Effects of GSK3β inhibition on the migration and invasion of pancreatic cancer cells.

<p>(A) Upper panels show the time course for PANC-1 cell migration in a wound-healing assay in the presence of DMSO or AR-A014418 (AR). The lower panel shows the relative widths of wounds measured as a percentage of the initial gap at time zero. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418. (B) Migrating cells through uncoated transwell and invading cells through matrigel-coated transwell were scored for PANC-1 and MIA PaCa-2 cells treated with DMSO or AR-A014418 (AR) for 22 hrs. Representative photomicroscopic findings in each assay are shown below the columns. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418.</p

Combined effect of gemcitabine or ionizong radiation and GSK3β inhibitor against cancer cells and xenografts.

<p>(A) The influence of AR-A014418 on the effect of gemcitabine was analyzed using the isobologram <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Tallarida1" target="_blank">[21]</a> by plotting the IC₅₀ of the combination therapy (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289.s002" target="_blank">Fig. S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289.s007" target="_blank">Table S4</a>). (B) The combined effect of ionizing radiation and AR-A014418 was tested in PANC-1 and MIA PaCa-2 cells by colony formation assay. *<i>p</i><0.05, statistically significant difference between cells treated with DMSO or AR-A014418. (C) The combined effect of gemcitabine and AR-A014418 was tested in PANC-1 xenografts. Athymic mice with PANC-1 xenograft were assigned to four groups for treatment with intraperitoneal injection (twice a week) of DMSO (control; 8 mice), gemcitabine (GEM; 20 mg/kg body weight; 9 mice) and AR-A014418 (AR; 2 mg/kg body weight; 8 mice), alone or in combination (GEM+AR; 9 mice). At the time after treatment for 10 weeks, tumor volume (cm³) was calculated using the formula 0.5×S²×L, where S is the smallest tumor diameter (cm) and L is the largest (cm) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Shakoori2" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055289#pone.0055289-Mai1" target="_blank">[12]</a>. The mean tumor volume was compared between the 4 groups. *<i>p</i><0.05, statistically significant difference between data.</p

Changes in expression and phosphorylation of the proteins in cancer cells following GSK3β inhibition.

<p>(A) Immunoblotting analysis compares the expression of Rb, CDK4, CDK6 and cyclin D1, and the phosphorylation of Rb at S780 and S807/811 residues (p-Rb^S780, p-Rb^S807/811) between cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 24 hrs. (B) Changes in levels of p-Rb^S780 and p-Rb^S807/811 and expression of Rb and cyclin D1 were examined in MIA PaCa-2 cells at the indicated time points after treatment with 10 µM AR-A014418. (C) Expression of Rb, cyclin D1, GSK3α and GSK3β proteins and levels of Rb phosphorylation (p-Rb^S780) were examined and compared between the same pancreatic cancer cells transfected with non-specific siRNA (NS) or GSK3β-specific siRNA (S) (10 nM each). (A–C) β-actin expression was monitored as a loading control.</p