University Children's Hospital, Ulm, Germany.
Gut. 2011 Feb;60(2):225-37. doi: 10.1136/gut.2009.202325. Epub 2010 Sep 28.
Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated.
Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics.
c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis.
Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.
胰腺癌细胞逃避细胞凋亡是其主要特征之一。然而，其中的机制还不是很清楚，可能涉及抗凋亡蛋白如 c-FLIP。本研究旨在探讨 c-FLIP 在调控胰腺癌细胞死亡受体诱导凋亡中的作用。
采用免疫组化方法检测胰腺癌患者癌组织、癌旁组织和胰腺癌细胞系中 c-FLIP 的表达；通过 RNAi 干扰技术下调 c-FLIP 全长及短型异构体的表达和过表达 c-FLIP，观察对 TRAIL 和 CD95 诱导的 caspase 激活和凋亡的影响；用化疗药物 5-氟尿嘧啶（5-FU）、顺铂和吉西他滨预处理胰腺癌细胞后观察对 c-FLIP 表达和细胞凋亡的影响；用免疫共沉淀检测化疗药物处理前后 DISC 中 caspase-8 的募集和激活情况。
c-FLIP 在胰腺上皮内瘤变（PanIN）和胰腺导管腺癌中表达，而正常胰腺导管上皮中不表达 c-FLIP。用 RNAi 技术同时下调 c-FLIP 全长和短型异构体的表达，或单独下调 c-FLIP 任一异构体的表达，均能明显增强 TRAIL 和 CD95 诱导的 caspase 激活和凋亡。化疗药物预处理能下调 c-FLIP 的表达并增强胰腺癌细胞对死亡受体诱导的凋亡的敏感性，而且，5-FU 或顺铂预处理能增强 TRAIL 诱导的原代胰腺癌细胞的凋亡。机制研究表明，5-FU 下调 c-FLIP 表达能促进 DISC 中 caspase-8 的募集和激活，导致 caspase-3 的激活和凋亡。过表达 c-FLIP（L）能挽救 5-FU 或顺铂介导的 TRAIL 诱导的细胞凋亡，提示 c-FLIP 的下调是化疗药物增强 TRAIL 敏感性的关键。此外，同时中和 c-FLIP 和 XIAP 能协同增强 TRAIL 诱导的凋亡。
c-FLIP 的长型和短型异构体都是胰腺癌细胞死亡受体诱导凋亡的关键调节因子，并且能被化疗药物所抑制。靶向 c-FLIP（L）或 c-FLIP（S）都能促进胰腺癌细胞死亡受体诱导的凋亡。这些发现为设计基于 TRAIL 的联合治疗方案治疗胰腺癌提供了理论依据。
