Triggering of eryptosis, the suicidal erythrocyte death, by phenoxodiol

  • Madeline Fink
  • Abdulla Al Mamun Bhuyan
  • Bernd Nürnberg
  • Caterina Faggio
  • Florian LangEmail author
Original Article


Phenoxodiol is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. At least in theory, phenoxodiol could similarly stimulate suicidal erythrocyte death or eryptosis. Eryptosis is characterized by cell shrinkage and breakdown of cell membrane asymmetry with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), formation of reactive oxygen species (ROS), and increase of ceramide abundance at the cell surface. The present study explored whether phenoxodiol induces eryptosis and whether it modifies Ca2+ entry, ROS, and ceramide. Using flow cytometry, phosphatidylserine exposure at the cell surface was quantified from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, ROS from DCFDA-dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48-h exposure of human erythrocytes to phenoxodiol (100 μg/ml [416 μM]) significantly increased the percentage of annexin V binding cells, significantly decreased average forward scatter and Fluo3 fluorescence and significantly increased ceramide abundance, but did not significantly modify DCFDA fluorescence. The effect of phenoxodiol on annexin V binding tended to decrease following removal of extracellular Ca2+, an effect, however, not reaching statistical significance. In conclusion, phenoxodiol triggers eryptosis, an effect paralleled by increase of ceramide abundance.


Phosphatidylserine Eryptosis Staurosporine Ceramide Oxidative stress Calcium Red blood cells 



The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic.

Authors’ contribution

BN, CF, and FL conceived and designed research. MF and AMB conducted experiments and analyzed data. FL wrote the manuscript. All authors read and approved the manuscript.


The study was supported by the Deutsche Forschungsgemeinschaft, DFG grant “Gi proteins and platelets” (NU 53/13-1) and Deutscher Akademischer Austaauschdienst (DAAD).


  1. Abed M, Towhid ST, Mia S, Pakladok T, Alesutan I, Borst O, Gawaz M, Gulbins E, Lang F (2012) Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells. Am J Physiol Cell Physiol 303:C991–C999CrossRefGoogle Scholar
  2. Aguero MF, Facchinetti MM, Sheleg Z, Senderowicz AM (2005) Phenoxodiol, a novel isoflavone, induces G1 arrest by specific loss in cyclin-dependent kinase 2 activity by p53-independent induction of p21WAF1/CIP1. Cancer Res 65:3364–3373CrossRefGoogle Scholar
  3. Aguero MF, Venero M, Brown DM, Smulson ME, Espinoza LA (2010) Phenoxodiol inhibits growth of metastatic prostate cancer cells. Prostate 70:1211–1221CrossRefGoogle Scholar
  4. Alvero AB, O’Malley D, Brown D, Kelly G, Garg M, Chen W, Rutherford T, Mor G (2006) Molecular mechanism of phenoxodiol-induced apoptosis in ovarian carcinoma cells. Cancer 106:599–608CrossRefGoogle Scholar
  5. Alvero AB, Brown D, Montagna M, Matthews M, Mor G (2007) Phenoxodiol-topotecan co-administration exhibit significant anti-tumor activity without major adverse side effects. Cancer Biol Ther 6:612–617CrossRefGoogle Scholar
  6. Alvero AB, Kelly M, Rossi P, Leiser A, Brown D, Rutherford T, Mor G (2008) Anti-tumor activity of phenoxodiol: from bench to clinic. Future Oncol 4:475–482CrossRefGoogle Scholar
  7. Bissinger R, Bhuyan AAM, Qadri SM, Lang F (2019) Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases. FEBS J 286:826–854CrossRefGoogle Scholar
  8. Briglia M, Rossi MA, Faggio C (2017) Eryptosis: ally or enemy. Curr Med Chem 24:937–942CrossRefGoogle Scholar
  9. Chen Y, Cass SL, Kutty SK, Yee EM, Chan DS, Gardner CR, Vittorio O, Pasquier E, Black DS, Kumar N (2015) Synthesis, biological evaluation and structure-activity relationship studies of isoflavene based Mannich bases with potent anti-cancer activity. Bioorg Med Chem Lett 25:5377–5383CrossRefGoogle Scholar
  10. Choueiri TK, Mekhail T, Hutson TE, Ganapathi R, Kelly GE, Bukowski RM (2006a) Phase I trial of phenoxodiol delivered by continuous intravenous infusion in patients with solid cancer. Ann Oncol 17:860–865CrossRefGoogle Scholar
  11. Choueiri TK, Wesolowski R, Mekhail TM (2006b) Phenoxodiol: isoflavone analog with antineoplastic activity. Curr Oncol Rep 8:104–107CrossRefGoogle Scholar
  12. De Luca T, Bosneaga E, Morre DM, Morre DJ (2008) Downstream targets of altered sphingolipid metabolism in response to inhibition of ENOX2 by phenoxodiol. Biofactors 34:253–260CrossRefGoogle Scholar
  13. De Luca T, Morre DM, Morre DJ (2010) Reciprocal relationship between cytosolic NADH and ENOX2 inhibition triggers sphingolipid-induced apoptosis in HeLa cells. J Cell Biochem 110:1504–1511CrossRefGoogle Scholar
  14. de Souza PL, Russell PJ, Kearsley JH, Howes LG (2010) Clinical pharmacology of isoflavones and its relevance for potential prevention of prostate cancer. Nutr Rev 68:542–555CrossRefGoogle Scholar
  15. Foller M, Bobbala D, Koka S, Huber SM, Gulbins E, Lang F (2009) Suicide for survival--death of infected erythrocytes as a host mechanism to survive malaria. Cell Physiol Biochem 24:133–140CrossRefGoogle Scholar
  16. Gamble JR, Xia P, Hahn CN, Drew JJ, Drogemuller CJ, Brown D, Vadas MA (2006) Phenoxodiol, an experimental anticancer drug, shows potent antiangiogenic properties in addition to its antitumour effects. Int J Cancer 118:2412–2420CrossRefGoogle Scholar
  17. Herst PM, Petersen T, Jerram P, Baty J, Berridge MV (2007) The antiproliferative effects of phenoxodiol are associated with inhibition of plasma membrane electron transport in tumor cell lines and primary immune cells. Biochem Pharmacol 74:1587–1595CrossRefGoogle Scholar
  18. Herst PM, Davis JE, Neeson P, Berridge MV, Ritchie DS (2009) The anti-cancer drug, phenoxodiol, kills primary myeloid and lymphoid leukemic blasts and rapidly proliferating T cells. Haematologica 94:928–934CrossRefGoogle Scholar
  19. Isono M, Sato A, Asano T, Okubo K, Asano T (2018) Evaluation of therapeutic potential of phenoxodiol, a novel isoflavone analog, in renal cancer cells. Anticancer Res 38:5709–5716CrossRefGoogle Scholar
  20. Kamsteeg M, Rutherford T, Sapi E, Hanczaruk B, Shahabi S, Flick M, Brown D, Mor G (2003) Phenoxodiol--an isoflavone analog--induces apoptosis in chemoresistant ovarian cancer cells. Oncogene 22:2611–2620CrossRefGoogle Scholar
  21. Kluger HM, McCarthy MM, Alvero AB, Sznol M, Ariyan S, Camp RL, Rimm DL, Mor G (2007) The X-linked inhibitor of apoptosis protein (XIAP) is up-regulated in metastatic melanoma, and XIAP cleavage by Phenoxodiol is associated with carboplatin sensitization. J Transl Med 5:6CrossRefGoogle Scholar
  22. Lang PA, Kaiser S, Myssina S, Wieder T, Lang F, Huber SM (2003) Role of Ca2+-activated K+ channels in human erythrocyte apoptosis. Am J Physiol Cell Physiol 285:C1553–C1560CrossRefGoogle Scholar
  23. Lang PA, Kempe DS, Myssina S, Tanneur V, Birka C, Laufer S, Lang F, Wieder T, Huber SM (2005) PGE(2) in the regulation of programmed erythrocyte death. Cell Death Differ 12:415–428CrossRefGoogle Scholar
  24. Lang E, Modicano P, Arnold M, Bissinger R, Faggio C, Abed M, Lang F (2013) Effect of thioridazine on erythrocytes. Toxins (Basel) 5:1918–1931CrossRefGoogle Scholar
  25. Lang E, Bissinger R, Gulbins E, Lang F (2015) Ceramide in the regulation of eryptosis, the suicidal erythrocyte death. Apoptosis 20:758–767CrossRefGoogle Scholar
  26. Lang F, Bissinger R, Abed M, Artunc F (2017) Eryptosis - the neglected cause of anemia in end stage renal disease. Kidney Blood Press Res 42:749–760CrossRefGoogle Scholar
  27. Li Y, Huang X, Huang Z, Feng J (2014) Phenoxodiol enhances the antitumor activity of gemcitabine in gallbladder cancer through suppressing Akt/mTOR pathway. Cell Biochem Biophys 70:1337–1342CrossRefGoogle Scholar
  28. Mahoney S, Arfuso F, Rogers P, Hisheh S, Brown D, Millward M, Dharmarajan A (2012) Cytotoxic effects of the novel isoflavone, phenoxodiol, on prostate cancer cell lines. J Biosci 37:73–84CrossRefGoogle Scholar
  29. Mahoney S, Arfuso F, Millward M, Dharmarajan A (2014) The effects of phenoxodiol on the cell cycle of prostate cancer cell lines. Cancer Cell Int 14:110CrossRefGoogle Scholar
  30. McPherson RA, Galettis PT, de Souza PL (2009) Enhancement of the activity of phenoxodiol by cisplatin in prostate cancer cells. Br J Cancer 100:649–655CrossRefGoogle Scholar
  31. Mischitelli M, Jemaa M, Almasry M, Faggio C, Lang F (2016a) Ca2+ entry, oxidative stress, ceramide and suicidal erythrocyte death following diosgenin treatment. Cell Physiol Biochem 39:1626–1637CrossRefGoogle Scholar
  32. Mischitelli M, Jemaa M, Almasry M, Faggio C, Lang F (2016b) Triggering of erythrocyte cell membrane scrambling by emodin. Cell Physiol Biochem 40:91–103CrossRefGoogle Scholar
  33. Miyamoto M, Takano M, Aoyama T, Soyama H, Ishibashi H, Kato K, Iwahashi H, Takasaki K, Kuwahara M, Matuura H, Sakamoto T, Yoshikawa T, Furuya K (2018) Phenoxodiol increases cisplatin sensitivity in ovarian clear cancer cells through XIAP down-regulation and autophagy inhibition. Anticancer Res 38:301–306Google Scholar
  34. Mor G, Fu HH, Alvero AB (2006) Phenoxodiol, a novel approach for the treatment of ovarian cancer. Curr Opin Investig Drugs 7:542–548Google Scholar
  35. Mor G, Montagna MK, Alvero AB (2008) Modulation of apoptosis to reverse chemoresistance. Methods Mol Biol 414:1–12Google Scholar
  36. Morre DJ, Chueh PJ, Yagiz K, Balicki A, Kim C, Morre DM (2007) ECTO-NOX target for the anticancer isoflavene phenoxodiol. Oncol Res 16:299–312CrossRefGoogle Scholar
  37. Pretorius E, du Plooy JN, Bester J (2016) A comprehensive review on eryptosis. Cell Physiol Biochem 39:1977–2000CrossRefGoogle Scholar
  38. Saif MW, Tytler E, Lansigan F, Brown DM, Husband AJ (2009) Flavonoids, phenoxodiol, and a novel agent, triphendiol, for the treatment of pancreaticobiliary cancers. Expert Opin Investig Drugs 18:469–479CrossRefGoogle Scholar
  39. Sapi E, Alvero AB, Chen W, O’Malley D, Hao XY, Dwipoyono B, Garg M, Kamsteeg M, Rutherford T, Mor G (2004) Resistance of ovarian carcinoma cells to docetaxel is XIAP dependent and reversible by phenoxodiol. Oncol Res 14:567–578CrossRefGoogle Scholar
  40. Silasi DA, Alvero AB, Rutherford TJ, Brown D, Mor G (2009) Phenoxodiol: pharmacology and clinical experience in cancer monotherapy and in combination with chemotherapeutic drugs. Expert Opin Pharmacother 10:1059–1067CrossRefGoogle Scholar
  41. Straszewski-Chavez SL, Abrahams VM, Funai EF, Mor G (2004) X-Linked inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-mediated apoptosis. Mol Hum Reprod 10:33–41CrossRefGoogle Scholar
  42. Wu LY, De Luca T, Watanabe T, Morre DM, Morre DJ (2011) Metabolite modulation of HeLa cell response to ENOX2 inhibitors EGCG and phenoxodiol. Biochim Biophys Acta 1810:784–789CrossRefGoogle Scholar
  43. Yagiz K, Wu LY, Kuntz CP, James Morre D, Morre DM (2007) Mouse embryonic fibroblast cells from transgenic mice overexpressing tNOX exhibit an altered growth and drug response phenotype. J Cell Biochem 101:295–306CrossRefGoogle Scholar
  44. Yao C, Wu S, Li D, Ding H, Wang Z, Yang Y, Yan S, Gu Z (2012) Co-administration phenoxodiol with doxorubicin synergistically inhibit the activity of sphingosine kinase-1 (SphK1), a potential oncogene of osteosarcoma, to suppress osteosarcoma cell growth both in vivo and in vitro. Mol Oncol 6:392–404CrossRefGoogle Scholar
  45. Yu F, Watts RN, Zhang XD, Borrow JM, Hersey P (2006) Involvement of BH3-only proapoptotic proteins in mitochondrial-dependent phenoxodiol-induced apoptosis of human melanoma cells. Anti-Cancer Drugs 17:1151–1161CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Madeline Fink
    • 1
  • Abdulla Al Mamun Bhuyan
    • 1
  • Bernd Nürnberg
    • 1
  • Caterina Faggio
    • 2
  • Florian Lang
    • 3
    • 4
    Email author
  1. 1.Department of Phamacology and Experimental TherapyEberhard-Karls-University of TuebingenTübingenGermany
  2. 2.Department of Chemical, Biological, Pharmaceutical and Environmental SciencesUniversity of MessinaMessinaItaly
  3. 3.Department of Internal Medicine IIIEberhard-Karls-University of TuebingenTübingenGermany
  4. 4.Department of Vegetative & Clinical Physiology, University of TübingenTübingenGermany

Personalised recommendations