title: Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN).
abstract: BACKGROUND
  Current chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects.
  
  
  OBJECTIVE
  The acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines.
  
  
  METHODS
  We assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo.
  
  
  RESULTS
  Combination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program.
  
  
  CONCLUSIONS
  We suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.
authors.affiliation: Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada. syedshahidul@yahoo.com.|Cancer Biology and Experimental Therapeutics Section, Division of Molecular Oncology, King Faisal Specialist Hospital and Research Centre, MBC# 03-66, PO Box 3354, Riyadh, 11211, Saudi Arabia. syedshahidul@yahoo.com.
authors.forename: S S
authors.initials: SS
authors.lastname: Islam
authors.affiliation: Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: R B
authors.initials: RB
authors.lastname: Mokhtari
authors.affiliation: Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Akbari
authors.affiliation: Faculty of Medicine, Charles University in Prague, Karlovaska, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Hatina
authors.affiliation: Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: H
authors.initials: H
authors.lastname: Yeger
authors.affiliation: Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: W A
authors.initials: WA
authors.lastname: Farhat
mesh_terms: D000086:Acetazolamide / Q000008:administration & dosage; D000818:Animals; D016588:Anticarcinogenic Agents / Q000008:administration & dosage; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005260:Female; D006801:Humans; D017879:Isothiocyanates / Q000008:administration & dosage; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011897:Random Allocation; D013454:Sulfoxides; D015996:Survival Rate; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays
chemical_list: D016588:Anticarcinogenic Agents; D017879:Isothiocyanates; D013454:Sulfoxides; C016766:sulforaphane; D000086:Acetazolamide