Oxidative stress induces FAK and AKT activation in human neuroblastoma cells

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Purpose: Vascular endothelial growth factor receptor-3 (VEGFR-3) and focal adhesion kinase (FAK) are protein kinases that are important in angiogenesis and cell survival pathways. Oxidative stress has been shown to activate VEGFR-3 in human tumor cells. We hypothesize that neuroblastoma cells express VEGFR-3 and when subjected to oxidative stress, FAK and subsequent downstream survival pathways will be phosphorylated (activated) through VEGFR-3 binding. Methods: For our experiments, we employed SK-N-AS (AS) human neuroblastoma cells. Immunoblotting is utilized to document baseline VEGFR-3 expression. Immunoprecipitation techniques (IP) are used to demonstrate binding between VEGFR-3 and FAK. Hydrogen peroxide (H2O2) is utilized to provide oxidative stress to the cells, and the cells are cultured with H2O2 (1mM) at varying times. The expression of FAK, phosphorylated FAK (pFAK), Akt, and phosphorylated Akt (pAkt) is measured by immunoblotting. β-actin expression serves as a control to assure equal protein loading. The experiments are repeated in triplicate. Results: Representative IP’s and immunoblots are below. The SK-N-AS neuroblastoma cell line does express VEGFR-3 (A.). Immunoprecipitation (IP) with VEGFR-3 and Western blot (WB) for FAK shows that VEGFR-3 and FAK are binding partners in these cells (B.). IP with MOPC serves as a negative control, and IP with FAK with WB for FAK serves as a positive control (B.). Expression of total FAK and Akt are unchanged in the neuroblastoma cells by oxidative stress (C.). Control cells express little phosphorylated FAK or Akt (C.). However, oxidative stress results in phosphorylation (activation) of both anti-apoptotic protein kinases, FAK and Akt, in a time dependent fashion (C.). Conclusions: SK-N-AS neuroblastoma cells express VEGFR-3, and VEGFR-3 and FAK are binding partners in these cells. Exposure of SK-N-AS neuroblastoma cells to H2O2 results in the phosphorylation of FAK and Akt, providing a cellular survival mechanism for the cells under oxidative stress. Targeting the binding between VEGFR-3 and FAK may provide a molecular mechanism for the treatment of neuroblastoma.