Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/β-Catenin Pathways
3-Cl-AHPC decreased expression of IGF-1R, cyclin D1, β-catenin, and cleaved Notch-1 and increased levels of cleaved-caspase-3 in CD44+/CD24+ spheres. (a) IGF-1R, cyclin D1, and β-catenin expression decreased cleaved-caspase-3 increased with no change in Notch-1 protein levels in spheres following exposure to 3-Cl-AHPC. Pancreatic cancer cells and PANC-1 spheres were exposed to 1.0 μM 3-Cl-AHPC for 7 days. Western blots were prepared as described in Materials and Methods. (b) mRNA expression of GLI1, GLI2, and Ptch1 in PANC-1 cells. Cells were grown in the presence of 1 μM 3-Cl-AHPC or vehicle alone (control). (c) Knockdown of IGF-1R expression by sh-IGF-1R inhibited sphere formation and enhanced ARR inhibition of sphere formation. The error bars represent the mean of three separate determinations +/− the standard deviation. •• was significantly different between spheres comprised of sh-vector cells treated with vehicle and 3-Cl-AHPC or AHP3. ♦♦ was significantly different between spheres comprised of sh-vector and IGF-1R-KD1 or IGF-1R-KD 2 at 7 and 14 days, respectively. ** was significantly different in comparison between IGF-1R-KDl/IGF-1R-KD2 spheres (vehicle treated) and IGF-1R-KD1/IGF-1R-KD2 spheres treated with 3-Cl-AHPC or AHP3. Data were analyzed by ANOVA, Tukey HSD test for multiple comparisons, ♦♦, ••, and **. (d) Knockdown (KD) of IGF-1R enhanced AHP3- and 3-Cl-AHPC-mediated apoptosis in the PANC-1 cells and IGF-IR protein expression in IGF-1R knockdown cells. Apoptosis was assessed using acridine orange/ethidium bromide staining as described in Section 2.
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