Sorafenib Down Regulates Akt and STAT3 Survival Pathways and Induces Apoptosis in a Human Neuroblastoma Cell Line
H Chai, A Luo, P Weerasinghe, RE Brown. University of Texas- Medical School, Houston, TX
Background: Neuroblastoma ranks second among solid tumors in children, and its tumorigenicity is enhanced by the expression of survival pathways such as Akt, Nuclear Factor (NF)-kappaB and signal transducer and activator of transcription (STAT)3 and the expression of the anti-apoptotoic protein, Mcl-1. Sorafenib is a multi-kinase inhibitor that also inhibits STAT3 signaling and induces apoptosis in association with downregulation of Mcl-1. In this study, we examine the efficacy of sorafenib on a human neuroblastoma cell line and also investigate its possible mechanisms.
Design: A neuroblastoma cell line, SK-N-AS, was purchased from ATCC. After reaching 50-60% confluence, cells were treated with various concentrations of sorafenib (0, 0.1, 1, 5, 10 and 20 μM) for different period of times (2, 6, 24, 48 and 72 hours). The cell viability was determined by MTT colorimetric assay. The apoptotic responses were measured with TUNEL assay. Phosphorylation of Akt1/2/3 (pAkt1/2/3[Thr308]), AMP-activated protein kinase alpha1 subunit (p--AMPKa1[Thr172]), STAT3 (p-STAT3[Ser727]), and phospholipase D2 (PLD2) protein expression levels were determined with Western blot assay.
Results: As early as 2 hours post-treatment, cell viability was significantly decreased at 10 μM concentration. In 24 hours or longer treatment groups, sorafenib at 5 μM and above concentrations significant decreased cell viability. TUNEL assay showed similar results. A significant increased of apoptosis was observed in 5 and 20 μM treatment groups 24 hours after treatment. Western blots showed a decrease of p-Akt1/2/3 and p-STAT3 expression levels in sorafenib treatment groups. A slight increase of p-AMPKa1 was also observed. PLD2 levels remained unchanged.
Conclusions: Our results indicate that sorafenib significantly decreased cell viability and increased apoptosis in a human neuroblastoma cell line in association with down regulation of p-Akt and p-STAT3 survival pathways. These data suggested a potential clinical application of sorafenib in the treatment of neuroblastoma cases with constitutive activation of Akt and STAT3.
Monday, March 22, 2010 1:00 PM
Poster Session II # 197, Monday Afternoon