, Volume 12, Issue 1, pp 113–123 | Cite as

Increased ROS generation and p53 activation in α-lipoic acid-induced apoptosis of hepatoma cells

  • G. Simbula
  • A. Columbano
  • G. M. Ledda-Columbano
  • L. Sanna
  • M. Deidda
  • A. Diana
  • M. Pibiri


α-lipoic acid (α-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, α-LA induces apoptosis in different cancer cells types. However, whether α-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated whether α-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that α-LA inhibits the growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased levels of the cyclin/CDKs inhibitors, p27Kip1 and p21Cip1. Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that α-LA induces apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests that it may prove useful in liver cancer therapy.


Apoptosis p53 phosphorylation α-lipoic acid ROS Hepatoma cells 



α-lipoic acid




c-jun N-terminal kinase


protein kinase B/Akt


dichlorofluorescein diacetate


reactive oxygen species



Vit C

Vitamin C


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Gackowski D, Banaszkiewicz Z, Rosalski R, Jawien A, Olinski R (2002) Persistent oxidative stress in colorectal carcinoma patients. Int J Cancer 101:395–97CrossRefPubMedGoogle Scholar
  2. 2.
    Glaab WE, Hill RB, Skopek TR (2001) Suppression of spontaneous and hydrogen peroxide-induced mutagenesis by the antioxidant ascorbate in mismatch repair-deficient human colon cancer cells. Carcinogenesis 21:1709–713CrossRefGoogle Scholar
  3. 3.
    Forsberg L, de Faire U, Morgenstwern R (2001) Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys 389:84–3CrossRefPubMedGoogle Scholar
  4. 4.
    Paganelli GM, Biasco G, Brandi G et al (1992) Effect of vitamin A, C and E supplementation on rectal cell proliferation in patients with colorectal adenomas. J Natl Cancer Inst 84:47–1CrossRefPubMedGoogle Scholar
  5. 5.
    Monghadasian MH, Freeman HJ, Godin DV (1996) Endogenous antioxidant status in neoplastic and adjacent tissues in 1,2-dimethylhidrazine-induced colon cancer in rats: effects of olsalazine. Carcinogenesis 17:983–87CrossRefGoogle Scholar
  6. 6.
    Packer L, Witt EH, Trischler HJ (1995) α-lipoic acid as a biological antioxidant. Free Radic Biol Med 19:227–50CrossRefPubMedGoogle Scholar
  7. 7.
    Mantovani G, Maccio A, Madeddu C et al (2003) The impact of different antioxidant agents alone or in combination on reactive oxygen species, antioxidant enzymes and cytokines in a series of advanced cancer patients at different sites: correlation with disease progression. Free Radic Res 37:213–23CrossRefPubMedGoogle Scholar
  8. 8.
    Mantovani G, Maccio A, Madeddu C et al (2003) Reactive oxygen species, antioxidant mechanisms, and serum cytokines levels in cancer patients: impact of an antioxidant treatment. J Environ Pathol Toxicol Oncol 22:17–8CrossRefPubMedGoogle Scholar
  9. 9.
    Bludovska M, Kotyzova D, Koutensky J, Eybl V (1999) The influence of α-lipoic acid on the toxicity of cadmium. Gen Physiol Biophys 18:28–2PubMedGoogle Scholar
  10. 10.
    Bustamante J, Lodge JK, Marcocci L, Tritscheler HJ, Packer L, Rihn BH (1998) α-lipoic acid in liver metabolism and disease. Free Radic Biol Med 24:1023–039CrossRefPubMedGoogle Scholar
  11. 11.
    Marley R, Holt S, Fernando B et al (1999) Lipoic acid prevents development of hyperdynamic circulation in anesthetized rats with biliary cirrhosis. Hepatology 29:1358–363CrossRefPubMedGoogle Scholar
  12. 12.
    Biewenga GP, Haenen GR, Bast A (1997) The pharmacology of the antioxidant lipoic acid. Gen Pharmacol 29:315–31PubMedGoogle Scholar
  13. 13.
    Packer L, Roy S, Sen CK (1997) α lipoic acid: a metabolic antioxidant and potential redox modulator of transcription. Adv Pharmacol 38:79–01CrossRefPubMedGoogle Scholar
  14. 14.
    Moini H, Packer L, Saris NEL (2002) Antioxidant and pro-oxidant activities of a-lipoic acid and dihydrolipoic acid. Toxicol Appl Pharmacol 182:84–0CrossRefPubMedGoogle Scholar
  15. 15.
    Kuo P-L, Chang LC, Lin C-C (2002) Resveratrol-induced apoptosis is mediated by p53-dependent pathway in HepG2 cells. Life Sci 72:23–4CrossRefPubMedGoogle Scholar
  16. 16.
    Rao C, Riven V, Simi AB, Reddy BS (1995) Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound. Cancer Res 55:259–66PubMedGoogle Scholar
  17. 17.
    Li M, Zhang Z, Hill DL, Chen X, Wang H, Zhang R (2005) Genistein, a dietary isoflavone, down-regulates the MDM2 oncogene at both transcriptional and posttranslational levels. Cancer Res 65:8200–208CrossRefPubMedGoogle Scholar
  18. 18.
    Wenzel U, Nickel A, Daniel H (2005) α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant O- 2 generation. Apoptosis 10:359–68CrossRefPubMedGoogle Scholar
  19. 19.
    Sen CK, Roy S, Packer L (1999) Fas mediated apoptosis of human Jurkat T-cells: intracellular events and potentiation by redox-active alpha-lipoic acid. Cell Death Differ 6:481–91CrossRefPubMedGoogle Scholar
  20. 20.
    Van De Mark K, Chen J, Steliou K, Perrine SP, Faller D (2003) α-lipoic acid induces p27kip1-dependent cell cycle arrest in non transformed cell lines and apoptosis in tumor cell lines. J Cell Physiol 194:325–40CrossRefPubMedGoogle Scholar
  21. 21.
    Piotrowski P, Wierzbicka K, Smialek M (2001) Neuronal death in the rat hippocampus in experimental diabetes and cerebral ischaemia treated with antioxidants. Folia Neuropathol 39:147–54.PubMedGoogle Scholar
  22. 22.
    Pierce RH, Campbell JS, Stephenson AB et al (2000) Disruption of redox homeostasis in tumor necrosis factor-alpha-induced apoptosis in a murine hepatocyte cell line. Am J Pathol 157:221–36PubMedGoogle Scholar
  23. 23.
    Bosch FX (1997) In: Okuda K, Tabor E (eds) Liver cancer. Churchill Livingston, New York, pp 13–8Google Scholar
  24. 24.
    Ching YP, Wong CM, Chan SF et al (2003) Deleted in liver cancer (DLC) 2 encodes a RhoGAP protein with growth suppressor function and is underexpressed in hepatocellular carcinoma. J Biol Chem 278:10824–0830CrossRefPubMedGoogle Scholar
  25. 25.
    Borenfreund E, Puerner JA (1986) Cytotoxicity of metals, metal-metal and metal-chelator combinations assayed in vitro. Toxicology 39:121–34CrossRefPubMedGoogle Scholar
  26. 26.
    Karasavvas N, Zakeri Z (1999) Relationships of apoptotic signalling mediated by ceramide and TNF-a in U937 cells. Cell Death Differ 6:115–23CrossRefPubMedGoogle Scholar
  27. 27.
    Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–54CrossRefPubMedGoogle Scholar
  28. 28.
    Jiao H-L, Zhao BL (2002) Cytotoxic effect of peroxisomal proliferator fenofibrate on human HepG2 hepatoma cell line and relevant mechanisms. Toxicol Appl Pharm 185:172–79CrossRefGoogle Scholar
  29. 29.
    Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Ann Rev Biochem 68:383–24CrossRefPubMedGoogle Scholar
  30. 30.
    Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–75CrossRefPubMedGoogle Scholar
  31. 31.
    Van Gool L, Meyer R, Tobiasch E et al (1997) Overexpression of human poly(ADP-ribose)polymerase in transfected hamster cells leads to increased poly(ADP-ribosyl)ation and cellular sensitisation to gamma irradiation. Eur J Biochem 244:15–0CrossRefPubMedGoogle Scholar
  32. 32.
    Nosseri C, Coppola S, Ghibelli L (1994) Possibile involvement of poly(ADP-ribosyl)polymerase in triggering stress-induced apoptosis. Exp Cell Res 212:367–73CrossRefPubMedGoogle Scholar
  33. 33.
    Lazebnik YA, Kauffmann SH, Desnoyers S, Poirier GG, Earnshaw WC (1994) Cleavage of poly(ADP-ribose)polymerase by a proteinase with properties like ICE. Nature 371:6784–793CrossRefGoogle Scholar
  34. 34.
    Kim JW, Kim K, Kang K, Joe CO (2000) Inhibition of homodimerization of poly(ADP-ribose)polymerase by its C-terminal cleavage products produced during apoptosis. J Biol Chem 275:8121–125CrossRefPubMedGoogle Scholar
  35. 35.
    Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptotic gene, survivin, expressed in cancer and lymphoma. Nature Medicine 3:917–21CrossRefPubMedGoogle Scholar
  36. 36.
    Velculescu VE, Madden SL, Zhang L et al (1999) Analysis of human transcriptomes. Nat Genet 23:287–88CrossRefGoogle Scholar
  37. 37.
    Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N (1998) Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 58:5071–074PubMedGoogle Scholar
  38. 38.
    Sarela AI, Macadam RC, Farmery SM, Markham AF, Guillou PJ (2000) Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut 46:645–50CrossRefPubMedGoogle Scholar
  39. 39.
    Ito R, Asami S, Motohashi S et al (2005) Significance of survivin mRNA expression in prognosis of neuroblastoma. Biol Pharm Bull 28:565–68CrossRefPubMedGoogle Scholar
  40. 40.
    Chiodino C, Cesinaro AM, Ottani D et al (1999) Expression of the novel inhibitor of apoptosis survivin in normal and neoplastic skin. J Invest Dermatol 113:415–18CrossRefPubMedGoogle Scholar
  41. 41.
    Shinohara ET, Gonzalez A, Massion PP et al (2005) Nuclear survivin predicts recurrence and poor survival in patients with resected non small cell lung carcinoma. Cancer 103:1685–692CrossRefPubMedGoogle Scholar
  42. 42.
    Tanaka T, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N (2000) Expression of survivin and its relationship with apoptosis in breast carcinomas. Clin Cancer Res 6:127–34PubMedGoogle Scholar
  43. 43.
    Rodel C, Haas J, Groth A, Grabenbauer GG, Sauer R, Rodel F (2003) Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys 55:1341–347PubMedGoogle Scholar
  44. 44.
    Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132–136CrossRefPubMedGoogle Scholar
  45. 45.
    Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309–312CrossRefPubMedGoogle Scholar
  46. 46.
    Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Green DR (2004) Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303:1010–014CrossRefPubMedGoogle Scholar
  47. 47.
    Gao CF, Ren S, Zhang K et al (1998) Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis. Exp Cell Res 265:145–51Google Scholar
  48. 48.
    Nakano K, Vousden KH (2001) PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7:683–94CrossRefPubMedGoogle Scholar
  49. 49.
    Oda E, Ohki R, Murasawa H et al (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288:1053–058CrossRefPubMedGoogle Scholar
  50. 50.
    Hollander MC, Alamo I, Jackman J, Wang MG, McBride OW, Fornace AJ Jr (1993) Analysis of the mammalian gadd45 gene and its response to DNA damage. J Biol Chem 268:24385–4393PubMedGoogle Scholar
  51. 51.
    Myashita T, Reed JC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–99CrossRefGoogle Scholar
  52. 52.
    Thornborrow EC, Manfred JJ (2001) The tumor suppressor protein p53 requires a cofactor to activate transcriptionally the human Bax promoter. J Biol Chem 276:15598–5608CrossRefPubMedGoogle Scholar
  53. 53.
    Kennedy SG, Wagner AJ, Conzen SD et al (1997) The PI3 kinase/Akt signalling pathway delivers an antiapoptotic signal. Genes Dev 11:701–13CrossRefPubMedGoogle Scholar
  54. 54.
    Chen YR, Meyer CF, Tan TH (1996) Persistent activation of c-jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis. J Biol Chem 271:631–34CrossRefPubMedGoogle Scholar
  55. 55.
    Sarker KP, Bisswas KK, Yamakuchi M et al (2003) ASK1-p38 MAPK/JNK signalling cascade mediates anandamide-induced PC12 cell death. J Neurochem 85:50–1PubMedCrossRefGoogle Scholar
  56. 56.
    Bachelder RE, Ribick MJ, Marchetti A et al (1999) p53 inhibits alpha6 beta4 integrin survival signalling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol 147:1063–072CrossRefPubMedGoogle Scholar
  57. 57.
    Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunol Today 15:7–0CrossRefPubMedGoogle Scholar
  58. 58.
    Curtin JF, Donovan M, Cotter TG (2002) Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 265:49–2CrossRefPubMedGoogle Scholar
  59. 59.
    Aruoma OI, Halliwell B, Hoey BM, Butler J (1989) The antioxidant action of N-acetyl cysteine: its reaction with hydrogen peroxide, hydroxylic radical, superoxide and hydrochlorous acid. Free Radical Biol Med 6:127–31CrossRefGoogle Scholar
  60. 60.
    Meister A, Anderson ME, Hwang O (1986) Intracellular cysteine and glutathione delivery systems. J Am College Nutrit 5:137–51Google Scholar
  61. 61.
    Liebler DC (1993) The role of metabolism in the antioxidant function of Vitamin E. Critical Rev Toxicol 23:147–69CrossRefGoogle Scholar
  62. 62.
    Mayer M, Noble M (1994) N-acetyl-l-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro. Proc Natl Acad Sci USA 91:7496–500CrossRefPubMedGoogle Scholar
  63. 63.
    Teichert J, Tuemmers T, Achenbach H et al (2005) Pharmacokinetics of alpha-lipoic acid in subjects with severe kidney damage and end-stage renal disease. J Clin Pharmacol 45:313–28CrossRefPubMedGoogle Scholar
  64. 64.
    Ziegler D (2004) Thioctic acid for patients with symptomatic diabetic polyneuropathy: a critical review. Treat Endocrinol 3:173–89CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • G. Simbula
    • 1
  • A. Columbano
    • 1
  • G. M. Ledda-Columbano
    • 1
  • L. Sanna
    • 1
  • M. Deidda
    • 1
  • A. Diana
    • 2
  • M. Pibiri
    • 1
  1. 1.Department of ToxicologyOncology and Molecular Pathology UnitItaly
  2. 2.Department of CytomorphologyUniversity of CagliariItaly

Personalised recommendations