Neurotoxicity Research

, Volume 32, Issue 3, pp 368–380 | Cite as

Evaluation of the Protective Effects of Sarains on H2O2-Induced Mitochondrial Dysfunction and Oxidative Stress in SH-SY5Y Neuroblastoma Cells

  • Rebeca Alvariño
  • Eva Alonso
  • Marie-Aude Tribalat
  • Sandra Gegunde
  • Olivier P. Thomas
  • Luis M. Botana


Sarains are diamide alkaloids isolated from the Mediterranean sponge Haliclona (Rhizoniera) sarai that have previously shown antibacterial, insecticidal and anti-fouling activities. In this study, we examined for the first time the neuroprotective effects of sarains 1, 2 and A against oxidative stress in a human neuronal model. SH-SY5Y cells were co-incubated with sarains at concentrations ranging from 0.01 to 10 μM, and the well-known oxidant hydrogen peroxide at 150 μM for 6 h and the protective effects of the compounds were evaluated. Among the sarains tested, sarain A was the most promising compound, improving mitochondrial function and decreasing reactive oxygen species levels in human neuroblastoma cells treated with the compound at 0.01, 0.1 and 1 μM. This compound was also able to increase the activity of the antioxidant enzymes superoxide dismutases by inducing the translocation of the nuclear factor E2-related factor 2 (Nrf2) to the nucleus at the lower concentrations tested (0.01 and 0.1 μM). Moreover, sarain A at 0.1 and 1 μM blocked the mitochondrial permeability transition pore (mPTP) opening through cyclophilin D inhibition. These results suggest that the protective effects produced by the treatment with sarain A are related with its ability to block the mPTP and to enhance the Nrf2 pathway, indicating that sarain A may be a candidate compound for further studies in neurodegenerative diseases.


Sarains Oxidative stress Nrf2 mPTP Cyclophilin D Neuroprotection 



The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD and ITC-20161072. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Baines CP et al (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434:658–662. doi: 10.1038/nature03434 CrossRefPubMedGoogle Scholar
  2. Blanquer-Rossello MD, Hernandez-Lopez R, Roca P, Oliver J, Valle A (2017) Resveratrol induces mitochondrial respiration and apoptosis in SW620 colon cancer cells. Biochim Biophys Acta 1861:431–440. doi: 10.1016/j.bbagen.2016.10.009 CrossRefPubMedGoogle Scholar
  3. Blihoghe D, Manzo E, Villela A, Cutignano A, Picariello G, Faimali M, Fontana A (2011) Evaluation of the antifouling properties of 3-alyklpyridine compounds. Biofouling 27:99–109. doi: 10.1080/08927014.2010.542587 CrossRefPubMedGoogle Scholar
  4. Burchell VS, Gandhi S, Deas E, Wood NW, Abramov AY, Plun-Favreau H (2010) Targeting mitochondrial dysfunction in neurodegenerative disease: part I. Expert Opin Ther Targets 14:369–385. doi: 10.1517/14728221003652489 CrossRefPubMedGoogle Scholar
  5. Butterfield DA, Hardas SS, Lange ML (2010) Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Alzheimer’s disease: many pathways to neurodegeneration. J Alzheimers Dis 20:369–393. doi: 10.3233/jad-2010-1375 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Calkins MJ et al (2009) The Nrf2/ARE pathway as a potential therapeutic target in neurodegenerative disease. Antioxid Redox Signal 11:497–508. doi: 10.1089/ARS.2008.2242 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Caprioli V, Cimino G, De Giulio A, Madaio A, Scognamiglio G, Trivellone E (1992) Selected biological activities of saraines. Comp Biochem Physiol B 103:293–296CrossRefPubMedGoogle Scholar
  8. Cardoso S, Seica RM, Moreira PI (2017) Mitochondria as a target for neuroprotection: implications for Alzheimer’s disease. Expert Rev Neurother 17:77–91 doi: 10.1080/14737175.2016.1205488
  9. Chakraborty TR, Cohen J, Yohanan D, Alicea E, Weeks BS, Chakraborty S (2016) Estrogen is neuroprotective against hypoglycemic injury in murine N38 hypothalamic cells. Mol Med Rep 14:5677–5684. doi: 10.3892/mmr.2016.5952 CrossRefPubMedGoogle Scholar
  10. Cimino G, Stefano SD, Scognamiglio G, Sodano G, Trivellone E (1986) Sarains: a new class of alkaloids from the marine sponge Reniera Sarai. Bulletin des Sociétés Chimiques Belges 95:783–800. doi: 10.1002/bscb.19860950907 CrossRefGoogle Scholar
  11. Cimino G, Mattia CA, Mazzarella L, Putili R, Scognamiglio G, Spinella A, Trivellone E (1989) Unprecedented alkaloid skeleton from the mediterranean sponge reniera sarai: X-ray structure of an acetate derivative of sarain-a. Tetrahedron 45:3863–3872 doi: 10.1016/S0040-4020(01)89245-0
  12. Clarke SJ, McStay GP, Halestrap AP (2002) Sanglifehrin A acts as a potent inhibitor of the mitochondrial permeability transition and reperfusion injury of the heart by binding to cyclophilin-D at a different site from cyclosporin. A J Biol Chem 277:34793–34799. doi: 10.1074/jbc.M202191200 CrossRefPubMedGoogle Scholar
  13. Coccini T, Manzo L, Bellotti V, De Simone U (2014) Assessment of cellular responses after short- and long-term exposure to silver nanoparticles in human neuroblastoma (SH-SY5Y) and astrocytoma (D384) cells. ScientificWorldJournal 2014:259765. doi: 10.1155/2014/259765 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Connern CP, Halestrap AP (1994) Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel. Biochem J 302(Pt 2):321–324CrossRefPubMedPubMedCentralGoogle Scholar
  15. Corona JC, Duchen MR (2015) Impaired mitochondrial homeostasis and neurodegeneration: towards new therapeutic targets? J Bioenerg Biomembr 47(1–2):89–99. doi: 10.1007/s10863-014-9576-6 CrossRefPubMedGoogle Scholar
  16. Crompton M, Costi A (1988) Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. A potential mechanism for mitochondrial dysfunction during cellular Ca2+ overload. Eur J Biochem 178:489–501CrossRefPubMedGoogle Scholar
  17. Defant A, Mancini I, Raspor L, Guella G, Turk T, Sepčić K (2011) New structural insights into Saraines A, B, and C, macrocyclic alkaloids from the Mediterranean sponge Reniera (Haliclona) sarai. Eur J Org Chem 2011:3761–3767. doi: 10.1002/ejoc.201100434 CrossRefGoogle Scholar
  18. Di Domenico F, Barone E, Perluigi M, Butterfield DA (2015) Strategy to reduce free radical species in Alzheimer’s disease: an update of selected antioxidants. Expert Rev Neurother 15:19–40. doi: 10.1586/14737175.2015.955853 CrossRefPubMedGoogle Scholar
  19. Du H, Yan SS (2010) Mitochondrial permeability transition pore in Alzheimer’s disease: cyclophilin D and amyloid beta. Biochim Biophys Acta 1802:198–204. doi: 10.1016/j.bbadis.2009.07.005 CrossRefPubMedGoogle Scholar
  20. Du H et al (2008) Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer’s disease. Nat Med 14:1097–1105. doi: 10.1038/nm.1868 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Eliseev RA, Filippov G, Velos J, Van Winkle B, Goldman A, Rosier RN, Gunter TE (2007) Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition. Neurobiol Aging 28:1532–1542. doi: 10.1016/j.neurobiolaging.2006.06.022 CrossRefPubMedGoogle Scholar
  22. Esteras N, Dinkova-Kostova AT, Abramov AY (2016) Nrf2 activation in the treatment of neurodegenerative diseases: a focus on its role in mitochondrial bioenergetics and function. Biol Chem 397:383–400. doi: 10.1515/hsz-2015-0295 CrossRefPubMedGoogle Scholar
  23. Fischer G, Bang H, Berger E, Schellenberger A (1984) Conformational specificity of chymotrypsin toward proline-containing substrates. Biochim Biophys Acta 791:87–97CrossRefPubMedGoogle Scholar
  24. Gandhi S, Abramov AY (2012) Mechanism of oxidative stress in neurodegeneration. Oxidative Med Cell Longev 2012:428010. doi: 10.1155/2012/428010 CrossRefGoogle Scholar
  25. Gregory MA et al (2011) Preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family. Antimicrob Agents Chemother 55:1975–1981. doi: 10.1128/aac.01627-10 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Grosso C, Valentão P, Ferreres F, Andrade PB (2014) Bioactive marine drugs and marine biomaterials for brain diseases. Mar Drugs 12:2539–2589. doi: 10.3390/md12052539 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Halliwell B, Whiteman M (2004) Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol 142:231–255. doi: 10.1038/sj.bjp.0705776 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Hansson MJ, Persson T, Friberg H, Keep MF, Rees A, Wieloch T, Elmer E (2003) Powerful cyclosporin inhibition of calcium-induced permeability transition in brain mitochondria. Brain Res 960:99–111CrossRefPubMedGoogle Scholar
  29. Holmstrom KM et al (2013) Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration. Biol Open 2:761–770. doi: 10.1242/bio.20134853 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Huang SL, He HB, Zou K, Bai CH, Xue YH, Wang JZ, Chen JF (2014) Protective effect of tomatine against hydrogen peroxide-induced neurotoxicity in neuroblastoma (SH-SY5Y) cells. J Pharm Pharmacol 66:844–854. doi: 10.1111/jphp.12205 CrossRefPubMedGoogle Scholar
  31. Itoh K, Ye P, Matsumiya T, Tanji K, Ozaki T (2015) Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria. J Clin Biochem Nutr 56:91–97. doi: 10.3164/jcbn.14-134 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kharroubi W, Nury T, Ahmed SH, Andreoletti P, Sakly R, Hammami M, Lizard G (2017) Induction by arsenate of cell-type-specific cytotoxic effects in nerve and hepatoma cells. Hum Exp Toxicol. doi: 10.1177/0960327116687893
  33. Kumar A, Singh A (2015) A review on mitochondrial restorative mechanism of antioxidants in Alzheimer’s disease and other neurological conditions. Front Pharmacol 6:206. doi: 10.3389/fphar.2015.00206 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lee J (2016) Mitochondrial drug targets in neurodegenerative diseases. Bioorg Med Chem Lett 26:714–720. doi: 10.1016/j.bmcl.2015.11.032 CrossRefPubMedGoogle Scholar
  35. Leirós M et al (2015) Gracilins: spongionella-derived promising compounds for Alzheimer disease. Neuropharmacology 93:285–293. doi: 10.1016/j.neuropharm.2015.02.015 CrossRefPubMedGoogle Scholar
  36. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795. doi: 10.1038/nature05292 CrossRefPubMedGoogle Scholar
  37. Lu MC, Ji JA, Jiang ZY, You QD (2016) The Keap1-Nrf2-ARE pathway as a potential preventive and therapeutic target: an update. Med Res Rev 36:924–963. doi: 10.1002/med.21396 CrossRefPubMedGoogle Scholar
  38. Magesh S, Chen Y, Hu L (2012) Small molecule modulators of Keap1-Nrf2-ARE pathway as potential preventive and therapeutic agents. Med Res Rev 32:687–726. doi: 10.1002/med.21257 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Malhotra D et al (2010) Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis. Nucleic Acids Res 38:5718–5734. doi: 10.1093/nar/gkq212 CrossRefPubMedPubMedCentralGoogle Scholar
  40. McBean GJ, Lopez MG, Wallner FK (2016) Redox-based therapeutics in neurodegenerative disease. Br J Pharmacol. doi: 10.1111/bph.13551 Google Scholar
  41. Moreira PI et al (2010) Mitochondria: a therapeutic target in neurodegeneration. Biochim Biophys Acta 1802:212–220. doi: 10.1016/j.bbadis.2009.10.007 CrossRefPubMedGoogle Scholar
  42. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefPubMedGoogle Scholar
  43. Nguyen TT, Stevens MV, Kohr M, Steenbergen C, Sack MN, Murphy E (2011) Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore. J Biol Chem 286:40184–40192. doi: 10.1074/jbc.M111.243469 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Ouazia D, Levros LC Jr, Rassart E, Desrosiers RR (2014) Dopamine down-regulation of protein L-isoaspartyl methyltransferase is dependent on reactive oxygen species in SH-SY5Y cells. Neuroscience 267:263–276. doi: 10.1016/j.neuroscience.2014.03.001 CrossRefPubMedGoogle Scholar
  45. Park SY, Kim DY, Kang JK, Park G, Choi YW (2014) Involvement of activation of the Nrf2/ARE pathway in protection against 6-OHDA-induced SH-SY5Y cell death by alpha-iso-cubebenol. Neurotoxicology 44:160–168. doi: 10.1016/j.neuro.2014.06.011 CrossRefPubMedGoogle Scholar
  46. Pertega-Gomes N et al (2015) A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy. J Pathol 236:517–530. doi: 10.1002/path.4547 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F (1999) Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76:725–734. doi: 10.1016/s0006-3495(99)77239-5 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Ramsey CP et al (2007) Expression of Nrf2 in neurodegenerative diseases. J Neuropathol Exp Neurol 66:75–85. doi: 10.1097/nen.0b013e31802d6da9 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rao VK, Carlson EA, Yan SS (2014) Mitochondrial permeability transition pore is a potential drug target for neurodegeneration. Biochim Biophys Acta 1842:1267–1272. doi: 10.1016/j.bbadis.2013.09.003 CrossRefPubMedGoogle Scholar
  50. Sasaki S, Tozawa T, Van Wagoner RM, Ireland CM, Harper MK, Satoh T (2011) Strongylophorine-8, a pro-electrophilic compound from the marine sponge Petrosia (strongylophora) corticata, provides neuroprotection through Nrf2/ARE pathway. Biochem Biophys Res Commun 415:6–10. doi: 10.1016/j.bbrc.2011.09.114 CrossRefPubMedGoogle Scholar
  51. Singh A et al (2013) Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis. J Clin Invest 123:2921–2934. doi: 10.1172/jci66353 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Tricarico PM, de Oliveira Franca RF, Pacor S, Ceglia V, Crovella S, Celsi F (2016) HIV protease inhibitors apoptotic effect in SH-SY5Y neuronal cell line. Cell Physiol Biochem 39:1463–1470. doi: 10.1159/000447849 CrossRefPubMedGoogle Scholar
  53. Vega-Avila E, Pugsley MK (2011) An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proc West Pharmacol Soc 54:10–14PubMedGoogle Scholar
  54. Wang X, Wang W, Li L, Perry G, Lee HG, Zhu X (2014) Oxidative stress and mitochondrial dysfunction in Alzheimer’s disease. Biochim Biophys Acta 1842:1240–1247. doi: 10.1016/j.bbadis.2013.10.015 CrossRefPubMedGoogle Scholar
  55. Weidinger A, Kozlov AV (2015) Biological activities of reactive oxygen and nitrogen species: oxidative stress versus signal transduction. Biomol Ther 5:472–484. doi: 10.3390/biom5020472 Google Scholar
  56. Yan MH, Wang X, Zhu X (2013) Mitochondrial defects and oxidative stress in Alzheimer disease and Parkinson disease. Free Radic Biol Med 62:90–101. doi: 10.1016/j.freeradbiomed.2012.11.014 CrossRefPubMedGoogle Scholar
  57. Yan W et al (2015) Identification, synthesis and pharmacological evaluation of novel anti-EV71 agents via cyclophilin A inhibition. Bioorg Med Chem Lett 25:5682–5686. doi: 10.1016/j.bmcl.2015.11.002 CrossRefPubMedGoogle Scholar
  58. Zhang HA et al (2012) Salvianolic acid A protects human SH-SY5Y neuroblastoma cells against H(2)O(2)-induced injury by increasing stress tolerance ability. Biochem Biophys Res Commun 421:479–483. doi: 10.1016/j.bbrc.2012.04.021 CrossRefPubMedGoogle Scholar
  59. Zhou X, Zheng W, Nagana Gowda GA, Raftery D, Donkin SS, Bequette B, Teegarden D (2016) 1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell. J Steroid Biochem Mol Biol 163:147–156. doi: 10.1016/j.jsbmb.2016.04.022 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Rebeca Alvariño
    • 1
  • Eva Alonso
    • 1
  • Marie-Aude Tribalat
    • 2
  • Sandra Gegunde
    • 1
  • Olivier P. Thomas
    • 2
    • 3
  • Luis M. Botana
    • 1
  1. 1.Departamento de Farmacología, Facultad de VeterinariaUniversidad de Santiago de CompostelaLugoSpain
  2. 2.Géoazur UMR Université Nice Sophia AntipolisNiceFrance
  3. 3.Marine Biodiscovery, School of ChemistryNational University of Ireland GalwayGalwayIreland

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