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. 2016 Feb 10;7(12):14220–14240. doi: 10.18632/oncotarget.7303

Figure 6. Effects of IGF-1R inhibitor AEW541 on AR−/PSA− PC3 and Du145 cells and PSA−/lo LNCaP cells.

Figure 6

A-D. PC3 and Du145 cells are sensitive to AEW541. In A and C, PC3 (A) or Du145 (C) cells (2,000/well) were plated in 96-well plates. After cells attached to the plate (12 h), vehicle control (Ctrl; DMSO) or AEW541 (10 μM final concentration) was added to the wells and relative live cell number was determined by WST-1 assays at day 0, 5, and 8 (see Supplementary Methods). Presented are fold changes in cell numbers (**P<0.05; ***P<0.001). Apoptosis assays were performed using the Vybrant kit and shown in B and D are representative histograms showing AEW541-induced cell death. E. Dose response of AEW541 in LNCaP cells, as measured by WST-1 assays. F-G. The PSA−/lo LNCaP cells are susceptible to AEW541. Purified PSA+/PSA−/lo LNCaP cells were utilized in WST-1 cell growth assays (F) and Vybrant apoptosis assays (G) in the presence of DMSO (Ctrl) or AEW541 (10 μM). **P<0.05; ***P<0.001. H-J. AEW541 inhibits LNCaP sphere formation. Bulk LNCaP cells were plated at the clonal level in methylcellulose based sphere assays in the presence of DMSO (Ctrl) or AEW541 (10 μM). 2 weeks later, sphere number and size were measured (***P<0.001). Shown in H are representative images. K-L. AEW541 is more effective than CDSS+MDV3100 in killing LNCaP and VCaP (E) cells. Bulk LNCaP or VCaP cells were plated at 2,000 cells/well in triplicate in 96-well plates and cultured in RPMI-7% FBS. Cell number in each well was assessed by WST-1 assays in the presence of DMSO (Ctrl). Cells were then treated with DMSO (ctrl), CDSS+MDV3100 (20 μM), AEW541 (10 μM), or combination. Live cells were determined at the indicated time points, and results are presented as fold increase. In K, the difference between Ctrl vs. other conditions at D2-D12 was all statistically significant (P<0.01). In L, the difference between Ctrl vs. the other three conditions at D2-D12 was statistically significant (P<0.001). The difference between CDSS+MDV3100 vs. AEW541 or combination at D2-D12 was also statistically significant (P<0.05).