Wilson Timothy R, McLaughlin Kirsty M, McEwan Miranda, Sakai Hidekazu, Rogers Katherine M A, Redmond Kelly M, Johnston Patrick G, Longley Daniel B
Drug Resistance Group, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, Northern Ireland.
Cancer Res. 2007 Jun 15;67(12):5754-62. doi: 10.1158/0008-5472.CAN-06-3585.
c-FLIP is an inhibitor of apoptosis mediated by the death receptors Fas, DR4, and DR5 and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. We found that small interfering RNA (siRNA)-mediated silencing of c-FLIP induced spontaneous apoptosis in a panel of p53 wild-type, mutant, and null colorectal cancer cell lines and that this apoptosis was mediated by caspase-8 and Fas-associated death domain. Further analyses indicated the involvement of DR5 and/or Fas (but not DR4) in regulating apoptosis induced by c-FLIP siRNA. Interestingly, these effects were not dependent on activation of DR5 or Fas by their ligands tumor necrosis factor-related apoptosis-inducing ligand and FasL. Overexpression of c-FLIP(L), but not c-FLIP(S), significantly decreased spontaneous and chemotherapy-induced apoptosis in HCT116 cells. Further analyses with splice form-specific siRNAs indicated that c-FLIP(L) was the more important splice form in regulating apoptosis in HCT116, H630, and LoVo cells, although specific knockdown of c-FLIP(S) induced more apoptosis in the HT29 cell line. Importantly, intratumoral delivery of c-FLIP-targeted siRNA duplexes induced apoptosis and inhibited the growth of HCT116 xenografts in BALB/c severe combined immunodeficient mice. In addition, the growth of c-FLIP(L)-overexpressing colorectal cancer xenografts was more rapid than control xenografts, an effect that was significantly enhanced in the presence of chemotherapy. These results indicate that c-FLIP inhibits spontaneous death ligand-independent, death receptor-mediated apoptosis in colorectal cancer cells and that targeting c-FLIP may have therapeutic potential for the treatment of colorectal cancer.
c-FLIP是一种由死亡受体Fas、DR4和DR5介导的凋亡抑制剂，以长（c-FLIP(L)）和短（c-FLIP(S)）剪接形式表达。我们发现，小干扰RNA（siRNA）介导的c-FLIP沉默在一组p53野生型、突变型和缺失型结直肠癌细胞系中诱导了自发凋亡，且这种凋亡由半胱天冬酶-8和Fas相关死亡结构域介导。进一步分析表明，DR5和/或Fas（而非DR4）参与调节c-FLIP siRNA诱导的凋亡。有趣的是，这些效应不依赖于其配体肿瘤坏死因子相关凋亡诱导配体和FasL对DR5或Fas的激活。c-FLIP(L)的过表达而非c-FLIP(S)的过表达显著降低了HCT116细胞中的自发凋亡和化疗诱导的凋亡。用剪接形式特异性siRNAs进行的进一步分析表明，c-FLIP(L)是调节HCT116、H630和LoVo细胞凋亡中更重要的剪接形式，尽管特异性敲低c-FLIP(S)在HT29细胞系中诱导了更多凋亡。重要的是，在BALB/c重度联合免疫缺陷小鼠中，瘤内递送靶向c-FLIP的siRNA双链体诱导了凋亡并抑制了HCT116异种移植瘤的生长。此外，过表达c-FLIP(L)的结直肠癌异种移植瘤的生长比对照异种移植瘤更快，在化疗存在的情况下这种效应显著增强。这些结果表明，c-FLIP抑制结直肠癌细胞中自发的、不依赖死亡配体的、死亡受体介导的凋亡，靶向c-FLIP可能对结直肠癌的治疗具有治疗潜力。
