Inhibition of hexokinase-2 with targeted liposomal 3-bromopyruvate in an ovarian tumor spheroid model of aerobic glycolysis

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 mu m by day 5. HK2 enzyme activity levels under hypoxic conditions were found to be higher than under normoxic conditions (P < .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 mu M than the aqueous solution form at 3, 6, and 24 hours post administration. Similarly, the cytotoxic activity 3-BPA at various concentrations (10 mu M-100 mu M) 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 mu M and 25 mu M concentrations. Conclusion: 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.