Ferroptosis is a type of regulated cell death dependent on iron and reactive oxygen species (ROS), and is characterized by lipid peroxidation [1]. The role of antioxidant events has also received wide attention in the process of ferroptotic cancer cell death. Ferroptosis is initiated by inhibition of system Xc or GPX4 activity, ultimately leading to cell death.
Ferroptosis differs from programmed cell death (such as the aforementioned apoptosis) in several ways.
The impact of iron overload and ferroptosis on reproductive disorders in humans: implications for preeclampsia.
The role of autophagy
Abstract
Introduction
Tumor suppressive factors are negatively regulated by mTOR and AMPK, resulting in the induction of autophagy and suppression of cancer initiation [16]. Beclin1 is important in phagophore formation, suggesting that Beclin1 functions as a tumor suppressor [19]. Knockdown of autophagy-related (ATG)5 and ATG7 limited erastin-stimulated ferroptosis by reducing intracellular ferrous iron concentration and lipid peroxidation [30].
Ferritinophagy is the process of autophagic degradation of the iron storage protein ferritin, which is crucial for the regulation of iron levels in cells.
Results
Furthermore, cell viability was decreased after treatment with 5 μM erastin for 8 and 24 h in wild-type cells but not in BECN1+/– and LC3B–/– fibroblastic cells ( Fig. 3B ). A similar level of cell death was observed in BECN1+/–, LC3B–/– and wild-type fibroblastic cells 24 h after auranofin administration (Figure 3D), suggesting that. These fibroblast cells were treated with vehicle or different doses of erastin (2, 5, or 10 μM) and total proteins were harvested 24 h after cell treatment.
Wild-type or autophagy-deficient LC3B−/− fibroblastic cells were treated with vehicle or erastin and harvested total protein or 10 h after treatment. The protein levels of FTH1 were found to decrease over time in wild-type fibroblastic cells in the presence of erastine (Figure 6B). However, pretreatment with NAC or mito-TEMPO had no significant effect on erastine-induced cell death in BECN1+/– and LC3B–/– fibroblastic cells (Figure 7A and 7B).
Wild-type fibroblast cells were treated with vehicle, erastin (5μM), or erastin plus autophagy inhibitors (CQ or Baf A1) for 24 h, and cytosolic ROS and lipid peroxidation were measured (C, D). Wild-type, BECN1+/−, or LC3B−/− fibroblast cells were treated with vehicle or erastin (5μM) in the absence or presence of the ROS scavenger, NAC, for 24 h, and intracellular iron levels were assessed using a commercial assay (A). Wild-type fibroblast cells were treated with vehicle or erastin (5μM) in the absence or presence of the autophagy inhibitor, 3-MA (10μM).
Wild-type, BECN1+/−, or LC3B−/− fibroblast cells were treated with vehicle, DFP (5 μM), or erastin (5 μM) in the absence or presence of DFP for 24 h, and cell viability was assessed (D). Wild-type fibroblast cells were treated with vehicle or erastin (5 μM) in the absence or presence of the ROS scavenger, NAC (A) or mito-TEMPO (B), the iron chelator, DFO (C), or the ferroptosis inhibitor, ferrostatin 1 (D).
Discussion
Regarding the role of autophagy in ferroptosis, they demonstrated that autophagy regulates ferroptosis by regulating cellular iron homeostasis and cellular ROS generation [ 32 ]. However, the levels of iron in the cell must be carefully regulated, as an excess of iron can have harmful effects due to the formation of ROS [53]. In particular, autophagy can lead to the degradation of cellular iron stem protein ferritin, thereby causing an increase in cellular labile iron levels via NCOA4-mediated autophagy pathway, termed ferritinophagy.
High levels of cellular labile iron allow for rapid accumulation of cellular ROS, which is essential for ferroptosis [32]. This study showed that the levels of intracellular iron were lower in the autophagy-deficient cells compared to wild-type fibroblastic cells (Figure 6A). Consistently, the heavy subunit of ferritin, ferritin heavy chain 1 (FTH1), was degraded by erastin in wild-type cells, but not in autophagy-deficient cells, or in wild-type cells in the presence of the autophagy inhibitor, 3MA (Figure 6B and 6C), which indicates that autophagy regulates intracellular iron levels during erastin-induced ferroptotic process.
The transferrin receptor is a carrier protein for transferrin, a plasma protein that binds iron, thus controlling the level of intracellular iron levels [47]. Interestingly, transferrin receptor (TfR1) protein levels were increased by erastin in wild-type cells, but not in autophagy-deficient cells and autophagy inhibitor-treated wild-type cells ( Figure 6C ). Thus, our results indicate that autophagy regulates intracellular iron levels through ferritin degradation and transferrin receptor induction.
Although autophagy has been reported during ferroptosis, the mediator of autophagy induction was not known. Ferroptosis and autophagy-induced cell death occur independently after siramesin and lapatinib treatment in breast cancer cells. PLoS One.
The role of PGC1α
To investigate the role of PGC1α in erastin-induced ferroptotic cell death, HT1080 cells were treated with vehicle or erastin (10μM) and total protein was collected at the indicated times and 10 hours). To check the regulation of PGC1α expression in HT1080 cells transfected with PGC1α shRNA, compared with HT1080 cells transfected with control shRNA. PGC1α protein and mRNA levels were decreased in HT1080 cells transfected with PGC1a shRNA compared with HT1080 cells transfected with control shRNA ( Figure 2A and 2B ).
And then, HT1080 cells transfected with PGC1α shRNA or control shRNA treated with vehicle or erastin (10μM) cell cytotoxicity was confirmed using lactate dehydrogenase (LDH) release assay. To investigate the role of PGC1α in erastin-induced lipid peroxidation, HT1080 cells were treated with vehicle or erastin (10μM) in the absence or presence of the PGC1α inhibitor, SR18292 (20μM), and lipid peroxidation was assessed using flux fluorometry. - THE BODY. Similarly, HT1080 cells transfected with PGC1α shRNA or control shRNA were treated with erastin (10μM) and lipid peroxidation was assessed by flow cytometry using C11-BODIPY fluorescent probes.
Erastin treatment increased lipid peroxidation compared with vehicle in control shRNA-transfected HT1080 cells but not in PGC1α shRNA-transfected HT1080 cells (Figure 3B). When HT1080 cells were treated with erastin (10 μM) for 12 h, oxidized signals gradually increased (Figure 3C, middle panels). In addition, erastin-induced intracellular iron ion in HT1080 cells was found to be reduced in the presence of the PGC1α inhibitor, SR18292 (20 μM) (Figure 3D).
However, erastin-induced mitochondrial ROS levels were disrupted in PGC1α shRNA-transfected HT1080 cells ( Figure 4C ). When HT1080 cells were treated with erastin (10μM) for 12 h, green fluorescence signals gradually increased (Figure 5B, middle panels), compared to vehicle (Figure 5B, upper panels). Erastin-induced cell death was reduced by downregulation of HO-1 expression in the presence of PGC1α inhibitor, SR18292.
Similarly, the effects of HT1080 cell death were reduced in PGC1α shRNA-transfected HT1080 cells in the presence.
Material & Method
그리고 표적 세포 사멸 과정은 일반적인 암 치료 방법으로 알려져 있습니다. 그 중 페롭토시스(ferroptosis)는 최근에 발견된 세포 사멸의 한 유형으로, 철 매개 지질 과산화에 의해 발생하는 세포 사멸이다. 이 과정에서 활성산소가 과도하게 생성되면 산화 스트레스가 증가해 세포사멸이 일어나게 되는데 이를 페로프토시스(ferroptosis)라고 한다.
먼저, autophagy의 대표적인 표지 유전자인 LC3B가 erastin에 의해 개시되는 ferroptotic cell death에서 증가됨을 확인하였다. HT1080 세포에 LC3B shRNA를 형질감염시켰을 때, 에라스틴 강화 세포사멸 수준이 감소했습니다. 둘째, 우리는 에라스틴 개시 페롭토시스 세포 사멸에서 PGC1α의 역할을 연구했습니다.
에라스틴에 의해 페롭토시스가 유도될 때 단백질 수준에서 PGC1α의 발현이 증가함을 확인하였고, PGC1α의 억제제로 알려진 SR18292가 페롭토시스 세포사를 보호함을 MTS 및 LDH 분석을 통해 확인하였다. Mito-ferrin 단백질 발현과 미토콘드리아 특이 지질 과산화의 변화는 에라스틴에 의해 유도된 ferroptosis 세포 사멸이 미토콘드리아 의존적임을 시사합니다. 마지막으로, 이러한 결과는 이전에 발표된 HO-1 의존성 페롭토시스 세포 사멸에 영향을 미치는 것으로 보입니다.
따라서 이번 연구에서는 에라스틴에 의한 페로토시스 세포사멸과 또 다른 세포사멸인 자가포식과 미토콘드리아의 큰 분자인 PGC1α와의 관계를 처음으로 규명했다. 위의 연구는 암 치료에 사용되는 표적 세포 사멸을 대표합니다.
