nM Aconcentrations ranging from 6.4 nM to 20,000 nM. All experiments were done in triplicate. After 72 hours incubation, cell FAK signaling viability was evaluated, and the IC50 was calculated for each cell line, and averaged for repeat experiments. As shown in Table 1, the IC50 for NVPBKM120 and LY294002 inhibition ranged from 716 nM to 1265 nM and 4123 nM to 11925 nM, respectively. To test whether growth inhibition inflicted by NVP BKM120 and LY294002 are specific or at least enhanced in malignant compared to normal cells, we used an immortalized, nontransformed bronchoaveolar cell line, HBE135 E6E7. These cells were derived from normal bronchial epithelium taken from a man undergoing lobectomy for squamous cell carcinoma.
NVPBKM120 and LY294002 had little effect on the normal bronchoaveolar cells at concentrations approximately 10 and 5 fold the average IC50 of these two drugs, respectively, Hesperadin in NSCLC cells. 13 growth inhibition was seen at 10 mM NVP BKM120 but an IC50 could not be reached at higher concentrations, and a mere 25 growth inhibition was obtained with 50 mM LY294002 treatment. Given that the expression levels of drug targets sometimes predict drug sensitivity, we studied the association between the degree of sensitivity resistance to NVP BKM120 and LY294002 and PI3K levels. We assessed the two PI3K subunits and total and phosphorylated AKT by immunoblotting. No clear association was found between pretreatment levels of PI3K or total and phosphorylated AKT and sensitivity resistance to NVPBKM120 or LY294002.
Synergism between PI3K and mTOR inhibitors Resistance to PI3K inhibition has been noted in different diseases and attributed to numerous mechanisms. Constitutive PI3K pathway activation could result from AKT activation by mTOR C2 or mTOR activation by MAP kinase pathway members despite specific PI3K drug inhibition. Due to the coexpression between p110a and mTOR seen in our clinical specimens, we studied synergism between the mTOR inbitor rapamycin and NVP BKM120 and rapamycin and LY294002. Concentrations of 1000 and 500 nM of NVP BKM120 were combined with a range of concentrations of rapamycin in six NSCLC cell lines. Synergism was seen in five of the six cell lines at all concentrations of NVP BKM120 with all three concentrations of rapamycin, and in the sixth cell line, synergy was seen at higher concentrations of NVP BKM120.
We then studied synergism between LY294002 and rapamycin. Concentrations of 5000 and 2500 nM of LY294002, were combined with a range of concentrations of rapamycin in six lung cell lines. Synergism was seen in all six cell lines at all concentrations of LY294002 with all three concentrations of rapamycin as shown in Activity of a dual PI3K mTOR inhibitor in NSCLC cell lines Given the synergism seen between PI3K inhibitors and rapamycin in lung cancer cell lines, a dual PI3K mTOR inhibitor that has been given to solid tumor patients in phase I clinical trials, NVP BEZ235, was studied. In all six lung cancer cell lines the