Anti-cancer activity of glucosamine through inhibition of N-linked glycosylation
© Chesnokov et al.; licensee BioMed Central Ltd. 2014
Received: 19 June 2013
Accepted: 13 May 2014
Published: 28 May 2014
We have reported that the glucosamine suppressed the proliferation of the human prostate carcinoma cell line DU145 through inhibition of STAT3 signaling. DU145 cells autonomously express IL-6 and the IL-6/STAT3 signaling is activated. IL-6 receptor subunits are subject to N-glycosylation, a posttranslational modification which is important for protein stability and function. We speculated that the inhibition of STAT3 phosphorylation by glucosamine might be a functional consequence of the reduced N-glycosylation of gp130.
The human prostate cancer cell lines DU145 and PC-3 and human melanoma cell line A2058 were used in this study. Glucosamine effects on N-glycosylation of glycoproteins were determined by Western blot analysis. IL-6 binding to DU145 cells was analyzed by flow cytometry. The cell proliferation suppression was investigated by colorimetric Janus green staining method.
In DU145 cells glucosamine reduced the N-glycosylation of gp130, decreased IL-6 binding to cells and impaired the phosphorylation of JAK2, SHP2 and STAT3. Glucosamine acts in a very similar manner to tunicamycin, an inhibitor of protein N-glycosylation. Glucosamine-mediated inhibition of N-glycosylation was neither protein- nor cell-specific. Sensitivity of DU145, A2058 and PC-3 cells to glucosamine-induced inhibition of N-glycosylation were well correlated to glucosamine cytotoxicity in these cells.
Our results suggested that the glucosamine-induced global inhibition of protein N-glycosylation might be the basic mechanism underlying its multiple biochemical and cellular effects.
In the previous paper , we reported that glucosamine suppressed the proliferation and induced the death of human prostate carcinoma DU145 cells. This anti-cancer activity was associated with a decreased DNA synthesis, cell cycle arrest in G1 phase, and induction of apoptosis. We demonstrated for the first time that glucosamine inhibited the phosphorylation of signal transducer and activator of transcription (STAT) 3 at Tyr705 residue, thereby inhibiting the STAT3 activity in DU145 . STAT3, a member of the STAT family, is activated by phosphorylation and mediates cellular responses to cytokines and growth factors . Activated STAT3 promotes angiogenesis, inflammation, proliferation and survival of cancer cells and is frequently detected in numerous human tumors . Suppression of the STAT3 signaling pathway [4–6] therefore represents a validated target for cancer therapy [3, 7]. In DU145 cells, IL-6 is autonomously expressed and activates the IL-6/STAT3 signaling pathway by an autocrine mechanism . IL-6 first binds to the two non-signaling α-receptor subunits (IL-6Rα/gp80) and the resultant complex recruits the two signal transducing receptor subunits (gp130) to form the functional ternary receptor complex. Janus kinases (JAKs) associated with gp130 are activated by phosphorylation and then STAT3 becomes activated by the phosphorylated JAKs .
Almost all receptors on the cell membrane are N-linked glycosylated proteins . N-linked glycosylation (N-glycosylation) is a posttranslational modification; a preassembled core oligosaccharide moiety (glycan) is transferred to asparagine (N) residues at potential glycosylation sites of newly synthesized polypeptides and the attached N-linked glycans (N-glycans) are subjected to remodeling and branching . N-glycosylation plays an important role for protein stability and functions [10, 12]. Taking into account that gp130 is a highly N-glycosylated protein  and that glucosamine was proposed as an inhibitor of N-glycosylation , we speculated that the inhibition of STAT3 phosphorylation by glucosamine might be a functional consequence of the reduced N-glycosylation of gp130.
In this paper, we revealed that in DU145 cells glucosamine reduced the N-glycosylation of gp130, decreased IL-6 binding to cells and impaired the phosphorylation of JAK2 on Tyr1007/1008, SHP2 on Tyr542 and STAT3 on Tyr705 residues. Treatment with tunicamycin, an inhibitor of protein N-glycosylation , demonstrated all the effects of glucosamine supporting our speculation. We also showed that glucosamine-mediated inhibition of N-glycosylation was neither protein- nor cell-specific. Our results suggested that the glucosamine-induced global inhibition of protein N-glycosylation might be the basic mechanism underlying its multiple biochemical and cellular effects.
Glucosamine reduces the molecular mass of gp130 in DU145 cells by the inhibition of co-translational N-glycosylation
Glucosamine-induced inhibition of N-glycosylation of gp130 represses the IL6/JAK/STAT3 signaling in DU145 cells
Glucosamine affects on multiple N-glycosylated proteins
Sensitivity of cells to glucosamine-induced inhibition of N-glycosylation is correlated to glucosamine anti-cancer activity
Glucosamine, an amino monosaccharide has been widely used as a dietary supplement to relieve discomfort of rheumatoid arthritis or osteoarthritis for more than fifty years . Glucosamine has also been reported as an inhibitor of tumor growth both in vivo and in vitro. However, the mechanism for the anti-cancer activity is still not fully understood to explore the potentials of glucosamine as an anti-cancer agent. Previously , we have proposed our hypothesis that glucosamine could inhibit the N-glycosylation of IL-6 receptor in human prostate carcinoma DU145 cells, thereby reducing the activity of the IL-6/JAK/STAT3 signaling pathway. This pathway is activated and contributes to carcinogenesis in many different tumors. This study focused on proving our hypothesis and examining the mechanism by which the STAT3 activity is suppressed by glucosamine. When DU145 cells were treated with glucosamine, Western blot analysis showed that several gp130 proteins with a lower molecular mass appeared in a time and dose dependent manner (Figure 1). In the following experiments, we demonstrated that digestion of the extracts from the untreated and glucosamine-treated DU145 cells with peptide-N-glycosidase F (PNGase F), which removed all N-linked glycans , provided proteins with the same mobility on Western blot gels regardless of the mobility of the proteins before the digestion. These results indicated that gp130 proteins with a lower molecular mass had less N-glycosylated sites and therefore, were not truncated products of translation step. Furthermore, the glucosamine treatment of DU145 cells in the presence of cycloheximide did not produce any gp130 proteins with a lower molecular mass suggesting that glucosamine inhibited N-glycosylation of the newly synthesized protein but did not remove N-glycans from the mature gp130 (Figure 2). IL-6 receptor is composed of four subunits, two gp80 subunits containing the IL-6 binding domain and two gp130 subunits containing the signal transducing domain. Flow cytometry binding assays showed that IL-6 bound weakly to cells treated with glucosamine. We speculated that the gp130 subunit deficient in N-glycosylation could not form the intact IL-6 receptor with the gp80 subunit and thereby the binding of IL-6 to cells was suppressed to reduce the signal transduction. It was shown that N-glycosylation-deficient gp130 after tunicamycin treatment lost the ability to form a heterodimer with the leukemia inhibitory factor (LIF) binding subunit, and lost the signal transduction in response to LIF in neuroepithelial cells . To further confirm that glucosamine acts in a similar manner to tunicamycin, we carried out the same experiments described above using tunicamycin in place of glucosamine. Tunicamycin is a proven inhibitor of protein N-glycosylation which blocks the transfer of N-acetylglucosamine to dolichol in the first step of oligosaccharide synthesis . Indeed, tunicamycin lowered the molecular mass of the normally expressed gp130, suppressed IL-6 binding to cells and reduced the activity of the IL-6/JAK/STAT3 signaling pathway in a dose dependent manner like glucosamine (Figure 1C, 3). Obviously, various proteins are N-glycosylated and the modification of proteins by N-glycosylation is not specific to gp130. To confirm that these N-glycosylated proteins are substrates for glucosamine, we carried out the Western blot analysis of several glycoproteins, EGFR, c-MET, CD44, clusterin and MRP1 after the glucosamine treatment. In addition to gp130, the treatment produced these glycoproteins with a lower molecular mass in a different extent (Figure 5A). Since most of cell-surface receptors are N-glycosylated , it was expected that multiple signaling pathways activated from these receptors were affected by glucosamine. Indeed, Western blot analysis showed that in addition to STAT3, the phosphorylation (activity) of other proteins playing key signaling roles in cancer cells, AKT and ERK1/2, were suppressed after the treatment with glucosamine in DU145 cells (Figure 5B). These results suggest that glucosamine-induced inhibition of global protein N-glycosylation might be the basic mechanism of multiple biochemical and cellular events of its anti-cancer action. In fact, the sensitivity of different cells to glucosamine measured by de-N-glycosylation of clusterin was in a good agreement with their viability (Figure 6). As it was proposed, targeting multiple signaling pathways by disrupting posttranslational N-glycosylation in a combination with other agents might be an effective strategy for cancer therapy . For example, tunicamycin treatment enhanced the susceptibility of human non-small cell lung cancer cells to EGFR tyrosine kinase inhibitor erlotinib , and also stimulated TRAIL–induced apoptosis in human prostate cancer PC-3 cells . Unfortunately, tunicamycin is too toxic to use for the human treatment . Alternatively, glucosamine could be a candidate for clinical use as an inhibitor of posttranslational N-glycosylation because of its low toxicity to the normal cells and enhanced uptaken by tumor cells due to “Warburg effect” . We will investigate this possibility and study the mechanism underlying inhibition of N-glycosylation by glucosamine.
Current study found that, in human prostate carcinoma cell line DU145, glucosamine reduced N-glycosylation of IL-6 receptor subunit gp130 by inhibiting co-translational N-glycosylation, resulting in less IL-6 binding to cells and less phosphorylation (activation) of down-stream JAK2 and STAT3 proteins. Our results demonstrated that glucosamine-mediated inhibition of N-glycosylation was neither protein- nor cell-specific. Glucosamine induced global inhibition of protein N-glycosylation in all three cancer cell lines which were used in this study. Several important signals, such as STAT3, AKT and ERK1/2, were activated by N-glycosylated membrane receptors. The treatment of glucosamine led to the suppression of these signals, suggesting that the glucosamine-induced inhibition of N-glycosylation might be the basic mechanism underlying its biochemical and cellular effects.
Cell culture, chemical compounds and biological reagents
The human prostate cancer DU145 and PC3 cells and human melanoma A2058 cells were obtained from the American Type Culture Collection. Cells were cultured in RPMI medium 1640 supplemented with glutamine, essential amino acids, 10% fetal bovine serum and antibiotics (100 units/ml penicillin G and 100 ug/ml streptomycin sulfates). Cells were incubated at 37°C in 5% CO2, and the medium was changed every 3-4 days. Cells were passaged at 70% confluent using trypsin/EDTA. Treatments of cells with D-glucosamine hydrochloride, tunicamycin, cycloheximide, cytochalasin B and interleukin-6 (IL-6) were performed in 6-well plates (Corning, Inc., Corning, NY). D-glucosamine hydrochloride, tunicamycin, cycloheximide, propidium iodide and cytochalasin B were purchased from Sigma Chemical Co., (St. Louis, MO; G-1514, T-7765, C-4859, C-6762, P-1607, and C2743). IL-6 and Human IL-6 Flow cytometry Kit were purchased from R & D Systems (Minneapolis, MN; 206-IL, NF600). Peptide: N-glycanase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA; PO704). Antibodies used for immunoblot (Western) analysis included anti-gp130, anti-clusterin (Millipore, Danvers, MA; 06-291, 05-354), anti-STAT3, anti-phospho (Tyr705)-STAT3, anti-phospho (Tyr1007/1008)-JAK2, anti-phospho (Tyr542)-SHP2, anti-EGFR, anti-phospho (Tyr845)-EGFR, anti-phospho (Ser473)-AKT, anti-phospho (Thr202/Tyr204)-ERK1/2, anti-c-Met, anti-CD44, anti-mouse IgG HRP-linked and anti-rat IgG HRP-linked from Cell Signaling Technology (Beverly, MA; 9132, 9145, 3771, 3751, 2232, 2231, 4060, 4370, 3127, 5640, 7076, 7077), anti-actin and anti-MRP1 from Santa Cruz Biotechnology (Santa Cruz, CA; 1615, 59607). Additionally, Janus Green whole-cell stain was purchased from ThermoScientific-Pierce (Rockford, IL; 62203).
After removing the culture medium cells in 6-well plates were washed with 1× PBS and then lysed in the wells with 0.1 ml of RIPA lysis and extraction buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% Sodium deoxycholate, 0.1% SDS, pH 7.6, (G-Bioscience, St. Louis, MO; 786-489,) supplemented with protease and phosphatase inhibitor cocktail (ThermoScientific-Pierce, Rockford, IL; 78440) for 15 min at 4°C. Lysates were then transferred to 1.5 ml microcentrifuge tubes, vortexed at maximum speed for 15 sec to shear DNA and centrifuged at 12000 g for 10 min at 4°C. Supernatants were quantified for protein concentrations by BCA protein assay kit (ThermoScientific-Pierce, Rockford, IL; 23227) and stored at -80°C in aliquots or used immediately for SDS-PAGE. The samples for SDS-PAGE were prepared by incubating with 4% SDS at 37°C for 30 min for solubilizing N-glycosylation deficient proteins and then boiling in the sample buffer (50 mM Tris-HCI pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue) for 5 min. Equal amounts of proteins were then loaded onto 8% precast SDS-PAGE gels (ThermoScientific-Pierce, Rockford, IL; 25200). Immunoblotting was performed after the electrophoretic transfer of proteins onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA; 162-0115). The proteins were detected using protein-specific first antibodies, horseradish peroxidase-conjugated second antibodies and chemiluminescent detection reagent (Denville Scientific, Metuchen, NJ; E2500) according to the manufacturer’s conditions. Protein sizes on nitrocellulose membranes were detected by protein markers (Bio-Rad Laboratories, Hercules, CA; 161-0374). To reuse, nitrocellulose membranes were washed with western blot stripping buffer (ThermoScientific-Pierce, Rockford, IL; 46430) and blocked again.
Peptide: N-glycanase F (PNGase F) treatment
To remove N-glycans from glycoproteins in vitro aliquots of whole cell extracts in RIPA buffer were incubated with Peptide:N-glycanase F (40 μg/μl) at 37°C for 4 h and then stored at -80°C or used immediately for Western blot analysis.
Evaluation of IL-6 binding to DU145 cells by flow cytometry
The Human IL-6 Flow Cytometry Kit (R & D Systems) was utilized for analysis of IL-6 binding according to the manufacturer’s protocol with slight modifications. Briefly, DU145 cells were harvested by the treatment with 1 mM EDTA for 3-5 min, washed two times with phosphate-buffered saline (PBS), resuspended in PBS (4 × 106/ml) and followed by the incubation of 50 μl of cells for 1 hour at 4°C with either 20 μl of biotinylated IL-6 or 20 μl of biotinylated negative control reagent. 20ul of Avidin-fluorescein was then added to each set of cells and incubation was continued for a further 30 minutes at 4°C. Cells were then washed two times with the supplied wash buffer, resuspended with 300 μl of wash buffer containing 10 ug/ml propidium iodide (PI) to identify dead cells and examined by flow cytometry. A total of 40000 cells were counted for each analysis. PI-positive/dead cells were subsequently excluded from the IL-6 binding examination. Fluorescence was measured using the CyAn System with Summit software (Beckman Coulter, Brea, CA).
Cell growth suppression analysis
DU145, PC-3 or A2058 cells were plated in 96-well plates in a complete culture medium with or without different amount of glucosamine. Two days later, the relative cell numbers per well were determined by the colorimetric Janus green whole-cell stain method. Briefly, culture medium was removed from cell layers, then cells were fixed with 4% formaldehyde (Thermo Scientific, Rockford, IL; 28906) for 30 min at room temperature, and followed by vacuum aspiration of the fixative. Subsequently, fixed cell layers were stained with Janus Green Whole-Cell Stain solution (Thermo Scientific, Rockford, IL; 62203) for 5 min at room temperature. Then the excess stain was removed by vacuum aspiration and cells were washed 4 times with water. The dyes associated with cell layers were then eluted by the addition of 100 μl of 0.5 N HCl per well and shaking the plates at room temperature for 1 h. The colored dye solutions were read in a microplate reader (ASYS UVM340 Microplate Reader, Cambridge UK) at 615 nm. Results were expressed as a percentage of the control without glucosamine. Data were represented as mean +/- standard deviation (SD) from quadruplicate wells. Each cell line was analyzed in three independent experiments.
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