the nM range while no effect was seen in HBE135 E6E7 cells, even at concentrations as high as 1000 nM or twenty times the average IC50 observed in NSCLC cell lines. fgfr signaling These results suggest that NSCLC growth and survival mediated by a broad range of molecular factors are selectively sensitive to inhibition of PI3K by NVP BEZ235. Targets of NVP BEZ235 were determined by immunoblot analysis in cells with up to 24 hours drug exposure. Any drug exposure beyond 24 hours resulted in significant amount of dead cells and debris and would have limited the interpretability. pAKT, pP70S6K and pS6 decreased with exposure to the drug in a time dependent fashion, as shown by immunoblot analysis in Figure 4, panel A for H2170 and HCC2935 cell lines.
Relative to untreated cells, pAKT, pP70S6K, and pS6 were downregulated with NVP BKM120, LY294002, and NVP BEZ235. NVP BEZ235 was previously shown to cause PARP cleavage and induce apoptosis through activation of caspase 2, but not caspases 8, 9, and 10. We therefore studied the effect of NVP BEZ235 on PARP cleavage and caspase 2 activation in two most sensitive lung cancer cell Hesperidin lines, HCC2935 and H2170. As shown in Figure 4, panel B NVP BEZ235 treatment results in PARP cleavage and modest caspase 2 activation. Cleaved caspase 2 was not detected by western blotting in this assay. Synergism between the dual PI3K mTOR inhibitor NVPBEZ235 and the EGFR inhibitor erlotinib in NSCLC cell lines Our results so far show that mTOR blockade is necessary to enhance the inhibition of the PI3K pathway.
Given that inhibiting only one or two steps of the survival and proliferation pathways often proves to be therapeutically insufficient due to the various escape mechanisms, we studied the efficacy of multi level targeting by adding EGFR tyrosine kinase inhibition to dual PI3K mTOR inhibition in the same set of six NSCLC cell lines. Cells were treated with the PI3K inhibitor NVP BEZ235 alone or combined with erlotinib, at their respective single drug IC50, IC25 or IC10 concentrations, and viability was measured at 72 hours with the Cell Titer Glo assay. As shown in Table S6, synergism was seen in all six cell lines studied, including SW900 and SK MES 1 which are highly resistant to erlotinib alone. Notably, in all four erlotinib sensitive cell lines synergy was relatively more enhanced at higher concentrations of erlotinib.
A graphic example of the combined effect of these two drugs in H2170 and HCC2935 is shown in Figure 5 and Table S7. Of note, the majority of erlotinib concentrations used in these experiments are well below the reported steady state level of erlotinib achieved in actual patients which has been reported as 1200 2620 ng ml. This translates into 2797.2 21445.2 nM of erlotinib. Discussion Deregulation of the PI3K AKT mTOR signaling pathway has been demonstrated in NSCLC. In this study, we assessed the expression of PI3K subunits p85 and p110a in NSCLC tumor specimens. In the two