Mechanism Underlying NaF-induced Apoptosis and Cell Cycle Arrest on G361 Human Melanoma Cell Line
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불화나트륨(sodium fluoride, NaF)은 치아우식증을 예방하기 위한 치과용 물질로 널리 사용되어 왔다. 치아우식증 예방을 위한 불소의 장기간 섭취가 인체에 미치는 영향에 대해 알려진 것이 거의 없고, 과복용 시심각하고 치명적인 독성을 일으킬 수 있다는 보고가 있으나 불소의 안전성은 여전히 논쟁요소로 남아 있다. 그러나 불소는 구강건강을 위한 주요 물질로서 인정받고 있는 것이 사실이다. NaF은 몇 가지의 정상세포들에서 세포자멸사를 유도하고, 얼마간의 암세포에서 세포주기 정지와 세포자멸사를 유도한다고 알려져 있다. 이 연구의 목표는 사람구강편평상피세포암종세포(G361 세포)에 NaF 적용 후, 세포자멸사의 유도와 세포자멸사에 대한 분자생물학적 기전을 밝히는 것이다. 사람흑색종세포의 생존율은 MTT법으로, G361 세포의 성장억제능의 측정은 clonogenic assay를 사용하였다. NaF 적용시, G361 세포에서 세포자멸사가 유도되는 것을 확인하기 위해서 Hemacolor 염색법, Hoechst 염색법,DNA 전기영동법 및 TUNEL법을 사용하였다. 그리고 G361 세포에 NaF을 적용한 후, Western blot 분석, 세포면역화학염색, 공초점레이져주사현미경 검경, FACScan flow cytometry, 사립체막 전위변화, proteasome 활성도 측정등을 시행하였다. NaF로 처리된 G361 세포는 시간 및 용량의존적인 세포생존율 감소, 용량의존적인 성장억제 및 세포자멸사에의한 세포죽음을 보였다. 그리고 NaF가 적용된 G361 세포에서 핵농축, DNA 조각남, 사립체막전위와 proteasome 활성도의 감소, DNA양의 감소, cytochrome c의 사립체에서의 세포질로의 유리, AIF와 DFF40 (CAD)의 핵으로의 이동, Bax와 Bcl-2의 분율 변화, 그리고 caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C, DFF45(ICAD)의 활성화와 같은 다양한 세포자멸사 증거를 보였다. 더 나아가서 NaF에 처리된 G361 세포에서 G1 세포주기에 관계하는 Cyclin D1, Cyclin D3, Cdk2 그리고 Cdk4의 발현의 감소와 p53의 탁월한 발현의 증가를 보였다. 이 연구를 통하여 NaF가 G1 세포주기정지 단백질의 변형 그리고 proteasome, 사립체 및 caspase 경로의 연속반응을 통해 G361 세포의 세포자멸사를 유도함을 명확하게 증명하였다.
Abstract
Fluoride is widely used in dentistry to prevent dental caries, even though the safety of fluoride is a controversial issue. There are no known adverse effects of long-term fluoride ingestion for caries prevention, but an overdose can cause serious acute toxicity. Nevertheless it is accepted that fluoride is an important material for oral health. This study was undertaken to investigate the modulation of cell cycle-related proteins and apoptosis induction underlying mechanism by NaF treatment on G361 human melanoma cell line. The viability of G361 cells and the growth inhibition of G361 cells were assessed by MTT assay and clonogenic assay respectively. Hoechst staining, DNA electrophoresis and TUNEL staining were conducted to observe G361cells undergoing apoptosis. G361 cells were treated with NaF, and Western blotting, immunocytochemistry, confocal microscopy, FACScan flow cytometry, MMP activity and proteasome activity were performed. NaF treatment in G361 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. And tested G361 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, a significant shift of Bax/Bcl-2 ratio, and the activation of caspase-9, caspase-7, caspase-6, caspase-3, PARP, Lamin A/C and DFF45 (ICAD). Furthermore, NaF resulted in down-regulation of the G1 cell cycle-related proteins, and up-regulation of p53. Taken collectively, our present findings demonstrate that NaF strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins and induces apoptosis via proteasome, mitochondria and caspase cascades in G361 cells.
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