Effect Of FoxO3a On Gefitinib Resistance By Inhibiting PDK4 In PC9 Cells

Epidermal growth factor tyrosine kinase inhibitors?EGFR TKIs?have been widely used in tumor biotargeting therapy,such as Gefinitib.However,it is difficult to avoid drug resistance when using EGFR TKI continuously,which is the problem that limits its further application.Inhibition of glycolysis can induce cell death or restore the sensitivity of tumor cells to chemotherapy and radiotherapy in a variety of tumors.There is a close correlation between the EGFR pathway and the metabolic processes of tumor cells.Inhibiting glycolysis increases the sensitivity of non-small cell lung cancer?NSCLC?cells to EGFR TKIs.Pyruvate dehydrogenase kinase isoform 4?PDK4?promotes glycolysis by blocking the effects of pyruvate dehydrogenase complex?PDH?.And PDK4 is associated with drug resistance and survival in many tumor cells.The transcription factor FoxO3a can regulate a variety of cellular processes,including metabolism,and act as a tumor inhibitor.Here,we explored the mechanisms by which FoxO3a reverses the resistance of EGFR TKIs through regulating glycolysis in NSCLC cells,and provided new treatment strategies and experimental data support for solving the problem of clinical EGFR TKIs resistance.ObjectivesTo study the effect and mechanism of FoxO3a on EGFR TKIs resistance and PDK4 in NSCLC and to explore new treatment strategies that specifically inhibit PDK4 in EGFR TKIs resistance,molecular biological methods such as recombinant plasmid over-expressing genes and siRNA-specific silencing genes were used in vitro.Methods1 Detection of Cell Sensitivity to Gefitinib by MTTRecombinant plasmids and siRNAs were transfected into NSCLC cells by calcium phosphate transfection and lipofection,respectively.The sensitivity of NSCLC cells to Gefitinib before and after FoxO3a and PDK4 expression changes was measured by MTT assay.Growth inhibition curves were drawn and the IC₅₀ was calculated.2 Detection of the effects of FoxO3a on glucose consumption and lactic acid production by colorimetryRecombinant plasmids and siRNAs were transfected into NSCLC cells by calcium phosphate transfection and lipofection,respectively.Subsequently,colorimetry was used to detect glucose consumption and lactate production in NSCLC cells before and after FoxO3a and PDK4 expression changes.3 Exploring the relationship between FoxO3a and PDK4 in vitroIn order to explore the mechanism of FoxO3a regulation of glycolysis,after high expression and silencing of FoxO3a,the expressions of genes related to glycolysis,including PKM,LDHA,LDHB,PDK3,and PDK4,were detected at the protein level and mRNA level by Western blot and RT-PCR,respectively.4 The effect of PDK4 on cell resistance in non-small cell lung cancer cellsTo investigate the effects of PDK4 on drug resistance in non-small cell lung cancer,we tested the sensitivity of cells to Gefitinib after high expression and silencing of PDK4 by MTT assay.5 Exploring the regulation of FoxO3a to PDK4 in vitroIn order to further explore the specific mechanism of FoxO3a as a transcription factor regulating PDK4,we detected the promoter sequence of 2000 bp upstream of the PDK4 transcription initiation site through the NCBI database.The JASPAR CORE Vertebrata database was then used to predict the possible binding sites for FOXO3 in the PDK4 promoter region.According to these binding sites,the corresponding RT-PCR primers were designed to detect the results of chromatin immunoprecipitation assays,which further demonstrated whether FoxO3a exerts transcriptional regulation through direct binding to the PDK4 promoter region.Results1 Changes of FoxO3a,Cell Resistance and Glucose Metabolism in NSCLC Cell LinesPC9 cells are sensitive to Gefitinib with an IC₅₀ of 1.137±0.021 nM;H1975 and H1299 cells were resistant to Gefitinib with IC₅₀ of 2184.711±95.286 nM and15474.795±420.671 nM,respectively,P<0.0001.The protein and mRNA expression levels of FoxO3a in H1975 and H1299 cells were significantly lower than those of FoxO3a in PC9 cells?protein level P<0.0001 H1299 v.s.PC9,P=0.0092 H1975 v.s.PC9;mRNA level P<0.0001 v.s.PC9?.In the drug-resistant cell lines H1975 and H1299,both glucose consumption?15.454±0.041 mM and 2.317±0.006 mM,P<0.0001,respectively?and lactic acid production?16.838±0.060 mM and17.000±0.053 mM,respectively,P<0.0001?were increased compared to the sensitive cell line PC9?glucose consumption was 3.178±0.025 mM,lactate production was3.818±0.003 mM?.2 FoxO3a is associated with cell resistance and glucose metabolism in NSCLC cell linesAfter high expression of FoxO3a,the IC₅₀ of PC9 cells to Gefitinib was reduced to 0.566±0.009 nM?P<0.0001?,glucose consumption and lactic acid production also decreased,being 11.931±0.024 mM?P<0.0001?and 1.924±0.004 mM?P<0.0001?,respectively.The IC₅₀ of H1975 cells against Gefitinib also decreased to407.889±2.696 nM?P<0.0001?,glucose consumption and lactate production also decreased,being 14.272±0.048 mM?P<0.0001?and 2.722±0.018 mM?P<0.0001?,respectively.After silencing FoxO3a,the IC₅₀ of PC9 cells against Gefitinib increased to4.061±0.046 nM?P<0.0001?,glucose consumption and lactic acid production also increased,with 18.131±0.060 mM?P<0.0001?and 2.878±0.009 mM?P<0.0001?,respectively.The IC₅₀ of H1975 cells against Gefitinib also increased to5091.710±57.514 nM?P<0.0001?,glucose consumption and lactic acid production also increased,with 17.562±0.215 mM?P=0.0049?and 3.337±0.007 mM?P<0.0001?,respectively.3 Effect of FoxO3a on glycolysis-related genes in NSCLC cellsOn protein and mRNA levels,FoxO3a significantly inhibited PKM and PDK4,but had no significant effect on LDHA,LDHB,and PDK3.4 The effect of PDK4 on cell resistance in non-small cell lung cancer cellsAfter high expression of PDK4,the IC₅₀ of PC9 cells against Gefitinib was increased to 9.735±0.223 nM?P<0.0001?,and the IC₅₀ of H1975 cells against Gefitinib was also increased to 3985.821±157.505 nM?P<0.0001?.After PDK4 was silenced,the IC₅₀ of PC9 cells against Gefitinib was reduced to 0.284±0.008 nM?P<0.0001?,and the IC₅₀ of H1975 cells against Gefitinib was also reduced to923.185±14.672 nM?P<0.0001?.5 FoxO3a inhibits glycolysis by influencing transcription and inhibiting PDK4Comparing the multiples of the enrichment efficiency of the binding sites in the FoxO3a group,we found that the enrichment factor at the GTGAACAA binding site was 6.13-fold,and the difference was statistically significant.FoxO3a may exert its inhibitory effect on PDK4 by directly binding to the 5'-GTGAACAA-3'sequence in the PDK4 promoter region.Conclusion1 FoxO3a is highly expressed in Gefitinib-sensitive NSCLC cells,and PDK4 is highly expressed in Gefitinib-resistant NSCLC cells.2 FoxO3a enhances the sensitivity of NSCLC cells to Gefitinib.3 FoxO3a can inhibit glucose consumption and lactate production in NSCLC cells.4 FoxO3a may exert its inhibitory effect on PDK4 by directly binding to the5'-GTGAACAA-3'sequence in the PDK4 promoter region.