Modulation Of ALDHA1 Activity Reverses Stem-like Cell-mediated EGFR-TKI Resistance In NSCLC

Objective:to study all trans retinoic acid（ATRA）could whether reverse stem-like cell-mediated epidermal growth factor receptor tyrosine kinase inhibitor（EGFR-TKI）resistance in non small cell lung cancer（NSCLC）and explore its potential mechanism.Methods:using amplification refractory mutation system（ARMS）method for the detection of NSCLC cell gene mutation status;using crystalviolet staining method to detect the growth of inhibiting to gefitinib and ATRA in NSCLC cells;flow cytometry was used to detect NSCLC cells aldehyde dehydrogenase A1（ALDHA1）activity and expression CD44.Results:H1650 cells and the fourth generation residual living cells after induction with high-concentration gefitinib have EGFR 19-Del gene mutation;EGFR gene excon mutation including Exon-18,Exon-19,Exon-20 and Exon-21mutation are not detected on A549 cells and the fourth generation residual living cells after induction with high-concentration gefitinib.NSCLC cells have not secondary EGFR gene mutation after induction with high-concentration gefitinib in a short period.Use high-concentration gefitinib to induce the fourth generation H1650 cells and build gefitinib tolerant cells（H1650/GR）;compared with parental generation H1650 cells,IC₅₀0 of H1650/GR cells are 1.61 times of that of parental generation H1650 cells,and the ability to tolerate gefitinib is enhanced significantly（P<0.001）,ALDHA1 activity and CD44 expression increase significantly（P<0.05）;use high-concentration gefitinib to induce the fourth generation A549 cells and build gefitinib tolerant cells（A549/GR）;compared with parental generation A549 cells,IC₅₀0 of A549/GR cells are 1.35times of that of parental generation A549 cells,and the ability to tolerate gefitinib is enhanced significantly（P<0.001）,and ALDHA1 activity and CD44expression increase significantly（P<0.05）.Indicates the fourth generation NSCLC cells are enriched with stem cell-like markers including ALDHA1 and CD44 after gefitinib induction.Use IC₂₀0 concentration ATRA to induce H1650/GR for one,three and five days respectively;compared with the group without adding ATRA,its ALDHA1 activity and CD44 expression decrease significantly（P<0.05）（ALDHA1 activity decreases by 6.81 times,and the proportion of CD44 expression decreases by 2.44 times on the fifth day）;after cultivation with sequential gefitinib for 72 hours,the result shows compared with the group without ATRA induction,the IC₅₀0 of ATRA induction group decreases significantly（P<0.05）（the group with ATRA induction for 5 days has decreased by 1.49 times）,and the reaction of the group with ATRA induction to gefitinib increases significantly;use IC₂₀0 concentration ATRA to induce A549/GR for one,three and five days respectively;compared with the group without adding ATRA,its ALDHA1 activity and CD44 expression decrease significant（P<0.05）（ALDHA1 activity decreases by 9.75 times,and the proportion of CD44 expression decreases by 2.48 times on the fifth day）;after cultivation with sequential gefitinib for 72 hours,the result shows compared with the group without ATRA induction,the IC₅₀0 of ATRA induction group decreases significantly（P<0.05）（the group with ATRA induction for 5 days has decreased by 1.28 times）,the reaction of the group with ATRA induction to gefitinib increases significantly.ALDHA1 and CD44 are positively related（P<0.01）,which indicates ALDHA1 and CD44 can be the co-expression stem cell-like markers of NSCLC.Conclusion:short time and high concentration gefitinib can induce NSCLC to enrich ALDHA1^hi CD44^hi stem cell-like cells.ATRA can reverse stem-like cell-mediated EGFR-TKI resistance in NSCLC by down regulating ALDHA1 activity.