Apoptosis

, Volume 10, Issue 4, pp 777–786 | Cite as

Intracellular free iron and acidic pathways mediate TNF-induced death of rat hepatoma cells

  • R. Autelli
  • S. Crepaldi
  • D. De Stefanis
  • M. Parola
  • G. Bonelli
  • F. M. Baccino
Article

Rat hepatoma HTC cells are intrinsically resistant to various apoptosis-inducing agents. Strategies to induce death in hepatoma cells are needed and the present experimental study was aimed to investigate the sensitivity of HTC cells to TNF and to clarify the mechanisms of action of this cytokine. Cells were treated with TNF and death mechanisms characterized employing an integration of morphological and biochemical techniques. HTC cells, sensitized to TNF toxicity with cycloheximide, died in a caspase-independent apoptosis-like manner. Although we found no evidence for a direct involvement of lysosomal cathepsins, bafilomycin A1 and ammonium chloride significantly attenuated TNF toxicity. Also desferrioxamine mesylate, an iron chelator, partly protected the cells from TNF, while a complete protection was afforded by combining ammonium chloride and iron chelator. Moreover, HTC were protected from TNF also by lipophylic antioxidants and diphenylene iodonium chloride, a NADPH oxidase inhibitor. These data depict a novel mechanism of TNF-mediated cytotoxicity in HTC cells, in which the endo-lysosomal compartment, NADPH oxidase and an iron-mediated pro-oxidant status contribute in determining a caspase-independent, apoptosis-like cell death.

Keywords

apoptosis-like death hepatoma iron chelation lysosomes TNF 

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References

  1. 1.
    Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281: 1305–1308.PubMedGoogle Scholar
  2. 2.
    Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998; 17: 1675–1687.CrossRefPubMedGoogle Scholar
  3. 3.
    Leist M, Jaattela M. Four deaths and a funeral: From caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001; 2: 589–598.PubMedGoogle Scholar
  4. 4.
    Jones BE, Lo CR, Liu H, et al. Hepatocytes sensitized to tumor necrosis factor-alpha cytotoxicity undergo apoptosis through caspase-dependent and caspase-independent pathways. J Biol Chem 2000; 275: 705–712.CrossRefPubMedGoogle Scholar
  5. 5.
    Deas O, Dumont C, MacFarlane M, et al. Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes. J Immunol 1998; 161: 3375–3383.PubMedGoogle Scholar
  6. 6.
    Akerman P, Cote P, Yang SQ, et al. Antibodies to tumor necrosis factor-alpha inhibit liver regeneration after partial hepatectomy. Am J Physiol 1992; 263: G579–G585.PubMedGoogle Scholar
  7. 7.
    Bradham CA, Plumpe J, Manns MP, Brenner DA, Trautwein C. Mechanisms of hepatic toxicity. I. TNF-induced liver injury. Am J Physiol 1998; 275: G387–G392.PubMedGoogle Scholar
  8. 8.
    Czaja MJ, Weiner FR, Flanders KC, et al. In vitro and in vivo association of transforming growth factor-beta 1 with hepatic fibrosis. J Cell Biol 1989; 108: 2477–2482.CrossRefPubMedGoogle Scholar
  9. 9.
    McClain CJ, Cohen DA. Increased tumor necrosis factor production by monocytes in alcoholic hepatitis. Hepatology 1989; 9: 349–351.PubMedGoogle Scholar
  10. 10.
    Cao G, Kuriyama S, Du P, et al. Complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor alpha gene transfer. Gastroenterology 1997; 112: 501–510.CrossRefPubMedGoogle Scholar
  11. 11.
    Evans-Storms RB, Cidlowski JA. Dominant suppression of lymphocyte apoptosis by hepatoma cells. Exp Cell Res 1997; 230: 121–132.CrossRefPubMedGoogle Scholar
  12. 12.
    Ruiz-Vela A, Gonzalez de B, Martinez A. Implication of calpain in caspase activation during B cell clonal deletion. EMBO J 1999; 18: 4988–4998.CrossRefPubMedGoogle Scholar
  13. 13.
    Isidoro C, Demoz M, De Stefanis D, Baccino FM, Bonelli G. High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma. Invasion Metastasis 1995; 15: 116–124.PubMedGoogle Scholar
  14. 14.
    Kornberg A. Lactic dehydrogenase of muscle. Methods Enzymol 1955; 1: 441–443.CrossRefGoogle Scholar
  15. 15.
    Duriez PJ, Wong F, Dorovini-Zis K, Shahidi R, Karsan A. A1 functions at the mitochondria to delay endothelial apoptosis in response to tumor necrosis factor. J Biol Chem 2000; 275: 18099–18107.CrossRefPubMedGoogle Scholar
  16. 16.
    Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis. Cancer Res 1993; 53: 3976–3985.PubMedGoogle Scholar
  17. 17.
    Smiley ST, Reers M, Mottola-Hartshorn C, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 1991; 88: 3671–3675.PubMedGoogle Scholar
  18. 18.
    Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998; 60: 619–642.CrossRefPubMedGoogle Scholar
  19. 19.
    Canuto RA, Muzio G, Maggiora M, et al. Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro. Cell Death Differ 2003; 4: 224–232.CrossRefGoogle Scholar
  20. 20.
    Guicciardi ME, Deussing J, Miyoshi H, et al. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 2000; 106: 1127–1137.PubMedGoogle Scholar
  21. 21.
    Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J 2001; 15: 2286–2287.PubMedGoogle Scholar
  22. 22.
    Deiss LP, Galinka H, Berissi H, Cohen O, Kimchi A. Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J 1996; 15: 3861–3870.PubMedGoogle Scholar
  23. 23.
    Tessitore L, Bonelli G, Cecchini G, Amenta JS, Baccino FM. Regulation of protein turnover versus growth state: Ascites hepatoma as a model for studies both in the animal and in vitro. Arch Biochem Biophys 1987; 255: 372–384.CrossRefPubMedGoogle Scholar
  24. 24.
    Deng J, Rudick V, Dory L. Lysosomal degradation and sorting of apolipoprotein E in macrophages. J Lipid Res 1995; 36: 2129–2140.PubMedGoogle Scholar
  25. 25.
    Radisky DC, Kaplan J. Iron in cytosolic ferritin can be recycled through lysosomal degradation in human fibroblasts. Biochem J 1998; 336: 201–205.PubMedGoogle Scholar
  26. 26.
    Breuer W, Epsztejn S, Cabantchik ZI. Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II). J Biol Chem 1995; 270: 24209–24215.CrossRefPubMedGoogle Scholar
  27. 27.
    Sakaida I, Kyle ME, Farber JL. Autophagic degradation of protein generates a pool of ferric iron required for the killing of cultured hepatocytes by an oxidative stress. Mol Pharmacol 1990; 37: 435–442.PubMedGoogle Scholar
  28. 28.
    Yu Z, Persson HL, Eaton JW, Brunk UT. Intralysosomal iron: A major determinant of oxidant-induced cell death. Free Radic Biol Med 2003; 34: 1243–1252.CrossRefPubMedGoogle Scholar
  29. 29.
    Garner B, Roberg K, Brunk UT. Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress. Free Radic Res 1998; 29: 103–114.PubMedGoogle Scholar
  30. 30.
    Rauen U, Kerkweg U, Weisheit D, Petrat F, Sustmann R, de Groot H. Cold-induced apoptosis of hepatocytes: Mitochondrial permeability transition triggered by nonmitochondrial chelatable iron. Free Radic Biol Med 2003; 35: 1664–1678.CrossRefPubMedGoogle Scholar
  31. 31.
    Talley AK, Dewhurst S, Perry SW, et al. Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: Protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA. Mol Cell Biol 1995; 15: 2359–2366.PubMedGoogle Scholar
  32. 32.
    Schulze-Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem 1992; 267: 5317–5323.PubMedGoogle Scholar
  33. 33.
    O’Donnell VB, Spycher S, Azzi A. Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: Role for lipoxygenase pathway but not mitochondrial respiratory chain. Biochem J 1995; 310: 133–141.PubMedGoogle Scholar
  34. 34.
    Zinck R, Cahill MA, Kracht M, Sachsenmaier C, Hipskind RA, Nordheim A. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1. Mol Cell Biol 1995; 15: 4930–4938.PubMedGoogle Scholar
  35. 35.
    Tang D, Lahti JM, Grenet J, Kidd VJ. Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism. J Biol Chem 1999; 274: 7245–7252.CrossRefPubMedGoogle Scholar
  36. 36.
    Kwok JC, Richardson DR. Examination of the mechanism(s) involved in doxorubicin-mediated iron accumulation in ferritin: Studies using metabolic inhibitors, protein synthesis inhibitors, and lysosomotropic agents. Mol Pharmacol 2004; 65: 181–195.CrossRefPubMedGoogle Scholar
  37. 37.
    Natoli G, Costanzo A, Guido F, et al. Nuclear factor kB-independent cytoprotective pathways originating at tumor necrosis factor receptor-associated factor 2. J Biol Chem 1998; 273: 31262–31272.CrossRefPubMedGoogle Scholar
  38. 38.
    DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M. A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB. Nature 1997; 388: 548–554.CrossRefPubMedGoogle Scholar
  39. 39.
    Wyllie AH, Golstein P. More than one way to go. Proc Natl Acad Sci USA 2001; 98: 11–13.CrossRefPubMedGoogle Scholar
  40. 40.
    Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol 1995; 146: 3–15.PubMedGoogle Scholar
  41. 41.
    Lockshin RA, Zakeri Z. Caspase-independent cell deaths. Curr Opin Cell Biol 2002; 14: 727–733.PubMedGoogle Scholar
  42. 42.
    De Duve C. The significance of lysosomes in pathology and medicine. Proc Inst Med Chic 1966; 26: 73–76.PubMedGoogle Scholar
  43. 43.
    Stoka V, Turk B, Schendel SL, et al. Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J Biol Chem 2001; 276: 3149–3157.CrossRefPubMedGoogle Scholar
  44. 44.
    Monney L, Olivier R, Otter I, Jansen B, Poirier GG, Borner C. Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis. Eur J Biochem 1998; 251: 295–303.CrossRefPubMedGoogle Scholar
  45. 45.
    Bidere N, Lorenzo HK, Carmona S, et al. Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem 2003; 278: 31401–31411.CrossRefPubMedGoogle Scholar
  46. 46.
    Jaattela M, Cande C, Kroemer G. Lysosomes and mitochondria in the commitment to apoptosis: A potential role for cathepsin D and AIF. Cell Death Differ 2004; 11: 135–136.CrossRefPubMedGoogle Scholar
  47. 47.
    Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM. Inhibition of autophagy abrogates tumour necrosis factor alpha induced apoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol 1997; 98: 673–685.CrossRefPubMedGoogle Scholar
  48. 48.
    Doulias PT, Christoforidis S, Brunk UT, Galaris D. Endosomal and lysosomal effects of desferrioxamine: Protection of HeLa cells from hydrogen peroxide-induced DNA damage and induction of cell-cycle arrest. Free Radic Biol Med 2003; 35: 719–728.CrossRefPubMedGoogle Scholar
  49. 49.
    Persson HL, Yu Z, Tirosh O, Eaton JW, Brunk UT. Prevention of oxidant-induced cell death by lysosomotropic iron chelators. Free Radic Biol Med 2003; 34: 1295–1305.CrossRefPubMedGoogle Scholar
  50. 50.
    Lloyd JB, Cable H, Rice-Evans C. Evidence that desferrioxamine cannot enter cells by passive diffusion. Biochem Pharmacol 1991; 41: 1361–1363.CrossRefPubMedGoogle Scholar
  51. 51.
    Persson HL, Nilsson KJ, Brunk UT. Novel cellular defenses against iron and oxidation: Ferritin and autophagocytosis preserve lysosomal stability in airway epithelium. Redox Rep 2001; 6: 57–63.CrossRefPubMedGoogle Scholar
  52. 52.
    Morel I, Lescoat G, Cillard J, Pasdeloup N, Brissot P, Cillard P. Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures. Involvement of free radicals. Biochem Pharmacol 1990; 39: 1647–1655.CrossRefPubMedGoogle Scholar
  53. 53.
    Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci USA 1995; 92: 8115–8119.PubMedGoogle Scholar
  54. 54.
    Chen CS. Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes. BMC Cell Biol 2002; 3: 21–32.CrossRefPubMedGoogle Scholar
  55. 55.
    Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J 1993; 12: 3095–3104.PubMedGoogle Scholar
  56. 56.
    Jacobson MD. Reactive oxygen species and programmed cell death. Trends Biochem Sci 1996; 21: 83–86.CrossRefPubMedGoogle Scholar
  57. 57.
    Majander A, Finel M, Wikstrom M. Diphenyleneiodonium inhibits reduction of iron-sulfur clusters in the mitochondrial NADH-ubiquinone oxidoreductase (Complex I). J Biol Chem 1994; 269: 21037–21042.PubMedGoogle Scholar
  58. 58.
    Frey RS, Rahman A, Kefer JC, Minshall RD, Malik AB. PKCzeta regulates TNF-alpha-induced activation of NADPH oxidase in endothelial cells. Circ Res 2002; 90: 1012–1019.CrossRefPubMedGoogle Scholar
  59. 59.
    Liao F, Andalibi A, deBeer FC, Fogelman AM, Lusis AJ. Genetic control of inflammatory gene induction and NF-kappa B-like transcription factor activation in response to an atherogenic diet in mice. J Clin Invest 1993; 91: 2572–2579.PubMedGoogle Scholar
  60. 60.
    Bowie AG, Moynagh PN, O’Neill LA. Lipid peroxidation is involved in the activation of NF-kappaB by tumor necrosis factor but not interleukin-1 in the human endothelial cell line ECV304. Lack of involvement of H2O2 in NF-kappaB activation by either cytokine in both primary and transformed endothelial cells. J Biol Chem 1997; 272: 25941–25950.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • R. Autelli
    • 1
    • 2
  • S. Crepaldi
    • 1
  • D. De Stefanis
    • 1
  • M. Parola
    • 1
  • G. Bonelli
    • 1
  • F. M. Baccino
    • 1
  1. 1.Department of Experimental Medicine and OncologyUniversity of TurinTurinItaly
  2. 2.Dipartimento di Medicina ed Oncologia SperimentaleSezione Patologia GeneraleTorinoItaly

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