Tumor Biology

, Volume 36, Issue 3, pp 1871–1879 | Cite as

A549 cells adapted to high nitric oxide show reduced surface CEACAM expression and altered adhesion and migration properties

  • Madeeha Aqil
  • Kim M. Elseth
  • Ashok Arjunakani
  • Philip Nebres
  • Courtney P. Amegashie
  • Devang H. Thanki
  • Premal B. Desai
  • James A. Radosevich
Research Article


The migration and adhesion properties of tumors affect their metastatic rate. In the present study, we investigated carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1, 5, and 6 expression in high nitric oxide (HNO)-adapted lung cancer cells compared to parent cells. We observed high transcript levels of CEACAM 1 (4S, 4L), CEACAM 5, and CEACAM 6 in HNO cells compared to parent cells. However, the surface expression was low in HNO cells. Interestingly, the intracellular protein levels were high for these three CEACAMs. We confirmed these results with immunohistochemical experiments. Further, the adhesion and migration assays showed reduced clumping in HNO-adapted A549 (A549-HNO) cells and faster migration rates, respectively. These results document the altered adhesion and migration properties of cells adapted to HNO. Further, our studies also indicate a dynamic regulation of CEACAM protein expression and surface transport in HNO cells.


Lung adenocarcinoma High nitric oxide CEACAM Adhesion Migration 


Conflict of interest



  1. 1.
    Beauchemin N, Arabzadeh A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev. 2013;32(3–4):643–71.CrossRefPubMedGoogle Scholar
  2. 2.
    Gold P, Freedman SO. Specific carcinoembryonic antigens of the human digestive system. J Exp Med. 1965;122(3):467–81.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hammarstrom S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol. 1999;9(2):67–81.CrossRefPubMedGoogle Scholar
  4. 4.
    Kodera Y, Isobe K, Yamauchi M, Satta T, Hasegawa T, Oikawa S, et al. Expression of carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA) in gastrointestinal cancer; the correlation with degree of differentiation. Br J Cancer. 1993;68(1):130–6.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wirth T, Soeth E, Czubayko F, Juhl H. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth. Clin Exp Metastasis. 2002;19(2):155–60.CrossRefPubMedGoogle Scholar
  6. 6.
    Gemei M, Mirabelli P, Di Noto R, Corbo C, Iaccarino A, Zamboli A, et al. CD66c is a novel marker for colorectal cancer stem cell isolation, and its silencing halts tumor growth in vivo. Cancer. 2013;119(4):729–38.CrossRefPubMedGoogle Scholar
  7. 7.
    Witzens-Harig M, Hose D, Junger S, Pfirschke C, Khandelwal N, Umansky L, et al. Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6. Blood. 2013;121(22):4493–503.CrossRefPubMedGoogle Scholar
  8. 8.
    Tsang JY, Kwok YK, Chan KW, Ni YB, Chow WN, Lau KF, et al. Expression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in breast cancers. Breast Cancer Res Treat. 2013;142(2):311–22.CrossRefPubMedGoogle Scholar
  9. 9.
    Blumenthal RD, Leon E, Hansen HJ, Goldenberg DM. Expression patterns of CEACAM5 and CEACAM6 in primary and metastatic cancers. BMC Cancer. 2007;7:2.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Laack E, Nikbakht H, Peters A, Kugler C, Jasiewicz Y, Edler L, et al. Expression of CEACAM1 in adenocarcinoma of the lung: a factor of independent prognostic significance. J Clin Oncol. 2002;20(21):4279–84.CrossRefPubMedGoogle Scholar
  11. 11.
    Thies A, Moll I, Berger J, Wagener C, Brummer J, Schulze HJ, et al. CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease. J Clin Oncol. 2002;20(10):2530–6.CrossRefPubMedGoogle Scholar
  12. 12.
    Toffalorio F, Belloni E, Barberis M, Bucci G, Tizzoni L, Pruneri G, et al. Gene expression profiling reveals GC and CEACAM1 as new tools in the diagnosis of lung carcinoids. Br J Cancer. 2014;110(5):1244–9.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kuespert K, Pils S, Hauck CR. CEACAMs: their role in physiology and pathophysiology. Curr Opin Cell Biol. 2006;18(5):565–71.CrossRefPubMedGoogle Scholar
  14. 14.
    Vesper BJ, Elseth KM, Tarjan G, Haines 3rd GK, Radosevich JA. Long-term adaptation of lung tumor cell lines with increasing concentrations of nitric oxide donor. The Open Lung Cancer J. 2009;2:35–44.CrossRefGoogle Scholar
  15. 15.
    Bentz BG, Simmons RL, Haines 3rd GK, Radosevich JA. The yin and yang of nitric oxide: reflections on the physiology and pathophysiology of NO. Head Neck. 2000;22(1):71–83.CrossRefPubMedGoogle Scholar
  16. 16.
    Bentz BG, Hammer ND, Radosevich JA, Haines 3rd GK. Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line. J Carcinog. 2004;3(1):16.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Aqil M, Elseth KM, Vesper BJ, Deliu Z, Aydogan B, Xue J, et al. Part I—mechanism of adaptation: high nitric oxide adapted A549 cells show enhanced DNA damage response and activation of antiapoptotic pathways. Tumour Biol. 2014;35(3):2403–15.CrossRefPubMedGoogle Scholar
  18. 18.
    Aqil M, Deliu Z, Elseth KM, Shen G, Xue J, Radosevich JA. Part II—mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints. Tumour Biol. 2014;35(3):2417–25.CrossRefPubMedGoogle Scholar
  19. 19.
    Paradise WA, Vesper BJ, Goel A, Waltonen JD, Altman KW, Haines GK, et al. Nitric oxide: perspectives and emerging studies of a well known cytotoxin. Int J Mol Sci. 2010;11(7):2715–45.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bentz BG, Hammer ND, Milash B, Klein S, Burnett DM, Radosevich JA, et al. The kinetics and redox state of nitric oxide determine the biological consequences in lung adenocarcinoma. Tumour Biol. 2007;28(6):301–11.CrossRefPubMedGoogle Scholar
  21. 21.
    Cubillos-Rojas M, Amair-Pinedo F, Tato I, Bartrons R, Ventura F, Rosa JL. Simultaneous electrophoretic analysis of proteins of very high and low molecular mass using Tris-acetate polyacrylamide gels. Electrophoresis. 31(8):1318–21.Google Scholar
  22. 22.
    Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2):329–33.CrossRefPubMedGoogle Scholar
  23. 23.
    Sienel W, Dango S, Woelfle U, Morresi-Hauf A, Wagener C, Brummer J, et al. Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 promotes progression of non-small cell lung cancer. Clin Cancer Res. 2003;9(6):2260–6.PubMedGoogle Scholar
  24. 24.
    Singer BB, Scheffrahn I, Kammerer R, Suttorp N, Ergun S, Slevogt H. Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells. PLoS One. 2010;5(1):e8747.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Muturi HT, Dreesen JD, Nilewski E, Jastrow H, Giebel B, Ergun S, et al. Tumor and endothelial cell-derived microvesicles carry distinct CEACAMs and influence T-cell behavior. PLoS One. 2013;8(9):e74654.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Davis FM, Stewart TA, Thompson EW, Monteith GR. Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol Sci. 2014.Google Scholar
  27. 27.
    De Sanctis F, Sandri S, Ferrarini G, Pagliarello I, Sartoris S, Ugel S, et al. The emerging immunological role of post-translational modifications by reactive nitrogen species in cancer microenvironment. Front Immunol. 2014;5:69.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Vesper BJ, Onul A, Haines 3rd GK, Tarjan G, Xue J, Elseth KM, et al. Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines. Tumour Biol. 2013;34(1):203–14.CrossRefPubMedGoogle Scholar
  29. 29.
    De Vitto H, Mendonca BS, Elseth KM, Vesper BJ, Portari EA, Gallo CV, et al. Part II. Mitochondrial mutational status of high nitric oxide adapted cell line BT-20 (BT-20-HNO) as it relates to human primary breast tumors. Tumour Biol. 2013;34(1):337–47.CrossRefPubMedGoogle Scholar
  30. 30.
    De Vitto H, Mendonca BS, Elseth KM, Onul A, Xue J, Vesper BJ, et al. Part III. Molecular changes induced by high nitric oxide adaptation in human breast cancer cell line BT-20 (BT-20-HNO): a switch from aerobic to anaerobic metabolism. Tumour Biol. 2013;34(1):403–13.CrossRefPubMedGoogle Scholar
  31. 31.
    Onul A, Elseth KM, De Vitto H, Paradise WA, Vesper BJ, Tarjan G, et al. Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide. Tumour Biol. 2012;33(3):739–48.CrossRefPubMedGoogle Scholar
  32. 32.
    Yarmolyuk YR, Vesper BJ, Paradise WA, Elseth KM, Tarjan G, Haines 3rd GK, et al. Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines. Tumour Biol. 2011;32(1):77–85.CrossRefPubMedGoogle Scholar
  33. 33.
    Tarjan G, Haines 3rd GK, Vesper BJ, Xue J, Altman MB, Yarmolyuk YR, et al. Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines. Tumour Biol. 2011;32(1):87–98.CrossRefPubMedGoogle Scholar
  34. 34.
    Vesper BJ, Elseth KM, Tarjan G, Haines 3rd GK, Radosevich JA. Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide. Tumour Biol. 2010;31(4):267–75.CrossRefPubMedGoogle Scholar
  35. 35.
    Bentz BG, Chandra R, Haines 3rd GK, Robinson AM, Shah P, Radosevich JA. Nitric oxide and apoptosis during human head and neck squamous cell carcinoma development. Am J Otolaryngol. 2002;23(1):4–11.CrossRefPubMedGoogle Scholar
  36. 36.
    Chandra R, Haines 3rd GK, Bentz BG, Shah P, Robinson AM, Radosevich JA. Expression of nitric oxide synthase type 3 in reflux-induced esophageal lesions. Otolaryngol Head Neck Surg. 2001;124(4):442–7.CrossRefPubMedGoogle Scholar
  37. 37.
    Bentz BG, Haines 3rd GK, Radosevich JA. Increased protein nitrosylation in head and neck squamous cell carcinogenesis. Head Neck. 2000;22(1):64–70.CrossRefPubMedGoogle Scholar
  38. 38.
    Bentz BG, Haines 3rd GK, Lingen MW, Pelzer HJ, Hanson DG, Radosevich JA. Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck. Ann Otol Rhinol Laryngol. 1999;108(8):781–7.CrossRefPubMedGoogle Scholar
  39. 39.
    Bentz BG, Haines 3rd GK, Hanson DG, Radosevich JA. Endothelial constitutive nitric oxide synthase (ecNOS) localization in normal and neoplastic salivary tissue. Head Neck. 1998;20(4):304–9.CrossRefPubMedGoogle Scholar
  40. 40.
    Zhang L, Liu J, Wang X, Li Z, Zhang X, Cao P, et al. Upregulation of cytoskeleton protein and extracellular matrix protein induced by stromal-derived nitric oxide promotes lung cancer invasion and metastasis. Curr Mol Med. 2014.Google Scholar
  41. 41.
    Oronsky B, Fanger GR, Oronsky N, Knox S, Scicinski J. The implications of hyponitroxia in cancer. Transl Oncol. 2014;7(2):167–73.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Lawson EL, Mills DR, Brilliant KE, Hixson DC. The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity. PLoS One. 2012;7(1):e29606.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Kobayashi M, Miki Y, Ebina M, Abe K, Mori K, Narumi S, et al. Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma. Br J Cancer. 2012;107(10):1745–53.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Strickland LA, Ross J, Williams S, Ross S, Romero M, Spencer S, et al. Preclinical evaluation of carcinoembryonic cell adhesion molecule (CEACAM) 6 as potential therapy target for pancreatic adenocarcinoma. J Pathol. 2009;218(3):380–90.CrossRefPubMedGoogle Scholar
  45. 45.
    Han SU, Kwak TH, Her KH, Cho YH, Choi C, Lee HJ, et al. CEACAM5 and CEACAM6 are major target genes for Smad3-mediated TGF-beta signaling. Oncogene. 2008;27(5):675–83.CrossRefPubMedGoogle Scholar
  46. 46.
    Hokari M, Matsuda Y, Wakai T, Shirai Y, Sato M, Tsuchiya A, et al. Tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 potentates the anchorage-independent growth of human hepatoma HepG2 cells. Life Sci. 2007;81(4):336–45.CrossRefPubMedGoogle Scholar
  47. 47.
    Dango S, Sienel W, Schreiber M, Stremmel C, Kirschbaum A, Pantel K, et al. Elevated expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) is associated with increased angiogenic potential in non-small-cell lung cancer. Lung Cancer. 2008;60(3):426–33.CrossRefPubMedGoogle Scholar
  48. 48.
    Liu W, Wei W, Winer D, Bamberger AM, Bamberger C, Wagener C, et al. CEACAM1 impedes thyroid cancer growth but promotes invasiveness: a putative mechanism for early metastases. Oncogene. 2007;26(19):2747–58.CrossRefPubMedGoogle Scholar
  49. 49.
    Dery KJ, Gaur S, Gencheva M, Yen Y, Shively JE, Gaur RK. Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M. J Biol Chem. 2011;286(18):16039–51.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Singer BB, Scheffrahn I, Heymann R, Sigmundsson K, Kammerer R, Obrink B. Carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes. J Immunol. 2002;168(10):5139–46.CrossRefPubMedGoogle Scholar
  51. 51.
    Fiori V, Magnani M, Cianfriglia M. The expression and modulation of CEACAM1 and tumor cell transformation. Ann Ist Super Sanita. 2012;48(2):161–71.CrossRefPubMedGoogle Scholar
  52. 52.
    Sappino AP, Buser R, Seguin Q, Fernet M, Lesne L, Gumy-Pause F, et al. The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage. Oncogenesis. 2012;1:e7.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Tiernan JP, Perry SL, Verghese ET, West NP, Yeluri S, Jayne DG, et al. Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting. Br J Cancer. 2013;108(3):662–7.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Thomas P, Forse RA, Bajenova O. Carcinoembryonic antigen (CEA) and its receptor hnRNP M are mediators of metastasis and the inflammatory response in the liver. Clin Exp Metastasis. 2011;28(8):923–32.CrossRefPubMedGoogle Scholar
  55. 55.
    Zheng C, Feng J, Lu D, Wang P, Xing S, Coll JL, et al. A novel anti-CEACAM5 monoclonal antibody, CC4, suppresses colorectal tumor growth and enhances NK cells-mediated tumor immunity. PLoS One. 2011;6(6):e21146.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Kim KS, Kim JT, Lee SJ, Kang MA, Choe IS, Kang YH, et al. Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer. Clin Chim Acta. 2013;415:12–9.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Madeeha Aqil
    • 1
  • Kim M. Elseth
    • 1
  • Ashok Arjunakani
    • 2
  • Philip Nebres
    • 2
  • Courtney P. Amegashie
    • 2
  • Devang H. Thanki
    • 3
  • Premal B. Desai
    • 3
  • James A. Radosevich
    • 1
    • 4
  1. 1.Department of Oral Medicine and Diagnostic Sciences, College of DentistryUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Illinois Mathematics and Science AcademyAuroraUSA
  3. 3.Metea Valley High SchoolAuroraUSA
  4. 4.Jesse Brown VAMCChicagoUSA

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