2-Acetyl-5-tetrahydroxybutyl imidazole (THI) protects 661W cells against oxidative stress

  • Carlotta Fabiani
  • Aida Zulueta
  • Fabiola Bonezzi
  • Josefina Casas
  • Riccardo Ghidoni
  • Paola Signorelli
  • Anna Caretti
Original Article


Retinal degeneration and in particular retinitis pigmentosa (RP) is associated to ceramide (Cer) accumulation and cell death induction. Cer and sphingosine-1-phosphate (S1P) belong to the sphingolipids class and exert a pro-apoptotic and pro-survival activity, respectively. Our aim is to target sphingolipid metabolism by inhibiting S1P lyase that regulates one of the S1P degradation pathways, to reduce retinal photoreceptor damage. The murine 661W cone-like cell line was pretreated with THI, an inhibitor of S1P lyase and exposed to H2O2-induced oxidative stress. 661W cell viability and apoptosis were evaluated by Trypan Blue and TUNEL assay, respectively. Protein expression of mediators of the survival/death pathway (ERK1/2, Akt, Bcl-2, Bax) was analyzed by Western blotting. RT-PCR was performed to establish HO-1 transcript changes and LC-MS analysis to measure Cer intracellular content. THI rescues inhibitory H2O2-effect on 661W cell viability and impairs H2O2-induced apoptosis by increasing Bcl-2/Bax ratio. THI administration counteracts the oxidative stress effects of H2O2 on 661W cells by activating the Nrf2/HO-1 pathway, regulating ERK and Akt phosphorylation levels, and decreasing Cer intracellular content. We conclude that sphingolipid metabolism manipulation can be considered a therapeutic target to promote photoreceptor survival.


Sphingolipids Oxidative stress Photoreceptor Apoptosis 

Supplementary material

210_2017_1374_MOESM1_ESM.pdf (379 kb)
Supplemental file 1(PDF 378 kb)


  1. Acharya U, Patel S, Koundakjian E, Nagashima K, Han X, Acharya JK (2003) Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration. Science 299:1740–1743. doi:10.1126/science.1080549 CrossRefPubMedGoogle Scholar
  2. Arocho A, Chen B, Ladanyi M, Pan Q (2006) Validation of the 2-DeltaDeltaCt calculation as an alternate method of data analysis for quantitative PCR of BCR-ABL P210 transcripts. Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B 15:56–61CrossRefGoogle Scholar
  3. Benzie IF, Strain JJ (1999) Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 299:15–27CrossRefPubMedGoogle Scholar
  4. Berson EL et al (2010) Clinical trial of lutein in patients with retinitis pigmentosa receiving vitamin A. Arch Ophthalmol 128:403–411. doi:10.1001/archophthalmol.2010.32 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Betito S, Cuvillier O (2006) Regulation by sphingosine 1-phosphate of Bax and Bad activities during apoptosis in a MEK-dependent manner. Biochem Biophys Res Commun 340:1273–1277. doi:10.1016/j.bbrc.2005.12.138 CrossRefPubMedGoogle Scholar
  6. Bigaud M, Guerini D, Billich A, Bassilana F, Brinkmann V (2014) Second generation S1P pathway modulators: research strategies and clinical developments. Biochim Biophys Acta 1841:745–758. doi:10.1016/j.bbalip.2013.11.001 CrossRefPubMedGoogle Scholar
  7. Caretti A, Bianciardi P, Sala G, Terruzzi C, Lucchina F, Samaja M (2010) Supplementation of creatine and ribose prevents apoptosis in ischemic cardiomyocytes. Cellular Physiology and Biochemistry: International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology 26:831–838. doi:10.1159/000323992 CrossRefGoogle Scholar
  8. Chen J, Flannery JG, LaVail MM, Steinberg RH, Xu J, Simon MI (1996) bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 93:7042–7047CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chen H, Tran JT, Anderson RE, Mandal MN (2012) Caffeic acid phenethyl ester protects 661W cells from H2O2-mediated cell death and enhances electroretinography response in dim-reared albino rats. Mol Vis 18:1325–1338PubMedPubMedCentralGoogle Scholar
  10. Cieslik M, Czapski GA, Strosznajder JB (2015) The molecular mechanism of amyloid beta42 peptide toxicity: the role of sphingosine kinase-1 and mitochondrial sirtuins. PLoS One 10:e0137193. doi:10.1371/journal.pone.0137193 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Crawford MJ et al (2001) Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation. Biochem Biophys Res Commun 281:1304–1312. doi:10.1006/bbrc.2001.4501 CrossRefPubMedGoogle Scholar
  12. Czubowicz K, Strosznajder R (2014) Ceramide in the molecular mechanisms of neuronal cell death. The role of sphingosine-1-phosphate. Mol Neurobiol 50:26–37. doi:10.1007/s12035-013-8606-4 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dong SQ, Xu HZ, Xia XB, Wang S, Zhang LX, Liu SZ (2012) Activation of the ERK 1/2 and STAT3 signaling pathways is required for 661W cell survival following oxidant injury. International journal of ophthalmology 5:138–142. doi:10.3980/j.issn.2222-3959.2012.02.04 PubMedPubMedCentralGoogle Scholar
  14. van Echten-Deckert G, Zschoche A, Bar T, Schmidt RR, Raths A, Heinemann T, Sandhoff K (1997) cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons. J Biol Chem 272:15825–15833CrossRefPubMedGoogle Scholar
  15. Elsinghorst PW, di Salvo ML, Parroni A, Contestabile R (2015) Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI). Journal of enzyme inhibition and medicinal chemistry 30:336–340. doi:10.3109/14756366.2014.915396 CrossRefPubMedGoogle Scholar
  16. Faioni EM et al (2014) Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency. Thromb Haemost 112:1182–1189. doi:10.1160/TH14-04-0352 CrossRefPubMedGoogle Scholar
  17. Fan J, Xu G, Jiang T, Qin Y (2012) Pharmacologic induction of heme oxygenase-1 plays a protective role in diabetic retinopathy in rats. Invest Ophthalmol Vis Sci 53:6541–6556. doi:10.1167/iovs.11-9241 CrossRefPubMedGoogle Scholar
  18. Farrell SM, Groeger G, Bhatt L, Finnegan S, O’Brien CJ, Cotter TG (2011) bFGF-mediated redox activation of the PI3K/Akt pathway in retinal photoreceptor cells. Eur J Neurosci 33:632–641. doi:10.1111/j.1460-9568.2010.07559.x CrossRefPubMedGoogle Scholar
  19. Froger N et al (2014) Taurine: the comeback of a neutraceutical in the prevention of retinal degenerations. Prog Retin Eye Res 41:44–63. doi:10.1016/j.preteyeres.2014.03.001 CrossRefPubMedGoogle Scholar
  20. German OL, Insua MF, Gentili C, Rotstein NP, Politi LE (2006) Docosahexaenoic acid prevents apoptosis of retina photoreceptors by activating the ERK/MAPK pathway. J Neurochem 98:1507–1520. doi:10.1111/j.1471-4159.2006.04061.x CrossRefPubMedGoogle Scholar
  21. German OL, Agnolazza DL, Politi LE, Rotstein NP (2015) Light, lipids and photoreceptor survival: live or let die? Photochemical & photobiological sciences: Official journal of the European Photochemistry Association and the European Society for Photobiology 14:1737–1753. doi:10.1039/c5pp00194c CrossRefGoogle Scholar
  22. Giussani P, Brioschi L, Bassi R, Riboni L, Viani P (2009) Phosphatidylinositol 3-kinase/AKT pathway regulates the endoplasmic reticulum to golgi traffic of ceramide in glioma cells: a link between lipid signaling pathways involved in the control of cell survival. J Biol Chem 284:5088–5096. doi:10.1074/jbc.M808934200 CrossRefPubMedGoogle Scholar
  23. Gorshkova IA et al (2013) Inhibition of sphingosine-1-phosphate lyase rescues sphingosine kinase-1-knockout phenotype following murine cardiac arrest. Life Sci 93:359–366. doi:10.1016/j.lfs.2013.07.017 CrossRefPubMedGoogle Scholar
  24. Hannun YA, Obeid LM (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nature reviews. Molecular cell biology 9:139–150. doi:10.1038/nrm2329 PubMedGoogle Scholar
  25. Hartong DT, Berson EL, Dryja TP (2006) Retinitis pigmentosa. Lancet 368:1795–1809. doi:10.1016/S0140-6736(06)69740-7 CrossRefPubMedGoogle Scholar
  26. Hoffman DR et al (2014) Four-year placebo-controlled trial of docosahexaenoic acid in X-linked retinitis pigmentosa (DHAX trial): a randomized clinical trial. JAMA ophthalmology 132:866–873. doi:10.1001/jamaophthalmol.2014.1634 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kanan Y, Moiseyev G, Agarwal N, Ma JX, Al-Ubaidi MR (2007) Light induces programmed cell death by activating multiple independent proteases in a cone photoreceptor cell line. Invest Ophthalmol Vis Sci 48:40–51. doi:10.1167/iovs.06-0592 CrossRefPubMedGoogle Scholar
  28. Klyachkin YM et al (2015) Pharmacological elevation of circulating bioactive phosphosphingolipids enhances myocardial recovery after acute infarction. Stem Cells Transl Med 4:1333–1343. doi:10.5966/sctm.2014-0273 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kolomeyer AM, Zarbin MA (2014) Trophic factors in the pathogenesis and therapy for retinal degenerative diseases. Surv Ophthalmol 59:134–165. doi:10.1016/j.survophthal.2013.09.004 CrossRefPubMedGoogle Scholar
  30. Li Z et al (2013) Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phase II enzymes through activation of PI3K/Akt. Mol Vis 19:1656–1666PubMedPubMedCentralGoogle Scholar
  31. Liu C, Peng M, Laties AM, Wen R (1998) Preconditioning with bright light evokes a protective response against light damage in the rat retina. The Journal of neuroscience : the official journal of the Society for Neuroscience 18:1337–1344Google Scholar
  32. Maceyka M et al (2005) SphK1 and SphK2, sphingosine kinase isoenzymes with opposing functions in sphingolipid metabolism. J Biol Chem 280:37118–37129. doi:10.1074/jbc.M502207200 CrossRefPubMedGoogle Scholar
  33. Maceyka M, Harikumar KB, Milstien S, Spiegel S (2012) Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol 22:50–60. doi:10.1016/j.tcb.2011.09.003 CrossRefPubMedGoogle Scholar
  34. Mackey AM, Sanvicens N, Groeger G, Doonan F, Wallace D, Cotter TG (2008) Redox survival signalling in retina-derived 661W cells. Cell Death Differ 15:1291–1303. doi:10.1038/cdd.2008.43 CrossRefPubMedGoogle Scholar
  35. Merrill AH Jr, Sullards MC, Allegood JC, Kelly S, Wang E (2005) Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods 36:207–224. doi:10.1016/j.ymeth.2005.01.009 CrossRefPubMedGoogle Scholar
  36. Miranda GE, Abrahan CE, Politi LE, Rotstein NP (2009) Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors. Invest Ophthalmol Vis Sci 50:4416–4428. doi:10.1167/iovs.09-3388 CrossRefPubMedGoogle Scholar
  37. Mo MS, Li HB, Wang BY, Wang SL, Zhu ZL, Yu XR (2013) PI3K/Akt and NF-kappaB activation following intravitreal administration of 17beta-estradiol: neuroprotection of the rat retina from light-induced apoptosis. Neuroscience 228:1–12. doi:10.1016/j.neuroscience.2012.10.002 CrossRefPubMedGoogle Scholar
  38. Mustafi D, Engel AH, Palczewski K (2009) Structure of cone photoreceptors. Prog Retin Eye Res 28:289–302. doi:10.1016/j.preteyeres.2009.05.003 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Nguyen-Tran DH et al (2014) Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy. Dis Model Mech 7:41–54. doi:10.1242/dmm.013631 CrossRefPubMedGoogle Scholar
  40. Ohtoyo M, Tamura M, Machinaga N, Muro F, Hashimoto R (2015) Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency. Mol Cell Biochem 400:125–133. doi:10.1007/s11010-014-2268-z CrossRefPubMedGoogle Scholar
  41. Perusek L, Maeda T (2013) Vitamin A derivatives as treatment options for retinal degenerative diseases. Nutrients 5:2646–2666. doi:10.3390/nu5072646 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Piano I, Novelli E, Gasco P, Ghidoni R, Strettoi E, Gargini C (2013) Cone survival and preservation of visual acuity in an animal model of retinal degeneration. Eur J Neurosci 37:1853–1862. doi:10.1111/ejn.12196 CrossRefPubMedGoogle Scholar
  43. Portera-Cailliau C, Sung CH, Nathans J, Adler R (1994) Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa. Proc Natl Acad Sci U S A 91:974–978CrossRefPubMedPubMedCentralGoogle Scholar
  44. Prestera T, Talalay P, Alam J, Ahn YI, Lee PJ, Choi AM (1995) Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE). Mol Med 1:827–837PubMedPubMedCentralGoogle Scholar
  45. Rotstein NP, Miranda GE, Abrahan CE, German OL (2010) Regulating survival and development in the retina: key roles for simple sphingolipids. J Lipid Res 51:1247–1262. doi:10.1194/jlr.R003442 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Schwab SR, Pereira JP, Matloubian M, Xu Y, Huang Y, Cyster JG (2005) Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309:1735–1739. doi:10.1126/science.1113640 CrossRefPubMedGoogle Scholar
  47. Shelley EJ, Madigan MC, Natoli R, Penfold PL, Provis JM (2009) Cone degeneration in aging and age-related macular degeneration. Arch Ophthalmol 127:483–492. doi:10.1001/archophthalmol.2008.622 CrossRefPubMedGoogle Scholar
  48. Signorelli P et al (2015) Natural grape extracts regulate colon cancer cells malignancy. Nutr Cancer 67:494–503. doi:10.1080/01635581.2015.1004591 CrossRefPubMedGoogle Scholar
  49. Stone J et al (1999) Mechanisms of photoreceptor death and survival in mammalian retina. Prog Retin Eye Res 18:689–735CrossRefPubMedGoogle Scholar
  50. Strettoi E, Gargini C, Novelli E, Sala G, Piano I, Gasco P, Ghidoni R (2010) Inhibition of ceramide biosynthesis preserves photoreceptor structure and function in a mouse model of retinitis pigmentosa. Proc Natl Acad Sci U S A 107:18706–18711. doi:10.1073/pnas.1007644107 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Tanaka J, Nakanishi T, Ogawa K, Tsuruma K, Shimazawa M, Shimoda H, Hara H (2011) Purple rice extract and anthocyanidins of the constituents protect against light-induced retinal damage in vitro and in vivo. J Agric Food Chem 59:528–536. doi:10.1021/jf103186a CrossRefPubMedGoogle Scholar
  52. Terraneo L et al (2013) Expression of carbohydrate-antigen sialyl-Lewis a on colon cancer cells promotes xenograft growth and angiogenesis in nude mice. Int J Biochem Cell Biol 45:2796–2800. doi:10.1016/j.biocel.2013.10.003 CrossRefPubMedGoogle Scholar
  53. Toker A, Marmiroli S (2014) Signaling specificity in the Akt pathway in biology and disease. Advances in biological regulation 55:28–38. doi:10.1016/j.jbior.2014.04.001 CrossRefPubMedGoogle Scholar
  54. Trifunovic D et al (2012) Neuroprotective strategies for the treatment of inherited photoreceptor degeneration. Curr Mol Med 12:598–612CrossRefPubMedGoogle Scholar
  55. Tuson M, Marfany G, Gonzalez-Duarte R (2004) Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet 74:128–138. doi:10.1086/381055 CrossRefPubMedGoogle Scholar
  56. al-Ubaidi MR, Font RL, Quiambao AB, Keener MJ, Liou GI, Overbeek PA, Baehr W (1992) Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. J Cell Biol 119:1681–1687CrossRefPubMedGoogle Scholar
  57. Wright AF, Chakarova CF, Abd El-Aziz MM, Bhattacharya SS (2010) Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. Nat Rev Genet 11:273–284. doi:10.1038/nrg2717 CrossRefPubMedGoogle Scholar
  58. Yang P, Peairs JJ, Tano R, Jaffe GJ (2006) Oxidant-mediated Akt activation in human RPE cells. Invest Ophthalmol Vis Sci 47:4598–4606. doi:10.1167/iovs.06-0140 CrossRefPubMedGoogle Scholar
  59. Yasuo M, Mizuno S, Allegood J, Kraskauskas D, Bogaard HJ, Spiegel S, Voelkel NF (2013) Fenretinide causes emphysema, which is prevented by sphingosine 1-phoshate. PLoS One 8:e53927. doi:10.1371/journal.pone.0053927 CrossRefPubMedPubMedCentralGoogle Scholar
  60. Yung HW, Charnock-Jones DS, Burton GJ (2011) Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner. PLoS One 6:e17894. doi:10.1371/journal.pone.0017894 CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zarban A, Taheri F, Chahkandi T, Sharifzadeh G, Khorashadizadeh M (2009) Antioxidant and radical scavenging activity of human colostrum, transitional and mature milk. J Clin Biochem Nutr 45:150–154. doi:10.3164/jcbn.08-233 CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Health Sciences, University of MilanSan Paolo HospitalMilanItaly
  2. 2.Research Unit on Bioactive Molecules, Department of Biomedicinal ChemistryCatalan Institute of Advanced Chemistry (IQAC/CSIC)BarcelonaSpain

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