• Tuberculosis and Respiratory Diseases
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• v.61 no.4
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• pp.366-373
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• 2006

Background: Paraquat is extremely toxic chemical material, which generates reactive oxygen species (ROS), causing multiple organ failure. In particular, paraquat leads to irreversible progressive pulmonary fibrosis. Exaggerated cell deaths exceeding the normal repair of type II pneumocytes leads to mesenchymal cells proliferation and fibrosis. This study examined the followings; i) whether or not paraquat induces cell death in lung epithelial cells; ii) whether or not paraquat-induced cell deaths are apoptosis or necrosis; and iii) the effects of N-acetylcysteine, dexamethasone, and bcl-2 on paraquat-induced cell deaths. Methods: A549 and BEAS-2B lung epithelial cell lines were used. The cell viability and apoptosis were evalluated using a MTT assay, Annexin V staining was monitored by fluorescence microscopy, The level of bcl-2 inhibition was examined by establishing stable A549 pcDNA3-bcl-2 cell lines throung the transfection of pcDNA3-bcl-2 with the mock. Results: Paraquat decreased the cell viability in A549 and BEAS-2B cells in a dose and time dependent manner. The Annexin V assay showed that apoptosis was the type of paraquat-induced cell death. Paraquat-induced cell deaths was significantly inhibited by N-acetylcysteine, dexamethasone, and bcl-2 overexpression. The cell viability of A549 cells treated with N-acetylcysteine, and dexamethasone on the paraquat-induced cell deaths were increased significantly by 10 ~ 20%, particularly at high doses. In addition, the cell viability of A549 pcDNA3-bcl-2 cells overexpressing bcl-2 was significantly higher than the untransfected A549 cells. Conclusion: Paraquat induces apoptotic cell deaths in lung epithelial cells in a dose and time dependent manner. The paraquat-induced apoptosis of lung epithelial cells might occur through the mitochondrial pathway.

• Journal of Life Science
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• v.26 no.3
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• pp.376-386
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• 2016

Apoptosis induction has been proposed as an efficient mechanism by which malignant tumor cells can be removed following chemotherapy. The intrinsic mitochondria-dependent apoptotic pathway is frequently implicated in chemotherapy-induced tumor cell apoptosis. Since DNA-damaging agent (DDA)-induced apoptosis is mainly regulated by the tumor suppressor protein p53, and since more than half of clinical cancers possess inactive p53 mutants, microtubule-damaging agents (MDAs), of which apoptotic effect is mainly exerted via p53-independent routes, can be promising choice for cancer chemotherapy. Recently, we found that the apoptotic signaling pathway induced by MDAs (nocodazole, 17α-estradiol, or 2-methoxyestradiol) commonly proceeded through mitotic spindle defect-mediated prometaphase arrest, prolonged Cdk1 activation, and subsequent phosphorylation of Bcl-2, Mcl-1, and Bim in human acute leukemia Jurkat T cells. These microtubule damage-mediated alterations could render the cellular context susceptible to the onset of mitochondria-dependent apoptosis by triggering Bak activation, Δψm loss, and resultant caspase cascade activation. In contrast, when the MDA-induced Bak activation was inhibited by overexpression of anti-apoptotic Bcl-2 family proteins (Bcl-2 or Bcl-xL), the cells in prometaphase arrest failed to induce apoptosis, and instead underwent mitotic slippage and endoreduplication cycle, leading to formation of populations with 8N and 16N DNA content. These data indicate that cellular apoptogenic mechanism is critical for preventing polyploid formation following MDA treatment. Since the formation of polyploid cells, which are genetically unstable, may cause acquisition of therapy resistance and disease relapse, there is a growing interest in developing new combination chemotherapies to prevent polyploidization in tumors after MDA treatment.

• Journal of Plant Biotechnology
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• v.43 no.4
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• pp.450-456
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• 2016

Rhizoctonia leaf blight (large patch) has become a serious problem in Korean lawn grass, which is extremely hard to treat and develops mostly from the roots of lawn grass to wither it away. Rhizoctonia leaf blight (large patch) is caused by Rhizoctonia solani AG2-2 (IV). To develop zoysia japonica with strong disease tolerance against this pathogenic bacterium, ${\beta}-1,3-glucanase$ was cloned from zoysia japonica, which is one of the PR-Proteins known to play a critical role in plant defense reaction. ${\beta}-1,3-glucanase$ is known to be generated within the cells when plant tissues have a hypersensitive reaction due to virus or bacterium infection and secreted outside the cells to play mainly the function of resistance against pathogenic bacteria in the space between the cells. This study utilized the commonly preserved part in the sequence of corn, wheat, barley, and rice which had been researched for their disease tolerance among the ${\beta}-1,3-glucanase$ monocotyledonous plants. Based on the part, degenerate PCR was performed to find out the sequence and full-length cDNA was cloned. E.coli over-expression was conducted in this study to mass purify target protein and implement in vitro activation measurement and antibacterial test. In addition, to interpret the functions of ZjGlu1 gene, each gene-incorporating plant transformation vectors were produced to make lawn grass transformant. Based on ZjGlu1 protein, antibacterial activity test was conducted on 9 strains. As a result, R. cerealis, F. culmorum, R.solani AG-1 (1B), and T. atroviride were found to have antibacterial activity. The gene-specific expression amount in each organ showed no huge difference in the organs based upon the transformant and against 18s gene expression amount.

• Radiation Oncology Journal
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• v.20 no.1
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• pp.73-80
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• 2002

Purpose : Tumor growth in a given neoplasm is the net result of cell proliferation and cell loss, and apoptosis is the most significant component of continuous cell loss in most tumors. In this study, we examined non-Hodgkin's lymphoma (NHL, n=67) immunohistochemically for the presence of Bcl-2 oncoprotein and P53 protein and compared apoptotic indices (Als) and Ki-67 proliferative indices (percentages of Ki-67 positive cells). Materials and Methods : 67 patients with NHL were evaluated : 3 low-grade and 64 intermediate-grade. The phenotype was determined in 65 cases : 47 $(70\%)$ were B cell type and 18 $(27\%)$ were T ceil type. Als and Ki-67 proliferative indices were determined immunohistochemically and the overexpression of P53 and Bcl-2 protein were also evalutated. Results : The overexpressions of Bcl-2 protein and P53 protein were found in $40\%$ (26/65) and $31\%$ (20/65). The Al ranged from $0\%\;to\;15\%$ (mean 2.16, median 1.2). Cellular Bcl-2, which counteracts apoptosis, was significantly (p=0.005) associated with Als. Ki-67 proliferative indices ranged from $1\%\;to\;91\%$ (mean 55.4), and P53 was significantly (p=0.000) associated with Ki-67 proliferative indices. A positive correlation between Als and Ki-67 proliferative indices was revealed (p=0.012) in Bcl-2 positive patients. Conclusion : In NHL, we observed a correlation between Als and Bcl-2 expression, between Ki-67 proliferative indices and P53 expression, and between Als and Ki-67 proliferative indices in Bcl-2 positive patients. Our results suggest that cell apoptosis may be inseparable from cell proliferation during tumor growth.

• Microbiology and Biotechnology Letters
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• v.32 no.1
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• pp.72-77
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• 2004

Previously, we synthesized a novel Cyclin-dependent kinase inhibitor, MCS-5A. Also, we investigated the involvement of cell cycle regulatory events during MCS-5A-mediated apoptosis in HL-60(+p16/-p53) cells with up-regulation of p16 protein expression. In contrast, apoptosis was not observed in A549(-p16/+p53) cells. Therefore we propose that $p16^{INK4A}$ is a key enzyme for inducing apoptosis. In the present studies, we have explored the mechanism of $p16^{INK4A}$ -mediated cytotoxicity and the role of p16.sup INK4A/ overexpression in the induction of apoptosis in human tumor cells. The tumor suppressor gene $p16^{INK4A}$ is known as a cyclin-dependent kinase inhibitor (CKI) and cell cycle regulator. We expressed wild type $p16^{INK4A}$ in pcDNA3.1 vector and then transfected into non-small cell lung cancer (NSCLC) cell expressing different statue of p16$^{INK4A}$, p53 gene〔A549(-p16/+p53), H1299(-p16/-p53) and HeLa(+pl6/+p53) cell line〕. TUNEL assay (including propidium iodide staining following transfection of these cell line with pcDNA3.1-pl6) indicate that p16$^{INK4A}$-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating an induction of caspase 3 cleavage due to the transfection of A549, H1299 and HeLa cells with pcDNA3.1-pl6. These results suggest that p16$^{INK4A}$ has a new function of inducing apoptosis which is not related with the function of tumor suppressor gene p53.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.2
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• pp.89-98
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• 2010

Sex and its tropic hormones influence the lacrimal system, corneal anatomy and disease, aqueous humor dynamics and glaucoma, crystalline lens and cataract, and retinal disease. Dry eye occurs especially frequently during pregnancy, oral contraceptive use, and after menopause, during which androgen levels decrease. Androgen control development, differentiation, and lipid production of sebaceous glands throughout the body, and androgen deficiency also leads to meibomian gland dysfunction and evaporative dry eye. On the other hand, estrogen causes a reduction in size, activity, and lipid production of sebaceous glands. Sex and its tropic hormones also influence the corneal anatomy and disease, and corneal thickening occurred on the second day of the menstrual cycle and around the time of ovulation and appeared to be related to estrogen levels. Fuchs' dystrophy is more commonly seen in postmenopausal women than men and may be linked to hormonal changes that occur with aging. In addition, overexpression of estrogen and progesterone receptors in the conjunctiva of vernal keratoconjunctivitis patients. Serum progesterone levels also may be associated with intraocular pressure especially in pregnant women, and for the women. For women with cataracts, hormone levels were typical of menopause, and there was a significant negative correlation between estradiol and follicular stimulating hormone levels. In addition, serum testosterone levels are associated with the development of diabetic retinopathy. Although the role of sex hormones on the eye is largely unknown, and the results should be interpreted with caution until replicated, the functions of sex hormones in ocular disease remains to be investigated, because they may be involved in structure and function of the ocular components, which are important in the pathogenesis of ocular disease.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.4 s.48
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• pp.439-444
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• 2004

UV irradiation produces free radicals and related reactive oxygen species (ROS), and these are injury to all most of organisms of skin cells and extracellular matrix (ECM). In addition, free radicals and ROS stimulate the overexpression of matrix metalloproteinases (MMPs) that can degrade most components of ECM such as collagen. Since collagen constitutes almost of skin connective tissue, their disarrangement causes wrinkle formation and droop of skin. Therefore, scavenging activity on free radicals, ROS and suppression of MMP-1 is expected to prevent skin photoaging. In this study, to investigate the relationship between photoaging and Draconis sanguis, we examined the effects of antioxidant, in vitro MMP inhibition and expression of UVA-induced MMP-1 in human dermal fibroblasts. Draconis sanguis was found to show scavenging activities of radicals and ROS with the $IC_{50}$ values of $183{\;}{\mu}g/mL$ against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and $30{\;}{\mu}g/mL$ against superoxide radicals in the xanthine/xanthine oxidase system, respectively. Draconis sunguis inhibited the activities of MMP-1 in a does-dependent manner and the $IC_{50}$ value calculated from semi-log plots was $200{\;}{\mu}g/mL$. Also, UVA induced MMP expression was reduced $74\%$ by treatment with Draconis sanguis, and MMP-1 mRNA expression was reduced in a dose-dependent manner. Therefore Draconis sanguis was able to significantly inhibit MMP expression in protein and mRNA level. All these results suggested that Draconis sanguis may act as an anti-photoaging agent by antioxidation and reducing UVA-induced MMP-1 production.

• Tuberculosis and Respiratory Diseases
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• v.79 no.3
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• pp.143-152
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• 2016

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the accumulation of excessive fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor ${\beta}1$ (TGF-${\beta}1$)-induced epithelial-to-mesenchymal transition (EMT) is thought to be a possible source of fibroblasts/myofibroblasts in IPF lungs. We have previously reported that apolipoprotein A1 (ApoA1) has anti-fibrotic activity in experimental lung fibrosis. In this study, we determine whether ApoA1 modulates TGF-${\beta}1$-induced EMT in experimental lung fibrosis and clarify its mechanism of action. Methods: The A549 alveolar epithelial cell line was treated with TGF-${\beta}1$ with or without ApoA1. Morphological changes and expression of EMT-related markers, including E-cadherin, N-cadherin, and ${\alpha}$-smooth muscle actin were evaluated. Expressions of Smad and non-Smad mediators and TGF-${\beta}1$ receptor type 1 ($T{\beta}RI$) and type 2 ($T{\beta}RII$) were measured. The silica-induced lung fibrosis model was established using ApoA1 overexpressing transgenic mice. Results: TGF-${\beta}1$-treated A549 cells were changed to the mesenchymal morphology with less E-cadherin and more N-cadherin expression. The addition of ApoA1 inhibited the TGF-${\beta}1$-induced change of the EMT phenotype. ApoA1 inhibited the TGF-${\beta}1$-induced increase in the phosphorylation of Smad2 and 3 as well as that of ERK and p38 mitogen-activated protein kinase mediators. In addition, ApoA1 reduced the TGF-${\beta}1$-induced increase in $T{\beta}RI$ and $T{\beta}RII$ expression. In a mouse model of silica-induced lung fibrosis, ApoA1 overexpression reduced the silica-mediated effects, which were increased N-cadherin and decreased E-cadherin expression in the alveolar epithelium. Conclusion: Our data demonstrate that ApoA1 inhibits TGF-${\beta}1$-induced EMT in experimental lung fibrosis.

• Molecules and Cells
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• v.37 no.2
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• pp.178-186
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• 2014

Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) bound directly to these sites and activated transcription. Exposure to the hypoxia-mimetic compound $CoCl_2$, incubation under 1% $O_2$ conditions, or overexpression of HIF-$1{\alpha}$ enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with $CoCl_2$ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-$1{\alpha}$ to HRE sites and is epigenetically controlled by methylation of its promoter region.

• Journal of Life Science
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• v.18 no.11
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• pp.1499-1506
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• 2008

To examine antitumor activity of the edible plant Zanthoxylum schinifolium, the cytotoxic effect of various organic solvent extracts of its stems on human acute leukemia Jurkat T cells was investigated. Among these extracts such as methanol extract (SS-7), methylene chloride extract (SS-8), ethyl acetate extract (SS-9), n-butanol extract (SS-10), and residual fraction (SL-11), SS-8 exhibited the most cytotoxic activity against Jurkat T cells. The methylene chloride extract (SS-8) possessed the apoptogenic activity capable of inducing sub-G1 peak along with apoptotic DNA fragmentation in Jurkat T cells. Western blot analysis revealed that SS-8 induced apoptosis via mitochondrial cytochrome c release into cytoplasm, subsequent activation of caspase-9 and caspase-3, and cleavage of PARP, which could be blocked by overexpression of Bcl-xL. Jurkat T cell clone I2.1 $FADD^{-/-}$) and Jurkat T cell clone I9.2 (caspase-$8^{-/-}$ were as sensitive as was the wild-type Jurkat T cell clone A3 to the cytotoxic effect of SS-8, suggesting no contribution of Fas/FasL system to the SS-8-mediated apoptosis. The GC-MS analysis of SS-8 showed that it was composed of 16 ingredients including 9,12-octadecanoic acid (18.62%), 2,4-dihydro-5-methyl-4- (1-methylethylidene)- 2-(4-nitrophenyl)-3H- pyrazol-3-one (14.97%), hexadecanoic acid (14.23%), (z,z)-6,9-pentadecadien- 1-ol (13.73%), 5,6-dimethoxy-2-methyl benzofuran (10.95%), and 4-methoxy-2-methylcinnamic acid (5.38%). These results demonstrate that the methylene chloride extract of the stems of Z. schinifolium can induce apoptotic cell death in Jurkat T cells via intrinsic mitochondria-dependent caspase cascade regulated by Bcl-xL without involvement of the Fas/FasL system.