Cellular Uptake of MCT1 Inhibitors AR-C155858 and AZD3965 and Their Effects on MCT-Mediated Transport of L-Lactate in Murine 4T1 Breast Tumor Cancer Cells

Abstract

AR-C155858 and AZD3965, pyrrole pyrimidine derivatives, represent potent monocarboxylate transporter 1 (MCT1) inhibitors, with potential immunomodulatory and chemotherapeutic properties. Currently, there is limited information on the inhibitory properties of this new class of MCT1 inhibitors. The purpose of this study was to characterize the concentration- and time-dependent inhibition of L-lactate transport and the membrane permeability properties of AR-C155858 and AZD3965 in the murine 4T1 breast tumor cells that express MCT1. Our results demonstrated time-dependent inhibition of L-lactate uptake by AR-C155858 and AZD3965 with maximal inhibition occurring after a 5-min pre-incubation period and prolonged inhibition. Following removal of AR-C155858 or AZD3965 from the incubation buffer, inhibition of L-lactate uptake was only fully reversed after 3 and 12 h, respectively, indicating that these inhibitors are slowly reversible. The uptake of AR-C155858 was concentration-dependent in 4T1 cells, whereas the uptake of AZD3965 exhibited no concentration dependence over the range of concentrations examined. The uptake kinetics of AR-C155858 was best fitted to a Michaelis-Menten equation with a diffusional clearance component, P (K_m = 0.399 ± 0.067 μM, V_max = 4.79 ± 0.58 pmol/mg/min, and P = 0.330 ± 0.088 μL/mg/min). AR-C155858 uptake, but not AZD3965 uptake, was significantly inhibited by alpha-cyano-4-hydroxycinnamic acid, a known nonspecific inhibitor of MCTs 1, 2, and 4. AR-C155858 demonstrated a trend toward higher uptake at lower pH, a characteristic of proton-dependent MCT1. These findings provide evidence that AR-C155858 and AZD3965 exert slowly reversible inhibition of MCT1-mediated L-lactate uptake in 4T1 cells, with AR-C155858 representing a potential substrate of MCT1.

Keywords: AR-C155858; AZD3965; L-lactate; MCT1 inhibitor; Monocarboxylate transporter 1.

Fig. 1.

Chemical structure of AR-C155858 (a) and AZD3965 (b)

Fig. 2.

The time-dependent inhibition of L-lactate (0.5 mM) uptake by AR-C155858 (a) and AZD3965 (b) at 37 °C, pH 6.0, at a concentration of 200 nM. Data are presented as mean ± SD, n = 3

Fig. 3.

Inhibition of L-lactate uptake by AR-C15585 and AZD3965 at pH 6.0 and 7.4 in the 4T1 cells. The effect of L-lactate (0.5 mM) uptake under various AR-C155858 (a) and AZD3965 (b) (200 nM) treatment conditions: (1) uptake of L-lactate alone, (2) pretreatment with the inhibitor followed by uptake of L-lactate, (3) pretreatment with the inhibitor followed by washing the cells three times with ice-cold buffer and uptake of L-lactate. The inhibition of L-lactate uptake by AR-C155858 (c) and AZD3965 (d) (200 nM) at various times after the removal of the inhibitors, as in treatment condition (3). Dotted line represents time to return back to the baseline of L-lactate uptake at pH 6.0 baseline of L-lactate uptake alone at pH 6.0 (c) and (d). The reference line for AR-C155858 (c) was based on the baseline from the study with AZD3965 (d). ****P < 0.0001 compared to the control at pH 6.0; ^##P < 0.01, ^###P < 0.001 compared to the control at pH 7.4. Two-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD, n = 3

Fig. 4.

Inhibition of L-lactate uptake by AR-C15585 at pH 6.0 and 7.4 in the HCC1937 cells that express both MCT1 and MCT4. The effect of L-lactate (0.5 mM) uptake under various AR-C155858 (a) (200 nM) treatment conditions: (1) uptake of L-lactate alone, (2) pretreatment with the inhibitor followed by uptake of L-lactate, (3) pretreatment with the inhibitor followed by washing the cells three times with ice-cold buffer and uptake of L-lactate. The inhibition of L-lactate uptake by AR-C155858 (200 nM) at various times after the removal of the inhibitor, as in treatment condition (3). Dotted line represents time return back to the baseline of L-lactate uptake alone at pH 6.0 (b). ****P < 0.0001 compared to the control at pH 6.0; ^###P < 0.001 compared to the control at pH 7.4. Two-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD, n = 3

Fig. 5.

The AR-C155858 and AZD3965 uptake in the 4T1 cells at physiological pH (serum-free medium). The time-dependent uptake of AR-C155858, 30 nM (a) and AZD3965, 50 nM (b). The concentration-dependent uptake of AR-C155858 (c) and AZD3965 (d). The uptake of AR-C-155858 and AZD3965 was carried out for 2 and 5 min, respectively. The concentration-dependent uptake of AR-C155858 was best fitted to a Michaelis-Menten equation with a diffusional uptake clearance component (P). Observed mean data are shown by symbols and the line represents model fitted results. Data are presented as mean ± SD, n = 3–6

Fig. 6.

The effect of CHC on the cellular uptake of AR-C155858 and AZD3965 in 4T1 cells. The uptake of AR-C155858 (a) and AZD3965 (b) in the absence and presence of CHC (20 mM) at physiological pH (serum-free medium). Cells were pre-incubated with CHC for 30 min at 37 °C followed by uptake of AR-C155858 or AZD3965 at RT for 2 or 5 min, respectively. NS nonsignificant; **P < 0.01; ***P < 0.001; ****P < 0.0001 compared to the uptake AR-C155858 or AZD3965 in the absence of CHC. Two-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD, n = 3

Fig. 7.

The effect of pH and sodium on the uptake of AR-C155858 in 4T1 cells. The uptake of AR-C155858 (30 nM) at various pH (a) and in the presence or absence of sodium (b). *P < 0.05; ***P < 0.001. One-way ANOVA followed by Dunnett’s post hoc test. Data are presented as mean ± SD, n = 3