EA 4427 SeRAIC, Faculty of Pharmacy, University of Rennes 1, IFR 140 GFAS, Rennes F-35043, France.
Eur J Cancer. 2010 May;46(8):1445-55. doi: 10.1016/j.ejca.2010.01.034. Epub 2010 Mar 4.
In human colon cancer cells, cisplatin-induced apoptosis involves the Fas death receptor pathway independent of Fas ligand. The present study explores the role of ezrin and actin cytoskeleton in relation with Fas receptor in this cell death pathway. In response to cisplatin treatment, a rapid and transient actin reorganisation is observed at the cell membrane by fluorescence microscopy after Phalloidin-FITC staining. This event is dependent on the membrane fluidification studied by electron paramagnetic resonance and necessary for apoptosis induction. Moreover, early after the onset of cisplatin treatment, ezrin co-localised with Fas at the cell membrane was visualised by membrane microscopy and was redistributed with Fas, FADD and procaspase-8 into membrane lipid rafts as shown on Western blots. In fact, cisplatin exposure results in an early small GTPase RhoA activation demonstrated by RhoA-GTP pull down, Rho kinase (ROCK)-dependent ezrin phosphorylation and actin microfilaments remodelling. Pretreatment with latrunculin A, an inhibitor of actin polymerisation, or specific extinction of ezrin or ROCK by RNA interference prevents both cisplatin-induced actin reorganisation and apoptosis. Interestingly, specific extinction of Fas receptor by RNA interference abrogates cisplatin-induced ROCK-dependent ezrin phosphorylation, actin reorganisation and apoptosis suggesting that Fas is a key regulator of cisplatin-induced actin remodelling and is indispensable for apoptosis. Thus, these findings show for the first time that phosphorylation of ezrin by ROCK via Fas receptor is involved in the early steps of cisplatin-induced apoptosis.
在人类结肠癌细胞中，顺铂诱导的细胞凋亡涉及 Fas 死亡受体途径，而不依赖 Fas 配体。本研究探讨 ezrin 和肌动蛋白细胞骨架在该细胞死亡途径中与 Fas 受体的关系。通过荧光显微镜观察鬼笔环肽-FITC 染色后细胞膜上的快速和瞬时肌动蛋白重排，发现顺铂处理后会发生这种情况。该事件依赖于电子顺磁共振研究的膜流动性，并且是诱导细胞凋亡所必需的。此外，在顺铂处理开始后早期，通过细胞膜显微镜观察到 Fas 与 ezrin 在细胞膜上共定位，并在 Fas、FADD 和 procaspase-8 重分布到膜脂筏中，如 Western blot 所示。事实上，顺铂暴露导致早期小 GTPase RhoA 激活，通过 RhoA-GTP 下拉、Rho 激酶（ROCK）依赖性 ezrin 磷酸化和肌动蛋白微丝重塑来证明。用细胞松弛素 A（一种肌动蛋白聚合抑制剂）预处理或通过 RNA 干扰特异性消除 ezrin 或 ROCK，可防止顺铂诱导的肌动蛋白重排和细胞凋亡。有趣的是，通过 RNA 干扰特异性消除 Fas 受体可阻断顺铂诱导的 ROCK 依赖性 ezrin 磷酸化、肌动蛋白重排和细胞凋亡，表明 Fas 是顺铂诱导肌动蛋白重塑的关键调节剂，对于细胞凋亡是不可或缺的。因此，这些发现首次表明，ROCK 通过 Fas 受体对 ezrin 的磷酸化参与了顺铂诱导的细胞凋亡的早期步骤。
