Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2 cells

Sun,Lei; Wang,Hua; Yu,Shanshan; Zhang,Lin; Jiang,Jue; Zhou,Qi

doi:10.3892/ijmm.2021.5072

February-2022 Volume 49 Issue 2

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February-2022 Volume 49 Issue 2

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Article Open Access

Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2 cells

Authors:
- Lei Sun
- Hua Wang
- Shanshan Yu
- Lin Zhang
- Jue Jiang
- Qi Zhou
View Affiliations / Copyright

Affiliations: Department of Ultrasound, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China

Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Article Number: 17
|
Published online on: December 16, 2021

https://doi.org/10.3892/ijmm.2021.5072
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Abstract

Ferroptosis has been previously implicated in the pathological progression of cardiomyopathy. Herceptin (trastuzumab), which targets HER2, is commonly applied for the treatment of HER2⁺ breast cancer. However, its clinical use is limited by its cardiotoxicity. Therefore, the present study aimed to investigate if targeting ferroptosis could protect against Herceptin‑induced heart failure in an in vitro model of H9c2 cells after treatment of Herceptin, Herceptin + ferroptosis inhibitor ferrostatin‑1 (Fer‑1) or Herceptin + Deferoxamine. H9c2 cell viability was measured by MTT assay. Reactive oxygen species (ROS) levels were detected by measuring the ﬂuorescence of DCFH‑DA‑A and MitoSOX™ Red. Glutathione (GSH)/oxidized glutathione (GSSG) ratio was measured using the GSH/GSSG Ratio Detection Assay kit. Mitochondrial membrane potential and ATP content were evaluated by JC‑1 staining and bioluminescent assay kits, respectively. Protein expressions of glutathione peroxidase 4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1‑1/2, mitofusin, Acyl‑CoA synthetase long chain family member 4, cytochrome c, voltage‑dependent anion‑selective channel, dynamin‑related protein, mitochondrial fission 1 protein and mitochondrial ferritin were evaluated by western blotting. It was found that Herceptin reduced H9c2 cell viability whilst increasing intracellular and mitochondrial ROS levels in a dose‑ and time‑dependent manner. Furthermore, Herceptin decreased glutathione peroxidase (GPX) protein expression and the GSH/ GSSG ratio in H9c2 cells in a dose‑ and time‑dependent manner. The Fer‑1 abolished this Herceptin‑induced reduction in cell viability, GSH/GSSG ratio, mitochondrial membrane potential and ATP content. Fer‑1 also reversed the suppressive effects of Herceptin on the protein expression levels of GPX4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1‑1/2 and mitofusin in H9c2 cells. Subsequently, Fer‑1 was found to reverse the Herceptin‑induced increase in mitochondrial ROS and iron levels in H9c2 cells, as well as the increased protein expression levels of Acyl‑CoA synthetase long chain family member 4, cytochrome c, voltage‑dependent anion‑selective channel, dynamin‑related protein, mitochondrial fission 1 protein and mitochondrial ferritin in H9c2 cells. However, compared with deferoxamine, an iron chelator, the effects of Fer‑1 were less effective. Collectively, these findings provided insights into the pathogenic mechanism that underlie Herceptin‑induced cardiomyopathy, which potentially provides a novel therapeutic target for the prevention of cardiotoxicity in HER2⁺ breast cancer treatment.

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1	Fuchs Y and Steller H: Programmed cell death in animal development and disease. Cell. 147:742–758. 2011. View Article : Google Scholar : PubMed/NCBI
2	Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, Patel DN, Bauer AJ, Cantley AM, Yang WS, et al: Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell. 149:1060–1072. 2012. View Article : Google Scholar : PubMed/NCBI
3	Angeli JPF, Shah R, Pratt DA and Conrad M: Ferroptosis inhibition: Mechanisms and opportunities. Trends Pharmacol Sci. 38:489–498. 2017. View Article : Google Scholar : PubMed/NCBI
4	Fang X, Wang H, Han D, Xie E, Yang X, Wei J, Gu S, Gao F, Zhu N, Yin X, et al: Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci USA. 116:2672–2680. 2019. View Article : Google Scholar : PubMed/NCBI
5	Linkermann A, Skouta R, Himmerkus N, Mulay SR, Dewitz C, De Zen F, Prokai A, Zuchtriegel G, Krombach F, Welz PS, et al: Synchronized renal tubular cell death involves ferroptosis. Proc Natl Acad Sci USA. 111:16836–16841. 2014. View Article : Google Scholar : PubMed/NCBI
6	Yang WS and Stockwell BR: Ferroptosis: Death by lipid peroxidation. Trends Cell Biol. 26:165–176. 2016. View Article : Google Scholar :
7	Loibl S and Gianni L: HER2-positive breast cancer. Lancet. 389:2415–2429. 2017. View Article : Google Scholar
8	Slamon D, Eiermann W, Robert N, Pienkowski T, Martin M, Press M, Mackey J, Glaspy J, Chan A, Pawlicki M, et al: Adjuvant trastuzumab in HER2-positive breast cancer. N Engl J Med. 365:1273–1283. 2011. View Article : Google Scholar : PubMed/NCBI
9	Jerusalem G, Lancellotti P and Kim SB: HER2⁺ breast cancer treatment and cardiotoxicity: Monitoring and management. Breast Cancer Res Treat. 177:237–250. 2019. View Article : Google Scholar : PubMed/NCBI
10	Barish R, Gates E and Barac A: Trastuzumab-induced cardiomyopathy. Cardiol Clin. 37:407–418. 2019. View Article : Google Scholar : PubMed/NCBI
11	Whelan RS, Kaplinskiy V and Kitsis RN: Cell death in the pathogenesis of heart disease: Mechanisms and significance. Annu Rev Physiol. 72:19–44. 2010. View Article : Google Scholar : PubMed/NCBI
12	Pantopoulos K, Porwal SK, Tartakoff A and Devireddy L: Mechanisms of mammalian iron homeostasis. Biochemistry. 51:5705–5724. 2012. View Article : Google Scholar : PubMed/NCBI
13	Ward RJ, Zucca FA, Duyn JH, Crichton RR and Zecca L: The role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol. 13:1045–1060. 2014. View Article : Google Scholar : PubMed/NCBI
14	Rouault TA: Iron metabolism in the CNS: Implications for neurodegenerative diseases. Nat Rev Neurosci. 14:551–564. 2013. View Article : Google Scholar : PubMed/NCBI
15	Reichert CO, de Freitas FA, Sampaio-Silva J, Rokita-Rosa L, Barros PL, Levy D and Bydlowski SP: Ferroptosis mechanisms involved in neurodegenerative diseases. Int J Mol Sci. 21:87652020. View Article : Google Scholar :
16	Torti SV and Torti FM: Iron and cancer: More ore to be mined. Nat Rev Cancer. 13:342–355. 2013. View Article : Google Scholar : PubMed/NCBI
17	Chen Y, Fan Z, Yang Y and Gu C: Iron metabolism and its contribution to cancer (Review). Int J Oncol. 54:1143–1154. 2019.PubMed/NCBI
18	Lapice E, Masulli M and Vaccaro O: Iron deficiency and cardiovascular disease: An updated review of the evidence. Curr Atheroscler Rep. 15:3582013. View Article : Google Scholar : PubMed/NCBI
19	Kobayashi M, Suhara T, Baba Y, Kawasaki NK, Higa JK and Matsui T: Pathological roles of iron in cardiovascular disease. Curr Drug Targets. 19:1068–1076. 2018. View Article : Google Scholar : PubMed/NCBI
20	Wood JC: History and current impact of cardiac magnetic resonance imaging on the management of iron overload. Circulation. 120:1937–1939. 2009. View Article : Google Scholar : PubMed/NCBI
21	Valko M, Jomova K, Rhodes CJ, Kuča K and Musílek K: Redox- and non-redox-metal-induced formation of free radicals and their role in human disease. Arch Toxicol. 90:1–37. 2016. View Article : Google Scholar
22	Ichikawa Y, Ghanefar M, Bayeva M, Wu R, Khechaduri A, Naga Prasad SV, Mutharasan RK, Naik TJ and Ardehali H: Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J Clin Invest. 124:617–630. 2014. View Article : Google Scholar : PubMed/NCBI
23	Gujja P, Rosing DR, Tripodi DJ and Shizukuda Y: Iron overload cardiomyopathy: Better understanding of an increasing disorder. J Am Coll Cardiol. 56:1001–1012. 2010. View Article : Google Scholar : PubMed/NCBI
24	Kurokawa YK, Shang MR, Yin RT and George SC: Modeling trastuzumab-related cardiotoxicity in vitro using human stem cell-derived cardiomyocytes. Toxicol Lett. 285:74–80. 2018. View Article : Google Scholar : PubMed/NCBI
25	Baba Y, Higa JK, Shimada BK, Horiuchi KM, Suhara T, Kobayashi M, Woo JD, Aoyagi H, Marh KS, Kitaoka H and Matsui T: Protective effects of the mechanistic target of rapamycin against excess iron and ferroptosis in cardiomyocytes. Am J Physiol Heart Circ Physiol. 314:H659–H668. 2018. View Article : Google Scholar :
26	Wu C, Zhao W, Yu J, Li S, Lin L and Chen X: Induction of ferroptosis and mitochondrial dysfunction by oxidative stress in PC12 cells. Sci Rep. 8:5742018. View Article : Google Scholar : PubMed/NCBI
27	Xu T, Ding W, Ji X, Ao X, Liu Y, Yu W and Wang J: Oxidative stress in cell death and cardiovascular diseases. Oxid Med Cell Longev. 2019:90305632019. View Article : Google Scholar : PubMed/NCBI
28	Chen S, Zhang Z, Qing T, Ren Z, Yu D, Couch L, Ning B, Mei N, Shi L, Tolleson WH and Guo L: Activation of the Nrf2 signaling pathway in usnic acid-induced toxicity in HepG2 cells. Arch Toxicol. 91:1293–1307. 2017. View Article : Google Scholar :
29	Mohan N, Jiang J and Wu WJ: Implication of autophagy and oxidative stress in trastuzumab-mediated cardiac toxicities. Austin Pharmacol Pharm. 2:10052017.
30	Lee H, Ki J, Lee SY, Park JH and Hwang GS: Processed panax ginseng, sun ginseng, decreases oxidative damage induced by tert-butyl hydroperoxide via regulation of antioxidant enzyme and anti-apoptotic molecules in HepG2 cells. J Ginseng Res. 3:248–255. 2012. View Article : Google Scholar
31	Anderson ME: Glutathione: An overview of biosynthesis and modulation. Chem Biol Interact. 111-112:1–14. 1998. View Article : Google Scholar : PubMed/NCBI
32	Townsend DM, Tew KD and Tapiero H: The importance of glutathione in human disease. Biomed Pharmacother. 57:145–155. 2003. View Article : Google Scholar : PubMed/NCBI
33	Ma W, Wei S, Zhang B and Li W: Molecular mechanisms of cardiomyocyte death in drug-induced cardiotoxicity. Front Cell Dev Biol. 8:4342020. View Article : Google Scholar : PubMed/NCBI
34	Oudit GY, Sun H, Trivieri MG, Koch SE, Dawood F, Ackerley C, Yazdanpanah M, Wilson GJ, Schwartz A, Liu PP and Backx PH: L-type Ca2⁺ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med. 9:1187–1194. 2003. View Article : Google Scholar : PubMed/NCBI
35	Pennell DJ, Udelson JE, Arai AE, Bozkurt B, Cohen AR, Galanello R, Hoffman TM, Kiernan MS, Lerakis S, Piga A, et al: Cardiovascular function and treatment in beta-thalassemia major: A consensus statement from the American heart association. Circulation. 128:281–308. 2013. View Article : Google Scholar : PubMed/NCBI
36	Munzel T, Gori T, Keaney JF Jr, Maack C and Daiber A: Pathophysiological role of oxidative stress in systolic and diastolic heart failure and its therapeutic implications. Eur Heart J. 36:2555–2564. 2015. View Article : Google Scholar : PubMed/NCBI
37	Lei G, Zhang Y, Koppula P, Liu X, Zhang J, Lin SH, Ajani JA, Xiao Q, Liao Z, Wang H and Gan B: The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression. Cell Res. 30:146–162. 2020. View Article : Google Scholar : PubMed/NCBI
38	Seibt TM, Proneth B and Conrad M: Role of GPX4 in ferroptosis and its pharmacological implication. Free Radic Biol Med. 133:144–152. 2019. View Article : Google Scholar
39	Koppula P, Zhang Y, Zhuang L and Gan B: Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer. Cancer Commun (Lond). 38:122018. View Article : Google Scholar
40	Cheng J, Fan YQ, Liu BH, Zhou H, Wang JM and Chen QX: ACSL4 suppresses glioma cells proliferation via activating ferroptosis. Oncol Rep. 43:147–158. 2020.
41	Yuan H, Li X, Zhang X, Kang R and Tang D: Identification of ACSL4 as a biomarker and contributor of ferroptosis. Biochem Biophys Res Commun. 478:1338–1343. 2016. View Article : Google Scholar : PubMed/NCBI
42	Aon MA, Bhatt N and Cortassa SC: Mitochondrial and cellular mechanisms for managing lipid excess. Front Physiol. 5:2822014. View Article : Google Scholar : PubMed/NCBI
43	Kadenbach B, Ramzan R, Moosdorf R and Vogt S: The role of mitochondrial membrane potential in ischemic heart failure. Mitochondrion. 11:700–706. 2011. View Article : Google Scholar : PubMed/NCBI
44	Opie LH: Metabolism of the heart in health and disease. I Am Heart J. 76:685–698. 1968. View Article : Google Scholar
45	Ventura-Clapier R, Garnier A and Veksler V: Energy metabolism in heart failure. J Physiol. 555:1–13. 2004. View Article : Google Scholar
46	Chen H and Chan DC: Mitochondrial dynamics-fusion, fission, movement, and mitophagy-in neurodegenerative diseases. Hum Mol Genet. 18:R169–R176. 2009. View Article : Google Scholar : PubMed/NCBI
47	Nguyen D, Alavi MV, Kim KY, Kang T, Scott RT, Noh YH, Lindsey JD, Wissinger B, Ellisman MH, Weinreb RN, et al: A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics. Cell Death Dis. 2:e2402011. View Article : Google Scholar : PubMed/NCBI
48	Varikmaa M, Bagur R, Kaambre T, Grichine A, Timohhina N, Tepp K, Shevchuk I, Chekulayev V, Metsis M, Boucher F, et al: Role of mitochondria-cytoskeleton interactions in respiration regulation and mitochondrial organization in striated muscles. Biochim Biophys Acta. 1837:232–245. 2014. View Article : Google Scholar
49	Hara Y, Yanatori I, Tanaka A, Kishi F, Lemasters JJ, Nishina S, Sasaki K and Hino K: Iron loss triggers mitophagy through induction of mitochondrial ferritin. EMBO Rep. 21:e502022020. View Article : Google Scholar : PubMed/NCBI
50	Wang H, Zhang W, Yu J, Wu C, Gao Q, Li X, Li Y, Zhang J, Tian Y, Tan T, et al: Genetic screening and multipotency in rhesus monkey haploid neural progenitor cells. Development. 145:dev1605312018. View Article : Google Scholar : PubMed/NCBI