Identification of c-FLIPL and c-FLIPS as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells
Identification of c-FLIPL and c-FLIPS as critical regulators of death receptor-induced apoptosis in pancreatic cancer cells
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DOI：
10.1136/gut.2009.202325
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发表时间：
2011-02-01
期刊：
影响因子：
24.5
通讯作者：
Fulda, Simone
中科院分区：
文献类型：
--
作者：
Haag, Christian;Stadel, Dominic;Fulda, Simone
背景：逃避细胞凋亡是胰腺癌的一个标志。然而，潜在的机制仍然只有部分了解，并可能涉及抗凋亡蛋白，如c-FLIP。方法通过基因敲除、过表达研究和化疗的调控，分析c-flipl和c-flips在胰腺癌组织、胰腺癌细胞系和原代肿瘤细胞中的表达及其对死亡受体诱导的细胞凋亡的调节作用。结果c-flip在胰腺上皮内瘤变(Panin)病变和胰腺导管腺癌中均有表达，而正常胰腺导管中c-flip始终为阴性。同时下调c-FLIPL和c-Flips，以及通过RNA干扰单独敲除这两种异构体均可显著增强TRAIL(肿瘤坏死因子相关凋亡诱导配体)和CD95诱导的caspase激活和caspase依赖的细胞凋亡。此外，化疗药物--即5-氟尿嘧啶(5-FU)、顺铂或吉西他滨--可下调c-flip的表达，使细胞对死亡受体引发的细胞凋亡敏感。同样，原代培养的胰腺癌细胞通过预先暴露于5-FU或顺铂而准备好TRAIL诱导的细胞凋亡。机制研究表明，5-FU介导的c-Flip抑制导致TRAIL诱导的死亡诱导信号复合体(DISC)上caspase-8的募集和激活增加，导致caspase-3激活和caspase依赖的细胞死亡。C-FLIPL的过表达将细胞从5-FU或顺铂介导的TRAIL诱导的凋亡敏感性中解救出来，表明c-FLIP抑制是化疗介导的TRAIL敏感性的关键事件。结论抗凋亡蛋白c-FLIP的长、短异构体都是死亡受体诱导胰腺癌细胞凋亡的关键调节因子，化疗药物对其有抑制作用。靶向c-flipl或c-flips足以促进死亡受体诱导的胰腺癌细胞的凋亡。这些发现对设计基于TRAIL的胰腺癌联合治疗方案具有重要意义。
Background 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.Methods Expression of c-FLIPL and c-FLIPS 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.Results 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-FLIPL and c-FLIPS 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-FLIPL 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.Conclusions 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-FLIPL or c-FLIPS 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.