PNA-TPP Activates ROS-Induced Autophagy To Promote Apoptosis Of A549 Stem Cells

In recent decades,lung Cancer incidence and mortality are rapidly growing,which has become one of the most common malignant tumors.Through the efforts of researchers,the mortality rate of lung cancer has been significantly decreased,but 5-year survival rate still less than 18%.The poor prognosis of lung cancer is mainly due to the lack of understanding of tumor occurrence and drug resistance mechanisms.Recent studies showed that CSCs are regarded as the major cause of the failure in anticancer therapy.Our previous study found that the concentration of ROS in A549 stem cells was significantly lower than that in A549 cells,it helps protect lung cancer stem cells from chemotherapeutic damage and apoptosis,so Increasing the concentration of reactive oxygen species become one of the important strategies to enhance chemosensitivity.Autophagy,a process of cellular self-digestion and response to stress,has a role in tumor formation and progression,and it may play a dual role in CSCs-related resistance to anticancer therapy,However,the role of lung cancer stem cell resistance remains controversial.Autophagy is induced by ROS and functions to mitigate oxidative stress,but very high ROS levels may result in apoptosis.Our previous studies have shown that the compound of peptide nucleic acids(PNAs)coupled with triphenylphosphonium(TPP)can significantly activate ROS concentration in lung cancer stem cells and reduce the high drug resistance.Our study aims to construct antisense peptide nucleic acid-triphenylphosphine complex(PNA-TPP),activate ROS and mediate autophagy in lung cancer stem cells,and provide theoretical basis for future clinical application.Objective:1.To clarify the role of lower basal autophagy in high-resistance in A549 stem cells.2.PNA-TPP induces autophagic apoptosis of A549 stem cells by increasing ROS concentration via pi3k/akt/mTOR pathway,and further reduces the chemical resistance of lung cancer stem cells.Methods:1?A549 stem cells were formed by serum-free medium(SFM);Peptide nucleic acid-triphenyl phosphonium(PNA-TPP)complexes were constructed and synthesized by solid phase method;2?The changes of autophagosomes in A549 stem cells after PNA-TPP transfection were observed by laser confocal microscopy.3?The transfection efficiency of the fragmented siATG5 and simTOR transfected A549 stem cells was verified by Western-blot and RT-PCR.4?Detection of ROS levels of PNA-TPP transfected A549 stem cells by reactive oxygen species kit.5?CCK-8 kit to detect the proliferation level of A549 stem cells transfected byPNA-TPP and interference fragments.6?AnnexinV-FITC/PI double staining was used to detect the apoptosis level of A549 stem cells transfected by PNA-TPP and interference fragments.7?Western-blot blotting was used to detect the expression of autophagic proteins LC3 II and P62 and the expression of pi3 k,p-akt/akt and p-mTOR/mTOR proteins in PNA-TPP transfected A549 stem cells.Results:1?The expression of autophagy,ROS content and apoptosis rate of A549 stem cells were lower than those of A549 cells;2?A549 stem cells were transfected with the PNA-TPP complex,which has been identified as unimodal and of good purity.The number of autophagosomes and fluorescent dot-like aggregates increased,even the expression of autophagy protein LC3 II,ROS content and apoptosis were increased in A549 stem cells transfected with PNA-TPP;3?siATG5 can reverse the growth inhibition and apoptosis-promoting effect of NA-TPP on A549 stem cells;while simTOR can increase the growth nhibition and apoptosis-promoting effects of A549 stem cells.4?NAC can reverse the high ROS concentration of PNA-TPP transfected A549 tem cells,high autophagy protein LC3 II expression,and growth inhibition nd apoptosis.5?The results of Western blot showed that the expression of the phosphorylation evels of AKT and mTOR protein were attenuated when treated by PNA-TPP,although the levels of AKT and mTOR did not exhibit significant changes.The addition of chloroquine and NAC could reverse the inhibition of hosphorylation levels of AKT and mTOR protein on A549 stem cells by NA-TPP.Conclusion:1.The levels of autophagy,ROS and apoptosis of A549 stem cells were lower than those of A549 cells,indicating that low basal autophagy is one of the reasons for leading high drug resistance of A549 stem cells2.PNA-TPP induces autophagic apoptosis of A549 stem cells via pi3k/akt/mTOR pathway by increasing ROS concentration.