• 대한한의학회지
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• 제25권4호
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• pp.79-89
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• 2004

Saussurea lappa and Taraxacum mongolicum have been used for herbal medicinal treatments against cancers in East Asia. We performed this study to understand the molecular basis underlying the anti-tumor effects of two herbs and analyzed the effects of these medicinal herbs on proliferation and on expression of cell growth/apoptosis related molecules by using an AGS gastric cancer cell line. The treatments of Saussurea lappa dramatically reduced cell viabilities in a dose and time-dependent manner, but Taraxacum mongolicum did not. FACS analysis and Annexin V staining assay also showed that Saussurea lappa induces apoptotic cell death of AGS. Expression analyses via RT-PCR and Western blots revealed that Saussurea lappa increased expression of the p53 and its downstream effector p21/sup Waf1/, and that the both increased expression of apoptosis related Bax and cleavage of active caspase-3 protein. We also confirmed the translocation of Bax to mitochondria Collectively, our data demonstrate that Saussurea lappa induces growth inhibition and apoptosis of human gastric cancer cells, and these effects are correlated with down- and up-regulation of growth-regulating apoptotic and tumor suppressor genes, respectively.

• 동의생리병리학회지
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• 제17권2호
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• pp.394-402
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• 2003

This study was designed to investigate the protective effect of Bojungmyunyuk-dan(BJMY-Dan) on the cisplatin-induced cytotoxicity of primary rat astrocytes. BJMY-Dan is an oriental herbal prescription for its ability to recover protective effects against anti-cancer chemotherapies. After astrocytes were treated cisplatin, MTT assay was performed for cell viability test. To explore the mechanism of cytotoxicity, I used the several measures of apoptosis to determine whether this processes was involved in cisplatin-induced cell damage in astrocytes. Also, astrocytes were treated with BJMY-Dan and then, followed by the addition of cisplatin. Cisplatin decreased the viability of astrocytes in a dose and time-dependent manner. BJMY-Dan increased the viability of astrocytes treated cisplatin. Astrocytes treated cisplatin were revealed as apoptosis characterized by nuclear staining and flow cytometry. BJMY-Dan protected astrocytes from cisplatin-induced nuclear fragmentation and chromatin condensation. Also, caspase-3 and caspase-9 proteases were activated in astrocytes by cisplatin. BJMY-Dan inhibited the activation of caspase proteases in cisplatin-treated astrocytes. Cleavage of [poly(ADP-ribose) polymerase](PARP) was occurred at 12hr after treatment of cisplatin in astrocytes. BJMY-Dan recovered the cleavage of PARP in cisplatin-treated astrocytes. Also, BJMY-Dan inhibited the activation of pro-apoptotic factor, Bak by cisplatin. Lastly, astrocytes stained with JC-1 and Rhodamine 123 were photographed by fluorescence microscope to visualize changes of mitochondrial membrane permeability transition(MPT) during treatment with cisplatin for 24hr. BJMY-Dan recovered the change of MPT by cisplatin in astrocytes. According to above results, BJMY-Dan may protect astrocytes from cytotoxicity induced by chemotherapeutic agents, including cisplatin.

• 대한한방내과학회지
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• 제28권3호
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• pp.409-420
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• 2007

Objectives : The purpose of this study was to identify the anti-tumor effects of realgar on various cancer cells through molecular biologic and cellular biologic methods. Materials & Methods : We used 5 kinds of cancer cell lines:stomach cancer cell (AGS), glioma cells (T98G, A172, SNU-489) and prostate cancer cells (LNCaP). We injected the boiled extract of realgar. $50{\mu}$g/ml and $100{\mu}$g/ml to culture media (ml) for 24 hours. We examined the morphological changes under an inverted microscope and a fluorescence microscope. We measured the suppressive effect on viability of 5 kinds of cancer cells via XTT assay. We examined the effect on the revelation of PARP cleavage, Bcl-2 protein and Bax protein by western blot analysis. Results : The extract of realgar caused markedly morphological changes on AGS, T98G, SNU-489, and LNCaP. All of them showed withdrawn and floating appearance. The suppressive effect on viability of AGS, T98G, A172, SNU-489, and LNCaP showed that each test group had more suppressive effect on viability of AGS, T98G, A172, SNU-489, and LNCaP than the control group, which was statistically significantly (p<0.01). The extract of realgar did not induce PARP cleavage in AGS, T98G, A 172, SNU-489, or LNCaP. In the revelation of protein related to apoptosis, the protein levels of Bcl-2 decreased and the protein levels of Bax increased in AGS, T98G, SNU-489, and LNCaP treated with realgar. The protein levels of Bcl-2 decreased and the protein levels of Bax did not change in A172 treated with realgar. Conclusions : This experiment showed that realgar has anti-tumor effect on stomach cancer cells (AGS), glioma cells (T98G, SNU-489L and prostate cancer cells (LNCaP)

• 동의생리병리학회지
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• 제17권5호
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• pp.1243-1250
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• 2003

This study was designed to elucidate the synergistic cytotoxic mechanisms of the co-treatment of adriamycin and Paljinhangahm-dan in human hepatoma malignant cancer cell line, HepG2. The combination of adriamycin and the ethanol extract of Paljinhangahm-dan synergistically augmented the cytotoxicity of Adriamycin and Paljinhangahm-dan in HepG2 cells. The cytotoxicity of two drugs was revealed as apoptosis characterized by DNA fragmentaton in agarose gel electrophoresis. The apoptotic cytotoxicity of adriamycin and Paljinhangahm-dan was accompanied by the cleavage of procaspase -3 protease and PARP. Of note, anti apoptotic Bcl2 protein was obviously decreased, but Fas was dramatically increased in HepG2 cells co-treated with Adriamycin and Paljinhangahm -dan. In addition, through 2-D gel electorphoresis, we observed that the expression levels of a lot of proteins were obviously changed by the status of drug treatments. This results suggest that the synergistic cytotoxicity of the co-treatment of adriamycin and Paljinhangahm-dan might be caused by the changes of the expression levels of a lot of proteins which play pivotal roles in cell survival or death.

• Journal of Applied Biological Chemistry
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• 제43권3호
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• pp.147-151
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• 2000

A gene, nprM, from Bacillus megaterium ATCC 14945 was obtained by PCR using primers synthesized based on two nprM sequences from two different strains, and cloned into Escherichia coli. The gene nprM encoded an extracellular neutral protease, and the molecular mass of the expressed enzyme was estimated to be approximately 36kDa on a denaturating gel. The enzyme was activated by $Ca^{2+}$, and the optimum concentration of $Ca^{2+}$ was 5 mM. The enzyme was inhibited by EDTA but not by PMSF. The optimal pH and temperature of the cloned enzyme were $50^{\circ}C and pH 7.5-8.0, respectively, and were similar to those of the enzyme from the gene gonor cell. The cloned NprM caused internal cleavage of the native endoglucanase of B. subtilis BSE616 as a model foregin protein, and resulted in a small truncated but still active endoglucanase.

• 대한한방내과학회지
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• 제31권4호
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• pp.693-705
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• 2010

Objectives : The water extract of Daechilgi-tang(DCGT) has traditionally been used for treatment of qi stagnation(氣滯), which is considered to be one of the important causes of neuronal disease in oriental medicine. However, little is known about the mechanism by which DCGT protects neuronal cells from brain cell damages. Methods and Results : The author tested the mechanism of the cytoprotective effect of DCGT on glutamate -stimulated rat C6 glial cells. DCGT significantly protected C6 glial cells from glutamate in MTT assay. Pre-treatment of C6 glial cells with DCGT markedly inhibited the DNA fragmentation of C6 cells induced by glutamate. Glutamate increased the generation of reactive oxygen species(ROS) and intracellular calcium level in C6 glial cells. However, pre-treatment with DCGT markedly suppressed the increase of ROS generation and intracellular calcium accumulation induced by glutamate. Among apoptosis signaling mediators, DCGT markedly increased the expression level of Bcl2 in glutamate-treated cells. It also inhibited the cleavage of caspase-3 and PARP proteins by glutamate in C6 glial cells. Conclusions : These results suggest that DCGT protects brain cells from glutamate cytotoxicity through inhibition of ROS generation and activation of apoptosis signaling pathway as well as induction of the anti-oxidant system.

• 대한한의학회지
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• 제26권2호
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• pp.1-12
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• 2005

Objectives: Malignant gliomas are often treated with cisplatin (cis-diamminedichloroplatinum(II), CDDP) and radiation but results remain unsatisfactory. Since malignant glioma displays moderate resistance to conventional therapy, a new treatment modality is needed to improve the outcome of patients with these tumors. The aim of this study was to investigate the effects of the combined use of Jongjihwan(JJH) and cisplatin(CDDP) on cultured malignant glioma cells, A172. Methodss & Results: The combined use of cisplatin and Jeongjihwan had synergistic effects on Al72 cells during 24 hr-incubation, This treatment resulted in a decrease of cell viability, Which was revealed as apoptosis Characterized by activation of caspase-3 protease as well as cleavage of poly ADP-ribose polymerase (PARP) with change of mitochondria membrane potential transition. The expression of members of the Bcl-2 protein family was modulated during co-treatment with Jeongjihwan and cisplatin. Activation of caspase-3 and mitochondrial alterations were central to co-treatment with Jeongjihwan and cisplatin-induced apoptosis. Conclusions: We conclude that co-treatment with Jeongjihwan and cisplatin-induced activation of the mitochondrial pathway enables cell death. Also, we suggest the combined theory of JJH and cisplatin could be a useful method for glioblastoma.

• 동의생리병리학회지
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• 제27권2호
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• pp.202-211
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• 2013

The purpose of this study is to investigate the apoptotic effect of Phellodendri Cortex water extract (PCWE) on pancreatic cancer cells and to find out the regulating mechanisms. Human-derived pancreatic cancer cell line, MIA PaCa-2 cells were treated by PCWE with various concentrations and the cytotoxicity was determined by MTT assay. The activation of Annexin V, DNA fragmentation, cell cycle arrest and caspase activation were observed to investigate the role of PCWE in pancreatic cancer cells. Also, to find out the regulating mechanisms, we examined the ROS production. The treatment of PCWE induced the cell death in both concentration and time dependent manner. The treatment of PCWE also increased the expression of Annexin V, DNA fragmentation, cell cycle arrest, and cleavage of caspase, which means cell-death PCWE induced was apoptosis but not necrosis. The ROS production was increased by PCWE treatment and the blockade of ROS inhibited the PCWE-induced cell death. These results could suggest that PCWE induced apoptosis via ROS release in pancreatic cancer cell.

• 대한한의학방제학회지
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• 제13권1호
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• pp.71-83
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• 2005

Chelidonii Herba (Baekgulchae in Korean: CHE), a commonly used herb in Korea, Japan and China, is widely used in the treatment of stomach cancer, jaundice, gastric ulcer, edema and pain of stomach. In the present study, we demonstrated that CHE induces apoptosis in AGS cells, human stomach adenocarcinoma cell line. One of the most important recent advances in cancer research is the recognition that apoptosis plays a major role in both tumor formation and treatment response, In this study, CHE caused a decrease of viability in AGC cells. When AGS cells were treated with CHE, cells showed dose-dependent manner apoptotic cell death. Increased apoptotic cell death, exposured to CHE, resulted from induction of Bad translocation to mitochondria, cytochrome-c release from mitochondria to cytosol, activation of caspase-3, 8, 9, and PARP cleavage. These results suggest that CHE may be potential therapeutic approach in the clinical management of stomach adenocarcinoma.

• 대한한방내과학회지
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• 제35권1호
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• pp.79-91
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• 2014

Objectives : To investigate the anti-cancer effect of Palbohoichoon-tang (PBHCT) extracts. Methods : The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MMT) assay and cell morphological changes were microscopically analyzed after staining with $10{\mu}M$ 2-[4-amidinophenyl]-6-indolecarbamidine dihydrochloride (DAPI) and TUNEL. We also analyzed expression of Bcl2, $Bcl_{xL}$, Bax, procaspase-3, procaspase-9, and procyclic acidic repetitive protein (PARP) by western blot method. Results : Observations showed that PBHCT induced the apoptotic cell death proved by increased sub-G1 phase cell population, apoptotic body formation and chromatin condensation. Western blot analysis of total cell lysates revealed that the PBHCT induced cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP). In addition, PBHCT dose-dependently increased the activity of caspase-9, caspase-3 and PARP-1. Furthermore, PBHCT reduced anti-apoptotic Bcl2, $Bcl_{xL}$ expression which contributed to the loss of mitochondrial membrane potential and the activations of caspase-9 and caspase-3. Conclusions : These findings suggest that PBHCT exerts anti-cancer effects on human neuroblastoma SH-SY5Y cells by inducing apoptotic death via down-regulation of anti-apoptotic proteins such as Bcl2 and $Bcl_{xL}$, up-regulation of pro-apoptotic proteins such as Bax, and activation of caspase cascades and PARP-1.