Effect And Mechanism Of Dual-target Inhibitor BEZ235Overcome Gefitinib Resistance In Lung Adenocarcinoma H1975Cell Lines

Part ⅠIdentification of characterization on lung adenocarcinoma H1975cell lines with gefitinib resistanceObjective:This study is to verify lung adenocarcinoma H975cell lines whether carried the T790M mutations and was resistant to gefitinib.Methods:The gefitinib resistant H1975cell lines was identified whether it carried the T790M and L858R mutations by gene sequencing. MTT assay was used to detect the effect of gefitinib to the proliferation of H1975cell lines to ascertain whether it was resistant to gefitinib.Results:Gene sequencing showed that the H1975cell lines contained the EGFR T790M and L858R mutations. IC50value of gefitinib alone in H1975cell lines was39.176μM, that was consistent with the describes of gefitinib acquired resistant cell lines. In the cell proliferation experiments.Conclusion:The lung adenocarcinoma H975cell lines contain the T790M and L858R mutations and conform with the characteristics of the cell lines that is resistant to gefitinib. Part IIEffect and mechanism of dual-target inhibitor BEZ235on lung adenocarcinoma H1975cell lines in virtoObjective:Through cell experiments, the sensitivity of the H1975cell lines to dual target PI3K/mTOR inhibitor BEZ235was preliminarily assessed. The effect of the dual-target inhibitor BEZ235to lung adenocarcinoma H975cell lines with gefitinib resistance and its role to PI3K/Akt/mTOR signaling pathway in H975cell lines, and try to clarify whether BEZ235can inhibit PI3K/Akt/mTOR signaling pathway and overcome gefitinib resistance caused by the EGFR T790M mutation.Methods:The inhibiting effect of BEZ235alone or combation with gefitinib on the proliferation of lung adenocarcinoma H975cells were studied. The1975cells were devided into four groups treated with DMSO as control, BEZ235, gefitinib and combin-ation with BEZ235and gefitinib respectively. The influence of different combinations of BEZ235and gefitinib on H975cell migration was evaluated through cell scratch test. Transwell chamber invasion test is used to assess the effect of different combinations with BEZ235and gefitinib to the ability of H1975cell invasion. Flow cytometry was employed to measure the distribution of cell cycle and apoptosis of H975cell intervened by BEZ235and(or) gefitinib. The transcrip levels of PIK3CA, mTOR and VEGF were measured by reverse transcription-PCR assay in H975cell lines intervened by BEZ235and(or) gefitinib. Timmunofluorescence and western blotting assay were applied to detect the expressing level of phosphorylated protein in the PI3K/Akt/mTOR signaling pathway,such as p-mTOR, p-Akt p-S6and,as well as Akt and S6.Results:The IC50value of BEZ235alone was0.327μM, and showed a concentration-dependent. When it was combined with gefitinib, the IC50value was0.073μ.M, there were significant differences between the two groups(P<0.05). The abilities of migration and invasion of H1975cell lines that were treated BEZ235alone or combination with gefitinib were inhibited to be compared with gefitinib alone, the difference was statistically significant, but no significant difference in G1phase arrest between the four groups. The expression of PIK3CA were up-regulated and that of VEGF were inhibited at the transcriptional level by BEZ235alone or combination with gefitinib, while the expression of mTOR was down-regulated either BEZ235or gefitinib. Western blot showed that BEZ235could inhibit the phosphorylation levels of p-Akt and p-S6in PI3K/Akt/mTOR signaling pathway, while the expression of total protein were not significant down-regulated as Akt and S6in any group.Conclusion:The dual inhibitor BEZ235may inhibit the proliferation of H1975cell lines, and combine with gefitinib to show a synergistic effect on proliferation inhibition. BEZ235can inhibit the proliferation of H1975cell lines that are resistant to gefitinib through down-regulating the phosphorylated protein in PI3K/Akt/mTOR signal pathways, and can effectively inhibit tumor growth signals are passed to downstream of PI3K/Akt/mTOR signal pathway, and can overcome the gefitinib acquired resistance caused by EGFR T790M mutation. BEZ235can inhibit the abilities of migration and invasion of human lung adenocarcinoma H1975cell lines and increase the proportion of apoptotic and necrotic cells in vitro. Part ⅢEffect and mechanism of BEZ235on xenograft of lung adenocar-cinoma H1975cell lines in BALB/c nude miceObjective:Through xenograft models of BALB/c nude mice,we explored the effect of the PI3K/mTOR dual-target inhibitor BEZ235to lung adenocarcinoma H1975cell line with gefitinib resistance and its role to PI3K/Akt/mTOR signaling pathway in H1975cell lines, and try to clarify whether BEZ235can inhibit PI3K/Akt/mTOR signaling pathway and overcome gefitinib resistance caused by the EGFR T790M mutation and suppressed both tumor growth and angiogenesis in BALB/c nude mice.Methods:32nude mice were devided into four groups(N=8) treated with DMSO as control, gefitinib, BEZ235and combination with BEZ235and gefitinib respectively. Xenograft model of BALB/c nude mice were established by H1975cell lines transplanted subcutaneously. Western blot assay of xenograft intervened by BEZ235and(or) gefitinib were applied to detect the expressing level of phosphorylated protein p-mTOR,as well as VEGF. The growth curve of transplanted tumor in BALB/c nude mice were drawn to compare whether the tumor growth were different in four groups. When BALB/c nude mice were treated by BEZ235and/or gefitinib, the time to tumor formation and tumor volume were compared to decide whether they were different. After the intervention was finished, the tumor weight of BALB/c nude mice were evaluated to decide the effect of intervention whether there are differences in four groups. MR imaging was used to evaluate whether tumor growth is affected by the intervention or no. The expression of VEGF and CD31in tumor tissues of xenograft in BALB/c nude were detected by IHC to decide whether BEZ235could inhibit angiogenesis.Results:The study of xenograft in BALB/c nude mice have shown that the tumor growth was inhibited, when BALB/c nude mice were treated by BEZ235alone or combination with gefitinib, the combination group was more significant. The volume and weight of tumor were compared with both control and GEF groups were significant difference (P<0.01). Western blot assay of tumor tissue in BALB/c nude mice showed that the expression of p-mTOR and VEGF were down-regulated in BEZ235alone or combination with gefitinib, which was similar with the results in vitro. Immunohisto-chemical assay showed that the expression of p-S6,VEGF and CD31in tumor tissue were significantly decreased in BEZ235alone or combination with gefitinib.Conclusion:BEZ235can down-regulate the expression of CD31and VEGF, and thus inhibit tumor angiogenesis in xenograft of H1975cell lines in BALB/c nude mice, and can inhibit the transplanted tumor growth of H1975cell lines in BALB/c nude and has synergistic effect with gefitinib in vivo.