The MCF10A normal breast epithelial cells were obtained from Dr E. Saunderson and Dr J. Gomm of St Bartholomews Hospital (London, UK). The ER⁻ pII cell line was established in our laboratory (Profs. Luqmani and Khajah, College of Pharmacy, Kuwait City, Kuwait) by transfection of MCF7 cells originally obtained from ATCC with ER-directed short hairpin (sh)RNA plasmid as previously described (33,34). YS1.2 was also derived from MCF7 cells transfected with the shRNA plasmid but failed to downregulate ER, therefore this cell line was used as a transfected control for pII which behaves similarly to the parental MCF7 cells. For routine culture, all cancer cell lines were maintained as monolayers in advanced Dulbecco's minimum essential medium (DMEM) containing phenol red and supplemented with 5% fetal bovine serum (FBS), 600 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 6 ml/500 ml 100 × non-essential amino acids (all from Invitrogen; Thermo Fisher Scientific, Inc.), and were grown at 37°C in an incubator gassed with an atmosphere of 5% CO₂ and maintained at 95% humidity. MCF10A cells were cultured in DMEM F12 (cat. no. SH30023.01; Cytiva) supplemented with 5% horse serum, 1× pen/strep, 20 ng/ml mouse epidermal growth factor (EGF), 0.5 µg/ml hydrocortisone, 100 ng/ml cholera toxin and 10 µg/ml insulin (all from Invitrogen; Thermo Fisher Scientific, Inc.) (4).

Cells were cultured in 6-well plates to 80-90% confluency. The medium was subsequently aspirated off, and cell monolayers were harvested by scraping and re-suspended into 300 µl of lysis buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA 1% Triton X, 100 µg/ml phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin and 10 µg/ml leupeptin stored at −80°C. Protein concentration was determined by the Bradford assay using BSA as standard. A total of 8 µg protein lysate was mixed with an equal volume of 2× SDS and heated at 90°C for 10 min. Samples were loaded onto a 10% SDS-polyacrylamide gel and electrophoresed at 150 V for 1 h. Proteins were transferred to a nitrocellulose membrane and blocked with 2% BSA at 4°C for 1 h before being incubated overnight (16 h) at 4°C with primary antibodies (prepared in 2% BSA) against β-actin (loading control; 1:1,000 dilution; cat. no. 4970; Cell Signaling Technology, Inc.) and phospho- to total- p38 MAPK, AKT, ERK1/2, Src, NFĸB, E-cadherin and vimentin (1:1,000 dilution; cat. nos. 4695, 9101, 9212, 9211, 9272, 9271, 14243, 8242, 3195 and 5741, respectively; Cell Signaling Technology, Inc.). The membrane was then washed (with 0.1% v/v Tween 20) and incubated with anti-HRP-conjugated secondary antibody (1:500 dilution; cat. no. 7074; Cell Signaling Technology, Inc.) at 4°C for 1 h, developed with Super Signal ECL (Thermo Fisher Scientific, Inc.) and visualized with a Cell bio-imager (ChemiDoc MP System; Bio-Rad Laboratories, Inc.) (4).

Cells were cultured to a density of ~10⁶ cells in 6-well plates. The culture medium was removed, and protein concentration was estimated using the Bradford assay. The extracellular lactate was measured in aliquots using the EnzyChrom L-Lactate Assay Kit ECLC-100 (BioAssay Systems), following the manufacturer's protocol. Standards were prepared by dilution of a stock solution of 100 mM L-lactate in serum-free media, and 20 µl samples or standards were transferred into 96-well plates. Two reactions were performed for each sample: One with both enzymes A and B, and another without enzyme A (control). The working reagent was prepared freshly by mixing 60 µl Assay Buffer, 1 µl enzyme A, 1 µl enzyme B, 10 µl NAD and 14 µl MTT. For control, enzyme A was omitted from the reagent mix, and 80 µl working reagent was added to each sample well and mixed by pipetting up and down. The background optical density at 650 nm was measured using a plate reader at ‘zero’ time (OD₀) and after 20 min incubation (OD₂₀) at room temperature and subtracted from that at 565 nm. For the standard curve, the corrected OD₀ was subtracted from OD₂₀. For samples with no enzyme A control, the ΔOD_{no enzA} value was subtracted from ΔOD_sample. The ΔΔOD values were used to determine sample L-lactate concentration from the standard curve.

Intra- and extra-cellular lactate levels were measured. Intracellular lactate was estimated by measuring the amount released into 300 µl lysis buffer volume. However, it is noteworthy that this does not represent the actual concentration inside the cells, which will be substantially higher since the intracellular volume will be low. Since the intracellular volume could not be determined, and since the absolute concentration is not a necessary parameter for the present study, the data for the treated samples were presented either as a percentage of the control or as one cell line compared with another, providing a relative comparison (4).

Quantichrom LDH Kit (cat. no. DLDH-100; BioAssay Systems) was used following the manufacturer's protocol for the same samples that were prepared for the lactate assay. Freshly prepared assay reagent, containing 14 µl MTT solution, 8 µl NAD solution and 170 µl substrate buffer, were aliquoted into a 96-well plate at room temperature, and 10 µl sample was added to start the reaction. Control wells contained 200 µl H₂O (for OD_H2O) and 200 µl calibrator (for OD_Cal). The absorbance of the solution at 565nm was determined using a plate reader spectrophotometer at OD₀ and again after 25 min (OD₂₅). LDH activity was calculated according to the equations provided in the protocol. As was explained for lactate, the intracellular LDH activity was expressed as a percentage of the control or one cell line versus another rather than in absolute units (4). In a separate experimental setup, LDH activity was measured on cell lysate rather than on live cells.

Cells were cultured in 6-well plates with complete DMEM to 80-90% confluency (on both sides of the scratch). A scratch was created in the cell monolayer using a sterile p1000 pipette tip and an image of the scratched area was immediately taken (0 h). The plates were then placed at 37°C and 5% CO₂. After overnight (16 h) incubation, another image was taken of the same scratched area using light microscopy (magnification, 10×). The width of the scratch at 24 h was calculated as a percentage of the width at 0 h; a minimum of three areas along the scratch were measured (4). The following drugs were used: Quercetin (range, 100-500 µM; cat. no. Q4951; Sigma-Aldrich; Merch KGaA), oxamate (range, 100 µM-60 mM; cat. no. O2751; Sigma-Aldrich; Merch KGaA), lonidamine (range, 1-100 µM; cat. no. L4900; Sigma-Aldrich; Merch KGaA) and GNE-140 (range, 1-300 µM; cat. no. BCP37998; Shanghai Biochempartner Co., Ltd).

Cells were routinely seeded into 24-well culture plates and allowed to grow to 30-35% confluency. Cell density was determined either immediately (day 0) or after 1 and 4 days of cultivation. For the measurement, medium was removed and replaced with 500 µl MTT reagent (0.5 mg/ml; Sigma-Aldrich; Merck KGaA) at 37°C for 2 h. The MTT solution was then removed, and 200 µl acidic isopropanol was added to dissolve the blue formazan crystals that had formed. Plates were scanned at 595 and 650 nm (for background subtraction) using a Multiscan spectrum (Molecular Devices, LLC) spectrophotometer, and absorbance was compared between samples as a measure of proliferation (4).

Cell invasion was also assessed using Cultrex^® 24-well BME cell invasion assay (Trevigen, Inc.; Bio-Techne) according to the manufacturer's instructions. All reagents were provided with the kit. In brief, the invasion chamber was coated with 100 µl 1× BME solution and incubated overnight (16 h) at 37°C. A total of 1×10⁶ pII cells that had been serum-starved overnight (16 h) at 37°C/5% CO₂, were re-suspended to a total of 10⁶ cells/ml in DMEM (control) or DMEM containing various doses of quercetin (500 µM), oxamate (60 mM), GNE-140 (200-300 µM) or lonidamine (1-10 µM), and 100 µl suspension was loaded into the upper chamber. The lower chamber was loaded with 500 µl DMEM supplemented with 10% FBS as a chemoattractant. Cells were incubated at 37°C/5% CO₂ and allowed to invade from the top chamber to the bottom. After 48 h, liquid from both top and bottom chambers was removed by aspiration, and chambers were gently washed with 1× cell wash buffer, provided by the supplier. Calcein-acetomethylester (AM)/cell dissociation solution complex was added to the bottom chamber and left for 1 h at 37°C/5% CO₂. Cells internalize calcein-AM and intracellular esterases cleave the AM moiety generating free fluorescent calcein. Invading cells were determined by recording the fluorescence emission using a microplate reader with a filter set of excitation/emission at 485/535 nm (35).

The means of the experimental groups were compared with those of controls using an unpairedStudent's t-test or one-way ANOVA followed by the Bonferroni post hoc test. GraphPad Instat software (version 6; Dotmatics) was used. GraphPad Prism (version 6; Dotmatics) was used to plot graphs. P<0.05 was considered to indicate a statistically significant difference.

Breast cancer cells were treated with various doses of LDH/LTIs, and their effect on intracellular LDH activity and extracellular lactate levels, as well as on cell proliferation, motility and invasion was measured. There is no evidence to suggest that any of the inhibitors studied have any direct effect on the gene expression of LDH; they were purely used as inhibitors of LDH activity/transport.

As shown in Fig. 1, treatment of pII cells with quercetin (range, 200-500 µM) reduced intracellular LDH activity (Fig. 1A) and extracellular lactate levels (Fig. 1B) in a dose-dependent manner, with significant effects observed. Treatment of pII cells with oxamate also reduced intracellular LDH activity (Fig. 1C) and extracellular lactate levels (Fig. 1D) in a dose-dependent manner but a higher dose was required (10-60 mM) compared with that of quercetin. To further confirm the LDH inhibitory effects of quercetin and oxamate, pII lysates (not live cells; Fig. 1A-D) were treated with these drugs, and it was shown that they inhibited LDH activity (range, 10-1,000 µM; Fig. 1E). Previous studies (18,22,23) have considered quercetin as an inhibitor of lactate transport. The intra and extracellular lactate levels were measured in pII cells treated with either quercetin (range, 100-500 µM) or oxamate (range, 100 µM-60 mM), and it was shown that these two agents act by directly inhibiting LDH activity rather than affecting the lactate transport machinery since there was no significant increase in the intracellular lactate level observed upon treatment with either quercetin or oxamate (Fig. 1F). Next, the effect of quercetin and oxamate on pII cell proliferation was measured using the MTT assay. Treatment with quercetin (500 µM) and oxamate (range, 40-60 mM) significantly inhibited pII cell viability on day 4 (Fig. 1G) but not on day 1 (Fig. 1H) post treatment. However, a 24-h treatment with quercetin or oxamate significantly inhibited both pII cell motility (Fig. 1A and B) and invasion (Fig. 1C). Thus, the latter effects were not due to inhibition in cell proliferation which occurred later. The effect of these inhibitors was also tested the on the ER⁺ YS1.2 cells. As shown in Fig. 2, quercetin treatment inhibited YS1.2 cell proliferation in a dose-dependent manner on day 4 (Fig. 2D), but not on day 1 (Fig. 2E). Oxamate inhibited YS 1.2 cell proliferation but at a lesser degree compared with its effect on pII cells (Fig. 2D). Both inhibitors also reduced YS1.2 motility in a dose-dependent manner (Fig. 2F and G). Since it was previously shown that ER⁺ breast cancer cells are motile but not invasive compared with pII cells (35), invasion assays were not performed on YS1.2 cells. Treatment with quercetin did not inhibit the proliferation of the normal breast epithelial cell line MCF10A at the same dose range effective against cancer cells (Fig. 2H), suggesting a preferential growth inhibitory effect. On the other hand, oxamate treatment did inhibit MCF10A as well as pII and YS1.2 cell viability at similar dose ranges (Fig. 2H). A motility assay for MCF10A cells was performed and it was shown that these cells are not motile (data not shown), and therefore these inhibitors were not used in the motility assay for MCF10A cells. The effect of a combination regimen of quercetin (range, 100-500 µM) with oxamate (40 mM) was also tested the on the proliferation and motility of pII cells. As shown in Fig. 2I-K, combination treatment significantly inhibited cell proliferation and motility on days 1 and 4 compared with treatment with either inhibitor.

As shown in Fig. 3, treatment of pII cells with the other LDH inhibitor, lonidamine, inhibited intracellular LDH activity (Fig. 3A) and extracellular lactate levels (Fig. 3B) in a dose dependent manner with a significant effect observed at concentration range of 1-100 µM. Treatment with lonidamine (starting concentration 1 µM) also reduced pII cell proliferation after 4 days of treatment (Fig. 3C), but not on day 1 (Fig. 3D). pII cell motility and invasion were also inhibited following lonidamine treatment (Fig. 3E-G). Furthermore, treatment of the ER⁺ YS1.2 breast cancer cells with lonidamine inhibited their viability (Fig. 3H) and migrative ability (Fig. 3J and K). Lonidamine did not inhibit proliferation of the normal MCF10A cells (Fig. 3L), which indicates preferential effect on breast cancer cells.

As shown in Fig. 4, in pII cells, the LDH-A inhibitor GNE-140 inhibited LDH activity (range, 200-300 µM; Fig. 4A), reduced extracellular lactate levels (Fig. 4B) and inhibited cell proliferation on day 4 but not on day 1 (Fig. 4C and D), migration (Fig. 4E and F) and invasion (Fig. 4G). GNE-140 also inhibited YS 1.2 cell proliferation on day 4 but not on day 1 (range, 100-300 µM; Fig. 4H and I) and motility (Fig. 4J). It also inhibited MCF10-A proliferation (Fig. 4K).

To investigate the possible mechanisms used by LDH inhibitors to exert their actions, their effect on the level of several key intermediates of the intracellular mitogenic signaling pathway activated by EGF were studied, previously shown to be a potent stimuli of breast cancer cell proliferation, motility and invasion (35). Fig. 5A shows a time course of ERK1/2 phosphorylation after addition of EGF (100 ng/ml) to pII cells to determine the optimum stimulation conditions, which was subsequently used to determine the effect of the four inhibitors. As shown in Fig. 5B, pretreatment with quercetin but not oxamate or lonidamine inhibited EGF-induced p38 MAPK phosphorylation without affecting the expression of total p38 MAPK. GNE-140 reduced both p38 MAPK phosphorylation as well as expression of total p38 MAPK. Oxamate and quercetin but not lonidamine and GNE-140 inhibited EGF-induced ERK1/2 phosphorylation. Pretreatment with either oxamate, lonidamine or GNE-140 inhibited EGF-induced AKT phosphorylation. None of the LDH inhibitors affected the EGF-induced phosphorylation level of Src or NF-κB.

It was also tested if treatment with LDH inhibitors modulate the expression profile of epithelial and mesenchymal markers in pII cells. As shown in Fig. 5C, pII cells which have a mesenchymal phenotype express the epithelial marker E-cadherin at low levels and the mesenchymal marker vimentin at high levels. Treatment with either quercetin or oxamate for 24 h increased the expression of E-cadherin and reduced the expression of vimentin compared with those in the control (untreated) cells. These data suggest that reduced pII cell motility and invasion in response to treatment with LDH inhibitors is in part due to modulation in the expression of epithelial and mesenchymal markers, and the possible reversibility of the epithelial-mesenchymal transition (EMT) process.