The Research Of Enhancing Chemotherapy Sensitive Of Lung Adenocarcinoma Stem Cells By PNA-TPP Regulating Glutathione Antioxidant System

Objective:1.To detect the levers of mRNA and protein expression of glutathione-related and apoptosis-related genes in lung adenocarcinoma A549 cells and A549 cell spheres.2.To observe the effect of peptide nucleic acid-triphenylphosphine(PNA-TPP)on the anti-oxidation system of glutathione and cisplatin in lung adenocarcinoma A549 cell spheres.3.we preliminarily study on the regulation mechanism of PNA-TPP regulating glutathione antioxidant system.Methods:1.Intensive dilution method to selects tight monoclonal cells and serum-free medium(SFM)method to cultures them to form A549 cell spheres;2.Using CCK8 kit detects the proliferation inhibition rate of A549 cells and A549 stem cell spheres under different concentrations of cisplatin and calculates the IC50 half of the cisplatin(maximum inhibitory concentration).3.The kits were used to detect the levels of ATP,oxidized glutathione GSSH,reduced glutathione GSH and reactive oxygen species in A549 cells and A549 cell spheres.4.The expression of GCLC,GCLM,GSS and X-CT in A549 and A549 stem cell spheres were detected by Western Blot.The expressions of GCLC,GCLM,GSS and xCT mRNA were detected by RT-PCR.5.The antisense peptide nucleic acid-triphenylphosphine complex(antisense PNA-TTP)was constructed by solid phase peptide method.Western blot was used to detect the expression of glutathione synthesis related genes after PNA-TPP transfection in A549 and A549 sphere cells.6.Flow cytometry was used to detect changes in apoptosis levels after PNA-TPP with or without cisplatin treatment of A549 cell spheres.Western blot was used to detect the expression of apoptosis-related protein after PNA-TPP with or without cisplatin treatment of A549 cell spheres.7.The kits were used to detect the changes of cytoplasmic ATP,reduced glutathione GSH and reactive oxygen species after PNA-TPP with or without cisplatin treatment of A549 cell spheres.8.GCLC overexpression plasmid and SiRNA-GCLC was transfected into A549 cell spheres.The transfection efficiency was observed by fluorescence microscopy.The expression levels of GCLC mRNA and protein were detected by Western blot and RT-PCR respectively.The transfection efficiency was verified and the optimal interference fragment was screened.9.The protein levels of GCLC of A549 cell spheres before and after transfection with Overexpression or interference GLCL plasmid with or without PNA-TPP were measured by Western Blot.The glutathione levels in each group were detected by kit.The ROS levels in each group were detected by microplate reader.Results:1.Compact monoclonals were Picked out Successfully and A549 cell spheres were induced from the A549 cells by serum-free medium.An inverted phase contrast microscope observed that there were hundreds to thousands of cells in each sphere with good light transmission.2.With the concentration of cisplatin increased,the proliferation inhibition rate and the expression levels of Capase3 and BaX in A549 cells and A549 cell spheres were increased,while the expression level of Bcl-2 decreased.The proliferation inhibition rate of A549 cells was generally higher than that of A549.The IC50 was higher in the A549 cell spheres than in the A549 cells(9.191±0.4638 VS 5.109±0.1608)mg/ml.3.The PNA-TPP complex was synthesized successfully,which is identified as a single peak and no other impurity peaks,with good purity.4.Compared with A549 cells,the content of GSSG,GSH and total GSH in A549 cell spheres were increased,and the contents of GSH/GSSH,ROS and ATP were decreased.While GCLC,GSS,x-CT mRNA and protein expression were increased,and GCLM mRNA and protein expression were decreased.After PNA-TPP was transfected into A549 cells and A549 cell spheres,the expression levels of GCLM,GSS and x-CT were increased,while the expression of GCLC protein was decreased.5.Compared with the control group,the apoptosis rate and expression of Capase3?Bax were increased,while the expression of Bcl-2 were decreased with A549 cell spheres were treated with PNA-TPP or cisplatin alone.There was no statistical difference between the two groups.After treatment of A549 stem cell spheres with PNA-TPP combined with cisplatin,the apoptosis rate and expression of Capase3 and Bax were significantly increased,while the expression of Bcl-2 was significantly decreased.Compared with the single-drug treatment group,the change was obvious.The difference is statistically significant.6.Compared with control group,the content of GSH and ATP were decreased after A549 cell spheres were treated with PNA-TPP or cisplatin alone,Accompanied with the increased ROS content.After treated with PNA-TPP combined with cisplatin,the content of GSH and ATP was significantly decreased,Accompanying the ROS content was significantly increased.Compared with the single-drug treatment group,the change was obvious,and the difference was statistically significant.7.siRNA GCLC can down-regulate the expression of mRNA and protein of GCLC while overexpression of shRNA can up-regulatethe expression of mRNA and protein of GCLC in A549 cell spheres.After transfection of A549 stem cell spheres with siRNA GCLC,the expression of GCLC can be further down-regulated by PNA-TPP treatment.After transfection of A549 stem cell spheres with overexpressing plasmid GCLC,PNA-TPP treatment can inhibit the up-regulation of GCLC.Conclusion:1.Compared with A549 cells,the biosynthesis of glutathione in A549 stem cells is enhanced.2.PNA-TPP targeting into mitochondria inhibits the biosynthesis of glutathione in A549 stem cells by reducing the expression of catalytic subunit GCLC of GCL enzyme,and promotes apoptosis of cancer stem cells,thereby enhancing the sensitivity of cisplatin chemotherapy.