell line, and the apoptotic mechanism was identified as Fas-mediated apoptosis and decreased bcl-2 expression.

• Proceedings of the SCSK Conference
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• 2003.09b
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• pp.32-43
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• 2003

UV irradiation on a skin brings about the qualitative and quantitative alterations of the extracellular matrix. Repeated-UV irradiation suppressed the synthesis of collagen and activated the expression of the matrix metalloprotease (MMP). In this paper, on the purpose of development of novel anti-aging agents from natural sources, effects of several natural products on in vitro MMP-1 activity and UVA induced MMP-1 synthesis in human dermal fibroblast (HDF) culture were studied. We measured MMP-1 activities by fluorescence assay using gelatin as substrates. As a result, the extract of Dicentra spectabilis, and flower buds of Tussilago farfara showed strong inhibitory effect. Among them, the extract of flower buds of Tussilago fartara and Dicentra spectabilis inhibited MMP-1 activity by 92% and 87% at 0.05% (w/v). And UVA induced MMP-1 expression were analyzed by enzyme-linked immunosorbent assay (ELISA) and gelatin-based zymography in HDF culture. The extract of flower buds of Tussilago farfara and Dicentra spectabilis suppressed the UVA induced expression of MMP-1 by similar level of Vitamin C 200$\mu$M at 0.1% (w/v). These results suggest that the extract of Dicentra spectabilis, and flower buds of Tussilago farfara effectively prevent skin from the UV-induced photoaging. So the extracts are thought to have potential as effective raw materials for anti-aging cosmetics.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1627-1635
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• 2013

Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (${\Delta}{\psi}m$) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ${\Delta}{\psi}m$ in HFF cells when administered 24 h post-infection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.

• Ocean Science Journal
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• v.43 no.1
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• pp.31-37
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• 2008

As a part of an ongoing search for antioxidants from marine sources, antioxidant activities of 24 kinds of seaweeds (4 green algae, 8 brown algae, and 12 red algae) were investigated. The seaweeds were extracted by acetone/dichloromethane and methanol, respectively. The antioxidant properties of both extracts were evaluated using four different activity tests, including degree of occurrence of intracellular reactive oxygen species (ROS), NO, lipid peroxidation, and GSH (glutathione) in mouse macrophage Raw 264.7 cells. The levels of intracellular reactive oxygen species (ROS) and GSH were measured using 2',7'-dichlorofluorescin diacetate (DCFDA) and monobromobimane as fluorescence probe, respectively. Moreover, the generation of NO and lipid peroxidation products were determined by each method based on the Griess reaction and TBARS assay. Solvent extracts from seaweeds such as Scytosiphon lomentaria, Prionitis cornea, Laruencia okamurae, Callophyllis japonica, Sargassum horneri, Dictyopteris divaricata, Lomentaria catenata, Corallina confuse, Ishige okamurae, and Ahnfeltiopsis flabelliformi exhibited high antioxidant activities in cellular oxidizing systems.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.76-78
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• 2002

Heat shock proteins (HSPs) are induced in most living cells by mild heat treatment, ethanol, heavy metal ions and hypoxia. In yeast Saccharomyces cerevisiae, mild heat pretreatment strongly induces Hsp104 and thus provide acquired thermotolerance. The ability of hsp104 deleted mutant $({\triangle}hsp104)$ to acquire tolerance to extreme temperature is severely impaired. In providing thermotolerance, two ATP binding domains are indispensible, as demonstrated in ClpA and ClpB proteases of E. coli. The mechanisms by which Hsp104 protects cells from severe heat stress are not yet completely elucidated. We have investigated regulation of mitochondrial metabolic pathways controlled by the functional Hsp104 protein using $^{13}C_NMR$ spectroscopy and observed that the turnover rate of TCA cycle was enhanced in the absence of Hsp104. Production of ROS, which are toxic to kill cells radiply via oxidative stress, was also examined by fluorescence assay. Mitochondrial dysfunction was manifested in increased ROS levels and higher sensitivity for oxidative stress in the absence of Hsp104 protein expressed. Finally, we have identified mitochondrial complex I and Ferritin as binding protein(s) of Hsp104 by yeast two hybrid experiment. Based on these observations, we suggest that Hsp104 protein functions as a protector of oxidative stress via either keeping mitochondrial integrity, direct binding to mitochonrial components or regulating metal-catalyzed redox chemistry.

• Journal of Life Science
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• v.10 no.2
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• BLOOD RESEARCH
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• v.53 no.4
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• pp.299-306
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• 2018

Background IgG-mediated anaphylaxis occurs after infusion of certain monoclonal antibody-based therapeutics. New in vitro tests are urgently needed to diagnose such reactions. We investigated whether allergens trigger neutrophil oxidative burst (OB) and if neutrophil OB occurs due to allergen-specific IgG (sIgG). Methods Neutrophil OB was measured by dihydrorhodamine 123 flow cytometry using a leukocyte suspension spiked with a very small patch of the allergen crude extract, Dermatophagoides farinae (Der f). The mean fluorescence intensity ratio of stimulated to unstimulated samples was calculated as the neutrophil oxidative index (NOI). Results The Der f-specific NOI (Der f-sNOI) showed a time-dependent increase after Der f extract addition. At 15 min activation, higher Der f-sIgG levels were associated with lower Der f-sNOI values in 31 subjects (P<0.05). This inverse relationship occurs due to the initial blocking effect of free Der f-sIgG. Additionally, neutrophil OB was nearly absent (Der f-sNOI of -1) in two cases: a subject with undetectable Der f-sIgG levels and washed leukocyte suspensions deprived of Der f-sIgG. Conclusion Allergens can trigger neutrophil OB via preexisting allergen-sIgG. Neutrophil OB can be easily measured in a leukocyte suspension spiked with the allergen. This assay can be used to diagnose IgG-mediated anaphylaxis.