Investigational New Drugs

, Volume 25, Issue 1, pp 21–29 | Cite as

Proteomic analysis of prodigiosin-induced apoptosis in a breast cancer mitoxantrone-resistant (MCF-7 MR) cell line

  • Marta Monge
  • Marta Vilaseca
  • Vanessa Soto-Cerrato
  • Beatriz Montaner
  • Ernest Giralt
  • Ricardo Pérez-Tomás


Prodigiosin (PG) is a bacterial, red-pigmented antibiotic with immunosuppressive and apoptotic activities. To better understand its mechanisms of action, we tried to identify proteins associated with apoptosis induced by PG. For this purpose, the variation of protein expression on exposure to apoptotic concentrations of PG was examined, by high-resolution two-dimensional gel electrophoresis (2D-E), in the MCF-7 cancer cell line resistant to mitoxantrone (MCF-7-MR). Six PG apoptosis-associated protein spots were further characterized by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on a matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer. The proteins identified were involved in various cellular functions, including cell defence, DNA repair and cellular organization. Our data provide novel information on cell response to PG, a new apoptotic drug with interesting anticancer activity.


Apoptosis Prodigiosin Proteomics Breast cancer 





formic acid


glutathione S-transferase


intermediate filaments


Isoelectric focusing


isoelectric point


matrix-assisted laser desorption ionization-time-of-flight/time-of-flight


MCF-7 breast cancer cell line resistant to mitoxantrone


mitogen-activated protein kinase


multidrug resistance


multidrug resistance protein




trifluoroacetic acid


two-dimensional gel electrophoresis


vacuolar H+-ATPase


α-cyano-4-hydroxycinnamic acid


2,5-dihydroxybenzoic acid


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We want to thank Miguel Abal for critical and comprehensive reading of the manuscript. We also want to thank Dr. Eliandre de Oliveira and David Bellido from “Plataforma de Proteòmica” University of Barcelona for technical support. M. Monge was a recipient of a fellowship from the University of Barcelona.


  1. 1.
    Montaner B, Perez-Tomas R (2003) The prodigiosins: A new family of anticancer drugs. Curr Cancer Drug Targets 3:57–65PubMedCrossRefGoogle Scholar
  2. 2.
    Llagostera E, Soto-Cerrato V, Joshi R, Montaner B, Gimenez-Bonafe P, Perez-Tomas R (2005) High cytotoxic sensitivity of the human small cell lung doxorubicin-resistant carcinoma (GLC4/ADR) cell line to prodigiosin through apoptosis activation. Anticancer Drugs 16:393–399PubMedCrossRefGoogle Scholar
  3. 3.
    Montaner B, Navarro S, Pique M, Vilaseca M, Martinell M, Giralt E, Gil J, Perez-Tomas R (2000) Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br J Pharmacol 131:585–593PubMedCrossRefGoogle Scholar
  4. 4.
    Montaner B, Perez-Tomas R (2001) Prodigiosin-induced apoptosis in human colon cancer cells. Life Sci 68:2025–2036PubMedCrossRefGoogle Scholar
  5. 5.
    Diaz-Ruiz C, Montaner B, Perez-Tomas R (2001) Prodigiosin induces cell death and morphological changes indicative of apoptosis in gastric cancer cell line HGT-1. Histol Histopathol 16:415–421PubMedGoogle Scholar
  6. 6.
    Sato T, Konno H, Tanaka Y, Kataoka T, Nagai K, Wasserman HH, Ohkuma S (1998) Prodigiosins as a new group of H+/Cl- symporters that uncouple proton translocators. J Biol Chem 273:21455–21462PubMedCrossRefGoogle Scholar
  7. 7.
    Yamamoto C, Takemoto H, Kuno K, Yamamoto D, Tsubura A, Kamata K, Hirata H, Yamamoto A, Kano H, Seki T, Inoue K (1999) Cycloprodigiosin hydrochloride, a new H(+)/Cl(-) symporter, induces apoptosis in human and rat hepatocellular cancer cell lines in vitro and inhibits the growth of hepatocellular carcinoma xenografts in nude mice. Hepatology 30:894–902PubMedCrossRefGoogle Scholar
  8. 8.
    Perez-Tomas R, Montaner B (2003) Effects of the proapoptotic drug prodigiosin on cell cycle-related proteins in Jurkat T cells. Histol Histopathol 18:379–385PubMedGoogle Scholar
  9. 9.
    Melvin MS, Wooton KE, Rich CC, Saluta GR, Kucera GL, Lindquist N, Manderville RA (2001) Copper-nuclease efficiency correlates with cytotoxicity for the 4-methoxypyrrolic natural products. J Inorg Biochem 87:129–135PubMedCrossRefGoogle Scholar
  10. 10.
    Montaner B, Perez-Tomas R (2002) The cytotoxic prodigiosin induces phosphorylation of p38-MAPK but not of SAPK/JNK. Toxicol Lett 129:93–98PubMedCrossRefGoogle Scholar
  11. 11.
    Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Greenberger L, Cole SP, Doyle LA (1999) A typical multidrug resistance: Breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 91:429–433PubMedCrossRefGoogle Scholar
  12. 12.
    Taylor CW, Dalton WS, Parrish PR, Gleason MC, Bellamy WT, Thompson FH, Roe DJ, Trent JM (1991) Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line. Br J Cancer 63:923–929PubMedGoogle Scholar
  13. 13.
    Soto-Cerrato V, Llagostera E, Montaner B, Scheffer GL, Perez-Tomas R (2004) Mitochondria-mediated apoptosis operating irrespective of multidrug resistance in breast cancer cells by the anticancer agent prodigiosin. Biochem Pharmacol 68:1345–1352PubMedCrossRefGoogle Scholar
  14. 14.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021PubMedGoogle Scholar
  15. 15.
    Heukeshoven J, Dernick R (1988) Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels. Electrophoresis 9:28–32PubMedCrossRefGoogle Scholar
  16. 16.
    Erdjument-Bromage H, Lui M, Lacomis L, Grewal A, Annan RS, McNulty DE, Carr SA, Tempst P (1998) Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J Chromatogr A 826:167–181PubMedCrossRefGoogle Scholar
  17. 17.
    Laugesen S, Roepstorff P (2003) Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis. J Am Soc Mass Spectrom 14:992–1002PubMedCrossRefGoogle Scholar
  18. 18.
    Reed JC, Tomaselli KJ (2000) Drug discovery opportunities from apoptosis research. Curr Opin Biotechnol 11:586–592PubMedCrossRefGoogle Scholar
  19. 19.
    He QY, Chiu JF (2003) Proteomics in biomarker discovery and drug development. J Cell Biochem 89:868–886PubMedCrossRefGoogle Scholar
  20. 20.
    Thiede B, Rudel T (2004) Proteome analysis of apoptotic cells. Mass Spectrom Rev 23:333–349PubMedCrossRefGoogle Scholar
  21. 21.
    Perez-Tomas R, Montaner B, Llagostera E, Soto-Cerrato V (2003) The prodigiosins, proapoptotic drugs with anticancer properties. Biochem Pharmacol 66:1447–1452PubMedCrossRefGoogle Scholar
  22. 22.
    Morrow CS, Smitherman PK, Townsend AJ (2000) Role of multidrug-resistance protein 2 in glutathione S-transferase P1-1-mediated resistance to 4-nitroquinoline 1-oxide toxicities in HepG2 cells. Mol Carcinog 29:170–178PubMedCrossRefGoogle Scholar
  23. 23.
    Cullen KJ, Newkirk KA, Schumaker LM, Aldosari N, Rone JD, Haddad BR (2003) Glutathione S-transferase pi amplification is associated with cisplatin resistance in head and neck squamous cell carcinoma cell lines and primary tumors. Cancer Res 63:8097–8102PubMedGoogle Scholar
  24. 24.
    Harbottle A, Daly AK, Atherton K, Campbell FC (2001) Role of glutathione S-transferase P1, P-glycoprotein and multidrug resistance-associated protein 1 in acquired doxorubicin resistance. Int J Cancer 92:777–783PubMedCrossRefGoogle Scholar
  25. 25.
    Depeille P, Cuq P, Mary S, Passagne I, Evrard A, Cupissol D, Vian L (2004) Glutathione S-transferase M1 and multidrug resistance protein 1 act in synergy to protect melanoma cells from vincristine effects. Mol Pharmacol 65:897–905PubMedCrossRefGoogle Scholar
  26. 26.
    Tchorzewski M, Krokowski D, Rzeski W, Issinger OG, Grankowski N (2003) The subcellular distribution of the human ribosomal “stalk” components: P1, P2 and P0 proteins. Int J Biochem Cell Biol 35:203–211PubMedCrossRefGoogle Scholar
  27. 27.
    Brockstedt E, Rickers A, Kostka S, Laubersheimer A, Dorken B, Wittmann-Liebold B, Bommert K, Otto A (1998) Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J Biol Chem 273:28057–28064PubMedCrossRefGoogle Scholar
  28. 28.
    Grabowski DT, Pieper RO, Futscher BW, Deutsch WA, Erickson LC, Kelley MR (1992) Expression of ribosomal phosphoprotein PO is induced by antitumor agents and increased in Mer- human tumor cell lines. Carcinogenesis 13:259–263PubMedGoogle Scholar
  29. 29.
    Montaner B, Castillo-Avila W, Martinell M, Ollinger R, Aymami J, Giralt E, Perez-Tomas R (2005) DNA interaction and dual topoisomerase I and II inhibition properties of the anti-tumor drug prodigiosin. Toxicol Sci 85:870–879PubMedCrossRefGoogle Scholar
  30. 30.
    Nishida J, Shiratsuchi A, Nadano D, Sato TA, Nakanishi Y (2002) Structural change of ribosomes during apoptosis: Degradation and externalization of ribosomal proteins in doxorubicin-treated Jurkat cells. J Biochem (Tokyo) 131:485–493Google Scholar
  31. 31.
    Steinert PM, Roop DR (1988) Molecular and cellular biology of intermediate filaments. Annu Rev Biochem 57:593–625PubMedCrossRefGoogle Scholar
  32. 32.
    Oshima RG (2002) Apoptosis and keratin intermediate filaments. Cell Death Differ 9:486–492PubMedCrossRefGoogle Scholar
  33. 33.
    Caulin C, Salvesen GS, Oshima RG (1997) Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 138:1379–1394PubMedCrossRefGoogle Scholar
  34. 34.
    Ku NO, Liao J, Omary MB (1997) Apoptosis generates stable fragments of human type I keratins. J Biol Chem 272:33197–33203PubMedCrossRefGoogle Scholar
  35. 35.
    Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, Marberger M, Biven K, Shoshan MC, Linder S (2004) Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res 64:1751–1756PubMedCrossRefGoogle Scholar
  36. 36.
    Stigbrand T (2001) The versatility of cytokeratins as tumor markers. Tumour Biol 22:1–3PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • Marta Monge
    • 1
  • Marta Vilaseca
    • 2
  • Vanessa Soto-Cerrato
    • 1
  • Beatriz Montaner
    • 1
  • Ernest Giralt
    • 3
  • Ricardo Pérez-Tomás
    • 1
    • 4
  1. 1.Department of Pathology and Experimental Therapeutic, Cancer Cell Biology Research GroupUniversity of BarcelonaBarcelonaSpain
  2. 2.Servei Espectrometria de Masses-Serveis CientificotècnicsUniversity of BarcelonaBarcelonaSpain
  3. 3.Departament de Química Orgànica, IRBB-PCBUniversity of BarcelonaBarcelonaSpain
  4. 4.Dept. Patologia i Terapèutica Experimental. CCBR GroupBarcelonaSpain

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