• 한국식품영양과학회지
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• 제38권12호
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• pp.1679-1684
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• 2009

본 연구에서는 천년초선인장 열매의 총 폴리페놀과 플라보노이드 함량을 분석하고, 열매의 추출물이 인간의 유방상피조직에서 유래한 암세포인 MCF-7의 증식과 세포사멸에 미치는 영향을 조사하였다. 천년초선인장 열매의 총 폴리페놀 함량은 약 4.49 g/100 g, 플라보노이드 함량은 약 1.31g/100 g로 나타났다. 열매의 물 추출물 100과 500 ${\mu}$g/mL 농도에서 정상세포인 BJ에 대한 세포독성을 나타내지 않으면서 MCF-7 세포의 살아있는 세포수를 농도 의존적으로 감소시키는 효과를 보였다. 또한 세포배양액에 열매의 물 추출물을 첨가한 경우, G1 arrest가 일어나 세포주기 진행이 지연되었으나, apoptosis에는 영향을 주지 않음이 확인되어 천년초선인장 열매 물 추출물이 apoptosis보다는 G1 arrest를 유도하여 유방암세포를 억제한다는 것을 규명하였다. 이러한 결과들은 천년초선인장 열매의 물 추출물이 세포독성에는 영향을 주지 않으면서 유방암세포의 증식과 세포주기 진행을 억제하는 효과적인 항암물질이 될 수 있는 기능성 소재로 활용 가능하다는 것을 암시한다.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• Clinical and Experimental Pediatrics
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• 제52권2호
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• pp.213-219
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• 2009

목 적:철은 세포 성장과 분화, 전자 전달 반응, 산소 전달, 해독작용 등 여러 가지 중요한 생체 반응에 반드시 필요한 요소로서 종양세포의 성장과 증식에도 절대적으로 필요하다. 최근에 철킬레이트제인 deferoxamine이 악성 구강 각질세포의 성장을 억제하고 세포자멸사를 유도하며, 난소암세포의 증식을 억제하고 세포자멸사를 유도하여 난소암의 성장을 억제하였다고 보고되었다. 그러므로 반복적인 수혈에 의하여 헤모시데린침착증이 발생한 소아 종양 환아에서 deferoxamine이 철을 제거 할 뿐만 아니라 암세포의 세포자멸사를 유도하는지에 대하여 알아보고 세포자멸사를 유도한다면 그 경로에 대하여 알아보고자 하였다. 방 법:골육종세포인 Saos-2에서 deferoxamine에 대한 효과를 알아보기 위하여 크리스탈 바이올렛과 트리판 블루 염색으로 세포의 성장 및 증식을 측정하였고, DNA 분획, 핵 응축, 세포주기분석으로 세포자멸사를 분석하였고, 세포자멸사와 관련된 분자들의 발현을 Western hybridization으로 분석하였다. 결 과:Deferoxamine은 Saos-2 세포에 대하여 시간과 농도에 의존적으로 세포 증식 억제 효과를 나타내었다. 이러한 세포 증식 억제 효과는 DNA 단편화, 핵 응축, 세포주기 분석에서의 $A_{0}$ 기의 증가, PARP의 활성도의 증가 등 세포자멸사가 유도되었음을 알 수 있었다. 또한 Saos-2 세포에서 deferoxamine 처리 후 Akt/PKB의 활성화가 억제되어 caspase 9의 활성화, 그 하류의 caspase 3의 활성화로 이어지는 미토콘드리아 매개되는 경로로 세포자멸사가 유도되었다. 결 론:결론적으로 철킬레이트제인 deferoxamine이 세포증식을 억제시키고 세포자멸사를 유도시킴으로써 골육종 세포의 증식을 억제하는 것을 보여주었다. 따라서 반복적 대량 수혈에 의한 철 과부하에 따른 장기손상이 우려되는 각종 소아 종양환자들에서 deferoxamine은 체내 축적된 철을 제거할 뿐만 아니라 종양 환자의 치료에 있어서 항암제 치료의 효과를 증가시킬 수 있는 새로운 치료법으로 개발될 수 있을 것이다.

• Clinical and Experimental Pediatrics
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• 제52권12호
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• pp.1337-1347
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• 2009

목 적:타우린은 술폰 기를 산기로 하는 황 아미노산의 일종이며 뇌, 망막, 심장, 근육에 많이 분포되어 있다. 최근 국소적 뇌허혈에 대한 타우린의 신경보호효과에 관한 연구들이 발표되고 있으나 대부분 연구가 성인의 뇌졸중의 치료에 대한 연구이며 신생아시기의 저산소성 손상에 대한 효과를 구체적으로 연구한 바가 없다. 본 연구에서는 타우린이 저산소 상태로 유발된 뇌세포 배양과 신생 백서의 저산소성 허혈성 뇌손상에서 항세포사멸사를 통한 뇌보호 효과가 있는지를 알아보고자 실험하였다. 방 법:재태기간 18일된 태아 흰쥐의 대뇌피질 세포를 배양하여 1% $O_2$ 배양기에서 저산소 상태로 뇌세포 손상을 유도하여 저산소군, 손상 전 후 타우린 투여군($30{\mu}g/mL$)으로 나누어 정상산소군과 비교하였다. 세포사멸사와 관련을 알아보기 위해 Bcl-2, Bax, caspase-3 primer와 항체로 실시간 중합효소연쇄반응과 western blotting을 하였다. 또한, 생후 7일된 백서의 좌측 총 경동맥을 결찰한 후 저산소(8% $O_2$) 상태로 2시간 노출시켜서, 저산소성 허혈성 뇌 손상을 유발하였고, 뇌손상 전 후 30분에 타우린을 체중 kg당 30 mg을 투여하였다. 저산소성 허혈성 뇌손상 후 1일, 3일, 1주, 2주, 4주 째 뇌를 적출하여 Bcl-2, Bax, caspase-3 primer를 이용하여 실시간 중합효소연쇄반응을 하였고, 동일 항체로 western blotting하였다. 결 과:저산소로 유발된 뇌세포 배양에서 정상군에 비해 저산소군에서 뇌세포 손상이 많았고 저산소 손상전 타우린 투여군에서 뇌세포 손상이 회복되었으며 저산소 손상 후 타우린 투여군에서는 저산소 손상 전 투여군보다 회복력이 떨어졌다. 실시간 중합효소연쇄반응과 western blotting을 이용한 저산소 상태의 태아 백서 뇌세포 배양 실험뿐만 아니라 저산소성 허혈성 뇌손상 동물 모델에서도 타우린을 투여한 경우 Bcl-2의 발현은 증가하고, Bax/Bcl-2의 비율, Bax와 caspase-3의 발현은 감소함을 보였다. 결 론:본 연구에서 타우린은 주산기 저산소성 허혈성 뇌손상에서 Bcl-2 발현 감소, Bax와 caspase-3 발현 증가를 유발시켜 항 세포사멸사 기전을 통한 신경보호 역할을 하는 것을 알 수 있었다. 그리고 이것은 저산소 손상 후 1주와 2주째에 가장 효과가 있었다.

• 대한한의학회지
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• 제35권4호
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• pp.10-23
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• 2014

Objectives: This study evaluated the effects of Guibi-tang (GBT) on neuronal apoptosis and cognitive impairment induced by beta amyloid ($A{\beta}$), (1-42) injection in the hippocampus of ICR mice. Methods: $A{\beta}$ (1-42) was injected unilaterally into the lateral ventricle using a Hamilton syringe and micropump ($2{\mu}g/3{\mu}{\ell}$, $0.6{\mu}{\ell}/min$). Water extract of GBT was administered orally once a day (500 mg/kg) for 3 weeks after the $A{\beta}$ (1-42) injection. Acquisition of learning and retention of memory were tested using the Morris water maze. Neuronal damage and $A{\beta}$ accumulation in the hippocampus was observed using cresyl violet and Congo red staining. The anti-apoptotic effect of GBT was evaluated using TUNEL labeling in the hippocampus. Results: GBT significantly shortened the escape latencies during acquisition training trials. GBT significantly increased the number of target headings to the platform site, the swimming time spent in the target quadrant, and significantly shortened the time for the 1st target heading during the retention test trial. GBT significantly attenuated the reduction in thickness and number of CA1 neurons, and $A{\beta}$ accumulation in the hippocampus produced by $A{\beta}$ (1-42) injection. GBT significantly reduced the number of TUNEL-labeled neurons in the hippocampus. Conclusion: These results suggest that GBT improved cognitive impairment by reducing neuronal apoptosis and $A{\beta}$ accumulation in the hippocampus. GBT may be a beneficial herbal formulation in treating cognitive impairment including Alzheimer's disease.

• 한국식품영양과학회지
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• 제44권9호
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• pp.1270-1278
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• 2015

시료 된장의 pH, 아미노태, 암모니아태 수치는 각 군 간 특별한 차이는 보이지 않았다. 검정콩 된장이 가장 높은 폴리페놀 농도, DPPH를 이용한 항산화 효과를 보였다. 된장 추출물은 0.1~0.5 mg/mL 범위까지는 RAW 264.7 cells의 증식을 억제하지 않았으며, HT-29를 이용한 MTT에서 BD 군이 가장 높은 암세포 성장 억제율을 보였다. HT-29에서 pro-inflammatory cytokine인 $TNF-{\alpha}$, IL-6와 염증관련 인자 COX-2의 mRNA 발현은 시료 처리군에서 대조군에 비해 낮은 수치를 나타냈으며 BD군에서 가장 낮은 수치를 보였다(P<0.05). 세포 증식에 관여하는 p21, p53과 cyclin D1의 mRNA 발현은 p21과 p53가 BD군에서 발현이 증가하였고, cyclin D1은 BD군에서 낮아졌다. Apoptosis 관련 유전자인 Bcl-2는 BD군이 가장 낮은 발현을 보였다. 이상의 결과로 BD군은 CD, SD군에 비해 높은 폴리페놀 농도, 항산화 효과, 대장암세포에서 pro-inflammatory cytokines과 세포 증식에 관여하는 유전자 등을 조절한다. 이 결과는 아마도 서목태 된장의 높은 총 페놀화합물의 양과 안토시아닌의 함량으로 얻어진 결과로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.926-936
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• 2003

Overexpression of human Bcl-2 protein in recombinant Chinese hamster ovary (rCHO) cells producing humanized antibody (SH2-0.32) considerably suppressed sodium butyrate (NaBu)-induced apoptosis during batch culture by using commercially available serum-free medium, which extended the culture longevity. Due to the extended culture longevity provided by the anti-apoptotic effect of Bcl-2 overexpression, the final antibody concentration of 14C6-bcl-2 culture (Bcl-2 high producer, $23\;\mu\textrm{g}\;ml^{-1}$) was 2 times higher than that of the $SH2-0.32-{\Delta}bcl-2$ culture (cells transfected with bcl-2-deficient plasmid, $10.5\;\mu\textrm{g}\;ml^{-1}$) in the presence of NaBu. To determine the effect of NaBu/Bcl-2 overexpression on the molecular integrity of protein products, antibodies purified from 14C6-bcl-2 and $SH2-0.32-{\Delta}bcl-2$ cultures in the presence of NaBu were characterized by using various molecular assay systems. For comparison, antibody purified from the parental rCHO cell culture (SH2-0.32) in the absence of NaBu was also characterized. No significant changes in molecular weight of antibodies could be observed by SDS-PAGE. From GlycoSep-N column analysis, it was found that the core oligosaccharide structure ($GlcNAc_2Man_3GlcNAc_2$) was not affected by NaBu/Bcl-2 overexpression, while the microheterogeneity of N-linked oligosaccharide structure was slightly affected. Compared with the antibody produced in the absence of NaBu, the proportion of neutral oligosaccharides was increased from 10% (14C6-bcl-2) to 16% ($SH2-0.32-{\Delta}bcl-2$) in the presence of NaBu, which was accompanied by the reduced proportion of acidic oligosaccharides, especially of monosialylated and disialylated forms. The changes in microheterogeneous oligoformal structures of antibody in turn affected the mobility of antibody isoforms in isoelectric focusing (IEF), resulting in the occurrence of some more basic antibody isoforms produced in the presence of NaBu. However, the antigen-antibody binding properties were not changed by alteration of glycosylation pattern. The competitive enzyme-linked immunosorbent assay (ELISA) showed that the antibody produced by NaBu/Bcl-2 overexpression maintained its antigen-antibody binding properties with binding affinity of about $2.5{\times}10^9{\;}M^{-1}$. Taken together, no significant effects of NaBu/Bcl-2 overexpression on the molecular integrity of antibodies, produced by using serum-free medium, could be observed by the molecular assay systems.

• Tuberculosis and Respiratory Diseases
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• 제75권1호
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• pp.9-17
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• 2013

Background: In cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells. Methods: NCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control. Results: Monensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-$G_1$ phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process. Conclusion: Our findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

• 한국안광학회지
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• 제13권3호
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• pp.95-101
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• 2008

목적: 활성산소종의 하나인 $H_2O_2$는 DNA, RNA에 직접적인 손상을 주어 유전자를 변형시키고 세포고사를 일으켜 각종 질환을 일으키는 발병인자로 알려져 있다. 본 연구는 결막 세포주에서 일어나는 $H_2O_2$에 의한 세포고사에서 EGCG의 보호효과를 알아보고자 실험하였다. 방법: 세포의 생존율은 MTT assay로 확인하였고, 세포고사의 형태적인 특징은 DNA 단절화을 통해 확인하였으며, EGCG의 자유라디칼 제거능력을 측정하기 위해 DPPH free radical 소거능을 실시하고, 세포내 활성산소량의 평가를 위한 DCFH-DA assay를 시행하였다. 세포내의 mRNA 발현은 RTPCR을 시행하여 밴드 양상을 확인하였다. 결과: 세포생존율과 프리라디칼 소거능은 EGCG의 처리 농도 의존적으로 증가하였고, DNA 분절화와 세포내 활성산소량은 감소하였다. mRNA 합성에서 bcl-2, bcl-xL의 발현은 증가하였고, bax 발현은 감소되었다. 결론: EGCG는 $H_2O_2$로 세포고사를 유도한 결막 세포주에서 항산화효과를 나타냈으며, EGCG가 anti-apoptosis 관련한 mRNA의 발현을 증가시켜 결막 세포주의 산화적 손상을 억제함으로서 세포보호효과를 나타냈음을 확인할 수 있었다.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2000년도 International Meeting 2000
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• pp.23-36
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• 2000

Japanese encephalitis virus (JEV) is the causative agent of a mosquito-borne encephalitis and is transmitted to human via persistently infected mosquito vectors. Although the virus is known to cause only acute infection, there were reports that showed neurological sequelae, latent infection in peripheral mononuclear cells, and recurrence of the disease after acute encephalitis. Innate resistance of certain cell lines, abnormal SN1 expression of the virus, and anti-apoptotic effect of cullular bcl-2 have been suggested as probable causes of JEV persistence even in the absence of defective interfering (DI) particles. Although possible involvement of DI particles in JEV persistence was suggested, neither has a direct evidence for DI presence nor its molecular characterization been made. Two questions asked in this study are whether the DI virus plays any role in JEV persistent infection if it is associated with and what type of change(s) can be made in persistently infected cells to avoid apoptosis even with the continuous virus replication, DI-free standard stock of JEV was infected in BHK-21, Vero, and SW13 cells and serial high multiplicity passages were performed in order to generate DI particles. There different-sized DI RNA species which were defective in both structural and nonstructural protein coding genes. Rescued ORFs of the DI genome maintained in-frame and the presence of replicative intermediate or replicative form RNA of the DI particles confirmed their replication competence. On the other hand, several clones with JEV persistent infection were established from the cells survived acute infections during the passages. Timing of the DI virus generation during the passages seemed coincide to the appearance of persistently infected cells. The DI RNAs were identified in most of persistently infected cells and were observed throughout the cell maintenance. One of the cloned cell line maintained the viral persistence without DI RNA coreplication. The cells with viral persistence released the reduced but continuous infectious JEV particle for up to 9 months and were refractory to homologous virus superinfection but not to heterologous challenges. Unlike the cells with acute infection these cells were devoid of characteristic DNA fragmentation and JEV-induced apoptosis with or without homologous superinfection. Therefore, the DI RNA generated during JEV undiluted serial passage on mammalian cells was shown to be biologically active and it seemed to be responsible, at least in part, for the establishment and maintenance of the JEV persistence in mammalian cells. Viral persistence without DI RNA coreplication, as in one of the cell clones, supports that JEV persistent infection could be maintained with or without the presence of DI particles. In addition, the fact that the cells with JEV persistence were resistant against homologous virus superinfection, but not against heterologous one, suggests that different viruses have their own and independent pathway for cytopathogenesis even if viral cytopathic effect could be converged to an apoptosis after all.