| ObjectiveFerroptosis is a newly characterized iron-dependent form of non-apoptotic regulated cell death triggered by Lipid reactive oxygen species(ROS).The underlying molecular mechanisms about ferroptotic initialization are closely related to the dysfuntion of oxidation-reduction homeostasis:When the GPX4 activity is inhibited directly or indirectly,ferroptosis would be initialized and subsequently resulted in ferroptotic process.Increasing evidence suggest the ferroptosis plays an essential negative role in the tumorigenesis and development.Thus,it is a potential strategy to activate ferroptosis for tumor therapy.While the main pathway of ferroptosis has been established,the reported regulators and pathways of ferroptosis are limited and the exact regulatory network is still largely unknown.Peroxiredoxin 6(PRDX6)is a member of the non-selenium peroxidases family and uses GSH as the physiological reductant to exert its peroxidase activities.Several studies have demonstrated that PRDX6 plays a major role in the repair of peroxidized cell membranes through its three enzymatic activities,including of phospholipid hydroperoxidase,Ca2+-independent phospholipase A2 and lysophospholipid acyl transferase.Thus,PRDX6s show a great similarity with GPX4 in physiological reductant,substrates and enzymatic activity.However,there is no correlative report about PRDX6 in ferroptosis.In this study,we firstly investigated the effect of PRDX6 on ferroptosis and clarified the underlying molecular mechanisms of PRDX6 in the regulation of ferroptosis.The aim of this study is to discover a new regulator of ferroptosis and clarify its molecular mechanisms,which would enrich the regulation net of ferroptosis.Besides,it would provide potential targets and new insights for ferroptosis-based chemotherapy.MethodsNon-small cell lung cancer(NSCLC)cell lines were used in this study.(1)Flow cytometer combined with the tetracycline-inducible lentiviral knockdown system was performed to investigate the effect of PRDX6 on the celluar Lipid ROS or ferroptotic Lipid ROS;(2)Two shRNA plasmids targeting PRDX6(shPRDX6#1 and#2)were designed and introduced into NSCLC cell lines,and(3)Flow cytometer was performed to detect the effect of PRDX6 silencing on ferroptosis;(4)SRB assay was used to the effect of PRDX6 silencing on Erastin-induced cell proliferation-inhibition;(5)Flow cytometer was performed to detect the effect of PRDX6 overexpression on ferroptosis;(6)SRB assay was used to the effect of PRDX6 overexpression on Erastin-induced cell proliferation-inhibition;(7)Intracellular GSH level was examined using a kit when PRDX6 was silenced during ferroptosis;(8)Western blot was used to detect the effect of PRDX6 silencing on the expression of NRF2 protein during ferroptosis;(9)Real time PCR was used to analyze the NRF2 mRNA level when PRDX6 was silenced during ferroptosis;(10)Western blot was used to detect the effect of PRDX6 silencing on the expression of Keapl protein during ferroptosis;(11)Real time PCR was used to analyze the mRNA levels of NRF2 downstream when PRDX6 was silenced during ferroptosis;(12)Flow cytometer was performed to detect the effect of HMOX-1 overexpression on ferroptosis;(13)Flow cytometer was performed to investigate the effect of MJ33 on Lipid ROS during ferroptosis.Results(1)The effect of PRDX6 protein level decline on ferroptosis initialization in tumor cells(Using Tet-on-shPRDX6 system;in this system,the expression level of intracellular PRDX6 is tightly controlled by doxycycline).Doxycycline-induced PRDX6 silencing could not result in upregulation of Lipid ROS in H1299 cells.It suggests that PRDX6 silencing may be not an initial factor for inducing ferroptosis.(2)The effect of PRDX6 protein level decline on ferroptosis in tumor cells(Using Tet-on-shPRDX6 system).? PRDX6 silencing significantly increased Erastin-induced Lipid ROS in H1299 cells,from 24.33 ± 2.76%to 49.95 ± 2.70%.? PRDX6 silencing increased different doses of RSL-3-induced Lipid ROS in H1299 cells.? Ferrostatin-1(10 ?M)treatment completely reversed Erastin-induced Lipid ROS accumulation in control and PRDX6 knockdown cells.(3)The effect of PRDX6 protein level decline on ferroptosis in tumor cells(Using shRNA lentivirus system;in this system,PRDX6 expression is transiently silenced by lentivirus and another sequence of shPRDX6 is added;subsequent experiments use this system).?PRDX6 transient knockdown using lentivirus significantly increases all three ferroptotic inducers(Erastin,RSL-3 and Sorafenib)-induced Lipid ROS in H1299 cells.In Erastin group,induced Lipid ROS were increased from 19.37 ± 4.55%(shRNA control)to 85.48 ± 6.06%(shPRDX6#1)and 76.44 ±7.05%(shPRDX6#2),respectively.And Ferrostatin-1(10 ?M)treatment completely reversed Erastin-induced Lipid ROS accumulation in control and PRDX6 knockdown cells,while Necrostatin-1 and Z-VA-FMK could not.In RLS-3 group,induced Lipid ROS were increased from 8.86 ± 1.31%(shRNA control)to 69.68 ± 1.51%(shPRDX6#1)and 64.34 ± 6.40%(shPRDX6#2),respectively.In Sorafenib group,induced Lipid ROS were increased from 22.70 ± 2.07%(shRNA control)to 65.98 ± 2.65%(shPRDX6#1)and 28.06 ± 0.89%(shPRDX6#2),respectively.?PRDX6 transient knockdown using lentivirus significantly increases Erastin-induced Lipid ROS in A549 cells,from 11.74 ± 6.67%(shRNA control)to 35.59 ± 8.01%(shPRDX6#1)and 51.16 ± 5.94%(shPRDX6#2),respectively.?PRDX6 transient knockdown using lentivirus significantly increases Erastin-induced cell proliferation-inhibition in both H1299 and A549 cells.And Ferrostatin-1(10 ?M)treatment completely reversed Erastin-induced cell proliferation-inhibition in H1299 control and PRDX6 knockdown cells.These results suggest that PRDX6 is a negative regulator of ferroptotic cell death.(4)The effect of PRDX6 protein overexpression on ferroptosis in tumor cells.?PRDX6 overexpression by gene transfection significantly decreases ferroptotic inducers(Erastin and RSL-3)-induced Lipid ROS in H1299 cells.In Erastin group,induced Lipid ROS were decreased from 46.17 ± 1.80%to 29.65 ± 3.49%.In RSL-3 group,induced Lipid ROS were decreased from 61.97 ± 3.93%to 47.51±2.93%.? PRDX6 overexpression by gene transfection significantly decreases Erastin-induced cell proliferation-inhibition in H1299 cells.(5)The effect of PRDX6 on intracellular GSH level during ferroptosis in tumor cells.?PRDX6 knockdown did not affect intracellular GSH level in H1299 cells.?PRDX6 knockdown could not further decrease GSH levels compared to control knockdown after Erastin treatment in H1299 cells.These results suggest that PRDX6 negatively regulating ferroptosis is independent of GSH depletion.(6)The effect of PRDX6 on the expression of NRF2 protein and the mRNA expression of its target genes in tumor cells.?PRDX6 silencing significantly upregulated the expression of NRF2 after Erastin treatment in H1299 cells.?NRF2 mRNA expression is not obviously changed in PRDX6 silencing cells treated with or without erastin.?The Keapl protein level significantly decreased when PRDX6 was silenced in both control and erastin groups.?In H1299 cells,the mRNA expression of HMOX1 was significantly further upregulated in the PRDX6 knockdown cells compared with control knockdown cells after Erastin treatment,from 7.2 fold change(shRNA control)to 19.5 fold change(shPRDX6#1)and 18.3 fold change(shPRDX6#2),respectively.However,the mRNA expression of NQO-1,FTH1,FTL,GCLC and GCLM were not affected.?In A549 cells,the mRNA expression of HMOX1 was significantly further upregulated in the PRDX6 knockdown cells compared with control knockdown cells after Erastin treatment,from 8.7 fold change(shRNA control)to 20.2 fold change(shPRDX6#1)and 16.9 fold change(shPRDX6#2),respectively.?Treatment of deferoxamine(DFO),an iron chelator,completely blocked erastin-induced Lipid ROS both in the control and PRDX6 silencing cells.? HMOX-1 overexpression by gene transfection significantly increases ferroptotic inducers(Erastin and RSL-3)-induced Lipid ROS in H1299 cells.In Erastin group,induced Lipid ROS were increased from 25.63 ?0.59%to 41.77 ± 4.74%.In RSL-3 group,induced Lipid ROS were increased from 25.74 ± 2.27%to 38.97 ± 4.82%.These results suggest that PRDX6 silencing promotes the NRF2-mediated transcriptionally activation of HMOX1.(7)The effect of MJ33 on ferroptosis in tumor cells.?When blocking the iPLA2 activity of PRDX6 using MJ33,there is no upregulation of Lipid ROS.?MJ33(5?M)significantly increased Erastin-induced Lipid ROS in H1299 cells,from 30.76± 1.24%to 45.40 ± 6.01%.And increased Lipid ROS could be completely reversed by Ferrostatin-1(10 ?M).?MJ33 cannot further enhance the erastin-induced Lipid ROS in the PRDX6 silencing cells,while MJ33 can still enhance Erastin-induced Lipid ROS in the control cells.?Erastin-induced Lipid ROS were gradually increased with increased doses of MJ33(5,10,15,20 and 25 ?M)in A549 cells,from 17.23%to 21.54%,29.29%,33.14%,38.21%and 47.81%.These results suggest that PRDX6 removes a part of Lipid ROS through its iPLA2 activity during the ferrototic process.ConclusionWe firstly report that PRDX6 is an essential negative regulator of sensitivity to ferroptosis in tumor cells,especially in non-small cell lung cancer cell lines.The knockdown of PRDX6 significantly enhances ferroptosis inducers(Erastin,RSL-3 and Sorafenib)-triggered Lipid ROS and ferroptotic cell death in various cancer cell lines.Mechanistically,PRDX6 not only suppresses the NRF2-HMOX-1 pathway resulting in decreasing iron levels and subsequent Fenton Chemistry,but also removes a part of induced-Lipid ROS through its phospholipase A2 activity during ferroptosis.In this study,we reveal an essential role of PRDX6 in ferroptosis and enrich the regulation net of ferroptosis.Besides,we provide a potential target to improve the antitumor activity of ferroptosis-based chemotherapy. |
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