title: Activity of aaptamine and two derivatives, demethyloxyaaptamine and isoaaptamine, in cisplatin-resistant germ cell cancer.
abstract: We analyzed the effects of all three marine alkaloids aaptamine, demethyloxyaaptamine and isoaaptamine in NT2-R, a cisplatin-resistant subline of the human embryonal carcinoma cell line NT2. All aaptamines were found to be equally effective in both cell lines, excluding cross-resistance between aaptamines and cisplatin in vitro. At the inhibitory concentration (IC50), aaptamine exerted an antiproliferative effect, whereas demethyloxyaaptamine and isoaaptamine were strong inducers of apoptosis. We analyzed the changes in the proteome of NT2-R cells treated with these compounds. 16-22 proteins were found to be significantly altered, of which several were validated by Western blotting and two-dimensional Western blotting analysis. Changes in the proteome pattern frequently resulted from post-transcriptional protein modifications, i.e. phosphorylation or hypusination in the case of eIF5A. Although the lists of altered proteins were heterogeneous and compound-specific, gene ontology analyses identified rather similar profiles regarding the affected molecular functions. Ingenuity pathway analysis by IPA put the following factors in a central position of the hypothetical networks: myc and p53 for aaptamine; tumor necrosis factor (TNF) for demethyloxyaaptamine; and all three, myc, p53, and TNF for isoaaptamine. Our results represent an important step towards a better understanding of the molecular basis underlying the observed bioactivity of these promising marine compounds.
  
  
  CONCLUSIONS
  We characterized the mode of action of three aaptamines, marine natural compound with anti-tumor activity, using a functional proteomics approach and the cisplatin-resistant pluripotent human embryonal carcinoma cell line NT2-R. The manuscript is of particular scientific interest, as we could reveal the similarities and differences of the modes of action. Furthermore, we were able to identify several new targets of these promising compounds. We found hypusination of eIF5A to be a prominent feature exclusively of aaptamine treatment, as this was not observed upon treatment with demethyloxyaaptamine or isoaaptamine. Our results are a step towards unraveling the mode of action of these interesting compounds.
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Laboratory of Marine Natural Products Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Dyshlovoy
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Greifswald, Germany; Interfacultary Institute of Genetics and Functional Genomics, Department of Functional Genomics, University of Greifswald, Greifswald, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Venz
authors.affiliation: Laboratory of Marine Natural Products Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.
authors.forename: Larisa K
authors.initials: LK
authors.lastname: Shubina
authors.affiliation: Laboratory of Marine Natural Products Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Fedorov
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, University of Greifswald, Greifswald, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Walther
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Jacobsen
authors.affiliation: Laboratory of Marine Natural Products Chemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.
authors.forename: Valentin A
authors.initials: VA
authors.lastname: Stonik
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Bokemeyer
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Division of Hematology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Balabanov
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address: friedemann.honecker@zetup.ch.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Honecker
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002945:Cisplatin; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D009287:Naphthyridines / Q000493:pharmacokinetics / Q000494:pharmacology*; D009373:Neoplasms, Germ Cell and Embryonal / Q000188:drug therapy* / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D009287:Naphthyridines; C417553:isoaaptamine; C043986:aaptamine; D002945:Cisplatin
keywords: 2-D WB; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Aaptamine; Cis-diamminedichloroplatinum (II); Cisplatin; Cisplatin resistance; Eukaryotic initiation factor 5A-1; Germ cell cancer; Heat shock protein 70kDa; Hsp70; MTT; Marine alkaloids; Mini-2D Western blotting analysis; Proteome analysis; eIF5A