Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3β/Tyr216 Accumulation

Figure 4

ABZ treatment downregulates the snail expression in melanoma cells by increasing the accumulation of phosphorylated GSK-3β/Tyr216. (a) The relative transcription levels of Snail in the ABZ-treated (0.4 μM) and control groups of A375 (left) and B16-F10 (right) melanoma cells were measured by RT-qPCR, with β-actin as the internal control. (b) The expression of transcription factor Snail in A375 (left) and B16-F10 (right) cells was detected by western blot analysis, with β-actin as the internal reference protein. (c–d) The expression levels of cytoplasmic proteins AKT, pAKT, GSK-3β, pGSK-3β (Ser9/Tyr216) and Snail, and nuclear protein pSnail in A375 and B16-F10 cells were also determined by western blotting, with β-actin and PCNA as the internal controls for the cytoplasmic and nuclear proteins, respectively. The histograms show the relative density of AKT/pAKT, GSK-3β/pGSK-3β (Ser9/Tyr216), and Snail/p-Snail. (e) A375 cells were cotreated with or without MG132 and 0.4 μM ABZ for 24 h western blot (up) was used to detect the expression levels of AKT, pGSK-3β/Tyr216, Snail, N-cadherin, and E-cadherin in the cytoplasm of A375 cells. The histogram (bottom) shows the relative density of AKT, pGSK-3β/Tyr216, Snail, E-cadherin, and N-cadherin. (f) Histogram showing the relative expression intensity of pGSK-3β (Ser9/Tyr216) and pAKT after immunohistochemical staining of mouse metastatic lung cancer tissues. Scale bars = 100 and 20 μm. Each point in each group is calculated as the average score for five randomly selected areas in one lung slice and represents an IHC index of one mouse. All data are expressed as means ± SD. All experiments were performed thrice. ; ; ns, not significant.