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Probucol Protects Neuronal Cells Against Peroxide-Induced Damage and Directly Activates Glutathione Peroxidase-1

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Abstract

Experimental evidence has shown that probucol, a hypocholesterolemic agent, is also able to increase glutathione peroxidase (GPx) activity. However, there is a lack of knowledge about the mechanism(s) involved in this event. In this study, in vitro experiments with purified GPx1 from bovine erythrocytes and cultured SH-SY5Y neuroblastoma cells, as well as in silico studies with GPx1, were performed in order to elucidate mechanisms mediating the stimulatory effect of probucol on GPx activity and to investigate the relevance of this event in terms of susceptibility against peroxide-induced cytotoxicity. In vitro experiments with purified GPx1 showed a direct stimulatory effect of probucol on the activity of GPx1, which was related to an increase in Vmax with no changes in KM. Probucol also increased GPx activity in cultured SH-SY5Y neuroblastoma cells, while the levels of GPx1 expression were not changed, corroborating the results found with the purified enzyme. In addition, probucol rendered SH-SY5Y cells more resistant to hydroperoxide-induced cytotoxicity, and this event was abolished in GPx1 knocked-down cells. In silico studies with GPx1 pointed to a potential binding site for probucol at the close vicinity of the GSH pocket. Collectively, the results presented herein indicate that GPx1 plays a central role in the probucol-induced protective effects against peroxide toxicity. This highlights a novel target (GPx1) and a new mechanism of action (direct activation) for an “old drug.” The relevance of such results for in vivo conditions deserves further investigation.

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Abbreviations

CuOOH:

Cumene hydroperoxide

DCFH-DA:

2′,7′-Dichlorodihydrofluorescein diacetate

DMSO:

Dimethyl sulfoxide

GPx:

Glutathione peroxidase

GR:

Glutathione reductase

GSH:

Glutathione

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Na2SeO3 :

Sodium selenite

PB:

Probucol

ROS:

Reactive oxygen species

tBuOOH:

tert-Butyl hydroperoxide

References

  1. Forman HJ, Augusto O, Brigelius-Flohe R, Dennery PA, Kalyanaraman B, Ischiropoulos H, Mann GE, Radi R et al (2015) Even free radicals should follow some rules: a guide to free radical research terminology and methodology. Free Radic Biol Med 78:233–235

    Article  CAS  PubMed  Google Scholar 

  2. Sies H (2014) Role of metabolic H2O2 generation: redox signaling and oxidative stress. J Biol Chem 289:8735–8741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lubos E, Loscalzo J, Handy DE (2011) Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal 15:1957–1997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Vlahos R, Bozinovski S (2013) Glutathione peroxidase-1 as a novel therapeutic target for COPD. Redox report: communications in free radical research 18:142–149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rajendran P, Nandakumar N, Rengarajan T, Palaniswami R, Gnanadhas EN, Lakshminarasaiah U, Gopas J, Nishigaki I (2014) Antioxidants and human diseases. Clinica chimica acta; international journal of clinical chemistry 436:332–347

    Article  CAS  PubMed  Google Scholar 

  6. Brigelius-Flohe R, Maiorino M (2013) Glutathione peroxidases. Biochim Biophys Acta 1830:3289–3303

    Article  CAS  PubMed  Google Scholar 

  7. Mills GC (1957) Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J Biol Chem 229:189–197

    Article  CAS  PubMed  Google Scholar 

  8. Nascimento V, Alberto EE, Tondo DW, Dambrowski D, Detty MR, Nome F, Braga AL (2012) GPx-like activity of selenides and selenoxides: experimental evidence for the involvement of hydroxy perhydroxy selenane as the active species. J Am Chem Soc 134:138–141

    Article  CAS  PubMed  Google Scholar 

  9. Ibrahim M, Muhammad N, Naeem M, Deobald AM, Kamdem JP, Rocha JB (2015) In vitro evaluation of glutathione peroxidase (GPx)-like activity and antioxidant properties of an organoselenium compound. Toxicology in vitro: an international journal published in association with BIBRA 29:947–952

    Article  CAS  Google Scholar 

  10. Barbosa NV, Nogueira CW, Nogara PA, de Bem AF, Aschner M, Rocha JBT (2017) Organoselenium compounds as mimics of selenoproteins and thiol modifier agents. Metallomics: integrated biometal science 9:1703–1734

    Article  CAS  Google Scholar 

  11. Bartolini D, Piroddi M, Tidei C, Giovagnoli S, Pietrella D, Manevich Y, Tew KD, Giustarini D et al (2015) Reaction kinetics and targeting to cellular glutathione S-transferase of the glutathione peroxidase mimetic PhSeZnCl and its D,L-polylactide microparticle formulation. Free Radic Biol Med 78:56–65

    Article  CAS  PubMed  Google Scholar 

  12. Cheng YY, Qian PC (1990) The effect of selenium-fortified table salt in the prevention of Keshan disease on a population of 1.05 million. Biomedical and environmental sciences: BES 3:422–428

    CAS  PubMed  Google Scholar 

  13. Baliga MS, Wang H, Zhuo P, Schwartz JL, Diamond AM (2007) Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage. Biol Trace Elem Res 115:227–242

    Article  CAS  PubMed  Google Scholar 

  14. Schnabel R, Lubos E, Messow CM, Sinning CR, Zeller T, Wild PS, Peetz D, Handy DE et al (2008) Selenium supplementation improves antioxidant capacity in vitro and in vivo in patients with coronary artery disease the selenium therapy in coronary artery disease patients (SETCAP) study. Am Heart J 156(1201):e1201–e1211

    Google Scholar 

  15. Hernandez-Montes E, Pollard SE, Vauzour D, Jofre-Montseny L, Rota C, Rimbach G, Weinberg PD, Spencer JP (2006) Activation of glutathione peroxidase via Nrf1 mediates genistein’s protection against oxidative endothelial cell injury. Biochem Biophys Res Commun 346:851–859

    Article  CAS  PubMed  Google Scholar 

  16. Jornot L, Junod AF (1997) Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism. The Biochemical journal 326(Pt 1):117–123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yamashita S, Hbujo H, Arai H, Harada-Shiba M, Matsui S, Fukushima M, Saito Y, Kita T et al (2008) Long-term probucol treatment prevents secondary cardiovascular events: a cohort study of patients with heterozygous familial hypercholesterolemia in Japan. J Atheroscler Thromb 15:292–303

    Article  PubMed  Google Scholar 

  18. Liu J, Li M, Lu H, Qiao W, Xi D, Luo T, Xiong H, Guo Z (2015) Effects of probucol on restenosis after percutaneous coronary intervention: a systematic review and meta-analysis. PLoS One 10:e0124021

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Colle D, Hartwig JM, Soares FA, Farina M (2012) Probucol modulates oxidative stress and excitotoxicity in Huntington’s disease models in vitro. Brain Res Bull 87:397–405

    Article  CAS  PubMed  Google Scholar 

  20. Santos DB, Peres KC, Ribeiro RP, Colle D, dos Santos AA, Moreira EL, Souza DO, Figueiredo CP et al (2012) Probucol, a lipid-lowering drug, prevents cognitive and hippocampal synaptic impairments induced by amyloid beta peptide in mice. Exp Neurol 233:767–775

    Article  CAS  PubMed  Google Scholar 

  21. Ribeiro RP, Moreira EL, Santos DB, Colle D, Dos Santos AA, Peres KC, Figueiredo CP, Farina M (2013) Probucol affords neuroprotection in a 6-OHDA mouse model of Parkinson’s disease. Neurochem Res 38:660–668

    Article  CAS  PubMed  Google Scholar 

  22. Santos DB, Colle D, Moreira EL, Peres KC, Ribeiro RP, dos Santos AA, de Oliveira J, Hort MA et al (2015) Probucol mitigates streptozotocin-induced cognitive and biochemical changes in mice. Neuroscience 284:590–600

    Article  CAS  PubMed  Google Scholar 

  23. Caballero B, Olguin N, Campos F, Farina M, Ballester F, Lopez-Espinosa MJ, Llop S, Rodriguez-Farre E et al (2017) Methylmercury-induced developmental toxicity is associated with oxidative stress and cofilin phosphorylation. Cellular and human studies Neurotoxicology 59:197–209

    Article  CAS  PubMed  Google Scholar 

  24. Colle D, Santos DB, Moreira EL, Hartwig JM, dos Santos AA, Zimmermann LT, Hort MA, Farina M (2013) Probucol increases striatal glutathione peroxidase activity and protects against 3-nitropropionic acid-induced pro-oxidative damage in rats. PLoS One 8:e67658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Farina M, Campos F, Vendrell I, Berenguer J, Barzi M, Pons S, Sunol C (2009) Probucol increases glutathione peroxidase-1 activity and displays long-lasting protection against methylmercury toxicity in cerebellar granule cells. Toxicological sciences: an official journal of the Society of Toxicology 112:416–426

    Article  CAS  Google Scholar 

  26. Siveski-Iliskovic N, Kaul N, Singal PK (1994) Probucol promotes endogenous antioxidants and provides protection against adriamycin-induced cardiomyopathy in rats. Circulation 89:2829–2835

    Article  CAS  PubMed  Google Scholar 

  27. Asiri YA (2010) Probucol attenuates cyclophosphamide-induced oxidative apoptosis, p53 and Bax signal expression in rat cardiac tissues. Oxidative Med Cell Longev 3:308–316

    Article  Google Scholar 

  28. Duan SB, Liu GL, Wang YH, Zhang JJ (2012) Epithelial-to-mesenchymal transdifferentiation of renal tubular epithelial cell mediated by oxidative stress and intervention effect of probucol in diabetic nephropathy rats. Ren Fail 34:1244–1251

    Article  CAS  PubMed  Google Scholar 

  29. Xiao X, Hou H, Lin V, Ho D, Tran K, Che B, May A, Zhang J et al (2017) Probucol protects rats from cardiac dysfunction induced by oxidative stress following cardiopulmonary resuscitation. Oxidative Med Cell Longev 2017:1284804

    Article  CAS  Google Scholar 

  30. Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:325–333

    Article  CAS  PubMed  Google Scholar 

  31. Vendrell I, Carrascal M, Vilaro MT, Abian J, Rodriguez-Farre E, Sunol C (2007) Cell viability and proteomic analysis in cultured neurons exposed to methylmercury. Human & experimental toxicology 26:263–272

    Article  CAS  Google Scholar 

  32. Petegnief V, Friguls B, Sanfeliu C, Sunol C, Planas AM (2003) Transforming growth factor-alpha attenuates N-methyl-D-aspartic acid toxicity in cortical cultures by preventing protein synthesis inhibition through an Erk1/2-dependent mechanism. J Biol Chem 278:29552–29559

    Article  CAS  PubMed  Google Scholar 

  33. Wardman P (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 43:995–1022

    Article  CAS  PubMed  Google Scholar 

  34. Engel D, Zomkowski AD, Lieberknecht V, Rodrigues AL, Gabilan NH (2013) Chronic administration of duloxetine and mirtazapine downregulates proapoptotic proteins and upregulates neurotrophin gene expression in the hippocampus and cerebral cortex of mice. J Psychiatr Res 47:802–808

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  36. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera--a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  PubMed  Google Scholar 

  38. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general amber force field. J Comput Chem 25:1157–1174

    Article  CAS  PubMed  Google Scholar 

  39. Ali ST, Jahangir S, Karamat S, Fabian WM, Nawara K, Kona J (2010) Theoretical study on the redox cycle of bovine glutathione peroxidase GPx1: pKa calculations, docking, and molecular dynamics simulations. J Chem Theory Comput 6:1670–1681

    Article  CAS  PubMed  Google Scholar 

  40. Wallace AC, Laskowski RA, Singh J, Thornton JM (1996) Molecular recognition by proteins: protein-ligand interactions from a structural perspective. Biochem Soc Trans 24:280–284

    Article  CAS  PubMed  Google Scholar 

  41. Chaudiere J, Wilhelmsen EC, Tappel AL (1984) Mechanism of selenium-glutathione peroxidase and its inhibition by mercaptocarboxylic acids and other mercaptans. J Biol Chem 259:1043–1050

    Article  CAS  PubMed  Google Scholar 

  42. Koshland DE (1958) Application of a theory of enzyme specificity to protein synthesis. Proc Natl Acad Sci U S A 44:98–104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li C, Deng X, Zhang W, Xie X, Conrad M, Liu Y, Angeli JPF, Lai L (2019) Novel allosteric activators for ferroptosis regulator glutathione peroxidase 4. J Med Chem 62:266–275

    Article  CAS  PubMed  Google Scholar 

  44. Du Y, Zhang X, Ji H, Liu H, Li S, Li L (2012) Probucol and atorvastatin in combination protect rat brains in MCAO model: upregulating Peroxiredoxin2, Foxo3a and Nrf2 expression. Neurosci Lett 509:110–115

    Article  CAS  PubMed  Google Scholar 

  45. Sheng L, Jiao B, Shao L, Bi S, Cheng C, Zhang J, Jiang Y (2013) Probucol inhibits hydrogen peroxide to induce apoptosis of vascular smooth muscle cells. Mol Med Rep 7:1185–1190

    Article  CAS  PubMed  Google Scholar 

  46. Zhou Z, Liu C, Chen S, Zhao H, Zhou K, Wang W, Yuan Y, Li Z et al (2017) Activation of the Nrf2/ARE signaling pathway by probucol contributes to inhibiting inflammation and neuronal apoptosis after spinal cord injury. Oncotarget 8:52078–52093

    Article  PubMed  PubMed Central  Google Scholar 

  47. Poirier J, Miron J, Picard C, Gormley P, Théroux L, Breitner J, Dea D (2014) Apolipoprotein E and lipid homeostasis in the etiology and treatment of sporadic Alzheimer’s disease. Neurobiol Aging 35:S3–S10

  48. Colle D, Santos DB, Hartwig JM, Godoi M, Engel DF, de Bem AF, Braga AL, Farina M (2016) Succinobucol, a lipid-lowering drug, protects against 3-nitropropionic acid-induced mitochondrial dysfunction and oxidative stress in SH-SY5Y cells via upregulation of glutathione levels and glutamate cysteine ligase activity. Mol Neurobiol 53:1280–1295

    Article  CAS  PubMed  Google Scholar 

  49. Buxbaum E (1999) Co-operative binding sites for transported substrates in the multiple drug resistance transporter Mdr1. Eur J Biochem 265:64–70

    Article  CAS  PubMed  Google Scholar 

  50. Chada S, Whitney C, Newburger PE (1989) Post-transcriptional regulation of glutathione peroxidase gene expression by selenium in the HL-60 human myeloid cell line. Blood. 74:2535–2541

    Article  CAS  PubMed  Google Scholar 

  51. Heeg JF, Tachizawa H (1980) Plasma levels of probucol in man after single and repeated oral doses. Nouv Press Med 9:2990–2994

    CAS  Google Scholar 

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Funding

The financial supports by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) are gratefully acknowledged. We are also grateful to the Laboratório Multiusuário de Estudos em Biologia at the Universidade Federal de Santa Catarina (LAMEB/UFSC) for providing its infrastructure for carrying out biochemical analysis.

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Authors and Affiliations

  1. Department of Biochemistry, Federal University of Santa Catarina, Florianopolis, Santa Catarina, 88040-900, Brazil

    Danúbia B. Santos, Karin Santos, Guilherme Razzera & Marcelo Farina

  2. Department of Clinical Analyses, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil

    Dirleise Colle

  3. Department of Physiological Sciences, Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil

    Eduardo L. G. Moreira

  4. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA

    Alessandra A. Santos

  5. Institute of Biological Sciences, Federal University of Rio Grande, Rio Grande, Rio Grande do Sul, Brazil

    Mariana A. Hort

  6. Institute of Bioscience, Sector of Biochemistry, Federal University of Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, Brazil

    Jean P. Oses

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  1. Danúbia B. Santos
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Correspondence to Danúbia B. Santos or Marcelo Farina.

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Highlights

•Probucol increased GPx1 Vmax in in vitro conditions with purified enzyme.

•Probucol increased GPx activity in cultured SH-SY5Y cells without changing GPx1 protein levels.

•Probucol-treated SH-SY5Y cells were more resistant to hydroperoxide toxicity.

•Probucol’s protective effect against hydroperoxide toxicity is abolished in GPx1 knocked-down cells.

Electronic Supplementary Material

Supplemental Figure 1

Effect of probucol on the immunoreactivity of GPx1 in human neuroblastoma cells. Cells were pretreated for 6 days with probucol (PB, 3 or 10 μM), sodium selenite (Na2SeO3, positive control, 30 nM) or vehicle. Representative microscopic images of GPx-1 in SH-SY5Y evaluated by immunoreactivity (scale bar = 200 μm). Left panels = GPx1 antibody; Central panels = nuclei stain; Right panels = merge (TIF 1467 kb)

High resolution image (PNG 933 kb)

Supplemental Figure 2

Standardization of the transfection assay. Cells were transfected with 3 siRNA targets for GPx1, negative control (scrambled) and transfection control (TYE ™ 563 DS). The photographs were performed at 24 h, 3 and 6 days after transfection with siRNA positive and negative controls (TIF 1678 kb)

High resolution image (PNG 683 kb)

Supplemental Figure 3

Messenger RNA expression, GPx1 activity and GPx1 immunoreactivity (Western Blot). After the transfection assays (see Materials and Methods; GPx1 knockdown, initial standardization), GPx1 mRNA expression (A), activity (B) and protein levels (C and D) were determined. Cells were collected at 36 h (mRNA expression) or at 72 h (enzyme activity and Western Blot) after transfection. Fig. D is a representative Western Blot image of Fig. C data. Data (A-C) are mean ± S.E.M. expressed as percentages of control cells (transfected with scrambled) n = 4. * indicate significant (p < 0.05) differences compared to control by one-way ANOVA, followed by Tukey’s post hoc test. (TIFF 93 kb)

High resolution image (PNG 816 kb)

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Santos, D.B., Colle, D., Moreira, E.L.G. et al. Probucol Protects Neuronal Cells Against Peroxide-Induced Damage and Directly Activates Glutathione Peroxidase-1. Mol Neurobiol 57, 3245–3257 (2020). https://doi.org/10.1007/s12035-020-01963-w

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