Machilin A Inhibits Tumor Growth and Macrophage M2 Polarization Through the Reduction of Lactic Acid

Lactate dehydrogenase A (LDHA) is an important enzyme responsible for cancer growth and energy metabolism in various cancers via the aerobic glycolytic pathway. Here, we report that machilin A (MA), which acts as a competitive inhibitor by blocking the nicotinamide adenine dinucleotide (NAD) binding site of LDHA, suppresses growth of cancer cells and lactate production in various cancer cell types, including colon, breast, lung, and liver cancers. Furthermore, MA markedly decreased LDHA activity, lactate production, and intracellular adenosine triphosphate (ATP) levels induced by hypoxia-induced LDHA expression in cancer cells, and significantly inhibited colony formation, leading to reduced cancer cell survival. In mouse models inoculated with murine Lewis lung carcinoma, MA significantly suppressed tumor growth as observed by a reduction of tumor volume and weight; resulting from the inhibition of LDHA activity. Subsequently, the suppression of tumor-derived lactic acid in MA-treated cancer cells resulted in decrease of neovascularization through the regulation of alternatively activated macrophages (M2) polarization in macrophages. Taken together, we suggest that the reduction of lactate by MA in cancer cells directly results in a suppression of cancer cell growth. Furthermore, macrophage polarization and activation of endothelial cells for angiogenesis were indirectly regulated preventing lactate production in MA-treated cancer cells.

. Isothermal titration calorimetry (ITC) analysis of the LDHA and NADH. The NADH was titrated into the LDHA solution. LDHA: lactate dehydrogenase A; NADH: 1,4-dihydronicotinamide adenine dinucleotide.   Tetrameric structures of LDHA with malonate/NADH are shown. Chains A, B, C and D are colored green, yellow, blue, and cyan, respectively. The LDHA molecules are color coded with C in green, O in red, and N in blue. The 2Fo-Fc electron density maps of LDHA with malonate and NADH are contoured at 1σ (gray) (C) Superposition of the LDHA complexes with malonate and NADH is shown. (D) Superposition of LDHA complexes with machilin A and NADH is shown. LDHA: lactate dehydrogenase A; NADH: 1,4-dihydronicotinamide adenine dinucleotide.   under normoxic and hypoxic conditions. The activity of LDH was investigated using cell lysates. Data are presented as mean ± SD. Statistic testing was performed using a Student's t-test. *** p < 0.001 compared to the control under normoxia (first column). ## p < 0.01 compared to the control under hypoxia (third column). (B) LLC cells were treated with the indicated concentrations of machilin A for 48 h. Cell viability was evaluated using an MTT assay. Results of three independent replicates are shown as means ± SD. Statistic testing was performed using a oneway ANOVA. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared to the control. LDHA: lactate dehydrogenase A; SD: standard deviation.
. Figure S9. The effects of machilin A on liver or kidney functioning. (A) LLC and (B) CT26 cells were inoculated subcutaneously into mice. After inoculation, the indicated dosages of machilin A were intraperitoneally injected into mice daily. Thirteen days after inoculation of tumor. The blood of each mouse was collected from the retro-orbital plexus, and then the sera from blood were prepared. To investigate the toxicity of machilin A on liver and kidney of mice, biochemical analysis on the aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and blood urea nitrogen (BUN) were performed by commercial service by Green Cross Co.  To determine effect of machilin A on lactate production in cancer cells using NMR spectroscopy, HT29 colorectal cancer cells (5 × 10 5 cells/2mL) were treated in presence of machilin A (30 μM) in normoxia and hypoxia conditions. Production of lactate was measured by NMR spectroscopy. The results from 3 independent experiments are shown as the means ± SD. * p < 0.05 compared to the normoxia control (1st column). ** p < 0.01 compared to the hypoxia control (3rd column). (C) Raw 264.7 macrophage cells were treated with indicated concentration of lactic acid for 24 h. The expressions of Arg-1 and CD206 mRNAs were determined by RT-PCR. GAPDH mRNA expression was used for internal control. (D) Raw 264.7 macrophage cells were treated with lactic acid or LPS for 24 h. The expressions of Arg-1, CD206, YM1, and VEGF mRNAs were determined by RT-PCR. GAPDH mRNA expression was used for internal control. (E) The HT29 cancer cells were treated with or without machilin A (30 μM), and incubated in hypoxic condition. At 48 h after incubation, the conditioned medium (1 mL) harvested from cancer cells was added to bone marrow-derived macrophage in the presence or absence of lactate (5 mM). The expressions of ARG-1 and CD206 proteins as M2 markers and the expressions of CD86 and iNOS proteins as M1 markers were determined by FACS. Data are presented as mean ± SD. Data were also statistically compared using the Student's t-test. *** p < 0.001 compared to No.1. ## p < 0.01 and ### p < 0.001 compared to No.2. § § p < 0.01 and § § § p < 0.001 compared to No.3.  Values in parentheses are for the highest resolution shell. a Rmerge = ∑|Ii Im|/∑Ii, where Ii is the intensity of the measured reflection and Im is the mean value of all symmetry-related reflections. b Rcryst = Σ||Fobs|−|Fcalc||/Σ|Fobs|, where Fobs and Fcalc denotes the observed and calculated structure factor amplitude. Rfree = ∑T||Fobs|−|Fcalc||/ΣT|Fobs|, here T is a test data set of about 5% of the total reflections randomly chosen and set aside prior to refinement. LDHA: lactate dehydrogenase A; NADH: 1,4-dihydronicotinamide adenine dinucleotide.

Electron Microscopy
Purified lactate dehydrogenase A (LDHA) (concentration, 0.5 mg/mL) was applied to a glowdischarged copper grid covered with a continuous carbon film. The sample negative stained with 1% (w/v) uranyl acetate for 1 min. Images were collected at a nominal magnification of 67,000x, 110,000x and 220,000x by a Tecnai G2 Spirit electron microscope.

Sample Preparation of NMR Spectroscopy
350 μL of media sample was transferred to micro centrifuge tube containing 350 μL of deuterated sodium phosphate buffer containing 4 mM of TSP-d4(3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt). 630 μL of cell lysate sample was transferred to micro centrifuge tube containing 70 μL of deuterated sodium phosphate buffer containing 20 mM of TSP-d4. Each sample was transferred to 5-mm nuclear magnetic resonance (NMR) tube. Trimethylsilylpropanoic acid (TSP) was used as a quantitative reference (2 mM) and standard of chemical shift (0.00 ppm). Each sample was transferred to 5-mm NMR tube.

NMR Spectroscopy
All spectra were acquired at 600.167 MHz Agilent NMR spectrometer. CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence was used because of suppression of water and high molecular mass compounds. The acquisition time was 1.704 sec, relaxation delay was 1.5 sec and 128 transient were collected. Lactate was quantified using Chenomx NMR Suite 7.1 professional with the Chenomx 600 MHz library database (Chenomx Inc., Edmonton, Alberta, Canada,).

Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) measurements were carried out using MicroCal auto_iTC200 (GE healthcare, Chicago, IL, USA) at 25 °C. The purified LDHA protein was dialyzed in buffer containing 50 mM Tris-HCl (pH 8.0) and 200 mM NaCl at a concentration of 0.1 mM. NADH was solubilized in the same buffer at concentrations of 1 to 2 mM. The NADH was titrated into the LDHA solution onto an ITC200 microcalorimeter cell. The raw data were integrated and normalized using MicroCal Origin 7.0 software (GE healthcare). The determined binding affinity (KA) and enthalpy changes (ΔH) values were used to calculate ΔS from the standard thermodynamic equation.

Flow Cytometry
For generation of bone marrow-derived macrophage (BMDM), bone marrow cells were extracted from C57BL/6 mice by surgery, and treated with M-CSF (10ng/ml) for 7days. BMDM cells were incubated with the indicated concentration of culture media with MA and lactate or not. The cells were washed with phosphate buffered saline (PBS) and fixed in 3.7% formaldehyde in PBS for 1h. After washing two times with PBS, the samples were incubated with primary antibody against CD206 (ab64693, Abcam, Cambridge, UK), Arg-1 (#9819, Cell Signaling, Danvers, MA, USA), CD86 (sc-28347, SantaCruz, Clara, CA, USA), or iNOS (#482728, Calbiochem, St. Louis, MO, USA) in 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The cells were washed again and incubated with secondary antibody for 1 h. The samples were examined on a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).