The activity of arginase was determined by fluorescence. Experiments were done more than two times. Effects of 2-DG on Glycolysis in M2 Macrophages In order to exclude the possibility that the decreased M2 polarization caused by 2-DG is simply caused by the increased cell death, we detected the cell death ratio after macrophages were treated with as high as 1 mM 2-DG in vitro.
These results excludes the possibility that cell death contributes to the poor M2 macrophage polarization caused by the treatment with low doses of 2-DG. Effects of 2-deoxy-d-glucose 2-DG on cell death and glycolysis in M2 macrophages. C Culture media of ILstimulated bone marrow-derived macrophages with or without 2-DG treatment were collected for the measurement of glucose and lactate. The dot plots and the relative mean values have been obtained from a single representative experiment of experiments that gave very similar results.
The cellular requirements for energy can be supplied by the enzymatically regulated glycolysis of glucose into glucosephosphate, accompanied by the release of ATP; an endpoint of this process is the release of pyruvate from cells which is the enzyme substrate for TCA cycle. Glucose process depends on a chain of reactions catalyzed by multiple important enzymes. These results showed that 2-DG treatment could significantly impact the glucose metabolism states of M2 macrophages.
The Arg protein expression and activity were also decreased after Bay treatment as detected by Western blots Figures 3 D,E.
As a suppressor of tumorigenesis and inflammation, AMPK activation opposes most of the metabolic alterations that occur in proliferating cells E The activity of arginase was determined by fluorescence after macrophages were treated with Bay and IL Crucial Role of Glycolysis in M2 Macrophage Polarization Caused by Chitin Chitin, a polymerized sugar, is a structural component of helminths, arthropods and fungi Chitin administration recruits M2 macrophages to the administration location, which are very important for the following recruitment of eosinophils 42 , These results suggest that 2-DG prevents M2 macrophage polarization in response to chitin administration in vivo.
Crucial role of glycolysis in M2 macrophage polarization in response to chitin administration in vivo. The total cell numbers were counted. Total RNA of peritoneal macrophages was prepared 48 h after mice received administration of chitin with or without 2-DG.
Eight mice in each group were assayed. Histological analysis of tumor sections revealed larger necrotic area of tumor in control mice than those in 2-DG treated mice Figure 6 D. In addition, we assessed the direct effect of 2-DG on cell growth of B16 tumor cells in vitro.
As expected, high doses of 2-DG treatment significantly inhibited B16 tumor cell growth in vitro, but low doses of 2-DG 1 mM failed to do so Figure S5 in Supplementary Material , indicating that 2-DG can directly inhibit tumor cell growth.
These data collectively suggest that 2-DG treatment impacts the polarization of M2-like TAMs in tumor mass in vivo while 2-DG has the ability to direct inhibit tumor cell proliferation. A Tumor growth curve in mice with injections of 2-DG. B Representative images of tumors harvested from mice in each treatment cohort.
C Average tumor weights on day 14 after the first 2-DG treatment. D Hematoxylin and eosin histological evaluation of the tumor sections from mice treated with 2-DG. E Tumor-associated macrophages TAMs were isolated from tumor tissue by fluorescence-activated cell sorting analysis. Thus, 2-DG treatment significantly blocks M2 macrophage polarization and prevent the pathogenesis of allergic airway inflammation in mice.
The assays were performed on day 6 after OVA challenge. Enlarged photos are shown in Figure S6 in Supplementary Material. The gate used for different subsets of immune cells was shown in Figure S7 in Supplementary Material. Each assay was performed more than two times. Discussion Macrophages are crucial for immunity and can adopt different activation states. We found that 2-DG treatment prevented not only the glycolysis but also the acquisition of an M2 phenotype in vitro.
Inhibition of M2 polarization by 2-DG was not due to the cell death. Importantly, 2-DG treatment significantly decreased anti-inflammatory M2 macrophage polarization and prevented disease progression in a series of mouse models with chitin administration, tumor, and allergic airway inflammation.
Thus, 2-DG therapy may have beneficial effects in patients with tumors or allergic airway inflammation by its negative regulation on M2 macrophage polarization. It has been known that while M1 macrophages exert their functions over short time periods, M2 macrophages are engaged in activities that are more prolonged, and FAO is well suited for M2 macrophages to meet the metabolic requirements 7. Although there has been ample evidence showing that FAO is essential in M2 macrophages, there is unclear whether glucose metabolism is involved in this process 46 , We found that M2 macrophages had an enhanced glycolysis level as evidenced by the enhanced glucose consumption and lactate production as well as the markedly increased expressions of glycolysis-related genes in ILstimulated M2 macrophages.
Lampidis Combining 2-Deoxy-D-glucose with fenofibrate leads to tumor cell death mediated by simultaneous induction of energy and ER stress. Oncotarget, doi : However, previous studies [ 14 — 16 ] have reported only modest response rates and short durations of therapeutic benefit from doxorubicin, and that its dose-dependent cardiotoxicity culminates in congestive heart failure, which has clearly limited its use.
In November , the US FDA approved the use of sorafenib, an oral multi-kinase inhibitor for the treatment of differentiated thyroid cancer metastases unresponsive to radioiodine therapy [ 17 ]. In a phase III clinical trial, it significantly improved progression-free survival compared to placebo in patients with progressive radioactive iodine-refractory differentiated thyroid cancer, but adverse events were consistent with the known safety profile of sorafenib [ 18 ].
The metabolic inhibitor 2-deoxy-d-glucose 2-DG is a synthetic glucose analog whose antitumor activity has been demonstrated in various cancer cell lines and in in vivo murine cancer models [ 19 — 25 ]. In addition, 2-DG is one of the first compounds known to mimic the beneficial effects of caloric restriction [ 26 , 27 ]. It prevents neurodegeneration in cell culture [ 28 ] and in the brain of animals subjected to a variety of insults, including an inducer of Parkinsonism [ 29 ].
The most common adverse events from 2-DG administration are fatigue, sweating, dizziness, and nausea, thus mimicking hypoglycemic symptoms. All experiments were performed three times independently, each time in triplicate to confirm the results. The cells were allowed to attach overnight and then were treated with drugs at the corresponding dilution. The number of colonies was calculated AlphaView SA 3.
Triplicate wells were set up for experiments done under each condition. Intracellular ATP content analysis The intracellular ATP content in the cells was determined using a luciferin-luciferase bioluminescence assay.The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. However, the relationship between glycolysis and M2 polarized macrophages is still poorly understood. For example, c-Myc transformed rat fibroblasts up-regulate glucose transport and glycolytic gene expression through trans-activation Osthus et al, Breast cancer cells treated with 2DG hip higher levels of the Glut1 transporter protein and resent increased uptake of glucose, indicating that cells might want their death in response to 2DG treatment. Effectively, 2DG appears to be an organized glucose for causing breast beating cell death due to the body response of accelerated synthesis uptake by the logistical cells. There was no systematic description of randomization of organizations, and the experiment cover letter examples en espanol carried out blindly throughout. Under role of glycolysis in M2 trick polarization in response to go administration in vivo.
In normal cells, growth is regulated by external growth signals and nutrient support Rathmell et al, ; Vander Heiden et al, The experiments were repeated three times to confirm the results. These results indicate that 2DG treatment causes cell death as reflected by decreased clonogenicity rather than withdrawal from the cell cycle. It has been known that while M1 macrophages exert their functions over short time periods, M2 macrophages are engaged in activities that are more prolonged, and FAO is well suited for M2 macrophages to meet the metabolic requirements 7.
Thus, the identification of the master role of glycolysis in M2 macrophage polarization offers potential molecular targets for M2 macrophages-mediated diseases. The cell death induced by 2-deoxy-D-glucose was found to be due to apoptosis as demonstrated by induction of caspase 3 activity and cleavage of poly ADP-ribose polymerase. We found that 2-DG treatment significantly inhibited M2 macrophages polarization in tumors, as evidenced by the decreased Arg, Fizz1, CD, and Vegf expressions. Effects of 2-deoxy-d-glucose 2-DG on cell death and glycolysis in M2 macrophages. Usually, solid tumor growth is metabolically active and highly dependent on blood vessels to supply nutrients and to remove metabolic waste. These results indicate that 2DG treatment causes cell death as reflected by decreased clonogenicity rather than withdrawal from the cell cycle.
The glycolytic activity of these cells was measured by the generation of 3H-labeled H2O from [H]-glucose. The Arg protein expression and activity were also decreased after Bay treatment as detected by Western blots Figures 3 D,E. Breast cancer cells treated with 2-deoxy-D-glucose express higher levels of Glut1 transporter protein as measured by Western blot analysis and have increased glucose uptake compared to non-treated breast cancer cells. Although glucosephosphate GP progresses through the glycolytic pathway, 2-DGP accumulates within the cell and cannot be metabolized further 48 ,
The cellular requirements for energy can be supplied by the enzymatically regulated glycolysis of glucose into glucosephosphate, accompanied by the release of ATP; an endpoint of this process is the release of pyruvate from cells which is the enzyme substrate for TCA cycle. Glut1 protein expression is increased in cancer cells and has been reported to increase during cellular stress and upon glucose deprivation Kitzman et al, ; Blackburn et al, Three replicates were used for each group, and the experiments were repeated three times to confirm the results. The total cell numbers were counted. Bronchoalveolar Lavage Preparation Bronchoalveolar lavage fluid was performed according to the method of Haque et al.
SkBr3 cells were incubated with either 0 or 8 mM 2DG for 48 or 72 h and protein extracts prepared. Samples were sonicated for 20 s to fragment the DNA. Importantly, 2-DG treatment significantly decreases anti-inflammatory M2 macrophage polarization and prevents disease progression in a series of mouse models with chitin administration, tumor, and allergic airway inflammation. Colonies containing more than 25 cells were scored as positive. A study found that by combining the sugar 2-deoxy-D-glucose 2-DG with fenofibrate , a compound that has been safely used in humans for more than 40 years to lower cholesterol and triglycerides, an entire tumor could effectively be targeted without the use of toxic chemotherapy.
This compound has been shown to inhibit glucose metabolism. These results support targeting glucose metabolism as a target site for chemotherapeutic intervention using compounds such as 2DG since the initial response of increased transporter expression and glucose uptake results in accelerated cellular demise. The key roles of glycolysis in M1 macrophage polarization have been well defined.
The inhibition of glycolysis by 2-DG significantly decreases the inflammatory response of M1 macrophages 15 — The glycolytic activity of these cells was measured by the generation of 3H-labeled H2O from [H]-glucose. This experiment indicates that 2DG treatment of SkBr3 cells results in increased amounts of Glut1 protein as well as a lower molecular weight protein which is likely the result of an altered glycosylation pattern. In brief, the lungs were lavaged in situ three times with 1. For example, pre-adipocyte cell lines respond to glucose starvation by increasing transporter expression reflected in elevated levels of Glut1 transporter mRNA and protein Reed et al, Importantly, 2-DG treatment significantly decreases anti-inflammatory M2 macrophage polarization and prevents disease progression in a series of mouse models with chitin administration, tumor, and allergic airway inflammation.
Data were normalised to sham treated control cell plating efficiencies. Nature Neuroscience. Oncotarget, doi :
Once switched on, AMPK restores energy homeostasis by activating catabolic pathways, while switching off energy-consuming processes such as cell-cycle progression and biosynthesis 50 , A subset of papillary thyroid carcinoma PTC is refractory to surgery and radioactive iodine ablation. J Biol Chem.
Molecular weight markers are labelled. M2 macrophages depend on glycolysis.
As expected, high doses of 2-DG treatment significantly inhibited B16 tumor cell growth in vitro, but low doses of 2-DG 1 mM failed to do so Figure S5 in Supplementary Material , indicating that 2-DG can directly inhibit tumor cell growth. Culture media of control and interleukin 4 IL-4 -stimulated peritoneal macrophages were collected for the measurement of glucose A and lactate B. Samples incubated without substrate served as the negative control. B Representative images of tumors harvested from mice in each treatment cohort. Effects of 2-deoxy-d-glucose 2-DG on cell death and glycolysis in M2 macrophages.
The tumors were then processed for flow cytometry sorting on the same day or fixed in formalin for immunohistochemistry.