• 동의생리병리학회지
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• 제23권4호
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• pp.888-894
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• 2009

It has been reported that silibinin, a natural polyphenolic flavonoid, induces cell death in various cancer cell types. However, the underlying mechanisms by which silibinin induces apoptosis in human glioma cells are poorly understood. The present study was therefore undertaken to examine the effect of silibinin on glioma cell apoptosis and to determine its underlying mechanism in human glioma cells. Apoptosis was estimated by FACS analysis. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (${\Psi}m$) were measured using fluorescence dyes DCFH-DA and $DiOC_6$(3), respectively. Cytochrome c release from mitochondria and caspase-3 activation were estimated by Western blot analysis using specific antibodies. Exposure of cells to 30 mM silibinin induced apoptosis starting at 6 h, with increasing effects after 12-48h in a time-dependent manner. Silibinin caused ROS generation and disruption of ym, which were associated with the silibinin-induced apoptosis. The silibinin-induced ROS generation and disruption in ym were prevented by inhibitors of mitochondrial electron transport chain. The hydrogen peroxide scavenger catalase blocked ROS generation and apoptosis induced by silibinin. Silibinin induced cytochrome c release into cytosolic fraction and its effect was prevented by catalase and cyclosporine A. Silibinin treatment caused caspase-3 activation, which was inhibited by DVED-CHO and cyclosporine A. Pretreatment of caspase inhibitors also protected against the silibinin-induced apoptosis. These findings indicate that ROS generation plays a critical role in the initiation of the silibinin-induced apoptotic cascade by mediation of the mitochondrial apoptotic pathway including the disruption of ${\Psi}m$, cytochrome c release, and caspase-3 activation.

• Bulletin of the Korean Chemical Society
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• 제34권2호
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• pp.406-410
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• 2013

Tetrahyropapaveroline (THP) is compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegenerative disorder, such as Parkinson's disease (PD). The aim of this study was to evaluate the potential of THP to cause oxidative damage on the structure of cytochrome c (cyt c). Our data showed that THP led to protein aggregation and the formation of carbonyl compound in protein aggregates. THP also induced the release of iron from cyt c. Reactive oxygen species (ROS) scavengers and iron specific chelator inhibited the THP-mediated cyt c modification and carbonyl compound formation. The results of this study show that ROS may play a critical role in THP-induced cyt c modification and iron releasing of cyt c. When cyt c that has been exposed to THP was subsequently analyzed by amino acid analysis, lysine, histidine and methionine residues were particularly sensitive. It is suggested that oxidative damage of cyt c by THP might induce the increase of iron content in cells and subsequently led to the deleterious condition. This mechanism is associated with the deterioration of organs under neurodegenerative disorder such as PD.

• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.225-236
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• 2021

The epipyrone (EPN) biosynthetic gene cluster of Epicoccum nigrum is composed of epnC, epnB, and epnA, which encode cytochrome P450 oxidase, glycosyltransferase, and highly reducing polyketide synthase, respectively. Gene inactivation mutants for epnA, epnB, and epnC were previously generated, and it was found that all of them were incapable of producing EPN and any of its related compounds. It was also reported that epnB inactivation abolished epnA transcription, generating ΔepnAB. This study shows that the introduction of native epnC readily restored EPN production in ΔepnC, suggesting that epnC is essential for polyketide release from EpnA and implies that EpnC works during the polyketide chain assembly of EpnA. Introduction of epnC promoter-epnA restored EPN production in ΔepnA. The ΔepnB genotype was prepared by introducing the epnA expression vector into ΔepnAB, and it was found that the resulting recombinant strain did not produce any EPN-related compounds. A canonical epnB inactivation strain was also generated by deleting its 5'-end. At the deletion point, an Aspergllus nidulans gpdA promoter was inserted to ensure the transcription of epnA, which is located downstream of epnB. Examination of the metabolite profile of the resulting ΔepnB mutant via LC-mass spectrometry verified that no EPN-related compound was produced in this strain. This substantiates that C-glycosylation by EpnB is a prerequisite for the release of EpnA-tethered product. In conclusion, it is proposed that cytochrome P450 oxidase and glycosyltransferase work in concert with polyketide synthase to generate EPN without the occurrence of any free intermediates.

• 생명과학회지
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• 제13권1호
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• pp.118-127
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• 2003

Phenylalanine의 구조유사체인 p-fluotophenylalanine (FPA)은 인체 급성백혈병세포주인 Jurkat T 세포의 세포자살을 유도한다. FPA에 의한 세포자살에 미치는 Bcl-2 또는 Bcl-xL의 영향을 조사하기 위해, Bcl-2 또는 Bcl-xL을 stable transfection하거나 empty vectors만을 Transfection한 Jurkat 세포를 이용하여 FPA의 세포독성과 FPA에 의한 세포내 세포자살 신호전달경로를 비교 분석하였다. Jurkt T 세포에 0.63∼3.0 mLf의 FPA를 처리하였을 때 세포의 생육도는 농도에 비례하여 감소하였다. 또한 세포자살관련 DNA fragmentation, caspase-8 activatoin, Bid cleavage, mitochondria로 부터의 cytochrome c 방출, caspase-9 및 -3 activation, PARP degradation 등이 유도되었다. 한편, FPA에 의해 유도되는 이러한 일련의 생화학적 현상들은 Bcl-2 또는 Bcl-xL의 overexpression에 의해 현저히 저해되었다. 이상의 결과들은 caspase-8 activation, Bid cleavage, mitochondnal cytochrome c 방출에 의해 활성화되는 casuase cascade 등의 현상이, Bcl-2 또는 Bcl-xL에 의해 억제됨을 나타내며 FPA에 의해 유도되는 세포자살에 필요한 과정임을 시사한다.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.316.2-316.2
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• 2002

Manassantin B classified into dineolignans have been isolated from Saururus chinensis Manassantin B was found to induce apoptosis in human promyelocytic leukaemia HL -60 cells with characteristic apoptotic features like increase of nucleosomalladder. apoptotic body ormation. flipping of membrane phosphatidylserine. Manassantin B induced FAS and FAS ligand expression, and activated caspase 8 which cleaved bid to tbid in cytosol. The release of cytochrome c to sytosol was accompanied with decrease of bcl-2 protein and incresase of tbid and bax protein in mitochondria. Released xytochrome c activated caspase 9 and-3. but these effects were completely attenuated by the treatment of broad caspses ingibitor. Z-VAD fmk. These results indicate that manassatin B induce apoptosis through upregulation of FAS. caspase family and mitochondria-related proteins.

• 동의생리병리학회지
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• 제22권2호
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• pp.328-332
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• 2008

The purpose of this study was to identify the protective effect of Codonopis pilosula extract on cell death induced by $H_2O_2$ in SK-N-MC neuroblastoma cells. We measured the antioxidant effect by DPPH radical scavenging analysis, BSA analyssis and examined the cell viability by crystal violet and cytochrome C, Bax, Bcl-2, p53, p21 by using Western blot analysis. Codonopis pilosula extract scavenged DPPH radical in a dose-dependent manner and shown direct free radical scavenging effect, suggested that Codonopis pilosula extract have antioxidant effect in vitro. Treatment of cells with hydrogen peroxide, a reactive oxygen species, was to induce cell death and pretreatment with Codonopis pilosula extract attenuated the occurrence of $H_2O_2-induced$ cell death. To elucidate the protective mechanisms of action of Codonopis pilosula extract, Western blot analyses for Bcl-2 and Bax expression and cytochrome c release were carried out. Pretreatment with Codonopis pilosula extract induced the expression of Bcl-2 and suppressed the release of cytochrome c and Bax into the cytosol, thereby arresting $H_2O_2-induced$ apoptotic cell death. Especially p21 and p53 were decreased prior to $H_2O_2$ treatment. These results suggest that Codonopis pilosula extract is associated with the cell cycle and anti-apoptotic cell death.

• Animal cells and systems
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• 제6권2호
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• pp.171-180
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• 2002

Nitric oxide has high affinity for iron, and thus it can cause intracellular iron loss. We tested the idea that intracellular iron can be the primary target of NO toxicity by comparing the signaling mechanisms involved in cell death caused by iron depletion and that caused by NO. Treatment of HL-60 cells with a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), decreased the intracellular iron level rapidly as that observed with the iron chelator deferoxamine (DFO). Iron chelators such as DFO and mimosine could induce death of human leukemic HL-60 cells by a mechanism requiring activation of p38 kinase, c-Jun N-terminal kinase, caspase-3 and caspase-8. DFO and SNAP also caused release of cytochrome c from mitochondria. Inhibition of p38 kinase by a selective inhibitor, SB203580, abolished the NO and DFO-induced cell death, release of cytochrome c, and activation of caspase-3 and caspase-8, thus indicating that p38 kinase lies upstream in the cell death processes. In a parallel situation, the cells that are sensitive to NO showed similar sensitivity to DFO. Moreover, simultaneous addition of ferric citrate, an iron-containing compound, inhibited the SNAP and DFO-induced activation of caspases and also blocked the NO-mediated cell cycle arrest at $G_1$ phase. Collectively, our data implicate that the NO-induced cell death of tumor cells including HL-60 cells is mediated by depletion of iron and further suggest that activation of p38 kinase lies upstream of cytochrome c release and caspase activation involved in this apoptotic process.

• Biomolecules & Therapeutics
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• 제16권1호
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• pp.1-8
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• 2008

Mitochondria are important sensor of apoptosis. $H_2O_2-induced$ cell death rate was enhanced by serum deprivation. In this study, we investigated whether serum deprivation using 0.5 or 3 % FBS induces apoptotic cell death through mitochondrial enzyme activation as compared to 10 % FBS. Apoptotic cell death was observed by chromosome condensation and the increase of sub-G0/G1 population. Serum deprivation reduced cell growth rate, which was confirmed by the decrease of S-phase population in cell cycle. Serum deprivation significantly increased caspase-9 activity and cytochrome c release from mitochondria into cytosol. Serum deprivation-induced mitochondrial changes were also indicated by the increase of ROS production and the activation of mitochondrial enzyme, succinate dehydrogenase. Mitochondrial enzyme activity increased by serum deprivation was reduced by the treatment with rotenone, mitochondrial electron transport inhibitor. In conclusion, serum deprivation induced mitochondrial apoptotic cell death through the elevation of mitochondrial changes such as ROS production, cytochrome c release and caspase-9 activation. It suggests that drug sensitivity could be enhanced by the increase of mitochondrial enzyme activity in serum-deprived condition.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.158.2-158.2
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• 2003

Vitamin K-related analogs induce growth inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1, 4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyeloid leukemic HL-60 cells. DDN induced cytochrome c release, cleavage of Bid, and activation of caspases -8, -9 and -3. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor zVAD-fmk, whereas cytochrome c release was not affected by zVAD-fmk. (omitted)

• Journal of Preventive Medicine and Public Health
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• 제39권3호
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• pp.199-204
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• 2006

Objectives: Mercury is a hazardous organ-specific environmental contaminant. It exists in a wide variety of physical and chemical states, each of which has unique characteristics for the target organ specificity. Exposure to mercury vapor and to organic mercury compounds specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds. Methods: In this study, mercury chloride $(HgCl_2)$ was studied in a renal derived cell system, i.e., the tubular epithelial Madin-Darby canine kidney (MDCK) cell line, which has specific sensitivity to the toxic effect of mercury. MDCK cells were cultured for 6-24 hr in vitro in various concentrations (0.1-100 M) of $HgCl_2$, and the markers of apoptosis or cell death were assayed, including DNA fragmentation, caspase-3 activity andwestern blotting of cytochrome c. The influence of the metal on cell proliferation and viability were evaluated by the conventional MTT test. Results: The cell viability was decreased in a time and concentration dependent fashion: decreases were noted at 6, 12 and 24 hr after $HgCl_2$, exposure. The increases of DNA fragmentation were also observed in the concentrations from 0.1 to 10 M of $HgCl_2$ at 6 hr after exposure. However, we could not observe DNA fragmentation in the concentrations more than 25 M because the cells rapidly proceeded to necrotic cell death. The activation of caspase-3 was also observed at 6 hr exposure in the $HgCl_2$ concentrations from 0.1 to 10 M. The release of cytochrome c from the mitochondria into the cytosol, which is an initiator of the activation of the caspase cascade, was also observed in the $HgCl_2-treated$ MDCK cells. Conclusions: These results suggest that the activation of caspase-3 was involved in $HgCl_2-induced$ apoptosis. The release of cytochrome c from the mitochondria into the cytosol was also observed in the $HgCl_2-treated$ MDCK cells. These findings indicate that in MDCK cells, $HgCl_2$ is a potent inducer of apoptosis via cytochrome c release from the mitochondria.