Neuroblastoma is a rare developmental cancer of the sympathetic nervous system found primarily in infants and young children under 5. Its unique presentation and tumor biology present challenges for effective treatment regimens, notably in patients diagnosed as high risk (>MYCN expression) [
6,
27,
28]. In recent years, research has focused on inhibition of the GKS-3 signaling pathway in cancers [
16‐
18,
29]. Studies exploring GSK-3 inhibitors in cancer treatment have been published by us and others within the last several years; however, only Tideglusib and AR-A011418 as a selective GSK-3 inhibitor in neuroblastoma can be found [
19,
23]. We have reported that treatment of neuroblastoma cell lines with AR-A014418 reduced the growth and associated with reduction in the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr219. Recently, Tideglusib, another GSK3 inhibitor, has been shown to reduce growth in vitro in neuroblastoma IMR32 cells. Despite this, clinical trials for the GSK-3 inhibitors are very limited. This may be due to the non-specific nature and extensive side effects of general GSK-3 inhibitors. However, the toxicity profile of LY2090314, a selective GSK-3 inhibitor, in combination with carboplatin was reported as similar to the reported cisplatin treatment alone [
24]. In this study, we tested 3 different human neuroblastoma cell lines NGP, SK-N-AS, and SH-SY-5Y. These cell lines have different genetic groups; SH-SY-5Y (Non-amplified MYCN or single copy, TP53 WT, ALK mutation (F1174 L)), NGP 1p alteration (t(1p), MYCN amplified, ALK wild type, TP53 mutated-MDM2 amplified), and SK-N-AS (1p deletion, MYCN single copy, TP53 mutation (H168R), and ALK WT). These cell lines showed a clear trend in declining cellular growth in increasing concentrations of LY2090314 as well as a continuous decrease of growth found at longer incubation periods (48, 72, and 96 h). In summary, we found that LY2090314 was highly effective at much lower concentrations in the nanomolar range compared to the micromolar range needed for similar reduction using Tideglusib. Tideglusib was demonstrated to have an anti-proliferative effect in neuroblastoma cell line IMR32 but has not been studied in other NB cell lines [
22,
23]. Mathuram et al. used lithium as standard GSK-3 inhibitor to compare the efficiency of growth inhibition by Tideglusib [
23]. Therefore, we compared LY2090314 with the Tideglusib, both used in clinics. Our findings indicated that LY2090314 is more potent at nM concentration as an anticancer agent for neuroblastoma, suggesting that it may be used in clinic to treat patients with neuroblastoma disease. Mechanistically, consistent with our prior findings, LY2090314 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr219 [
19]. In addition, inactive phosphorylation of both isoforms (ser21 for α and ser 9 for β) is significantly reduced in SH-SY-5Y cells compared to NGP and SK-N-AS cells. This may be due to cell line specificity. Akt is upstream of GSK-3 and showed minimal to no effect on its phosphorylation, suggesting that LY2090314 is more specific to GSK-3 protein. Furthermore, LY2090314 treatment induced apoptosis, as evidenced by increase in cleaved caspase-3, cleaved PARP and reduced cyclinD1 and possibly survivin and Mcl-1. The mechanism by which GSK-3 acts on cyclin D1 degradation is still controversial [
30,
31]. As toxicity remains a common problem with chemotherapy treatments, our results indicate LY2090314 might be a useful alternative to other potential drugs. Our findings thus demonstrate that further preclinical studies are needed to explore the efficacy of LY2090314 in NB. Further, LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study completed looking at the effect of LY2090314 in NB cell lines in vitro. In summary, effective inhibition of NB cell growth and contribution of LY2090314 to the clinical benefit observed in other cancer types indicated potential use of LY2090314 is predicted for patients with NB in future.