Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares Madrid Spain

Department of Biology Faculty of Medicine and Dentistry Palacky University Olomouc Hnevotinska 3 77515 Olomouc Czech Republic

Department of Biotechnology Chemistry and Pharmacy Laboratory of Pharmacology and Toxicology University of Siena Via A Moro 2 53100 Siena Italy

Department of Dermatology and Allergology School of Medicine Technical University of Munich Biedersteinerstr 29 80802 München Germany

Department of Dermatology Eberhard Karls University of Tübingen 72074 Tübingen Germany

Physiologisches Institut Abteilung für Vegetative Und Klinische Physiologie Eberhard Karls University of Tübingen 72074 Tübingen Germany

Psoriasis Center Department of Dermatology University Medical Center Schleswig Holstein Campus Kiel Rosalind Franklin Str 7 24105 Kiel Germany

Unidad de Investigación Hospital Santa Cristina Instituto de Investigación Sanitaria Princesa Madrid Spain

Zobrazit více v PubMed

Jin X, Wang C, Wang L. Costunolide inhibits osteosarcoma growth and metastasis via suppressing STAT3 signal pathway. Biomed Pharmacother. 2020;121:109659. PubMed

Butturini E, Cavalieri E, de Prati AC, et al. Two naturally occurring terpenes, dehydrocostuslactone and costunolide, decrease intracellular GSH content and inhibit STAT3 activation. PLoS ONE. 2011;6:e20174. PubMed PMC

Choi YK, Cho SG, Woo SM, et al. Saussurea lappa clarke-derived costunolide prevents TNF alpha -induced breast cancer cell migration and invasion by inhibiting NF- kappa B activity. Evid Based Complement Alternat Med. 2013;2013:936257. PubMed PMC

Li Q, Wang Z, Xie Y, Hu H. Antitumor activity and mechanism of costunolide and dehydrocostus lactone: two natural sesquiterpene lactones from the Asteraceae family. Biomed Pharmacother. 2020;125:109955. PubMed

Park E, Song JH, Kim MS, Park SH, Kim TS. Costunolide, a sesquiterpene lactone, inhibits the differentiation of pro-inflammatory CD4(+) T cells through the modulation of mitogen-activated protein kinases. Int Immunopharmacol. 2016;40:508–516. PubMed

Kim JH, Yang YI, Lee KT, Park HJ, Choi JH. Costunolide induces apoptosis in human endometriotic cells through inhibition of the prosurvival Akt and nuclear factor kappa B signaling pathway. Biol Pharm Bull. 2011;34:580–585. PubMed

Park JB, Lee CK, Park HJ. Anti-Helicobacter pylori effect of costunolide isolated from the stem bark of Magnolia sieboldii. Arch Pharm Res. 1997;20:275–279. PubMed

Peng Z, Wang Y, Gu X, Guo X, Yan C. Study on the pharmacokinetics and metabolism of costunolide and dehydrocostus lactone in rats by HPLC-UV and UPLC-Q-TOF/MS. Biomed Chromatogr. 2014;28:1325–1334. PubMed

Jeong SJ, Itokawa T, Shibuya M, et al. Costunolide, a sesquiterpene lactone from Saussurea lappa, inhibits the VEGFR KDR/Flk-1 signaling pathway. Cancer Lett. 2002;187:129–133. PubMed

Saraswati S, Alhaider AA, Abdelgadir AM. Costunolide suppresses an inflammatory angiogenic response in a subcutaneous murine sponge model. APMIS. 2018;126:257–266. PubMed

He Y, Moqbel S, Xu L, et al. Costunolide inhibits matrix metalloproteinases expression and osteoarthritis via the NF-κB and Wnt/β-catenin signaling pathways. Mol Med Rep. 2019;20:312–322. PubMed PMC

Liu B, Rong Y, Sun D, et al. Costunolide inhibits pulmonary fibrosis via regulating NF-kB and TGF-beta1/Smad2/Nrf2-NOX4 signaling pathways. Biochem Biophys Res Commun. 2019;510:329–333. PubMed

Ge MX, Liu HT, Zhang N, et al. Costunolide represses hepatic fibrosis through WW domain-containing protein 2-mediated Notch3 degradation. Br J Pharmacol. 2020;177:372–387. PubMed PMC

Kim YE, Choi HC, Nam G, Choi BY. Costunolide promotes the proliferation of human hair follicle dermal papilla cells and induces hair growth in C57BL/6 mice. J Cosmet Dermatol. 2019;18:414–421. PubMed PMC

Liu ZL, He Q, Chu SS, Wang CF, Du SS, Deng ZW. Essential oil composition and larvicidal activity of Saussurea lappa roots against the mosquito Aedes albopictus (Diptera: Culicidae) Parasitol Res. 2012;110:2125–2130. PubMed

Choi JH, Ha J, Park JH, et al. Costunolide triggers apoptosis in human leukemia U937 cells by depleting intracellular thiols. Jpn J Cancer Res. 2002;93:1327–1333. PubMed PMC

Ghashghaeinia M, Giustarini D, Koralkova P, et al. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11–7082, parthenolide and dimethyl fumarate. Sci Rep. 2016;6:28754. PubMed PMC

Rostami-Yazdi M, Clement B, Schmidt TJ, Schinor D, Mrowietz U. Detection of metabolites of fumaric acid esters in human urine: implications for their mode of action. J Invest Dermatol. 2009;129:231–234. PubMed

Orr JW, Keranen LM, Newton AC. Reversible exposure of the pseudosubstrate domain of protein kinase C by phosphatidylserine and diacylglycerol. J Biol Chem. 1992;267:15263–15266. PubMed

Flint AJ, Paladini RD, Koshland DE., Jr Autophosphorylation of protein kinase C at three separated regions of its primary sequence. Science. 1990;249:408–411. PubMed

Trubiani O, Guarnieri S, Diomede F, et al. Nuclear translocation of PKCalpha isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells. Cell Sign. 2016;28:1631–1641. PubMed

Wang Y, Zhu L, Kuokkanen S, Pollard JW. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17beta is mediated by a PKC-ERK1/2-mTOR signaling pathway. Proc Natl Acad Sci USA. 2015;112:E1382–1391. PubMed PMC

Alisi A, Spagnuolo S, Napoletano S, Spaziani A, Leoni S. Thyroid hormones regulate DNA-synthesis and cell-cycle proteins by activation of PKCalpha and p42/44 MAPK in chick embryo hepatocytes. J Cell Physiol. 2004;201:259–265. PubMed

Valdes-Rives SA, de la Fuente-Granada M, Velasco-Velazquez MA, Gonzalez-Flores O, Gonzalez-Arenas A. LPA1 receptor activation induces PKCalpha nuclear translocation in glioblastoma cells. Int J Biochem Cell Biol. 2019;110:91–102. PubMed

Takami M, Katayama K, Noguchi K, Sugimoto Y. Protein kinase C alpha-mediated phosphorylation of PIM-1L promotes the survival and proliferation of acute myeloid leukemia cells. Biochem Biophys Res Commun. 2018;503:1364–1371. PubMed

Kim CW, Asai D, Kang JH, Kishimura A, Mori T, Katayama Y. Reversal of efflux of an anticancer drug in human drug-resistant breast cancer cells by inhibition of protein kinase Calpha (PKCalpha) activity. Tumour Biol. 2016;37:1901–1908. PubMed

Fine RL, Chambers TC, Sachs CW. P-glycoprotein, multidrug resistance and protein kinase C. Stem Cells. 1996;14:47–55. PubMed

Morrison MM, Young CD, Wang S, et al. mTOR Directs breast morphogenesis through the PKC-alpha-Rac1 signaling axis. PLoS Genet. 2015;11:e1005291. PubMed PMC

Li W, Zhang J, Flechner L, et al. Protein kinase C-alpha overexpression stimulates Akt activity and suppresses apoptosis induced by interleukin 3 withdrawal. Oncogene. 1999;18:6564–6572. PubMed

Yun BR, Lee MJ, Kim JH, Kim IH, Yu GR, Kim DG. Enhancement of parthenolide-induced apoptosis by a PKC-alpha inhibition through heme oxygenase-1 blockage in cholangiocarcinoma cells. Exp Mol Med. 2010;42:787–797. PubMed PMC

Jasinski P, Zwolak P, Terai K, Borja-Cacho D, Dudek AZ. PKC-alpha inhibitor MT477 slows tumor growth with minimal toxicity in in vivo model of non-Ras-mutated cancer via induction of apoptosis. Invest New Drugs. 2011;29:33–40. PubMed

de Jong K, Rettig MP, Low PS, Kuypers FA. Protein kinase C activation induces phosphatidylserine exposure on red blood cells. Biochemistry. 2002;41:12562–12567. PubMed

Klarl BA, Lang PA, Kempe DS, et al. Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion. Am J Physiol Cell Physiol. 2006;290:C244–253. PubMed

Lang KS, Lang PA, Bauer C, et al. Mechanisms of suicidal erythrocyte death. Cell Physiol Biochem. 2005;15:195–202. PubMed

Lang E, Lang F. Triggers, inhibitors, mechanisms, and significance of eryptosis: the suicidal erythrocyte death. Biomed Res Int. 2015;2015:513518. PubMed PMC

Ghashghaeinia M, Wesseling MC, Ramos E, et al. Trifluoperazine-induced suicidal erythrocyte death and s-nitrosylation inhibition, reversed by the nitric oxide donor sodium nitroprusside. Cell Physiol Biochem. 2017;42:1985–1998. PubMed

Ghashghaeinia M, Cluitmans JC, Akel A, et al. The impact of erythrocyte age on eryptosis. Br J Haematol. 2012;157:606–614. PubMed

Myssina S, Huber SM, Birka C, et al. Inhibition of erythrocyte cation channels by erythropoietin. J Am Soc Nephrol. 2003;14:2750–2757. PubMed

Vota DM, Maltaneri RE, Wenker SD, Nesse AB, Vittori DC. Differential erythropoietin action upon cells induced to eryptosis by different agents. Cell Biochem Biophys. 2013;65:145–157. PubMed

Fink M, Al Mamun Bhuyan A, Zacharopoulou N, Lang F. Stimulation of eryptosis, the suicidal erythrocyte death, by costunolide. Cell Physiol Biochem. 2018;50:2283–2295. PubMed

Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990;172:993–999. PubMed

Andrews DA, Yang L, Low PS. Phorbol ester stimulates a protein kinase C–mediated agatoxin-TK–sensitive calcium permeability pathway in human red blood cells. Blood. 2002;100:3392–3399. PubMed

Chmura SJ, Dolan ME, Cha A, Mauceri HJ, Kufe DW, Weichselbaum RR. In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo. Clin Cancer Res. 2000;6:737–742. PubMed

Shi B, Li S, Ju H, Liu X, Li D, Li Y. Protein kinase C inhibitor chelerythrine attenuates partial unilateral ureteral obstruction induced kidney injury in neonatal rats. Life Sci. 2019;216:85–91. PubMed

Ghashghaeinia M, Bobbala D, Wieder T, et al. Targeting glutathione by dimethylfumarate protects against experimental malaria by enhancing erythrocyte cell membrane scrambling. Am J Physiol Cell Physiol. 2010;299:C791–804. PubMed

Schumacker PT. Reactive oxygen species in cancer: a dance with the devil. Cancer Cell. 2015;27:156–157. PubMed

Ghashghaeinia M, Koberle M, Mrowietz U, Bernhardt I. Proliferating tumor cells mimick glucose metabolism of mature human erythrocytes. Cell Cycle. 2019;18:1316–1334. PubMed PMC

Gardos G. The role of calcium in the potassium permeability of human erythrocytes. Acta Physiol Acad Sci Hung. 1959;15:121–125. PubMed

Grinstein S, Furuya W, Bianchini L. Protein kinases, phosphatases, and the control of cell volume. Physiology. 1992;7:232–237.

Kahle KT, Khanna AR, Alper SL, et al. K-Cl cotransporters, cell volume homeostasis, and neurological disease. Trends Mol Med. 2015;21:513–523. PubMed PMC

Smirnova GV, Oktyabrsky ON. Glutathione in bacteria. Biochemistry (Moscow) 2005;70:1199–1211. PubMed

Franco R, Schoneveld OJ, Pappa A, Panayiotidis MI. The central role of glutathione in the pathophysiology of human diseases. Arch Physiol Biochem. 2008;113:234–258. PubMed

Rana SV, Allen T, Singh R. Inevitable glutathione, then and now. Indian J Exp Biol. 2002;40:706–716. PubMed

Van’t Erve TJ, Wagner BA, Ryckman KK, Raife TJ, Buettner GR. The concentration of glutathione in human erythrocytes is a heritable trait. Free Radical Biol Med. 2013;65:742–749. PubMed PMC

Griffith OW. Glutathione turnover in human erythrocytes. Inhibition by buthionine sulfoximine and incorporation of glycine by exchange. J Biol Chem. 1981;256:4900–4904. PubMed

Jowett M, Quastel JH. The glyoxalase activity of the red blood cell: the function of glutathione. Biochem J. 1933;27:486–498. PubMed PMC

Ghashghaeinia M, Wieder T, Duszenko M, et al. Common features of oxidative stress and metabolic impairements in human erythrocytes and nucleated cells. In: Dichi I, et al., editors. Role of oxidative stress in chronic diseases. Boca Raton: CRC Press; 2014. pp. 421–478.

Cimen MY. Free radical metabolism in human erythrocytes. Clin Chim Acta. 2008;390:1–11. PubMed

Łapiński R, Siergiejuk M, Worowska A, Gacko M. Oxidants and antioxidants of erythrocytes. Prog Health Sci. 2014;4:211–219.

Hill AS, Jr, Haut A, Cartwright GE, Wintrobe MM. The role of nonhemoglobin proteins and reduced glutathione in the protection of hemoglobin from oxidation in vitro. J Clin Invest. 1964;43:17–26. PubMed PMC

Simoni J, Villanueva-Meyer J, Simoni G, Moeller JF, Wesson DE. Control of oxidative reactions of hemoglobin in the design of blood substitutes: role of the ascorbate-glutathione antioxidant system. Artif Organs. 2009;33:115–126. PubMed

Kehr S, Jortzik E, Delahunty C, Yates JR, 3rd, Rahlfs S, Becker K. Protein S-glutathionylation in malaria parasites. Antioxid Redox Signal. 2011;15:2855–2865. PubMed PMC

May JM. Ascorbate function and metabolism in the human erythrocyte. Front Biosci. 1998;3:d1–10. PubMed

May JM, Qu Z, Morrow JD. Mechanisms of ascorbic acid recycling in human erythrocytes. Biochim Biophys Acta. 2001;1528:159–166. PubMed

Raftos JE, Whillier S, Kuchel PW. Glutathione synthesis and turnover in the human erythrocyte: alignment of a model based on detailed enzyme kinetics with experimental data. J Biol Chem. 2010;285:23557–23567. PubMed PMC

Anderson ME, Meister A. Transport and direct utilization of gamma-glutamylcyst(e)ine for glutathione synthesis. Proc Natl Acad Sci USA. 1983;80:707–711. PubMed PMC

Minnich V, Smith MB, Brauner MJ, Majerus PW. Glutathione biosynthesis in human erythrocytes. I. Identification of the enzymes of glutathione synthesis in hemolysates. J Clin Invest. 1971;50:507–513. PubMed PMC

Srivastava SK, Beutler E. The transport of oxidized glutathione from human erythrocytes. J Biol Chem. 1969;244:9–16. PubMed

Staal GE, Visser J, Veeger C. Purification and properties of glutathione reductase of human erythrocytes. Biochim Biophys Acta. 1969;185:39–48. PubMed

Worthington DJ, Rosemeyer MA. Human glutathione reductase: purification of the crystalline enzyme from erythrocytes. Eur J Biochem. 1974;48:167–177. PubMed

Rall TW, Lehninger AL. Glutathione reductase of animal tissues. J Biol Chem. 1952;194:119–130. PubMed

Kuhajda FP, Jenner K, Wood FD, et al. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci USA. 1994;91:6379–6383. PubMed PMC

Flavin R, Peluso S, Nguyen PL, Loda M. Fatty acid synthase as a potential therapeutic target in cancer. Future Oncol. 2010;6:551–562. PubMed PMC

Chen L, Zhang Z, Hoshino A, et al. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat Metab. 2019;1:404–415. PubMed PMC

Lewis CA, Parker SJ, Fiske BP, et al. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell. 2014;55:253–263. PubMed PMC

Govekar R, Zingde S. Protein kinase C isoforms in human erythrocytes. Ann Hematol. 2001;80:531–534. PubMed

Lang PA, Kempe DS, Tanneur V, et al. Stimulation of erythrocyte ceramide formation by platelet-activating factor. J Cell Sci. 2005;118:1233–1243. PubMed

Giustarini D, Dalle-Donne I, Milzani A, Rossi R. Detection of glutathione in whole blood after stabilization with N-ethylmaleimide. Anal Biochem. 2011;415:81–83. PubMed

Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc. 2013;8:1660–1669. PubMed

Di Iorio EE. Preparation of derivatives of ferrous and ferric hemoglobin. Methods Enzymol. 1981;76:57–72. PubMed

Beutler E, Blume KG, Kaplan JC, Lohr GW, Ramot B, Valentine WN. International committee for standardization in haematology: recommended methods for red-cell enzyme analysis. Br J Haematol. 1977;35:331–340. PubMed

Cell death signaling in human erythron: erythrocytes lose the complexity of cell death machinery upon maturation

Apoptosis and eryptosis: similarities and differences