사람의 신경교모세포종 기원 세포에서 레스베라트롤에 의한 활성산소종 생성 증가와 MAPK 활성화를 통한 세포 사멸 효과 Resveratrol Induces Cell Death through ROS-dependent MAPK Activation in A172 Human Glioma Cells원문보기
다형성 신경교모세포종은 사람의 원발성 뇌종양 중 가장 흔하면서 악성이 높은 종양의 하나로 수술과 방사선치료, 화학치료 등 집중적 치료에도 불구하고 사망률이 높은 종양이다. 레스베라트롤은 다양한 자연산 물질에 포함되어 있는 폴리페놀의 일종으로 여러 종류의 암세포들에서 항암 작용이 있음이 보고되어 있으나 그 기전은 명확하게 밝혀져 있지 않다. 본 연구에서는 사람의 신경교모세포종 기원 세포인 A172 세포에서 레스베라트롤에 의한 세포 사멸 효과와 그 기전을 확인하고자 하였다. 레스베라트롤은 A172세포에서 활성산소종의 생성을 촉진하였으며 N-acetylcystein 혹은 catalase 등의 항산화제들을 전처치시 레스베라트롤의 세포 사멸 효과가 차단되었다. 레스베라트롤은 ERK와 p38 kinase, JNK 등의 인산화를 촉진하였으며 이들 인산화 효소의 억제제들을 전처치하면 레스베라트롤의 세포 사멸 효과가 차단되었다. Caspase 억제제를 전처치시 레스베라트롤에 의한 caspase-3의 활성화와 세포 사멸이 차단되었으며, N-acetylcystein을 전처치시 레스베라트롤에 의한 ERK의 활성화와 caspase-3의 활성화가 차단되었다. 이들 결과를 종합하면 레스베라트롤은 A172 세포에서 활성산소종의 생성을 촉진하며 이는 ERK와, p38 kinase, JNK 등의 활성화를 통해 caspase-의존성 기전으로 세포사멸을 유도하는 것으로 사료된다.
다형성 신경교모세포종은 사람의 원발성 뇌종양 중 가장 흔하면서 악성이 높은 종양의 하나로 수술과 방사선치료, 화학치료 등 집중적 치료에도 불구하고 사망률이 높은 종양이다. 레스베라트롤은 다양한 자연산 물질에 포함되어 있는 폴리페놀의 일종으로 여러 종류의 암세포들에서 항암 작용이 있음이 보고되어 있으나 그 기전은 명확하게 밝혀져 있지 않다. 본 연구에서는 사람의 신경교모세포종 기원 세포인 A172 세포에서 레스베라트롤에 의한 세포 사멸 효과와 그 기전을 확인하고자 하였다. 레스베라트롤은 A172세포에서 활성산소종의 생성을 촉진하였으며 N-acetylcystein 혹은 catalase 등의 항산화제들을 전처치시 레스베라트롤의 세포 사멸 효과가 차단되었다. 레스베라트롤은 ERK와 p38 kinase, JNK 등의 인산화를 촉진하였으며 이들 인산화 효소의 억제제들을 전처치하면 레스베라트롤의 세포 사멸 효과가 차단되었다. Caspase 억제제를 전처치시 레스베라트롤에 의한 caspase-3의 활성화와 세포 사멸이 차단되었으며, N-acetylcystein을 전처치시 레스베라트롤에 의한 ERK의 활성화와 caspase-3의 활성화가 차단되었다. 이들 결과를 종합하면 레스베라트롤은 A172 세포에서 활성산소종의 생성을 촉진하며 이는 ERK와, p38 kinase, JNK 등의 활성화를 통해 caspase-의존성 기전으로 세포사멸을 유도하는 것으로 사료된다.
Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor in humans. Despite intensive treatment, including surgery, radiation, and chemotherapy, most patients die of the disease. Although the anti-cancer activity of resveratrol has been demonstrated in various cance...
Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor in humans. Despite intensive treatment, including surgery, radiation, and chemotherapy, most patients die of the disease. Although the anti-cancer activity of resveratrol has been demonstrated in various cancer cell types, its underlying mechanism in glioma cells is not fully elucidated. The present study was undertaken to investigate the effect of resveratrol on cell viability and to determine the molecular mechanism in A172 human glioma cells. Resveratrol caused the generation of reactive oxygen species (ROS), and resveratrol-induced cell death was prevented by antioxidants (N-acetylcysteine and catalase), suggesting that an oxidative mechanism is responsible for resveratrol-induced cell death. Resveratrol-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK), and resveratrol-induced cell death were prevented by inhibitors of these kinases. Resveratrol-induced activation of caspase-3 and cell death were prevented by the caspase inhibitors. ERK activation and caspase-3 activation induced by resveratrol was blocked by N-acetylcysteine. Taken together, these results suggest that resveratrol causes a caspase-dependent cell death via activation of ERK, p38, and JNK, mediated by ROS generation, in human glioma cells.
Glioblastoma multiforme is the most common and most aggressive type of primary brain tumor in humans. Despite intensive treatment, including surgery, radiation, and chemotherapy, most patients die of the disease. Although the anti-cancer activity of resveratrol has been demonstrated in various cancer cell types, its underlying mechanism in glioma cells is not fully elucidated. The present study was undertaken to investigate the effect of resveratrol on cell viability and to determine the molecular mechanism in A172 human glioma cells. Resveratrol caused the generation of reactive oxygen species (ROS), and resveratrol-induced cell death was prevented by antioxidants (N-acetylcysteine and catalase), suggesting that an oxidative mechanism is responsible for resveratrol-induced cell death. Resveratrol-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK), and resveratrol-induced cell death were prevented by inhibitors of these kinases. Resveratrol-induced activation of caspase-3 and cell death were prevented by the caspase inhibitors. ERK activation and caspase-3 activation induced by resveratrol was blocked by N-acetylcysteine. Taken together, these results suggest that resveratrol causes a caspase-dependent cell death via activation of ERK, p38, and JNK, mediated by ROS generation, in human glioma cells.
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제안 방법
To determine whether ROS generation is involved in the resveratrol-induced cell death, the effect of antioxidants NAC and catalase on the cell viability was evaluated. The resveratrol-induced cell death was prevented by these antioxidants (Fig.
To evaluate whether activation of MAPKs is responsible for the resveratrol-induced cell death, the effect of inhibitors of these kinases on the cell death was evaluated. As shown in Fig.
To evaluate whether resveratrol induces ROS generation in A172 cells, the cells were exposed to resveratrol and changes in DCF fluorescence were measured using flow cytometry. Resveratrol caused an increase in ROS generation as evidenced by an increase in the proportion of cells with higher fluorescence intensity (Fig.
대상 데이터
A172 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained by serial passages in 75-cm2 culture flasks (Costar, Cambridge, MA, USA). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Invitrogen, Carsbad, CA, USA) containing 10% heat inactivated fetal bovine serum (Hy-Clone, Logan, UT, USA) at 37oC in humidified 95% air/5% CO2 incubator.
Resveratrol, N-acetylcysteine (NAC), catalase, Hoechst 33258, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). U0126, SB203580, SP600125, z-DEVD-FMK, and DEVD-CHO were purchased from Calbiochem (CA, USA).
Louis, MO, USA). U0126, SB203580, SP600125, z-DEVD-FMK, and DEVD-CHO were purchased from Calbiochem (CA, USA). 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) is obtained from Molecular Probes (Eugene, OR, USA).
데이터처리
t-test. Multiple group comparison was done using one-way analysis of variance followed by the Tukey post hoc test using SPSS v10.1 (SPSS Inc., Chicago, Illinois, USA). A probability level of 0.
The data are expressed as means ± SEM and the difference between two groups was evaluated by unpaired Student’s t-test. Multiple group comparison was done using one-way analysis of variance followed by the Tukey post hoc test using SPSS v10.
이론/모형
Cells were treated with various concentrations of resveratrol for 24 and 48 hr (A) and 100 μM resveratrol for various times (B). Cell viability was determined by MTT assay. Data are mean ± SEM of four independent experiments performed in duplicate.
Cells were exposed to 100 μM resveratrol for 48 hr in the presence or absence of each 10 μM of DEVD-CHO (CHO) and z-VAD-FMK (FMK). Cell viability was estimated by MTT assay. Data are mean ± SEM of five independent experiments performed in duplicate.
Cell viability was evaluated using MTT assay. After washing the cells, culture medium containing 0.
성능/효과
Cells were exposed to 100 μM resveratrol for various times. (A) Representative expression of phospho-ERK1/2 (p-ERK), phosph-p38 (p-p38), and phospho-JNK (p-JNK) was evaluated using the specific antibodies. Total ERK (t-ERK) and GAPDH were employed as a loading control.
In conclusion, the present study demonstrated that resveratrol results in human glioma cell death through a cas-pase-dependent mechanism involving up-regulation of ERK, p38, and JNK. These events are initiated by ROS generation.
후속연구
Our results suggest that resveratrol may be considered a potential candidate for induction of apoptosis in glioblastoma. Further investigation is needed to validate the contribution of resveratrol to tumor therapy in vivo.
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