title: Chemoresistance is associated with increased cytoprotective autophagy and diminished apoptosis in bladder cancer cells treated with the BH3 mimetic (-)-Gossypol (AT-101).
abstract: BACKGROUND
  Acquired resistance to standard chemotherapy causes treatment failure in patients with metastatic bladder cancer. Overexpression of pro-survival Bcl-2 family proteins has been associated with a poor chemotherapeutic response, suggesting that Bcl-2-targeted therapy may be a feasible strategy in patients with these tumors. The small-molecule pan-Bcl-2 inhibitor (-)-gossypol (AT-101) is known to induce apoptotic cell death, but can also induce autophagy through release of the pro-autophagic BH3 only protein Beclin-1 from Bcl-2. The potential therapeutic effects of (-)-gossypol in chemoresistant bladder cancer and the role of autophagy in this context are hitherto unknown.
  
  
  METHODS
  Cisplatin (5637(r)CDDP(1000), RT4(r)CDDP(1000)) and gemcitabine (5637(r)GEMCI(20), RT4(r)GEMCI(20)) chemoresistant sub-lines of the chemo-sensitive bladder cancer cell lines 5637 and RT4 were established for the investigation of acquired resistance mechanisms. Cell lines carrying a stable lentiviral knockdown of the core autophagy regulator ATG5 were created from chemosensitive 5637 and chemoresistant 5637(r)GEMCI(20) and 5637(r)CDDP(1000) cell lines. Cell death and autophagy were quantified by FACS analysis of propidium iodide, Annexin and Lysotracker staining, as well as LC3 translocation.
  
  
  RESULTS
  Here we demonstrate that (-)-gossypol induces an apoptotic type of cell death in 5637 and RT4 cells which is partially inhibited by the pan-caspase inhibitor z-VAD. Cisplatin- and gemcitabine-resistant bladder cancer cells exhibit enhanced basal and drug-induced autophagosome formation and lysosomal activity which is accompanied by an attenuated apoptotic cell death after treatment with both (-)-gossypol and ABT-737, a Bcl-2 inhibitor which spares Mcl-1, in comparison to parental cells. Knockdown of ATG5 and inhibition of autophagy by 3-MA had no discernible effect on apoptotic cell death induced by (-)-gossypol and ABT-737 in parental 5637 cells, but evoked a significant increase in early apoptosis and overall cell death in BH3 mimetic-treated 5637(r)GEMCI(20) and 5637(r)CDDP(1000) cells.
  
  
  CONCLUSIONS
  Our findings show for the first time that (-)-gossypol concomitantly triggers apoptosis and a cytoprotective type of autophagy in bladder cancer and support the notion that enhanced autophagy may underlie the chemoresistant phenotype of these tumors. Simultaneous targeting of Bcl-2 proteins and the autophagy pathway may be an efficient new strategy to overcome their "autophagy addiction" and acquired resistance to current therapy.
authors.affiliation: Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. jens.mani@kgu.de.
authors.forename: Jens
authors.initials: J
authors.lastname: Mani
authors.affiliation: Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. stefan.vallo@kgu.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Vallo
authors.affiliation: Experimental Neurosurgery, Neuroscience Center, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. stefanie.rakel@kgu.de.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Rakel
authors.affiliation: Experimental Neurosurgery, Neuroscience Center, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. antonietti@med.uni-frankfurt.de.
authors.forename: Patrick
authors.initials: P
authors.lastname: Antonietti
authors.affiliation: Experimental Neurosurgery, Neuroscience Center, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. f.gessler@med.uni-frankfurt.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Gessler
authors.affiliation: Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. blaheta@em.uni-frankfurt.de.
authors.forename: Roman
authors.initials: R
authors.lastname: Blaheta
authors.affiliation: Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. georg.bartsch@kgu.de.
authors.forename: Georg
authors.initials: G
authors.lastname: Bartsch
authors.affiliation: Institute for Medical Virology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. m.michaelis@kent.ac.uk.|School of Biosciences, The University of Kent, Canterbury, Kent, CT2 7NZ, UK. m.michaelis@kent.ac.uk.
authors.forename: Martin
authors.initials: M
authors.lastname: Michaelis
authors.affiliation: Institute for Medical Virology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. cinatl@em.uni-frankfurt.de.
authors.forename: Jindrich
authors.initials: J
authors.lastname: Cinatl
authors.affiliation: Department of Urology, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. axel.haferkamp@kgu.de.
authors.forename: Axel
authors.initials: A
authors.lastname: Haferkamp
authors.affiliation: Experimental Neurosurgery, Neuroscience Center, Goethe University Hospital, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. koegel@em.uni-frankfurt.de.
authors.forename: Donat
authors.initials: D
authors.lastname: Kögel
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000008:administration & dosage; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006072:Gossypol / Q000008:administration & dosage / Q000031:analogs & derivatives*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D010446:Peptide Fragments / Q000008:administration & dosage; D011518:Proto-Oncogene Proteins / Q000008:administration & dosage; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D000093542:Gemcitabine
chemical_list: D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; C474007:Bax protein (53-86); D000071186:Beclin-1; D008565:Membrane Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D003841:Deoxycytidine; D006072:Gossypol; D002945:Cisplatin; C028178:gossypol acetic acid; D000093542:Gemcitabine