title: The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells.
abstract: BACKGROUND
  Abietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells.
  
  
  METHODS
  The cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry.
  
  
  RESULTS
  Parvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-xL did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation.
  
  
  CONCLUSIONS
  Parvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Burmistrova
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Perdomo
authors.affiliation: Faculdade de Farmácia da Universidade de Lisboa, iMed.ULisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
authors.forename: M Fátima
authors.initials: MF
authors.lastname: Simões
authors.affiliation: Faculdade de Farmácia da Universidade de Lisboa, iMed.ULisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal; C BIOS, Universidade Lusófona, Campo Grande 376, 1749-024 Lisboa, Portugal.
authors.forename: Patricia
authors.initials: P
authors.lastname: Rijo
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Quintana
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain. Electronic address: festevez@dbbf.ulpgc.es.
authors.forename: Francisco
authors.initials: F
authors.lastname: Estévez
mesh_terms: D045784:Abietanes / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D018922:HL-60 Cells / Q000187:drug effects; D006801:Humans; D020014:K562 Cells / Q000187:drug effects; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D015394:Molecular Structure; D031346:Plectranthus / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C000603753:2-(4-hydroxy)-benzoyloxy-11-hydroxy-5,7,9(11),13-abietatetraen-12-one; D045784:Abietanes; D000972:Antineoplastic Agents, Phytogenic; D017382:Reactive Oxygen Species; D020169:Caspases
keywords: Abietane diterpenes; Apoptosis; Cytotoxicity; Parvifloron D; Plectranthus ecklonii