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

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

Apoptosis and eryptosis: similarities and differences