The Role Of Apatinib Reverses The Resistance Of Alectinib In EML4-ALK Positive Lung Cancer Cell Line H3122

Objective:Anaplastic lymphoma tyrosine kinase inhibitors(ALK-TKIs)changed the treatment strategy of the non-small cell lung cancer patients who harbouring EML4-ALK fusion gene mutations.The patients can get obvious survival benefit from it.However,almost all of the patients are inevitably arose resistance within one year after the initial treatment.The emergence of acquired resistance is the main obstacles for the greater clinical benefit.Bypass signaling pathways activating is one of the resistance mechanisms in targeted therapy of NSCLC.Mesylate apatinib is a multitargeted anti-cancer drugs.It can binding to the ATP site of vascular endothelial growth factor-2 efficiently and specificity.So it can inhibite the activation of VEGFR-2 and block the downstream signal transduction.It indicated that apatinib has the potential to improve the curative effect and has the ability of overcoming the acquired resistance that associated with angiogenesis.Of this study was to explore whether the insulin-like growth factor-1(IGF-1)induced resistance to Alectinib by activating the bypass signaling pathway and to explore the role of apatinib in alectinib resistance ulteriorly in H3122 lung cancer cell line which contains EML4-ALK fusiongene.Methods: 1.To detect the expression of IGF-1R in H3122 cell line by qRT– PCR.2.CCK-8 assay was used to detect the the sensibility of H3122 cell line(contain EML4-ALK fusion gene variant 1).(1)detect the proliferation inhibition of apatinib to H3122 cell line(2)detect the proliferation inhibition of alectinib to H3122 cell line(1)measure the proliferation ability of H3122 under different concentrations of alectinib.(2)detect the alteration of proliferation inhibition effect of H3122 cell when in presence of different concentrations of hIGF-1(50,100,150 ng/mL).(3)detect the cell growth inhibition of 10 umol/L apatinib on H3122-IGF-CR cell which triggered by hIGF-1.Calculating the half maximal inhibitory concentrations(IC50)of alectinib for H3122 cells and the resistance index of H3122-IGF-CR cells by GraphPad Prism 5 software.And the reversal index of apatinib for H3122-IGF-CR cells was calculated.3.The apoptosis rate was detected by Annexin V /7-PE AAD double staining methods.Selecting 0.03 umol/L alectinib and 10 umol/L apatinib as experimental concentration according to the result of CCK-8 assay.(1)detecting the apoptosis rate of H3122 cells after treating with alectinib for 48 h.(2)detecting the alteration of apoptosis rate of H3122 cell when in presence of100 ng/mL hIGF-1.(3)detecting the apoptosis rate of 10 umol/L apatinib on H3122-IGF-CR cells.4.Scratch assay and colony formation assay was used to evaluate hIGF-1and Apatinib effect on migration and proliferation ability of H3122 cells.Experiment group cells were treated with 100 ng/mL hIGF-1 or combination with 10 ?mol/L apatinib.5.The molecular mechanism of resistance was detected by western blot.EML4-ALK positive cell line H3122 was treated with alectinib alone or in combination with apatinib after induced by 100 ng/mL.The protein expression levels of PI3K/AKT,Ras-Raf-MEK-ERK/MAPK,JAK3/STAT3 and angiogenesis-associated signaling pathways HIF-VEGF-VEGFR were examined.Results: 1.The ct value of the IGF-1R in H3122 cell and internal reference was(20.90±0.06)and(12.48±0.12),respectively.And the ?ct value < 12,P <0.05,suggesting that IGF-1R gene was high expression in H3122 cell line.2.H3122 cells were sensitive to apatinib in a relative high concentrations.It cannot inhibit the proliferation of H3122 cells when its concentrations below10 umol/L,with IC50 values of 42.97 umol/L.3.The viability of H3122 cell was hypersensitive to alectinib and inhibited by alectinib in a dose-dependent manner,with IC50 values of(0.01737±0.17)?mol/L.4.However,under the exposure of hIGF-1,H3122 cells exhibited insensitivity to alectinib,with IC50 values of(0.2618±0.03)?mol/L.The resistance index was 15.07.While the addition of apatinib,the sensitivity of the resistant cell lines to alectinib was significantly increased,with IC50 values of(0.0905±0.07)?mol/L.The reversing multiple was 6.5.The differences in sensitivity to alectinib between the sensitive and the resistant cell H3122-IGF-CR were significant(P < 0.05).5.Alectinib induces apoptosis in H3122 cells,the apoptosis rates was(19.77±0.93)% when treated with 0.03 umol/L alectinib for 48 h.While combined with 100 ng/ml hIGF-1 for 48 h,the apoptosis rate was(8.35±0.37)%,significantly lower than alectinib monotherapy(P <0.05).Apatinib utilizedtogether with alectinib had a synergistic effect of enhancing the apoptosis of H3122-IGF-1-CR cells,with the apoptosis rate of(14.5667± 0.56)%,significantly higher than alectinib plus hIGF-1 treatment group(P <0.05).6.Scratch assay and colony formation assay was used to evaluate hIGF-1and apatinib effect on migration and proliferation ability of H3122 cells.In comparison with the blank group,cell migration was reduced in the Apatinib plus hIGF-1 group and was enhanced in the hIGF-1 group(P < 0.05).When stimulating with 100 ng/ml hIGF-1,the wound healing rate and the colony forming efficincy was increased.And combination with Apatinib significantly reduced the wound healing and colony formation in H3122 cell line.The wound healing rate was(80.79±1.28)vs(56.18±0.96),and the colony-forming efficiency was(31.5%±1.037)vs(16.5%±0.165)in hIGF-1 groupand the combination group,respectively.n=3,P< 0.05.7.The results from the Western Blot analysis showed that treating with 0.03umol/L alectinib for 4 hours could suppress the expression of the EML4-ALK gene downstream signaling protein.IGF-1R phos-phorylation was elevated in the hIGF-1 triggered alectinib resistance cells,together with upregulated expression of phosphorylated proteins,including AKT,mTOR,P70S6 K,ERK,STAT3.However,the drugs exhibited negligible inhibition on the total proteins.In addition,after exogenous hIGF-1 stimulated,the expression of angiogenesis-associated protein(HIF-1a,VEGFR2 and p-VEGFR2)were increased.The levels of p-AKT,p-P70S6 K,p-mTOR,p-ERK,p-STAT3 were remarkably decreased when combination with apatinib(P <0.05).Conclusion: hIGF-1 can induced EML4-ALK positive non-small cell lung cancer cells H3122 aquiring resistance by means of activating the bypass signaling pathway.Meanwhile,angiogenesis-associated signaling pathway wasactivated.Simultaneously inhibition of ALK and VEGF/VEGF receptor 2,by combination alectinib with apatinib,may be useful for controlling progression of EML4-ALK gene fusion lung cancer by reversing ALK-TKI resistance and for inhibiting angiogenesis.