Role Of MTOR Signaling Pathway In 17-DMAG Overcoming Acquire Resistance To Alectinib In EML4–ALK Positive Lung Cancer Cell Line H3122

Objective: To observe the change of mTOR signaling pathway in17-DMAG overcoming acquire resistance to alectinib induced by bypass signal pathway activation in EML4-ALK fusion gene positive lung cancer cell line H3122 and to explore its potential regulatory mechanisms.Methods: By adding transforming growth factor alpha(TGF-α)and epidermal growth factor(EGF)(100ng/ml)activating bypass pathway to induce H3122 cells resistant to alectinib,finally observing the resistance whether can be circumvented after joining in 17-DMAG.Cell viabilities were assessed after72 h by CCK-8 assay.Cell appotosises were evaluated by the AnnexinⅤ-PE assay.Finally,Western blotting was used to detect the expression of ALK,EGFR and phosphorylated proteins,and to determine the expression and activation levels of the key proteins in the mTOR signaling pathway.Results: Alectinib inhibited the viability of H3122 cells in a dose-dependent manner for 72 h,and which IC50 was 0.042 μmol/L.H3122 cells were insensitive to alectinib combined with TGF-α or EGF(100ng/ml),IC50 is more than 10 μmol/L.Only 17-DMAG inhibited the viability of H3122 cells in adose-dependent manner,and which IC50 was 0.245μmol/L.even in the presence of TGF-α or EGF,the viability of H3122 cells was also inhibited by17-DMAG.IC50 were 0.251μmol/L and 0.301 μmol/L,respectively.After treated with 0.05μmol/L alectinib for 72 h,the apoptosis rate of H3122 cells was(30.01 ±0.92)%;the apoptosis rate of alectinib combined with TGF-α or EGF were(6.36±0.14)% and(6.13±0.21)%,respectively,significantly lower than that of alectinib single treatment(P<0.001).After treated with 0.03μmol/L17-DMAG alone or combined with TGF-α or EGF for 72 h,the apoptosis rate of H3122 cells were(28.37 ±1.75)%,(26.69 ± 1.2)% and(26.62 ±0.72)%,respectively.the apoptosis rate between each other had no significant difference(P > 0.05).Alectinib inhibited p-ALK and p-mTOR,and the activation levels of the key proteins in upstream and downstream of mTOR pathway were also inhibited in H3122 cells.EGF significantly increased the expression of p-EGFR,p-mTOR and the activation levels of the key proteins in upstream and downstream of mTOR pathway in cells.Although alectinib inhibited p-ALK,but could not inhibit the expression of p-mTOR and the activation levels of the key proteins in upstream and downstream of mTOR pathway induced by EGF.17-DMAG inhibited the expression of ALK,EGFR,mTOR signaling pathway proteins and their activated state proteins in the cells,irrespective of EGF presence.Conclusions: The way of activating bypass signaling pathway mediated resistance to alectinib in EML4-ALK fusion gene positive lung cancer cell line H3122 is feasible.mTOR signaling pathway play a role in 17-DMAG overcoming acquire resistance to alectinib induced by activating bypass signaling pathway,which provides a basis for resistance and conquering resistance mechanism of alectinib.