Efron Philip A, Chen Mike K, Iyengar Meera, Dai Wei, Nagaram Abhilasha, Beierle Elizabeth A
Division of Pediatric Surgery, Department of Surgery, University of Florida College of Medicine, Box 100286, JHMHSC, Gainesville, FL 32610-0286, USA.
J Pediatr Surg. 2006 Jun;41(6):1072-80. doi: 10.1016/j.jpedsurg.2006.02.001.
In many human tumor cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through caspase activation, whereas activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway prevents apoptosis. We hypothesized that inhibition of PI3K/Akt would increase TRAIL-induced apoptosis in neuroblastoma cells.
SK-N-AS, SH-SY5Y, and IMR-32 neuroblastoma cells were cultured with either standard media or media with PI3K/Akt inhibitor for 24 hours. These cells were then exposed to 100 ng/mL of TRAIL for 90 minutes and harvested. Cells either underwent flow cytometric analysis of apoptosis, had protein extracted for Western blot, had RNA extracted for reverse transcription-polymerase chain reaction, or had cell lysates analyzed for caspase-3, -8, and -9.
Baseline expression of TRAIL receptors and Akt varied among the cell lines. Inhibition of PI3K/Akt decreased caspase-3 activation in the AS and SY cells, but did not alter TRAIL-induced apoptosis in any of the cell lines. Activity of caspase-8 and -9 was also unaffected by PI3K/Akt attenuation.
Inhibition of the PI3K/Akt pathway does not increase the sensitivity of neuroblastoma cell lines to TRAIL-induced apoptosis. Neuroblastoma is unique in that activation of the PI3K/Akt pathway is either not essential to its TRAIL resistance or counteracted because of the multiple repetitive pathways of TRAIL resistance.
在许多人类肿瘤细胞中，肿瘤坏死因子相关凋亡诱导配体（TRAIL）通过半胱天冬酶激活诱导凋亡，而磷脂酰肌醇3激酶（PI3K）/Akt途径的激活则可防止细胞凋亡。我们推测抑制PI3K/Akt会增加TRAIL诱导的神经母细胞瘤细胞凋亡。
将SK-N-AS、SH-SY5Y和IMR-32神经母细胞瘤细胞分别用标准培养基或含PI3K/Akt抑制剂的培养基培养24小时。然后将这些细胞暴露于100 ng/mL的TRAIL中90分钟，之后进行收获。对细胞进行凋亡的流式细胞术分析、提取蛋白质进行蛋白质印迹分析、提取RNA进行逆转录-聚合酶链反应分析，或对细胞裂解物进行半胱天冬酶-3、-8和-9分析。
TRAIL受体和Akt的基线表达在各细胞系中有所不同。PI3K/Akt的抑制降低了AS和SY细胞中半胱天冬酶-3的激活，但未改变任何细胞系中TRAIL诱导的凋亡。半胱天冬酶-8和-9的活性也不受PI3K/Akt衰减的影响。
抑制PI3K/Akt途径不会增加神经母细胞瘤细胞系对TRAIL诱导凋亡的敏感性。神经母细胞瘤的独特之处在于，PI3K/Akt途径的激活对其TRAIL抗性要么不是必需的，要么由于TRAIL抗性的多种重复途径而被抵消。
