Targeting MCT1 Blocks Tumor Angiogenesis Figure 9. Model illustrating the anti-angiogenic activity of MCT1 inhibitors targeting lactate-induced HIF-1 activation in endothelial cells. HIF-1 activity primarily depends on the stability of HIF-1a. When HIF prolylhydroxylase (PHD) is active as it is the case for normoxic endothelial cells (ECs) in oxidative tissues (left), HIF-1a is hydroxylated and addressed to the proteasome for degradation. PHD requires 2-oxoglutarate as a substrate and is competitively inhibited by pyruvate. Normoxic ECs in glycolytic tissues such as tumors (right) take up exogenous lactate originating from distant hypoxic cells, a process under the control of monocarboxylate transporter 1 (MCT1). ECs do not primarily use lactate as a fuel for oxidative phosphorylation (OXPHOS). Lactate oxidation by LDH-B rather increases the pool of intracellular pyruvate, now available to compete with 2-oxoglutarate from PHD, resulting in HIF-1a protein stabilization, HIF-1 activation, and angiogenesis. Because MCT1 is the main transporter for lactate uptake by ECs, targeting MCT1 inhibits lactate-induced angiogenesis in tumors. Abbreviations: asc., ascorbate; dehydroasc., dehydroascorbate; GLUT, glucose transporter. doi:10.1371/journal.pone.0033418.g009 containing Matrigel plugs (Figure 8A) and the apparent anti- vascular effects of MCT1 inhibition in vivo (Figure 8C) are related to changes in endothelial HIF-2 activity remains to be determined. The anti-angiogenic effects of MCT1 inhibitors that we document here offers an additional rationale for the antitumor effects of MCT1 inhibitors that we identified previously in several types of tumors and attributed to metabolic effects [26]. In a model of colon cancer xenograft involving the co-injection of HUVECs, a downregulation of MCT1 expression in ECs was indeed documented to significantly retard tumor growth [23]. In conclusion, we have shown that lactate activates HIF-1 in normoxic ECs and consecutively promotes the expression of bFGF and VEGFR2, the latter reinforcing the stimulation of the VEGF signaling pathway promoted by the lactate-induced VEGF expression occurring in tumor and stromal cells. Importantly, we also report that in ECs, MCT1 inhibition can thus drive direct anti-angiogenic effects through a reduction in HIF-1 activity, which, together with the antimetabolic effects of MCT1 inhibition on tumor cells, considerably enlarges the therapeutic potential of this class of drugs. Materials and Methods Cells and chemicals BAECs (Clonetics) were routinely cultured in EBM-EGM (Cambrex), HUVECs (Clonetics) in EBM2-MV (Cambrex), and LLc (LGC Standards, [54]) and TLT (originally obtained at from HS Taper, University of Louvain [55]) tumor cells in DMEM containing 4500 mg/liter glucose (Invitrogen) with 10% FBS at 5% CO2 and 21% O2, 37uC. Glucose-deprived DMEM was from Krackeler Scientific. Media were supplemented with 1% penicillin-streptomy- cin. All experiments were performed in pyruvate-free media buffered at pH 7.3 (3.7 g/liter NaHCO3). To minimize fluctuations due to unequal growth rates, all assays were performed on confluent cells. Hypoxia (1% O2) was achieved as previously described [56]. Cell death was quantified using the NucleoCounter device from ChemoMetec. Unless stated otherwise, all chemicals including CHC were from Sigma. Carboxylates were used as sodium salts. Determination of intracellular L-lactate and pyruvate For NMR experiments, HUVECs in EBM2 media were incubated during 6-h to 10 mM 13C-methyl-labeled lactate (Isotec, Sigma), washed twice with DPBS, lysed and collected in 0.9 M perchloric acid (1:10 in diH2O). 12C-(unlabeled)-lactate was used as control. Lysates were spun at 4uC for 10 min at 12,000 rpm. 13C and 1H NMR spectra acquisitions were performed on supernatants using a 500-MHz Varian Inova spectrometer equipped with a 5- mm tunable broadband probe. 13C-NMR spectra were acquired at 125.7 MHz with 45u flip angle, 0.8-second interpulse delay, a 1.334 s acquisition time, and at a temperature of 25uC. Control runs of known concentrations of lactate were used to assess peak PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33418