• Journal of Physiology & Pathology in Korean Medicine
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• v.21 no.1
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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.

• Development and Reproduction
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• v.15 no.3
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• pp.205-213
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• 2011

Ethanol treatment during the brain growth spurt period has been known to induce the death of Purkinje cells. The underlying molecular mechanisms and the role of reactive oxygen species (ROS) in triggering ethanol-induced Purkinje cell death are, however, largely unresolved. We undertook TUNEL staining, western blotting assay and immunohistochemistry for the cleaved forms of caspase-3 and -9, with calbindin D28K double immunostaining to identify apoptotic Purkinje cells. The possibility of ROS-induced Purkinje cell death was immunohistochemically determined by using anti-8-hydroxy-2'deoxyguanosine (8-OHdG), a specific cellular marker for oxidative damage. The results show that Purkinje cell death of PD 5 rat cerebellum following ethanol administration is mediated by the activation of caspase-3 and -9. However, unexpectedly, TUNEL staining did not reveal any positive Purkinje cells while there were some TUNEL-positive cells in the internal and external granular layer. 8-OHdG was detected in the Purkinje cell layers at 8 h, peaked at 12-24 h, but not at 30 h post-ethanol treatment. No 8-0HdG immunoreactive cells were detected in the internal and external granular layer. The lobule specific 8-OHdG staining patterns following ethanol exposure are consistent with that of ethanol-induced Purkinje cell loss. Thus, we suggest that ethanol-induced Purkinje cell death may not occur by the classical apoptotic pathway and oxidative damage is involved in ethanol-induced Purkinje cell death in the developing cerebellum.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.5
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• pp.1322-1329
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• 2008

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruits on cell viability in human glioma cells. The present study was undertaken to examine the effect of mulberry fruit (Mar; Fructus) on cell viability and to determine its underlying mechanism in human glioma cells. Cell viability and cell death were estimated by MTT assay and trypanblue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with $DiOC_6$(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Mori Fructus resulted in apoptotic cell death in a dose- and time-dependent manner. Mori Fructus increased ROS generation and the Mori Fructus-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in Mari Fructus-induced cell death. Western blot analysis showed that Mori Fructus treatment caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). Mori Fructus induced depolarization of mitochondrial membrane potential and its effect was inhibited by the antioxidants NAC and catalase. Mori Fructus induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by Mori Fructus and caspase inhibitors prevented the Mori Fructus-induced cell death. These findings suggest that Mori Fructus results in human glioma cell death through ROS-dependent mitochondrial pathway in human glioma cells.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.69-73
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• 1999

In plants as well as in other multicellular organisms, programmed cell death plays essential roles in the abortion or formation of specific cells and tissues during development to organize the plant [11, 15, 18]. A typical example of developmentally programmed cell death in plants is the death during differentiation of tracheary elements which are components of vessels and tracheids, a water-conducting system. The programming of cell death during tracheary element differentiation has been revealed to be unique to plant cells by using the in vitro Zinnia mesophyll cell culture system. In particular, new biosynthesis of autolysis-related enzymes such as cysteine proteases and nucleases, their accumulation of the vacuole and the programmed collapse of the vacuole are essential to the death of tracheary elements and differ greatly from the process of the apoptotic cell death in animals.

• Journal of Korean Medicine Rehabilitation
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• v.18 no.4
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• pp.1-11
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• 2008

Objectives : Cerebral ischemia resulting from transient or permanent occlusion of cerebral arteries leads to neuronal cell death and eventually causes neurological impairments. Scrophulariae radix is the roots of Scrophularia buergeria. In the present study, we investigated the effects of the aqueous extract of Scrophulariae radix on apoptotic cell death in the hippocampal dentate gyrus following transient global ischemia in gerbils. Methods : For this study, step-down avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were performed. Results : The present results showed that apoptotic cell death in the hippocampal dentate gyrus was significantly increased following transient global ischemia in gerbils. Treatment with the aqueous extract of Scrophulariae radix suppressed the ischemia-induced apoptosis in the dentate gyrus and thus facilitated the recovery of short-term memory impairment induced by ischemic cerebral injury. Conclusions : Here in this study, we have shown that Scrophulariae radix has a positive effect on-and possesses protective qualities against ischemia-induced apoptotic neuronal cell death, and it can be used for the treatment of ischemic brain diseases.

• Journal of Acupuncture Research
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• v.20 no.3
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• pp.45-62
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• 2003

Objective : To examine the effects of Beevenom on the cell proliferation of human breast carcinoma cell line MCF-7, we performed various experiments such as does-dependent effect of Beevenom on cell proliferation and viability, morphological changes, and alterations of apoptosis/cell cycle-regulatory gene products. Methods : Beevenom induced cell viability and proliferation of MCF-7 cells in a concentration-dependent manner. The anti-proliferative effect by Beevenom treatment in MCF-7 cells was associated with morphological changes such as membrance shrinking and cell rounding up. Results : Beevenom induced apoptotic cell death in a concentration-dependent manager, which was associated with degradation of ${\beta}$-catenin, an apoptotic target protein. Beevenom induced the Bax expressions, a pro-apoptotic gene, both in protein and mRNA levels, however, the levels of Bcl-$X_{S/L}$ expression, an anti-apoptotic gene, were down-regulated in Beevenom-treated cells. Western blot analysis and RT-PCT data revealed that the levels of cyclin of B1 protein and cyclin E mRNA were reduced by Beevenom treatment in MCF-7 cells, respectively, where as the expression of tumor suppressor p53 and cyclin dependent kinase inhibitor p21 mRNA were markedly increased in a concentration-dependent fashion. Conclusions : Taken together, these findings suggest that Beevenom induced inhibition of human breast cancer cell proliferation is associated with the induction of apoptotic cell death and Beevenom may have therapeutic potential in human breast cancer.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.1
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• pp.187-192
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• 2006

Herbal medicines have been utilized to treat a variety of diseases, including cancer. Several species of the family rubiaceae have been reported to have antitumor activity. In this study, we report the cytotoxicity and antitumor activity exhibited dy the methanol extracts prepared from Rubia radix (RRME), Uncaria gambir (UGME) and Oldenlandia diffusa (ODME) (family: Rubiaceae) against human promyleloid leukemia cell line, HL-60. The cytotoxicity of RRME (2~20 ${\mu}g/ml$), UGME (20~200 ${\mu}g/ml$) and ODME (20~200 ${\mu}g/ml$) were assessed dy the MTT reduction assay. IC50 values for RRME, UGME and ODME were 11.0, 99.5 and 106.1 ${\mu}g/ml$, respectively. When the HL-60 cells were treated with RRME (10 ${\mu}g/ml$), UGME (120 ${\mu}g/ml$) and ODME (140 ${\mu}g/ml$) for 24 h, several apoptotic characteristics such as DNA fragmentation and morphologic changes were observed. Furthermore, flow cytometric analysis was peformed to determine the percent of apoptotic cells. The poupulation of sub-G1 hypodiploid cells was increased 37.49% in RRME treatment, 12.49% in UGME treatment and 7.21% in ODME treatment compared with untreated control cells (2.64%). To further confirm apoptotic cell death, we assayed caspase-3, -8 and -9 activities in RRME, UGME and ODME-treated cells. After treatment of RRME, UGME and ODME for 12 h, caspase-3, -8 and -9 activities significantly increased.compared to untreated control cells. These results show that RRME, UGME and ODME induced apoptotic cell death in HL-60 cells and may have a possibility of potential antitumor activities.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.6
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• pp.1584-1592
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• 2006

Houttuynia cordata Thunb, well known as 'E-Sung-Cho' in Korea, is traditional medicinal plant generally used in Oriental medicine therapy. We previously reported that the water extract of H. cordata inhibited cell proliferation and induced apoptosis in human breast carcinoma cells. In the present study, we investigated the biochemical mechanisms of anti-proliferative effects by the methanol extract of H. cordata (MEHC) in human lung carcinoma A549 cells. It was found that MEHC could inhibit the cell growth in a dose-dependent manner, which was associated with morphological change and apoptotic cell death as determined by formation of apoptotic bodies, DNA fragmentation and increased populations of apoptotic-sub G1 phase cells. Apoptosis of A549 cells by MEHC was also connected with a down-regulation of anti-apoptotic Bcl-2 and inhibitor of apoptosis proteins (IAPs) expression. MEHC treatment induced the proteolytic activation of caspase-3, caspase-8 and caspase-9, and a concomitant inhibition of poly(ADP-ribose) polymerase (PARP), ${\beta}$-catenin and phospholipase (PLC)-${\gamma}$1 protein expression. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of H. cordata.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.4
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• pp.1147-1152
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• 2004

To investigate the anti-cancer effects of aqueous extract of Cheonkumwikyung-tang (CKWKT) on the growth of human lung carcinoma cell line A549, we performed various biochemical experiments such as the effects of CKWKT on the cell proliferation and viability, the morphological changes, the effects on expression of apoptosis and cell growth-regulatory gene products. Results obtained are as follow; CKWKT treatment declined the cell viability and proliferation of A549 cells in a concentration-dependent manner. The anti-proliferative effect by CKWKT treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. CKWKT treatment induced apoptotic cell death of A549 cells in a concentration-dependent manner, which was associated with inhibition and/or degradation of apoptotic target proteins such poly(ADP-ribose) polymerase, β-catenin and phospholipase C-γ1. Western blot analysis revealed that the levels cyclin-dependent kinase inhibitor p21 expression were induced by CKWKT treatment in A549 cells. Taken together, these findings suggest that CKWKT-induced inhibition of human lung cancer cell proliferation is associated with the induction of apoptotic cell death via regulation of several major growth regulatory gene products and CKWKT may have therapeutic potential in human lung cancer.

• IMMUNE NETWORK
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• v.14 no.2
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• pp.116-122
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• 2014

Macrophage death plays a role in several physiological and inflammatory pathologies such as sepsis and arthritis. In our previous work, we showed that simvastatin triggers cell death in LPS-activated RAW 264.7 mouse macrophage cells through both caspase-dependent and independent apoptotic pathways. Here, we show that the nuclear orphan receptor NR4A1 is involved in a caspase-independent apoptotic process induced by LPS and simvastatin. Simvastatin-induced NR4A1 expression in RAW 264.7 macrophages and ectopic expression of a dominant-negative mutant form of NR4A1 effectively suppressed both DNA fragmentation and the disruption of mitochondrial membrane potential (MMP) during LPS- and simvastatin-induced apoptosis. Furthermore, apoptosis was accompanied by Bcl-2-associated X protein (Bax) translocation to the mitochondria. Our findings suggest that NR4A1 expression and mitochondrial translocation of Bax are related to simvastatin-induced apoptosis in LPS-activated RAW 264.7 macrophages.