Previous studies showed a similar therapeutic effect of lithium, a non-specific and ATP-non-competitive GSK3 inhibitor, against ESCC46,47. biological rationale for GSK3 as a potential therapeutic target in ESCC. value Oglufanide of? ?0.05 considered to be statistically significant. IHC scores between normal tissues and tumors of ESCC patients were statistically analyzed by One-way ANOVA test using GraphPad Prism 5.0 (GraphPad Software, Inc. CA) and compared with clinical and pathologic characteristics by Chi-square test. Results Expression and phosphorylation-dependent activity of GSK3 in ESCC cells and patient tumors The expression levels of GSK3 and its Y216 phosphorylated portion (pGSK3Y216, active form) were higher in all ESCC cell lines compared to normal esophageal squamous TYNEK-3 cells (Fig.?1A), with less detectable S9 phosphorylation (pGSK3S9, inactive form). Increased expression and activity of GSK3 in ESCC cells was also supported by the finding that S641 phosphorylation ARF6 of GS (pGSS641, inactive form), the primary substrate of GSK315,16, was higher in ESCC than in TYNEK-3 cells (Supplementary Information, Fig. S2A). The levels of intracellular glycogen in ESCC cell lines were significantly lower than normal TYNEK-3 cells and were restored following treatment with GSK3 inhibitors (Supplementary Information, Fig. S2B). Open in a separate window Physique 1 Comparative analysis for the expression and phosphorylation of GSK3 in human ESCC cells (TE-1, TE-5, TE-8, TE-9, TE-10, TE-15, KES), normal esophageal squamous epithelial cells (TYNEK-3), and normal squamous mucosa and main tumors from ESCC patients. (A) Expression of GSK3 and of its phosphorylated forms (pGSK3S9, inactive form; pGSK3Y216, active form) were examined by Western blotting. -actin expression was monitored as a loading control in each sample. (B) Representative findings for the expression of GSK3 and its Y216 phosphorylated portion (pGSK3Y216) in the primary tumor and corresponding normal squamous mucosa of ESCC patients. The scale bar indicates 100?m in length. Immunohistochemical images were captured using Keyence BZ-X700 Analyzer (Version 1.3). The two right hand graphs generated using GraphPad Prism 5.0 (GraphPad Software, Inc. CA) show statistical comparison of the immunohistochemistry (IHC) scores for GSK3 and pGSK3Y216 between the main tumor (T) and normal mucosa (N) of ESCC patients. A horizontal bar in each group shows the imply value of IHC scores. (C) Expression of GSK3 mRNA in normal esophageal tissues (N) and main ESCC tumor tissues (T) based on the TCGA database. The data was generated using the analysis tool UALCAN (https://ualcan.path.uab.edu/)33. n, quantity of patients; **glycogen synthase, glycogen synthase kinase 3, lymph node, moderately differentiated SCC, poorly differentiated SCC, squamous cell carcinoma, well differentiated SCC. Oglufanide Effect of GSK3 Oglufanide inhibition on ESCC cell survival, proliferation and apoptosis To address our hypothesis of a putative tumor-promoting role for GSK3 in ESCC, the biological outcome resulting from GSK3 inhibition was examined in terms of tumor cell survival, proliferation and apoptosis. Treatment with the GSK3 inhibitors (AR-A014418, SB-216763) reduced viability of all ESCC cells in a dose- and time-dependent manner, while sparing normal TYNEK-3 cells (Fig.?2A, Supplementary Information, Fig. S4A). The IC50 values of both inhibitors at 48?h after treatment were within the reported pharmacological dose range (1C100?mol/L) for AR-A01441830 and SB-21676331. These GSK3 inhibitors decreased the number of EdU-positive proliferating cells (Fig.?2B, Supplementary Information, Fig. S5A) and increased the incidence of apoptosis in ESCC cells (Fig.?2C). Treatment with LY2090314 within the reported pharmacological dose range showed therapeutic effects against ESCC cells that were comparable to AR-A014418 and SB-216763. (Supplementary information, Fig. S6). Induction of apoptosis by GSK3 inhibition was further confirmed by increases in the portion of c-PARP (Fig.?2D) and the sub-G0/G1 portion in cell cycle analysis (Fig.?3A,B, Supplementary Information, Fig. S7A). Comparable effects were observed in ESCC cells following depletion of GSK3 by siRNA.