Page 656 - Read Online

Page 8 of 14 Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102

A GPR81 B MCT1 MCT4

C MCT1 MCT4

MCT1 MCT4
β-actin β-actin

Figure 3. GPR81 regulates expression of the gene involved in lactate import in MCF-7 epithelial breast cancer cell line. A: real-time qPCR
analysis of GPR81; B: MCT1 and MCT4 mRNA levels in MCF-7-siNT (control) and MCF-7-siGPR81 cultured in 3-dimensional Matrigel
with physiological modified medium at 72 and 96 h after transfection; C: Western blot analysis of cell lysates from MCF-7-siNT and MCF-
7-siGPR81 cells used to detect protein expression levels of MCT1 and MCT4. β-actin was used as a loading control. The bars represent
the mean ± S.E.M of three independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (ordinary one-way ANOVA)

A Intracellular Lactate B Extracellular Lactate

C Glucose Consumption

Figure 4. GPR81 regulates lactate uptake in MCF-7 epithelial breast cancer cell lines. A: intracellular lactate of MCF-7-siNT (control) and
MCF-7-siGPR81 measured using NMR analysis after 72 h of transfection; B: lactate concentration in the medium of MCF-7-siNT 10 nmol/L
and MCF-7-siGPR81 10 nmol/L were measured after 96 h of transfection; C: glucose consumption rate of MCF-7-siNT and MCF-7- siGPR81
were measured after 96 h of transfection. MCF-7-siNT and MCF-7-siGPR81 cells were cultured and transfected in 3-dimensional Matrigel
with physiological modified medium and measurements were taken after 24 h of culture in fresh medium. The bars represent the mean
± S.E.M of three independent experiments; *P < 0.05; **P < 0.01; (ordinary one-way ANOVA)

lactate importer, we hypothesized that the reduction of MCT1 expression may affect the lactate uptake of the
epithelial MCF-7 breast cancer cells. To test this hypothesis, we measured the intracellular lactate of non-
targeted control MCF-7-siNT and MCF-7-siGPR81 in MPM culture using NMR analysis. MCF-7-siGPR81 cells
had significant lower intracellular lactate compared to MCF-7-siNT cells [Figure 4A], indicating reduced
lactate uptake of MCF-7-siGPR81 cells. Additionally, GPR81 knockdown resulted in higher levels of lactate