title: Pazopanib and sunitinib trigger autophagic and non-autophagic death of bladder tumour cells.
abstract: BACKGROUND
  Tyrosine kinase inhibitors (TKI) such as sunitinib and pazopanib display their efficacy in a variety of solid tumours. However, their use in therapy is limited by the lack of evidence about the ability to induce cell death in cancer cells. Our aim was to evaluate cytotoxic effects induced by sunitinib and pazopanib in 5637 and J82 bladder cancer cell lines.
  
  
  METHODS
  Cell viability was tested by MTT assay. Autophagy was evaluated by western blot using anti-LC3 and anti-p62 antibodies, acridine orange staining and FACS analysis. Oxygen radical generation and necrosis were determined by FACS analysis using DCFDA and PI staining. Cathepsin B activation was evaluated by western blot and fluorogenic Z-Arg-Arg-AMC peptide. Finally, gene expression was performed using RT-PCR Profiler array.
  
  
  RESULTS
  We found that sunitinib treatment for 24 h triggers incomplete autophagy, impairs cathepsin B activation and stimulates a lysosomal-dependent necrosis. By contrast, treatment for 48 h with pazopanib induces cathepsin B activation and autophagic cell death, markedly reversed by CA074-Me and 3-MA, cathepsin B and autophagic inhibitors, respectively. Finally, pazopanib upregulates the α-glucosidase and downregulates the TP73 mRNA expression.
  
  
  CONCLUSIONS
  Our results showing distinct cell death mechanisms activated by different TKIs, provide the biological basis for novel molecularly targeted approaches.
authors.affiliation: Department of Medical Oncology, Polytechnic University of the Marche Region, 60121 Ancona, Italy. giorgio.santoni@unicam.it
authors.forename: M
authors.initials: M
authors.lastname: Santoni
authors.forename: C
authors.initials: C
authors.lastname: Amantini
authors.forename: M B
authors.initials: MB
authors.lastname: Morelli
authors.forename: S
authors.initials: S
authors.lastname: Liberati
authors.forename: V
authors.initials: V
authors.lastname: Farfariello
authors.forename: M
authors.initials: M
authors.lastname: Nabissi
authors.forename: L
authors.initials: L
authors.lastname: Bonfili
authors.forename: A M
authors.initials: AM
authors.lastname: Eleuteri
authors.forename: M
authors.initials: M
authors.lastname: Mozzicafreddo
authors.forename: L
authors.initials: L
authors.lastname: Burattini
authors.forename: R
authors.initials: R
authors.lastname: Berardi
authors.forename: S
authors.initials: S
authors.lastname: Cascinu
authors.forename: G
authors.initials: G
authors.lastname: Santoni
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D002294:Carcinoma, Squamous Cell / Q000473:pathology; D002295:Carcinoma, Transitional Cell / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D007191:Indazoles; D007211:Indoles / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009336:Necrosis / Q000139:chemically induced*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D017382:Reactive Oxygen Species; D013449:Sulfonamides / Q000494:pharmacology*; D000077210:Sunitinib; D001749:Urinary Bladder Neoplasms / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D007191:Indazoles; D007211:Indoles; D011743:Pyrimidines; D011758:Pyrroles; D017382:Reactive Oxygen Species; D013449:Sulfonamides; C516667:pazopanib; D011505:Protein-Tyrosine Kinases; D000077210:Sunitinib