Pathology & Oncology Research

, Volume 25, Issue 1, pp 391–399 | Cite as

Etoposide Upregulates Survival Favoring Sphingosine-1-Phosphate in Etoposide-Resistant Retinoblastoma Cells

  • Vinodh KakkasseryEmail author
  • S. Skosyrski
  • A. Lüth
  • B. Kleuser
  • M. van der Giet
  • R. Tate
  • J. Reinhard
  • A. Faissner
  • S. C. Joachim
  • N. Kociok
Original Article


Improved knowledge of retinoblastoma chemotherapy resistance is needed to raise treatment efficiency. The objective of this study was to test whether etoposide alters glucosyl-ceramide, ceramide, sphingosine, and sphingosine-1-phosphate (sphingosine-1-P) levels in parental retinoblastoma cells (WERI Rb1) or their etoposide-resistant subclones (WERI EtoR). WERI Rb1 and WERI EtoR were incubated with 400 ng/ml etoposide for 24 h. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-P were detected by Q-TOF mass spectrometry. Statistical analysis was done by ANOVA followed by Tukey post-hoc test (p < 0.05). The mRNA expression of sphingolipid pathways enzymes in WERI Rb1, WERI EtoR and four human retinoblastoma tissue samples was analyzed by quantitative real-time PCR. Pathways enzymes mRNA expression confirmed similarities of human sphingolipid metabolism in both cell lines and tissue samples, but different relative expression. Significant up-regulation of sphingosine was seen in both cell lines (p < 0.001). Only sphingosine-1-P up-regulation was significantly increased in WERI EtoR (p < 0.01), but not in WERI Rb1 (p > 0.2). Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation, but only WERI EtoR produces additional survival favorable sphingosine-1-P. These data may suggest a role of sphingosine-1-P in retinoblastoma chemotherapy resistance, although this seems not to be the only resistance mechanism.


Retinoblastoma Sphingosine-1-phosphate Chemotherapy resistance 



We thank C Gavranic, S Metzner, G Fels and N Wagner for their technical support in these experiments.


This study was supported by Jackstädt-Stiftung S 134–10.063 (V Kakkassery) and Deutsche Forschungsgemeinschaft DFG KL988/4–4 (B Kleuser).

Compliance with Ethical Standards

Conflict of Interest



  1. 1.
    Sanders BM, Draper GJ, Kingston JE (1988) Retinoblastoma in Great Britain 1969-80: incidence, treatment, and survival. Br J Ophthalmol 72(8):576–583CrossRefGoogle Scholar
  2. 2.
    Abramson DH, Schefler AC (2004) Update on retinoblastoma. Retina 24(6):828–848CrossRefGoogle Scholar
  3. 3.
    Schuler AO, Bornfeld N (2000) Current therapy aspects of intraocular tumors. Ophthalmologe 97(3):207–222CrossRefGoogle Scholar
  4. 4.
    Shields CL, De Potter P, Himelstein BP, Shields JA, Meadows AT, Maris JM (1996) Chemoreduction in the initial management of intraocular retinoblastoma. Arch Ophthalmol 114(11):1330–1338CrossRefGoogle Scholar
  5. 5.
    Friedman DL, Himelstein B, Shields CL, Shields JA, Needle M, Miller D et al (2000) Chemoreduction and local ophthalmic therapy for intraocular retinoblastoma. J Clin Oncol 18(1):12–17CrossRefGoogle Scholar
  6. 6.
    Toma NM, Hungerford JL, Plowman PN, Kingston JE, Doughty D (1995) External beam radiotherapy for retinoblastoma: II. Lens sparing technique. Br J Ophthalmol 79(2):112–117CrossRefGoogle Scholar
  7. 7.
    Hungerford JL, Toma NM, Plowman PN, Doughty D, Kingston JE (1997) Whole-eye versus lens-sparing megavoltage therapy for retinoblastoma. Front Radiat Ther Oncol 30:81–87CrossRefGoogle Scholar
  8. 8.
    Abramson DH, Marr BP, Dunkel IJ, Brodie S, Zabor EC, Driscoll SJ et al (2012) Intra-arterial chemotherapy for retinoblastoma in eyes with vitreous and/or subretinal seeding: 2-year results. Br J Ophthalmol 96(4):499–502CrossRefGoogle Scholar
  9. 9.
    Shields CL, Manjandavida FP, Lally SE, Pieretti G, Arepalli SA, Caywood EH et al (2014) Intra-arterial chemotherapy for retinoblastoma in 70 eyes: outcomes based on the international classification of retinoblastoma. Ophthalmology 121(7):1453–1460CrossRefGoogle Scholar
  10. 10.
    Munier FL, Beck-Popovic M, Balmer A, Gaillard MC, Bovey E, Binaghi S (2011) Occurrence of sectoral choroidal occlusive vasculopathy and retinal arteriolar embolization after superselective ophthalmic artery chemotherapy for advanced intraocular retinoblastoma. Retina 31(3):566–573CrossRefGoogle Scholar
  11. 11.
    Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Biol 9(2):139–150CrossRefGoogle Scholar
  12. 12.
    Li W, Yu CP, Xia JT, Zhang L, Weng GX, Zheng HQ et al (2009) Sphingosine kinase 1 is associated with gastric cancer progression and poor survival of patients. Clin Cancer Res 15(4):1393–1399CrossRefGoogle Scholar
  13. 13.
    French KJ, Schrecengost RS, Lee BD, Zhuang Y, Smith SN, Eberly JL et al (2003) Discovery and evaluation of inhibitors of human sphingosine kinase. Cancer Res 63(18):5962–5969Google Scholar
  14. 14.
    Johnson KR, Johnson KY, Crellin HG, Ogretmen B, Boylan AM, Harley RA et al (2005) Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue. J Histochem Cytochem 53(9):1159–1166CrossRefGoogle Scholar
  15. 15.
    Li J, Guan HY, Gong LY, Song LB, Zhang N, Wu J et al (2008) Clinical significance of sphingosine kinase-1 expression in human astrocytomas progression and overall patient survival. Clin Cancer Res 14(21):6996–7003CrossRefGoogle Scholar
  16. 16.
    Van Brocklyn JR, Jackson CA, Pearl DK, Kotur MS, Snyder PJ, Prior TW (2005) Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines. J Neuropathol Exp Neurol 64(8):695–705CrossRefGoogle Scholar
  17. 17.
    Kohno M, Momoi M, Oo ML, Paik JH, Lee YM, Venkataraman K et al (2006) Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation. Mol Cell Biol 26(19):7211–7223CrossRefGoogle Scholar
  18. 18.
    Kawamori T, Kaneshiro T, Okumura M, Maalouf S, Uflacker A, Bielawski J et al (2009) Role for sphingosine kinase 1 in colon carcinogenesis. FASEB J 23(2):405–414CrossRefGoogle Scholar
  19. 19.
    Bayerl MG, Bruggeman RD, Conroy EJ, Hengst JA, King TS, Jimenez M et al (2008) Sphingosine kinase 1 protein and mRNA are overexpressed in non-Hodgkin lymphomas and are attractive targets for novel pharmacological interventions. Leuk Lymphoma 49(5):948–954CrossRefGoogle Scholar
  20. 20.
    Ruckhaberle E, Rody A, Engels K, Gaetje R, von Minckwitz G, Schiffmann S et al (2008) Microarray analysis of altered sphingolipid metabolism reveals prognostic significance of sphingosine kinase 1 in breast cancer. Breast Cancer Res Treat 112(1):41–52CrossRefGoogle Scholar
  21. 21.
    Erez-Roman R, Pienik R, Futerman AH (2010) Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression. Biochem Biophys Res Commun 391(1):219–223CrossRefGoogle Scholar
  22. 22.
    Grammatikos G, Teichgraber V, Carpinteiro A, Trarbach T, Weller M, Hengge UR et al (2007) Overexpression of acid sphingomyelinase sensitizes glioma cells to chemotherapy. Antioxid Redox Signal 9(9):1449–1456CrossRefGoogle Scholar
  23. 23.
    Morita Y, Perez GI, Paris F, Miranda SR, Ehleiter D, Haimovitz-Friedman A et al (2000) Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat Med 6(10):1109–1114CrossRefGoogle Scholar
  24. 24.
    Lacour S, Hammann A, Grazide S, Lagadic-Gossmann D, Athias A, Sergent O et al (2004) Cisplatin-induced CD95 redistribution into membrane lipid rafts of HT29 human colon cancer cells. Cancer Res 64(10):3593–3598CrossRefGoogle Scholar
  25. 25.
    Akao Y, Banno Y, Nakagawa Y, Hasegawa N, Kim TJ, Murate T et al (2006) High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation. Biochem Biophys Res Commun 342(4):1284–1290CrossRefGoogle Scholar
  26. 26.
    Pchejetski D, Golzio M, Bonhoure E, Calvet C, Doumerc N, Garcia V et al (2005) Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models. Cancer Res 65(24):11667–11675CrossRefGoogle Scholar
  27. 27.
    Guillermet-Guibert J, Davenne L, Pchejetski D, Saint-Laurent N, Brizuela L, Guilbeau-Frugier C et al (2009) Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug. Mol Cancer Ther 8(4):809–820CrossRefGoogle Scholar
  28. 28.
    Baran Y, Salas A, Senkal CE, Gunduz U, Bielawski J, Obeid LM et al (2007) Alterations of ceramide/sphingosine 1-phosphate rheostat involved in the regulation of resistance to imatinib-induced apoptosis in K562 human chronic myeloid leukemia cells. J Biol Chem 282(15):10922–10934CrossRefGoogle Scholar
  29. 29.
    Stephan H, Boeloeni R, Eggert A, Bornfeld N, Schueler A (2008) Photodynamic therapy in retinoblastoma: effects of verteporfin on retinoblastoma cell lines. Invest Ophthalmol Vis Sci 49(7):3158–3163CrossRefGoogle Scholar
  30. 30.
    Mergler S, Cheng Y, Skosyrski S, Garreis F, Pietrzak P, Kociok N et al (2012) Altered calcium regulation by thermosensitive transient receptor potential channels in etoposide-resistant WERI-Rb1 retinoblastoma cells. Exp Eye Res 94(1):157–173CrossRefGoogle Scholar
  31. 31.
    Pewzner-Jung Y, Tavakoli Tabazavareh S, Grassme H, Becker KA, Japtok L, Steinmann J et al (2014) Sphingoid long chain bases prevent lung infection by Pseudomonas aeruginosa. EMBO Mol Med 6(9):1205–1214CrossRefGoogle Scholar
  32. 32.
    Japtok L, Schmitz EI, Fayyaz S, Kramer S, Hsu LJ, Kleuser B (2015) Sphingosine 1-phosphate counteracts insulin signaling in pancreatic beta-cells via the sphingosine 1-phosphate receptor subtype 2. FASEB J 29(8):3357–3369Google Scholar
  33. 33.
    Gulbins E, Palmada M, Reichel M, Luth A, Bohmer C, Amato D et al (2013) Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs. Nat Med 19(7):934–938CrossRefGoogle Scholar
  34. 34.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134CrossRefGoogle Scholar
  35. 35.
    Sobue S, Nemoto S, Murakami M, Ito H, Kimura A, Gao S et al (2008) Implications of sphingosine kinase 1 expression level for the cellular sphingolipid rheostat: relevance as a marker for daunorubicin sensitivity of leukemia cells. Int J Hematol 87(3):266–275CrossRefGoogle Scholar
  36. 36.
    Xia P, Gamble JR, Wang L, Pitson SM, Moretti PA, Wattenberg BW et al (2000) An oncogenic role of sphingosine kinase. Curr Biol: CB 10(23):1527–1530CrossRefGoogle Scholar
  37. 37.
    Sankala HM, Hait NC, Paugh SW, Shida D, Lepine S, Elmore LW et al (2007) Involvement of sphingosine kinase 2 in p53-independent induction of p21 by the chemotherapeutic drug doxorubicin. Cancer Res 67(21):10466–10474CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2017

Authors and Affiliations

  • Vinodh Kakkassery
    • 1
    • 2
    • 3
    Email author
  • S. Skosyrski
    • 1
  • A. Lüth
    • 4
  • B. Kleuser
    • 4
  • M. van der Giet
    • 5
  • R. Tate
    • 6
  • J. Reinhard
    • 7
  • A. Faissner
    • 7
  • S. C. Joachim
    • 2
  • N. Kociok
    • 1
  1. 1.Department of OphthalmologyCharité UniversitätsmedizinBerlinGermany
  2. 2.Department of OphthalmologyRuhr-University BochumBochumGermany
  3. 3.Department of OphthalmologyUniversity RostockRostockGermany
  4. 4.Department of Nutrition ScienceUniversity PotsdamPotsdamGermany
  5. 5.Department of Nephrology, Campus Benjamin FranklinCharité UniversitätsmedizinBerlinGermany
  6. 6.Strathclyde Institute of Pharmacy and Biomedical SciencesUniversity of StrathclydeGlasgowUK
  7. 7.Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology and BiotechnologyRuhr-University BochumBochumGermany

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