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Effect of 5-aminolevulinic acid on the expression of carcinogenesis-related proteins in cultured primary hepatocytes

  • P. R. Menezes
  • C. B. González
  • A. O. DeSouza
  • D. A. Maria
  • J. Onuki
Short Communication
  • 56 Downloads

Abstract

Acute intermittent porphyria (AIP) is a heme pathway disorder caused by a decrease in the activity and synthesis of porphobilinogen deaminase. Thus, the first heme precursor 5-aminolevulinic acid (ALA) accumulates in the liver. Reactive oxygen species (ROS) resulting from ALA oxidation may be correlated to a higher incidence of hepatocellular carcinoma (HCC) in AIP patients. However, the molecular mechanisms of this relationship have not been thoroughly elucidated to date. In this study, we investigated the effect of increasing levels of ALA on the expression of proteins related to DNA repair, oxidative stress, apoptosis, proliferation and lipid metabolism. Primary rat hepatocytes were isolated by the collagenase perfusion method, lipoperoxidation was evaluated by a TBA fluorimetric assay and Western blotting was used to assess protein abundance. The data showed that ALA treatment promoted a dose-dependent increase of p53 expression, downregulation of Bcl-2, HMG-CoA reductase and OGG1 and an increase in lipoperoxidation. There was no alteration in the expression of the transcription factor NF-κB, catalase and superoxide dismutase. ALA oxidation products induced protein regulation patterns, suggesting the interconnection of cellular processes, such as the intrinsic pathway of apoptosis, redox homeostasis, cell proliferation, lipid metabolism and DNA repair. This study helps to elucidate the molecular mechanisms of hepatotoxicity mediated by ALA pro-oxidant effects and supports the hypothesis that ALA accumulation correlates with a higher incidence of hepatic carcinogenic events.

Keywords

5-Aminolevulinic acid Acute intermittent porphyria Hepatocellular carcinoma Protein expression Reactive oxygen species Primary rat hepatocytes 

Abbreviations

Bcl-2

β-Cell lymphoma 2

ALA

5-Aminolevulinic acid

8-oxodGuo

8-Oxo-7,8-dihydro-2´-deoxyguanosine

OGG1

8-Oxoguanine DNA glycosylase I

AIP

Acute intermittent porphyria

CAT

Catalase

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

HCC

Hepatocellular carcinoma

HMG-CoAr

Hydroxymethylglutaryl-CoA reductase

NF-κB

Nuclear factor κB

ROS

Reactive oxygen species

SOD

Superoxide dismutase

p53

Tumor protein p53

Notes

Acknowledgements

This work was supported by the “Fundação de Amparo à Pesquisa do Estado de São Paulo” FAPESP (Grants: 07/01966-5 and 10/51068-6). PRM and CBG received fellowships from “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” - CAPES (Biotechnology Program-USP-33002010156PO and “Programa de Estudantes-Convênio de Pós-Graduação”-PEC-PG notice 042/2012)”.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The experimental procedure was approved by the animal ethics committee of Butantan Institute (CEUAIB 755/10) and was performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and the associated guidelines, the EU Directive 2010/63/EU for animal experiments and complied with the ARRIVE guidelines.

Research involving human participants

All authors declare that this article does not contain any studies with human participants performed by any of the authors.

References

  1. 1.
    Karim Z, Lyoumi S, Nicolas G et al (2015) Porphyrias: a 2015 update. Clin Res Hepatol Gastroenterol 39:412–425.  https://doi.org/10.1016/j.clinre.2015.05.009 CrossRefPubMedGoogle Scholar
  2. 2.
    Besur S, Schmeltzer P, Bonkovsky HL (2015) Acute porphyrias. J Emerg Med 49:305–312.  https://doi.org/10.1016/j.jemermed.2015.04.034 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bissell DM, Wang B (2015) Acute hepatic porphyria. J Clin Transl Hepatol 3:17–26.  https://doi.org/10.14218/JCTH.2014.00039 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sardh E, Wahlin S, Björnstedt M et al (2013) High risk of primary liver cancer in a cohort of 179 patients with acute hepatic porphyria. J Inherit Metab Dis 36:1063–1071.  https://doi.org/10.1007/s10545-012-9576-9 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Onuki J, Medeiros MH, Bechara EJ, Di Mascio P (1994) 5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions. Biochim Biophys Acta 1225:259–263CrossRefPubMedCentralGoogle Scholar
  6. 6.
    Fraga CG, Onuki J, Lucesoli F et al (1994) 5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2′-deoxyguanosine in DNA. Carcinogenesis 15:2241–2244CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Douki T, Onuki J, Medeiros MHG et al (1998) Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 5-aminolevulinic acid. FEBS Lett 428:93–96.  https://doi.org/10.1016/S0014-5793(98)00504-3 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fiedler DM, Eckl PM, Krammer B (1996) Does delta-aminolaevulinic acid induce genotoxic effects? J Photochem Photobiol B 33:39–44CrossRefPubMedCentralGoogle Scholar
  9. 9.
    Onuki J, Rech CM, Medeiros MHG et al (2002) Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the Salmonella/microsuspension mutagenicity assay and SOS chromotest. Environ Mol Mutagen 40:63–70.  https://doi.org/10.1002/em.10083 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Homedan C, Schmitt C, Laafi J et al (2015) Mitochondrial energetic defects in muscle and brain of a Hmbs−/− mouse model of acute intermittent porphyria. Hum Mol Genet 24:5015–5023.  https://doi.org/10.1093/hmg/ddv222 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ciccarese F, Ciminale V (2017) Escaping death: mitochondrial redox homeostasis in cancer cells. Front Oncol 7:117.  https://doi.org/10.3389/fonc.2017.00117 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Singh KK (2006) Mitochondria damage checkpoint, aging, and cancer. Ann N Y Acad Sci 1067:182–190.  https://doi.org/10.1196/annals.1354.022 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Onuki J, Chen Y, Teixeira PC et al (2004) Mitochondrial and nuclear DNA damage induced by 5-aminolevulinic acid. Arch Biochem Biophys 432:178–187.  https://doi.org/10.1016/j.abb.2004.09.030 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sies H (2017) Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress. Redox Biol 11:613–619.  https://doi.org/10.1016/j.redox.2016.12.035 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Onuki J, Teixeira PC, Medeiros MHG, Di Mascio P (2002) Danos ao DNA promovidos por ácido 5-aminolevulínico: Possível associação com o desenvolvimento de carcinoma hepatocelular em portadores de porfiria aguda intermitente. Quim Nova 25:594–608.  https://doi.org/10.1590/S0100-40422002000400015 CrossRefGoogle Scholar
  16. 16.
    Olinski R, Gackowski D, Rozalski R et al (2003) Oxidative DNA damage in cancer patients: a cause or a consequence of the disease development? Mutat Res 531:177–190CrossRefPubMedCentralGoogle Scholar
  17. 17.
    Rani V, Deep G, Singh RK et al (2016) Oxidative stress and metabolic disorders: pathogenesis and therapeutic strategies. Life Sci 148:183–193.  https://doi.org/10.1016/j.lfs.2016.02.002 CrossRefPubMedGoogle Scholar
  18. 18.
    Guguen-Guillouzo C, Guillouzo A (1996) No Title. In: Guillouzo A, Guguen-Guillouzo C (eds) Isolated and cultured hepatocytes. Les Editions INSERM and John Libbey Eurotext, Paris, pp 1–12Google Scholar
  19. 19.
    Onuki J, Teixeira PC, Medeiros MHG et al (2002) Is 5-aminolevulinic acid involved in the hepatocellular carcinogenesis of acute intermittent porphyria? Cell Mol Biol 48:17–26PubMedPubMedCentralGoogle Scholar
  20. 20.
    Noble JE (2014) Quantification of protein concentration using UV absorbance and coomassie dyes. Methods Enzymol 536:17–26.  https://doi.org/10.1016/B978-0-12-420070-8.00002-7 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mihara M, Uchiyama M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Hofseth LJ, Hussain SP, Harris CC (2004) p53: 25 years after its discovery. Trends Pharmacol Sci 25:177–181.  https://doi.org/10.1016/j.tips.2004.02.009 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49–53CrossRefGoogle Scholar
  24. 24.
    Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323–331CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Miyashita T, Krajewski S, Krajewska M et al (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9:1799–1805PubMedPubMedCentralGoogle Scholar
  26. 26.
    Kirkin V, Joos S, Zörnig M (2004) The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 1644:229–249.  https://doi.org/10.1016/j.bbamcr.2003.08.009 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chong SJF, Low ICC, Pervaiz S (2014) Mitochondrial ROS and involvement of Bcl-2 as a mitochondrial ROS regulator. Mitochondrion 19:39–48.  https://doi.org/10.1016/j.mito.2014.06.002 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Pierce RH, Vail ME, Ralph L et al (2002) Bcl-2 expression inhibits liver carcinogenesis and delays the development of proliferating foci. Am J Pathol 160:1555–1560.  https://doi.org/10.1016/S0002-9440(10)61101-7 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Takaki A, Yamamoto K (2015) Control of oxidative stress in hepatocellular carcinoma: helpful or harmful? World J Hepatol.  https://doi.org/10.4254/wjh.v7.i7.968 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Cabrera R, Limaye A, Cabrera R (2012) Hepatocellular carcinoma: current trends in worldwide epidemiology, risk factors, diagnosis, and therapeutics. Hepatic Med Evid Res 4:19.  https://doi.org/10.2147/HMER.S16316 CrossRefGoogle Scholar
  31. 31.
    Vairo G, Innes KM, Adams JM (1996) Bcl-2 has a cell cycle inhibitory function separable from its enhancement of cell survival. Oncogene 13:1511–1519PubMedPubMedCentralGoogle Scholar
  32. 32.
    Murphy KL, Kittrell FS, Gay JP et al (1999) Bcl-2 expression delays mammary tumor development in dimethylbenz(a)anthracene-treated transgenic mice. Oncogene 18:6597–6604.  https://doi.org/10.1038/sj.onc.1203099 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Um H-D (2016) Bcl-2 family proteins as regulators of cancer cell invasion and metastasis: a review focusing on mitochondrial respiration and reactive oxygen species. Oncotarget 7:5193–5203.  https://doi.org/10.18632/oncotarget.6405 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sun T, Sun B, Zhao X et al (2011) Promotion of tumor cell metastasis and vasculogenic mimicry by way of transcription coactivation by Bcl-2 and Twist1: a study of hepatocellular carcinoma. Hepatology 54:1690–1706.  https://doi.org/10.1002/hep.24543 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Hockenbery DM, Oltvai ZN, Yin XM et al (1993) Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251CrossRefPubMedCentralGoogle Scholar
  36. 36.
    Ellerby LM, Ellerby HM, Park SM et al (1996) Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2. J Neurochem 67:1259–1267CrossRefPubMedCentralGoogle Scholar
  37. 37.
    Emanuelli T, Pagel FW, Porciúncula LO, Souza DO (2003) Effects of 5-aminolevulinic acid on the glutamatergic neurotransmission. Neurochem Int 42:115–121.  https://doi.org/10.1016/S0197-0186(02)00074-8 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Laafi J, Homedan C, Jacques C et al (2014) Pro-oxidant effect of ALA is implicated in mitochondrial dysfunction of HepG2 cells. Biochimie 106:157–166.  https://doi.org/10.1016/j.biochi.2014.08.014 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Rossin D, Calfapietra S, Sottero B et al (2017) HNE and cholesterol oxidation products in colorectal inflammation and carcinogenesis. Free Radic Biol Med.  https://doi.org/10.1016/j.freeradbiomed.2017.01.017 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Douki T, Onuki J, Medeiros MHG et al (1998) DNA alkylation by 4,5-dioxovaleric acid, the final oxidation product of 5-aminolevulinic acid. Chem Res Toxicol 11:150–157.  https://doi.org/10.1021/tx970157d CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Goldstein JL, Brown MS (1990) Regulation of the mevalonate pathway. Nature 343:425–430.  https://doi.org/10.1038/343425a0 CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Garcia-Ruiz C, Mari M, Colell A et al (2009) Mitochondrial cholesterol in health and disease. Histol Histopathol 24:117–132.  https://doi.org/10.14670/HH-24.117 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Lo Sasso G, Celli N, Caboni M et al (2010) Down-regulation of the LXR transcriptome provides the requisite cholesterol levels to proliferating hepatocytes. Hepatology 51:1334–1344.  https://doi.org/10.1002/hep.23436 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Dang CV (2012) Links between metabolism and cancer. Genes Dev 26:877–890.  https://doi.org/10.1101/gad.189365.112 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Casey SC, Amedei A, Aquilano K et al (2015) Cancer prevention and therapy through the modulation of the tumor microenvironment. Semin Cancer Biol 35:S199–S223.  https://doi.org/10.1016/j.semcancer.2015.02.007 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Montero J, Morales A, Llacuna L et al (2008) Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma. Cancer Res 68:5246–5256.  https://doi.org/10.1158/0008-5472.CAN-07-6161 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Ribas V, García-Ruiz C, Fernández-Checa JC (2016) Mitochondria, cholesterol and cancer cell metabolism. Clin Transl Med 5:22.  https://doi.org/10.1186/s40169-016-0106-5 CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Maxfield FR, Tabas I (2005) Role of cholesterol and lipid organization in disease. Nature 438:612–621.  https://doi.org/10.1038/nature04399 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Borena W, Strohmaier S, Lukanova A et al (2012) Metabolic risk factors and primary liver cancer in a prospective study of 578,700 adults. Int J Cancer 131:193–200.  https://doi.org/10.1002/ijc.26338 CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Agren R, Mardinoglu A, Asplund A et al (2014) Identification of anticancer drugs for hepatocellular carcinoma through personalized genome-scale metabolic modeling. Mol Syst Biol 10:721CrossRefPubMedCentralGoogle Scholar
  51. 51.
    Singh S, Singh PP (2014) Statins for prevention of hepatocellular cancer: one step closer? Hepatology 59:724–726.  https://doi.org/10.1002/hep.26614 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Kryston TB, Georgiev AB, Pissis P, Georgakilas AG (2011) Role of oxidative stress and DNA damage in human carcinogenesis. Mutat Res 711:193–201.  https://doi.org/10.1016/j.mrfmmm.2010.12.016 CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Tanaka S, Miyanishi K, Kobune M et al (2013) Increased hepatic oxidative DNA damage in patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma. J Gastroenterol 48:1249–1258.  https://doi.org/10.1007/s00535-012-0739-0 CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Hikita H, Kodama T, Tanaka S et al (2015) Activation of the mitochondrial apoptotic pathway produces reactive oxygen species and oxidative damage in hepatocytes that contribute to liver tumorigenesis. Cancer Prev Res 8:693–701.  https://doi.org/10.1158/1940-6207.CAPR-15-0022-T CrossRefGoogle Scholar
  55. 55.
    Pan L, Zhu B, Hao W et al (2016) Oxidized guanine base lesions function in 8-oxoguanine DNA glycosylase-1-mediated epigenetic regulation of nuclear factor κB-driven gene expression. J Biol Chem 291:25553–25566.  https://doi.org/10.1074/jbc.M116.751453 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Ghosh S, Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109(Suppl):S81–S96CrossRefGoogle Scholar
  57. 57.
    Tonks NK (2005) Redox redux: revisiting PTPs and the control of cell signaling. Cell 121:667–670.  https://doi.org/10.1016/j.cell.2005.05.016 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Molecular BiologyButantan InstituteSão PauloBrazil
  2. 2.Department of Clinical and Toxicological Analyses, School of Pharmaceutical SciencesUniversity of São PauloSão PauloBrazil
  3. 3.Sección de Toxicología, Departamento de Ciencias ForensesOrganismo de Investigación JudicialHerediaCosta Rica

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