Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth

Abstract

Objective: Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma.

Methods: NGP and SH-5Y-SY cells were treated with 0-20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot.

Results: Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P<0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P<0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression.

Conclusion: Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.

Keywords: AR-A014418; GSK3; apoptosis; neuroblastoma.

Figure 1. Growth inhibition by GSK-3 inhibitor AR-A014418 in neuroblastoma cells. (A) NGP, (B) SH-5Y-SY cells were incubated with AR-A014418 for up to 6 days at various concentrations and cell viability was measured by both MTT assay and colony formation assay. Significant reduction in both number and size of the colonies were seen in AR-A014418 treatment (C and D).

Figure 2. Phosphorylation of GSK-3α reduction is associated with decreased expression of NE markers. Western analysis showing that decreased phosphorylation of GSK-3 α at Tyr 279 compared to GSK-3β phosphorylation at Tyr216 by AR-A014418. In addition, there is a reduction in GSK-3β phosphorylation at ser9. This decrease in phosphorylation reduced the expression of β-catenin, ASCL1, and CgA in NGP (A) and SH-5Y-SY (B) cells. D, DMSO-treated control. GAPDH was used as a loading control.

Figure 3. AR-A014418 attenuation of apoptosis inhibitor expression in NGP and SH-5Y-SY cells. Western blot analysis showed there is increase in cleaved PARP, a marker for apoptosis, This was associated with reduction in anti-apoptotic protein Mcl-1 and survivin. D, DMSO-treated control. GAPDH was used as loading control.

Figure 4. Continuous treatment of AR-A014418 is required for growth suppression of NGP and SH-5Y-SY cells. Cells were treated with AR-A014418 for 4 d and then media was changed to complete media without AR-A014418 for up to 4 d. Cell viability was measured by MTT assay (A) and western blot analysis for the levels of GSK-3 phosphorylation, β-catenin, and cyclin D1 (B). Significant reduction in cellular proliferation was observed with AR-A014418 treatment whereas cellular growth is increased compared to the treatment when the medium was changed to regular medium without AR-A014418 (A) in both cell lines. Importantly, reversal of phosphorylation of GSK-3α protein and increase in β-catenin and cyclin D1 protein was seen in withdrawal condition (B).