Curcumin And Osimertinib Synergistically Inhibit The Proliferation And Migration Of Osimertinib-Resistant Non-Small Cell Lung Cancer Cells Through Ferroptosis

Objective:To investigate the effect and mechanism of curcumin on the proliferation and migration of osimertinib-resistant non-small cell lung cancer cells（NSCLC）and the sensitization effect of curcumin on Osimertinib.To provide theoretical and experimental basis for curcumin combined with osimertinib in the treatment of osimertinib-resistant non-small cell lung cancer.Methods:Osimertinib-resistant cell line HCC827 was established by increasing concentration method.The effects of curcumin combined with osimertinib on the proliferation and migration of Osimertinib-resistant cell lines were observed by MTT,plate clone formation and Transwell assay.Total RNA was extracted from curcuin-treated and untreated Osimertinib-resistant cell lines,and the differential genes in curcuin-treated and untreated cells were compared by RNA seq.RT-q PCR and Western blot were used to detect the changes in the transcription and protein expression levels of ferroptosis-related genes in the osimertinib-resistant cells after the combined treatment of curcumin and osimertinib.The intracellular reactive oxygen species（ROS）levels in the osimertinib-resistant cells treated with curcumin and osimertinib were detected by flow cytometry.Transmission electron microscopy was used to observe the morphological changes of the subcellular structure of the osimertinib-resistant cells after the combination of curcumin and osimertinib.Results:MTT results showed that the IC50 of osimertinib in parental sensitive HCC827cells was 2.234×10^-2μM,and the IC50 of osimertinib-resistant cells was 19.1μM,and the RI index was 854.97,indicating that the osimertinib-resistant cell line HCC827-OR was successfully constructed.MTT assay,colony formation assay and Transwell assay showed that curcumin combined with osimertinib significantly inhibited the proliferation and migration of osimertinib-resistant HCC827-OR cells.RNA seq analysis showed that 189 genes were down-regulated and 79 genes were up-regulated after curcumin treatment compared with the control group.The differentially expressed genes were mainly enriched in ferroptosis pathway,MAPK signaling pathway,Protein digestion absorption and Flow shear stress and atherosclerosis signaling pathways.The results of RT-q PCR and Western blot showed that curcumin significantly up-regulated the expression of HMOX-1（heme oxygenase-1）and inhibited the expression of GPX4（glutathione peroxidase）,the key gene of ferroptosis,in a concentration-dependent manner.Compared with the sensitive cells,GPX4 expression was increased in the osimertinib-resistant cells.After curcumin combined with osimertinib,the transcription levels of HMOX-1,SAT1（spamidine/spamine N1-acetyltransferase 1）,FTL（light peptide ferritin）and SLC3A2（solute carrier family 3 member 2）were increased,the protein expression level of HMOX-1 was increased,and the protein expression level of GPX4 was decreased.Compared with the blank control group,the intracellular ROS level was increased in the osimertinib-resistant HCC827-OR cells treated with curcumin combined with osimertinib.Electron microscopy showed that mitochondrial volume was significantly reduced,mitochondrial membrane was damaged,and mitochondrial cristoid was reduced or disappeared.Conclusion:Curcumin can enhance the sensitivity of osimertinib to osimertinib in osimertinib-resistant NSCLC cell lines.Curcumin can regulate ferroptosis signaling pathway.Curcumin and osimertinib synergistically inhibit the proliferation and migration of osimertinib-resistant non-small cell lung cancer cells by regulating ferroptosis.