트로글리타존에 의한 두경부 암세포의 세포주기억제 및 세포사멸 연구

Abstract

Troglitazone (Rezulin)은 thiazolidinedione 화합물로써 성인 인슐린 비의존성 당뇨병의 치료제였었으나 심각한 간세포 독성 때문에 2000년 이후 판매가 중지되었다. 최근 Troglitazone이 세포주기 조절과 세포사멸 유도와 관련된 신호경로를 변화시킴으로써 다양한 암세포들의 사멸을 일으키는 것으로 보고되었다. 본 논문에서는 사람의 FaDu 두경부 암세포를 사용하여 세포생존과 사멸기전에 관련된 Troglitazone의 세포영향을 연구하였다. Troglitazone은 세포사멸을 초래하며 농도 의존적으로 세포성장을 억제하였다 (IC50=150 μM). 세포 성장의 억제는 G2/M에서 세포분열을 막는 것과 연관있으며다양한 세포주기 조절인자 및 억제자들의 발현양에 변화를 일으켰다. Western blotting 실험을 통해 Troglitazone이 세포주기에서 음성조절자인 p27Kip1의 발현을 증가시키는 반면, cdc2 (cdk1), cdk2, cdk4, cyclin B1, cyclin D1, cyclin E1의 발현을 억제시키는 것을 확인하였다. 세포사멸은 caspase-3와 caspase-7의 활성을 유도하였다. Troglitazone은 세포질에서 분비되는 proapoptotic 단백질인 Bax, Bad뿐만 아니라 세포질에서의 cytochrome C농도를 증가시켰으며 미토콘드리아 경로에 의한 세포사멸을 PPARγ 의존적으로 유도하였다. 뿐만 아니라 Troglitazone 처리는 빠른 시간 내에 p38 활성 억제와 세포외 신호조절인산화효소(ERK)의 발현을 증가시켰다. 결론적으로, Troglitazone은 PPARγ 의존적으로 세포성장의 억제와 세포사멸의 유도를 통해 FaDu 세포의 성장을 억제시켰다.|Troglitazone (Rezulin), a thiazolidinedione compound, was an antidiabetic agent that was originally approved to treat patient with adult-oneset non-insulin-dependent diabetes. However, Troglitazone was withdrawn from the market in 2000 due to its severe hepatotoxicity. Recent studies showed that Troglitazone caused cell death in a variety of tumor cell lines by altering signaling pathway associated with cell cycle regulation and apoptosis induction. In this study, we investigated the cellular effects of Troglitazone on cell survival and death mechanism in FaDu human pharyngeal squamous carcinoma cells. Troglitazone was dose-dependently toxicity on cell growth with IC50 at 150 μM and induced apoptosis. The growth inhibition was linked to the cell cycle arrest at G2/M checkpoint with two folds increase of DNA content at G2/M (from 14.34% to 28.03%). Cell cycle inhibition was further confirmed by altered expression of various cell cycle regulators and inhibitors. Western blotting studies showed that Troglitazone suppressed the expression of cdc2 (cdk1), cdk2, cdk4, cyclin B1, cyclin D1, cyclin E1, while upregulated the expression of p27Kip1, a negative regulator of cell cycle. Apoptosis induction was confirmed by DNA fragmentation and the activation of caspase-3 and caspase-7. Troglitazone increased the gene level as well as protein expression of pro-apoptotic protein Bax, Bad and cytochrome C release into cytoplasm, suggesting that Troglitazone induced apoptosis was related with mitochondrial pathway. Incubation of FaDu cells by Troglitazone led to the up-regulation of PPAR gamma (PPARγ) gene expression and this effect was PPARγ dependent. Further more, Troglitazone treatment up-regulated the expression of extra-cellular signal-regulated protein kinase (ERK) at early time points and inhibited p38 activation. Taken together, Troglitazone inhibited the growth of FaDu cells via the inhibition of cell growth and the induction of apoptosis in a PPARγ dependent manner.