We detected the effect of XAV939 on cell cycle in SH-SY5Y, SK-N-SH and IMR-32 cells. As shown in Figure 4A, the number of SH-SY5Y cells in the G0/G1 phase decreased, while those in the S and G2/M phase both increased after XAV939 treatment (Figure 4A). At 24 h after treatment, 58.25% of the DMSO-treated SH-SY5Y cells were at G0/G1, 28.02% at S and 13.73% at G2/M (Figure 4B, DNA Damage inhibitor P < 0.05). In comparison, 48.38% of the XAV939-treated SH-SY5Y cells were at G0/G1, 33.68% at S and 17.94% at G2/M (Figure 4B, P < 0.05). This trend was continued at later
time points (Figure 4B). Figure 4C, E indicated the cell cycle of SK-N-SH and IMR-32 cells respectively, and showed the same tendency with that of SH-SY5Y cells (Figure 4D, F, P < 0.05). This suggested that TNKS1 plays a role in cell cycle regulation and that TNKS1 inhibition induces an accumulation of NB cell lines at G2/M and S phase of the cell cycle. Figure 4 TNKS1 inhibition induces G2/M accumulation in SH-SY5Y, SK-N-SH and IMR-32 cells. A, C, E. The representative diagrams of distribution of stained SH-SY5Y, SK-N-SH and IMR-32 cells in control group and XAV939 group. B, D, F. The bar graph of the average percent cells of G0/G1, S and G2/M phases in control Sapitinib clinical trial group and XAV939 group for SH-SY5Y, SK-N-SH and IMR-32 cells click here respectively (P < 0.05). TNKS1 inhibition reduces the expression of anti-apoptotic proteins It has been reported that XAV939 could inhibit the proliferation
of DLD-1 cells growth by attenuating the expression of Wnt signaling . Western blot was performed to determine if TNKS1 inhibition induces apoptosis in SH-SY5Y and SK-N-SH cells and if so whether Wnt/β-catenin signalling and Bcl-2 plays a role. TNKS1 in SH-SY5Y and SK-N-SH cells was inhibited by XAV939 of 1 and 0.5 μM, or by specific shRNA to TNKS1. Following inhibition
of TNKS1 in SH-SY5Y and SK-N-SH cells, protein levels of Bcl-2 were both reduced (P < 0.05, Figure 5A, B; P < 0.01, Figure 5C, D). This suggests that TNKS1 PDK4 inhibition might induce apoptosis in NB cell lines in part by reducing expression of the anti-apoptotic protein Bcl-2. After treating with XAV939 or specific shRNA to TNKS1, we noted that the accumulation of β-catenin reduced as well as Cyclin D1 and c-Myc in both NB cell lines (Figure 5A, C). Quantification analysis revealed these results (P < 0.05, Figure 5B, D). As a result, the anti-apoptotic protein Bcl-2 also decreased. Figure 5 TNKS1 inhibition altered the expression of anti-apoptotic proteins. A, C. Western blot analysis of β-catenin, Cyclin D1, c-Myc and Bcl-2 proteins level in SH-SY5Y and SK-N-SH cells untransfected, XAV939 treatment, transfected with TNKS1 shRNA or control shRNA. β-actin was loading control. B, D. The bar graph showed mean ± SD of the ratio interest proteins/β-actin band intensity obtained by pooling the results from 3 independent experiments in SH-SY5Y and SK-N-SH cells respectively.