Chai Hong, Luo Annie Z, Weerasinghe Priya, Brown Robert E
Department of Pathology and Laboratory Medicine, University of Texas Health Science Center-Medical School at Houston, MSB 2.286, Houston, TX 77030, USA.
Int J Clin Exp Pathol. 2010 Apr 23;3(4):408-15.
Neuroblastoma is a common solid tumor in children and its tumorigenicity is enhanced by the expression of survival pathways such as Akt and signal transducer and activator of transcription 3 (STAT3). Sorafenib is a multikinase inhibitor that also inhibits STAT3 signaling and induces apoptosis. In this study, we will examine the efficacy of sorafenib on a human neuroblastoma cell line (SK-N-AS) and also investigate its possible mechanisms. After cells reached 50-60% confluence, they were treated with various concentrations of sorafenib (0, 0.1, 1, 5, 10 and 20 microM) for different periods of time. The cell viability and apoptosis were determined by MTS colorimetric assay and TUNEL, respectively. Phosphorylation of Akt1/2/3 (p-Akt1/2/3), extracellular signal-regulated kinase 1/2 (p-ERK1/2), STAT3 (p-STAT3), and AMP-activated protein kinase alpha subunit (p-AMPKalpha) were determined with Western blot. The results indicate that as early as 2 hours post-treatment, cell viability was significantly decreased at 10 microM concentration. In 24 hours or longer treatment groups, sorafenib at 5 microM and above significantly decreased cell viability. TUNEL assay showed a significant increased of apoptosis in 5 and 20 microM treatment groups 24 hours after treatment. Western blots showed a decrease of p-ERK1/2, p-Akt1/2/3, p-STAT3, and p-AMPKalpha expression levels in various sorafenib treatment groups. Our results indicate that sorafenib significantly decreased cell viability and increased apoptosis in human neuroblastoma cell line in association with down-regulation of p-ERK1/2, p-Akt, p-STAT3 survival pathways. These data suggested potential clinical application of sorafenib in the treatment of neuroblastoma.
神经母细胞瘤是儿童常见的实体瘤，其致瘤性因Akt和信号转导及转录激活因子3（STAT3）等生存通路的表达而增强。索拉非尼是一种多激酶抑制剂，它也能抑制STAT3信号传导并诱导细胞凋亡。在本研究中，我们将检测索拉非尼对人神经母细胞瘤细胞系（SK-N-AS）的疗效，并研究其可能的作用机制。当细胞达到50%-60%汇合度后，用不同浓度的索拉非尼（0、0.1、1、5、10和20微摩尔）处理不同时间。分别通过MTS比色法和TUNEL法测定细胞活力和凋亡情况。用蛋白质免疫印迹法检测Akt1/2/3（p-Akt1/2/3）、细胞外信号调节激酶1/2（p-ERK1/2）、STAT3（p-STAT3）和AMP激活的蛋白激酶α亚基（p-AMPKα）的磷酸化情况。结果表明，早在处理后2小时，10微摩尔浓度时细胞活力就显著降低。在24小时或更长时间的处理组中，5微摩尔及以上浓度的索拉非尼显著降低细胞活力。TUNEL检测显示，处理24小时后，5微摩尔和20微摩尔处理组的凋亡显著增加。蛋白质免疫印迹显示，各索拉非尼处理组中p-ERK1/2、p-Akt1/2/3、p-STAT3和p-AMPKα的表达水平降低。我们的结果表明，索拉非尼通过下调p-ERK1/2、p-Akt、p-STAT3生存通路，显著降低人神经母细胞瘤细胞系的细胞活力并增加凋亡。这些数据提示索拉非尼在神经母细胞瘤治疗中具有潜在的临床应用价值。
