Erastin, a ferroptosis-inducing agent, sensitized cancer cells to X-ray irradiation via glutathione starvation in vitro and in vivo

Autoři: Yuki Shibata aff001;  Hironobu Yasui aff001;  Kei Higashikawa aff001;  Naoki Miyamoto aff004;  Yuji Kuge aff001
Působiště autorů: Department of Biomedical Imaging, Graduate School of Biomedical Science and Engineering, Hokkaido University, Sapporo, Hokkaido, Japan aff001;  Central Institute of Isotope Science, Hokkaido University, Sapporo, Hokkaido, Japan aff002;  Laboratory of Radiation Biology, Department of Applied Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan aff003;  Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido, Japan aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0225931


High concentrations of antioxidants in cancer cells are huge obstacle in cancer radiotherapy. Erastin was first discovered as an inducer of iron-dependent cell death called ferroptosis accompanied by antioxidant depletion caused by cystine glutamate antiporter inhibition. Therefore, treatment with erastin is expected to potentially enhance cellular radiosensitivity. In this study, we investigated the influence of treatment with erastin on the radiation efficiency against cancers. The clonogenic ability, glutathione peroxidase 4 (GPX4) expression, and glutathione concentration were evaluated using HeLa and NCI-H1975 adenocarcinoma cell lines treated with erastin and/or X-ray irradiation. For in vivo studies, NCI-H1975 cells were transplanted in the left shoulder of nude mice, and then radiosensitizing effect of erastin and glutathione concentration in the cancer were evaluated. Treatment with erastin induced ferroptosis and decreased the concentration of glutathione and GPX4 protein expression levels in the two tumor cell lines. Moreover, erastin enhanced X-ray irradiation-induced cell death in both human tumor cell lines. Furthermore, erastin treatment of a tumor-transplanted mouse model similarly demonstrated the radiosensitizing effect and decrease in intratumoral glutathione concentration in the in vitro study. In conclusion, our study demonstrated the radiosensitizing effect of erastin on two adenocarcinoma cell lines and the tumor xenograft model accompanied by glutathione depletion, indicating that ferroptosis inducers that reduce glutathione concentration could be applied as a novel cancer therapy in combination with radiotherapy.

Klíčová slova:

Antioxidants – Cancer treatment – Cell death – Glutathione – HeLa cells – Protein expression – Radiation therapy – Adenocarcinoma cells


1. Deng Z, Manz DH, Torti SV, Torti FM. Iron-responsive element-binding protein 2 plays an essential role in regulating prostate cancer cell growth. Oncotarget. 2017;8(47):82231–43. doi: 10.18632/oncotarget.19288 29137259

2. Manz DH, Blanchette NL, Paul BT, Torti FM, Torti SV. Iron and cancer: recent insights. Ann N Y Acad Sci. 2016;1368(1):149–61. doi: 10.1111/nyas.13008 26890363

3. Torti SV, Manz DH, Paul BT, Blanchette-Farra N, Torti FM. Iron and Cancer. Annu Rev Nutr. 2018;38:97–125. doi: 10.1146/annurev-nutr-082117-051732 30130469

4. Muckenthaler MU, Galy B, Hentze MW. Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network. Annu Rev Nutr. 2008;28:197–213. doi: 10.1146/annurev.nutr.28.061807.155521 18489257

5. Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y. Regulators of Iron Homeostasis: New Players in Metabolism, Cell Death, and Disease. Trends Biochem Sci. 2016;41(3):274–86. doi: 10.1016/j.tibs.2015.11.012 26725301

6. Muckenthaler MU, Rivella S, Hentze MW, Galy B. A Red Carpet for Iron Metabolism. Cell. 2017;168(3):344–61. doi: 10.1016/j.cell.2016.12.034 28129536

7. Bystrom LM, Rivella S. Cancer cells with irons in the fire. Free Radic Biol Med. 2015;79:337–42. doi: 10.1016/j.freeradbiomed.2014.04.035 24835768

8. Elford HL, Freese M, Passamani E, Morris HP. Ribonucleotide reductase and cell proliferation. I. Variations of ribonucleotide reductase activity with tumor growth rate in a series of rat hepatomas. J Biol Chem. 1970;245(20):5228–33. 4319235

9. Högemann-Savellano D, Bos E, Blondet C, Sato F, Abe T, Josephson L, et al. The transferrin receptor: a potential molecular imaging marker for human cancer. Neoplasia. 2003;5(6):495–506. doi: 10.1016/s1476-5586(03)80034-9 14965443

10. Habashy HO, Powe DG, Staka CM, Rakha EA, Ball G, Green AR, et al. Transferrin receptor (CD71) is a marker of poor prognosis in breast cancer and can predict response to tamoxifen. Breast Cancer Res Treat. 2010;119(2):283–93. doi: 10.1007/s10549-009-0345-x 19238537

11. White S, Taetle R, Seligman PA, Rutherford M, Trowbridge IS. Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects. Cancer Res. 1990;50(19):6295–301. 2400993

12. Shen Y, Li X, Dong D, Zhang B, Xue Y, Shang P. Transferrin receptor 1 in cancer: a new sight for cancer therapy. Am J Cancer Res. 2018;8(6):916–31. 30034931

13. Traverso N, Ricciarelli R, Nitti M, Marengo B, Furfaro AL, Pronzato MA, et al. Role of glutathione in cancer progression and chemoresistance. Oxid Med Cell Longev. 2013;2013:972913. doi: 10.1155/2013/972913 23766865

14. Bansal A, Simon MC. Glutathione metabolism in cancer progression and treatment resistance. J Cell Biol. 2018;217(7):2291–8. doi: 10.1083/jcb.201804161 29915025

15. Tagde A, Singh H, Kang MH, Reynolds CP. The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma. Blood Cancer J. 2014;4:e229. doi: 10.1038/bcj.2014.45 25036800

16. Villablanca JG, Volchenboum SL, Cho H, Kang MH, Cohn SL, Anderson CP, et al. A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent/Refractory High-Risk Neuroblastoma. Pediatr Blood Cancer. 2016;63(8):1349–56. doi: 10.1002/pbc.25994 27092812

17. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060–72. doi: 10.1016/j.cell.2012.03.042 22632970

18. Gao M, Monian P, Quadri N, Ramasamy R, Jiang X. Glutaminolysis and Transferrin Regulate Ferroptosis. Mol Cell. 2015;59(2):298–308. doi: 10.1016/j.molcel.2015.06.011 26166707

19. Yang WS, Stockwell BR. Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem Biol. 2008;15(3):234–45. doi: 10.1016/j.chembiol.2008.02.010 18355723

20. Dixon SJ, Patel DN, Welsch M, Skouta R, Lee ED, Hayano M, et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife. 2014;3:e02523. doi: 10.7554/eLife.02523 24844246

21. Lu B, Chen XB, Ying MD, He QJ, Cao J, Yang B. The Role of Ferroptosis in Cancer Development and Treatment Response. Front Pharmacol. 2017;8:992. doi: 10.3389/fphar.2017.00992 29375387

22. Sontheimer H, Bridges RJ. Sulfasalazine for brain cancer fits. Expert Opin Investig Drugs. 2012;21(5):575–8. doi: 10.1517/13543784.2012.670634 22404218

23. Mooney MR, Geerts D, Kort EJ, Bachmann AS. Anti-tumor effect of sulfasalazine in neuroblastoma. Biochem Pharmacol. 2019.

24. Roh JL, Kim EH, Jang HJ, Park JY, Shin D. Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer. Cancer Lett. 2016;381(1):96–103. doi: 10.1016/j.canlet.2016.07.035 27477897

25. Yu Y, Xie Y, Cao L, Yang L, Yang M, Lotze MT, et al. The ferroptosis inducer erastin enhances sensitivity of acute myeloid leukemia cells to chemotherapeutic agents. Mol Cell Oncol. 2015;2(4):e1054549. doi: 10.1080/23723556.2015.1054549 27308510

26. Chen L, Li X, Liu L, Yu B, Xue Y, Liu Y. Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-γ-lyase function. Oncol Rep. 2015;33(3):1465–74. doi: 10.3892/or.2015.3712 25585997

27. Luo M, Wu L, Zhang K, Wang H, Zhang T, Gutierrez L, et al. miR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma. Cell Death Differ. 2018;25(8):1457–72. doi: 10.1038/s41418-017-0053-8 29348676

28. Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ, et al. RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature. 2007;447(7146):864–8. doi: 10.1038/nature05859 17568748

29. Sato M, Kusumi R, Hamashima S, Kobayashi S, Sasaki S, Komiyama Y, et al. The ferroptosis inducer erastin irreversibly inhibits system x c − and synergizes with cisplatin to increase cisplatin’s cytotoxicity in cancer cells. Sci Rep. 2018;8(1):968. doi: 10.1038/s41598-018-19213-4 29343855

30. Bridges CC, Kekuda R, Wang H, Prasad PD, Mehta P, Huang W, et al. Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 2001;42(1):47–54. 11133847

31. Gao M, Monian P, Pan Q, Zhang W, Xiang J, Jiang X. Ferroptosis is an autophagic cell death process. Cell Res. 2016;26(9):1021–32. doi: 10.1038/cr.2016.95 27514700

32. Yant LJ, Ran Q, Rao L, Van Remmen H, Shibatani T, Belter JG, et al. The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults. Free Radic Biol Med. 2003;34(4):496–502. doi: 10.1016/s0891-5849(02)01360-6 12566075

33. Huang X. Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal. Mutat Res. 2003;533(1–2):153–71. doi: 10.1016/j.mrfmmm.2003.08.023 14643418

34. Sleire L, Skeie BS, Netland IA, Førde HE, Dodoo E, Selheim F, et al. Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion. Oncogene. 2015;34(49):5951–9. doi: 10.1038/onc.2015.60 25798841

35. Rodman SN, Spence JM, Ronnfeldt TJ, Zhu Y, Solst SR, O'Neill RA, et al. Enhancement of Radiation Response in Breast Cancer Stem Cells by Inhibition of Thioredoxin- and Glutathione-Dependent Metabolism. Radiat Res. 2016;186(4):385–95. doi: 10.1667/RR14463.1 27643875

36. Bao Y, Jemth P, Mannervik B, Williamson G. Reduction of thymine hydroperoxide by phospholipid hydroperoxide glutathione peroxidase and glutathione transferases. FEBS Letters. 1997;410(2–3):210–2. doi: 10.1016/s0014-5793(97)00591-7 9237631

37. Vos O, Van Der Schans GP, Roos-Verhey WSD. Effects of BSO and DEM on thiol-level and radiosensitivity in HeLa cells. 1984;10(8):1249–53. doi: 10.1016/0360-3016(84)90327-4 6469746

38. Yi X, Ding L, Jin Y, Ni C, Wang W. The toxic effects, GSH depletion and radiosensitivity by BSO on retinoblastoma. International Journal of Radiation Oncology*Biology*Physics. 1994;29(2):393–6.

39. Carney DN, Mitchell JB, Kinsella TJ. In vitro radiation and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morphological variants. Cancer Res. 1983;43(6):2806–11. 6303568

40. Morstyn G, Russo A, Carney DN, Karawya E, Wilson SH, Mitchell JB. Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines. J Natl Cancer Inst. 1984;73(4):801–7. 6148444

41. Bristow RG, Hardy PA, Hill RP. Comparison between in vitro radiosensitivity and in vivo radioresponse of murine tumor cell lines I: parameters of in vitro radiosensitivity and endogenous cellular glutathione levels. International Journal of Radiation Oncology*Biology*Physics. 1990;18(1):133–45.

42. Sartor CI. Epidermal growth factor family receptors and inhibitors: Radiation response modulators. Seminars in Radiation Oncology. 2003;13(1):22–30. doi: 10.1053/srao.2003.50003 12520461

43. Yagishita S, Horinouchi H, Katsui Taniyama T, Nakamichi S, Kitazono S, Mizugaki H, et al. Epidermal Growth Factor Receptor Mutation Is Associated With Longer Local Control After Definitive Chemoradiotherapy in Patients With Stage III Nonsquamous Non–Small-Cell Lung Cancer. 2015;91(1):140–8. doi: 10.1016/j.ijrobp.2014.08.344 25442336

44. Amornwichet N, Oike T, Shibata A, Nirodi CS, Ogiwara H, Makino H, et al. The EGFR mutation status affects the relative biological effectiveness of carbon-ion beams in non-small cell lung carcinoma cells. Scientific Reports. 2015;5(1):11305.

45. Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014;156(1–2):317–31. doi: 10.1016/j.cell.2013.12.010 24439385

46. Stockwell BR, Friedmann Angeli JP, Bayir H, Bush AI, Conrad M, Dixon SJ, et al. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 2017;171(2):273–85. doi: 10.1016/j.cell.2017.09.021 28985560

47. Nagane M, Kanai E, Shibata Y, Shimizu T, Yoshioka C, Maruo T, et al. Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma. PLoS One. 2018;13(4):e0195151. doi: 10.1371/journal.pone.0195151 29649284

48. Cobler L, Zhang H, Suri P, Park C, Timmerman LA. xCT inhibition sensitizes tumors to γ-radiation via glutathione reduction. Oncotarget. 2018;9(64):32280–97. doi: 10.18632/oncotarget.25794 30190786

49. Healy BJ, van der Merwe D, Christaki KE, Meghzifene A. Cobalt-60 Machines and Medical Linear Accelerators: Competing Technologies for External Beam Radiotherapy. Clin Oncol (R Coll Radiol). 2017;29(2):110–5. doi: 10.1016/j.clon.2016.11.002 27908503

Článok vyšiel v časopise


2019 Číslo 12