title: Inhibition of hexokinase-2 with targeted liposomal 3-bromopyruvate in an ovarian tumor spheroid model of aerobic glycolysis.
abstract: BACKGROUND
  The objective of this study was to evaluate the expression levels of glycolytic markers, especially hexokinase-2 (HK2), using a three-dimensional multicellular spheroid model of human ovarian adenocarcinoma (SKOV-3) cells and to develop an epidermal growth factor receptor-targeted liposomal formulation for improving inhibition of HK2 and the cytotoxicity of 3-bromopyruvate (3-BPA).
  
  
  METHODS
  Multicellular SKOV-3 tumor spheroids were developed using the hanging drop method and expression levels of glycolytic markers were examined. Non-targeted and epidermal growth factor receptor-targeted liposomal formulations of 3-BPA were formulated and characterized. Permeability and cellular uptake of the liposomal formulations in three-dimensional SKOV-3 spheroids was evaluated using confocal microscopy. The cytotoxicity and HK2 inhibition potential of solution form of 3-BPA was compared to the corresponding liposomal formulation by using cell proliferation and HK2 enzymatic assays.
  
  
  RESULTS
  SKOV-3 spheroids were reproducibly developed using the 96-well hanging drop method, with an average size of 900 µm by day 5. HK2 enzyme activity levels under hypoxic conditions were found to be higher than under normoxic conditions (P<0.0001, Student's t-test, unpaired and two-tailed). Liposomal formulations (both non-targeted and targeted) of 3-BPA showed a more potent inhibitory effect (P<0.001, Student's t-test, unpaired and two-tailed) at a dose of 50 µM than the aqueous solution form at 3, 6, and 24 hours post administration. Similarly, the cytotoxic activity 3-BPA at various concentrations (10 µM-100 µM) showed that the liposomal formulations had an enhanced cytotoxic effect of 2-5-fold (P<0.0001, Student's t-test, unpaired and two-tailed) when compared to the aqueous solution form for both 10 µM and 25 µM concentrations.
  
  
  CONCLUSIONS
  SKOV-3 spheroids developed by the hanging drop method can be used as a tumor aerobic glycolysis model for evaluation of therapies targeting the glycolytic pathway in cancer cells. Encapsulation of 3-BPA in a liposomal formulation improved permeability, HK2 inhibition, and cytotoxicity in the multicellular spheroid model.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA, USA.
authors.forename: Srujan Kumar
authors.initials: SK
authors.lastname: Gandham
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA, USA.
authors.forename: Meghna
authors.initials: M
authors.lastname: Talekar
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Singh
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA, USA.
authors.forename: Mansoor M
authors.initials: MM
authors.lastname: Amiji
mesh_terms: D000332:Aerobiosis; D045744:Cell Line, Tumor; D005260:Female; D006019:Glycolysis / Q000187:drug effects; D006593:Hexokinase / Q000037:antagonists & inhibitors*; D006801:Humans; D008081:Liposomes / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
chemical_list: D008081:Liposomes; D011773:Pyruvates; C017092:bromopyruvate; D006593:Hexokinase
keywords: 3-bromopyruvate; aerobic glycolysis; epidermal growth factor receptor-targeted liposomes; hexokinase-2; ovarian cancer