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Inhibition of ErbB2 by Herceptin reduces viability and survival, induces apoptosis and oxidative stress in Calu-3 cell line

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Abstract

Human epidermal growth factor receptor 2 (ErbB2) amplification and overexpression has been seen in many cancer types including non-small cell lung cancer (NSCLC). Thus, ErbB2 is an important target for cancer therapies. Increased ErbB2 expression has been associated with drug resistance in cancer cells. Herceptin is a humanized monoclonal antibody that targets the extracellular domain of ErbB2. In this study, we aimed to block ErbB2 signaling with Herceptin and assess cytotoxicity and effects on apoptosis, oxidative stress, nuclear factor kappa-B (NF-kB), and Survivin expression in Calu-3 cell line. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay were used to assess cell viability as a marker of proliferation. Acridine orange/ethidium bromide (AO/EB) staining and caspase 3/7 activity were measured as the markers of apoptosis. The relative expressions of NF-kB-p50 and Survivin mRNAs were evaluated. Activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), and the levels of glutathione (GSH) and reactive oxygen species (ROS) were determined in a time- and dose-dependent manner. Our results show that Herceptin treatment inhibits cell proliferation and activates apoptosis but without effects on Survivin and NF-kB expression in Calu-3 cell line. Intracellular glutathione levels and SOD and CAT activities were decreased in a time- and dose-dependent manner associated with oxidative stress. Also, ROS were increased at 24 h. These results provide evidence that Herceptin can be used as a cytotoxic and apoptotic agent in NSCLC.

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References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer statistics. CA Cancer J Clin 58:71–96

    Article  PubMed  Google Scholar 

  2. Travis WD (2002) Pathology of lung cancer. Clin Chest Med 23:65–81

    Article  PubMed  Google Scholar 

  3. Lee SM (2006) Is EGFR expression important in non-small cell lung cancer? Thorax 61:98–99

    Article  CAS  PubMed  Google Scholar 

  4. Kruser TJ, Wheeler DL (2010) Mechanisms of resistance to HER family targeting antibodies. Exp Cell Res 316:1083–1100

    Article  CAS  PubMed  Google Scholar 

  5. Rajkumar T, Gullick WJ (1994) The type I growth factor receptors in human breast cancer. Breast Cancer Res Treat 29:3–9

    Article  CAS  PubMed  Google Scholar 

  6. Lonardo F, Di Marco E, King CR, Pierce JH, Segatto O, Aaronson SA, Di Fiore PP (1990) The normal ErbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol 11:992–1003

    Google Scholar 

  7. Graus-Porta D, Beerli RR, Daly JM, Hynes NE (1997) ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16:1647–1655

    Article  CAS  PubMed  Google Scholar 

  8. Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus-Porta D, Ratzkin BJ, Seger R, Hynes NE, Yarden Y (1996) ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 15:254–264

    CAS  PubMed  Google Scholar 

  9. Wang SC, Hung MC (2001) HER2 overexpression and cancer targeting. Semin Oncol 28(5 Suppl 16):115–224

    Article  CAS  PubMed  Google Scholar 

  10. Pauletti G, Godolphin W, Press MF et al (1996) Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene 13:63–72

    CAS  PubMed  Google Scholar 

  11. Hynes NE, Stern DF (1994) The biology of ErbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1198:165–184

    PubMed  Google Scholar 

  12. Nakamura H, Saji H, Ogata A, Hosaka M, Hagiwara M, Kawasaki N, Kato H (2003) Correlation between encoded protein overexpression and copy number of the HER2 gene with survival in non-small cell lung cancer. Int J Cancer 103:61–66

    Article  CAS  PubMed  Google Scholar 

  13. Swanton C, Futreal A, Eisen T (2006) Her2-targeted therapies in non-small cell lung cancer. Clin Cancer Res 12:4377–4383

    Article  Google Scholar 

  14. Goldenberg MM (1999) Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin Ther 21:309–318

    Article  CAS  PubMed  Google Scholar 

  15. Pietras RJ, Pegram MD, Finn RS, Maneval DA, Slamon DJ (1998) Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 17:2235–2249

    Article  CAS  PubMed  Google Scholar 

  16. Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J (1999) Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 58:2825–2831

    Google Scholar 

  17. Altundag K, Altundag O, Morandi P, Gunduz M (2005) Targeted therapy for targeted patients: trastuzumab in adjuvant treatment of non-small-cell lung cancer. J Clin Oncol 23:1325 (author reply 1326–1327)

    Google Scholar 

  18. Kalemkerian G (2005) Trastuzumab in the treatment of advanced non-small-cell lung cancer: is there a role? J Clin Oncol 23:1325–1326 (author reply 1326–1327)

    Google Scholar 

  19. Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, Hortobagyi GN, Hung MC, Yu D (2004) PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6:117–127

    Article  CAS  PubMed  Google Scholar 

  20. Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 3:917–921

    Article  CAS  PubMed  Google Scholar 

  21. Monzó M, Rosell R, Felip E, Astudillo J, Sánchez JJ, Maestre J, Martín C, Font A, Barnadas A, Abad A (1999) A novel anti-apoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol 17:2100–2104

    PubMed  Google Scholar 

  22. Ikehara M, Oshita F, Kameda Y, Ito H, Ohgane N, Suzuki R, Saito H, Yamada K, Noda K, Mitsuda A (2002) Expression of survivin correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep 9:835–838

    CAS  PubMed  Google Scholar 

  23. Falleni M, Pellegrini C, Marchetti A, Oprandi B, Buttitta F, Barassi F, Santambrogio L, Coggi G, Bosari S (2003) Survivin gene expression in early-stage non-small cell lung cancer. J Pathol 200:620–626

    Article  CAS  PubMed  Google Scholar 

  24. Hofmann HS, Simm A, Hammer A, Silber RE, Bartling B (2002) Expression of inhibitors of apoptosis (IAP) proteins in non-small cell human lung cancer. J Cancer Res Clin Oncol 128:554–560

    Article  CAS  PubMed  Google Scholar 

  25. Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK, Oh BH (2001) An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 40:1117–1123

    Article  CAS  PubMed  Google Scholar 

  26. Altieri DC (2003) Validating survivin as a cancer therapeutic target. Nat Rev Cancer 3:46–54

    Article  CAS  PubMed  Google Scholar 

  27. Tracey L, Pérez-Rosado A, Artiga MJ, Camacho FI, Rodríguez A, Martínez N, Ruiz-Ballesteros E, Mollejo M, Martinez B, Cuadros M, Garcia JF, Lawler M, Piris MA (2005) Expression of the NF-kappaB targets BCL2 and BIRC5/Survivin characterizes small B-cell and aggressive B cell lymphomas, respectively. J Pathol 206:123–134

    Article  CAS  PubMed  Google Scholar 

  28. Angileri FF, Aguennouz M, Conti A, La Torre D, Cardali S, Crupi R, Tomasello C, Germanò A, Vita G, Tomasello F (2008) Nuclear factor-kappaB activation and differential expression of survivin and Bcl-2 in human grade 2–4 astrocytomas. Cancer 112:2258–2266

    Article  CAS  PubMed  Google Scholar 

  29. Shishodia S, Aggarwal BB (2004) Nuclear factor-kappaB activation mediates cellular transformation, proliferation, invasion angiogenesis and metastasis of cancer. Cancer Treat Res 119:139–173

    Article  CAS  PubMed  Google Scholar 

  30. Aggarwal BB (2004) Nuclear factor-kappaB: the enemy within. Cancer Cell 6:203–208

    Article  CAS  PubMed  Google Scholar 

  31. Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J (1998) Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J Exp Med 188:211–216

    Article  CAS  PubMed  Google Scholar 

  32. Clutton S (1997) The importance of oxidative stress in apoptosis. Br Med Bull 53:662–668

    CAS  PubMed  Google Scholar 

  33. Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14:1511–1527

    Article  CAS  PubMed  Google Scholar 

  34. Temple MD, Perrone GG, Dawes IW (2005) Complex cellular responses to reactive oxygen species. Trends Cell Biol 15:319–326

    Article  CAS  PubMed  Google Scholar 

  35. Ribble D, Goldstein NB, Norris DA, Shellman YG (2005) A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol 5:12–19

    Article  PubMed  Google Scholar 

  36. Herderson LB, Cheppell B (1993) Dihydrorhodamine 123: a fluorescent probe for superoxide generation? Eur J Biochem 217:973–980

    Article  Google Scholar 

  37. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

    Article  CAS  PubMed  Google Scholar 

  38. Sun Y, Oberley LW, Li YA (1998) A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500

    Google Scholar 

  39. Anderson ME (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548–555

    Article  CAS  PubMed  Google Scholar 

  40. Marmor MD, Skaria KB, Yarden Y (2004) Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys 58:903–913

    Article  CAS  PubMed  Google Scholar 

  41. Asanuma H, Torigoe T, Kamiguchi K, Hirohashi Y, Ohmura T, Hirata K, Sato M, Sato N (2005) Survivin expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells. Cancer Res 65:11018–11025

    Article  CAS  PubMed  Google Scholar 

  42. Nahta R, Esteva FJ (2006) HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Res 8:215

    Article  PubMed  Google Scholar 

  43. Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL (2002) Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 62:4132–4141

    CAS  PubMed  Google Scholar 

  44. Zhu H, Zhang G, Wang Y, Xu N, He S, Zhang W, Chen M, Liu M, Quan L, Bai J, Xu N (2010) Inhibition of ErbB2 by Herceptin reduces survivin expression via the ErbB2-beta-catenin/TCF4-survivin pathway in ErbB2-overexpressed breast cancer cells. Cancer Sci 101(5):1156–1162

    Article  CAS  PubMed  Google Scholar 

  45. Longva KE, Pedersen NM, Haslekås C, Stang E, Madshus IH (2005) Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down-regulation of ErbB2. Int J Cancer 116:359–367

    Article  CAS  PubMed  Google Scholar 

  46. 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 7:38–47

    Article  CAS  PubMed  Google Scholar 

  47. Bunn PA Jr, Helfrich B, Soriano AF, Franklin WA, Varella-Garcia M, Hirsch FR, Baron A, Zeng C, Chan DC (2001) Expression of Her-2/neu in human lung cancer cell lines by immunohistochemistry and fluorescence in situ hybridization and its relationship to in vitro cytotoxicity by trastuzumab and chemotherapeutic agents. Clin Cancer Res 7:3239–3250

    CAS  PubMed  Google Scholar 

  48. O’Donovan N, Byrne AT, O’Connor AE, McGee S, Gallagher WM, Crown J (2010) Synergistic interaction between trastuzumab and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells. Invest New Drugs. doi.10.1007/s10637-010-9415-5

  49. Henson ES, Hu X, Gibson SB (2006) Herceptin sensitizes ErbB2-overexpressing cells to apoptosis by reducing antiapoptotic Mcl-1 expression. Clin Cancer Res 12:845–853

    Article  CAS  PubMed  Google Scholar 

  50. Kim EM, Lobocki C, Dubay L, Mittal VK (2009) A specific vascular endothelial growth factor receptor tyrosine kinase inhibitor enhances the antiproliferative effect of trastuzumab in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines. Am J Surg 197:331–336

    Article  CAS  PubMed  Google Scholar 

  51. Bijman MN, van Berkel MP, Kok M, Janmaat ML, Boven E (2009) Inhibition of functional HER family members increases the sensitivity to docetaxel in human ovarian cancer cell lines. Anticancer Drugs 20:450–460

    Article  CAS  PubMed  Google Scholar 

  52. Xia W, Bisi J, Strum J, Liu L, Carrick K, Graham KM, Treece AL, Hardwicke MA, Dush M, Liao Q, Westlund RE, Zhao S, Bacus S, Spector NL (2006) Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers. Cancer Res 66:1640–1647

    Article  CAS  PubMed  Google Scholar 

  53. Guo G, Wang T, Gao Q, Tamae D, Wong P, Chen T, Chen WC, Shively JE, Wong JY, Li JJ (2004) Expression of ErbB2 enhances radiation-induced NF-kappaB activation. Oncogene 23:535–545

    Article  CAS  PubMed  Google Scholar 

  54. Doroshow JH, Davies KJ (1986) Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical. J Biol Chem 26:3068–3074

    Google Scholar 

  55. Sawyer DB, Fukazawa R, Arstall MA, Kelly RA (1999) Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res 84:257–265

    CAS  PubMed  Google Scholar 

  56. Pentassuglia L, Timolati F, Seifriz F, Abudukadier K, Suter TM, Zuppinger C (2007) Inhibition of ErbB2/neuregulin signaling augments paclitaxel-induced cardiotoxicity in adult ventricular myocytes. Exp Cell Res 313:1588–1601

    Article  CAS  PubMed  Google Scholar 

  57. de Azambuja E, Bedard PL, Suter T, Piccart-Gebhart M (2009) Cardiac toxicity with anti-HER-2 therapies: what have we learned so far? Target Oncol 4:77–88

    Article  PubMed  Google Scholar 

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Acknowledgment

This study was supported by Gazi University Research Fund as a project with code number 01/2008-05.

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Authors and Affiliations

  1. Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, Besevler, 06500, Ankara, Turkey

    Irem Dogan & Abdullah Ekmekci

  2. Department of Medical Biochemistry, Faculty of Medicine, Gazi University, Besevler, Ankara, Turkey

    Ahmet Cumaoglu & Aysel Aricioglu

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  1. Irem Dogan
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  2. Ahmet Cumaoglu
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Correspondence to Irem Dogan.

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Dogan, I., Cumaoglu, A., Aricioglu, A. et al. Inhibition of ErbB2 by Herceptin reduces viability and survival, induces apoptosis and oxidative stress in Calu-3 cell line. Mol Cell Biochem 347, 41–51 (2011). https://doi.org/10.1007/s11010-010-0610-7

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