Kim Sunghoon, Kang Junghee, Qiao Jingbo, Thomas Robert P, Evers B Mark, Chung Dai H
Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77555-0536, USA.
J Pediatr Surg. 2004 Apr;39(4):516-21. doi: 10.1016/j.jpedsurg.2003.12.008.
BACKGROUND/PURPOSE: Recent findings have correlated neuroblastoma development with aberration of apoptosis. In particular, increased levels of survivin, a member of the inhibitor of apoptosis protein (IAP) family, have been associated with increased resistance to apoptosis in neuroblastomas. The purpose of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt pathway can alter the expression of survivin and facilitate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in neuroblastoma cells.
Human neuroblastoma cells (SK-N-SH, BE[2]C, LAN-1 and IMR-32) were treated with LY294002 or wortmannin, inhibitors of PI3K, for 24 hours. Transient transfection of a dominant negative PI3K expression plasmid, pCGNN-Deltap85, was performed to inhibit PI3K activation. Cells were treated with TRAIL, caspase-3, or pan-caspase inhibitors. RNase protection assay was performed to assess mRNA changes in IAP family members, including survivin. Western blot analysis was performed to measure changes in the levels of procaspases and survivin. Apoptosis was assessed by a DNA fragmentation ELISA assay.
The authors found that survivin and cIAP1 mRNA as well as protein expression were decreased after PI3K inhibition. Combination treatment with LY294002 and TRAIL increased apoptosis of SK-N-SH cells compared with TRAIL alone; these results were further corroborated by complete inhibition of apoptosis by caspase-3 or pan-caspase inhibitor.
The authors report that PI3K pathway inhibition down-regulates survivin expression and enhances TRAIL-mediated apoptosis in neuroblastomas. PI3K pathway may play a crucial role in neuroblastoma cell survival; therefore, treatment with inhibitors of PI3K may provide potential novel therapeutic options.
背景/目的：最近的研究结果表明神经母细胞瘤的发生与细胞凋亡异常有关。特别是，凋亡抑制蛋白（IAP）家族成员survivin水平的升高与神经母细胞瘤对细胞凋亡的抗性增加有关。本研究的目的是确定磷脂酰肌醇3激酶（PI3K）/Akt信号通路是否能改变survivin的表达，并促进肿瘤坏死因子（TNF）相关凋亡诱导配体（TRAIL）介导的神经母细胞瘤细胞凋亡。
用PI3K抑制剂LY294002或渥曼青霉素处理人神经母细胞瘤细胞（SK-N-SH、BE[2]C、LAN-1和IMR-32）24小时。进行显性负性PI3K表达质粒pCGNN-Deltap85的瞬时转染以抑制PI3K激活。细胞用TRAIL、半胱天冬酶-3或泛半胱天冬酶抑制剂处理。采用核糖核酸酶保护试验评估IAP家族成员（包括survivin）的mRNA变化。进行蛋白质免疫印迹分析以检测前半胱天冬酶和survivin水平的变化。通过DNA片段化ELISA试验评估细胞凋亡。
作者发现PI3K抑制后survivin和cIAP1的mRNA以及蛋白表达均降低。与单独使用TRAIL相比，LY294002和TRAIL联合处理增加了SK-N-SH细胞的凋亡；半胱天冬酶-3或泛半胱天冬酶抑制剂对凋亡的完全抑制进一步证实了这些结果。
作者报告PI3K信号通路抑制下调神经母细胞瘤中survivin的表达并增强TRAIL介导的凋亡。PI3K信号通路可能在神经母细胞瘤细胞存活中起关键作用；因此，用PI3K抑制剂治疗可能提供潜在的新治疗选择。
