Breast Cancer Research and Treatment

, Volume 132, Issue 1, pp 109–119 | Cite as

ErbB1/2 tyrosine kinase inhibitor mediates oxidative stress-induced apoptosis in inflammatory breast cancer cells

  • Katherine M. Aird
  • Jennifer L. Allensworth
  • Ines Batinic-Haberle
  • H. Kim Lyerly
  • Mark W. Dewhirst
  • Gayathri R. DeviEmail author
Preclinical study


Overexpression of epidermal growth factor receptors (ErbB) is frequently seen in inflammatory breast cancer (IBC). Treatment with ErbB1/2-targeting agents (lapatinib) mediates tumor apoptosis by downregulating ErbB1/2 phosphorylation and downstream survival signaling. In this study, using carboxy-H2DCFDA, DHE, and MitoSOX Red to examine changes in hydrogen peroxide radicals, cytoplasmic and mitochondrial superoxide, respectively, we observed that GW583340 (a lapatinib-analog) increases reactive oxygen species (ROS) in two models of IBC (SUM149, SUM190) that are sensitive to ErbB1/2 blockade. This significant increase in ROS levels was similar to those generated by classical oxidative agents H2O2 and paraquat. In contrast, minimal to basal levels of ROS were measured in a clonal population of GW583340-resistant IBC cells (rSUM149 and rSUM190). The GW583340-resistant IBC cells displayed increased SOD1, SOD2, and glutathione expression, which correlated with decreased sensitivity to the apoptotic-inducing effects of GW583340, H2O2, and paraquat. The ROS increase and cell death in the GW583340-sensitive cells was reversed by simultaneous treatment with a superoxide dismutase (SOD) mimic. Additionally, overcoming the high levels of antioxidants using redox modulators induced apoptosis in the GW583340-resistant cells. Taken together, these data demonstrate a novel mechanism of lapatinib-analog-induced apoptosis and indicate that resistant cells have increased antioxidant potential, which can be overcome by treatment with SOD modulators.


Reactive oxygen species SUM149 SUM190 IBC XIAP Lapatinib Superoxide SOD Antioxidants Therapeutic resistance Redox modulators Caspases 



AMP activated protein kinase




Estrogen receptor


Inflammatory breast cancer


Internal ribosomal entry site


Nuclear factor kappa B


Progesterone receptor


Reactive oxygen species


Superoxide dismutase


X-linked inhibitor of apoptosis protein



The authors would like to thank Sharon Peplinski for her technical help with flow cytometry-based experiments and Dr. Neil Spector and Dr. Michael Morse for helpful discussions during the preparation of the manuscript. This work was supported by funding from American Cancer Society RSG-08-290-01-CCE (GRD), Department of Defense Predoctoral award, W81XWH-08-1-0363 (KMA), Viral Oncology Training Grant, 5T32-CA009111-32 (JLA) and SPORE in breast cancer grant (5P50-CA068438) at Duke Comprehensive Cancer Center.

Conflicts of interest

All authors state that there is no conflict of interest.


  1. 1.
    Anderson WF, Schairer C, Chen BE, Hance KW, Levine PH (2005) Epidemiology of inflammatory breast cancer (IBC). Breast Dis 22:9–23PubMedGoogle Scholar
  2. 2.
    Woodward WA, Cristofanilli M (2009) Inflammatory breast cancer. Semin Radiat Oncol 19:256–265PubMedCrossRefGoogle Scholar
  3. 3.
    Van den Eynden GG, Van der Auwera I, Van Laere S, Colpaert CG, van Dam P, Merajver S, Kleer CG, Harris AL, Van Marck EA, Dirix LY et al (2004) Validation of a tissue microarray to study differential protein expression in inflammatory and non-inflammatory breast cancer. Breast Cancer Res Treat 85:13–22PubMedCrossRefGoogle Scholar
  4. 4.
    Xia W, Mullin RJ, Keith BR, Liu LH, Ma H, Rusnak DW, Owens G, Alligood KJ, Spector NL (2002) Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/erbB2 and downstream Erk1/2 and AKT pathways. Oncogene 21:6255–6263PubMedCrossRefGoogle Scholar
  5. 5.
    Xia W, Bisi J, Strum J, Liu L, Carrick K, Graham KM, Treece AL, Hardwicke MA, Dush M, Liao Q et al (2006) Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers. Cancer Res 66:1640–1647PubMedCrossRefGoogle Scholar
  6. 6.
    Dai CL, Tiwari AK, Wu CP, Su XD, Wang SR, Liu DG, Ashby CR Jr, Huang Y, Robey RW, Liang YJ et al (2008) Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2. Cancer Res 68:7905–7914PubMedCrossRefGoogle Scholar
  7. 7.
    Polli JW, Humphreys JE, Harmon KA, Castellino S, O’Mara MJ, Olson KL, John-Williams LS, Koch KM, Serabjit-Singh CJ (2008) The role of efflux and uptake transporters in [N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions. Drug Metab Dispos 36:695–701PubMedCrossRefGoogle Scholar
  8. 8.
    Spector NL, Yarden Y, Smith B, Lyass L, Trusk P, Pry K, Hill JE, Xia W, Seger R, Bacus SS (2007) Activation of AMP-activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells. Proc Natl Acad Sci USA 104:10607–10612Google Scholar
  9. 9.
    Johnston S, Trudeau M, Kaufman B, Boussen H, Blackwell K, LoRusso P, Lombardi DP, Ben Ahmed S, Citrin DL, DeSilvio ML et al (2008) Phase II study of predictive biomarker profiles for response targeting human epidermal growth factor receptor 2 (HER-2) in advanced inflammatory breast cancer with lapatinib monotherapy. J Clin Oncol 26:1066–1072PubMedCrossRefGoogle Scholar
  10. 10.
    Chen FL, Xia W, Spector NL (2008) Acquired resistance to small molecule ErbB2 tyrosine kinase inhibitors. Clin Cancer Res 14:6730–6734PubMedCrossRefGoogle Scholar
  11. 11.
    Aird KM, Ghanayem RB, Peplinski S, Lyerly HK, Devi GR (2010) X-linked inhibitor of apoptosis protein inhibits apoptosis in inflammatory breast cancer cells with acquired resistance to an ErbB1/2 tyrosine kinase inhibitor. Mol Cancer Ther 9:1432–1442PubMedCrossRefGoogle Scholar
  12. 12.
    Xia W, Bacus S, Hegde P, Husain I, Strum J, Liu L, Paulazzo G, Lyass L, Trusk P, Hill J et al (2006) A model of acquired autoresistance to a potent ErbB2 tyrosine kinase inhibitor and a therapeutic strategy to prevent its onset in breast cancer. Proc Natl Acad Sci USA 103:7795–7800PubMedCrossRefGoogle Scholar
  13. 13.
    Martin AP, Miller A, Emad L, Rahmani M, Walker T, Mitchell C, Hagan MP, Park MA, Yacoub A, Fisher PB et al (2008) Lapatinib resistance in HCT116 cells is mediated by elevated MCL-1 expression and decreased BAK activation and not by ERBB receptor kinase mutation. Mol Pharmacol 74:807–822PubMedCrossRefGoogle Scholar
  14. 14.
    Xia W, Bacus S, Husain I, Liu L, Zhao S, Liu Z, Moseley MA 3rd, Thompson JW, Chen FL, Koch KM et al (2010) Resistance to ErbB2 tyrosine kinase inhibitors in breast cancer is mediated by calcium-dependent activation of RelA. Mol Cancer Ther 9:292–299PubMedCrossRefGoogle Scholar
  15. 15.
    Hardie DG (2004) The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 117:5479–5487PubMedCrossRefGoogle Scholar
  16. 16.
    Shell SA, Lyass L, Trusk PB, Pry KJ, Wappel RL, Bacus SS (2008) Activation of AMPK is necessary for killing cancer cells and sparing cardiac cells. Cell Cycle 7:1769–1775PubMedCrossRefGoogle Scholar
  17. 17.
    Holcik M, Lefebvre C, Yeh C, Chow T, Korneluk RG (1999) A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nat Cell Biol 1:190–192PubMedCrossRefGoogle Scholar
  18. 18.
    Ott M, Gogvadze V, Orrenius S, Zhivotovsky B (2007) Mitochondria, oxidative stress and cell death. Apoptosis 12:913–922PubMedCrossRefGoogle Scholar
  19. 19.
    Agostinelli E, Seiler N (2006) Non-irradiation-derived reactive oxygen species (ROS) and cancer: therapeutic implications. Amino Acids 31:341–355PubMedCrossRefGoogle Scholar
  20. 20.
    Choi SL, Kim SJ, Lee KT, Kim J, Mu J, Birnbaum MJ, Soo Kim S, Ha J (2001) The regulation of AMP-activated protein kinase by H(2)O(2). Biochem Biophys Res Commun 287:92–97PubMedCrossRefGoogle Scholar
  21. 21.
    Gordon LI, Burke MA, Singh AT, Prachand S, Lieberman ED, Sun L, Naik TJ, Prasad SV, Ardehali H (2009) Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathways. J Biol Chem 284:2080–2087PubMedCrossRefGoogle Scholar
  22. 22.
    Aird KM, Ding X, Baras A, Wei J, Morse MA, Clay T, Lyerly HK, Devi GR (2008) Trastuzumab signaling in ErbB2-overexpressing inflammatory breast cancer correlates with X-linked inhibitor of apoptosis protein expression. Mol Cancer Ther 7:38–47PubMedCrossRefGoogle Scholar
  23. 23.
    Amantana A, London CA, Iversen PL, Devi GR (2004) X-linked inhibitor of apoptosis protein inhibition induces apoptosis and enhances chemotherapy sensitivity in human prostate cancer cells. Mol Cancer Ther 3:699–707PubMedGoogle Scholar
  24. 24.
    Eruslanov E, Kusmartsev S (2010) Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol 594:57–72PubMedCrossRefGoogle Scholar
  25. 25.
    Elorza A, Hyde B, Mikkola HK, Collins S, Shirihai OS (2008) UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis. J Biol Chem 283:30461–30470PubMedCrossRefGoogle Scholar
  26. 26.
    Miao L, St Clair DK (2009) Regulation of superoxide dismutase genes: implications in disease. Free Radic Biol Med 47:344–356PubMedCrossRefGoogle Scholar
  27. 27.
    Balendiran GK, Dabur R, Fraser D (2004) The role of glutathione in cancer. Cell Biochem Funct 22:343–352PubMedCrossRefGoogle Scholar
  28. 28.
    Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W (2000) Superoxide dismutase as a target for the selective killing of cancer cells. Nature 407:390–395PubMedCrossRefGoogle Scholar
  29. 29.
    Trachootham D, Alexandre J, Huang P (2009) Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 8:579–591PubMedCrossRefGoogle Scholar
  30. 30.
    Batinic-Haberle I (2002) Manganese porphyrins and related compounds as mimics of superoxide dismutase. Methods Enzymol 349:223–233PubMedCrossRefGoogle Scholar
  31. 31.
    Fernandes AS, Gaspar J, Cabral MF, Rueff J, Castro M, Batinic-Haberle I, Costa J, Oliveira NG (2010) Protective role of ortho-substituted Mn(III) N-alkylpyridylporphyrins against the oxidative injury induced by tert-butylhydroperoxide. Free Radic Res 44:430–440PubMedCrossRefGoogle Scholar
  32. 32.
    Saba H, Batinic-Haberle I, Munusamy S, Mitchell T, Lichti C, Megyesi J, MacMillan-Crow LA (2007) Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion. Free Radic Biol Med 42:1571–1578PubMedCrossRefGoogle Scholar
  33. 33.
    Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, Chan KW, Ciceri P, Davis MI, Edeen PT et al (2008) A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 26:127–132PubMedCrossRefGoogle Scholar
  34. 34.
    Chinnaiyan AM, Prasad U, Shankar S, Hamstra DA, Shanaiah M, Chenevert TL, Ross BD, Rehemtulla A (2000) Combined effect of tumor necrosis factor-related apoptosis-inducing ligand and ionizing radiation in breast cancer therapy. Proc Natl Acad Sci USA 97:1754–1759PubMedCrossRefGoogle Scholar
  35. 35.
    Jones RG, Thompson CB (2009) Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev 23:537–548PubMedCrossRefGoogle Scholar
  36. 36.
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527PubMedCrossRefGoogle Scholar
  37. 37.
    Contreras CM, Gurumurthy S, Haynie JM, Shirley LJ, Akbay EA, Wingo SN, Schorge JO, Broaddus RR, Wong KK, Bardeesy N et al (2008) Loss of Lkb1 provokes highly invasive endometrial adenocarcinomas. Cancer Res 68:759–766PubMedCrossRefGoogle Scholar
  38. 38.
    Hardie DG (2007) AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol 47:185–210PubMedCrossRefGoogle Scholar
  39. 39.
    Batinic-Haberle I, Reboucas JS, Spasojevic I (2010) Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential. Antioxid Redox Signal 13:877–918Google Scholar
  40. 40.
    Zhao Y, Chaiswing L, Oberley TD, Batinic-Haberle I, St Clair W, Epstein CJ, St Clair D (2005) A mechanism-based antioxidant approach for the reduction of skin carcinogenesis. Cancer Res 65:1401–1405PubMedCrossRefGoogle Scholar
  41. 41.
    Batinic-Haberle I, Spasojevic I, Tse HM, Tovmasyan A, Rajic Z, St Clair DK, Vujaskovic Z, Dewhirst MW, Piganelli JD (2010) Design of Mn porphyrins for treating oxidative stress injuries and their redox-based regulation of cellular transcriptional activities. Amino Acids. doi: 10.1007/s00726-010-0603-6
  42. 42.
    Kong Q, Beel JA, Lillehei KO (2000) A threshold concept for cancer therapy. Med Hypotheses 55:29–35PubMedCrossRefGoogle Scholar
  43. 43.
    Roninson IB, Brown JM, Bredesen DE (2008) Beyond apoptosis: cellular outcomes of cancer therapy. Informa Healthcare, New YorkGoogle Scholar
  44. 44.
    Ladas EJ, Jacobson JS, Kennedy DD, Teel K, Fleischauer A, Kelly KM (2004) Antioxidants and cancer therapy: a systematic review. J Clin Oncol 22:517–528PubMedCrossRefGoogle Scholar
  45. 45.
    Halliwell B, Gutteridge JMC (1985) Free radicals in biology and medicine. Clarendon Press, Oxford University Press, Oxford, New YorkGoogle Scholar
  46. 46.
    Li S, Yan T, Yang JQ, Oberley TD, Oberley LW (2000) The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase. Cancer Res 60:3927–3939PubMedGoogle Scholar
  47. 47.
    Landriscina M, Maddalena F, Laudiero G, Esposito F (2009) Adaptation to oxidative stress, chemoresistance, and cell survival. Antioxid Redox Signal 11:2701–2716PubMedCrossRefGoogle Scholar
  48. 48.
    Khan G, Merajver S (2009) Copper chelation in cancer therapy using tetrathiomolybdate: an evolving paradigm. Expert Opin Investig Drugs 18:541–548PubMedCrossRefGoogle Scholar
  49. 49.
    Heikkila RE, Cabbat FS, Cohen G (1976) In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate. J Biol Chem 251:2182–2185PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Katherine M. Aird
    • 3
    • 5
  • Jennifer L. Allensworth
    • 3
  • Ines Batinic-Haberle
    • 4
  • H. Kim Lyerly
    • 1
    • 2
    • 3
  • Mark W. Dewhirst
    • 2
    • 3
    • 4
  • Gayathri R. Devi
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of SurgeryDuke University Medical CenterDurhamUSA
  2. 2.Duke Comprehensive Cancer CenterDuke University Medical CenterDurhamUSA
  3. 3.Department of PathologyDuke University Medical CenterDurhamUSA
  4. 4.Department of Radiation OncologyDuke University Medical CenterDurhamUSA
  5. 5.Fox Chase Cancer CenterPhiladelphiaUSA

Personalised recommendations