Targeting MCT1 Blocks Tumor Angiogenesis Figure 1. Endothelial cells import exogenous lactate. (A and B) HUVECs were incubated during 6-h with 10 mM of 13C-methy-labeled sodium lactate (left panels) or 10 mM of 12C sodium lactate (right panels). 13C-lactate was detected using nuclear magnetic resonance (NMR) in cell lysates. (A) In 13C NMR spectra, a 13C-Lactate peak is detected at 19 ppm. (B) In 1H NMR spectra, the methyl doublets at 1.56 and 1.30 ppm arise from 13C- lactate whereas the doublet at 1.43 ppm arises from a coincidental overlap of endogenous 12C-Lactate and threonine. (C) HUVECs were incubated with 10 mM sodium L-lactate. L-Lactate was detected using an enzymatic assay in the lysate of cells collected at different time points. *p,0.05; n = 4. Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g001 pyruvate is preferentially catalyzed by the lactate dehydrogenase-B (LDH-B) gene product. We found that the LDH competitor oxamate [31] actually blocked lactate-induced HIF-1a protein expression in normoxic ECs, a property which was not shared by the structurally-related monocarboxylate acetate (Figure 4F, a representative blot is shown in Figure S4B). Oxamate inhibits both LDH-A and LDH-B. To confirm the involvement of LDH-B, LDH-B was silenced with a specific siRNA (see Figure S5A). This also prevented lactate-induced HIF-1a expression (Figure 4G and Figure S4C). Finally, we also provided evidence that pyruvate was a more potent inducer of HIF-1a than lactate in normoxic ECs (Figure 4H). Targeting MCT1 blocks lactate-induced HIF-1 activation in normoxic ECs The lactate anion can shuttle between extracellular and intracellular compartments through monocarboxylate transporters (MCTs) among which MCT subtypes 1–4 function as passive lactate-proton symporters [32,33]. Using quantitative PCR to compare mRNA levels of these 4 transporters, we identified MCT1 as the main transcript in HUVECs (Figure 5A). The correspond- ing protein was localized at the plasma membrane of the cells, as shown by immunocytochemistry in Figure 5B and in Figure S3B where MCT1, its anchor protein basigin/CD147 and the endothelial marker CD31 colocalized. Because MCT1 mediates the uptake of lactate in several cell types [26,32,33], we checked whether MCT1 was involved in lactate signaling in ECs. Silencing MCT1 with a specific siRNA (see Figure S5B for target extinction) prevented HIF-1a protein stabilization in HUVECs incubated with lactate (10 mM) for 24 h (Figure 5C). Consistent with the involvement of MCT1, a-cyano-4-hydroxycinnamate (CHC), a reversible inhibitor that has a 10-fold selectivity for MCT1 compared to other MCTs [34], prevented lactate-induced HIF-1a protein expression not only in HUVECs (Figure 5D) but also in BAECs (Figure 5E) without showing intrinsic EC toxicity (Figure S6). These results were verified using a series of known MCT1 inhibitors that all successfully blocked lactate-induced HIF-1a protein stabilization in BAECs (Figure S7). The functional PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33418