研究背景: 我們研究團隊發展一種以羧甲基纖維素修飾的零價鐵奈米藥物，名為 ZVI@CMC，先前研究發現在口腔癌模式中，ZVI@CMC 具有非典型細胞凋亡的抗癌潛力。這種抗癌能力是一種新穎的程序性細胞死亡路徑，名為 ferroptosis，此類的細胞死亡方式以鐵離子累積產生的活性氧化物造成細胞膜脂質過氧化為特徵。然而，ZVI@CMC 在其他癌症模式中的抗癌作用仍有待探索。
研究目的: 本研究旨在探討 ZVI@CMC 是否在非小細胞肺癌中造成 ferroptosis 鐵依賴細胞凋亡以及探討 ZVI@CMC 毒殺癌細胞的分子機制。此外，本研究探討 ZVI@CMC 是否有調節非小細胞肺癌的腫瘤微環境潛在作用。
研究結果: 在細胞實驗中，ZVI@CMC 針對多種非小細胞肺癌細胞株具有毒殺能力且造成細胞內活性氧化物激增和脂質過氧化，而正常肺細胞株則沒有損害。使用西方墨點法及轉錄聚合酶連鎖反應分析發現 ZVI@CMC 能抑制非小細胞肺癌細胞株抗氧化基因-轉錄因子 NRF2 (nuclear factor-erythroid 2-related factor 2) 蛋白表現，進而抑制 NRF2 調控的抗氧化基因表現，顯示 ZVI@CMC 抑制 NRF2 介導的氧化壓力恆定機制，並導致 ferroptosis 細胞死亡。免疫螢光染色的實驗結果顯示 ZVI@CMC 使非小細胞肺癌細胞株之蛋白降解因子 β-TrCP (β-transducing repeat-containing protein) 由細胞質轉位到細胞核以降解 NRF2 蛋白；西方墨點法證實 ZVI@CMC 促進 AMPK (AMP-activated protein kinase) 和 GSK3β (glycogen synthase kinase-3β) 磷酸化並且使磷酸化的mTOR表達降低。此外，我們使用 ZVI@CMC 對非小細胞肺癌的 IC50劑量處理骨髓源性巨噬細胞 (bone marrow-derived macrophages，BMDMs) 以及 T 淋巴細胞，細胞毒性分析結果顯示 ZVI@CMC 不影響 BMDMs跟T 淋巴細胞存活率；重要的是，透過反轉錄聚合酶連鎖反應和流式細胞儀檢測發現，ZVI@CMC 會使 BMDMs 朝向 M1 抗癌表型極化並活化毒殺性T細胞。值得注意的，ZVI@CMC 治療可以抑制同種移植肺癌小鼠的腫瘤生長以及異種移植人類大細胞肺癌株 H460 的腫瘤生長。腫瘤切片之免疫螢光染色顯示 ZVI@CMC 治療後增加腫瘤組織中抑癌型M1巨噬細胞以及活化毒殺性T細胞的浸潤，而 M2 巨噬細胞則分佈在腫瘤外圍。
結論： 本研究證明一種新的機制，即 ZVI@CMC 通過激活 p-AMPK /p-GSK3β 信號傳導並由此促進 p-GSK3/β-TrCP 降解NRF2的信號傳導路徑，使 ZVI@CMC 誘導非小細胞肺癌 ferroptosis 細胞死亡。另外， ZVI@CMC 可以透過調節腫瘤微環境中免疫細胞型態來抑制腫瘤生長。

Background: We have developed zero-valent iron nanoparticles coated with carboxymethylcellulose (ZVI@CMC) that exhibit anti-cancer potency by non-apoptotic cell death, namely ferroptosis, in head and neck cancers. Ferroptosis is characterized by the iron-dependent accumulation of lipid peroxidation. However, the anti-cancer effects of ZVI@CMC in other cancer models remain to be explored.
Purpose: This study aims to investigate whether ZVI@CMC induced ferroptosis cell death in non-small cell cancer (NSCLC) and explore molecular mechanisms involved in cytotoxic effects of ZVI@CMC. In addition, this study is designed to investigate the potential roles of ZVI@CMC NPs in modulating tumor microenvironment in NSCLC.
Results: ZVI@CMC induced cytotoxicity and intracellular reactive oxygen species surge including lipid peroxidation in various NSCLC cell lines but not in normal lung cell lines. Immunoblotting and RT-qPCR showed that expression of anti-ferroptosis protein, nuclear factor-E2-related factor 2 (NRF2) and its targeting antioxidant genes was decreased in NSCLC lines after ZVI@CMC treatment, suggesting that ZVI@CMC suppressed NRF2-mediated cytoprotective system and thus induced ferroptosis. Immunofluorescent results demonstrated that β-transducing repeat-containing protein (β-TrCP) translocated into nucleus along with reduced NRF2 protein expression after ZVI@CMC treatment. Immunoblotting results showed that ZVI@CMC induced phosphorylation of AMP-activated protein kinase (AMPK) and glycogen synthase kinase-3β (GSK3β), while reduced expression of phosphorylated mTOR after ZVI@CMC treatment. Moreover, to investigate the effects of ZVI@CMC NPs in immune cells, we used IC50 dose of ZVI@CMC on NSCLC to treat ex vivo isolated bone marrow derived macrophages (BMDMs) and splenic T lymphocytes. The MTT results showed that ZVI@CMC did not affect the cell viability of BMDMs and splenic T lymphocytes. Importantly, ZVI@CMC treatment skewed BMDMs polarization to anti-tumor M1 macrophages and increased cytotoxic CD8+ T cells by RT-qPCR and FLOW measurements. Notably, ZVI@CMC treatment inhibited the allograft growth of mouse Lewis lung carcinoma and xenograft growth of human large-cell lung carcinoma cell line, H460. Strikingly, multi-color fluorescent -immunohistochemistry demonstrated that the infiltrated M1 macrophages and cytotoxic CD8+T cells increased in tumor site whereas pro-tumor M2 macrophage located toward the tumor boundary after ZVI@CMC NPs treatment.
Conclusions: This study identified a novel mechanism that ZVI@CMC enhanced NRF2 degradation via induction of p-AMPK/p-GSK3β signaling and thus activation of p-GSK3/β-TrCP degradation pathway of NRF2 leading to ZVI@CMC-induced ferroptotic cell death. In addition, ZVI@CMC NPs modulated tumor associated immune cells to elicit an anti-tumor microenvironment leading to anti-tumor growth effects.

Abdalkader M, Lampinen R, Kanninen KM, Malm TM, Liddell JR (2018). Targeting NRF2 to suppress ferroptosis and mitochondrial dysfunction in neurodegeneration. Front Neurosci. 12:466.
Almeida JP, Figueroa ER, Drezek RA (2014). Gold nanoparticle mediated cancer immunotherapy. Nanomedicine. 10(3):503-514.
Anselmo AC, Mitragotri S (2019). Nanoparticles in the clinic: an update. Bioeng Transl Med. 4(3):e10143.
Antony JJ, Sithika MA, Joseph TA, Suriyakalaa U, Sankarganesh A, Siva D, Kalaiselvi S, Achiraman S (2013). In vivo antitumor activity of biosynthesized silver nanoparticles using Ficus religiosa as a nanofactory in DAL induced mice model. Colloids Surf B Biointerfaces. 108:185-190.
Arbour KC, Jordan E, Kim HR, Dienstag J, Yu HA, Sanchez-Vega F, Lito P, Berger M, Solit DB, Hellmann M, Kris MG, Rudin CM, Ni A, Arcila M, Ladanyi M, Riely GJ (2018). Effects of co-occurring genomic alterations on outcomes in patients with KRAS-mutant non-small cell lung cancer. Clin Cancer Res. 24(2):334-340.
Armstrong JL, Bonavaud SM, Toole BJ, Yeaman SJ (2001). Regulation of glycogen synthesis by amino acids in cultured human muscle cells. J Biol Chem. 276(2):952-956.
Bai X, Chen Y, Hou X, Huang M, Jin J (2016). Emerging role of NRF2 in chemoresistance by regulating drug-metabolizing enzymes and efflux transporters. Drug Metab Rev. 48(4):541-567.
Barta JA, Powell CA, Wisnivesky JP (2019). Global Epidemiology of Lung Cancer. Ann Glob Health. 85(1):8.
Basak P, Sadhukhan P, Sarkar P, Sil PC (2017). Perspectives of the Nrf-2 signaling pathway in cancer progression and therapy. Toxicol. Rep. 4:306–318.
Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y (2016). Regulators of iron homeostasis: new players in metabolism, cell death, and disease. Trends Biochem Sci. 41, 274–286.
Braun S, Hanselmann C, Gassmann MG, Auf Dem Keller U, Born-Berclaz C, Chan K, Kan YW, Werner S (2002). Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound. Mol Cell Biol. 22:5492–505.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Jemal (2018). Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 68(6): 394-424.
Bustamante-Marin XM, Ostrowski LE (2017). Cilia and mucociliary clearance. Cold Spring Harb Perspect Biol. 9(4):a028241.
Butts CA, Ding K, Seymour L, Twumasi-Ankrah P, Graham B, Gandara D, Johnson DH, Kesler KA, Green M, Vincent M, Cormier Y, Goss G, Findlay B, Johnston M, Tsao MS, Shepherd FA (2010). Randomized phase III trial of vinorelbine plus cisplatin compared with observation in completely resected stage IB and II non-small-cell lung cancer: updated survival analysis of JBR-10. J Clin Oncol. 28(1):29‐34.
Cao JY, Dixon SJ (2016). Mechanisms of ferroptosis. Cell Mol Life Sci. 73(11-12):2195-2209.
Carroll KJ, Hudgins DM, Spurgeon S, Kemner KM, Mishra B, Boyanov MI, Brown LW, Taheri ML, Carpenter EE (2010). One-Pot aqueous synthesis of Fe and Ag core/shell nanoparticles. Chem. Mater. 22: 6291-6296
Chakraborty B, Pal R, Ali M, Singh LM, Shahidur Rahman D, Kumar Ghosh S, Sengupta M (2016). Immunomodulatory properties of silver nanoparticles contribute to anticancer strategy for murine fibrosarcoma. Cell Mol Immunol. 13(2):191-205.
Chan K, Han XD, Kan YW (2001). An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proc Natl Acad Sci U S A. 98:4611–6.
Chattopadhyay S, Dash SK, Mandal D, Das B, Tripathy S, Dey A, Pramanik P, Roy S (2016). Metal based nanoparticles as cancer antigen delivery vehicles for macrophage based antitumor vaccine. Vaccine. 34(7):957-967.
Chen F, Zhuang X, Lin L, Yu P, Wang Y, Shi Y, Hu G, Sun Y (2015). New horizons in tumor microenvironment biology: challenges and opportunities. BMC Med. 13:45.
Chen J, Yu Y, Ji T, Ma R, Chen M, Li G, Li F, Ding Q, Kang Q, Huang D, Liang X, Lin H, Cai X (2016a). Clinical implication of Keap1 and phosphorylated Nrf2 expression in hepatocellular carcinoma. Cancer Med. 5(10):2678-2687.
Chen PL, Roh W, Reuben A, Cooper ZA, Spencer CN, Prieto PA, Miller JP, Bassett RL, Gopalakrishnan V, Wani K, De Macedo MP, Austin-Breneman JL, Jiang H, Chang Q, Reddy SM, Chen WS, Tetzlaff MT, Broaddus RJ, Davies MA, Gershenwald JE, Haydu L, Lazar AJ, Patel SP, Hwu P, Hwu WJ, Diab A, Glitza IC, Woodman SE, Vence LM, Wistuba II, Amaria RN, Kwong LN, Prieto V, Davis RE, Ma W, Overwijk WW, Sharpe AH, Hu J, Futreal PA, Blando J, Sharma P, Allison JP, Chin L, Wargo JA (2016b). Analysis of immune signatures in longitudinal tumor samples yields insight into biomarkers of response and mechanisms of resistance to immune checkpoint blockade. Cancer Discov. 6(8):827-837.
Cho NH, Cheong TC, Min JH, Wu JH, Lee SJ, Kim D, Yang JS, Kim S, Kim YK, Seong SY (2011). A multifunctional core-shell nanoparticle for dendritic cell-based cancer immunotherapy. Nat Nanotechnol. 6(10):675-682.
Costa da Silva M, Breckwoldt MO, Vinchi F, Correia MP, Stojanovic A, Thielmann CM, Meister M, Muley T, Warth A, Platten M, Hentze MW, Cerwenka A, Muckenthaler MU (2017). Iron Induces anti-tumor activity in tumor-associated macrophages. Front Immunol. 8:1479.
Dellacherie MO, Seo BR, Mooney DJ (2019). Macroscale biomaterials strategies for local immunomodulation. Nat Rev Mater. 4:379–397.
DeNardo DG, Ruffell B (2019). Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol. 19(6):369-382.
Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, Morrison B 3rd, Stockwell BR (2012). Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 149(5):1060‐1072.
Dixon SJ, Patel DN, Welsch M, Skouta R, Lee ED, Hayano M, Thomas AG, Gleason CE, Tatonetti NP, Slusher BS, Stockwell BR (2014). Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife. 3:e02523.
Dolma S, Lessnick SL, Hahn WC, Stockwell BR (2003). Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer Cell. 3(3):285-96.
Dreaden EC, Mackey MA, Huang X, Kang B, El-Sayed MA (2011). Beating cancer in multiple ways using nanogold. Chem Soc Rev. 40(7):3391-3404.
Duguet E, Vasseur S, Mornet S, Devoisselle JM (2006). Magnetic nanoparticles and their applications in medicine. Nanomedicine (Lond). 1(2):157-168.
Eling N, Reuter L, Hazin J, Hamacher-Brady A, Brady NR (2015). Identification of artesunate as a specific activator of ferroptosis in pancreatic cancer cells. Oncoscience. 2(5):517-532.
Evans ER, Bugga P, Asthana V, Drezek R (2018). Metallic Nanoparticles for Cancer Immunotherapy. Mater Today (Kidlington). 21(6):673-685.
Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P, Lozniewski A (2002). Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proc Natl Acad Sci U S A. 99:7610–5.
Fouad YA, Aanei C (2017). Revisiting the hallmarks of cancer. Am J Cancer Res. 27(5):1016-1036.
Frazer DM, Anderson GJ (2014). The regulation of iron transport. Biofactors 40, 206–214.
Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, Herbach N, Aichler M, Walch A, Eggenhofer E, Basavarajappa D, Rådmark O, Kobayashi S, Seibt T, Beck H, Neff F, Esposito I, Wanke R, Förster H, Yefremova O, Heinrichmeyer M, Bornkamm GW, Geissler EK, Thomas SB, Stockwell BR, O'Donnell VB, Kagan VE, Schick JA, Conrad M (2014). Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 16(12):1180-1191.
Gao M, Monian P, Quadri N, Ramasamy R, Jiang X (2015). Glutaminolysis and transferrin regulate ferroptosis. Mol. Cell. 59, 298–308.
Geiser M, Schurch S, Gehr P (2003). Influence of surface chemistry and topography of particles on their immersion into the lung's surface-lining layer. J Appl Physiol (1985). 94(5):1793-1801.
Gout PW, Buckley AR, Simms CR, Bruchovsky N (2001). Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)- cystine transporter: a new action for an old drug. Leukemia. 15:1633–1640.
Grinberg-Bleyer Y, Oh H, Desrichard A, Bhatt DM, Caron R, Chan TA, Schmid RM, Klein U, Hayden MS, Ghosh S (2017). NF-κB c-Rel is crucial for the regulatory T cell immune checkpoint in cancer. Cell. 170(6):1096-1108.e13.
Gu Z, Liu T, Tang J, Yang Y, Song H, Tuong ZK, Fu J, Yu C (2019). Mechanism of iron oxide-induced macrophage activation: The impact of composition and the underlying signaling pathway. J Am Chem Soc. 141(15):6122-6126.
Hamada C, Tsuboi M, Ohta M, Fujimura S, Kodama K, Imaizumi M, Wada H (2009). Effect of postoperative adjuvant chemotherapy with tegafur-uracil on survival in patients with stage IA non-small cell lung cancer: an exploratory analysis from a meta-analysis of six randomized controlled trials. J Thorac Oncol. 4(12):1511‐1516.
Hanahan D, Coussens LM (2012). Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 21(3):309-322.
Hanahan D, Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell. 144(5):646-674.
Havel JJ, Chowell D, Chan TA (2019). The evolving landscape of biomarkers for checkpoint inhibitor immunotherapy. Nat Rev Cancer. 19(3):133-150.
He F, Zhao D (2007). Manipulating the size and dispersibility of zerovalent iron nanoparticles by use of carboxymethyl cellulose stabilizers. Environ Sci Technol. 41(17):6216-6221.
Heath JR, (2015). Nanotechnologies for biomedical science and translational medicine. Proc Natl Acad Sci US A. 112(47):14436-14443.
Hinshaw DC, Shevde LA (2019). The tumor microenvironment innately modulates cancer progression. Cancer Res. 79(18):4557-4566.
Hosomi Y, Morita S, Sugawara S, Kato T, Fukuhara T, Gemma A, Takahashi K, Fujita Y, Harada T, Minato K, Takamura K, Hagiwara K, Kobayashi K, Nukiwa T, Inoue A; North-East Japan Study Group (2020). Gefitinib alone versus Gefitinib plus chemotherapy for non-small-cell lung cancer with mutated epidermal Growth Factor Receptor: NEJ009 Study. J Clin Oncol. 38(2):115‐123.
Huang KJ, Wei YH, Chiu YC, Wu SR, Shieh DB (2019). Assessment of zero-valent iron-based nanotherapeutics for ferroptosis induction and resensitization strategy in cancer cells. Biomater Sci. 7(4):1311-1322.
Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams BO, Guan KL (2006). TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell. 126(5):955-968.
Ishida Y, Agata Y, Shibahara K, Honjo T (1992). Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J. 11(11):3887-3895.
Isohookana J, Haapasaari KM, Soini Y, Karihtala P (2015). Keap1 expression has independent prognostic value in pancreatic adenocarcinomas. Diagn Pathol. 10:28.
Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999). Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13(1):76-86.
Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol. 2010;7(11):653-664.
Ji RR, Chasalow SD, Wang L, Hamid O, Schmidt H, Cogswell J, Alaparthy S, Berman D, Jure-Kunkel M, Siemers NO, Jackson JR, Shahabi V (2012). An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother. 61(7):1019-1031.
Jung M, Mertens C, Tomat E, Brüne B (2019). Iron as a central player and promising target in cancer progression. Int J Mol Sci. 2019;20(2):273.
Kagan VE, Mao G, Qu F, Angeli JP, Doll S, Croix CS, Dar HH, Liu B, Tyurin VA, Ritov VB, Kapralov AA, Amoscato AA, Jiang J, Anthonymuthu T, Mohammadyani D, Yang Q, Proneth B, Klein-Seetharaman J, Watkins S, Bahar I, Greenberger J, Mallampalli RK, Stockwell BR, Tyurina YY, Conrad M, Bayır H (2017). Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem Biol. 13(1):81‐90.
Kawasaki Y, Okumura H, Uchikado Y, Kita Y, Sasaki K, Owaki T, Ishigami S, Natsugoe S. (2014) Nrf2 is useful for predicting the effect of chemoradiation therapy on esophageal squamous cell carcinoma. Ann Surg Oncol. 21(7):2347-2352.
Kerins MJ, Ooi A (2018). The roles of NRF2 in modulating cellular iron homeostasis. Antioxid Redox Signal. 29(17):1756-1773.
Kheirolomoom A, Ingham ES, Mahakian LM, Tam SM, Silvestrini MT, Tumbale SK, Foiret J, Hubbard NE, Borowsky AD, Murphy WJ, Ferrara KW (2015). CpG expedites regression of local and systemic tumors when combined with activatable nanodelivery. J Control Release. 220(Pt A):253-264.
Kitamura H, Motohashi H (2018). NRF2 addiction in cancer cells. Cancer Sci. 109(4):900-911.
Kumar V, Patel S, Tcyganov E, Gabrilovich DI (2016). The nature of myeloid-derived suppressor cells in the tumor microenvironment. Trends Immunol. 37(3):208-220.
Kurutas EB (2016). The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J. 15(1):71.
Lachaier E, Louandre C, Godin C, Saidak Z, Baert M, Diouf M, Chauffert B, Galmiche A (2014). Sorafenib induces ferroptosis in human cancer cell lines originating from different solid tumors. Anticancer Res. 34:6417–6422.
Lee ES, Son DS, Kim SH, Lee J, Jo J, Han J, Kim H, Lee HJ, Choi HY, Jung Y, Park M, Lim YS, Kim K, Shim Y, Kim BC, Lee K, Huh N, Ko C, Park K, Lee JW, Choi YS, Kim J (2008). Prediction of recurrence-free survival in postoperative non-small cell lung cancer patients by using an integrated model of clinical information and gene expression. Clin Cancer Res. 14(22):7397-7404.
Lee JM, Calkins MJ, Chan K, Kan YW, Johnson JA (2003). Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem. 278(14):12029-12038.
Li R, Jia Z, Zhu H (2019). Regulation of Nrf2 signaling. React Oxyg Species (Apex). 8(24):312-322.
Li T, Li X, Zamani A, Wang W, Lee CN, Li M, Luo G, Eiler E, Sun H, Ghosh S, Jin J, Murali R, Ruan Q, Shi W, Chen YH (2020). c-Rel is a myeloid checkpoint for cancer immunotherapy. Nature Cancer. 1, 1-11.
Linkermann A, Skouta R, Himmerkus N, Mulay SR, Dewitz C, De Zen F, Prokai A, Zuchtriegel G, Krombach F, Welz PS, Weinlich R, Vanden Berghe T, Vandenabeele P, Pasparakis M, Bleich M, Weinberg JM, Reichel CA, Bräsen JH, Kunzendorf U, Anders HJ, Stockwell BR, Green DR, Krautwald S (2014). Synchronized renal tubular cell death involves ferroptosis. Proc Natl Acad Sci U S A. 111(47):16836-16841.
Liu VC, Wong LY, Jang T, Shah AH, Park I, Yang X, Zhang Q, Lonning S, Teicher BA, Lee C (2007). Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol. 178(5):2883-2892.
Lloyd RV, Hanna PM, Mason RP (1997). The origin of the hydroxyl radical oxygen in the Fenton reaction. Free Radic Biol Med. 22:885–888.
Louandre C, Ezzoukhry Z, Godin C, Barbare JC, Mazière JC, Chauffert B, Galmiche A (2013). Iron-dependent cell death of hepatocellular carcinoma cells exposed to sorafenib. Int J Cancer. 133(7):1732-42.
Lu B, Chen XB, Ying MD, He QJ, Cao J, Yang B (2018). The role of ferroptosis in cancer development and treatment response. Front Pharmacol. 8:992.
Magtanong L, Ko PJ, Dixon SJ (2016). Emerging roles for lipids in non-apoptotic cell death. Cell Death Differ. 23(7):1099-1109.
Matsushita M, Freigang S, Schneider C, Conrad M, Bornkamm GW, Kopf M (2015). T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. J Exp Med. 212(4):555-568.
Maurer U, Preiss F, Brauns-Schubert P, Schlicher L, Charvet C (2014). GSK-3 - at the crossroads of cell death and survival. J Cell Sci. 127(Pt 7):1369-1378.
Milling L, Zhang Y, Irvine DJ (2017). Delivering safer immunotherapies for cancer. Adv Drug Deliv Rev. 114:79-101.
Nabavi SF, Barber AJ, Spagnuolo C, Russo GL, Daglia M, Nabavi SM, Sobarzo-Sánchez E (2016). Nrf2 as molecular target for polyphenols: A novel therapeutic strategy in diabetic retinopathy. Crit Rev Clin Lab Sci. 53(5):293-312.
Onodera Y, Motohashi H, Takagi K, Miki Y, Shibahara Y, Watanabe M, Ishida T, Hirakawa H, Sasano H, Yamamoto M, Suzuki T (2014). NRF2 immunolocalization in human breast cancer patients as a prognostic factor. Endocr Relat Cancer. 21(2):241-252.
Pardoll DM (2012). The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 12(4):252-264.
Peng C, Zhao Q, Gao C (2010). Sustained delivery of doxorubicin by porous CaCO3 and chitosan/alginate multilayers-coated CaCO3 microparticles. Colloids Surf. A Physicochem. Eng. Asp. 353(2-3):132-139.
Perez VL, Van Parijs L, Biuckians A, Zheng XX, Strom TB, Abbas AK (1997). Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity. 6(4):411-417.
Perica K, Tu A, Richter A, Bieler JG, Edidin M, Schneck JP (2014). Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity. ACS Nano. 8(3):2252-2260.
Porto G, Sousa MD (2017). Iron overload and immunity. World J. Gastroenterol. 13(35): 4707-4715
Probst HC, McCoy K, Okazaki T, Honjo T, van den Broek M (2015). Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4. Nat Immunol. 6(3):280-286.
Rada P, Rojo AI, Chowdhry S, McMahon M, Hayes JD, Cuadrado A (2011). SCF/β-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner. Mol Cell Biol. 31(6):1121-1133.
Reck M, Rodríguez-Abreu D, Robinson AG, Hui R, Csőszi T, Fülöp A, Gottfried M, Peled N, Tafreshi A, Cuffe S, O'Brien M, Rao S, Hotta K, Leiby MA, Lubiniecki GM, Shentu Y, Rangwala R, Brahmer JR; KEYNOTE-024 Investigators (2016). Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med. 375(19):1823‐1833.
Ren D, Villeneuve NF, Jiang T, Wu T, Lau A, Toppin HA, Zhang DD. (2011) Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proc Natl Acad Sci U S A. 108(4):1433-1438.
René C, Lopez E, Claustres M, Taulan M, Romey-Chatelain MC (2010). NF-E2-related factor 2, a key inducer of antioxidant defenses, negatively regulates the CFTR transcription. Cell. Mol. Life Sci. 67, 2297–2309.
Ribas A, Wolchok JD (2018). Cancer immunotherapy using checkpoint blockade. Science. 359(6382):1350-1355.
Rojo AI, Sagarra MR, Cuadrado A (2008). GSK-3beta down-regulates the transcription factor Nrf2 after oxidant damage: relevance to exposure of neuronal cells to oxidative stress. J Neurochem. 105(1):192-202.
Romero R, Sayin VI, Davidson SM, Bauer MR, Singh SX, LeBoeuf SE, Karakousi TR, Ellis DC, Bhutkar A, Sánchez-Rivera FJ, Subbaraj L, Martinez B, Bronson RT, Prigge JR, Schmidt EE, Thomas CJ, Goparaju C, Davies A, Dolgalev I, Heguy A, Allaj V, Poirier JT, Moreira AL, Rudin CM, Pass HI, Vander Heiden MG, Jacks T, Papagiannakopoulos T (2017). Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis. Nat Med. 23(11):1362-1368.
Salimi M, Sarkar S, Fathi S, Alizadeh AM, Saber R, Moradi F, Delavari H (2018). Biodistribution, pharmacokinetics, and toxicity of dendrimer-coated iron oxide nanoparticles in BALB/c mice. Int J Nanomedicine. 13:1483-1493.
Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, Tamba M, Itoh K, Yamamoto M, Bannai S (2002). Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. J Biol Chem. 277(47):44765-44771.
Schmid O, Möller W, Semmler-Behnke M, Ferron GA, Karg E, Lipka J, Schulz H, Kreyling WG, Stoeger T (2009). Dosimetry and toxicology of inhaled ultrafine particles. Biomarkers. 14 Suppl 1:67-73.
Seiler A, Schneider M, Förster H, Roth S, Wirth EK, Culmsee C, Plesnila N, Kremmer E, Rådmark O, Wurst W, Bornkamm GW, Schweizer U, Conrad M (2008). Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab. 8(3):237-248.
Sequist LV, Yang JC, Yamamoto N, O'Byrne K, Hirsh V, Mok T, Geater SL, Orlov S, Tsai CM, Boyer M, Su WC, Bennouna J, Kato T, Gorbunova V, Lee KH, Shah R, Massey D, Zazulina V, Shahidi M, Schuler M (2013). Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol. 31(27):3327‐3334.
Seto T, Kato T, Nishio M, Goto K, Atagi S, Hosomi Y, Yamamoto N, Hida T, Maemondo M, Nakagawa K, Nagase S, Okamoto I, Yamanaka T, Tajima K, Harada R, Fukuoka M, Yamamoto N (2014). Erlotinib alone or with bevacizumab as first-line therapy in patients with advanced non-squamous non-small-cell lung cancer harbouring EGFR mutations (JO25567): an open-label, randomised, multicentre, phase 2 study. Lancet Oncol. 15(11):1236‐1244.
Sha JY, Zhou YD, Yang JY, Leng J, Li JH, Hu JN, Liu W, Jiang S, Wang YP, Chen C, Li W (2019). Maltol (3-hydroxy-2-methyl-4-pyrone) slows d-galactose-induced brain aging process by damping the Nrf2/HO-1-mediated oxidative stress in mice. J Agric Food Chem. 67(37):10342-10351.
Shah NH, Lazarus JH, Sheth PR, Jarowski CI (1981). Carboxymethylcellulose: effect of degree of polymerization and substitution on tablet disintegration and dissolution. J Pharm Sci. 70(6):611-613.
Shaw AT, Winslow MM, Magendantz M, Ouyang C, Dowdle J, Subramanian A, Lewis TA, Maglathin RL, Tolliday N, Jacks T (2011). Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress. Proc Natl Acad Sci U S A. 108(21):8773-8778.
Shevtsov MA, Nikolaev BP, Yakovleva LY, Parr MA, Marchenko YY, Eliseev I, Yudenko A, Dobrodumov AV, Zlobina O, Zhakhov A, Ischenko AM, Pitkin E, Multhoff G (2015). 70-kDa heat shock protein coated magnetic nanocarriers as a nanovaccine for induction of anti-tumor immune response in experimental glioma. J Control Release. 220(Pt A):329-340.
Shibata T, Saito S, Kokubu A, Suzuki T, Yamamoto M, Hirohashi S (2010). Global downstream pathway analysis reveals a dependence of oncogenic NF-E2-related factor 2 mutation on the mTOR growth signaling pathway. Cancer Res. 70(22):9095-9105.
Siegel RL, Miller KD, Jemal A (2019). Cancer statistics, 2019. CA Cancer J Clin. 69(1):7-34.
Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, Brock MV, Biswal S (2006). Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med. 3(10):e420.
Solis LM, Behrens C, Dong W, Suraokar M, Ozburn NC, Moran CA, Corvalan AH, Biswal S, Swisher SG, Bekele BN, Minna JD, Stewart DJ, Wistuba (2010). Nrf2 and Keap1 abnormalities in non-small cell lung carcinoma and association with clinicopathologic features. Clin Cancer Res. 16(14):3743-3753.
Sriram MI, Kanth SB, Kalishwaralal K, Gurunathan S (2010). Antitumor activity of silver nanoparticles in Dalton’s lymphoma ascites tumor model. Int J Nanomedicine. 5:753–62.
Sun Z, Wang W, Wang R, Duan J, Hu Y, Ma J, Zhou J, Xie S, Lu X, Zhu Z, Chen S, Zhao Y, Xu H, Wang C, Yang XD (2010). Aluminum nanoparticles enhance anticancer immune response induced by tumor cell vaccine. Cancer Nanotechnol. 1(1-6):63-69.
Tabata Y, Ikada Y (1988). Macrophage phagocytosis of biodegradable microspheres composed of L-lactic acid/glycolic acid homo- and copolymers. J Biomed Mater Res. 22(10):837-858.
Taguchi K, Shimada M, Fujii S, Sumi D, Pan X, Yamano S, Nishiyama T, Hiratsuka A, Yamamoto M, Cho AK, Froines JR, Kumagai Y (2008). Redox cycling of 9,10-phenanthraquinone to cause oxidative stress is terminated through its monoglucuronide conjugation in human pulmonary epithelial A549 cells. Free Radic Biol Med. 44(8):1645-1655.
Taiwan Ministry of Health and Welfare. (2019) General health statistics. https://www.mohw.gov.tw/lp-137-2.html
Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, Chen S, Klein AP, Pardoll DM, Topalian SL, Chen L (2012). Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 4(127):127ra37.
Tomida S, Takeuchi T, Shimada Y, Arima C, Matsuo K, Mitsudomi T, Yatabe Y, Takahashi T (2009). Relapse-related molecular signature in lung adenocarcinomas identifies patients with dismal prognosis. J Clin Oncol. 27(17):2793-2799.
Tonelli C, Chio IIC, David A, Tuveson DA (2018). Transcriptional regulation by Nrf2. Antioxid Redox Signal. 29(17):1727-1745.
Topalian SL, Drake CG, Pardoll DM (2015). Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell. 27(4):450-461.
Toraya-Brown S, Sheen MR, Zhang P, Chen L, Baird JR, Demidenko E, Turk MJ, Hoopes PJ, Conejo-Garcia JR, Fiering S (2014). Local hyperthermia treatment of tumors induces CD8(+) T cell-mediated resistance against distal and secondary tumors. Nanomedicine. 10(6):1273-1285.
Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG (2015). Introduction to the 2015 World Health Organization classification of tumors of the lung, pleura, thymus, and heart. J Thorac Oncol. 10(9):1240‐1242.
Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, Chmielowski B, Spasic M, Henry G, Ciobanu V, West AN, Carmona M, Kivork C, Seja E, Cherry G, Gutierrez AJ, Grogan TR, Mateus C, Tomasic G, Glaspy JA, Emerson RO, Robins H, Pierce RH, Elashoff DA, Robert C, Ribas A (2014). PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 515(7528):568-571.
Van Allen EM, Miao D, Schilling B, Shukla SA, Blank C, Zimmer L, Sucker A, Hillen U, Foppen MHG, Goldinger SM, Utikal J, Hassel JC, Weide B, Kaehler KC, Loquai C, Mohr P, Gutzmer R, Dummer R, Gabriel S, Wu CJ, Schadendorf D, Garraway LA (2015). Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science (New York, N.Y.), 350(6257), 207–211.
Ventola CL (2017). Progress in nanomedicine: approved and investigational nanodrugs. P T. 42(12):742-755.
Walunas TL, Bluestone JA (1998). CTLA-4 regulates tolerance induction and T cell differentiation in vivo. J Immunol. 160(8):3855-3860.
Wang AZ, Yuet K, Zhang L, Gu FX, Huynh-Le M, Radovic-Moreno AF, Kantoff PW, Bander NH, Langer R, Farokhzad OC (2010). ChemoRad nanoparticles: a novel multifunctional nanoparticle platform for targeted delivery of concurrent chemoradiation. Nanomedicine (Lond). 5(3):361-368.
Winer A, Bodor JN, Borghaei H (2018). Identifying and managing the adverse effects of immune checkpoint blockade. J Thorac Dis. 10(Suppl 3):S480-S489.
Woo JH, Shimoni Y, Yang WS, Subramaniam P, Iyer A, Nicoletti P, Rodríguez Martínez M, López G, Mattioli M, Realubit R, Karan C, Stockwell BR, Bansal M, Califano A (2015). Elucidating Compound Mechanism of Action by Network Perturbation Analysis. Cell. 162(2):441-451.
Wu KC, Cui JY, Klaassen CD (2011). Beneficial role of Nrf2 in regulating NADPH generation and consumption. Toxicol Sci. 123(2):590-600.
Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ, Wolpaw AJ, Smukste I, Peltier JM, Boniface JJ, Smith R, Lessnick SL, Sahasrabudhe S, Stockwell BR (2007). RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature. 447(7146):864-868.
Yamamoto M, Kensler TW, Motohashi H (2018). The KEAP1-NRF2 system: a thiol-Based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev. 98(3):1169-1203.
Yang LX, Wu YN, Wang PW, Huang KJ, Su WC, Shieh DB (2020). Silver-coated zero-valent iron nanoparticles enhance cancer therapy in mice through lysosome-dependent dual programed cell death pathways: triggering simultaneous apoptosis and autophagy only in cancerous cells. J. Mater. Chem. B. 8, 4122-4131.
Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, Cheah JH, Clemons PA, Shamji AF, Clish CB, Brown LM, Girotti AW, Cornish VW, Schreiber SL, Stockwell BR (2014). Regulation of ferroptotic cancer cell death by GPX4. Cell. 156(1-2):317-331.
Yang WS, Stockwell BR (2008). Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234–245.
Yang WS, Stockwell BR (2016). Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 26, 165–176.
Yao X, Panichpisal K, Kurtzman N, Nugent K (2007). Cisplatin nephrotoxicity: a review. Am J Med Sci. 334(2):115‐124.
You DG, Deepagan VG, Um W, Jeon S, Son S, Chang H, Yoon HI, Cho YW, Swierczewska M, Lee S, Pomper MG, Kwon IC, Kim K, Park JH (2016). ROS-generating TiO2 nanoparticles for non-invasive sonodynamic therapy of cancer. Sci Rep. 6:23200.
Zamay GS, Zamay TN, Lukyanenko KA, Kichkailo AS (2020). Aptamers increase biocompatibility and reduce the toxicity of magnetic nanoparticles used in biomedicine. Biomedicines. 8(3):59.
Zanganeh S, Hutter G, Spitler R, Lenkov O, Mahmoudi M, Shaw A, Pajarinen JS, Nejadnik H, Goodman S, Moseley M, Coussens LM, Daldrup-Link HE (2016). Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nat Nanotechnol. 11(11):986-994.
Zhang HH, Lipovsky AI, Dibble CC, Sahin M, Manning BD (2006). S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. Mol Cell. 24(2):185-197.
Zhou L, Thanh TL, Gong J, Kim JH, Kim EJ, Chang YS (2014). Carboxymethyl cellulose coating decreases toxicity and oxidizing capacity of nanoscale zerovalent iron. Chemosphere. 104:155-161.