The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters

Abstract

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki2.5 μM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevisoocytes with K0.5 and Hill coefficient values of 36-40 μM and 1.65-1.85 respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~ 7 μM) than other substrates including glutamate (IC50~ 20 μM). In isolated DB-1 melanoma cells 1-10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND onL-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC).

Keywords: bioenergetics; cancer; metabolism; mitochondrial pyruvate carrier; monocarboxylate transporter; tumour acidification.

Figure 1. Lonidamine inhibits pyruvate transport into mitochondria

In Panel A pyruvate transport into liver mitochondria was assayed directly at 9°C using [1-¹⁴C]-pyruvate as described under Methods. Data are presented as Means ± S.E.M. for 3 separate mitochondrial preparations. In each experiment, four replicates were performed at each LND concentration and the average value taken to calculate the extent of inhibition as percentage of control (no LND). Data were fitted to the standard inhibition equation (see Methods) to give a derived K_I value of 2.5 ± 0.1 μM. The absolute rate of pyruvate (60 μM) uptake in the absence of LND was 0.303 ± 0.032 nmol per mg protein in 45s. Panel B shows data for the inhibition of uncoupled pyruvate oxidation by isolated rat heart mitochondria at 30°C measured using an oxygen electrode as described under Methods. Mean data (± S.E.M.) are presented for 3 separate mitochondrial preparations. The absolute rate of pyruvate oxidation in the absence of LND was 87.9 ± 5.8 nmol O₂ · min⁻¹ per mg protein. The data were fitted to an equation that assumes oxidation of pyruvate is set by the activity PDH which in turn is controlled by the rate of pyruvate transport relative to that of PDH as described under Methods. The K_I value for LND of 2.5 μM derived from Panel A was employed and the V_MAX of PDH and MPC activity (expressed as % control rate of oxygen consumption and ± S.E.) were then calculated by least squares regression analysis to be 127 ± 6.3 and 233 ± 9.1, respectively

Figure 2. Lonidamine inhibits the proton-linked monocarboxylate carriers MCT1, MCT2 and MCT4

[U-¹⁴C]-L-lactate uptake into Xenopus laevis oocytes expressing the MCT isoform indicated was determined as described in Methods. Panel A shows the absolute rates of L-lactate uptake while Panels B–D show the effects of increasing concentrations of LND on rates of L-lactate uptake into oocytes expressing MCT1, MCT2 or MCT4 as indicated. Rates are expressed as a percentage of the control (no LND) after subtraction of the uptake by water-injected oocytes. Each data point represents mean data ± S.E.M. for 10–45 individual oocytes and data were fitted to the equation for cooperative inhibition using FigSys as described in Methods. The derived values for K_0.5 and the Hill Coefficient (n) are indicated on each plot (± S.E. of the fit shown).

Figure 3. Lonidamine inhibits oxidation of glutamate, 2-oxoglutarate and succinate by uncoupled rat heart and liver mitochondria less potnetly than pyruvate transport

Rates of oxidation by isolated rat heart (A) or liver (B) mitochondria at 30°C were measured using an oxygen electrode as described under Methods. Data are expressed as the percentage of rates in the absence of LND to allow better comparison between the different substrates and the lines drawn were fitted by FigSys using a Bezier Spline function. For heart mitochondria, data are presented for a single representative experiment with absolute rates of pyruvate + malate, glutamate + malate, 2-oxoglutarate + malate and succinate + rotenone oxidation in the absence of LND of 84, 83, 67 and 212 nmol O₂ · min⁻¹ per mg protein , respectively. For liver mitochondria (B), mean data (± S.E.M.) are presented for 3 separate mitochondrial preparations. The absolute rates of glutamate + malate and succinate (+ rotenone) oxidation in the absence of LND were 67.9 ± 3.0 and 109 ± 7.8 nmol O₂ · min⁻¹ per mg protein, respectively.

Figure 4. Concentration dependence of lonidamine effects on DB-1 melanoma cell L-lactate output and intracellular [L-lactate]

DB-1 cells were incubated with DMSO or LND at the indicated concentrations for 1 h. Levels of [L-lactate] in the culture medium (A) and intracellular [L-lactate] (B) were determined by LC-MS as described under Methods. The levels of [L-lactate] in the LND-treated group were normalized to the levels in DMSO controls. Data are presented as Means ± S.D. for 3 samples.