title: Jatrophane diterpenes and cancer multidrug resistance - ABCB1 efflux modulation and selective cell death induction.
abstract: BACKGROUND
  Modulation of P-glycoprotein (ABCB1) and evaluation of the collateral sensitivity effect are among the most promising approaches to overcome multidrug resistance (MDR) in cancer. In a previous study, two rare 12,17-cyclojatrophanes (1-2) and other novel jatrophanes (3-4), isolated from Euphorbia welwitschii, were screened for collateral sensitivity effect. Herein, the isolation of another jatrophane (5) is presented, being the broader goal of this work to investigate the role of euphowelwitschines A (1) and B (2), welwitschene (3), epoxywelwitschene (4) and esulatin M (5) as ABCB1 modulators and/or collateral sensitivity agents.
  
  
  METHODS
  Compounds 1-5 were evaluated for ABCB1 modulation ability through combination of transport and chemosensitivity assays, using a mouse T-lymphoma MDR1-transfected cell model. Moreover, the nature of interaction of compound 4 with ABCB1 was studied, using an ATPase assay. The MDR-selective antiproliferative activity of compound 5 was evaluated against gastric (EPG85-257) and pancreatic (EPP85-181) human cancer cells and their drug-selected counterparts (EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, EPP85-181RNOV). The drug induced cell death was investigated for compounds 4 and 5, using the annexin V/PI staining and the active caspase-3 assay.
  
  
  RESULTS
  The jatrophanes 1-5 were able to modulate the efflux activity of ABCB1, and at 2µM, 3-5 maintained the strong modulator profile. Structure activity results indicated that high conformational flexibility of the twelve-membered ring of compounds 3-5 favored ABCB1 modulation, in contrast to the tetracyclic scaffold of compounds 1 and 2. The effects of epoxywelwitschene (4) on the ATPase activity of ABCB1 showed it to interact with the transporter and to be able to reduce the transport of a second subtrate. Drug combination experiments also corroborated the anti-MDR potential of these diterpenes due to their synergistic interaction with doxorubicin (combination index <0.7). Esulatin M (5) showed a strong MDR-selective antiproliferative activity against EPG85-257RDB and EPP85-181RDB cells, with IC50 of 1.8 and 4.8 µM, respectively. Compounds 4 and 5 induced apoptosis via caspase-3 activation. A significant discrimination was observed between the resistant cell lines and parental cells.
  
  
  CONCLUSIONS
  This study strengthens the role of jatrophane diterpenes as lead candidates for the development of MDR reversal agents, higlighting the action of compounds 4 and 5.
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Avenue Prof. Gama Pinto, 1649-003 Lisbon, Portugal. Electronic address: malvesreis@ff.ulisboa.pt.
authors.forename: Mariana Alves
authors.initials: MA
authors.lastname: Reis
authors.affiliation: Institute of Pathology, University Hospital Charité, 10117 Berlin, Germany. Electronic address: omar.ahmed@charite.de.
authors.forename: Omar Bauomy
authors.initials: OB
authors.lastname: Ahmed
authors.affiliation: Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary. Electronic address: spengler.gabriella@med.u-szeged.hu.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Spengler
authors.affiliation: Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary. Electronic address: molnar.jozsef@med.u-szeged.hu.
authors.forename: Joseph
authors.initials: J
authors.lastname: Molnár
authors.affiliation: Institute of Pathology, University Hospital Charité, 10117 Berlin, Germany; Department of Pathology, Vivantes Clinics, Berlin, Germany. Electronic address: hermann.lage@vivantes.de.
authors.forename: Hermann
authors.initials: H
authors.lastname: Lage
authors.affiliation: Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Avenue Prof. Gama Pinto, 1649-003 Lisbon, Portugal. Electronic address: mjuferreira@ff.ulisboa.pt.
authors.forename: Maria-José U
authors.initials: MJ
authors.lastname: Ferreira
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004224:Diterpenes / Q000737:chemistry / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D028481:Euphorbia / Q000737:chemistry; D006801:Humans; D051379:Mice; D008968:Molecular Conformation; D013329:Structure-Activity Relationship
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000972:Antineoplastic Agents, Phytogenic; D004224:Diterpenes; C530345:jatrophane
keywords: 12,17-cyclojatrophane; ABCB1; Apoptosis; Collateral sensitivity; Jatrophane; Multidrug resistance