Fig. 2
Intracellular pathways and selective inhibition of PI3K/AKT axis. a, western blot analysis of intracellular transducers and their phosphorylation status at baseline conditions in parental versus resistant cell lines; AKT and its downstream effectors S6RP and 4EBP1 result activated preferentially in CM-RES clones. Tubulin was used for normalization of protein extract content. b, sensitivity of parental and resistant cell clones to treatment with the selective PI3Kα inhibitor pictilisib (GDC-0941) or the selective AKT1/2 inhibitor ipatasertib (GDC-0068) after 96-h treatment (range 0,05–10 μmol/L) evaluated for proliferation by MTT assay, as described in the Materials and Methods. All the results are average ± SD of 3 independent experiments, each done in triplicate. *: p < 0.05. c, combination of cetuximab and refametinib (BAY-86-9766), used at the fixed ratio 1 μg/ml:1 μmol/L, with pictilisib (GDC-0941), used at the IC50 ratio, as described in the Chou-Talalay model of synergism. Values on the X-axis refer to cetuximab and refametinib. Cetuximab:Refametinib:Pictilisib ratios are, respectively: 16 μg/ml:16 μmol/L:1 μmol/L for HCT116 CM-R, 150 μg/ml:150 μmol/L:1 μmol/L for HCT15 CM-R, 2 μg/ml:2 μmol/L:1 μmol/L for LOVO CM-R, 2 μg/ml:2 μmol/L:1 μmol/L for SW480 CM-R. d, Combination index (C.I.) analysis of the combination between cetuximab, refametinib and pictilisib in the resistant cell lines at different Effective Doses (EDs). CI < 1 indicates synergism, while CI < 0.5 indicates strong synergism