Figure 4

Dose response curves were generated for changes in P-AKT in LCC6 and LCC6^Her2 cells after treatment with QLT0267, docetaxel, or a fixed ratio combination of QLT0267 and docetaxel. LCC6 and LCC6^Her2 cells were treated for eight hours with increasing concentrations of QLT0267, docetaxel, or a fixed ratio combination of QLT0267 and docetaxel (50 μm:1 nm) to establish dose response curves based on an endpoint measuring suppression of P-AKT levels as determined by western blot analysis. Representative western blot images were collected using a fluorescence based imaging system (odyssey, Licor) where the colours (red or green) represent the secondary antibody used, show that increasing concentrations of (b) docetaxel (Dt) exerted no significant effect on the expression of integrin-linked kinase (ILK), protein kinase B (AKT), and phosphorylated protein kinase B (P-AKT) in either cell line. Treatment with increasing doses of (a) QLT0267 (267) alone or (c) in combination with Dt showed dose-dependent decrease in P-AKT. Densitometry assessment of western blots (n = 3) were used to estimate treatment response relative to controls (taken to be 100% P-AKT levels) and the resulting data was then analyzed by Calcusyn to determine estimated DRI. The DRI was then used to estimate the dose of 267 when used alone (black bars) or in combination with Dt (grey bars) needed to achieve a defined fraction affected (FA). The dose of drug required to achieve an FA of 0.5 is defined as the ED50 for the measured P-AKT suppression endpoint. The ED50 of 267 was about 30 μM in the (d) LCC6 cell line, while in the presence of (e) Dt the 267 ED50 was about 11 μM. In LCC6^Her2 cells, more 267 was required when used in combination to achieve FA similar to that of single agents. For example in the LCC6^Her2 cells, the 267 ED50 when used alone was about 30 μM, and this increased to 130 μM when 267 was used in combination with Dt.