The Acidic Modification Of Gefitinib Sensitizes It To Non-small Cell Lung Cancer

Background:Statistics worldwide showed that lung cancer remains the leading cause of cancer incidence and cancer mortality(18.4% of total cancer deaths)and the most commonly diagnosed cancer type(11.6% of total cancers)[1].Non-Small Cell Lung Cancer(NSCLC)is the most important type,accounting for 75–80% of all lung cancers [2].Epidermal growth factor receptor(EGFR)and mutational activation of its signaling pathways are the most common drivers of NSCLC.EGFR-targeted small molecule tyrosine kinase receptor inhibitors(TKIs)have achieved some good results in the treatment of non-small cell lung cancer;however no matter how significant the initial treatment effect is,most patients can only get benefits around half a year to one year and then there will be the drug resistance.Therefore,to clarity the specific mechanism of EGFR-TKIs drug resistance to non-small cell lung cancer,and to design corresponding strategies to reverse drug resistance is very important for NSCLC treatment.At present,according to the research findings,some corresponding strategies have been designed to reverse the drug resistance of NSCLC of EGFR-TKIs and achieved excellent curative effects.However,in this situation,the re-resistance is still inevitable,indicating that there are other unclear drug resistance mechanisms.Our previous research found that,whether it is the first-generation EGFR-TKI Gefitinib or the third-generation EGFR-TKIs,Osimertinib(AZD9291),lysosomal acidification of NSCLC cells is inhibited after treatment,causing inhibition of degradation of SQSTM1 via autophagy and the expression accumulates,resulting in NSCLC drug resistance to EGFR-TKIs.Knockdown of SQSTM1 can increase the effect of EGFR-TKIs on NSCLC by increasing apoptosis in vivo and in vitro.However,the specific mechanism of how EGFR-TKIs inhibit lysosomal acidification is not completely clear.Aim:By analyzing the molecular conformation of Gefitinib and AZD9291,we found that both are as basic as the lysosomal inhibitor chloroquine(CQ),which is because of its basicity that inhibits lysosomal acidification and hinders lysosomal function.In order to further determine whether EGFR-TKIs inhibited lysosomal acidification due to their alkalinity,which affected the autophagy degradation of SQSTM1 protein,resulting in NSCLC drug resistance to EGFR-TKIs,we take Gefitinib as an example to chemically modify Gefitinib to neutralize its alkalinity,and explore the connection between Gefitinib after acidification and NSCLC,and provide a new strategy for reversing the drug resistance of EGFR-TKIs.Method:First,we used a method of chemical synthesis to label rhodamine B(Gefi-RB)on Gefitinib to track the location of Gefitinib in the cell.At the same time,we used Lyso-tracker staining to locate the lysosome,and observed the location relationship between Gefitinib and lysosome.The chemical synthesis method was used to transform Gefitinib.Two acidic phenolic hydroxyl groups were introduced at the 7 position of Gefitinib to neutralize its basicity(Gefi-2OH).The efficacy of the modified Gefitinib derivatives was observed in NSCLC using MTS,immunofluorescence,and western blot.Results:Using Gefi-RB and Lyso-tracker colocalization experiments,we found that Gefitinib is indeed enriched in lysosomes,and that si RNA knockdown of EGFR expression does not affect the accumulation of Gefi-RB in lysosomes.It was confirmed by magnetic resonance that we did introduce 2 acidic phenolic hydroxyl groups at position 7 of Gefitinib.We found that the inhibitory effect of the modified Gefitinib derivative Gefi-2OH on the EGFR signal was not significantly changed compared to Gefitinib.Further experiments found that compared with Gefitinib,Gefi-2OH significantly increased the proliferation inhibition of NSCLC cells,significantly reduced the inhibition of lysosomal acidity,and significantly reversed the accumulation of SQSTM1 expression.Conclusion:We found that EGFR-TKIs may indeed be enriched in lysosomes due to their alkalinity,thereby inhibiting lysosomal acidification,hindering the degradation of SQSTM1 via autophagy and accumulating expression,resulting in NSCLC drug resistance to EGFR-TKIs.Acidification of Gefitinib can significantly reduce its effect on lysosomal acidity,thereby enhancing the efficacy of Gefitinib on NSCLC.Our research found that the physical and chemical properties of EGFR-TKIs,such as acidity and alkalinity,are likely to affect their sensitivity,leading to NSCLC drug resistance.Chemical modification of EGFR-TKIs may be a new way to increase the sensitivity of EGFR-TKIs and reverse drug resistance.