Hemolysis and eryptosis contribute to anemia encountered in patients undergoing chemotherapy. Eicosapentaenoic acid (EPA) is an omega-3 dietary fatty acid that has anticancer potential by inducing apoptosis in cancer cells, but its effect on the physiology and lifespan of red blood cells (RBCs) is understudied. Human RBCs were exposed to anticancer concentrations of EPA (10-100 ?M) for 24 h at 37 °C. Acetylcholinesterase (AChE) activity and hemolysis were measured by colorimetric assays whereas annexin-V-FITC and forward scatter (FSC) were employed to identify eryptotic cells. Oxidative stress was assessed by H2DCFDA and intracellular Ca2+ was measured by Fluo4/AM. EPA significantly increased hemolysis and K+ leakage, and LDH and AST activities in the supernatants in a concentration-dependent manner. EPA also significantly increased annexin-V-FITC-positive cells and Fluo4 fluorescence and decreased FSC and AChE activity. A significant reduction in the hemolytic activity of EPA was noted in the presence extracellular isosmotic urea, 125 mM KCl, and polyethylene glycol 8000 (PEG 8000), but not sucrose. In conclusion, EPA stimulates hemolysis and eryptosis through Ca2+ buildup and AChE inhibition. Urea, blocking KCl efflux, and PEG 8000 alleviate the hemolytic activity of EPA. The anticancer potential of EPA may be optimized using Ca2+ channel blockers and chelators to minimize its toxicity to off-target tissue. Keywords: EPA, Eryptosis, Hemolysis, Calcium, Anticancer.

Department of Clinical Laboratory Sciences College of Applied Medical Sciences King Saud University Riyadh Saudi Arabia

Zobrazit více v PubMed

Ashfaq W, Rehman K, Siddique MI, Khan QAA. Eicosapentaenoic acid and docosahexaenoic acid from fish oil and their role in cancer research. Food Rev Int. 2020;36:795–814. doi: 10.1080/87559129.2019.1686761. DOI

Gammone MA, Riccioni G, Parrinello G, D'orazio N. Omega-3 polyunsaturated fatty acids: Benefits and endpoints in sport. Nutrients. 2019;11:1–16. doi: 10.3390/nu11010046. PubMed DOI PMC

Volpato M, Hull MA. Omega-3 polyunsaturated fatty acids as adjuvant therapy of colorectal cancer. Cancer Metastasis Rev. 2018;37:545–555. doi: 10.1007/s10555-018-9744-y. PubMed DOI PMC

Kelavkar UP, Hutzley J, Dhir R, Kim P, Allen KGD, McHugh K. Prostate tumor growth and recurrence can be modulated by the ω-6:ω-3 ratio in diet: Athymic mouse xenograft model simulating radical prostatectomy. Neoplasia. 2006;8:112–24. doi: 10.1593/neo.05637. PubMed DOI PMC

Sawada N, Inoue M, Iwasaki M, Sasazuki S, Shimazu T, Yamaji T, et al. Consumption of n-3 fatty acids and fish reduces risk of hepatocellular carcinoma. Gastroenterology. 2012;142:1468–75. doi: 10.1053/j.gastro.2012.02.018. PubMed DOI

Aoyama T, Yoshikawa T, Ida S, Cho H, Sakamaki K, Ito Y, et al. Effects of perioperative eicosapentaenoic acid-enriched oral nutritional supplement on the long-term oncological outcomes after total gastrectomy for gastric cancer. Oncol Lett. 2022;23:1–7. doi: 10.3892/ol.2022.13272. PubMed DOI PMC

Kubota H, Matsumoto H, Higashida M, Murakami H, Nakashima H, Oka Y, et al. Eicosapentaenoic acid modifies cytokine activity and inhibits cell proliferation in an oesophageal cancer cell line. Anticancer Res. 2013;33:4319–24. PubMed

So WW, Liu WN, Leung KN. Omega-3 polyunsaturated fatty acids trigger cell cycle arrest and induce apoptosis in human neuroblastoma LA-N-1 cells. Nutrients. 2015;7:6956–73. doi: 10.3390/nu7085319. PubMed DOI PMC

Heimli H, Hollung K, Drevon CA. Eicosapentaenoic acid-induced apoptosis depends on acyl CoA-synthetase. Lipids. 2003;38:263–68. doi: 10.1007/s11745-003-1059-z. PubMed DOI

Ando N, Hara M, Shiga K, Yanagita T, Takasu K, Nakai N, et al. Eicosapentaenoic acid suppresses angiogenesis via reducing secretion of IL-6 and VEGF from colon cancer-associated fibroblasts. Oncol Rep. 2019;42:339–49. doi: 10.3892/or.2019.7141. PubMed DOI

Spencer L, Mann C, Metcalfe M, Webb M, Pollard C, Spencer D, et al. The effect of omega-3 FAs on tumour angiogenesis and their therapeutic potential. Eur J Cancer. 2009;45:2077–86. doi: 10.1016/j.ejca.2009.04.026. PubMed DOI

Giros A, Mike G, Sohn VR, Pons E, Morales JF, Xicola RM, et al. Regulation of Colorectal Cancer Cell Apoptosis by the n-3 Polyunsaturated Fatty Acids Docosahexaenoic and Eicosapentaenoic. Cancer Prev Res (Phila) 2008;2:732–42. doi: 10.1158/1940-6207.CAPR-08-0197. PubMed DOI PMC

Zhu S, Feng N, Lin G, Tong Y, Jiang X, Yang Q, et al. Metabolic Shift Induced by ω-3 PUFAs and Rapamycin Lead to Cancer Cell Death. Cell Physiol Biochem. 2018;48:2318–36. doi: 10.1159/000492648. PubMed DOI

Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, et al. Eicosapentaenoic acid in combination with EPHA2 inhibition shows efficacy in preclinical models of triple-negative breast cancer by disrupting cellular cholesterol efflux. Oncogene. 2019;38:2135–50. doi: 10.1038/s41388-018-0569-5. PubMed DOI PMC

Colquhoun A. Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro : Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction. J Urol. 2009;181:1885–93. doi: 10.1016/j.juro.2008.11.092. PubMed DOI

Zhang Y, Han L, Qi W, Cheng D, Ma X, Hou L, et al. Biochemical and Biophysical Research Communications Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS - Ca 2 + - JNK mitochondrial pathways. Biochem Biophys Res Commun. 2015;456:926–32. doi: 10.1016/j.bbrc.2014.12.036. PubMed DOI

Shirota T, Haji S, Yamasaki M, Iwasaki T, Hidaka T, Takeyama Y, et al. Apoptosis in human pancreatic cancer cells induced by eicosapentaenoic acid. Nutrition. 2005;21:1010–17. doi: 10.1016/j.nut.2004.12.013. PubMed DOI

Jayathilake AG, Senior PV, Su XQ. Krill oil extract suppresses cell growth and induces apoptosis of human colorectal cancer cells. BMC Complement Altern Med. 2016;16:1–11. doi: 10.1186/s12906-016-1311-x. PubMed DOI PMC

Mizoguchi K, Ishiguro H, Kimura M, Takahashi H. Induction of Apoptosis by Eicosapentaenoic Acid in Esophageal Squamous Cell Carcinoma. Anticancer Res. 2014;34:7145–49. PubMed

Heimli H, Giske C, Naderi S, Drevon CA, Hollung K. Eicosapentaenoic Acid Promotes Apoptosis in Ramos Cells via Activation of Caspase-3 and -9. Lipids. 2002;37:797–802. doi: 10.1007/s11745-002-0963-6. PubMed DOI

Mortaz E, Moloudizargari M, Khosravi A, Asghari MH, Vaezi MMM, Mohammad V, et al. EPA and DHA have selective toxicity for PBMCs from multiple myeloma patients in a partly caspase-dependent manner. Clin Nutr. 2019;37:2137–43. doi: 10.1016/j.clnu.2019.08.031. PubMed DOI

Ewaschuk JB, Newell M, Field CJ. Docosahexanoic Acid Improves Chemotherapy Efficacy by Inducing CD95 Translocation to Lipid Rafts in ER 2 Breast Cancer Cells. Lipids. 2012;47:1019–30. doi: 10.1007/s11745-012-3717-7. PubMed DOI

Fukui M, Kang KS, Okada K, Zhu BT. EPA, an omega-3 fatty acid, induces apoptosis in human pancreatic cancer cells: role of ROS accumulation, caspase-8 activation, and autophagy induction. J Cell Biochem. 2013;114:192–203. doi: 10.1002/jcb.24354. PubMed DOI

Arita K, Kobuchi H, Utsumi T, Takehara Y, Akiyama J, Hortone AA, et al. Mechanism of apoptosis in HL-60 cells induced by n-3 and n-6 polyunsaturated fatty acids. Biochem Pharmacol. 2001;62:821–28. doi: 10.1016/S0006-2952(01)00723-7. PubMed DOI

Tobberup R, Carus A, Rasmussen HH, Falkmer UG, Jorgensen MG, Schmidt EB, et al. Feasibility of a multimodal intervention on malnutrition in patients with lung cancer during primary anti-neoplastic treatment. Clin Nutr. 2021;40:525–33. doi: 10.1016/j.clnu.2020.05.050. PubMed DOI

Fietkau R, Lewitzki V, Kuhnt T, Hölscher T, Hess CF, Berger B, et al. A disease-specific enteral nutrition formula improves nutritional status and functional performance in patients with head and neck and esophageal cancer undergoing chemoradiotherapy: Results of a randomized, controlled, multicenter trial. Cancer. 2013;119:3343–53. doi: 10.1002/cncr.28197. PubMed DOI

McGlory C, Calder PC, Nunes EA. The Influence of Omega-3 Fatty Acids on Skeletal Muscle Protein Turnover in Health, Disuse, and Disease. Front Nutr. 2019;6:1–13. doi: 10.3389/fnut.2019.00144. PubMed DOI PMC

Sánchez-Lara K, Turcott JG, Juárez-Hernández E, Nuñez-Valencia C, Villanueva G, Guevara P, et al. Effects of an oral nutritional supplement containing eicosapentaenoic acid on nutritional and clinical outcomes in patients with advanced non-small cell lung cancer: randomized trial. Clin Nutr. 2014;33:1017–23. doi: 10.1016/j.clnu.2014.03.006. PubMed DOI

Lang E, Bissinger R, Qadri SM, Lang F. Suicidal death of erythrocytes in cancer and its chemotherapy: A potential target in the treatment of tumor-associated anemia. Int J Cancer. 2017;141:1522–28. doi: 10.1002/ijc.30800. PubMed DOI

Boulet C, Doerig CD, Carvalho TG. Manipulating Eryptosis of Human Red Blood Cells: A Novel Antimalarial Strategy? Front Cell Infect Microbiol. 2018;8:1–18. doi: 10.3389/fcimb.2018.00419. PubMed DOI PMC

Chang CF, Goods BA, Askenase MH, Hammond MD, Renfroe SC, Steinschneider AF, et al. Erythrocyte efferocytosis modulates macrophages towards recovery after intracerebral hemorrhage. J Clin Invest. 2018;128:607–24. doi: 10.1172/JCI95612. PubMed DOI PMC

Föller M, Lang F. Ion Transport in Eryptosis, the Suicidal Death of Erythrocytes. Front Cell Dev Biol. 2020;8:1–9. doi: 10.3389/fcell.2020.00597. PubMed DOI PMC

Lang E, Bissinger R, Gulbins E, Lang F. Ceramide in the regulation of eryptosis, the suicidal erythrocyte death. Apoptosis. 2015;20:758–67. doi: 10.1007/s10495-015-1094-4. PubMed DOI

Alghareeb S, Alsughayyir J, Alfhili M. Eriocitrin Disrupts Erythrocyte Membrane Asymmetry through Oxidative Stress and Calcium Signaling and the Activation of Casein Kinase 1 α and Rac1 GTPase. Pharmaceuticals (Basel) 2023;16:1–14. doi: 10.3390/ph16121681. PubMed DOI PMC

Alfhili M, Alsughayyir J. Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase. Toxicon. 2024:240. doi: 10.1016/j.toxicon.2024.107636. PubMed DOI

Alzoubi K, Alktifan B, Oswald G, Fezai M, Abed M, Lang F. Breakdown of phosphatidylserine asymmetry following treatment of erythrocytes with lumefantrine. Toxins (Basal) 2014;6:650–64. doi: 10.3390/toxins6020650. PubMed DOI PMC

Alghareeb S, Alfhili M, Alsughayyir J. Stimulation of Hemolysis and Eryptosis by β-Caryophyllene Oxide. Life (Basal) 2023;13:1–16. doi: 10.3390/life13122299. PubMed DOI PMC

Rossi AM, Taylor CW. Reliable measurement of free Ca2+ concentrations in the ER lumen using Mag-Fluo-4. Cell Calcium. 2020:87. doi: 10.1016/j.ceca.2020.102188. PubMed DOI PMC

Sicinska P, Kik K, Bukowska B. Human Erythrocytes Exposed to Phthalates and Their Metabolites Alter Antioxidant Enzyme Activity and Hemoglobin Oxidation. Int J Mol Sci. 2020;21:1–14. doi: 10.3390/ijms21124480. PubMed DOI PMC

Pohanka M, Hrabinova M, Kuca K, Simonato JP. Assessment of acetylcholinesterase activity using indoxylacetate and comparison with the standard Ellman's method. Int J Mol Sci. 2011;12:2631–40. doi: 10.3390/ijms12042631. PubMed DOI PMC

Zhbanov A, Yang S. Effects of Aggregation on Blood Sedimentation and Conductivity. PLoS One. 2015;10:1–25. doi: 10.1371/journal.pone.0129337. PubMed DOI PMC

Yang X, Sheng W, Sun GY, Lee JCM. Effects of fatty acid unsaturation numbers on membrane fluidity and α-secretase-dependent amyloid precursor protein processing. Neurochem Int. 2011;58:321–29. doi: 10.1016/j.neuint.2010.12.004. PubMed DOI PMC

Garcia D, Shaw RJ. AMPK: mechanisms of cellular energy sensing and restoration of Metabolic Balance. Mol Cell. 2018;66:789–800. doi: 10.1016/j.molcel.2017.05.032. PubMed DOI PMC

Wu Y, Zhang C, Dong Y, Wang S, Song P, Viollet B, et al. Activation of the AMP-activated protein kinase by eicosapentaenoic acid (EPA, 20:5 n-3) improves endothelial function in vivo. PLoS One. 2012;7:1–9. doi: 10.1371/journal.pone.0035508. PubMed DOI PMC

Lee MS, Kim IH, Kim Y. Effects of eicosapentaenoic acid and docosahexaenoic acid on uncoupling protein 3 gene expression in C2C12 muscle cells. Nutrients. 2013;5:1660–71. doi: 10.3390/nu5051660. PubMed DOI PMC

Kim N, Kang MS, Nam M, Kim SA, Hwang GS, Kim HS. Eicosapentaenoic Acid (EPA) modulates glucose metabolism by targeting AMP-Activated Protein Kinase (AMPK) pathway. Int J Mol Sci. 2019;20:1–16. doi: 10.3390/ijms20194751. PubMed DOI PMC

Dreischer P, Duszenko M, Stein J, Wieder T. Eryptosis: Programmed Death of Nucleus-Free, Iron-Filled Blood Cells. Cells. 2022;11:1–13. doi: 10.3390/cells11030503. PubMed DOI PMC

Saldanha C. Human erythrocyte acetylcholinesterase in health and disease. Molecules. 2017:22. doi: 10.3390/molecules22091499. PubMed DOI PMC

Pretorius E, Du Plooy JN, Bester J. A Comprehensive Review on Eryptosis. Cell Physiol Biochem. 2016;39:1977–2000. doi: 10.1159/000447895. PubMed DOI

Karl SL, Philipp AL, Bauer C, Duranton C, Wieder T, Huber SM, et al. Mechanisms of Suicidal Erythrocyte Death. Cell Physiol Biochem. 2005:195–202. doi: 10.1159/000086406. PubMed DOI

Heshmati J, Morvaridzadeh M, Maroufizadeh S. Omega-3 fatty acids supplementation and oxidative stress parameters: a systematic review and meta-analysis of clinical trials. Pharmacol Res. 2019:104462. doi: 10.1016/j.phrs.2019.104462. PubMed DOI

Barbosa S, Cecchini R, El Kadri MZ, Rodrı'guez MAM, Burini RC, Dichi I. Decreased Oxidative Stress in Patients With Ulcerative Colitis Supplemented With Fish Oil n-3 Fatty Acids. Nutrition. 2003;10:838–42. doi: 10.1016/S0899-9007(03)00162-X. PubMed DOI

Zylinska L, Lisek M, Guo F, Boczek T. Vitamin C Modes of Action in Calcium-Involved Signaling in the Brain. Antioxidants (Basal) 2023;12:1–14. doi: 10.3390/antiox12020231. PubMed DOI PMC