Correlation Between Erythropoietin And The Growth Of Non-Small Cell Lung Cancer Cell Lines In Simulated Hypoxic Environment

Objective:Recombinant human erythropoietin(rh EPO)is often used to treat anemia in patients with non-small cell lung carcinoma(NSCLC),but whether rh EPO enhances the malignant biological behavior of NSCLC cancer cells remains controversial.The purpose of this study was to determine whether the application of rh EPO has an effect on growth and immune escape indicators of non-small cell lung cancer by simulating the hypoxic environment of malignant tumor growth in vitro.Methods:The cells were cultured in 1%O2,5% CO2 and 94% N2 to simulate the hypoxic environment of tumor growth.A549 cell line with low expression of erythropoietin receptor(EPOR)and NCI-H838 cell line with high expression of EPOR were treated with rh EPO(0 U/ml,2 U/ml and 8 U/ml)under normal oxygen and hypoxic environment,respectively.The experiment was divided into 6 groups.Cell proliferation was detected using CCK-8 assay.The expression levels of vascular endothelial growth factor(VEGF),hypoxia-inducible factor-1α(HIF-1α)and programmed death-ligand 1(PD-L1)were determined by western blotting to measure tumor growth and immune escape.One-way ANOVA was used for cross-group statistical analysis,with P < 0.05 indicating a significant difference.Results:Hypoxia itself could decrease the survival rate of NSCLC cells.Under the hypoxic condition,rh EPO induced tumor cells proliferation,especially in the NCI-H838 cell line,where 2 U/ml rh EPO increased the total number of surviving cells(Hypoxia + rh EPO2U/ml vs Hypoxia,P <0.05).Western blot analysis showed oxygen-poor up-regulated a content of VEGF,HIF-1αand PD-L1 in NSCLC cell lines(Normoxia vs Hypoxia,P < 0.05),but may not be dependent on the expression levels of EPOR.Rh EPO decreased the expression levels of VEGF and HIF-1α.In A549 cells,it depended on the concentration of rh EPO and was particularly obvious in HIF-1α(Hypoxia vs Hypoxia + rh EPO2U/ml vs Hypoxia +rh EPO8U/ml,P < 0.05).Low concentration of rh EPO may not reduce VEGF expression.In NCI-H838 cell line,the effect of rh EPO on VEGF was more obvious,but it may be independent of rh EPO concentrations.The down-regulation of PD-L1 expression by rh EPO was only present in the A549 cell line and required higher rh EPO concentrations(Hypoxia+rh EPO8U/ml vs Hypoxia&Hypoxia+rh EPO2U/ml,P < 0.05).Conclusion:The effect of prolonged high concentrations of rh EPO under hypoxic condition resulted in accelerated cells proliferation of non-small cell lung cancer and was independent of EPOR expression levels on the cell lines surface.Hypoxia resulted in increased expression of VEGF,HIF-1α and PD-L1 on the NSCLC cell lines.Under normoxic condition,rh EPO did not affect the expression of VEGF,HIF-1α and PD-L1,but under hypoxic condition,the application of rh EPO reduced the expression of VEGF,HIF-1α and PD-L1,which had an impact on the biological behavior of tumor cells.