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Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.

Most malignant tumors exhibit the Warburg effect, which consists in increased glycolysis rates with production of lactate, even in the presence of oxygen. Monocarboxylate transporters (MCTs), maintain these glycolytic rates, by mediating the influx and/or efflux of lactate and are overexpressed in several cancer cell types. The lactate and pyruvate analogue 3-bromopyruvate (3-BP) is an inhibitor of the energy metabolism, which has been proposed as a specific antitumor agent. In the present study, we aimed at determining the effect of 3-BP in breast cancer cells and evaluated the putative role of MCTs on this effect. Our results showed that the three breast cancer cell lines used presented different sensitivities to 3-BP: ZR-75-1 ER (+)>MCF-7 ER (+)>SK-BR-3 ER (-). We also demonstrated that 3-BP reduced lactate production, induced cell morphological alterations and increased apoptosis. The effect of 3-BP appears to be cytotoxic rather than cytostatic, as a continued decrease in cell viability was observed after removal of 3-BP. We showed that pre-incubation with butyrate enhanced significantly 3-BP cytotoxicity, especially in the most resistant breast cancer cell line, SK-BR-3. We observed that butyrate treatment induced localization of MCT1 in the plasma membrane as well as overexpression of MCT4 and its chaperone CD147. Our results thus indicate that butyrate pre-treatment potentiates the effect of 3-BP, most probably by increasing the rates of 3-BP transport through MCT1/4. This study supports the potential use of butyrate as adjuvant of 3-BP in the treatment of breast cancer resistant cells, namely ER (-).

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1-18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H(2)O(2) scavenging effect to exogenous H(2)O(2), while lactate had no scavenging effect. 3BP induced H(2)O(2) production. Pyruvate protected against H(2)O(2)-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.