Evaluating the Glycolytic Pathway in Retinoblastoma: A Molecular Mechanistic Approach Using the Glycolytic Inhibitor 2-deoxy-D-glucose in vitro and in vivo

Abstract

Purpose: : To assess the molecular mechanism of glycolytic inhibition: (1) in vivo employing the LH_BETAT_AG retinoblastoma (RB) animal model, and (2) in vitro using two established human RB cell lines Y-79 and WERI-Rb-1.

Methods: : The study protocol was approved by the University of Miami Institutional Animal Care and Use Review Board Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. (1) To measure Hypoxia Inducible Factor-1α (HIF) and hexokinase II (HKII) expression in crude retinal tumor extracts, 16-week-old mice received one periocular injection of 2-deoxy-D-glucose (2-DG; 500 mg/kg). Two hours post injection, animals were euthanized and tumor extracts were analyzed for HIF and HKII expression using Western Blot. (2a) To measure basal levels of HIF and HKII in RB cells, Y-79 and WERI-Rb-1 cells were exposed to normoxia or hypoxia (0.5% O2) conditions for 24 hours. Cells were harvested and basal levels of HIF and HKII were measured using SDS-PAGE and Western Blot. (2b) To assay 2-DG cytotoxicity at increasing concentrations while under hypoxic conditions, Y-79 cells were treated with 0.1 mM, 0.5 mM, or 10 mM of 2-DG and placed in a chamber with 0.5% levels of oxygen. After 72 hours, viability and cytotoxicity were analyzed.

Results: : (1) Two hours post injection of 2-DG, there was a 41% reduction in HKII levels and 37% increase in HIF levels compared to the non-treated tumors (p≤0.05). (2a) Basal levels of HKII expression increased by two-fold in both cell lines (Y-79 and WERI-Rb-1) under hypoxic conditions when compared to normoxia (p≤0.05), while HIF levels remained the same (p=0.751). (2b) Following treatment with 2-DG under hypoxic conditions, Y-79 cell cytotoxicity increased by 29.3%, 27.4%, and 35.2% at 0.1 mM, 0.5 mM, and 10 mM concentrations of 2-DG when compared to non-treated control (p=0.028, p=0.055, and p=0.024, respectively).

Conclusions: : This study provides a better understanding of anaerobic glucose metabolism and its association with 2-DG efficacy in the LH_BETAT_AG retinoblastoma animal model. This study suggests for the first time that one mechanism of 2-DG tumor control is the change in HIF and HKII activity in retinoblastoma.

Keywords: retinoblastoma • transgenics/knock-outs • hypoxia