Study On Mechanisms Of Artesunate Induced Ferroptosis In The Ovarian Cancer

Background:Ovarian cancer(OC)is the leading cause of death in female reproductive system malignancies,with high mortality,recurrence,and low survival rates worldwide.Most patients with OC are found to be in the advanced stage,which seriously endangers women’s health.At present,the first-line chemotherapy regimen of paclitaxel combined with platinum drugs has serious side effects and drug resistance.As a derivative of artemisinin,a number of studies have shown that artesunate(ART)can efficiently kill a variety of tumor cells by producing a large number of reactive oxygen species(ROS)without causing serious side effects.The imbalance of intracellular redox caused by excessive ROS is one of the important factors of ferroptosis.ART has been considered as a new ferroptosis inducer.Purpose:To clarify the anti-OC effect of ART and reveal its role in inducing ferroptosis of OC cells.Moreover,clarify the scientific connotation behind it and provide candidate drugs for clinical treatment of OC.Methods:(1)CCK8 assay was used to detect the cell viability of OC cells after the intervention of different doses of ART.Combined with the staining of OC cell markers after the intervention of different doses of ART,the programmed cell death mode of OC cells induced by ART was preliminarily determined.CCK8 assay was used to detect the viability of OC cells treated with different programmed cell death inhibitors to further clarify the mode of programmed cell death induced by ART in OC cells.Subsequently,the levels of total ROS and lipid ROS,and the concentrations of MDA,GSH and iron ion in OC cells treated with different doses of ART were detected by kits.Western blot and RT-qPCR were used to detect the mRNA and protein expression levels of TFRC,SLC7A11,GPX4,FTH1 and OTUB1 in OC cells treated with different doses of ART.co-IP experiment was used to observe the ubiquitination level of SLC7A11.To identify the key regulatory factors of ART-induced ferroptosis in OC cells.Through the kits doses of ART intervention OC cells mitochondrial ROS level,the level of the Krebs cycle and mitochondrial membrane potential changes,combined with mitochondrial morphological changes under electron microscopy,reveal ART influence on mitochondrial function.(2)High-throughput gene sequencing technology was used to detect the changes in gene expression profiles in OC cells before and after ART intervention,and Western blot and RT-qPCR experiments were used to verify the upstream mechanism of SLC7A11 deubiquitination mediated by ART regulating OTUB1.The effect of STAT3 overexpression plasmid on OTUB1-luciferase activity was detected by dual luciferase assay and the mRNA level of OTUB1 was detected by ChIP-qPCR assay after STAT3 immunoprecipitation to clarify the transcriptional regulation of STAT3 on OTUB1.Through ART,ART+CCK8 method IL-6 and IL-6 intervention after OC cell vitality;Western blot was used to detect the protein changes of IL-6/STAT3 pathway and ferroptosis related indicators in OC cells after the above intervention,and to study the role of IL-6 signaling in ferroptosis of OC cells regulated by ART.(3)BRET and CETSA experiments,as well as molecular docking and molecular dynamics simulations were performed to determine the mode and site of ART binding to the IL-6/IL-6R/GP130 protein complex.The expression of IL-6/IL-6R/GP130 protein complex and ferroptosis related indicators after ART intervention were observed by transfection of GP130 point mutation plasmid to clarify the role of this binding site in ART-induced ferroptosis in OC cells.(4)The OC tumor-bearing mouse model was constructed for ART,ART+IL-6 and ART+OTUB1 plasmid treatment,and the inhibitory effects of the above treatments on tumor growth were compared.Combined with the detection of ferroptosis related indicators,the efficacy of ART in the treatment of OC was evaluated in vivo and the reliability of ART therapeutic targets was verified.The expression of IL-6,IL-6ST,OTUB1 and SLC7A11 in OC tissues was analyzed by bioinformatics,and the expression of OC tissues was detected by IHC to explore the clinical application value of ART.Results:(1)Treatment with 0,12.5,25 and 50 μM ART reduced the viability of SKOV-3 cells in a dose-dependent manner,and the IC50 of SKOV-3 cells was about 41.48 μM.Moreover,ART induced ferroptosis rather than apoptosis and necrosis in most OC cells.ART intervention significantly reduced the expression of SLC7A11 and GPX4 in SKOV-3 cells,increased the content of MDA,decreased the content of GSH,and increased the ubiquitination level of SLC7A11,indicating that ART induced ferroptosis by inhibiting the lipid peroxidation pathway in OC cells.ART treatment reduced mitochondrial volume,mitochondrial cristas disappeared,and mitochondrial outer membrane was disrupted,while increasing mitochondrial ROS level and decreasing the tricarboxylic acid cycle level and mitochondrial membrane potential,indicating that ART can damage mitochondrial structure and function.Restoration of OTUB1 expression could inhibit the proliferation of SKOV-3 cells,increase the ubiquitination level of SLC7A11,increase the levels of lipid ROS and MDA,and decrease the level of GSH,which induced ferroptosis.These results suggest that inhibition of OTUB1-SLC7A 11 interaction by ART may be one of the key regulatory mechanisms inducing ferroptosis in OC.(2)KEGG enrichment analysis showed that the differentially expressed genes before and after ART intervention were mainly enriched in the IL-6/JAK/STAT3 pathway.ART intervention significantly decreased the protein expression level of GP130 and further decreased its phosphorylation level,and also decreased the phosphorylation level of STAT3,indicating that ART inhibits the IL-6/JAK/STAT3 pathway in OC cells.Overexpression of STAT3 plasmid increased OTUB1-luciferase activity.The mRNA level of OTUB1 was significantly increased after STAT3 immunoprecipitation.All the above indicated that STAT3 could bind to the promoter region of OTUB1 to promote the transcription of OTUB1.The combination of IL-6 can protect OC cells from ferroptosis induced by ART by increasing OTUB1 mediated SLC7A11 deubiquitination,indicating that ART-inhibited IL-6 signaling may be the upstream signal of ART inhibiting OTUB1 mediated SLC7A11 deubiquitination.(3)BRET assay between IL-6 and GP130 showed that ART could interfere with the binding of IL-6 to GP130.CETSA assay showed that ART could bind to GP130.Molecular docking and molecular dynamics simulations suggested that Lys250 of GP130 may be the binding site of ART and further interfere with IL-6/IL-6R/GP130 protein complex formation.When GP130 Lys250 occurs after the point mutation,ART and its inhibition of IL-6 signal then on iron death related molecular regulation failure,all reveal GP130 Lys250 may be ART inhibition of IL-6/STAT3 signal transduction induced a key role in iron died OC cell site.(4)The tumor inhibition rate of ART treatment in OC tumor-bearing mice was 86.41%,and no obvious toxicity was observed in important organs such as liver and kidney.Treatment with ART+IL-6 and ART+OTUB1 plasmid relatively attenuated the effect of ART.IL-6 signaling was still inhibited in the tumor tissues of the ART treatment group,which resulted in the increase of SLC7A11 ubiquitination,lipid ROS and MDA levels and the decrease of GSH levels,while the above ferroptotic molecules were reversed in the tumor tissues of the ART+IL-6 treatment group and ART+OTUB1 plasmid treatment group.These results indicate that ART can still induce ferroptosis in OC cells in vivo by inhibiting IL-6 signaling and OTUB1 mediating SLC7A11 deubiquitination.Bioinformatics analysis and OC clinical samples of IHC validation showed that IL-6,OTUB1 and SLC7A11 in OC high expression of IL-6 st in OC low expression,this is can lower IL-6 and OTUB1 SLC7A11 expression level of ART and a theoretical basis was provided for the clinical treatment of OC.Conclusion:This study confirmed that ART could exert a good anti-tumor effect by inducing ferroptosis in OC cells at the in vivo,tissue,cell and molecular levels.The mechanism may be that ART binds to the Lys250 site of GP130 to interfere with the formation of IL-6/IL-6R/GP130 protein complex and inhibit IL-6/STAT3 signaling,thereby reducing STAT3 regulated OTUB1 transcription and OTUB1-mediated SLC7A11 deubiquitination.Finally,the ferroptosis of OC cells can be induced by inhibiting the lipid peroxidation pathway,thus producing a good anti-OC effect.At the same time,no obvious toxic and side effects are observed in the safety evaluation,which provides a certain theoretical basis and data support for the clinical treatment of OC by ART.