The Mechanism Study Of Inhibiting Lung Cancer Cells Growth Via Targeting Esterase D And Autophagy

Autophagy is an acute cellular response mechanism.It respond rapidly to maintain homeostasis via autophagy when the intracellular or extracellular microenvironment changes.Therefore,autophagy is critical for cell survival Autophagy is a double-edged sword in the process of tumor growth.When nutrition is abundant,autophagy would suppress tumor growth.However,autophagy could promote tumor survival at worsening envitronment.Many small chemical molecules can enter the cell quickly,have good selectivity and can reversibly bind proteins.These small molecules can bind to intracellular factors to regulate autophagy.In the clinic,these small chemical molecules that target autophagy are applied to treat patients with tumorsFPD5,a pyrazoline fluorescent chemical small molecule,is a novel activator of esterase D(ESD).ESD is a carboxylesterase that is highly conserved in evolution.It is widely distributed in organisms and mainly located in the cytoplasm.In many patients with cancer,including leukemia,lung adenocarcinoma and other diseases,ESD levels or activity have been found to decrease.It remains unclear that the relationship between ESD activity and tumor growth.To solve the scientific problem,the activator FPD5 was using as a tool to clarify the relationship between ESD and tumor growth in A549 cell lineIn this study,we found that FPD5 reduced the viability of tumor cell in a dose-dependent manner after the treatment with FPD5,and did not cause apoptosis and necrosis in lung cancer cell A549.FPD5 could inhibit tumor growth on chicken embryo membrane in vivo.To clarify how ESD regulate cancer cell growth,we further explored the relationship between ESD and autophagy.After activating ESD by FPD5,we found that ESD promoted autophagy in a time-and concentration-dependent manner,and it induced primary autophagy flux.When ESD level was knockdown by RNA interference(RNAi),FPD5 did not promote autophagy and induce autophagic flux.We also observed that FPD5 promoted autophagy in tumor sections.The activity of mTORC1(Mechanistic target of rapamycin)is closely associated with autophagy.The results indicated that FPD5 could inhibit the activity of mTORC1 after activating ESD.FPD5 could not inhibit the activity of mTORC1 when ESD was knockdown.FPD5 also failed to inhibit mTORC1 activity in the presence of mTORC1 activator 3BDO.To study the molecular mechanism of ESD regulating mTORC1,we screened out the interacting protein FKBP25(FK506 binding protein 25)of ESD by yeast two-hybrid.Combined with co-immunoprecipitation,immunofluorescence and mass spectrometry aasays,we valiated that ESD interacted with FKBP25.We also performed RNAi assay,and it did not affect the activity and level of ESD after FKBP25 was knockdown,thus confirming that FKBP25 was ESD client protein.We performed the deletion mutation assay and defined the interaction of the N-terminus of FKBP25 with ESD.Our data indicated ESD activated by FPD5 bond more FKBP25,reduced the polyubiquitin chain level of FKBP25,inhibited the FKBP25 degradation via proteasome pathway,and increased the protein level of FKBP25.In addition,we also found that FKBP25 could inhibit mTORC1 activity and promote autophagy.Based on the activator FPD5,we have discovered the molecular mechanism of autophagy via ESD and the signal pathway ESD/FKBP25/mTORCl in lung cancer cell A549.It is clear that ESD could inhibit lung cancer cell A549 growth by promoting autophagy.Autophagy plays a complex role in tumor growth.It varied in different types of tumors and different signaling pathways.3BDO is a novel inhibitor of autophagy.In our previous study,3BDO activated the mTORC1 complex to inhibit autophagy.It inhibited proliferation of smooth muscle cells,and promoted differentiation of human embryonic stem cell via cell cycle arrest and inhibition of autophagy.Chloroquine(CQ),a commonly used antimalarial drug,is now being developed for the treatment of tumors in clinic.High dose of CQ can significantly inhibit the growth of tumor cells,but it is highly toxic and exceeds the clinically accepted range.However,CQ at low has little the inhibitory effect on cancer cell growth.It also increases a large number of acidic membrane vesicles and causes lysosome vacuolization,lead to lysosome dysfunction.In this study,we combined the autophagy inhibitor 3BDO and CQ to inhibit the growth of tumor cell A549 without causing tumor cell necrosis.3BDO caused A549 cell cycle arrest.3BDO combined with CQ was able to reduce the accumulation of intracellular acidic vesicles and inhibited lysosomal vacuolation To investigate the mechanism of inhibiting tumor cell growth and lysosomal vacuolation,we found that the combination was able to inhibit Na+,K+-ATPase activity and reduced the expression level of long nocoding RNA TGFB2-OT1(LncRNA TGFB2-OT1).Through this part of the study,we have defined the role of LncRNA TGFB2-OT1 level in tumor cell growth.The study provides a preliminary research basis and new ideas for the clinical application of CQ.