Skip to main content
SpringerLink
Log in

In vitro optimization of retinoic acid–induced neuritogenesis and TH endogenous expression in human SH-SY5Y neuroblastoma cells by the antioxidant Trolox

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 μM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 μM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

RA:

Retinoic acid

ROS:

Reactive oxygen species

TH:

Tyrosine hydroxylase

PD:

Parkinson’s disease

CAT:

Catalase

SOD:

Superoxide dismutase

GPx:

Glutathione peroxidase

hESCs:

Human embryonic stem cells

iPS cells:

Induced pluripotent stem cells

MSCs:

Mesenchymal stem cells

References

  1. Bernheimer H, Birkmayer W, Hornykiewiez O, Jellinger K, Seitelberger F (1973) Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci 20(4):415–455

    Article  PubMed  CAS  Google Scholar 

  2. Hirsch E, Graybiel AM, Agid YA (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease. Nature 334:345–348

    Article  PubMed  CAS  Google Scholar 

  3. Björklund A, Lindvall O (2000) Cell replacement therapies for central nervous system disorders. Nat Neurosci 3:537–544

    Article  PubMed  Google Scholar 

  4. Perrier AL, Taba V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 34:12543–12548

    Article  Google Scholar 

  5. Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA 15:5856–5861

    Article  Google Scholar 

  6. Swistowski A, Peng J, Liu Q, Mali P, Rao MS, Cheng L, Zeng X (2010) Efficient generation of functional dopaminergic neurons from human induced pluripotent stem cells under defined conditions. Stem Cells 10:1893–1904

    Article  Google Scholar 

  7. Trzaska KA, Kuzhikandathil EV, Rameshwar P (2007) Specification of a dopaminergic phenotype from adult human mesenchymal stem cells. Stem Cells 11:2797–2808

    Article  Google Scholar 

  8. Chen S, Xianwen C, Dehua X, Zhenguo L, Lingfei X, Smith SW, Zhongcheng Z (2003) Behavioral correction of Parkinsonian rats following the transplantation of immortalized fibroblasts genetically modified with TH and GCH genes. Parkinsonism Relat Disord 9(2):91–97

    Article  Google Scholar 

  9. Zeidán-Chuliá F, Noda M (2009) “Opening” the mesenchymal stem cell tool box. Eur J Dent 3:240–249

    PubMed  Google Scholar 

  10. Arenas E (2010) Towards stem cell replacement therapies for Parkinson’s disease. Biochem Biophys Res Commun 396(1):152–156

    Article  PubMed  CAS  Google Scholar 

  11. Ross RA, Spengler BA, Biedler JL (1983) Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst 71:741–747

    PubMed  CAS  Google Scholar 

  12. Nicolini G, Miloso M, Zoia C, Di Silvestro A, Cavaletti G, Tredici G (1998) Retinoic acid differentiated SH-SY5Y human neuroblastoma cells: an in vitro model to assess drug neurotoxicity. Anticancer Res 8:2477–2481

    Google Scholar 

  13. Pahlman S, Hoehner JC, Nanberg E, Hedborg F, Fagerstrom S, Gestblom C, Johansson I, Larsson U, Lavenius E, Ortoft E, Söderholm H (1995) Differentiation and survival influences of growth factors in human neuroblastoma. Eur J Cancer 31A:453–458

    Article  PubMed  CAS  Google Scholar 

  14. Kim B, Leventhal PS, Saltiel AR, Feldman EL (1997) Insulin-like growth factor immediate neurite outgrowth in vitro requires mitogen-activated protein kinase activation. J Biol Chem 272:21268–21273

    Article  PubMed  CAS  Google Scholar 

  15. Encinas M, Iglesias M, Llecha N, Comella JX (1999) Extracellular-regulated kinases and phosphatidylinositol 3-kinase are involved in brain-derived neurotrophic factor mediated survival and neuritogenesis of the neuroblastoma cell line SH-SY5Y. J Neurochem 73:1409–1421

    Article  PubMed  CAS  Google Scholar 

  16. Olsson AK, Vadhammar K, Nanberg E (2000) Activation and protein kinase C dependent nuclear accumulation of ERK in differentiating human neuroblastoma cells. Exp Cell Res 256:454–467

    Article  PubMed  CAS  Google Scholar 

  17. Miloso M, Villa D, Crimi M, Galbiati S, Donzelli E, Nicolini G, Tredici G (2004) Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent. J Neurosci Res 75:241–252

    Article  PubMed  CAS  Google Scholar 

  18. Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141

    PubMed  CAS  Google Scholar 

  19. Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev 81:1269–1304

    PubMed  CAS  Google Scholar 

  20. Radominska-Pandya A, Chen G, Czernik PJ, Little JM, Samokyszyn VM, Carter CA, Nowak G (2000) Direct interaction of All-trans-retinoic acid with protein kinase C (PKC): implications for PKC signaling and cancer therapy. J Biol Chem 275:22324–22330

    Article  PubMed  CAS  Google Scholar 

  21. Frota MLC, Silva EG, Behr GA, Oliveira MR, Dal-Pizzol F, Klamt F, Moreira JCF (2006) All-trans retinoic acid induces free radical generation and modulate antioxidant enzyme activities in rat sertoli cells. Mol Cell Biochem 285:173–179

    Article  CAS  Google Scholar 

  22. Castro-Obregon S, Covarrubias L (1996) Role of retinoic acid and oxidative stress in embryonic stem cell death and neuronal differentiation. FEBS Lett 381:93–97

    Article  PubMed  CAS  Google Scholar 

  23. Delia D, Aiello A, Meroni L, Nicolini M, Reed JC, Pierotti MA (1997) Role of antioxidants and intracellular free radicals in retinamide-induced cell death. Carcinogenesis 18:943–948

    Article  PubMed  CAS  Google Scholar 

  24. Furuke K, Sasada T, Ueda-Taniguchi Y, Yamauchi A, Inamoto T, Yamaoka Y, Masutani H, Yodoi J (1997) Role of intracellular redox status in apoptosis induction of human T-cell leukemia virus type I- infected lymphocytes by 13 cis-retinoic acid. Cancer Res 57:4916–4923

    PubMed  CAS  Google Scholar 

  25. Wang H, Joseph JA (1999) Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 27:612–616

    Article  PubMed  CAS  Google Scholar 

  26. Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431

    Article  PubMed  CAS  Google Scholar 

  27. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

    Article  PubMed  CAS  Google Scholar 

  28. Bannister JV, Calabrese L (1987) Assays for SOD. Methods Biochem Anal 32:279–312

    Article  PubMed  CAS  Google Scholar 

  29. Flohé L, Günzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–121

    Article  PubMed  Google Scholar 

  30. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  31. Lowry OH, Rosebrough AL, Farr AL, Randal RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  32. Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Ceña V, Gallego C, Comella JX (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75:991–1003

    Article  PubMed  CAS  Google Scholar 

  33. Dunkley PR, Bobrovskaya L, Graham ME, von Nagy-Felsobuki EI, Dickson PW (2004) Tyrosine hydroxylase phosphorylation: regulation and consequences. J Neurochem 91:1025–1043

    Article  PubMed  CAS  Google Scholar 

  34. Fernández-Checa JC (2003) Redox regulation and signaling lipids in mitochondrial apoptosis. Biochem Biophys Res Commun 3:471–479

    Article  Google Scholar 

  35. Pantano C, Reynaert NL, van der Vliet A, Janssen-Heininger YM (2006) Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway. Antioxid Redox Signal 9–10:1791–1806

    Article  Google Scholar 

  36. Hansen JM (2006) Oxidative stress as a mechanism of teratogenesis. Birth Defects Res C Embryo Today 4:293–307

    Article  Google Scholar 

  37. Anilkumar N, Sirker A, Shah AM (2009) Redox sensitive signaling pathways in cardiac remodeling, hypertrophy and failure. Front Biosci 14:3168–3187

    Article  PubMed  CAS  Google Scholar 

  38. Inanami O, Watanabe Y, Syuto B, Nakano M, Tsuji M, Kuwabara M (1998) Oral administration of (-)catechin protects against ischemia-reperfusion-induced neuronal death in the gerbil. Free Radic Res 4:359–365

    Article  Google Scholar 

  39. Inanami O, Takahashi K, Kuwabara M (1999) Attenuation of caspase-3-dependent apoptosis by Trolox post-treatment of X-irradiated MOLT-4 cells. Int J Radiat Biol 2:155–163

    Article  Google Scholar 

  40. Niwa K, Inanami O, Yamamori T, Ohta T, Hamasu T, Kubawara M (2003) Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide. Antioxid Redox Signal 6:713–722

    Article  Google Scholar 

  41. Kuwabara M, Asanuma T, Niwa K, Inanami O (2008) Regulation of cell survival and death signals induced by oxidative stress. J Clin Biochem Nutr 2:51–57

    Article  Google Scholar 

  42. Numakawa Y, Numakawa T, Matsumoto T, Yagasaki Y, Kumamaru E, Kunugi H, Taguchi T, Niki E (2006) Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. J Neurochem 4:1191–1202

    Article  Google Scholar 

  43. López-Carballo G, Moreno L, Masiá S, Pérez P, Barettino D (2002) Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 28:25297–25304

    Article  Google Scholar 

  44. Kedar NP (2003) Can we prevent Parkinson’s and Alzheimer’s disease? J Postgrad Med 49:236–245

    PubMed  CAS  Google Scholar 

  45. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI (1995) Embryonic stem cells express neuronal properties in vitro. Dev Biol 2:342–357

    Article  Google Scholar 

  46. Ross SA, McCaffery PJ, Drager UC, De Luca LM (2000) Retinoids in embryonal development. Physiol Rev 80:1021–1054

    PubMed  CAS  Google Scholar 

  47. Duester G (2008) Retinoic acid synthesis and signaling during early organogenesis Gregg Duester. Cell 6:921–931

    Article  Google Scholar 

  48. Kamata H, Hirata H (1999) Redox regulation of cellular signalling. Cell Signal 11:1–14

    Article  PubMed  CAS  Google Scholar 

  49. Zhang L, Zhao B, Yew DT, Kusiak JW, Roth GS (1997) Processing of Alzheimer’s amyloid precursor protein during H2O2-induced apoptosis in human neuronal cells. Biochem Biophys Res Commun 235:845–848

    Article  PubMed  CAS  Google Scholar 

  50. Yamakawa H, Ito Y, Naganawa T, Banno Y, Nakashima S, Yoshimu S, Sawada M, Nishimura Y, Nozawa Y, Sakai N (2000) Activation of caspase-9 and 3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation. Neurol Res 22:556–564

    PubMed  CAS  Google Scholar 

  51. Wu H, Ichikawa S, Tani C, Zhu B, Tada M, Shimoishi Y, Murata Y, Nakamura Y (2009) Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells. Biochim Biophys Acta 1791:8–16

    PubMed  CAS  Google Scholar 

  52. Franco JL, Posser T, Gordon SL, Bobrovskaya L, Schneider JJ, Farina M, Dafre AL, Dickson PW, Dunkley PR (2010) Expression of tyrosine hydroxylase increases the resistance of human neuroblastoma cells to oxidative insults. Toxicol Sci 113(1):150–157

    Article  PubMed  CAS  Google Scholar 

  53. Kaplan DR, Stephens RM (1994) Neurotrophin signal transduction by the Trk receptor. J Neurobiol 25:1404–1417

    Article  PubMed  CAS  Google Scholar 

  54. Hoyos B, Imam A, Chua R, Swenson C, Tong GX, Levi E, Noy N, Hämmerling U (2000) The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors. J Exp Med 192:835–845

    Article  PubMed  CAS  Google Scholar 

  55. Aggarwal S, Kim SW, Cheon K, Tabassam F, Joon JH, Koo JS (2006) Nonclassical action of retinoic acid on the activation of the camp response element-binding protein in normal human bronchial epithelial cells. Mol Biol Cell 17:566–575

    Article  PubMed  CAS  Google Scholar 

  56. Masiá M, Alvarez S, de Lera AR, Barettino D (2007) Rapid, nongenomic actions of retinoic acid on phosphatidylinositol-3-kinase signaling pathway mediated by the retinoic acid receptor. Mol Endocrinol 21(10):2391–2402

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by CNPq, CAPES, FAPERGS, PROPESQ-UFRGS and “Rede Instituto Brasileiro de Neurociência” (IBM-Net) # 01.06.0842.00.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Center of Oxidative Stress Research (CEEO), Department of Biochemistry, Institute of Basic Health Sciences (ICBS), Federal University of Rio Grande do Sul (UFRGS), Rio Grande do Sul, Rua Ramiro Barcelos 2600—ANEXO, Porto Alegre, RS, 90035-003, Brazil

    Mario Luiz Conte da Frota Junior, André Simões Pires, Fares Zeidán-Chuliá, Ivi Juliana Bristot, Fernanda M. Lopes, Matheus Augusto de Bittencourt Pasquali, Alfeu Zanotto-Filho, Guilherme Antônio Behr, Fabio Klamt, Daniel Pens Gelain & José Cláudio Fonseca Moreira

Authors
  1. Mario Luiz Conte da Frota Junior
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. André Simões Pires
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fares Zeidán-Chuliá
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ivi Juliana Bristot
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fernanda M. Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matheus Augusto de Bittencourt Pasquali
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alfeu Zanotto-Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guilherme Antônio Behr
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fabio Klamt
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel Pens Gelain
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. José Cláudio Fonseca Moreira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mario Luiz Conte da Frota Junior.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frota Junior, M.L.C.d., Pires, A.S., Zeidán-Chuliá, F. et al. In vitro optimization of retinoic acid–induced neuritogenesis and TH endogenous expression in human SH-SY5Y neuroblastoma cells by the antioxidant Trolox. Mol Cell Biochem 358, 325–334 (2011). https://doi.org/10.1007/s11010-011-0983-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-011-0983-2

Keywords

Search

Navigation