Each experiment included triplicate wells and the experiment was repeated twice. Statistics Statistical analyses were carried out using a two sided t test and ANOVA with post hoc test. P 0. 05 was selleck chem considered significant. Results Differential effects of RAD001 and AZD8055 on proliferation and signalling in acquired endocrine resistant models The allosteric mTOR inhibitor RAD001 was a relatively poor inhibitor of growth measured over seven days in MCF7 derived tamoxifen resistant cells with an IC50 of 950 nM. In long term oestrogen deprived resistant MCF 7 cells, RAD001 was found to be even less potent with an IC50 1 uM. In contrast, the mTOR kinase inhibitor AZD8055 at 10 to 100 nM was a very Inhibitors,Modulators,Libraries effective inhibitor of growth in TamR cells with an IC50 of 18 nM.
AZD8055 10 to 100 nM also substantially inhibited growth of the MCF7 X cell model, with an IC50 of 24 nM, although MCF7 X cells were significantly less sensitive than the TamR cells to AZD8055 when examined at 25 nM and 50 nM. Additional studies performed in a tamoxifen Inhibitors,Modulators,Libraries resistant T47D derived model further confirmed that AZD8055 was highly growth inhibitory in acquired endocrine resistant cells. Despite having a poorer effect on cell growth, one hour treatment with RAD001 was still shown to inhibit mTORC1 associated signalling pathways with TamR cells being slightly more sensitive to RAD001 than MCF7 X cells. In both cell lines, RAD001 at 100 nM caused a reduction in mTOR phosphorylation at s2448, which has previously been described as the site predominantly associated with the mTORC1 complex.
Downstream p70S6K phos phorylation in TamR was undetectable after treatment with 1 nM RAD001 and pS6 activity was inhibited with RAD001 10 nM. These mTORC1 downstream pathways were less sensitive to RAD001 in MCF7 X cells, but phos phorylation of p70S6K and pS6 were still inhibited by 100 nM RAD001. However, in both models, there was no impact Inhibitors,Modulators,Libraries of one hour treatment with RAD001 on pPRAS40. RAD001 was a poor inhibitor of mTORC2 pathways in both TamR and MCF7 X cells, indicated by its failure to signifi cantly reduce both s473 Akt phosphorylation and mTOR phosphorylation at s2481, a site known to be associated with mTORC2. In both TamR and MCF7 X cells, RAD001 also failed to inhibit 4EBP 1 phosphorylation on the t37 46 site which has previously been described as rapamycin insensitive.
In contrast to RAD001, one hour treatment with AZD8055 inhibited pathways Inhibitors,Modulators,Libraries associated with both mTORC1 and mTORC2 signalling in both TamR and MCF7 X endocrine resistant cell lines. At con centrations from 1 to 100 nM, the inhibition of mTORC1 pathway elements, p p70s6kinase and p S6 ribosomal protein was similar or superior with AZD8055 to that seen with RAD0001. While inhibition of p PRAS40 was Inhibitors,Modulators,Libraries not detected after one STI571 hour treatment with RAD001, PRAS40 phosphorylation was eliminated by 100 nM AZD8055 in both TamR and MCF7 X cells.