• 동의생리병리학회지
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• 제21권1호
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• pp.117-125
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• 2007

Neuronal ischemia is a pathological process caused by a lack of oxygen (anoxia) and glucose (hypoglycemia), resulting in neuronal death. It is believed that apoptosis is one of the mechanisms involved in ischemic cell death. Neuronal apoptosis is a process characterized by nuclear DNA fragmentation, changes of plasma membrane organization. To elucidate the mechanism of neuronal death following ischemic insult and to develop neuroprotective effects of Daebowonjeon(DBWJ) against ischemic damage, in vitro models are used. In vitro models of cell death have been devloped with pheochromocytoma (PC12) cell, which have become widely used as neuronal models of oxidative stress, trophic factor, serum deprivation and chemical hypoxia. Using a special ischemic device and PC12 cultures, we investigated an in vitro model of ischemia based on combined Oxygen and Glucose Deprivation (OGD) insult, followed by reoxygenation, mimicking the pathological conditions of ischemia. In this study, Daebowonjeon rescued PC12 cells from Oxygen-Glucose Deprivation (OGD)-induced cell death in a dose-dependent manner The nuclear staining of PC12 cells clearly showed that DBWJ attenuated nuclear condensation and fragmentation which represent typical neuronal apoptotic characteristics. DBWJ also prevents the LDH release and induction of Hypoxia Inducing Factor (HIF)-1 by OGD-exposed PC12 cells. Furthermore, DBWJ reduced the activation of polyADP-ribose polymerase (PARP) by OGO-exposed PC12 cells. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that oriental medicine, such as DBWJ, may prevent PC12 cell from OG D-induced neuronal death by inhibiting the apoptotic process.

• Biomolecules & Therapeutics
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• 제17권4호
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• pp.438-445
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• 2009

We investigated global gene expression from both mouse liver and mouse hepatic cell lines treated with acetaminophen (APAP) in order to compare in vivo and in vitro profiles and to assess the feasibility of the two systems. During our analyses of gene expression profiles, we picked up several down-regulated genes, such as the cytochrome P450 family 51 (Cyp51), sulfotransferase family cytosolic 1C member 2 (Sult1c2), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1), and several genes that were up-regulated by APAP, such as growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1) and zinc finger protein 688 (Zfp688). For validation of gene function, synthesized short interfering RNAs (siRNAs) for these genes were transfected in a mouse hepatic cell line, BNL CL.2, for investigation of cell viability and mRNA expression level. We found that siRNA transfection of these genes induced down-regulation of respective mRNA expression and decreased cell viability. siRNA transfection for Cyp51 and others induced morphological alterations, such as membrane thickening and nuclear condensation. Taken together, siRNA transfection of these six genes decreased cell viability and induced alteration in cellular morphology, along with effective inhibition of respective mRNA, suggesting that these genes could be associated with APAP-induced toxicity. Furthermore, these genes may be used in the investigation of hepatotoxicity, for better understanding of its mechanism.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.97-97
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• 2003

Embryonic stem (ES) cells, derived from preimplantation embryos, are able to differentiate into various types of cells consisting the whole body, or pluripotency. In addition to the plasticity, ES cells are expected to be different from terminally differentiated cells in very many ways, such as patterns of gene expressions, ability and response of the cells in confronting environmental stimulations, metabolism, and growth rate. As a model system to differentiate these two types of cells, human ES (hES, MB03) cells and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is $H_2 O_2$. Ratio of dying cells as determined by the relative amount of dye neutral red entrapped within the cells after the exposures. Cell death rates were not significantly different when either MB03 or HeLa were exposed up to 0.4 mM $H_2 O_2$. However, relative amount of dye entrapped within the cells sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM $H_2 O_2$, while it was approximately 54% in MB03. Pretreatment of cells with BSO (GSH chelator) and measurement of GSH content results suggest that cellular GSH is the major defensive mechanism of hES cells. Induction of apoptosis in hES cell was confirmed by DNA laddering, induction of Bax, and chromatin condensation. In summary, hES cells 1) are extremely resistant to oxidative stress, 2) utilize GSH as a major defensive mechanism. and 3) experience apoptosis upon exposure to oxidative stress.

• 동의생리병리학회지
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• 제18권6호
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• pp.1661-1665
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• 2004

Herba Patriniae(HP) has been known to exert anti-tumoral activity in Korea. However, its molecular mechanism, of action is not understood. In this study, we found that HP induced apoptosis in androgen-dependent prostate cancer DU145 cells as evidenced by DNA fragmentation and chromatine condensation in hoechst dye staining. Our data demonstrated that HP-induced apoptotic cell death was accompanied by activation of caspase-3 and cleavages of its substrates, poly(ADP-ribose) polymerase(PARP) in a time- and concentration-dependent manner. Taken together, these results suggest that HP induces the activation of caspase-3, degradation of PARP, and eventually leads to apoptotic cell death.

• 동의생리병리학회지
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• 제18권6호
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• pp.1733-1739
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• 2004

The water extract of Hwangryunhaedog-tang(HRHDT} has been traditionally used for treatment of ischemic heart and brain damage in oriental medicine. However, little is known about the mechanism by which the water extract of HRHDT rescues cells from these damages. This study was designed to investigate the protective mechanisms of HRHDT on hypoxia-induced cytotoxicity in H9c2 cardiomyoblast cells. Hypoxia, markedly decreased the viability of H9c2 cells, which was characterized with apparent apoptptic features such as chromatin condensation as well as fragmentation of genomic DNA and nuclei. However, HRHDT significantly reduced hypoxia-induced cell death and apoptotic characteristics. Also, HRHDT prevented the mitochondrial dysfunction including the disruption of mitochondria membrane permeability transition (MPT) and an increase in expression of anti-apoptotic Bcl-2 proteins in hypoxia-H9c2 cells. Taken together, this study suggests that the protective effects of the water extract of HRHDT against hypoxic damages may be mediated by the modulation of Bcl-2 and Bak expression.

• 한국수정란이식학회지
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• 제13권2호
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• pp.179-190
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• 1998

Prolactin (PRL) surge in cycling rats at proestrous afternoon has previously been reported as an inducer of apoptotic cell death of luteal cells. This death-inducing action of PRL seeins unusual, because PRL can he categorized as a cell-survival factor, if other known physiological functions of PRL are taken into account. In this study, the apoptotic action of PRL was assessed in cultured cells prepared from rat luteal tissue and underlying molecular /cellular mechanism of PRL-induced luteolysis was analyzed. The latest crop of corpora lutea (CLs) were enucleated from rat ovaries at 18:00 h on the proestrous day before the next ovulation. Donor rats were pretreated with CB154, a dopamine agonist, in order to he exempted from the endogenous PRL surge. The harvested GLs were dispersed and cultured with or without PRL (2$\mu$g /ml) for 24 or 48 h. An addition of PRL to the culture medium changed the parameters indicative of cell death via apoptosis: a decrease in cell viability (MTT) and an increase in chromatin condensation. Most of the DNA breakdown in nuclei induced by PRL occurred in steroidogenic cells which were identified by 3$\beta$-HSD activity staining, and the number of 3$\beta$-HSD-positivecells were significantly decreased. Interestingly, most of the cells with an apoptotic nucleus adhered to one or more intact and seemingly non-steroidogenic cells. Because the expression of Fas has heen shown to be abundant in murine ovary, and Fas is known to have an exact physiological role in occurrence of apoptotic cell death, the membrane form-Fas ligand (rnFasL) was quantified in the cell lysate. An addition of PRL increased expression of mFasL. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, in place of PRL, enhanced the apoptotic parameters. Cumulatively, the apoptotic PRL action was addressed to cells unknown than steroidogenic lute~ cells. The most prohable candidate for the direct target cells is Tcells in the luteal tissue that can express mFasL in response to PRL.

• Biomolecules & Therapeutics
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• 제18권4호
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• pp.454-462
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• 2010

The present study investigated the neuroprotective effect of Carpinus tschonoskii MAX and its intracellular protective mechanism on 6-hydroxydopamine (6-OHDA)-induced oxidative damage in PC12 cells. We found that pretreatment of PC12 cells with C. tschonoskii extract significantly inhibited the cell death induced by 6-OHDA in a dose dependent manner. C. tschonoskii extract decreased 6-OHDA-induced apoptotic events such as chromatin condensation, DNA fragmentation, the decrease of Bcl-2/Bax ratio, caspase-3 activation and PARP cleavage. C. tschonoskii extract also reduced generation of 6-OHDA-induced reactive oxygen species and nitric oxide. Furthermore, C. tschonoskii extract up-regulated the myocyte enhancer factor 2 D (MEF2D), a critical transcription factor for neuronal survival, and Akt activity, whereas it inhibited the activity of ERK1/2 and JNK. The results suggest that C. tschonoskii extract decreases 6-OHDA-induced oxidative stress and could prevent PC12 cell apoptosis induced by 6-OHDA via the up-regulation of MEF2D and Akt activity, and thus may have application in developing therapeutic agents for Parkinson's disease.

• Journal of Korean Neurosurgical Society
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• 제46권6호
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• pp.552-557
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• 2009

Objective : Interferon-$\beta$, (IFN-$\beta$) has been used in the treatment of cancers. Inhibition of the enzyme cyclooxygenase (COX) with celecoxib had a significantly suppressive effect on tumor growth, angiogenesis, and metastasis in a variety of tumors. The aim of this study was to elucidate the antiglioma effect of combined treatment with IFN-$\beta$ and celecoxib in U87 glioma model. Methods : The in vitro effects of IFN-$\beta$ (50-1,000 IU/mL) and celecoxib ($50-250\;{\mu}M$) alone or combination of both on the proliferation and apoptosis of U87 cells were tested using MTT assay, FACS analysis and DNA condensation. To determine the in vivo effect, nude mice bearing intracerebral U87 xenograft inoculation were treated with IFN-$\beta$ intraperitoneally ($2{\times}10^5\;IU/day$ for 15 days), celecoxib orally (5, 10 mg/kg) or their combination. Results : IFN-$\beta$ or celecoxib showed an inhibitory effect on the proliferation of U87 cells. When U87 cells were treated with IFN-$\beta$ and celecoxib combination, it seemed that IFN-$\beta$ interrupted the antiproliferative and apoptotic activity of celecoxib. No additive effect was observed on the survival of the tumor bearing mice by the combination of IFN-$\beta$ and celecoxib. Conclusion : These results suggest that IFN-$\beta$ seems to inhibit the antiglioma effect of celecoxib, therefore combination of IFN-$\beta$ and celecoxib may be undesirable in the treatment of glioma.

• BMB Reports
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• 제34권3호
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• pp.189-193
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• 2001

Curcumin a yellow pigment from Curcuma Tonga, has been known to possess antioxidative and anticarcinogenic properties, as well as to induce apoptosis in some cancer cells. There have been, however, several contradictory reports that hypothesized curcumin (a hydrophobic molecule) can bind a membrane Gpid bilayer and induce nonspecific cytotoxicity in some cell lines. Why curcumin shows these contradictory effects is unknown. In A-431 cells, growth inhibition by curcumin is due mostly to the specific inhibition of the intrinsic tyrosine kinase activity of the epidermal growth factor receptor, as reported earlier by Korutla et al. Thus, we assumed that the cell death of A-431 by curcumin might be due to the specific induction of apoptosis. In this paper we clearly show that curcumin induces apoptosis in A-431 cells. The cureumin-induced cell death of A-431 exhibited various apoptotic features, including DNA fragmentation and nuclear condensation. Furthermore, the curcumin-induced apoptosis of A-431 cells involved activation of caspase-3-like cysteine protease. Involvement of caspase-3 was further confirmed by using a caspase-3 specific inhibitor, DEVD-CHO. In another study, decreased nitric oxide (NO) production was also shown in A-431 cells treated with curcumin, which seems to be the result of the inhibition of the iNOS expression by curcumin, as in other cell lines. However, 24 h after treatment of curcumin there was increased NO production in A-431 cells. This observation has not yet been clearly explained. We assumed that the increased NO production may be related to denitrosylation of the enzyme catalytic site in caspase-3 when activated. Taken together, this study shows that the cell death of A-431 by curcumin is due to the induction of apoptosis, which involves caspase-3 activation.

• Journal of Korean Neurosurgical Society
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• 제42권5호
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• pp.392-399
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• 2007

Objective : Arsenic trioxide ($As_2O_3$) has been used as an anticancer agent in traditional Chinese medicine for thousand years and berberine is an isoquinoline alkaloid present that has indicated significant antimicrobial activity. We have examined the combined anticancer effects of $As_2O_3$ and berberine against the human neuroblastoma (HNB) SH-SY5Y cells in vitro, and to elucidate underlying molecular mechanism. Methods : HNB SH-SY5Y cells were treated with $2\;{\mu}M\;As_2O_3$ and $75\;{\mu}g/ml$ berberine, and their survival, cell death mechanism as well as synergistic cytotoxic effects were estimated by using MTT assay, DAPI staining, agarose gel electrophoresis, flow cytometric analysis, and western blot analysis. Results : The combined treatment of two drugs also markedly decreased cell viability. The cytotoxic effects of two drugs were revealed as apoptosis characterized by chromatin condensation, DNA fragmentation, and the loss of mitochondrial membrane potential. The apoptotic cytotoxicity was accompanied by activation of caspase-3 protease as well as decreased the expression of Bcl-2, Bid, and Bcl-x/L. In addition, the cells treated with combination of two drugs also showed significantly increased intracellular reactive oxygen species levels and lipid peroxidation compared to cells $As_2O_3$or berberine only. Conclusion : Combined treatment of $As_2O_3$ with berberine induced activation of apoptotic signaling pathways in HNB SH-SY5Y cells. These results suggest that the possibility of the combined treatment of two chemotherapeutic agents with low concentration improving cytotoxic effect for cancer cells with minimal side effects.