Phosphatidylinositol 3-kinase/AKT pathway is involved in retinoic acid-induced neuroblastoma cell differentiation

Abstract

The overall prognosis for pediatric patients with neuroblastoma remains poor, largely due to aggressive undifferentiated tumor behavior and high recurrence rate. Recently, much focus has been directed to the use of differentiating agents, such as retinoid acid (RA), for the treatment of aggressive neuroblastomas; the cellular mechanisms involved in this process are not clearly defined. The phosphatidylinositol 3-kinase (PI3-K) is an important signaling pathway for cell survival and proliferation; however, a little is known about its role in cellular differentiation. Therefore, the purpose of this study was to determine whether PI3-K pathway plays a crucial role in the RA-induced differentiation of neuroblastoma cells. Methods: Human neuroblastoma cell lines (SK-N-SH and BE(2)-C) were treated with RA (5 μM) for 24 h to induce differentiation. Cell differentiation was observed with light microscopy. To determine whether the PI3-K pathway is involved in this process, cells were treated with RA with or without LY294002 (20 μM), a specific inhibitor of PI3-K. The phosphorylation of Akt, a downstream effector for PI3-K, was measured by Western blot analysis. The effects of RA on cell cycle progression were assessed by flow cytometry. Cell survival and DNA fragmentation were analyzed by ELISA. Results: Treatment with RA resulted in differentiation of human neuroblastoma cells as demonstrated by marked neurite-like extensions when compared to cells treated with vehicle alone (control); this effect was blocked by LY294002 treatment. RA treatment also increased Akt phosphorylation; this increase was attenuated with LY294002. Additionally, cells were arrested in the G1 phase and the expression of cyclin dependent kinase inhibitors p21Cip and p27Kip were significantly increased with RA treatment; this induction was prevented by LY294002. RA treatment also resulted in an approximately 50% increase in cell survival and 60% decrease in DNA fragmentation. Conclusions: Our results demonstrate that RA-induced neuroblastoma cell differentiation occurs via activation of the PI3-K pathway. Moreover, RA-induced differentiation appears to involve regulation of cell cycle arrest. RA-induced differentiation is also associated with enhanced cell survival and increased apoptosis in neuroblastoma cells. These findings suggest that PI3-K is a critical signaling pathway involved in RA-induced neuroblastoma differentiation and may provide insights into developing novel therapeutic strategies targeted at key cellular signaling.