title: Paclitaxel-hyaluronan hydrosoluble bioconjugate: mechanism of action in human bladder cancer cell lines.
abstract: OBJECTIVE
  A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells.
  
  
  METHODS
  Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on β-tubulin organization was carried out by confocal microscopy.
  
  
  RESULTS
  The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the β-tubulin microtubular mesh with subsequent cell death.
  
  
  CONCLUSIONS
  Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.
authors.affiliation: Istituto Oncologico Veneto IRCCS, Padova, Italy.
authors.forename: Isabella Monia
authors.initials: IM
authors.lastname: Montagner
authors.forename: Alessandra
authors.initials: A
authors.lastname: Banzato
authors.forename: Gaia
authors.initials: G
authors.lastname: Zuccolotto
authors.forename: Davide
authors.initials: D
authors.lastname: Renier
authors.forename: Monica
authors.initials: M
authors.lastname: Campisi
authors.forename: Pierfrancesco
authors.initials: P
authors.lastname: Bassi
authors.forename: Paola
authors.initials: P
authors.lastname: Zanovello
authors.forename: Antonio
authors.initials: A
authors.lastname: Rosato
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport; D001896:Boron Compounds / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D017239:Paclitaxel / Q000737:chemistry* / Q000494:pharmacology*; D011485:Protein Binding; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000970:Antineoplastic Agents; D001896:Boron Compounds; D005456:Fluorescent Dyes; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D017239:Paclitaxel