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. 2002 Nov 1;367(Pt 3):729–740. doi: 10.1042/BJ20020752

Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide.

Suwei Wang 1, Srigiridhar Kotamraju 1, Eugene Konorev 1, Shasi Kalivendi 1, Joy Joseph 1, Balaraman Kalyanaraman 1
PMCID: PMC1222928  PMID: 12139490

Abstract

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor kappaB (NF-kappaB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-kappaB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-kappaB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H(2)O(2), significantly decreased DOX-induced NF-kappaB activation and apoptosis. Inhibition of DOX-induced NF-kappaB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-kappaB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IkappaB mutant vector, another NF-kappaB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-kappaB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-kappaB activation is anti-apoptotic. Removal of intracellular H(2)O(2) protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-kappaB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.

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Selected References

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  1. Aggarwal B. B. Apoptosis and nuclear factor-kappa B: a tale of association and dissociation. Biochem Pharmacol. 2000 Oct 15;60(8):1033–1039. doi: 10.1016/s0006-2952(00)00393-2. [DOI] [PubMed] [Google Scholar]
  2. Aoki M., Nata T., Morishita R., Matsushita H., Nakagami H., Yamamoto K., Yamazaki K., Nakabayashi M., Ogihara T., Kaneda Y. Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension. 2001 Jul;38(1):48–55. doi: 10.1161/01.hyp.38.1.48. [DOI] [PubMed] [Google Scholar]
  3. Arlt A., Vorndamm J., Breitenbroich M., Fölsch U. R., Kalthoff H., Schmidt W. E., Schäfer H. Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin. Oncogene. 2001 Feb 15;20(7):859–868. doi: 10.1038/sj.onc.1204168. [DOI] [PubMed] [Google Scholar]
  4. Baeuerle P. A., Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol. 1994;12:141–179. doi: 10.1146/annurev.iy.12.040194.001041. [DOI] [PubMed] [Google Scholar]
  5. Bian X., McAllister-Lucas L. M., Shao F., Schumacher K. R., Feng Z., Porter A. G., Castle V. P., Opipari A. W., Jr NF-kappa B activation mediates doxorubicin-induced cell death in N-type neuroblastoma cells. J Biol Chem. 2001 Oct 25;276(52):48921–48929. doi: 10.1074/jbc.M108674200. [DOI] [PubMed] [Google Scholar]
  6. Boland M. P., Foster S. J., O'Neill L. A. Daunorubicin activates NFkappaB and induces kappaB-dependent gene expression in HL-60 promyelocytic and Jurkat T lymphoma cells. J Biol Chem. 1997 May 16;272(20):12952–12960. doi: 10.1074/jbc.272.20.12952. [DOI] [PubMed] [Google Scholar]
  7. Buzdar A. U., Marcus C., Smith T. L., Blumenschein G. R. Early and delayed clinical cardiotoxicity of doxorubicin. Cancer. 1985 Jun 15;55(12):2761–2765. doi: 10.1002/1097-0142(19850615)55:12<2761::aid-cncr2820551206>3.0.co;2-p. [DOI] [PubMed] [Google Scholar]
  8. Chan H., Bartos D. P., Owen-Schaub L. B. Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment. Mol Cell Biol. 1999 Mar;19(3):2098–2108. doi: 10.1128/mcb.19.3.2098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen F., Castranova V., Shi X., Demers L. M. New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clin Chem. 1999 Jan;45(1):7–17. [PubMed] [Google Scholar]
  10. Crow J. P. Manganese and iron porphyrins catalyze peroxynitrite decomposition and simultaneously increase nitration and oxidant yield: implications for their use as peroxynitrite scavengers in vivo. Arch Biochem Biophys. 1999 Nov 1;371(1):41–52. doi: 10.1006/abbi.1999.1414. [DOI] [PubMed] [Google Scholar]
  11. Davies K. J., Doroshow J. H. Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase. J Biol Chem. 1986 Mar 5;261(7):3060–3067. [PubMed] [Google Scholar]
  12. Day B. J., Shawen S., Liochev S. I., Crapo J. D. A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro. J Pharmacol Exp Ther. 1995 Dec;275(3):1227–1232. [PubMed] [Google Scholar]
  13. Gavrieli Y., Sherman Y., Ben-Sasson S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992 Nov;119(3):493–501. doi: 10.1083/jcb.119.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Golstein P. Controlling cell death. Science. 1997 Feb 21;275(5303):1081–1082. doi: 10.1126/science.275.5303.1081. [DOI] [PubMed] [Google Scholar]
  15. Green D. R., Reed J. C. Mitochondria and apoptosis. Science. 1998 Aug 28;281(5381):1309–1312. doi: 10.1126/science.281.5381.1309. [DOI] [PubMed] [Google Scholar]
  16. Heermeier K., Leicht W., Palmetshofer A., Ullrich M., Wanner C., Galle J. Oxidized LDL suppresses NF-kappaB and overcomes protection from apoptosis in activated endothelial cells. J Am Soc Nephrol. 2001 Mar;12(3):456–463. doi: 10.1681/ASN.V123456. [DOI] [PubMed] [Google Scholar]
  17. Hettmann T., DiDonato J., Karin M., Leiden J. M. An essential role for nuclear factor kappaB in promoting double positive thymocyte apoptosis. J Exp Med. 1999 Jan 4;189(1):145–158. doi: 10.1084/jem.189.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hsu S. C., Gavrilin M. A., Lee H. H., Wu C. C., Han S. H., Lai M. Z. NF-kappa B-dependent Fas ligand expression. Eur J Immunol. 1999 Sep;29(9):2948–2956. doi: 10.1002/(SICI)1521-4141(199909)29:09<2948::AID-IMMU2948>3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]
  19. Ji C., Kozak K. R., Marnett L. J. IkappaB kinase, a molecular target for inhibition by 4-hydroxy-2-nonenal. J Biol Chem. 2001 Mar 16;276(21):18223–18228. doi: 10.1074/jbc.M101266200. [DOI] [PubMed] [Google Scholar]
  20. Joyce D., Bouzahzah B., Fu M., Albanese C., D'Amico M., Steer J., Klein J. U., Lee R. J., Segall J. E., Westwick J. K. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J Biol Chem. 1999 Sep 3;274(36):25245–25249. doi: 10.1074/jbc.274.36.25245. [DOI] [PubMed] [Google Scholar]
  21. Kalish H., Camp J. E., Stepień M., Latos-Grazyński L., Balch A. L. Reactivity of mono-meso-substituted iron(II) octaethylporphyrin complexes with hydrogen peroxide in the absence of dioxygen. Evidence for nucleophilic attack on the heme. J Am Chem Soc. 2001 Nov 28;123(47):11719–11727. doi: 10.1021/ja011545b. [DOI] [PubMed] [Google Scholar]
  22. Kalivendi S. V., Kotamraju S., Zhao H., Joseph J., Kalyanaraman B. Doxorubicin-induced apoptosis is associated with increased transcription of endothelial nitric-oxide synthase. Effect of antiapoptotic antioxidants and calcium. J Biol Chem. 2001 Sep 28;276(50):47266–47276. doi: 10.1074/jbc.M106829200. [DOI] [PubMed] [Google Scholar]
  23. Kalyanaraman B., Perez-Reyes E., Mason R. P. Spin-trapping and direct electron spin resonance investigations of the redox metabolism of quinone anticancer drugs. Biochim Biophys Acta. 1980 Jun 5;630(1):119–130. doi: 10.1016/0304-4165(80)90142-7. [DOI] [PubMed] [Google Scholar]
  24. Konorev E. A., Zhang H., Joseph J., Kennedy M. C., Kalyanaraman B. Bicarbonate exacerbates oxidative injury induced by antitumor antibiotic doxorubicin in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2000 Nov;279(5):H2424–H2430. doi: 10.1152/ajpheart.2000.279.5.H2424. [DOI] [PubMed] [Google Scholar]
  25. Kotamraju S., Konorev E. A., Joseph J., Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem. 2000 Oct 27;275(43):33585–33592. doi: 10.1074/jbc.M003890200. [DOI] [PubMed] [Google Scholar]
  26. Laurent G., Jaffrézou J. P. Signaling pathways activated by daunorubicin. Blood. 2001 Aug 15;98(4):913–924. doi: 10.1182/blood.v98.4.913. [DOI] [PubMed] [Google Scholar]
  27. Li Q., Sanlioglu S., Li S., Ritchie T., Oberley L., Engelhardt J. F. GPx-1 gene delivery modulates NFkappaB activation following diverse environmental injuries through a specific subunit of the IKK complex. Antioxid Redox Signal. 2001 Jun;3(3):415–432. doi: 10.1089/15230860152409068. [DOI] [PubMed] [Google Scholar]
  28. Liu R. Y., Fan C., Olashaw N. E., Wang X., Zuckerman K. S. Tumor necrosis factor-alpha-induced proliferation of human Mo7e leukemic cells occurs via activation of nuclear factor kappaB transcription factor. J Biol Chem. 1999 May 14;274(20):13877–13885. doi: 10.1074/jbc.274.20.13877. [DOI] [PubMed] [Google Scholar]
  29. Manna S. K., Aggarwal B. B. Lipopolysaccharide inhibits TNF-induced apoptosis: role of nuclear factor-kappaB activation and reactive oxygen intermediates. J Immunol. 1999 Feb 1;162(3):1510–1518. [PubMed] [Google Scholar]
  30. Misko T. P., Highkin M. K., Veenhuizen A. W., Manning P. T., Stern M. K., Currie M. G., Salvemini D. Characterization of the cytoprotective action of peroxynitrite decomposition catalysts. J Biol Chem. 1998 Jun 19;273(25):15646–15653. doi: 10.1074/jbc.273.25.15646. [DOI] [PubMed] [Google Scholar]
  31. Moellering D., McAndrew J., Jo H., Darley-Usmar V. M. Effects of pyrrolidine dithiocarbamate on endothelial cells: protection against oxidative stress. Free Radic Biol Med. 1999 May;26(9-10):1138–1145. doi: 10.1016/s0891-5849(98)00300-1. [DOI] [PubMed] [Google Scholar]
  32. Myers C. E., McGuire W. P., Liss R. H., Ifrim I., Grotzinger K., Young R. C. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science. 1977 Jul 8;197(4299):165–167. doi: 10.1126/science.877547. [DOI] [PubMed] [Google Scholar]
  33. Nakamura T., Ueda Y., Juan Y., Katsuda S., Takahashi H., Koh E. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats: In vivo study. Circulation. 2000 Aug 1;102(5):572–578. doi: 10.1161/01.cir.102.5.572. [DOI] [PubMed] [Google Scholar]
  34. Nathens A. B., Bitar R., Davreux C., Bujard M., Marshall J. C., Dackiw A. P., Watson R. W., Rotstein O. D. Pyrrolidine dithiocarbamate attenuates endotoxin-induced acute lung injury. Am J Respir Cell Mol Biol. 1997 Nov;17(5):608–616. doi: 10.1165/ajrcmb.17.5.2661. [DOI] [PubMed] [Google Scholar]
  35. Nicholson D. W., Thornberry N. A. Caspases: killer proteases. Trends Biochem Sci. 1997 Aug;22(8):299–306. doi: 10.1016/s0968-0004(97)01085-2. [DOI] [PubMed] [Google Scholar]
  36. Pieper G. M., Olds C., Hilton G., Lindholm P. F., Adams M. B., Roza A. M. Antioxidant treatment inhibits activation of myocardial nuclear factor kappa B and inhibits nitrosylation of myocardial heme protein in cardiac transplant rejection. Antioxid Redox Signal. 2001 Feb;3(1):81–88. doi: 10.1089/152308601750100542. [DOI] [PubMed] [Google Scholar]
  37. Qin Z. H., Chen R. W., Wang Y., Nakai M., Chuang D. M., Chase T. N. Nuclear factor kappaB nuclear translocation upregulates c-Myc and p53 expression during NMDA receptor-mediated apoptosis in rat striatum. J Neurosci. 1999 May 15;19(10):4023–4033. doi: 10.1523/JNEUROSCI.19-10-04023.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rong Y., Doctrow S. R., Tocco G., Baudry M. EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology. Proc Natl Acad Sci U S A. 1999 Aug 17;96(17):9897–9902. doi: 10.1073/pnas.96.17.9897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Salvemini D., Wang Z. Q., Stern M. K., Currie M. G., Misko T. P. Peroxynitrite decomposition catalysts: therapeutics for peroxynitrite-mediated pathology. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2659–2663. doi: 10.1073/pnas.95.5.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sawyer D. B., Fukazawa R., Arstall M. A., Kelly R. A. Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res. 1999 Feb 19;84(3):257–265. doi: 10.1161/01.res.84.3.257. [DOI] [PubMed] [Google Scholar]
  41. Schmidt K. N., Amstad P., Cerutti P., Baeuerle P. A. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol. 1995 Jan;2(1):13–22. doi: 10.1016/1074-5521(95)90076-4. [DOI] [PubMed] [Google Scholar]
  42. Schreck R., Albermann K., Baeuerle P. A. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic Res Commun. 1992;17(4):221–237. doi: 10.3109/10715769209079515. [DOI] [PubMed] [Google Scholar]
  43. Shi X., Dong Z., Huang C., Ma W., Liu K., Ye J., Chen F., Leonard S. S., Ding M., Castranova V. The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB. Mol Cell Biochem. 1999 Apr;194(1-2):63–70. doi: 10.1023/a:1006904904514. [DOI] [PubMed] [Google Scholar]
  44. Singal P. K., Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med. 1998 Sep 24;339(13):900–905. doi: 10.1056/NEJM199809243391307. [DOI] [PubMed] [Google Scholar]
  45. Singal P. K., Li T., Kumar D., Danelisen I., Iliskovic N. Adriamycin-induced heart failure: mechanism and modulation. Mol Cell Biochem. 2000 Apr;207(1-2):77–86. doi: 10.1023/a:1007094214460. [DOI] [PubMed] [Google Scholar]
  46. Somerville L., Cory J. G. Enhanced roscovitine-induced apoptosis is mediated by a caspase-3-like activity in deoxyadenosine-resistant mouse leukemia L1210 cells. 2000 Sep- OctAnticancer Res. 20(5B):3347–3355. [PubMed] [Google Scholar]
  47. Wang L., Ma W., Markovich R., Chen J. W., Wang P. H. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res. 1998 Sep 7;83(5):516–522. doi: 10.1161/01.res.83.5.516. [DOI] [PubMed] [Google Scholar]
  48. Wang S., Leonard S. S., Castranova V., Vallyathan V., Shi X. The role of superoxide radical in TNF-alpha induced NF-kappaB activation. Ann Clin Lab Sci. 1999 Jul-Sep;29(3):192–199. [PubMed] [Google Scholar]
  49. Ye J., Ghosh P., Cippitelli M., Subleski J., Hardy K. J., Ortaldo J. R., Young H. A. Characterization of a silencer regulatory element in the human interferon-gamma promoter. J Biol Chem. 1994 Oct 14;269(41):25728–25734. [PubMed] [Google Scholar]

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