Journal of Oncology / 2012 / Article / Fig 7

Research Article

Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/β-Catenin Pathways

Figure 7

3-Cl-AHPC mediated inhibition of the activation of TCF/LEF in Wnt/β-catenin pathway and decreased of β-catenin nuclear localization. (a) 3-Cl-AHPC decreased nuclear β-catenin as indicated by Western blot using nuclear extracts and densitometric quantification. (b) Nuclear β-catenin in control- (i) and 3-Cl-AHPC- (ii) treated PANC-1 cells using confocal fluorescent microscope (magnification 40X). Cells were grown in eight chambered slides and then treated with 3-Cl-AHPC for 24 h. Slide was prepared as described in Section 2. DAPI was used for nuclear staining for 1 min and mounted the slide with prolong gold antifade kit. (c) 3-Cl-AHPC inhibited TCF/LEF activity in Wnt/β-catenin signaling in stably transfected Cignal TCF/LEF-Luc reporter PANC-1 cell lines and 50 mM LiCl was used as a positive control. For CD44/CD24 cells, TCF/LEF stably transfected cells were sorted by flow cytometry and followed the procedure same as wild type (Wt) stable cell line. Luciferase promoter activity values are expressed as fold using a total protein concentration for internal normalization. The error bars represent the mean of three separate determinations ± the standard deviation (SD). (d) 3-Cl-AHPC decreased Wnt/β-catenin signaling responsive c-Myc protein. ((e) and (f)) Knock down of β-catenin inhibited cell proliferations and enhanced more apoptosis in sh-β-catenin knockdown (KD) PANC-1 cell lines. Proliferation inhibition was evaluated after 72 h of seeding the cells by MTT assay and expressed as absorbance measured at 570 nm. The error bars represent the mean of three separate determinations ± the standard deviation (SD). ** (<0.01) was significantly different in comparison between sh-vector and Catenin-KDl/Catenin-KD2 and also between sh-vector and catenin-KD1/Catenin-KD2 treated with 3-Cl-AHPC, respectively.

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