• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.35-43
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• 2009

Objectives: Seminal concentration of tumor necrosis factor-alpha (TNF-${\alpha}$) relevant to sperm nuclear DNA integrity has not been studied. The present study aimed to evaluate seminal concentration of TNF-${\alpha}$ in correlation with sperm parameters and nuclear DNA integrity in asymptomatic healthy donors. Methods: Semen samples were obtained by masturbation from forty-five healthy donors. Results: Sperm quality was assessed by computer-assisted semen analysis and nuclear DNA integrity measured by the TUNEL assay in raw semen. TNF-${\alpha}$ concentrations were measured by ELISA in frozen-thawed seminal plasmas. Sperm DNA fragmentation rates were ranged between 1.9% and 53.0% (mean${\pm}$SD, 12.4${\pm}$9.6%). Univariate analysis revealed that DNA fragmentation rate was not associated with sperm concentration or motility but had a correlation with linearity negatively (r=-0.325, p=0.03) and age positively (r=0.484, p=0.001). The mean seminal concentration of TNF-${\alpha}$ was 4.9 pg/mL with a range from 1.1 to 22.6 pg/mL. The TNF-${\alpha}$ concentration had no correlation with clinically relevant parameters of sperm quality or nuclear DNA fragmentation rate. Conclusion: Our results indicate that sperm nuclear DNA fragmentation may be not associated with seminal TNF-${\alpha}$ level or sperm quality in asymptomatic healthy donors.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2015-2020
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• 2012

Oleanolic acid (OA) is a naturally occurring triterpenoid in food materials and is a component of the leaves and roots of Olea europaea, Viscum album L., Aralia chinensis L. and more than 120 other plant species. There are several reports validating its antitumor activity against different cancer cells apart from its hepatoprotective activity. However, antitumor activity against skin cancer has not beed studied well thus far. Hence the present study of effects of OA against HaCaT (immortalized keratinocyte) cells - a cell-based epithelial model system for toxicity/ethnopharmacology-based studies - was conducted. Radical scavenging activity ($DPPH{\cdot}$) and FRAP were determined spectrophotometrically. Proliferation was assessed by XTT assay at 24, 48 and 72 hrs with exposure to various concentrations (12.5-200 ${\mu}M$) of OA. Apoptotic induction potential of OA was demonstrated using a cellular DNA fragmentation ELISA method. Morphological studies were also carried out to elucidate its antitumor potential. The results revealed that OA induces apoptosis by altering cellular morphology as well as DNA integrity in HaCaT cells in a dose-dependent manner, with comparatively low cytotoxicity. The moderate toxicity observed in HaCaT cells, with induction of apoptosis, possibly suggests greater involvement of programmed-cell death-mediated mechanisms. We conclude that OA has relatively low toxicity and has the potential to induce apoptosis in HaCaT cells and hence provides a substantial and sound scientific basis for further validation studies.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.699-705
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• 1997

Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying excitatory amino acid-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which L-glutamate, an excitatory amino acid neurotransmitter, induces cell death in PC12 cell lines. To characterize cell death, we employed sandwich enzyme-linked immunosorbent assay(ELISA) method for cellular DNA fragmentation, DNA agarose gel electrophoresis and chromatin staining by acridine orange and ethidium bromide after treating the PC12 cells with L-glutamate. L-Glutamate caused dose-dependent cell death with a maximum at 24 hrs after the treatment. These cellular fragmentation was blocked by pretreatment of MK-801, a noncompetitive N-methyl-D-aspartic acid(NMDA) receptor antagonist, and nerve growth factor(NGF). Analysis of DNA integrity from L-glutamate-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. The PC12 cells that were induced to die by L-glutamate treatment exhibited classical chromatin condensation under the light microscopy after acridine orange and ethidium bromide staining. These results suggest that apoptosis is one of the key features that are involved in L-glutamate-induced excitotoxic cell death in PC12 cells, and these cell death are mediated by NMDA receptor and depend on NGF.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2729-2734
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• 2012

Despite clinical advances in anticancer therapy, there is still a need for novel anticancer metabolites, with higher efficacy and lesser side effects. Oroxylum indicum (L.) Vent. is a small tree of the Bignoniaceae family which is well known for its food and medicinal properties. In present study, the chemopreventive properties of O. indicum hot and cold non-polar extracts (petroleum ether and chloroform) were investigated with MDA-MB-231 (cancer cells) and WRL-68 (non-tumor cells) by XTT assay. All the extracts, and particularly the petroleum ether hot extract (PHO), exhibited significantly (P<0.05) higher cytotoxicity in MDA-MB-231 when compared to WRL-68 cells. PHO was then tested for apoptosis induction in estrogen receptor (ER)-negative (MDA-MB-231) and ER-positive (MCF-7) breast cancer cells by cellular DNA fragmentation ELISA, where it proved more efficient in the MDA-MB-231 cells. Further, when PHO was tested for anti-metastatic potential in a cell migration inhibition assay, it exhibited beneficial effects. Thus non-polar extracts of O. indicum (especially PHO) can effectively target ER-negative breast cancer cells to induce apoptosis, without harming normal cells by cancer-specific cytotoxicity. Hence, it could be considered as an extract with candidate precursors to possibly harness or alleviate ER-negative breast cancer progression even in advanced stages of malignancy.

• Genomics & Informatics
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• v.4 no.2
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• pp.77-79
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• 2006

In order to identify novel proapoptotic genes, we screened approximately 1,000 hypothetical genes whose functions are completely unknown. After these genes were transiently expressed in HeLa cells, their nuclei images were captured using automated high-speed fluorescence microscope, through which the ratio of apoptotic nuclei was estimated. We selected genes that induce greater than 3-fold increase in apoptotic nuclei compared to that of the vector control. The candidate proapoptotic genes were sequenced and their effects on cell death were further confirmed by the additional assay, DNA fragmentation ELISA. Finally, we were able to identify 4 full-length hypo-thetical genes with proapoptotic activity.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.5
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• pp.1281-1291
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• 2005

Chungpae-tang is suggested to have the antitumor activity on lung cancer. This study was peformed to investigate apoptotic effect in vitro and antitumor effect and immune response after injection of B16-F10 melanoma cells and Chungpae-tang into a tail vein of C57BL/6 mice and administratition of Chungpae-tang in A549 human lung cancer cell line in vivo, respectively. Experimental studies were obtained by measuring the median survival time and cytokine expression through RT-PCR, and ELISA assay. The results were summarized as follows: 5 mg/ml of Chungpae-tang causing DNA fragmentation, caspase-3 enzyme activation, PARP fragmentation, and cytochrome c release, suggested that Chungpae-tang has in vitro apoptotic effect in A549 human lung cancer cell line in the apoptosis-induced experiment. The median survival time of the Chungpae-tang treated group was 21 days and that of control group was 22 days, suggesting that the median survival time between the Chungpae-tang treated group and the control group was not significant. Cytokine expression between the Chungpae-fang treated group and the control group was noticeable, but was not significant in the RT-PCR. In the ELISA assay, IL-2 productivity in the Chungpae-tang treated group was to increase more than that in the normal group (p<0.05) and was no significant between the Chungpae-tang treated group and the control group. $INF-\gamma$ productivity of the control group decreased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group increased more than that of the control group (p<0.05). IL-12 productivity of the control group increased more than that of the normal group (p<0.05) and that of the Chungpae-tang-treated group decreased more than that of the control group (p<0.05) and the normal group. IL-4 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). IL-10 productivity of the Chungpae-tang-treated group increased more than that of the normal group and the control group (p<0.05). Accordingly the results show Chungpae-tang could induce apoptosis in A549 human lung cancer cell line and bring to antitumor effect and immune response against injection of B16-F10 melanoma cells into a tail vein of C57BL/6 mice but it needs more research on the precise mechanism of such effects.

• 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 Life Science
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• v.33 no.4
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• pp.305-314
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• 2023

Ultraviolet B (UVB) irradiation causes skin diseases by inducing cellular oxidative stress, photoaging, and inflammation. This study aimed to investigate the protective effects of morin against UVB-induced oxidative stress in normal human dermal fibroblasts (NHDFs). Morin has been reported to be a potential therapeutic candidate for oxidative stress-mediated diseases, neurodegenerative diseases, and inflammation. Since morin has been identified as a potential antioxidant, we speculated that morin could alleviate UVB-induced apoptosis in NHDFs. Cell viability and intracellular reactive oxygen species (ROS) levels were measured using the MTT assay, H2DCFDA, and the DHE staining method, respectively. Lipid peroxidation and protein carbonyl formation were tested using ELISA kits. DNA fragmentation and comet assay were used to assess DNA damage. Apoptotic bodies were analyzed using Hoechst 33342 staining and TUNEL assay. The expression of apoptosis-related proteins was examined using Western blot analysis. Morin showed a cyto-protective effect by scavenging UVB-induced ROS, increasing the expression of antioxidant-related proteins and inhibiting UVB-induced oxidative alterations such as lipid peroxidation, protein carbonylation, and DNA damage. Morin protects against UVB-induced cell apoptosis by inhibiting Bcl-2-associated X protein, caspase-9, and caspase-3 expression, while increasing the expression of the anti-apoptotic protein Bcl-2. These effects of morin were conferred through decreased phosphorylation of p38 and c-Jun N-terminal kinase 1/2. The results demonstrated that morin may be developed as a preventive/therapeutic drug to be used to prevent UVB-induced skin damage.

• Advances in Traditional Medicine
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• v.10 no.4
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• pp.254-262
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• 2010

Artemisia capillaris (A. capillaries) is known to play roles in many cellular events, such as cell proliferation, differentiation, and apoptosis. We investigated the antifibrogenic efficacy of A. capillaris in the immortalized human hepatic stellate cell line LX2. Cell proliferation was determined by the MTT assay. Cell cycle was analyzed by the flow cytometry. Apoptotic cells were measured using a cell death detection ELISA. Caspase activity was detected by a colorimetric assay. The mRNA level of Bcl-2 and Bax mRNA were measured by real-time PCR. MEK and ERK protein were detected by Western blot analysis. We provide evidence that A. capillaris induces cell cycle arrest, apoptosis, and potently inhibits the mitogen-activated protein kinase pathway. A. capillaris inhibited cell proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle analysis with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 mRNA levels and an increase in Bax expression. Exposure of LX2 cells to A. capillaris induced caspase-3 activation, but co-treatment of A. capillaris with the pan-caspase inhibitor Z-VAD-FMK, and the caspase-3 inhibitor Z-DEVE-FMK, blocked apoptosis. A. capillaris down-regulated Mcl-1 protein levels and inhibited phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.

• Biomolecules & Therapeutics
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• v.18 no.1
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• pp.56-64
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• 2010

Pentoxifylline (PTX) has been used for the local reduction of fat tissue in the clinical setting. However, its safety and efficacy have not been proven. The aim of this study was to evaluate the effects of PTX on cell lines established from fat tissue. Newly cultured human preadipocytes and adipocytes from subcutaneous abdominal fat in addition to purchased human lung fibroblasts and keratinocytes were treated with PTX at different concentrations. Cell viability was determined using the Cell counting kit (CCK)-8 assay and lipolysis was evaluated using an Elisa kit. DNA fragmentation, Western blot analysis, Hoechst and Propidium Iodide (PI) staining and fluorescence activated cell scanning analysis were performed to confirm apoptosis. The viability of adipocytes, preadipocytes, keratinocytes and fibroblasts was markedly decreased at concentrations of PTX above 20 mM. Apoptosis was induced at concentrations of PTX over 40 mM in all cell lines. Lipolysis was increased by 60% at concentrations of PTX of 20 mM compared to the control. In conclusion, the results of this study showed that 20 mM of PTX induced lipolysis. At concentrations over 20 mM, PTX reduced the viability of all cells studied including: adipocytes, preadipocytes, fibroblasts and keratinocytes, in a non-specific manner.