Lactate dehydrogenase (LDH) is a tetrameric enzyme composed of four subunits. The two most common subunits are LDHA and LDHB, and five isoenzymes of LDH are known: LDH-5, A4; LDH-4, A3B1; LDH-3, A2B2; LDH-2, A1B3; LDH-1, B4 [1
]. LDH-5 is the most efficient isoenzyme for catalyzing the transformation of pyruvate to lactate [1
]. Furthermore, it is known that the levels of LDHA expression are higher in many cancers than in normal tissues. Thus, the enhanced production of LDH-5 with four LDHA subunits in cancer cells leads to an increase in the glycolytic metabolism in the presence or absence of oxygen [1
]. Interestingly, most cancer cells take up more glucose than normal cells, and depend on lactate fermentation by LDHA, which catalyzes conversion of pyruvate to lactate during the aerobic glycolysis known as ‘Warburg effect’ instead of oxidative phosphorylation for adenosine triphosphate (ATP) production and cell proliferation [3
]. The 1,4-dihydronicotinamide adenine dinucleotide (NADH)-dependent enzyme LDHA mediates a redox reaction for the reduction of pyruvate to lactate at the end of glycolysis when the cytosolic NADH/nicotinamide adenine dinucleotide (NAD+) ratio is high, which is a crucial step for regenerating NAD+, and is needed to maintain glycolysis for the survival of cancer cells [6
]. Metabolic changes of the Warburg effect are promoted by up-regulating the expression of the glycolytic gene LDHA
during malignant transformation through oncogenic signals such as phosphoinositide 3-kinase (PI-3K) and 5’ adenosine monophosphate-activated protein kinase (AMPK) and oncogenic transcription factors such as Myc and Hif-1 [2
]. Furthermore, lactate secreted by cancer cells into the tumor microenvironment is strongly associated with tumor-associated inflammation, immune evasion, and tumor angiogenesis for tumor progression [11
]. In a previous study on genetic and pharmacologic targeting of LDHA, a knock-down of LDHA expression resulted in a decrease of mitochondrial membrane potential and suppressed carcinogenicity. Inhibition of LDHA and LDHA short hairpin ribonucleic acid (shRNA) induced oxidative stress, inhibited tumor progression, and suppressed tumor-initiating cell survival and proliferation [2
]. Thus, LDHA may be a potential target for cancer therapeutics due to the suppression of the lactate metabolism.
In the present study, we found out that machilin A (MA) as the strongest inhibitor on enzymatic activity of LDHA among 480 compounds in a natural product library. MA inhibited the LDHA activity through the suppression of an NADH cofactor binding to lactate dehydrogenase, based on structural and molecular investigations. This compound also suppressed the growth of tumor cells in in vitro and in vivo experiments by regulating LDHA activity. Furthermore, angiogenesis mediated by lactate produced and secreted from cancer was inhibited by MA. From these results, we suggest that MA, a novel LDHA inhibitor, may be candidates for anti-cancer drugs.
The activation of oncogenic proteins stimulates glycolytic metabolism in tumor tissue [2
]. Furthermore, increasing Hif-1 levels under non-hypoxic conditions can elicit the Warburg effect in cancer cells [3
]. Hypoxia also increases stability of the main factor Hif-1 for adaptation to hypoxic stress, a common feature in microenvironment of solid tumors, which induces the expression of glycolytic enzymes including LDHA (due to the Warburg effect), which again results in an increase of lactate production and cancer cell survival [2
]. Thus, LDHA affects initiation, maintenance, and progression of cancers [8
]. In the present study, we screened 480 chemical compounds for their potential inhibitory effects on LDHA activity. Interestingly, MA prohibited NADH from binding and inhibiting LDHA, and therefore inhibited growth of various cancer cell types. Furthermore, our findings demonstrated that the suppression of lactate production and secretion in cancers treated with MA under hypoxic conditions resulted in an inhibition of angiogenesis through the regulation of macrophage polarization in the tumor microenvironment.
Many cancer cells use the aerobic glycolysis pathway instead of oxidative phosphorylation for ATP production, rapid macromolecule biosynthesis, and cell growth [14
]. Furthermore, under hypoxic conditions, accelerated glycolysis ensures that the produced ATP levels meet the demands of fast proliferating tumor cells in low-oxygen environments [14
]. In contrast, depletion of the glycolytic enzyme LDHA and of the LDHA inhibitor FX11 reduced ATP levels and induced significant oxidative stress and cell death [2
]. Furthermore, reduction of LDHA resulted in a stimulation of mitochondrial respiration and a decrease of mitochondrial membrane potential, and interfered with carcinogenicity of malignant cells [14
]. Previous studies reported that the mechanism involved in the induction of apoptosis by LDHA inhibition were as associated with the production of mitochondrial ROS [2
]. Our results showed that the inhibition of LDHA activity by MA treatment reduced lactate and ATP production under normoxic and hypoxic conditions, leading to the suppression of tumor growth in vitro and in vivo (Figure 3
and Figure 4
). Furthermore, MA induced apoptosis and production of mitochondrial ROS via an inhibition of LDHA activity (Figure 3
E). However, MA did not affect LDHB activity when used in concentrations of up to 500 μM (data not shown).
Previously, MA have been reported that it has anticancer effect by inhibiting phospholipase Cγ1 [22
] and by activating caspase-3 activity [23
]. MA also has activity of inhibiting melanin synthesis [24
], protecting liver damage [25
], and activating osteoblast differentiation [26
]. In addition, MA has selective inhibitory effect on drug metabolism via directly interacting with human liver cytochrome P450 1A1 (CYP1A1) and 2B6 [27
]. These enzymes are involved with phenacetin O
-deethylation and bupropion hydroxylation, respectively. Although these enzymes are not directly related with survival or malignancy of cancer, these enzymes are involves in the bioactivation and detoxification of carcinogen [28
]. Thus, to identify a selective LDHA inhibitor, more extensive studies on MA and its derivatives is need.
In our structural study, the X-ray crystal structure of purified human LDHA showed a tetrameric complex consisting of 4 LDHA subunits (LDH-5). However, MA did not disrupt the formation of active tetrameric LDHA to inhibit LDHA activity (Supplementary Figure S4
). In addition, MA did not prevent LDHA tetramer formation, as observed using a purified human LDHA crosslinking assay (Figure 1
D). For LDHA activity, the NADH cofactor binds to LDHA. Subsequently, pyruvate interacts as a substrate with a binary LDHA/NADH complex, which catalyzes the conversion of pyruvate to lactate [29
]. In this study, LDHA interacted with NADH biomimetic beads. However, MA inhibited LDHA binding to the NADH mimic (Figure 1
C). Additionally, surface plasmon resonance (SPR) biosensor and isothermal titration calorimetry (ITC) analysis were performed to measure the binding ability of LDHA to MA/NADH (Figure 2
A and Figure S2
). We found that MA physically bound to LDHA with an apparent KD
of 29 μM and NADH bound to LDHA with an apparent KD
= 10 μM.
To examine the conformation of LDHA tetramers, we performed electron microscopy on MA binding to LDHA (Supplementary Figure S4
). Images of negatively stained samples were assessed, and the complexes were identified as tetramer-like shapes. In the present study, we tested interactions and the molecular basis of full-length LDHA and its natural inhibitor MA. In addition, molecular characterizations of LDHA with NADH and malonate were compared. These results showed that MA binds to LDHA in an open inactive conformation (Figure 2
D). Binding of MA to LDHA can decrease LDHA activity in lactate production by suppressing the binding between LDHA and the cofactor NADH. Interestingly, the oxygen of MA binds to S183 residue of LDHA through hydrogen bond (Figure 2
C). In the more active form of the enzyme, phosphorylase a
, specific Ser residues, one on each subunit, are phosphorylated [30
]. LDHA with MA may make inactive conformation by blocking phosphorylation by binding of MA with Ser183. In complexes of LDHA and NADH or malonate, the polar LDHA amino acids N137 and H192 are important residues for interactions with substrates through hydrogen bonding. Interestingly, the structures of LDHA with malonate and NADH were shown with all active site loops in closed conformation, whereas when LDHA was associated with the inhibitor MA, the active site loops were in an open conformation. In a previous study on molecular dynamics of LDHA inhibitors, Shi et al. suggested that closure of the mobile loop is not necessarily required for ligand binding, and certain S-site binders may force the loop open when binding [31
]. Compared to the LDHA structure with malonate or NADH, the structure of LDHA associated with MA showed substantial conformational changes in the active-site loop. The residues Q100–N107 in the loop region of the LDHA active-site with MA migrated to the open form over more than 12 Å from the closed form. These results suggest that considerable conformational changes of LDHA occurred in the active-site loop regions exposed to solvents in an open MA-binding form. These results provide insights into LDHA enzyme mechanisms and inhibition and provide framework for structure-assisted drug design that may contribute to new cancer therapies.
Lactate produced by cancer cells due to the Warburg effect results in acidification of the cancer’s microenvironment; lactate is also transported to neighboring cancer cells, immune cells, and vascular endothelial cells which promotes cancer development, tumor inflammation, angiogenesis, and metastasis [11
]. In previous studies, elevated lactate levels in cancer cells due to enhanced expression of LDHA during hypoxia substantially increased the N-myc downstream regulated gene (NDRG)-3 protein expression, which induced the Raf-extracellular signal regulated kinase (Raf-ERK) pathway and thus promoted angiogenesis and hypoxic cell growth [32
]. Furthermore, lactate metabolized by tumors modulates the phenotype expression of endothelial cells and thereby affects tumor vascular development [33
]. It was previously reported that lactate produced by LDHA in cancer cells is a potent inhibitor of the function and survival of T and Natural killer (NK) cells, leading to tumor immune evasion [34
]. Colegio et al. demonstrated that tumor-derived lactate affected M2 phenotype polarization of tumor-associated macrophages, which play an important role in tumor growth [19
]. Moreover, it was shown that tumor-conditioned media or lactic acid up-regulated the expression of VEGF angiogenic factor and Arg-1 M2 phenotype marker in macrophages [19
]. In line with these results, we found that tumor-derived lactate up-regulated the expressions of Arg-1
, and YM1
genes in macrophages, and an MA-mediated decrease of tumor-derived lactate resulted in a reduction of Arg-1
, and YM1
mRNA levels in macrophages (Figure 5
D). In contrast, the reduced expression of Arg-1
, and YM1
mRNAs mediated by MA was reduced by the addition of 5 mM lactate (Figure 5
D). These results clearly suggest that MA inhibited M2-like phenotype polarization of macrophages through the suppression of lactate production by regulating cancer cell metabolism under hypoxic conditions.
In the tumor microenvironment, immune cells are recruited into the tumor site for tumor progression [35
]. Among them, macrophages are particularly abundant and are strongly associated with tumor initiation, progression, and malignancy although macrophages are also important for anti-tumor immunity [20
], indicating the importance of macrophage M2 polarization by various factors in the tumor microenvironment. Tumor-associated macrophages (TAMs) accumulated in hypoxic regions of tumor [20
] generally show various characteristics of the alternatively activated M2-like phenotype, and also play an important role in vascular programming of tumors by enhancing the production of the VEGF for promotion of angiogenesis [36
]. In this study, the increase of angiogenic factors including VEGF and Arg-1 in macrophages due to tumor-derived lactate under hypoxic conditions affected the activation of endothelial cells for neovascularization and M2-like polarization of macrophages in vitro and in vivo (Figure 5
), whereas the reduction of tumor-derived lactate by MA treatment under hypoxic conditions showed an inhibition of neovascularization and M2-like polarization of macrophages by suppressing VEGF and Arg-1 expression (Figure 5
In the previous study, we demonstrated that 1-(phenylseleno)-4-(trifluoromethyl)benzene (PSTMB) inhibited LDHA activity through reducing its binding to substrate, pyruvate, without affecting the expression of LDHA. In addition, PSTMB induced mitochondria-mediated apoptosis of cancer cells [38
]. Consistent to the study, in this study we show that another LDHA inhibitor, MA, successfully suppressed their own growth of cancer cells. In addition, we further demonstrated that as a result of regulation of the Warburg effect, MA also inhibited the production of lactic acid released from cancer cells and suppressed the macrophage-mediated neovascularization.
Several small molecule LDHA inhibitors, such as FX11, galloflavin, gossypol, and GNE140, are under estimating for developing novel and effective anticancer agents [39
]. However, due to the limitation of their intracellular uptake, pharmacokinetic profile, and in vivo activity, still no LDHA inhibitor used for anticancer agents in clinic [39
]. Because MA has a unique structural feature compared with previously reported LDHA inhibitors, we suggest that MA or its derivatives might be good candidates for developing a novel anticancer drug through improvement in selectivity, activity, delivery, and pharmacokinetic profiles. In addition, several reports demonstrated that Warburg effect is closely related with aggressiveness and drug resistance of cancer cells [42
]. Furthermore, resistance to LDHA inhibitor was also overcome by addition of phenformin, an inhibitor of mitochondrial complex I [41
]. From these results, MA might be developed as a combinative therapeutic agent to resistant cancers.
4. Materials and Methods
A chemical library of 480 compounds was obtained from National Development Institute of Korean Medicine (NIKOM). Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (sc-32233, Dallas, TX, USA). The anti-LDHA antibody was obtained from Santa Cruz Biotechnology (sc-137243). All chemicals and reagents, including NADH, pyruvate, Cibacron Blue agarose, 1,3-benzodioxole, piperonyl alcohol, and nordihydroguaiaretic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4.2. Assay Compounds
Compounds such as MA, 1,3-benzodioxole, piperonyl alcohol, and nordihydroguaiaretic acid are typically dissolved in dimethyl sulfoxide (DMSO). Compounds with limited aqueous solubility were dissolved in DMSO before adding to the respective medium or buffer, and the final concentration of DMSO in cell culture media and assays was 0.1% (v/v).
4.3. LDH Activity Assay
A purification of recombinant LDHA was performed as described previously [6
]. LDH activity was determined by measuring the decrease of absorbance at 340 nm due to the oxidation of NADH in 20 mM HEPES-K+
, 0.05% bovine serum albumin (BSA), 20 μM NADH, and 2 mM pyruvate (Sigma-Aldrich, St. Louis, MO, USA) at a pH of 7.2 using a spectrofluorometer (Spectramax M2, Molecular Devices, San Jose, CA, USA; excitation 340 nm; emission 460 nm), as described previously [6
]. Ten nanograms of recombinant LDHA protein were used in the in vitro LDH activity assay. One microgram of total protein from cell or tissue lysates was used for intracellular or in vivo LDH assays.
4.4. NADH Binding Assay
An NADH binding assay was performed as described previously [6
]. The NADH binding ability of LDHA was determined by measuring the affinity of LDHA to agarose-immobilized Cibacron Blue 3GA, which resembles NADH. Then, 400 ng of purified recombinant human LDHA (rhLDHA) was incubated with NADH or MA, followed by incubation with 30 µL of Cibacron Blue agarose at 4 °C for 2 h. After a washing step with 20 mM Tris-HCl (pH 8.6), LDHA bound to beads was eluted in PBS using a sodium dodecyl sulfate (SDS) gel running buffer and was then subjected to–SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Standardized amounts of protein were used in each reaction.
4.5. Crosslinking Assay for Tetramer Formation of LDHA
The purified rhLDHA (100 ng) was incubated with or without MA at room temperature for 5 min in the presence of 0.0025% glutaraldehyde containing 5% glycerol, 10 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT). After this, SDS sample buffer was added and the samples were subjected to SDS-PAGE. The same amounts of non-crosslinking proteins were used in each reaction.
4.6. Surface Plasmon Resonance (SPR) Biosensor Analysis
The apparent dissociation constants (KD) between LDHA and Machilin A were measured using a Biacore T100 biosensor (GE Healthcare, Chicago, IL, USA). The purified LDHA was covalently bound to the Series S sensor chip CM5 using an amine coupling method as suggested by the manufacturer. A total of 150 μL LDHA (50 μg/mL) in 10 mM sodium acetate (pH 5.5) was coupled via injection for 15 min at 10 μL/min, followed by injection of 1 M ethanolamine to deactivate the residual amines. To measure kinetics at 25 °C, chemical compounds with concentrations ranging from 500 to 62 μM were prepared by dilution in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% v/v surfactant P20, and 1% DMSO) at a pH of 7.4. The immobilized ligand was regenerated by injecting 10 μL of 50 mM NaOH at a rate of 10 μL/min during the cycles.
4.7. Expression and Purification of LDHA for Structural Study
Full-length LDHA (residues 1–331) was obtained from cDNA by polymerase chain reaction (PCR) and cloned between the NdeI and XhoI sites of the pET-28a (Novagen, Burlington, MA, USA) vector. The successfully cloned plasmid, as confirmed by DNA sequencing (performed by SolGent, Daejeon, Korea), was used to transform E. coli BL21 (DE3) cells. A single colony was cultured overnight at 37 °C. This culture was inoculated placed in Luria-Bertani medium containing kanamycin (100 mg/mL), and the cells were grown at 37 °C. Expression of LDHA was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 16 h at 25 °C. The cells were centrifuged for harvesting, and the cell pellets were resuspended in lysis buffer A (50 mM Tris-HCl at pH 7.5 and 200 mM NaCl). The cells were subsequently disrupted by sonication on ice, after which the soluble LDHA supernatant was loaded onto a Ni-NTA column (Amersham Pharmacia Biotech, Buckinghamshire, UK). The column was washed with buffer A containing 20 mM imidazole, after which the bound protein was eluted using buffer A containing 200 mM imidazole. Purified fractions of the LDHA protein from the Ni-NTA column were mixed and purified using gel-filtration chromatography and fast protein liquid chromatography (FPLC) using a Superdex 200 10/300 GL column (Amersham Pharmacia Biotech, Buckinghamshire, UK) equilibrated in buffer A, after which the fractions were collected. Finally, the purified protein was concentrated to about 10 mg/mL. All purification steps were assessed using 15% SDS-PAGE and Coomassie Blue staining (Sigma-Aldrich, St. Louis, MO, USA). The purified LDHA was confirmed by a single band with a molecular weight of 39 kDa using SDS-PAGE.
4.8. Crystallization of LDHA, LDHA-NADH, and LDHA-MA Complexes
LDHA crystals were grown in hanging drops using a reservoir buffer of 200 mM sodium malonate and polyethylene glycol (PEG; 3.350). LDHA crystals with compounds were grown in hanging drops using a reservoir buffer (Tacsimate, pH 7.0, 20% PEG 3.350) with 50 mM compounds. The compounds used in this study were NADH and MA (Supplementary Figure S1
). Crystals appeared after three days and grew to maximum size within two weeks (Supplementary Figure S2
) at 20 °C.
4.9. Data Collection, Structure Determination, and Refinement
Data for full-length LDHA, LDHA-NADH, and LDHA-MA were collected at the Pohang Accelerator Laboratory (Pohang, Republic of Korea). Before data collection, each crystal was briefly soaked in a cryoprotectant solution consisting of the precipitant solution and 20% glycerol. The crystal was shock-cooled in a liquid-nitrogen stream at 100 K. Diffraction data were then produced and were processed using the HKL
-2000 software package. The structures of LDHA, LDHA-NADH, and LDHA-MA complexes were determined using the molecular replacement method of the software programs AMoRe, CCP4i
]. The model was improved by iterative model building using the software O
]. LDHA (amino acids 2–332; PDB entry 1I10) was used as an initial search model [48
]. As shown in Table S1
, final refinements were performed using the CNS
software packages [49
]. Structural visualizations shown in Figure 2
and Figure 3
were generated using MolScript, GRASP, Raster3D, and PyMOL [50
]. Ramachandran statistics were calculated using Procheck. The atomic coordinates and structure factors (codes 5ZJE, 5ZJD, and 5ZJF) have been deposited in the Protein Data Bank.
4.10. Cell Culture, Hypoxia Condition and Determination of Cell Viability
Human colon cancer cells (WiDr, DLD-1, RKO, and HT29) and murine Lewis lung carcinoma (LLC) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Human breast cancer MCF7 cells, human hepatocellular carcinoma cells (Huh-7, HepG2, and Hep3B), and human lung cancer cells (HCC-95 and NCI-H1793) were provided by the Korean Cell Line Bank (Seoul, Korea). The HCC-95 and NCI-H1793 cells were grown in a Roswell park memorial institute medium (RPMI) 1640 medium (HyCloneTM; GE Healthcare, Chicago, IL, USA). All other cells were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with L-glutamine (200 mg/L), 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator at 37 °C and with 5% CO2 before the experiments. For induction of hypoxia condition (1% oxygen), the cells were cultured were cultured in the mixture of 94% nitrogen and 5% CO2/air at 37 °C for 24 h in cell culture incubator. Cytotoxicity of MA was evaluated using an MTT assay. Briefly, cells were incubated in 24-well plates with specified concentrations of MA for 24 or 48 h. Then, MTT solution (2.0 mg/mL) was added to each well. After 4 h of incubation at 37 °C and 5% CO2 in a cell culture incubator, the conditioned media were removed, and the amount of formazan crystals formed in living cells was estimated by measuring the absorbance at 540 nm using a microplate reader (Spectramax M2; Molecular Devices, CA, USA). The respective percentages of living cells were compared with a control treatment.
4.11. Colony Formation Assay
Base agar (0.6%) was placed at the bottom of the 6-well culture plate. After 30 min at room temperature, top agar (0.4%) mixed with HT29 (1 × 104 cells) or Hep3B (1 × 104 cells) cells was added to the base agar. HT29 and Hep3B cells were incubated at 37 °C and 5% CO2 for three weeks. The colonies were stained using 0.005% crystal violet for 1 h after which they were photographed and counted.
4.12. Lactate Production Assay
Cellular lactate production was measured using a lactate fluorometric assay kit (Biovision, Milpitas, CA, USA) as described previously [6
]. Phenol red-free DMEM medium without FBS was added to a six-well plate containing subconfluent cells which were then incubated for 1 h at 37 °C. After incubation, the medium of each well was tested using a lactate assay kit (Biovision, Milpitas, CA, USA).
4.13. Measurement of Intracellular ATP
Intracellular ATP concentration was measured using an ATP bioluminescent somatic cell assay kit (Sigma-Aldrich) as previously described [6
]. A total of 1 × 106
cells were trypsinized and resuspended in ultrapure water. Luminescence was measured using a spectrofluometer (Spectramax M2, Molecular Devices, CA, USA) immediately after the addition of an ATP enzyme mix to the cell suspension.
4.14. Western Blot Analyses
Total protein was extracted from each cell using 1% NP-40 lysis buffer (150 mM NaCl, 10 mM HEPES (pH 7.45), 1% NP-40, 5 mM Na pyrophosphate, 5 mM NaF, 2 mM Na3VO4) containing protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Equal amounts (20 μg) of protein from each sample were fractionated using SDS-PAGE and the protein was then transferred onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Chicago, IL, USA) using electrophoresis. The membranes were blocked for 1 h at room temperature using 5% non-fat dry milk and were then incubated with primary antibodies specific to the target protein at 4 °C overnight. The membranes were washed three times, and incubated with the respective secondary antibodies conjugated with horseradish peroxidase. The specific bands of targeted proteins were detected using ECL Plus (GE Healthcare, Chicago, IL, USA).
4.15. Measurement of Mitochondrial Reactive Oxygen Species (ROS)
The production of mitochondrial ROS was determined by MitoSOX™ Red (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Briefly, 5 μM MitoSOX™ Red was added to each cells cultured in conditioned medium and incubated at 37 °C for 10 min. The cells were then washed twice with PBS and the fluorescence intensity was analyzed using a BD FACS Canto II (BD Biosciences, San Jose, CA, USA) measuring excitation/emission at wavelength of 510/580 nm.
4.16. Detection of Apoptotic Cells
HT29 cells were treated with the indicated concentrations of PSTMB for 48 h. Apoptotic cells were detected using the Annexin V-FITC Apoptosis Detection kit (Life Technologies, Carlsbad, CA, USA). The cells were suspended in 500 μL of buffer and treated with 5 μL of Annexin V-FITC. The fluorescence intensities were measured using BD FACSCANTO II (BD Biosciences, San Jose, CA, USA).
4.17. Tumor Allograft Model
LLC and CT26 cells were suspended separately in PBS (5 × 105 cells/100 µL PBS) and inoculated in subcutaneously on the back of 7-week-old C57BL/6 mice and BALB/c mice respectively (Orient Bio Inc., Seongnam, Korea; n = 7). One day after the inoculation with tumor cells, mice of the control group (n = 7) were intraperitoneally administrated 100 µL of PBS per day for 18 days. Each of the MA-treated mice were intraperitoneally administrated 0.02, 0.2, or 1 mg MA, respectively, in 100 µL PBS per 20 g body weight, each day for 18 days. All mice were euthanized 19 days after inoculation with the tumor cells, and the tumors were excised and weighed. Length (l) and width (w) of each tumor were measured using a caliper, and the respective volume (V) was calculated according to the formula V = [(l × w2)/2]. Tumors were immediately removed from euthanized mice and prepared for histological examinations. To test liver and kidney toxicity of MA, at the end of the in vivo allograft experiment blood was collected from each mouse and serum was separated. Biochemical analyses of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen (BUN) were performed using a commercial service (Green Cross Co., Yongin, Korea). All experimental procedures followed the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health of Korea, and were approved by the Institutional Animal Care and Use Committee of Pusan National University, Republic of Korea (PNU-2018-1954, Date of approval: 27th June 2018).
Tumor specimens were immediately removed from euthanized mice, fixed in PBS with 3.7% formalin, and embedded in paraffin for immunohistochemical analyses. The paraffin sections were immunostained using an antibody specific to the endothelial cell marker CD31 (ab28364, Abcam, Cambridge, UK), macrophage marker CD68 (ab125212, Abcam, Cambridge, UK), and M2 phenotype macrophage marker CD206 (ab64693, Abcam, Cambridge, UK). The staining was visualized using a Dako EnVision kit (Dako, Santa Clara, CA, USA), and counterstained with hematoxylin.
4.19. Reverse-Transcription (RT)-PCR and Quantitative Real-Time RT-PCR (qRT-PCR)
Total RNA of the cells was isolated using a GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of RNA was reverse-transcribed using a RevertAid reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), and single-stranded cDNA was PCR-amplified using AccPower® PCR PreMix (Bioneer, Daejeon, Korea). The qRT-PCR was performed using the QuantiNova™ SYBR®Green PCR Kit (Qiagen, GmbH, Hilden, Germany) and a Qiagen Rotor-Gene® Q Real-Time PCR Detection System according to the manufacturer’s instructions. PCR products were separated using electrophoresis on 1.5% agarose gel containing ethidium bromide in 1 × Tris-acetate buffer and were visualized under UV light.
4.20. Tube Formation Assay
To investigate the formation of a capillary-like network of human umbilical vein endothelial cells (HUVECs) through the activation of tumor associated macrophages (TAMs) by lactate secreted by cancer cells, a tube formation assay was performed according to a previous study [54
], with several modifications. For the preparation of conditioned media, HT29 colon cancer cells (3.5 × 105
cells) suspended in 2 mL of DMEM were seeded on 6-well plates, with or without addition of 30 µM MA, and incubated under normoxic or hypoxic conditions. After 48 h of incubation, 1 mL of conditioned medium harvested from cancer cells was added to 6-well plates seeded with or without RAW264.7 macrophage cells (1 × 106
cells), which were incubated at 37 °C and 5% CO2
. After 24 h, 500 µL of conditioned medium were harvested from each well to examine tube formation. For preparation of HUVECs, Matrigel (13.9 mg/mL) was thawed at 4 °C and mixed with endothelial cell medium-basal (Endothelial Cell Medium (ECM)-b, ScienCell Research Laboratories, Carlsbad, CA, USA) at a ratio of 1:1. Seventy microliters of ECM-b-diluted Matrigel (6.95 mg/mL) was added to each well of the 24-well culture plates and allowed to polymerize at 37 °C for 1 h. HUVECs (ScienCell Research Laboratories) were detached from the tissue culture plates, washed, resuspended in the ECM-b medium containing 1% FBS, and seeded in the Matrigel-coated wells (5 × 104 cells/500 µL in each well).The harvested conditioned medium was added to the seeded HUVECs. After incubation for 12 h at 37 °C and 5% CO2
, capillary-like tube formation of each well in the culture plates was photographed using a Nikon ECLIPSE TS100 light microscope (Nikon, Tokyo, Japan).
4.21. Statistical Analyses
Cell viability, LDHA activity, lactate production, and ATP production assays were calculated as a percentage of the respective value in control cells and expressed as mean ± standard deviation (SD). Differences between the mean values of the experimental groups were tested by a one-way analysis of variance (one-way ANOVA) and a student’s t-test using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical significance is reported at p < 0.05. Each experiment was performed in three independent replicates.