title: Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug resistance in neuroblastoma cells.
abstract: Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Aggressive cell growth and chemoresistance are notorious obstacles in neuroblastoma therapy. Exposure to the anticancer drug ellipticine inhibits efficiently growth of neuroblastoma cells and induces apoptosis in these cells. However, ellipticine induced resistance in these cells. The upregulation of a vacuolar (V)-ATPase gene is one of the factors associated with resistance development. In accordance with this finding, we found that levels of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI line than in the parental ellipticine-sensitive UKF-NB-4 cell line. Treatment of ellipticine-sensitive UKF-NB-4 and ellipticine-resistant UKF-NB-4ELLI cells with ellipticine-induced cytoplasmic vacuolization and ellipticine is concentrated in these vacuoles. Confocal microscopy and staining of the cells with a lysosomal marker suggested these vacuoles as lysosomes. Transmission electron microscopy and no effect of an autophagy inhibitor wortmannin ruled out autophagy. Pretreatment with a V-ATPase inhibitor bafilomycin A and/or the lysosomotropic drug chloroquine prior to ellipticine enhanced the ellipticine‑mediated apoptosis and decreased ellipticine-resistance in UKF-NB-4ELLI cells. Moreover, pretreatment with these inhibitors increased formation of ellipticine-derived DNA adducts, one of the most important DNA-damaging mechanisms responsible for ellipticine cytotoxicity. In conclusion, resistance to ellipticine in the tested neuroblastoma cells is associated with V-ATPase-mediated vacuolar trapping of this drug, which may be decreased by bafilomycin A and/or chloroquine.
authors.affiliation: Department of Pediatric Hematology and Oncology, Second Medical School, Charles University and University Hospital Motol, 150 06 Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Hrabeta
authors.affiliation: Department of Pediatric Hematology and Oncology, Second Medical School, Charles University and University Hospital Motol, 150 06 Prague, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Groh
authors.affiliation: Department of Pediatric Hematology and Oncology, Second Medical School, Charles University and University Hospital Motol, 150 06 Prague, Czech Republic.
authors.forename: Mohamed Ashraf
authors.initials: MA
authors.lastname: Khalil
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague, Czech Republic.
authors.forename: Jitka
authors.initials: J
authors.lastname: Poljakova
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, 613 00 Brno, Czech Republic.
authors.forename: Vojtech
authors.initials: V
authors.lastname: Adam
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, 613 00 Brno, Czech Republic.
authors.forename: Rene
authors.initials: R
authors.lastname: Kizek
authors.affiliation: Department of Histology and Embryology, Second Medical School, Charles University, 150 06 Prague, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Uhlik
authors.affiliation: Department of Pediatric Hematology and Oncology, Second Medical School, Charles University and University Hospital Motol, 150 06 Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Doktorova
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Cerna
authors.affiliation: Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), D-69 120 Heidelberg, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Frei
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborova
authors.affiliation: Department of Pediatric Hematology and Oncology, Second Medical School, Charles University and University Hospital Motol, 150 06 Prague, Czech Republic.
authors.forename: Tomas
authors.initials: T
authors.lastname: Eckschlager
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics* / Q000494:pharmacology; D017209:Apoptosis; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D018736:DNA Adducts / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004611:Ellipticines / Q000493:pharmacokinetics* / Q000494:pharmacology; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D009447:Neuroblastoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D018736:DNA Adducts; D004611:Ellipticines; D018942:Macrolides; C057620:bafilomycin A; C034192:ellipticine; D002738:Chloroquine; D025262:Vacuolar Proton-Translocating ATPases