研究背景: Ferroptosis是一種新穎的程序性死亡，此類的細胞死亡方式以鐵離子的堆積、活性氧化物累積以及脂質過氧化為特性。我們研究團隊發展了一種以零價鐵為核心的奈米藥物，名為ZVI@Ag，在口腔癌模式具有抗癌潛力。
研究目的: 本研究目的旨在探討ZVI@Ag是否在非小細胞肺癌中，不論是細胞或是活體的實驗，能夠誘導ferroptosis細胞死亡；並進一步探討ZVI@Ag毒殺癌細胞的分子機制。
研究結果: 在細胞實驗中ZVI@Ag對於不同非小細胞肺癌都具有明顯且專一的毒殺能力，相較於肺部正常細胞則沒有損害；ZVI@Ag也具有抑制癌症球體(sphere)生長以及降低癌幹細胞和促進血管新生基因表現的特性。重要的是，在活體實驗中，ZVI@Ag具有抑制從貼盤細胞以及癌症球體異種移植腫瘤(xenograft)的能力和減少血管內皮細胞的浸潤進而抑制肺轉移，並且對於活體本身無明顯毒性。為了證實ZVI@Ag能夠誘導ferroptosis而導致細胞死亡，穿透式電子顯微鏡發現ZVI@Ag改變了粒線體的結構並降低了粒線體膜電位、氧氣消耗速率以及ATP產量，進而使得粒線體活性降低；除此之外，ZVI@Ag造成癌細胞大量活性氧化物累積以及脂質的過氧化進而殺死癌細胞，在同時使用抗氧化劑或ferroptosis抑制劑時，確實能夠減緩ZVI@Ag所誘導的細胞死亡，證實ZVI@Ag能夠誘導ferroptosis細胞死亡的發生。在細胞內氧化還原的恆定中，轉錄因子NRF2 (nuclear factor-erythroid 2-related factor 2)扮演很重要的角色，且被視為ferroptosis的負向調控者；值得注意的是，本研究發現ZVI@Ag能夠抑制癌細胞中氧化還原恆定轉錄因子NRF2的蛋白表現量，進而抑制NRF2所調控的抗氧化基因。西方墨點及免疫螢光染色的實驗證實ZVI@Ag促進了GSK3β磷酸化NRF2，並增強β-TrCP對磷酸化的NRF2降解的現象；若同時使用GSK3β抑制劑，則看到NRF2蛋白不受ZVI@Ag抑制，顯示ZVI@Ag誘導GSK3β/β-TrCP路徑而降解NRF2，而使癌細胞內氧化還原失去恆定，最終導致ferroptosis細胞死亡。
結論：本研究發展了一個具有抗癌潛力的奈米粒子，且揭露了NRF2在以鐵為核心的奈米粒子所誘導的非小細胞肺癌ferroptosis細胞死亡的重要性。

Background: Ferroptosis is a novel programmed cell death, which is characterized by iron accumulation, reactive oxygen species (ROS) production and lipid peroxidation. Recently, we developed an iron-based nanoparticle named ZVI@Ag, which exerts potential anti-cancer effect in oral cancer model.
Purpose: This study aims to investigate whether ZVI@Ag induces ferroptosis cell death in non-small cell cancer (NSCLC) in vitro and in vivo. In addition, molecular mechanisms involved in cytotoxic effects of ZVI@Ag are explored.
Results: ZVI@Ag showed significant cancer-specific cytotoxicity in various NSCLC cells without apparent adverse effect toward to normal lung cells at the dose range examined by MTT assay. In addition, ZVI@Ag inhibited cancer sphere growth and expression of stemness genes and angiogenesis genes. Importantly, ZVI@Ag reduced in vivo tumor growth, endothelial cell infiltration and lung metastasis without detectable toxicity of treated animals. To confirm the ferroptotic death at the cellular level, transmission electron microscopy imaging and activity assays showed that ZVI@Ag damaged mitochondrial structure, increased ROS levels in mitochondrial and loss its membrane potential and decreased ATP production. Moreover, ZVI@Ag treatment increased strong intracellular ROS levels and lipid peroxidation. Importantly, addition of antioxidants or ferroptosis inhibitors rescued ZVI@Ag induced cell death, indicating that ZVI@Ag induced ferroptotic cell death. In response to oxidative stress, nuclear factor-E2-related factor 2 (NRF2), a central transcription factor in regulating cellular redox homeostasis, is recognized as a ferroptosis negative regulator. Notably, NRF2 protein expression and mRNA expression of its targeting antioxidant genes were significantly reduced after ZVI@Ag treatment. Immunoblotting and immunofluorescent results showed that NRF2 was phosphorylated by glycogen synthase kinase-3β (GSK3β), which enhanced recruitment of β-transducing repeat-containing protein (β-TrCP), an E3-ligase, for protein degradation of NRF2 upon ZVI@Ag treatment. Inversely, co-treatment with GSK3β inhibitor attenuated NRF2 protein expression suppressed by ZVI@Ag treatment, indicating that ZVI@Ag enhanced NRF2 degradation via induction of GSK-3β/β-TrCP signaling pathway.
Conclusions: This study develops a new nanoparticle-based anticancer drug and reveals that NRF2 plays a central role in ferroptosis cell death induced by this iron-core nanoparticle in NSCLC model.

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