Effects And Mechanisms Of Combined Treatment With Everolimus And Gefitinib On Non-small Cell Lung Cancer Cell Lines

Lung cancer is the leading course of mortality in oncology, and majority of the cases are non-small cell lung cancer (NSCLC), most of the NSCLC patients were advanced when diagnosis. Patients with EGFR gene non-mutation or EGFR mutation are treated with chemotherapy based on platinum or epidermal growth factor receptor tyrosine kinases inhibitor (EGFR-TKI) respectively. EGFR-TKI is an oral drug with less toxicity, but only 30% of all the NSCLC patients who harboring the EGFR mutation have the opportunity of benefiting from EGFR-TKI, however, patients who are sensitive to EGFR-TKI have a mean PFS of 11 months.mammalian target of rapamycin complex 1(mTORC1) is the target of Rapamycin, rapamycin and FK-binding protein(FKBP-12) combine to rapamycin-FKBP-12 complex, the resulting complex interacts with and inhibits mTOR and lead s to the downregulation of phosphorylated p70S6K and upregulation of 4EBP1.Rapamycin analogs tersirolimus (CCI-779) and everolimus (RAD001) have been approved by FDA for the treatment of advanced renal cell cancer. We speculate that combination of EGFR-TKI and mTOR inhibitor would be a useful strategy for patients who are resistant to EGFR-TKI. The aim of our experiments is to explore the effects of combination treatment of EGFR-TKI with mTOR inhibitor, and discover the mechanisms of combination treatment. Chapter 1 inhibit effect of combined treatment of gefitinib and everolimus in vitroMethodsCells were exposed to gefitinib, everolimus or the combination and the effects of inhibition were analyzed with MTT assay. Using CalcuSyn software to calculate the combination index (CI).Resultsthe IC50s of gefitinib were varying in different cell lines ranged from 0.02 to 14.9μmol/L,the IC50s of everolimus were similar ranged from 9.3-23.7μmol/L.cell lines were divided into gefitinib sensitive group and gefitinib resistant group by the cut-off value of IC50 1μmol/L.for the resistant group, cells were treated with concurrent and sequential regimens. Factor analysis was used to analyze the inhibit rates of different regimens. The combination treatments are superior to gefitinib or everolimus along(compared with gefitinib F=18.897,P<0.001; compared with everolimus (F=16.555,P<0.001). the comparisons between concurrent and sequential regimens were processed with factor analysis, for gefitinib-resistant cell lines, varying doses of gefitinib combined with 5μmol/L everolimus, there was significant difference between the two regimens(F=11.883,P=0.003); different doses of everolimus combined with 5μmol/L gefitinib, no significant different was observed (F=0.086,P=0.773).For the gefitinib-sensitive cell lines, no significant difference was observed between concurrent and sequential regimens when using the combination of varying doses of gefitinib and 5μmol/L everolimus. However, different doses of everolimus combined with 0.005 or 0.01μmol/L gefitinib, the responses of two regimens showed significant difference.The synergism can be observed in all three gefitinib-resistant cell lines when given lower doses of concurrent or sequential regimens, but these drugs just showed middle or weak synergistic effect. The inhibitory response increased with the accumulation of drugs doses, but the CI values surpass 1 and showed no synergism. In the gefitinib sensitive cell lines, the combination regimen showed synergistic effect with lower doses in HCC827, and showed strong synergistic effect in PC-9 cell lines.ConclusionThe combination treatment of gefitinib and everolimus achieved dose dependent enhanced proliferation inhibition. For gefitinib resistant cells, the concurrent regimens showed stronger inhibit effencicy and for the resistant cells, the everolimus→gefitinib sequential regimens were better.Chapter 2 effect on signaling pathway by the combined treatment of gefitinib and everolimusMethodsEstablishing models of cells treated with different drugs and the combination. Observing responses of signaling pathway moleculars with the methods of western blotting.ResultsBy the method of western blotting, we showed here the combination of gefitinib and everolimus showed stronger inhibitory of the p-mTOR and p-p70S6K expression than either of the single drug in gefitinib sensitive and resistant cell lines. Such inhibition showed a tendency of dose dependence and time dependence. Everolimus can effectively inhibit the phosphorylation of mTOR and p70S6K, but showed no apparent inhibition on p-Akt and p-MAPK or even activate such molecular.The PC-9 was exposed to varying doses of LY294002 for 24h. As a small molecular inhibitor of PI3K, LY294002 downregulated phosphorylation of EGFR, Akt, mTOR, p70S6K but not MAPK. However, gefitinib reduced the level of phosphorylated MAPK effectively.A gefitinib resistant cell line (PC-9GR) was established from PC-9, which showed a 20 times IC50 value compared with that of the primary cell line.0.1μmol/L Gefitinib totally inhibited p-EGFR in PC-9, but can only partly inhibited p-EGFR in PC-9GR.everolimus improved the inhibition of gefitinib and showed the value of combination treatment. ConclusionThe combination treatment could effectively inhibit dual p-mTOR and p-MAPK; through inhibit the PI3K/Akt/mTOR/p70S6K and Ras/Raf/MAPK/ERK pathway.