| objective: To investigate the induction of microtubule-associated protein 4-anaplastic lymphoma kinase(EML4-ALK)in echinoderma by using insulin-like growth factor-1(IGF-1).EML4-ALK fusion gene positive lung cancer H3122 cell acquired resistance to alectinib,and explored the mechanism of metformin restore H3122 cell sensitivity to alectinib by activating IGF-1R signaling pathway.methods: 1.The proliferation of EML4-ALK-positive lung cancer cells H3122 under alectinib was detected by CCK-8.According to the CCK-8 instruction manual,using different concentrations of alectinib,EML4-ALK-positive lung cancer cell line H3122 was treated for 48 hours,cell proliferation was detected.After exogenously adding metformin(5 mmol/L)or(and)IGF-1(100 ng/L),EML4-ALK-positive lung cancer cell H3122 was treated for 48 h,and the proliferation of H3122 cells was detected.2.Flow cytometry detection of apoptosisFlow cytometry was used to detect the apoptosis rate of H3122 cells by treated with 0.1?mol/L alectinib for 48 h,and the apoptosis rate of alectinib induced by IGF-1(100 ng/mL)for 48 h after H3122 cells was detected.Apoptotic rate of H3122 cells after exogenous addition of metformin(5 mmol/L)or(and)IGF-1(100 ng/L).3.Western blot to detect the expression of key protein molecules in the signaling pathwayH3122 cells treated with alectinib alone or in combination with IGF-1(100 ng/mL)and metformin(5 mmol/L),to detect expression level of IGF-1R,AKT,mTOR,p70s6 k,AMPK,ERK and corresponding phosphoproteins And explore its molecular mechanism.Results:1.H3122 cells showed high sensitivity to alectinib,after 48 h,the higher the concentration of alectinib,the lower the cell viability and which performed the dose-dependent phenomenon.The IC50 was 0.033 ?mol/L.2.After the action of exogenous IGF-1,alectinib can inhibit the growth of H3122 cells,which can reduce the inhibitory effect of alectinib on the viability of H3122 cells.3.After induction of exogenous IGF-1,metformin was added,and the growth curve of alectinib in inhibiting H3122 cells was shifted to the left compared with IGF-1 alone,and metformin could increase the inhibitory effect of alectinib.4.The apoptosis rate of H3122 cell line treated with 0.1?mol/L alectinib and 5mmol/L metformin for 48 hours was(3.84±1.25)%,(19.27±2.29)%,(11.96±2.38)% and alectinib,respectively.The apoptotic rate after combined with IGF-1 was(6.57±0.147)%,which was significantly lower than that of alectinib alone(P<0.05).The apoptotic rate of alectinib combined with metformin was(35.68±1.47)%.The apoptotic rate of alectinib combined with IGF-1 and metformin was(20.96±1.08)%.The difference between groups was statistically significant(P<0.05)..5.induced by IGF-1 factor,IGF-1 can significantly increase p-IGF-1R and its downstream p-AKT,P-mTOR and P-p70s6 k protein levels and p-ERK,but can significantly reduce p-AMPK,and alectinib,metformin alone can successfully inhibit IGF-1R and its downstream signaling pathways.In addition,alectinib combined with metformin can significantly inhibit the protein levels of p-AKT,P-mTOR,p-p70s6 k and p-ERK,and increase the p-AMPK protein level.Compared with alectinib combined with IGF-1 group,alectinib combined with IGF-1 and metformin can significantly inhibit the protein levels of p-IGF-1R,p-AKT,P-mTOR,P-p70s6 k and ERK.Conclusion: IGF-1 induced EML4-ALK-positive lung cancer cell H3122 is resistant to alectinib through activation of the related pathway of bypass signal pathway,and metformin can reverse IGF-1 acquired resistance to alectinib of H3122.The mechanism may be related to metformin blocking the IGF-1R signaling pathway. |
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