• Journal of Acupuncture Research
• /
• v.25 no.2
• /
• pp.59-74
• /
• 2008

목적 : 이 연구는 봉약침의 봉독과 그 주요성분인 멜리틴이 $NF-{\kappa}B$의 활성억제와 세포자멸사 관련 단백질의 발현 조절을 통하여 세포자멸사를 유도하고 전립선 암세포주인 LNCaP 세포의 성장을 억제하는지를 확인하고 해당 기전을 살펴보고자 하였다. 방법 : 봉독이나 멜리틴을 처리한 후 LNCaP의 성장억제를 관찰하기 위해 WST-1 assay, CCK-8 assay를 시행하였고, 세포자멸사의 관찰에는 DAPI, TUNEL staining assay를 시행하였으며, 세포자멸사 조절단백질의 변동 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 EMSA를 시행하였으며, LNCaP에서 봉독이나 멜리틴과 $NF-{\kappa}B$의 상호작용을 관찰하기 위해 transient transfection assay를 시행 시 세포생존율과 $NF-{\kappa}B$의 활성 변동을 측정하였다. 결과 : LNCaP 세포에 봉독이나 멜리틴을 처리한 후, 전립선암세포의 성장, 세포자멸사의 유발, 세포자멸사 관련 단백질의 발현, $NF-{\kappa}B$의 활성, $NF-{\kappa}B$의 p50 치환 후 $NF-{\kappa}B$의 활성과 LNCaP 세포 증식에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. LNCaP 세포에서 봉독이나 멜리틴을 처리한 후 세포자멸사가 유도되어 세포성장이 억제되었고, 세포자멸사 관련 단백질 중 분리된 PARP, caspase-9, Bax는 유의한 증가를, Bcl-2, p-Akt, MMP 13, XIAP, cXIAP는 유의한 감소를 나타내었다. 2. LNCaP 세포에서 봉독이나 멜리틴을 처리한 후 $NF-{\kappa}B$의 활성의 유의한 감소를 나타내었다. 3. LNCaP 세포에서 $NF-{\kappa}B$ p50를 치환하여 작용기를 없애고 봉독이나 멜리틴을 처리하였을 경우에도 $NF-{\kappa}B$의 활성의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 봉독이나 멜리틴이 $NF-{\kappa}B$의 활성 억제를 통하여 인간 전립선암세포주인 LNCaP의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것으로 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 도움이 될 것으로 기대된다. 다만 그 기전에서 봉독이나 멜리틴은 기존연구와 달리 $NF-{\kappa}B$ p50의 작용기와 직접적으로 상호작용을 하지는 않는 것으로 보이므로 심화 연구를 요한다.

• The Journal of Korean Obstetrics and Gynecology
• /
• v.21 no.4
• /
• pp.90-103
• /
• 2008

목적 : 본 연구는 현호색의 메탄올 추출물(Corydalis Tuber Extract:CTE)이 인간 유방암 세포인 MCF-7 세포의 자멸사에 미치는 영향을 알아보고자 하였다. 방법 : 현호색 메탄올 추출물로 인간유방암에서 유래된 MCF-7 세포를 처리하였다. CTE의 세포자멸사 유도 효과는 MTT, FACS, TUNEL, DNA laddering and immunoblot assay를 통하여 측정하였다. 결과: 본 연구에서 현호색 메탄올 추출물은 MCF-7 세포의 활성을 감소시켰다. CTE로 처리한 MCF-7 세포는 용량에 따라 세포 자멸사 과정이 증가했다. CTE에 노출하였을 때 Bcl-2와 $Bcl-_{XL}$의 발현을 억제하고, 미토콘드리아로부터 세포질로 cytochrome-c를 방출하며, caspase와 PARP의 절단을 유발하여 세포자멸사가 증가했다. 현호색 메탄올 추출물에 존재하는 성분중에서 coptisine이 세포 활성도를 효과적으로 감소시킨 것으로 사료된다. 결론: 실험결과 현호색 메탄올 추출물이 유방암의 임상 치료에 있어 가능성 있는 치료 방법이 될 수 있을 것으로 사료된다.

• Journal of Acupuncture Research
• /
• v.26 no.3
• /
• pp.39-48
• /
• 2009

목적 : 이 연구는 봉약침의 주요성분인 멜리틴이 NF-${\kappa}$B의 활성억제를 통하여 세포자멸사를 유도하고, 전립선 암세포주인 DU-145 세포의 성장을 억제하는지를 확인하고 멜리틴의 NF-${\kappa}$B 활성억제기전을 살펴보고자 하였다. 방법 : 멜리틴을 처리한 후 DU-145의 성장억제를 관찰하기 위해 WST-1 assay를 시행하였고, 세포자멸 사의 관찰에는 DAPI stairung assay를 통한 세포형태관찰을 시행하였으며, 염증관련유전자 발현 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 NF-${\kappa}$B의 활성 변화를 관찰하기 위해 EMSA와 luciferase assay를 시행하였으며, DU-145에서 멜리틴과 NF-${\kappa}$B의 상호작용을 관찰하기 위해 transient transfection assay를 시행 시 세포생존율과 NF-${\kappa}$B의 활성 변동을 측정하였다. 결과 : DU-145 세포에 멜리틴을 처리한 후, 전립선암세포의 성장, 세포자멸사의 유발, 염중관련유전자 발현 및 NF-${\kappa}$B의 활성, NF-${\kappa}$B의 p50 치환 후 NF-${\kappa}$B의 활성과 DU-145 세포 증식에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. DU-145 세포에서 멜리틴을 처리한 후 세포자멸사가 유도되어 세포성장이 억제되었다. 2. DU-145 세포에서 멜리틴을 처리한 후 염증관련유전자 발현 및 NF-${\kappa}$B의 활성에 유의한 감소를 나타내었다. 3. DU-145 세포에서 NF-${\kappa}$B의 p50와 IKK들을 치환하여 작용기를 없애고 멜리틴을 처리하였을 경우에도 세포활성 및 NF-${\kappa}$B의 활성의 유의한 감소를 나타내었다.

• Journal of Chest Surgery
• /
• v.34 no.6
• /
• pp.447-453
• /
• 2001

Background: Although pulmonary resection is the standard approach for the management of pulmonary metastases from soft tissue sarcoma, most of them are unresectable and chemotherapy remains the only option. The effectiveness of the cytotoxic drugs may be limited by the toxicities that occur before the therapeutic dose is reached. The regional administration of doxorubicin using pulmonary arterial perfusion in a rodent model can produce 10 to 25 times higher concentrations in the lung than systemic administration with minimal systemic toxicities. However, it is unclear whether a high concentration of doxorubicin has beneficial effects for killing cancer cells. Material and Method: We studied this to evaluate the dose-dependent cytotoxic and apoptotic effects of doxorubicin on methylcholanthrene-induced rat fibrosarcoma(MCA) cells. This study examined the cytotoxicity and apoptosis-related gene expressions(Fas, FasL, Bax, caspase 1, caspase 2, caspase 8, Bcl-2, Bcl-xL, Bcl-xS) in MCA cells after 24 hours exposure to various concentrations of doxorubicin such as 1, 5, 10, 50, and 100 $\mu$M. Result: Dose-dependent cytotoxicity was observed after 24 hours exposure to doxorubicin. However, peak apoptosis after 24 hours exposure was observed at 5 $\mu$M of doxorubicin. Above 5 $\mu$M, apoptotic activity was decreased with dose-increment. All mRNA levels of apoptosis-related genes after 24 hours exposure were up-regulated above the control level at 1 $\mu$M of doxorubicin and then decreased by doxorubicin dose-increment except caspase 8, which showed higher levels than the control level at 5 $\mu$M. Apoptosis-related protein levels were highest at 1 $\mu$M of doxorubicin and then decreased by doxorubicin dose-increment. However, Bax and Bcl-xL proteins steadily showed higher levels than the control throughout the different concentrations of doxorubicin. Conclusion: These results suggest that apoptosis is the main cytotoxic mechanism in low concentrations of doxorubicin in MCA cells and apoptosis-related genes, such as Bax, caspase 8, and Bcl-xL, are involved. At high concentrations, doxorubicin still can kill MCA cells, even when apoptosis is inhibited, and have its propriety for achieving much cytotoxicity against MCA cells.

• Journal of Gastric Cancer
• /
• v.6 no.2
• /
• pp.91-96
• /
• 2006

Purpose: Cancer is a genetic disease caused by alterations in key regulators of cell growth and cell turnover, We investigated apoptotic cell death and cell proliferation in gastric adenomas and adenocarcinomas. Materials and Methods: The TdT-mediated dUTP-biotin nick end labelling (TUNEL) method and immunohistochemistry for Ki-67 were peformed, using paraffin-embedded tissues of 41 gastric adenomas and 100 gastric adenocarcinomas. These results were compared with histopathologic parameters. Results: The Ki-67 labelling index was higher in adenocarcinomas than in adenomas and the apoptotic index was higher in adenomas than in adenocarcinomas. There were no significant difference between the apoptotic index/Ki-67 labelling index and clinicopathological parameters. Conclusion: We propose that cell proliferation is more closely associated with gastric adenocarcinomas than apoptosis is, but that neither has any clinical significance as a prognostic factor in gastric adenocarcinomas. (J Korean Gastric Cancer Assoc 2006;6:91-96)

• Korean Journal of Clinical Laboratory Science
• /
• v.39 no.2
• /
• pp.68-75
• /
• 2007

After reports on regression of cancer in humans and animals infected with microbial pathogens date back more than 100 years, much effort has been spent over the years in developing a wild type or attenuated bacterial and purified bacterial proteins for the treatment of cancer. Pseudomonas aeruginosa exotoxin A (ETA) is known to inhibit cell growth and trigger significant cell death in various cancer cells. Although ETA induces apoptosis of cancer cells, its exact mechanism of action is not known yet. Four different assays were performed in this study: morphological assessment of apoptotic cells, cell cytotoxity, annexin-V binding assay, and cell cycle analysis. The proliferation and survival of the K-562 cells treated with ETA were decreased in a dose dependent manner. In addition, the apoptotic body of K-562 cells was induced by ETA treatment in a dose dependent manner. The ETA-induced apoptosis was confirmed by annexin-V binding assay. Flow cytometric analysis was examined to ascertain whether ETA could arrest the cell cycle at the sub-G1 phase. Our results suggest that P. aeruginosa ETA inhibits cell growth and induces apoptosis in human leukemia K-562 cells.

• Applied Microscopy
• /
• v.40 no.2
• /
• pp.81-88
• /
• 2010

The fine structural characteristics of the apoptotic cells in the cutaneous epithelium of the anuran tadpole of the black-spotted frog, Rana nigromaculata was examined using the TUNEL (Terminal deoxynucleotidyl transferase-mediated biotinylated d-Uridine triphosphate Nick End Labeling) staining technique and TEM (transmission electron microscopy) observations. The cutaneous epithelium of the tadpole was composed of stratified cuboidal cells and the apoptotic cell death was observed continuously during the tail degeneration stages from the Shumway stage number 31 to 33. The early apoptotic cells shown in the epithelium demonstrated condensation and margination of the chromatin material at the nuclear periphery, and nuclear breakdown and cytoplasmic condensation were followed. Subsequent cytoplasmic degeneration of the apoptotic cell were produced by membrane-bounded cell fragments with relatively well preserved organelles. Following the processes of autophagic degradation, the late apoptotic cells being phagocytosed by other surrounding cells. These nearby cells, presumptive intraepithelial macrophages, contain a variety of lysosomal residual bodies which fuses with other cell organelles or other cytoplasmatic material to form secondary lysosomes. They are soon transformed into lamellar shaped vesicles and finally disappeared during the process of degradation.

• Proceedings of the KSAR Conference
• /
• 2001.10a
• /
• pp.32-36
• /
• 2001

난소의 재형성 과정은 난소 내 여러 조절인자들에 의해 조절되는 성장 및 퇴행 과정을 반복하는 특징을 가지고 있다. 황체는 주기적 성장과 퇴행을 보이며 과립세포의 세포자멸사 (apoptosis)를 통해 황체의 퇴행이 일어나게 된다지. 이러한 세포자멸사 과정은 난소의 정상 생리에 매우 중요하다. ATP 는 자율신경으로부터 세포외 유출을 통해 분비되어 근육 수축, 신경전달체계, 외분비 및 내분비 호르몬의 분비, 면역반응, 염증, 혈소판 응집, 동통 및 심장기능의 조절 등 매우 다양한 생물학적 기능에 영향을 미친다. 이러한 작용은 세포 표변에 존재하는 purinoceptor를 통해 이루어지는 것으로 알려져 있다. ATP는 일반적으로 세포 내에서는 에너지원으로서 작용하나 세포외부에 존재하는 ATP의 경우에는 조절물질로 작용하여 어떤 세포에 있어서는 세포용해를 일으키기도 하며, 어떤 세포에서는 세포자멸사를 유발하기도 한다. 세포 내에 존재하는 ATP는 세포의 주요한 에너지원으로 사용되며 살아있는 세포에서는 세포막을 통과하지 못하는 반면 세포 외에 존재하는 ATP는 말초신경계 혹은 중추신경계에 있어서 매우 중요한 신경전달물질로 작용하고 있다. (중략)

• The Journal of the Korean bone and joint tumor society
• /
• v.13 no.2
• /
• pp.88-95
• /
• 2007

Purpose: The purpose of this study was to compare the proapoptotic effects of matrix metal-loproteinase inhibitor (MMPI) and doxorubicin on wild-type p53 osteosarcoma cell line, socalled U2OS cell line. Materials and Methods: U2OS cells were treated with MMP inhibitor III (MMPI III) and doxorubicin, either respectively or simultaneously. In cells treated with doxorubicin, Fas-neutralizing antibody so called ZB4 was additionally treated to examine whether the doxorubicin played a role through the Fas/FasL pathway. Cells were analysed regarding to apoptosis and cell death by flow cytometry. Results: U2OS cells incubated with doxorubicin showed significant amount of cell death in dose-dependent manner. However, those incubated with MMPI III mostly remained viable state. In addition, there is no relationship between two drugs. Cells treated with doxorubicin and ZB4 at the same time did not show down regulation of apoptosis through inhibition of Fas/FasL pathway. Conclusion: It is important to re-examine MMP inhibitor's effect on other osteosarcoma cell line with wild-type p14 as well as wild-type p53 to evaluate its proapoptotic effect.

• Clinical and Experimental Pediatrics
• /
• v.46 no.7
• /
• pp.679-686
• /
• 2003

Purpose : Ataxia telangiectasia mutated(ATM) is involved in DNA damage responses at different cell cycle checkpoints, and signalling pathways associated with regulation of apoptosis in response to ionizing radiation(IR). However, the signaling pathway that underlies IR-induced apoptosis in ATM cells has remained unknown. The purpose of this study was, therefore, to investigate the apoptotic pathway that underlies IR-induced apoptosis in a CT-26 cells expressing dominant negative ATM (DN-ATM). Methods : We generated a replication-deficient recombinant adenovirus encoding the DN-ATM(Ad/DN-ATM) or control adenovirus encoding no transgene(Ad/GFP) and infected adenovirus to CT-26 cells. After infection, we examined apoptosis and apoptotic pathway by [$^3H$]-thymidine assay, DNA fragmentation, and Western immunoblot analysis. Results : DN-ATM gene served as the creation of AT phenotype in a CT-26 cells as revealed by decreased cell proliferations following IR. In addition, IR-induced apoptosis was regulated through the reduced levels of the anti-apoptotic protein Bcl-2, the increased levels of the apoptotic protein Bax, and the activation of caspase-9, caspase-3, and PARP. Conclusion : These results indicate that the pathway of IR-induced apoptosis in CT-26 cells expressing DN-ATM is mediated by mitochondrial signaling pathway involving the activation of caspase 9, caspase 3, and PARP.