Carter Yvette M, Kunnimalaiyaan Selvi, Chen Herbert, Gamblin T Clark, Kunnimalaiyaan Muthusamy
University of Wisconsin Endocrine Surgery Laboratory; Madison, WI USA.
University of Wisconsin Endocrine Surgery Laboratory; Madison, WI USA; Medical College of Wisconsin; Milwaukee, WI USA.
Cancer Biol Ther. 2014 May;15(5):510-5. doi: 10.4161/cbt.28015. Epub 2014 Feb 12.
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.
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.
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.
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.
神经母细胞瘤是一种常见的神经内分泌（NE）肿瘤，多在儿童早期出现，恶性率和复发率高。糖原合酶激酶-3（GSK-3）通路是一个潜在的治疗靶点，因为该通路已被证明在其他NE肿瘤的治疗中至关重要。然而，尚不清楚哪种同工型对于生长抑制是必需的。在本研究中，我们研究了GSK-3抑制剂AR-A014418对神经母细胞瘤中不同GSK-3同工型的影响。
用0 - 20 μM的AR-A014418处理NGP和SH-5Y-SY细胞，通过MTT法测定细胞活力。通过蛋白质免疫印迹分析NE标志物嗜铬粒蛋白A（CgA）和achaete-scute同源蛋白1（ASCL1）、GSK-3同工型以及凋亡标志物的表达水平。
用AR-A014418处理的神经母细胞瘤细胞在所有剂量和时间点的生长均显著降低（P<0.001）。在第6天，与对照相比, 10 μM处理（NGP为53% 对比0%，SH-5Y-SY为38% 对比0%，P<0.001）时细胞系生长出现降低，这与肿瘤标志物ASCL1和CgA表达的显著降低相对应。
与GSK-3β在Tyr216处的磷酸化相比，用AR-A014418处理神经母细胞瘤细胞系可降低GSK-3α在Tyr279处的磷酸化水平，并通过维持凋亡来减弱生长。本研究支持进一步研究以阐明GSK-3α抑制下调NE肿瘤标志物表达和神经母细胞瘤生长的机制。
