Background
Since the effect of glucosamine as an inhibitor of tumor growth was first reported by Quastel and Cantero, [
1] many
in vitro studies have shown that it interferes with the glycoslyation of glycoproteins, [
2,
3] decreases the rate of glycolysis and fructolysis, [
4,
5] and changes the component ratio of nucleotides in various carcinoma cell lines [
6,
7]. Results of a recent study indicated that glucosamine induces G1 cell-cycle arrest in mesangial cells and human cancer cells through a mechanism involving decreased expression of cyclin D1 and increased expression of p21
Waf1/Cip1, which are positive and negative regulators of cell cycle progression, respectively [
8,
9].
The PI3K/Akt pathway is often overactivated in various types of cancer cells. PI3K/Akt can transmit signals from RTKs and G-protein-coupled receptors that are activated by growth factors or cytokines; therefore, the PI3K/Akt signal transduction pathway regulates multiple cellular functions, including transcription, translation, and cell proliferation, cell cycle progression, and survival [
10‐
12]. Although the RTK-mediated signal transduction pathways overlap, PI3K-mediated activation of Akt specifically contributes to the anti-apoptotic activity of IGF-1R.
Recent studies have demonstrated that target proteins of glucosamine may exist in cancer cells [
13‐
16]. Glucosamine inhibits the growth of cancer cells by downregulating the phosphorylation of p70S6K, a regulator of protein translation [
15]. In addition, glucosamine inhibits HIF-1α by inhibiting protein translation through the reduction of phosphorylated p70S6K levels [
16]. Jang et al. reported that glucosamine hydrochloride inhibits N-glycosylation of COX-2 and enhances COX-2 protein turnover [
13]. Finally, glucosamine induces NF-κB inactivation by inhibiting transglutaminase 2 (TGase 2) activity [
14]. Together, these studies suggest that glucosamine has potential as an anticancer drug, although its mechanism of action remains poorly understood [
17]. Thus, we tested whether the IGF-1R/PI3K/Akt pathway, upstream of p70S6K and COX-2, is target of glucosamine. We also investigated the molecular mechanisms underlying the anticancer activity of glucosamine in NSCLC cells.
Methods
Cell lines and materials
Human NSCLC cell lines A549, H226B, H1299, and H460 were purchased from the American Type Culture Collection (Manassas, VA, USA).
The HA-Akt1 (T308D/S473D) expression vector was kindly provided by Dr. Gordon Mills (The University of Texas MD Anderson Cancer Center). The H226B-Babe cells were produced by infecting H226B NSCLC cells with a pBabe retroviral control vector. The H226B-Akt1-DD cells that possess a constitutively active form of Akt were produced by infecting H226B with a pBabe-HA-Akt1-DD construct harboring mutations that change Ser473 and Thr308 to aspartic acids. The H226B-Akt2-DD and The H226B-Akt3-DD cells were kindly provided by Dr. Ho-Young Lee (College of Pharmacy, Seoul National University, Seoul, Republic of Korea).
D-(+)-Glucosamine hydrochloride, MG132, and tunicamycin (TN) were purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies against pIGF-1R, pAkt, pERK1/2, Akt, PTEN, PARP, PDI, IRE1α, ATF4, GRP78, CHOP, and a/β-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against IGF-1R, COX-2, CDK2, CDK4, and β- ACTIN were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and the antibody against TGase 2 was obtained from Thermo Fisher Scientific, Inc. (Fremont, CA, USA).
Xenograft mouse tumor model
All animal experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) of National Cancer Center in Republic of Korea. To confirm antitumor effect of glucosamine in animal, we used xenograft tumor model. A549 cells (5 x 106 cells) were subcutaneously injected into flank region of BALB/c nude mice. After cancer cell injection, glucosamine (500 mg/kg body weight/day) was administered intrapenitorially to immuoncompromised mice. Tumor volume was measured using caliper and calculated according to the formula (L x W
2
)/2. All of the mice were sacrificed on Day 71, and tumor tissues were isolated from them. The results were represented as the mean of tumor volumes (n = 10) with SEM.
siRNA transfection
For RNA interference, A549, H1299, and H460 cells transfected with 40 nM siRNA. Double-stranded siRNAs designed to target IGF-1R (5′-CUG ACA UGG GCC UUU AAG A-3′), and a scrambled non-targeting siRNA were synthesized by Bionner (Seoul, South Korea). Cells were transfected with siRNAs using Lipofectamine reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol.
Semiquantitative RT-PCR
First strand cDNA was synthesized from 2 μg of extracted RNA using M-MLV reverse transcriptase (Invitrogen). RT-PCR was carried out with gene-specific primers for
IGF-1R, COX-2,
XBP1, GRP78, CHOP, ATF4, GAPDH, and
β-ACTIN (Table
1). Primers amplifying a region of
β-ACTIN or
GAPDH were used as an internal control.
Table 1
Primer sequences used for RT-PCR
IGF-1R
| F 5′-ACG CCA ATA AGT TCG TCC AC-3′ |
R 5′-TCC ATC CTT GAG GGA CTC AG-3′ |
COX-2
| F 5′-ATC TTT GGG GAG ACC ATG GTA GA-3′ |
R 5′-ACT GAA TTG AGG CAG TGT TGA TG-3′ |
XBP1
| F 5′-TTA CGA GAG AAA ACT CAT GGC C-3′ |
R 5′-GGG TCC AAG TTG TCC AGA ATG C-3′ |
GRP78
| F 5′-GGT ACA TTT GAT CTG ACT G-3′ |
R 5′-CAC TTC ACT AGA GTT TGC TG-3′ |
CHOP
| F 5′-CTT CAC TAC TCT TGA CCC TGC AT-3′ |
R 5′-ATG TGC ACT GGA GAT TGC TT-3′ |
ATF4
| F 5′-GTT CTC CAG CGA CAA GGC TA-3′ |
R 5′-ATC CTC CTT GCT GTT GTT GG-3′ |
GAPDH
| F 5′-GGT GAA GGT CGG TGT GAA CGG ATT T-3′ |
R 5′-ATT GCC AAA GTT GTC ATG GAT GAC C-3′ |
β-ACTIN
| F 5′-GTG GGG CGC CCC AGG CAC CA-3′ |
R 5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′ |
Western blot analysis
Preparation of whole-cell lysates from cancer cells, electrophoresis, and membrane transfer were performed as previously described [
18]. The membranes were then incubated overnight at 4°C with primary antibodies in TBS-T containing 5% bovine serum albumin. Membranes were washed with TBS-T and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody in 5% skim milk for 1 hour at room temperature.
Cell proliferation analysis
To determine the effects of glucosamine on the proliferation of various cancer cell lines, cells were seeded in 96-well plates (3,000 cells/well). On the following day, the medium was replaced with medium containing glucosamine, picropodophyllin, and A12 at the desired concentrations. After incubation for an additional 2 days, MTT assay was performed according to standard procedures. The bars represent SD of results.
Cell cycle analysis
For the cell cycle analysis, three human NSCLC cell lines were treated with the indicated concentration of glucosamine. Floating and attached cells were fixed in 70% ethanol for 1 hour at 4°C. After centrifugation, the cell pellet was washed twice with phosphate-buffered saline (PBS) and stained with propidium iodide (PI) containing RNase A (40 μg/ml) for 30 minutes at 4°C in the dark. The total cellular DNA content of each cancer cell line was quantified by flow cytometry.
Apoptosis analysis
To analyze the number of apoptotic cells after 2 days of glucosamine treatment, A549, H1299, and H460 cells were harvested and washed twice with PBS on ice. The cells were resuspended in 1 X binding buffer containing 5 μl fluorescein isothiocyanate (FITC)-conjugated Annexin V and 5 μl PI. Apoptotic events were detected by flow cytometry at 488 nm and 633 nm using the FITC Annexin V apoptosis detection kit I (BD Pharmingen, San Jose, CA). All procedures were carried out according to the manufacturer’s instructions.
Immunohistochemistry
Primary tumors from PBS or glucosamine treated animals were embedded in paraffin depending on the application. The 5 μm tumor tissue sections were prepared for immunohistochemistry. Paraffin sections were incubated overnight at 4°C with primary antibody against anti-phospho-Akt (Cell Signaling Technology; 1 : 100 dilution) and then processed for avidin – biotin immunohistochemistry according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). These sections counterstained with hematoxylin and eosin-Y (H&E). Immunohistochemical analysis were performed as previously described [
18].
Discussion
In this study, we showed that glucosamine effectively inhibits IGF-1R-mediated Akt signal transduction in various human carcinoma cell lines by both suppressing IGF-1-induced IGF-1R activation and reducing IGF-1R protein stability.
Some investigators have reported that glucosamine induces cell-cycle arrest at the G0/G1 phase in human cancer cells [
26,
27]. These studies have shown that this phenomenon is mediated by decreased cyclin D1 expression and increased p21
waf1/cip1 expression. Here, we showed that in three NSCLC cell lines, glucosamine could also downregulate CDK4 and CDK2 expression and that the extent of the glucosamine-mediated inhibition of these proteins reflected the proportion of cells arrested in the G0/G1 phase (Figure
1B and D). In addition, Lee et al. reported that expression of CDK4 is associated with PI3K/Akt and that the PI3K inhibitor LY294002 decreases the CDK4 level in corneal endothelial cells [
21]. In our study, the pAkt level was more effectively reduced by glucosamine in A549 and H226B cells, which exhibited more significant decreases in CDK levels than either the H1299 or H460 cells (Figure
2C and Figure
1D). Similarly, because pAkt is a positive regulator of cell survival and anti-apoptotic events, the glucosamine-induced increase in apoptosis and cleaved PARP were more evident in A549 cells than in the other cell lines (Figure
1C and D).
Interestingly, we found no significant differences in the total pAkt level in primary tumors derived from glucosamine treated animal although pIGF-1R level was significantly decreased (Figure
6C). However, we also confirmed the decrease of Akt activation in glucosamine treated tissue using histology analysis (Figure
6E). These conflicting findings were probably resulted from drug delivery system. Until 40 days after injection, glucosamine definitely reduced the primary tumor growth. However, after that, the effect of glucosamine was decreased (Figure
6A) and COX-2 level rather increased in the tumor mass (Figure
6C). According to tumor mass getting bigger and bigger, it is difficult that glucosamine could not penetrate into primary tumor. Thus, insufficient dose of glucosamine may partially decrease pAkt level. In addition, the complex tumor microenvironment and the presence of multiple redundant survival pathways at the primary tumor site may reduce the effect of glucosamine and compensate the pAkt activation.
Exogenous glucosamine can be transported across the hydrophobic cell membrane through facilitative glucose transporters (GLUTs) [
8,
28]. Once inside the cell, glucosamine is converted to UDP-N-acetylglucosamine (UDP-GlcNAc), the substrate for O-GlcNAc modification, through the hexosamine pathway [
8]. UDP-GlcNAc covalently modifies cytosolic and nuclear proteins, influencing the stability, localization, enzymatic activity, protein-protein interactions, and phosphorylation status of target proteins [
29]. Thus, intact O-GlcNAc modification is very important for proper cell cycle progression, and increasing O-GlcNAc modification of cell cycle regulators using the O-GlcNAcase inhibitor PUGNAc causes growth inhibition that is similar to G2/M arrest [
30]. Alteration of glycosylation on the surface of target proteins following glucosamine treatment could be responsible for the decreased CDK2 and CDK4 expression and increased cell cycle arrest. O-GlcNAc modification can also affect the protein turnover rate, and modified proteins are subject to proteasome degradation [
31]. We demonstrated that glucosamine influenced the IGF-1R protein stability and facilitated its proteasomal degradation (Figure
5B and C); therefore, the reduction in the IGF-1R half-life following glucosamine treatment may result from the alteration of glycoconjugate structures through O-linked glycosylation. Recent studies have shown that glucosamine inhibits COX-2
N-glycosylation and increases the COX-2 turnover rate [
13]. In this study, similar effects were also observed for the IGF-1R prototype, which showed a reduced molecular mass (Figure
5).
Although our results suggest that glucosamine effectively inhibits cell proliferation and tumor growth in A549, some of NSCLCs such as H460 relatively show glucosamine-resistant phenotype. Because the IGF-1R/Akt signaling axis includes many signal regulators, such as PI3K, PTEN, and p53, that can influence the pAkt level, [
32‐
35] reduction of pAkt by glucosamine could affect each cell line differently. Therefore, we will investigate whether mutation status of these genes affect glucosamine sensitivity in various types of cancer cell including NSCLCs.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors participated in design of the study. K-HS, J-HK, and H-YM performed the experimental work and wrote the manuscript. J-KW, J-SN, H-YL, and S-YK contributed to data analysis and interpretation. S-HO conceived of the study, participated in the experimental design, and helped to draft the manuscript. All authors read and approved the final manuscript.