Figure 1.
Proliferative effects on colorectal cancer cell lines plotted as IC50 following exposure to the PI3K/mTORi, PF-04691502 (0–5 umol/L).
Cells were plated in 96 well plates and allowed to adhere for 24 hours. Cells were exposed to increasing concentrations of compound for 72 hours and proliferation was assessed using CyQuant assay. Proliferaion assays were conducted in triplicate and IC50 values were calculated from the average proliferation. Mutational status of KRAS, BRAF, and PIK3CA are in colored boxes.
Figure 2.
Proliferative effects on colorectal cancer cell lines plotted as IC50 following exposure to the MEKi, PD-0325901 (0–1 umol/L).
Cells were plated in 96 well plates and allowed to adhere for 24 hours. Cells were exposed to increasing concentrations of compound for 72 hours and proliferation was assessed using SRB assay. Proliferation assays were conducted in triplicate and IC50 values were calculated from the average proliferation. Mutational status of KRAS, BRAF, and PIK3CA are in colored boxes.
Figure 3.
Growth inhibition of the PI3K/mTORi, PF-04691502 (PF-502) combined with the MEKi, PD-0325901 (PD-901) on four colorectal cancer cell lines.
LOVO (KRASG13D), HCT116 (KRASG13D/PIK3CAH1047R), WIDR (BRAFV600E/PIK3CAP449T), GEO (KRASG13D). Cells were exposed to various combinations of PF-502 and PD-901 for 72 hours. (A) Fraction inhibited was graphed following exposure to single agent and combination. (B) Bliss additivity was calculated and to assess combinatorial effects. Average bliss scores were graphed.
Figure 4.
Clonogenic analysis with the combination of the PI3K/mTORi, PF-04691502 (PF-502) combined with the MEKi, PD-0325901 (PD-901) on four colorectal cancer cell lines.
(A) LOVO, (B) HCT116, (C) WIDR, and (D) GEO were plated in 6 well plates and exposed to single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination for 72 hours. Drug was removed and replaced with media to allow for regrowth of clones. Cells were then stained with crystal violet and photographed. (E) The crystal violet was solubilized in 1% acetic acid and the absorbance was assessed on a plate reader. The data was normalized to control and graphed. All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *p,0.05, **p<0.01, ***p<0.001 vs vehicle, #p,0.05, ##p<0.01, ###p<0.001 vs PF-502, &p,0.05, &&p<0.01, &&&p<0.001 vs PD-901. All data represents three independent experiments.
Figure 5.
Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on downstream effector proteins assessed by immunoblotting.
Figure 6.
Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on Caspase 3/7 activity.
All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *p,0.05, **p<0.01, ***p<0.001 vs vehicle, #p,0.05, ##p<0.01, ###p<0.001 vs PF-502, &p,0.05, &&p<0.01, &&&p<0.001 vs PD-901. All data represents three independent experiments.
Figure 7.
Tumor growth rate analysis on patient-derived tumor xenograft models (PDTX). Tumor growth rates were determined for each individual tumor by fitting tumor volume data over the course of the treatment period to an exponential growth rate equation.
Each point represents a single tumor. Mean tumor growth rate ± standard deviation are represented by the bar and handles. All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *P<0.05 vs. Control; †P<0.05 vs. 901; ‡P<0.05 vs. 502.
Figure 8.
Effect of single agent the PI3K/mTORi, PF-04691502, the MEKi, PD-0325901 or the combination on downstream effector proteins assessed by antibody array.
Total protein was purified from PDTX at the end of treatment and assessed on a stress and apoptosis antibody array. Density of the spots were obtained, and graphed. All data presented as mean±SD, ANOVA Tukey’s adjusted p values: *p,0.05, **p<0.01, ***p<0.001 vs vehicle, #p,0.05, ##p<0.01, ###p<0.001 vs PF-502, &p,0.05, &&p<0.01, &&&p<0.001 vs PD-901. All data is a representative of three separate tumors from each group.