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Chios gum mastic에 의한 사람구강편평상피암종세포세포주기 정지와 세포자멸사 유도에 대한 연구

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis inYD9 Human Oral Squamous Carcinoma Cells

조수현|신상훈|이승은|정진|박봉수|박혜련|김철훈|김규천|김인령|곽현호|박재현|남창옥
대한체질인류학회|2008.03|해부·생물인류학 (Anatomy & Biological Anthropology)|제21권 제1호|pp. 55-68|DOI 10.11637/aba.2008.21.1.55

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키워드

ApoptosisCell cycle arrest세포자멸사세포주기정지chios gum mastic (CGM)사람구강편평상피암종세포Chios gum mastic (CGM)Human oral squamous carcinoma cell line
주제분류해부학

초록

식물인 Pistiacia lentiscus L. var. Chia.는 그리이스 키오스 섬의 남부지방에서만 서식하며, Chios gum mastic (CGM)으로 알려져 있는 수지를 만들어 낸다. Pistacia lentiscus 나무의 줄기와 잎에서 추출한 천연물질인 CGM은 과거 수세기 동안 지중해와 중동 지역 국가들에서 음식 첨가물과 치료약으로 광범히 하게 사용되어 왔었 다. 본 연구는 사람구강편평상피암종세포(YD9 cells)에서 CGM의 세포독성과 성장억제 효과, 그리고 세포주기의 변형과 세포자멸사(apoptosis)에 대한 분자생물학적 기전을 알기 위해서 수행하였다. YD9 세포와 사람정상각화세포(HaCaT)의 생존률 측정은 MTT법을 시행하였고, YD9 세포의 성장억제를 확인하 기 위해서는 clonogenic assay를 사용하였다. 세포자멸사가 유도되는 YD9 세포를 관찰하기 위해서 hoechst 염색법 과 DNA 전기영동법을 사용하였다. 그리고 YD9 세포에 CGM을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이저주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정 등을 시행하 였다. CGM으로 처리된 YD9 세포는 용량 의존적인 세포 성장억제와 세포자멸사에 의한 세포죽음을 보였고, caspase- 9, caspase-3, PARP 그리고 DFF45 (ICAD)의 파괴와 분절의 생성, DNA의 조각남, 핵 응축, 사립체막전위의 감소, Bax와 Bcl-2의 분율의 변화, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이 동과 같은 세포자멸사 증거를 보였다. Flow cytometry 분석에서는 cyclin D1, cyclin D3, Cdk2 그리고 Cdk4의 발현 의 감소와 p21WAF1/CIP1와 p53의 발현 증가와 관계있는 것으로 보여지는 G1 세포주기 정지를 보였다. 이러한 결과는 CGM이 세포주기 관련 단백질들의 변형을 유도한 G1 세포주기정지와 사립체와 caspase 경로를 통한 세포자멸사를 유도함을 명확하게 증명하고 있다. 이와 같은 강력한 세포주기 정지와 세포자멸사 유도능은 CGM이 사람구강편평세포암종의 새로운 치료전략으로서의 가능성을 높여 준다고 생각한다.

Abstract

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21WAF1/CIP1 and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.

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