• Title/Summary/Keyword: $S_{N}2$ pathway

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Water Extract of Allium sativum L. Induces Apoptosis in Human Leukemia U937 Cells through Reactive Oxygen Species Generation (마늘 열수 추출물의 활성산소종 생성을 통한 인체백혈병세포의 apoptosis 유발)

  • Choi, Woo-Young;Chung, Kyung-Tae;Yoon, Tae-Kyung;Choi, Byung-Tae;Lee, Yong-Tae;Lee, Won-Ho;Ryu, Chung-Ho;Choi, Yung-Hyun
    • Journal of Life Science
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    • v.17 no.12
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    • pp.1709-1716
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    • 2007
  • The health benefits of garlic (Allium sativum L.) are derived from a wide variety of components and from the different ways it is administered. The known health benefits of garlic include cardiovascular protective effects, stimulation of immune function, reduction of blood glucose level, protection against microbial, viral and fungal infections, as well as anticancer effects. In the present study, it was examined the effects of water extract of A. sativum (WEAS) on the growth of cultured human tumor cells in order to investigate its anti-proliferative mechanism. Treatment of WEAS to tumor cells resulted in the growth inhibition, especially in leukemia cells, which was associated with induction of G2/M arrest of the cell cycle and apoptosis. In order to further explore the critical events leading to apoptosis in WEAS-treated U937 human leukemia cells, the following effects of WEAS on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MMP), and the expression changes of Bcl-2 and IAP family proteins. The cytotoxic effect of WEAS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in U937 cells. The WEAS-induced apoptosis in U937 cells was correlated with the generation of intracellular ROS, collapse of MMP, activation of caspase-3 and down-regulation of anti-apoptotic proteins. The quenching of ROS generation with antioxidant N-acetyl-L-cysteine conferred significant protection against WEAS-elicited ROS generation, caspase-3 activation, G2/M arrest and apoptosis. In conclusion, the present study reveals that the cellular ROS generation plays a pivotal role in the initiation of WEAS-triggered apoptotic death in U937 cells.

Anti-inflammatory Effects of Houttuynia cordata and Lespedeza cuneata on Lipopolysaccharide-stimulated RAW264.7 Cells (마우스 대식세포 RAW264.7에서 어성초와 야관문의 항염증 효과)

  • Jeong Tae Kim;Chungwook Chung;Seong Ik Park;Man Hyo Lee;Joong Hee Roh;Ho Yong Sohn;Jong Sik Kim
    • Journal of Life Science
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    • v.33 no.1
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    • pp.73-81
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    • 2023
  • In the present study, we prepared hot water extracts and the subsequent organic solvent fractions of methanol extracts of Houttuynia cordata (HC) and Lespedeza cuneata (LC), and investigated their anti-inflammatory effects on lipopolysaccharide-stimulated RAW264.7 cells. Among the treated samples, hexane, chloroform, and ethyl-acetate fractions of HC and LC inhibited nitric oxide (NO) production in a dose-dependent manner, and decreased inducible nitric oxide synthase (iNOS) protein expression. And, we analyzed the flavonoid contents of the ethyl-acetate fraction of HC and LC, and chose apigenin for the further experiments because apigenin was one of flavonoids commonly found in HC and LC. Apigenin dramatically inhibited NO production in a dose-dependent manner without affecting cell viability and decreased iNOS and cyclooxygenase-2 (COX-2) expression. In addition, apigenin suppressed the phosphorylation of p38 and Jun N-terminal kinase (JNK) indicating that apigenin exerts anti-inflammatory activity via the mitogen-activated protein kinase (MAPK) signaling pathway. Subsequently, we conducted RNA-sequencing analysis to detect differentially expressed genes upon apigenin treatment. Among the down-regulated genes, four cytokine genes (interleukin (IL)-1α, IL-1β, IL-6, and colony stimulating factor 2 (CSF2)) were selected for the further analysis, and the reduction of their expression by apigenin was confirmed with quantitative real-time polymerase chain reaction. Overall, our results suggest that Houttuynia cordata and Lespedeza cuneata have the anti-inflammatory effects and apigenin can be the one of key molecules responsible for their anti-inflammatory activities.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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The Signal Sequence of Sporulation-Specific Glucoamylase Directs the Secretion of Bacterial Endo-1,4-β-D-Glucanase in Yeast (효모에서 포자형성 특이 글루코아밀라제의 분비서열에 의한 세균 endo-1,4-β-D-glucanase의 분비)

  • Ahn, Soon-Cheol;Kim, Eun-Ju;Chun, Sung-Sik;Cho, Yong-Kweon;Moon, Ja-Young;Kang, Dae-Ook
    • Journal of Life Science
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    • v.22 no.2
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    • pp.142-147
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    • 2012
  • The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4-${\beta}$-D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

Studies on Platelet Activation of Saikosaponin Isolated From Bupleuri Radix (시호 Saponin의 혈소판 활성화 작용에 관한 연구)

  • 박영현;송민주;김남수
    • Journal of Food Hygiene and Safety
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    • v.13 no.4
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    • pp.355-359
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    • 1998
  • Platelet activation is originated by the intracellular or/and extracellular $Ca^{2+}$. Agonist-induced $Ca^{2+}$ entry through a plasma-membrane pathway has been reported repeatedly, but the mechanisms has proven harder to elucidate. Recently, a number of natural products have been isolated from medicinal plants and marine organisms and have proved to be useful chemical tools for resolving the mechanism of cellular functions. In an attempt to understand the mechanism of platelet activation in Bupleuri Radix, we have studied some aspects of the isolation of active components and their dependence of external $Ca^{2+}$> on platelet activation. Acetone extract of Bupleuri Radix has the most activity on platelet activation and it's active components were identified as saikosaponin a and d. Their optimal concentration was respectively $20\;\mu\textrm{g}/ml$ and $5\;\mu\textrm{g}/ml$ and their platelet activation was not dependent on external $Ca^{2+}$>, whereas optimal concentration of each agonist was arachidonic acid ($10\;\mu\textrm{M}$), collagen ($10\;\mu\textrm{M}/ml$), thrombin (0.1 unit/mi), PAF (5 nM), PMA ($5\;\mu\textrm{M}$), ionophore A23187 ($2\;\mu\textrm{g}$) and their dependence of external $Ca^{2+}$> on platelet activation appeared to thrombin > collagen $\geq$ PAF > PMA > arachdonic acid> ionophore A23187. These results suggest that saikosaponin is different from each agonists in the dependence of external $Ca^{2+}$ on platelet activation.

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Effects of 17-DMAG Administration on Autophagy Flux in Mouse Skeletal Muscle (17-DMAG이 마우스 골격근에서 autophagy flux에 미치는 영향)

  • Ju, Jeong-sun;Lee, Yoo-Hyun
    • Journal of Life Science
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    • v.26 no.4
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    • pp.387-397
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    • 2016
  • The purpose of this study was to determine if heat shock proteins are involved in autophagy in skeletal muscle. We used the autophagy flux strategy, which is an LC3 II/p62 turnover assay conducted with and without an autophagy inhibitor, to determine whether 17-DMAG (an Hsp90 inhibitor/Hsp72 activator) stimulates autophagy in skeletal muscle. We treated C2C12 cells with 17-DMAG (500 nM) for 24 hr with and without the autophagy inhibitor (Bafilomycin A1, 200 ng/ml), and we injected C57BL/6 mice i.p. with 17-DMAG (10 mg/kg) daily for 7 days with and without colchicine as an autophagy inhibitor (0.4 mg/kg/day, administered on the last 2 days). C2C12 myotubes and tibialis anterior muscles were harvested for analysis of mTOR-dependent autophagy signaling pathway proteins and autophagic marker proteins (p62 and LC3 II) by Western blot analysis. The blots showed that 17-DMAG upregulated hsp72 and decreased Akt protein levels and S6 phosphorylation in C2C12 cells. However, an in vitro autophagic flux assay demonstrated that 17-DMAG did not increase LC3 II and p62 protein concentrations to a greater extent than Bafilomycin A1 treatment alone. Similarly, 17-DMAG increased Hsp72 protein levels and decreased the expression of Akt and the phosphorylation of S6 in mouse skeletal muscle. However, unlike the response seen in C2C12 myotubes, the p62 protein levels were significantly decreased in 17-DMAG-treated mouse skeletal muscle (~50%; p<0.05). The LC3 II protein levels in 17-DMAG-treated mice were increased ~2-fold more when degradation was inhibited by colchicine (p<0.01). This suggests that 17-DMAG stimulates basal autophagy in skeletal muscle but is not found in C2C12 myotubes.

Calculation of Damage to Whole Crop Corn Yield by Abnormal Climate Using Machine Learning (기계학습모델을 이용한 이상기상에 따른 사일리지용 옥수수 생산량에 미치는 피해 산정)

  • Ji Yung Kim;Jae Seong Choi;Hyun Wook Jo;Moonju Kim;Byong Wan Kim;Kyung Il Sung
    • Journal of The Korean Society of Grassland and Forage Science
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    • v.43 no.1
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    • pp.11-21
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    • 2023
  • This study was conducted to estimate the damage of Whole Crop Corn (WCC; Zea Mays L.) according to abnormal climate using machine learning as the Representative Concentration Pathway (RCP) 4.5 and present the damage through mapping. The collected WCC data was 3,232. The climate data was collected from the Korea Meteorological Administration's meteorological data open portal. The machine learning model used DeepCrossing. The damage was calculated using climate data from the automated synoptic observing system (ASOS, 95 sites) by machine learning. The calculation of damage was the difference between the dry matter yield (DMY)normal and DMYabnormal. The normal climate was set as the 40-year of climate data according to the year of WCC data (1978-2017). The level of abnormal climate by temperature and precipitation was set as RCP 4.5 standard. The DMYnormal ranged from 13,845-19,347 kg/ha. The damage of WCC which was differed depending on the region and level of abnormal climate where abnormal temperature and precipitation occurred. The damage of abnormal temperature in 2050 and 2100 ranged from -263 to 360 and -1,023 to 92 kg/ha, respectively. The damage of abnormal precipitation in 2050 and 2100 was ranged from -17 to 2 and -12 to 2 kg/ha, respectively. The maximum damage was 360 kg/ha that the abnormal temperature in 2050. As the average monthly temperature increases, the DMY of WCC tends to increase. The damage calculated through the RCP 4.5 standard was presented as a mapping using QGIS. Although this study applied the scenario in which greenhouse gas reduction was carried out, additional research needs to be conducted applying an RCP scenario in which greenhouse gas reduction is not performed.

Temperament and Social Support in Relation to Hope of Institutionalized Children (시설 보호 아동의 기질과 사회적 지지가 희망에 미치는 영향)

  • Lim, Jung Ha;Moon, Chi Yun
    • Korean Journal of Childcare and Education
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    • v.8 no.2
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    • pp.63-80
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    • 2012
  • This study examined the relationship among temperament, social support and hope of institutionalized children. A total of 198 children, aged 10 to 13, recruited from 20 institutions in Seoul participated. Demographics, temperament, social support from friends, institution-family and school teacher, and hope were assessed by self-report. Results showed that in bivariate level, temperament and social support were significantly associated with level of hope. However, hierarchical multiple regressions indicated that children's hope was predicted only by age and social support from friends, institution-family and school teacher. That is, temperament was not contributed to predict hope, whilst social support contributed unique variance to the prediction of hope, controlling for age. The more social support the children felt they had, the higher levels of hope they reported. Further analyses revealed that support from friends, institution-family and school teacher were influential factors in explaining hope agency, while support from institution-family and school teacher were influential factors in explaining hope pathway of institutionalized children, controlling for age.

Suppressive Effect of Maslinic Acid on PMA-induced Protein Kinase C in Human B-Lymphoblastoid Cells

  • Mooi, Lim Yang;Yew, Wong Teck;Hsum, Yap Wei;Soo, Khoo Kong;Hoon, Lim Saw;Chieng, Yeo Chew
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.4
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    • pp.1177-1182
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    • 2012
  • Protein kinase C (PKC) has been implicated in carcinogenesis and displays variable expression profiles during cancer progression. Studies of dietary phytochemicals on cancer signalling pathway regulation have been conducted to search for potent signalling regulatory agents. The present study was designed to evaluate any suppressive effect of maslinic acid on PKC expression in human B-lymphoblastoid cells (Raji cells), and to identify the PKC isoforms expressed. Effects of maslinic acid on PKC activity were determined using a PepTag$^{(R)}$ assay for non-radioactive detection of PKC. The highest expression in Raji cells was obtained at 20 nM PMA induced for 6 hours. Suppressive effects of maslinic acid were compared with those of four PKC inhibitors (H-7, rottlerin, sphingosine, staurosporine) and two triterpenes (oleanolic acid and ursolic acid). The $IC_{50}$ values achieved for maslinic acid, staurosporine, H-7, sphingosine, rottlerin, ursolic acid and oleanolic acid were 11.52, 0.011, 0.767, 2.45, 5.46, 27.93 and $39.29\;{\mu}M$, respectively. Four PKC isoforms, PKC ${\beta}I$, ${\beta}II$, ${\delta}$, and ${\zeta}$, were identified in Raji cells via western blotting. Maslinic acid suppressed the expression of PKC ${\beta}I$, ${\delta}$, and ${\zeta}$ in a concentration-dependent manner. These preliminary results suggest promising suppressive effects of maslinic acid on PKC activity in Raji cells. Maslinic acid could be a potent cancer chemopreventive agent that may be involved in regulating many downstream signalling pathways that are activated through PKC receptors.

Toxicogenomics Study on TK6 Human Lymphoblast Cells Treated with Mitomycin C

  • Kim, Joo-Hwan;Koo, Ye-Mo;Lee, Woo-Sun;Suh, Soo-Kyung;Kang, Jin-Seok;Han, Eui-Sik;Kim, Seung-Hee;Park, Sue-N.
    • Molecular & Cellular Toxicology
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    • v.3 no.3
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    • pp.165-171
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    • 2007
  • Mitomycin C (MMC), an antitumor antibiotic isolated from Streptomyces caespitosus, is used in chemotherapy of gastric, bladder and colorectal cancer. MMC is activated in vivo to alkylate and crosslink DNA, via G-G interstrand bonds, thereby inhibiting DNA synthesis and transcription. This study investigates gene expression changes in response to MMC treatment in order to elucidate the mechanisms of MMC-induced toxicity. MMC was admistered with single dose (0.32 and 1.6 ${\mu}M$) to TK6 cells. Applied Biosystem's DNA chips were used for identifying the gene expression profile by MMC-induced toxicity. We identified up- or down-regulated 90 genes including cyclin M2, cyclin-dependent kinase inhibitor 1A (p21, cip1), programmed cell death 1, tumor necrosis factor (ligand) superfamily, member 9, et al. The regulated genes by MMC associated with the biological pathways apoptosis signaling pathway. Further characterization of these candidate markers related to the toxicity will be useful to understand the detailed mechanism of action of MMC.