Investigational New Drugs

, Volume 32, Issue 1, pp 37–46 | Cite as

Rhodamine-marked bombesin: a novel means for prostate cancer fluorescence imaging

  • Alexander SturzuEmail author
  • Sumbla Sheikh
  • Hartmut Echner
  • Thomas Nägele
  • Martin Deeg
  • Bushra Amin
  • Christian Schwentner
  • Marius Horger
  • Ulrike Ernemann
  • Stefan Heckl


The gastrin releasing peptide receptor (GRPR) has been found to be strongly expressed in various types of cancers such as prostate and breast carcinomas. The GRPR ligands gastrin releasing peptide and bombesin can play a very significant role in cancer therapy and diagnostics. In this study we synthesized unlabeled bombesin BBN along with two conjugates in which the correct bombesin (BBN-Rhd) and a mutant bombesin (mBBN-Rhd) sequence was coupled to rhodamine, a fluorescent dye. These novel rhodamine fluorescent conjugates were used to study the targeting and uptake of bombesin on a cellular level. Nine different human cell lines including both tumor and healthy cells were examined using flow cytometry and confocal laser scanning microscopy. GRPR mRNA expression analysis was performed and it was found that the receptor is highly expressed in LNCaP and PC3 cells compared to the rest of other cell lines. Competition experiments were performed to verify the receptor dependence of the labeled conjugates using unmarked bombesin. The present study is a first attempt at direct fluorescence imaging of living cells using bombesin and its target, the GRPR. A rhodamine bombesin conjugate can be used as marker to differentiate between healthy cells and malignant cells such as prostate hyperplasia and prostate carcinoma in the early detection of cancer.


Gastrin releasing peptide receptor Bombesin Ligands Fluorescence imaging conjugates 



This study has been supported by the Wilhelm-Sander-Foundation and the Interdisciplinary Centre for Clinical Research, University of Tübingen.

Conflict of interest statement

The authors declare that they have no conflict of interest.


  1. 1.
    Reubi JC (2003) Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 24(4):389–427CrossRefPubMedGoogle Scholar
  2. 2.
    Liu S, Edwards DS (1999) 99mTc-Labeled small peptides as diagnostic radiopharmaceuticals. Chem Rev 99(9):2235–2268CrossRefPubMedGoogle Scholar
  3. 3.
    McAfee JG, Neumann RD (1996) Radiolabeled peptides and other ligands for receptors overexpressed in tumor cells for imaging neoplasms. Nucl Med Biol 23:673–676CrossRefPubMedGoogle Scholar
  4. 4.
    Okarvi SM (2004) Peptide-based radiopharmaceuticals: future tools for diagnostic imaging of cancers and other diseases. Med Res Rev 24:685–686CrossRefGoogle Scholar
  5. 5.
    Reubi JC (1995) Neuropeptide receptors in health and disease: the molecular basis for in vivo imaging. J Nucl Med 36:1825–1835PubMedGoogle Scholar
  6. 6.
    Villalba M, Bockaert J, Journot L (1997) Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. J Neurosci 17(1):83–90PubMedGoogle Scholar
  7. 7.
    Cattaneo MG, Amoroso D, Gussoni G, Sanguini AM, Vicentini LM (1996) A somatostatin analogue inhibits MAP kinase activation and cell proliferation in human neuroblastoma and in human small cell lung carcinoma cell lines. FEBS Lett 397(2–3):164–168CrossRefPubMedGoogle Scholar
  8. 8.
    Safavi A, Khazaeli MB, Qin H, Buchsbaum DJ (1997) Synthesis of bombesin analogues for radiolabeling with rhenium-188. Cancer 80:2354–2359CrossRefGoogle Scholar
  9. 9.
    Nagalla SR, Barry BJ, Falick AM, Gibson BW, Taylor JE, Dong JZ, Spindel ER (1996) There are three distinct forms of bombesin. Identification of [Leu13]bombesin,[Phe13]bombesin, and [Ser3, Arg10, Phe13]bombesin in the frog Bombina orientalis. J Biol Chem 271(13):7731–7737CrossRefPubMedGoogle Scholar
  10. 10.
    Anastasi A, Erspamer V, Bucci M (1971) Isolation and structure of bombesin and alytesin, 2 analogous active peptides from the skin of the European amphibians Bombina and Alytes. Experientia 27(2):166–167CrossRefPubMedGoogle Scholar
  11. 11.
    McDonald TJ, Jörnvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V (1979) Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 90(1):227–233CrossRefPubMedGoogle Scholar
  12. 12.
    Spindel ER (1986) Mammalian bombesin-like peptides. Trends Neurosci 9:130–133CrossRefGoogle Scholar
  13. 13.
    Lebacq-Verheyden AM, Trepel J, Sausville EA, Battey JF (1991) In: Sporn MB, Roberts AB (eds) Peptide growth factors and their receptors II. Springer Verlag, New York, pp 71–124Google Scholar
  14. 14.
    Li K, Nagalla SR, Spindel ER (1994) A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth. J Clin Invest 94(4):1605–1615PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Breeman WA, Hofland LJ, de Jong M, Bernard BF, Srinivasan A, Kwekkeboom DJ, Visser TJ, Krenning EP (1999) Evaluation of radiolabeled bombesin analogues for receptor targeted scintigraphy and radiotherapy. Int J Cancer 81:658–665CrossRefPubMedGoogle Scholar
  16. 16.
    Krenning EP, Kwekkeboom DJ, Bakker WH, Breeman WA, Kooij PP, Oei HY, van Hagen M, Postema PT, de Jong M, Reubi JC et al (1993) Somatostatin receptor scintigraphy with [111In-DTPA-D-Phe1]- and [123I-Tyr3]-octreotide: the Rotterdam experience with more than 1000 patients. Eur J Nucl Med 20(8):716–731CrossRefPubMedGoogle Scholar
  17. 17.
    Kwekkeboom D, Krenning EP, de Jong M (2000) Peptide receptor imaging and therapy. J Nucl Med 41(10):1704–1713PubMedGoogle Scholar
  18. 18.
    Bakker WH, Krenning EP, Reubi JC, Breeman WA, Setyono-Han B, de Jong M, Kooij PP, Bruns C, van Hagen PM, Marbach P et al (1991) In vivo application of [111In-DTPA-D-Phe1]-octreotide for detection of somatostatin receptor-positive tumors in rats. Life Sci 49(22):1593–1601CrossRefPubMedGoogle Scholar
  19. 19.
    Breeman WA, Hofland LJ, van der Pluijm M, van Koetsveld PM, de Jong M, Setyono-Han B, Bakker WH, Kwekkeboom DJ, Visser TJ, Lamberts SW (1994) A new radiolabelled somatostatin analogue [111In-DTPA-D-Phe1]RC-160: preparation, biological activity, receptor scintigraphy in rats and comparison with[111In-DTPA-D-Phe1]octreotide. Eur J Nucl Med 21(4):328–335CrossRefPubMedGoogle Scholar
  20. 20.
    Markwalder R, Reubi JC (1999) Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res 59:1152–1159PubMedGoogle Scholar
  21. 21.
    Plonowski A, Nagy A, Schally AV, Sun B, Groot K, Halmos G (2000) In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue AN-215. Int J Cancer 88(4):652–657CrossRefPubMedGoogle Scholar
  22. 22.
    Heimbrook DC, Saari WS, Balishin NL, Fischer TW, Friedman A, Kiefer DM, Rotberg NS, Wallen JW, Oliff A (1991) Gastrin releasing peptides antagonists with improved potency and stability. J Med Chem 34:2102–2107CrossRefPubMedGoogle Scholar
  23. 23.
    La Bella R, Garcia-Garayoa E, Langer M, Bläuenstein P, Beck-Sickinger AG, Schubiger PA (2002) In vitro and in vivo evaluation of a 99mTc(I)-labeled bombesin analogue for imaging of gastrin releasing peptide receptor-positive tumors. Nucl Med Biol 29(5):553–560CrossRefPubMedGoogle Scholar
  24. 24.
    Karra SR, Shibli R, Gali H, Katti KV, Hoffman TJ, Higginbotham C, Sieckman GL, Volkart WA (1999) 99mTc –labelling and in vivo studies of a bombesin analogue with a novel water soluble dithiadophosphine-based bifunctional chelating agent. Bioconjug Chem 10:254–260CrossRefPubMedGoogle Scholar
  25. 25.
    Mantey S, Frucht H, Coy DH, Jensen RT (1993) Characterisation of bombesin receptors using a novel, potent, radiolabelled antagonist that distinguishes bombesin receptor subtypes. Mol Pharmacol 43:762–774PubMedGoogle Scholar
  26. 26.
    Sun B, Halmos G, Schally AV, Wang X, Martinez M (2000) Presence of receptors for bombesin/gastrin-releasing peptide and mRNA for three receptor subtypes in human prostate cancers. Prostate 42(4):295–303CrossRefPubMedGoogle Scholar
  27. 27.
    Mahmoud S, Staley J, Taylor J, Bogden A, Moreau JP, Coy D, Avis I, Cuttitta F, Mulshine JL, Moody TW (1991) [Psi 13,14] Bombesin analogues inhibit growth of small cell lung cancer in vitro and in vivo. Cancer Res 51(7):1798–1802PubMedGoogle Scholar
  28. 28.
    Hoffman TJ, Gali H, Smith CJ, Sieckman GL, Hayes DL, Owen NK, Volkert WA (2003) Novel series of 111In-labeled bombesin analogs as potential radiopharmaceuticals for specific targeting of gastrin-releasing peptide receptors expressed on human prostate cancer cells. J Nucl Med 44(5):823–831PubMedGoogle Scholar
  29. 29.
    Reubi JC (1997) Regulatory peptide receptors as molecular targets for cancer diagnosis and therapy. Q J Nucl Med 41:63–70PubMedGoogle Scholar
  30. 30.
    Sancho V, Di Florio A, Moody TW, Jensen RT (2011) Bombesin receptor-mediated imaging and cytotoxicity: review and current status. Curr Drug Deliv 8(1):79–134PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Kelly KA, Reynolds F, Weissleder R, Josephson L (2004) Fluorescein isothiocyanate-hapten immunoassay for determination of peptide-cell interactions. Anal Biochem 330(2):181–185CrossRefPubMedGoogle Scholar
  32. 32.
    Sturzu A, Sheikh S, Echner H, Nägele T, Deeg M, Schwentner C, Horger M, Ernemann U, Heckl S (2013) Novel Bourgeonal fragrance conjugates for the detection of prostate cancer. Invest New Drugs in pressGoogle Scholar
  33. 33.
    Sturzu A, Sheikh S, Klose U, Echner H, Kalbacher H, Deeg M, Nägele T, Ernemann U, Horger M, Heckl S (2012) Using the neurotransmitter serotonin to target imaging agents to glioblastoma cells. Invest New Drugs 30(6):2141–2147CrossRefPubMedGoogle Scholar
  34. 34.
    Sramkoski RM, Pretlow TG 2nd, Giaconia JM, Pretlow TP, Schwartz S, Sy MS, Marengo SR, Rhim JS, Zhang D, Jacobberger JW (1999) A new human prostate carcinoma cell line, 22Rv1. Vitro Cell Dev Biol Anim 35(7):403–409CrossRefGoogle Scholar
  35. 35.
    Igawa T, Lin FF, Lee MS, Karan D, Batra SK, Lin MF (2002) Establishment and Characterization of androgen-independent human prostate cancer LNCaP cell model. Prostate 50(4):222–235CrossRefPubMedGoogle Scholar
  36. 36.
    Iwasa Y, Mizokami A, Miwa S, Koshida K, Namiki M (2007) Establishment and characterization of androgen-independent human prostate cancer cell lines, LN-REC4 and LNCaP-SF, from LNCaP. Int J Urol 14(3):233–239CrossRefPubMedGoogle Scholar
  37. 37.
    Vigna SR, Giraud AS, Soll AH, Walsh JH, Mantyh PW (1988) Bombesin receptors on gastrin cells. Ann N Y Acad Sci 547:131–137CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Alexander Sturzu
    • 1
    • 2
    Email author
  • Sumbla Sheikh
    • 2
  • Hartmut Echner
    • 2
  • Thomas Nägele
    • 1
  • Martin Deeg
    • 3
  • Bushra Amin
    • 2
  • Christian Schwentner
    • 4
  • Marius Horger
    • 5
  • Ulrike Ernemann
    • 1
  • Stefan Heckl
    • 1
  1. 1.Department of NeuroradiologyUniversity of TübingenTübingenGermany
  2. 2.Peptide Synthesis Laboratory, Interfaculty Institute of BiochemistryUniversity of TübingenTübingenGermany
  3. 3.Mass Spectrometry Laboratory, Medicinal Research CentreUniversity of TübingenTübingenGermany
  4. 4.Department of UrologyUniversity of TübingenTübingenGermany
  5. 5.Department of RadiologyUniversity of TübingenTübingenGermany

Personalised recommendations