Disruption of glycolytic enzyme activity and cellular energetics by the anti-tumor agent 3-bromopyruvate (3BrPA) Free

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BACKGROUND & METHODS: 3BrPA has been reported to eradicate advanced hepatocellular carcinoma in animals without any associated systemic toxicity. Although widely attributed to selective hexokinase (HK) inhibition and ATP depletion in glycolysis-dependent cancer cells, the molecular basis of this effect is poorly understood and specific interaction with HK has been only cursorily examined. To better understand both its mechanism(s) of action and its cytotoxic potential in non-tumor cells, we tested 3BrPA for the ability to directly alter glycolytic enzyme activity, lactate accumulation, ATP content, and cell viability in non-transformed renal epithelial cells. RESULTS: 3BrPA inhibited HK activity in cell-free lysates in a concentration-dependent manner (IC50 0.7 mM), an effect that was less potently mimicked by the alkylating agent iodoacetate (IAA; IC50 8 mM), but not by pyruvate, the 3BrPA analogue 3-fluoropyruvate (3FPA) or the alkylating agent iodoacetamide (IAM). A similar pattern and hierarchy of inhibition was observed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with 3BrPA representing a much more potent inhibitor of this enzyme than its classic antagonist IAA (IC50 0.0025 vs 0.15 mM). Monothioglycerol had no effect on basal HK activity but markedly reduced in vitro sensitivity to both 3BrPA and IAA inhibition. The effect of 3BrPA was also non-competitive with the HK substrates glucose (Glc) and ATP. When examined in intact cells, 3BrPA and IAA reduced both ATP content and lactate accumulation at micromolar concentrations that were orders of magnitude lower than those required for HK inhibition in vitro. Esterification of 3BrPA to maximize cell permeability did not enhance its effects. Cytotoxic LDH release was only observed following profound ATP depletion, which was uniformly greater for IAA. 3BrPA did not induce apoptosis at concentrations that were not overtly cytotoxic and where ATP levels were relatively well maintained. CONCLUSIONS: 3BrPA inhibits HK activity, ostensibly via selective alkylation of sulfhydryl groups important for enzymatic function, but not directly involved in Glc or ATP binding. However, glycolytic inhibitory potency appears to correlate better with GAPDH inhibition than with HK inhibition in non-tumor epithelial cells. It also corresponds closely to that described previously for cancer cells. The ability of IAA - but not 3FPA or IAM - to mimic 3BrPA suggests a specific shared mechanism of action, although the greater relative ATP depletion and cytotoxicity associated with IAA treatment also suggests actions additional to those shared with 3BrPA. These findings suggest that 3BrPA can affect glycolytic flux at multiple points in 'normal' cells' and thus raise significant questions regarding both the selectivity and specific determinants of 3BrPA action that warrant additional investigation.