Monocarboxylate transporter 4 involves in energy metabolism and drug sensitivity in hypoxia

Abstract

Metabolic reprogramming of cancer cells is a potential target for cancer therapy. It is also known that a hypoxic environment, one of the tumor microenvironments, can alter the energy metabolism from oxidative phosphorylation to glycolysis. However, the relationship between hypoxia and drug sensitivity, which targets energy metabolism, is not well known. In this study, A549 cells, a cell line derived from lung adenocarcinoma, were evaluated under normoxia and hypoxia for the sensitivity of reagents targeting oxidative phosphorylation (metformin) and glycolysis (α-cyano-4-hydroxycinnamic acid [CHC]). The results showed that a hypoxic environment increased the expression levels of monocarboxylate transporter (MCT) 4 and hypoxia-induced factor-1α (HIF-1α), whereas MCT1 and MCT2 expression did not vary between normoxia and hypoxia. Furthermore, the evaluation of the ATP production ratio indicated that glycolysis was enhanced under hypoxic conditions. It was then found that the sensitivity to metformin decreased while that to CHC increased under hypoxia. To elucidate this mechanism, MCT4 and HIF-1α were knocked down and the expression level of MCT4 was significantly decreased under both conditions. In contrast, the expression of HIF-1α was decreased by HIF-1α knockdown and increased by MCT4 knockdown. In addition, changes in metformin and CHC sensitivity under hypoxia were eliminated by the knockdown of MCT4 and HIF-1α, suggesting that MCT4 is involved in the phenomenon described above. In conclusion, it was shown that the sensitivity of reagents targeting energy metabolism is dependent on their microenvironment. As MCT4 is involved in some of these mechanisms, we hypothesized that MCT4 could be an important target molecule for cancer therapy.

Figure 1

Protein expression level of MCT1, MCT2, MCT4, and HIF-1α in normoxia and hypoxia. (A) Western blot analysis of MCT1, MCT2, MCT4, and HIF-1α in normoxia and hypoxia in A549 cells. The protein expression levels of MCT1 (B), MCT2 (C), MCT4 (D), and HIF-1α (E) were visualized, respectively. Data are presented as mean ± standard error of three independent experiments. *p < 0.05 compared with the normoxia using unpaired Student’s t test.

Figure 2

ATP production rate in normoxia and hypoxia. Mitochondrial respiratory activity was evaluated using a Seahorse XFp analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were calculated by normalizing the cell counts under normoxia (A) and hypoxia (B). Each mark represents the mean with a positive SE of three or four independent experiments. (C) ATP production rate under normoxia and hypoxia calculated from (A,B), respectively. Each mark represents the mean of three or four independent experiments.

Figure 3

Effects of metformin and CHC on cell viability with concentration dependency of each reagent and time dependency of hypoxic dependency. Concentration dependence of metformin 10 mM and 20 mM (A–C) and CHC 5 mM and 10 mM (D–F) for cell viability using the MTT assay. Time dependence of cell viability under hypoxic conditions (normoxia 72 h, normoxia 24 h + hypoxia 48 h, and hypoxia 72 h) using the MTT assay. Data are presented as the mean ± standard error of three independent experiments. *p < 0.05, compared with the control; ^†p < 0.05, compared with metformin 10 mM or CHC 5 mM using the Tukey–Kramer test.

Figure 4

Effects of MCT4 and HIF-1α siRNA on protein expression level and sensitivity of metformin and CHC. Western blot analysis after transfection with MCT4 and HIF-1α siRNA under normoxia (A) and hypoxia (B). Cell viability after treatment with 10 mM metformin under normoxia (C) and hypoxia (D) in A549 cells transfected with siRNA. Viability of CHC 5 mM under normoxia (E) and hypoxia (F) in A549 cells transfected with siRNA. Data are presented as the mean ± standard error of three independent experiments. *p < 0.05, compared with the control; ^†p < 0.05, compared with metformin 10 mM or CHC 5 mM using the Tukey–Kramer test.