• Journal of Acupuncture Research
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• 제23권3호
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• pp.177-190
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• 2006

Objectives : It has long been known about the anticancer effect of GRR-HAS, however, it has not been systemically determined the differentially regulated genes by GRR-HAS in cancer cells. The purpose of this study is to screen the GRR-HAS mediated differentially expressed genes in cancer cells such as HepG2 hepatoma cell lines. Oligonucleotide microarray and proteomic approaches were employed to screen the differential expression genes. Methods : GRR~HAS was prepared by boiling and stored at $-70^{\circ}C$ until use. Cells were treated with various concentrations of GRR-HAS (0.1, 0.5, 1.5, 10, $20mg/m{\ell}$) for 24 h. Cell toxicity was tested by MTT assay. To screen the differentially expressed genes in cancer cells, cells were treated with $1.5mg/m{\ell}$ of GRR-HAS. For oligonucleotide microarray assay, total RNA was used for gene expression analysis using oligonucleotide genechip (Human genome Ul33 Plus 2.0., Affimatrix Co.). For proteomic analysis, total protein was analyzed by 2D gel electrophoresis and Q-TOF mass spectrometer. Results : It has no cytotoxic effects on both HepG2 cells in all concentrations(0.1, 0.5, 1.5, 10,$20mg/m{\ell}$). In oligonucleotide microarray assay, the number of more than twofold differentially regulated known genes was 320 with 6 up-regulated and 314 down-regulated genes in HepG2 cells. In proteomic analysis, three spots were identified by 2D-gel electrophoresis and Q-TOF analysis. One down -regulated protein was protein disulfide isomerase and up-regulated proteins were fatty acid binding protein 1 and 14-3-3 gan1lTIa protein by $1.5mg/m{\ell}$ of CRR-HAS. Discussion : This study showed the comprehensive gene expression analysis using oligonucleotide microarray for the screening of GRR-HAS mediated differentially regulated genes. These results will provide a better application of GRR-HAS in cancer field and drug target development.

• 대한한의학방제학회지
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• 제22권1호
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• pp.151-165
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• 2014

Objectives : The aim of this study was to evaluate the efficacy of Aconiti Lateralis Preparata Radix (AP) and Acanthopanacis Cortex (AT) extracts in bone-derived adipocyte OP9 cell, osteoclast and osteoblast-like MG63 cells. Methods : MTT assay was used to evaluate the cytotoxicity of AP and AT extracts on OP9, osteoclast and MG63 cells. OP9 cells were treated with AP and AT, and the alterations in fat storage in the cells were determined by the Oil red O. To explain effects of RANKL-induced osteoclast differentiation in bone marrow macrophages, we performed the TRAP staining. The protein level of CAAAT/enhancer binding protein alpha ($C/EBP{\alpha}$) and peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) as a adipocyte differentiation marker, and adiponectin was examined using western blot in differentiated OP9 cells. Effects of related genes were confirmed by luciferase assay using reporter assay. Results : AP and AT was not toxic on OP9 and MG63 cells, but AT was a little cytotoxic to osteoclast at the dose of $100{\mu}g/m{\ell}$. They could inhibit differentiation of OP9 cells and osteoclast with results of oil red O staining and TRAP staining. By western blot, AP and AT decreased the expression of $PPAR{\gamma}$ and $C/EBP{\alpha}$ which is the key transcription factor in adipogenesis and adiponectin secretion. AT also inhibited the BMP-4 activity in luciferase assay. AP also inhibited BMP-4 and Wnt3a activity, stimulated ER-${\beta}$ activity but inhibited androgen receptor activity. Conclusions : These results show AP and AT can be useful in osteoporosis and obesity via inhibition of osteoclast and adipocyte differentiation.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• 한국미생물·생명공학회지
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• 제44권3호
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• pp.285-292
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• 2016

본 연구에서는 에탄올과 고압 열수 추출에 의해 준비된 벼세포 추출물의 창상 치유 효능 평가를 위해, 인간 섬유아세포와 각질세 포에 추출물 처리하여 세포 독성, 세포 이동능, collagen 단백질 및 유전자의 발현 양상에 대해 실험을 진행하였다. 그 결과 추출물은 처리한 범위에서 두 세포에 전혀 독성을 나타내지 않았으며, 대조구에 비해 증식을 최대 25% 촉진시키는 것으로 나타났다. 또한 인위적으로 세포에 창상을 낸 후 세포 이동능을 평가하였을 때, 비처리군에 비해 처리 농도에 비례하여 230−450% (섬유아세포), 170−350%(각질세포) 이동성이 향상되는 것을 확인할 수 있었다. 창상 치유의 중요한 역할을 하는 collagen의 유전자와 단백질 모두 추출물 처리시 농도의존적으로 증가하는 것을 확인할 수 있었다. 특히 에탄올 추출물 100 mg/ml 처리시에는 양성 대조군인 vitamin C (500 μM)와 유사한 효능을 나타내었다. 따라서 본 연구의 결과는 벼세포 추출물이 창상 치유제로서의 활용 가능성에 대해 제시하고 있다.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1097-1103
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• 2006

The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, $[B3(Fab)-toxin]_{2}$. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=l, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were $[Fab-S\;1,\;2,\;3-PE38]_{2}\;(=[Fab-SKPCIST-KAS(G_{4}S)nGGPE-PE38]_{2}\;(n=1,\;2,\;3))$, and they are derivatives of previously studied $[Fab-H2cys-PE38]_{2}\;(=[Fab-SKPCIST-KASGGPE-PE38]_{2})$. In $[Fab-Sl,\;2,\;3-PE38]_{2}$, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST- sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed $[Fab-Hlcys-PZ38]_{2}[7]$, 3-4-fold higher than $[Fab-ext-PZ38]_{2}[25]$. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of $[Fab-PE38]_{2}$ in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.

• Journal of Applied Biological Chemistry
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• 제52권2호
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• pp.45-50
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• 2009

본 연구에서는 개비자나무(Cephalotaxus koreana)를 기능성 생물소재로 개발하기 위해 개비자나무 추출물(Korean Plum-yem extract, KPE)의 항균활성, 암세포 증식 억제활성 및 독성을 확인하였고, 일반성분을 조사하였다. KPE의 농도에 따른 항균활성은 그램 양성균, 그램 음성균, 효모 및 사상균에 대하여 성장 저해현상을 나타내었으며, 특히 그램 음성균인 Pseudomonas aeruginosae가 KPE에 가장 민감하게 생육 저해현상을 보였고, 마찬가지로 그램 음성균인 Salmonella thyphimurim, Escherichia coli 순이었다. 그램 양성균이 그램 음성균에 비해 KPE에 의한 생육저해효과가 작은 것으로 나타났다. 혈액암 세포주를 이용한 KPE의 암세포 증식 억제활성을 분석한 결과 80 ${\mu}g/mL$의 농도에서 현저한 암세포 저해활성을 보였다. 또한, KPE를 섭취하였을 때 유발되는 용혈율을 측정하여 KPE의 적혈구 막지질 붕괴의 유무를 조사한 결과, KPE 중 hemolysis를 일으키는 물질은 KPE의 유효성분인 HHT 외에 다른 물질이 있는 것으로 판단되었으며, Ame's test에 의한 돌연변이원성을 갖지 않음을 확인하였다. 따라서 개비자나무 추출물은 항균과 암세포에 대한 세포독성 등 기능성을 갖는 천연물 소재로서 이를 응용한 기능성 식품 개발이 가능한 것으로 판단된다.

• 대한약리학회지
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• 제27권2호
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• pp.197-205
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• 1991

Adriamycin (Doxorubicin HCl)의 혈관밖 유출에 따른 조직의 손상, 특히 피부괴양 및 괴사 기전을 규명하기 위한 연구의 일환으로 흰쥐 피부세포를 이용한 in vitro 실험에서 adriamycin에 의한 산소라디칼 생성 및 그와 관련된 세포독성 기전으로 지질과산화를 검토하였다. Adriamycin은 흰쥐 태자 피부의 배양세포에서 lactic dehydrogenase(LDH) 유리를 용량 및 시간 의존적으로 증가 시켰으며, NADPH 및 NADH 첨가 조건에서 $superoxide\;anion(O^-\;_2{\cdot})$ 생성을 현저히 증가시켰다. Adriamycin은 지질과산화 반응의 척도인 malondialdehyde(MDA) 생성을 역시 NADPH, NADH 존재하에서 용량의존적으로 증가시켰고, 산소라디칼 제거물질들인 superoxide dismutase (SOD), catalase 및 thiourea와 항산화물질인 butylated hydroxytoluene(BHT), ${\alpha}-tocopherol$은 MDA 생성증가를 현저히 억제하였다. 1, 3,-bis(2-chloroethyl)-1-nitrosourea(BCNU)를 처리하여 산화성 공격에 대한 방어기전의 하나인 glutahione 체계를 억제할 경우 adriamycin에 의한 MDA 생성은 더욱 현저히 증가하였고, 이는 역시 항산화 물질들에 의하여 억제되었다. 이상의 연구성적에서 adriamycin은 산소라디칼 생성의 증가와 그에 따른 지질과산화를 촉진하므로서 피부세포에 손상을 줄 것으로 사료되었다.

• The Korean Journal of Physiology and Pharmacology
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• 제17권4호
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• pp.275-281
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• 2013

Astrocytes are reported to have critical functions in ischemic brain injury including protective effects against ischemia-induced neuronal dysfunction. Na-K ATPase maintains ionic gradients in astrocytes and is suggested as an indicator of ischemic injury in glial cells. Here, we examined the role of the Na-K ATPase in the pathologic process of ischemic injury of primary cultured astrocytes. Chemical ischemia was induced by sodium azide and glucose deprivation. Lactate dehydrogenase assays showed that the cytotoxic effect of chemical ischemia on astrocytes began to appear at 2 h of ischemia. The expression of Na-K ATPase ${\alpha}1$ subunit protein was increased at 2 h of chemical ischemia and was decreased at 6 h of ischemia, whereas the expression of ${\alpha}1$ subunit mRNA was not changed by chemical ischemia. Na-K ATPase activity was time-dependently decreased at 1, 3, and 6 h of chemical ischemia, whereas the enzyme activity was temporarily recovered to the control value at 2 h of chemical ischemia. Cytotoxicity at 2 h of chemical ischemia was significantly blocked by reoxygenation for 24 h following ischemia. Reoxygenation following chemical ischemia for 1 h significantly increased the activity of the Na-K ATPase, while reoxygenation following ischemia for 2 h slightly decreased the enzyme activity. These results suggest that the critical time for ischemia-induced cytotoxicity of astrocytes might be 2 h after the initiation of ischemic insult and that the increase in the expression and activity of the Na-K ATPase might play a protective role during ischemic injury of astrocytes.

• Applied Biological Chemistry
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• 제45권4호
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• pp.195-202
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• 2002

국내외에서 수집 재배한 29품종 유색미의 70% 에탄올 추출물로 세포독성, 변이원성, 화학적 직접변이원 mitomycin C, 4-nitroquinoline-N-oxide, 2,4,7-trinitro-9-fluorenone에 대한 항변이원성을 측정하였다. 세포 내재성 alkaline phosphatase활성을 지표로 유색미 추출물이 지시세포인 E. coli PQ 37의 성장에 미치는 영향을 조사한 결과 Jumlalocal, Jumlalocal-1을 포함하는 13종류의 유색미 추출물이 세포독성을 나타낸 반면 DK 1, SC-5, LK 1A-2-12-1-1및 wx 139-3-64-20-3-1등의 품종은 세포성장 촉진활성을 나타내었다. SOS chromotest 기법을 이용하여 유색미가 가지는 변이원성 및 항변이원성을 조사한 결과에서는 Jumlalocal-1, IR 17491-5-4-3-3 및 Jumlalocal 등의 품종이 변이원성을 유도하는 품종이었다. 화학적 직접변이원 mitomycin C, 2,4,7-trinitro-9-fluorenone 및 4-nitroquinoline-N-oxide 에 대해서 공통적으로 항변이원성 효과가 인정되는 유색미 품종은 LK1-3-6-12-1-1, Parnkhari 203, Jumlalocal, wx 139-3-64-20-3-1, Muthumanikam, HP 883-1-1-1-B-1-1, Jumla-local-1 등 7품종이었다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8503-8512
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• 2016

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline, WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.