The Research On The Mechanism Of Autophagy Activity Regulating Ferroptosis In Acute Lymphoblastic Leukemia Cells

Objective: Acute lymphoblastic leukemia(ALL)is the most prevalent malignancy in childhood.At present,multi-drug combined chemotherapy under the guidance of risk grading is the main treatment method,and relapse of chemotherapy resistance is the main reason affecting the survival rate of ALL.Most anti-leukemia chemotherapeutic drugs use apoptosis induction as the therapeutic mechanism,and apoptosis alone can easily lead to chemotherapy resistance.Therefore,killing leukemia cells by various death methods has gradually become a research hotspot.Ferroptosis as a non-apoptotic programmed cell death has attracted much attention.Autophagy plays an important regulatory role in the iron death.But the relationship between autophagy and iron death in the treatment of ALL remains unclear.Small molecule compound Erastin,as an activator of iron death,can induce iron death by changing the fluidity of mitochondrial voltage-dependent anion gated channels(VDACs),and is involved in a variety of tumor therapy studies.However,whether VDACs play a role in the mechanism of autophagy regulation of iron death remains unclear.Therefore,this study aims to investigate the regulatory relationship between autophagy and iron death induced by Erastin,which could provide a novel therapeutic target for chemotherapy resistanceMethods: 1.MTS and trypan blue staining assay were performed to detect cell viability and cell death of ALL cells including Reh,Jurkat and CCRF-CEM cells,respectively.2.The western blot assay was applied to detect protein expression levels of autophagy-related proteins in ALL cells including p62,LC3I/II,Ferritin,VDAC3 and Fbxw7.3.Labile iron pool(LIP)and ROS levels in ALL cells were detected by flow cytometry.4.The m RNA expression level of VDAC3 in ALL cells(REH,Jurkat,CCRF-CEM cells)was detected by RT-q PCR.5.Ubi Browser database predicted the Top10 E3 ligase with VDAC3 as substrate.6.The m RNA and protein expression levels of Fbxw7 and VDAC3 in 293 T cells after Fbxw7 silenced and Fbxw7 overexpressed were detected by RT-q PCR and western blot.7.The effects of silencing/overexpressing of Fbxw7 on the ubiquitination of VDAC3 protein were detected by immunoprecipitation(Co-IP)and western blot.Results:1.Reh cells were sensitive to ferroptosis activator Erastin,while Jurkat and CCRF-CEM cells were resistant to Erastin.2.The autophagy activator of rapamycin increased the sensitivity of Jurkat and CCRF-CEM cells to Erastin and promoted ferroptosis.The autophagy inhibitor chloroquine reduced the sensitivity of Reh cells to ferroptosis induced by Erastin and inhibited the occurrence of ferroptosis.3.Activated autophagy down-regulated Ferritin expression in Jurkat and CCRF-CEM cells,increased LIP level and ROS level;Inhibition of autophagy up-regulated Ferritin expression,decreased LIP level and ROS level in Reh cells.4.Autophagy only affected the protein expression of VDAC3,but not the gene expression.VDAC3 protein expression was upregulated in Jurkat and CCRF-CEM cells after autophagy activation.VDAC3 protein expression was down-regulated after autophagy inhibition.When proteasome activity was inhibited,the high expression of VDAC3 induced by autophagy activation did not change significantly,but the inhibition of autophagy-induced VDAC3 low expression was recovered.5.When autophagy was activated,the expression of Fbxw7 protein in Jurkat and CCRF-CEM cells was down-regulated,while the expression of Fbxw7 protein in autophagy Reh cells was up-regulated,which was contrary to the expression trend of VDAC3 protein,a substrate for autophagy activity regulation.6.The ubiquitination level of VDAC3 was increased after Fbxw7 expression silenced in 293 T cells,while overexpression of Fbxw7 reduced the ubiquitination of VDAC3.Conclusions: This study confirmed that the sensitivity of ALL cells to Erastin induced ferroptosis activator was different.And autophagy activity affected the sensitivity of ALL cells to ferroptosis activator Erastin.Autophagy activity regulates intracellular iron homeostasis by affecting Ferritin,ROS and LIP levels,and changes the sensitivity of ALL cells to ferroptosis activator Erastin.Autophagy activity only affects the protein stability of VDAC3 in ALL cells.VDAC3 specifically binds to the E3 ligase Fbxw7,and the expression of Fbxw7 is regulated by autophagy activity.But VDAC3 expression was opposite to autophagy activity,suggesting that autophagy activity is involved in posttranslational regulation of VDAC3.Autophagy activity changes the sensitivity of cells to ferroptosis activator Erastin by influencing Fbxw7 to regulate the degradation of VDAC3 ubiquitination.