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Cell cycle regulation by glucosamine in human pulmonary epithelial cells
Pulmonary Pharmacology & Therapeutics 26 (2013) 195e204
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Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
Cell cycle regulation by glucosamine in human pulmonary epithelial cells Kun-Han Chuang a, Chih-Shen Lu b, Yu Ru Kou a, Yuh-Lin Wu a, * a b
Department of Physiology, School of Medicine, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Beitou District, Taipei 112, Taiwan Department of Neurosurgery, Cheng Hsin General Hospital, No. 45, Cheng Hsin Street, Beitou District, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2012 Received in revised form 4 October 2012 Accepted 24 October 2012
Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G0/G1 phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G0/G1 phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Apoptosis Cell cycle Cell proliferation Epithelial cells Glucosamine
1. Introduction Airway epithelial cells are in direct contact with inhaled materials, including pollutants, allergens and microbes as well as other factors, and these factors are important to the development of inflammation in the respiratory system [1]. Inflammation may initiate genetic and epigenetic changes that lead to regulation of a variety of cellular functions, such as proliferation, survival and apoptosis [2]. In fact, inducible inflammatory mediators have been demonstrated to play significant roles in cell proliferation and cell cycle progression via the modulation of anti-apoptotic proteins and cell cycle regulators; these changes involve multiple signaling pathways in both human lung adenocarcinoma and bronchial epithelial cells [3]. Glucosamine is a natural amino monosaccharide involved in the formation of cartilage and it has been widely used to treat human osteoarthritis-related symptoms [4]. Glucosamine has been reported to have anti-inflammatory and immune-regulatory functions that are related to a reduced expression of inflammatory
* Corresponding author. Tel.: þ886 2 2826 7081; fax: þ886 2 2826 4049. E-mail address: [email protected] (Y.-L. Wu). 1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.10.007
mediators [5]. At the same time, glucosamine also has been demonstrated to induce cytotoxicity in pancreatic beta cells [6]. In addition, recent studies have noted that a suppression of proliferation occurs when various cancer cell types are treated with glucosamine [7,8]. However, the effects of glucosamine in cell proliferation of airway epithelial cells and the molecular mechanisms by which glucosamine acts remain unknown. The cell cycle is controlled by several cell cycle regulators, including various kinds of cyclin-dependent kinase inhibitors (CDKIs), such as p21 and p27 being well-recognized to inhibit the functions of most complexes of cyclins and cyclin-dependent kinases (CDKs), those are required for cell cycle progression [9,10]. In addition, induction of heme oxygenase isoform-1 (HO-1), a major kind of heme-degrading enzyme, has also been shown to be associated with the inhibition of the proliferation in several cell types, including human pulmonary epithelial cells [11e13]. Based on our previous studies, it is known that glucosamine is able to effectively suppress the LPS-induced expression and subsequent secretion of inflammatory mediators in primary human bronchial epithelial cells (HBECs) [14]. However, the impact of glucosamine on the cell growth, cell cycle and apoptosis of human primary cells or human airway epithelial cells has not been characterized. In this study, the aim was to investigate the potential
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impacts of glucosamine on cell proliferation, cell cycle progression as well as cell apoptosis in human respiratory epithelial cells at the molecular level and to determine how this occurs.
Table 1 Primers used and the sizes of PCR products. Gene
Sequence
Size (bp)
p21
Forward-50 GCC GCG ACT GTG ATG CGC TAA TG -30 Reverse-50 CCG GCG TTT GGA GTG GTA GA -30 Forward-50 AGA GGC GAG CCA GCG CAA -30 Reverse-50 CTG CTC CAC AGA ACC GGC A -30 Forward-50 CGG TTT CCG TCT GGG CTT CT -30 Reverse-50 GCA CCT CAA AGC TGT TCC GTC CC -30 Forward-50 CAG GCA GAG AAT GCT GAG TTC -30 Reverse-50 GAT GTT GAG CAG GAA CGC AGT -30 Forward-50 GGC ACC ACA CCT TCT ACA AT -30 Reverse-50 CGT CAT ACT CCT GCT TGC TG -30
377
2. Materials and methods p27
2.1. Chemicals and reagents Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT, USA). Glucosamine hydrochloride, MG132 and chloroquine were from Sigma (St. Louis, MO, USA). Reverse transcriptase and Taq polymerase were from Promega (Madison, WI, USA). Antibodies against p21, histone H1, Rb and p-Rb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against p27 was from Cell Signaling Technology (Danvers, MA, USA). Antibody against p53 was from NeoMarkers (Fremont, CA, USA). Antibody against HO-1 was purchased from Enzo Life Sciences International Inc. (Plymouth Meeting, PA, USA). Donkey anti-rabbit IgG secondary antibody was purchased from Amersham Life Science Inc. (Arlington Heights, Illinois, USA). Unless otherwise specified, all other chemicals and reagents used in this project were from Sigma. 2.2. Cell culture A human alveolar epithelial cell line (A549) was from the American Type Culture Collection (Rockville, MD, USA) and used as a model of human alveolar epithelial cells. Primary human bronchial epithelial cells (HBECs) were from Cell Application Inc. (San Diego, CA, USA). Cells were maintained at 37 C in a humidified 5% CO2 atmosphere in F12K medium (Sigma) containing 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were plated on the previous day and grown for 16e18 h prior to various treatments, which were carried out on the following day.
p53 HO-1
b-actin
400 497 555 833
USA). Data were processed using CXP FC500 software (Beckman Coulter, CA, USA). 2.5. Western blotting Overnight plated cells were treated with different concentrations of glucosamine for 24 or 48 h and harvested with lysis buffer (50 mM Tris, 5 mM EDTA, 300 mM NaCl, 1% Triton X-100, 1 mM PMSF, 100 ng/ml Aprotinin and 100 ng/ml Leupeptin-Hemisulfate). The harvested cell lysates were centrifuged at 12,000 rpm for 15 min at 4 C and the supernatant was collected as the total cellular protein sample. Nuclear proteins were obtained by Nonidet P-40 lysis buffer (10 mm HEPES, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 0.02% sodium azide, 0.5% Nonidet P-40, 100 mM PMSF, 0.1% Aprotinin and 0.1% Leupeptin-Hemisulfat), followed by
2.3. Determination of cell viability by trypan blue exclusion assay A549 cells and HBECs were seeded in 6-well plates at a density of 8 105 cells per well overnight. After serum-starvation for 24 h, cells were treated with different concentrations of glucosamine for 24 or 48 h. Cells were then trypsinized and mixed (1:1 vol/vol) with 0.4% trypan blue solution for staining and the suspension was added to a hemocytometer. Viable (unstained) and nonviable (blue) cells from each well were counted over eight microscopic fields. 2.4. Cell cycle and apoptosis analyses To determine the cell cycle distribution profiles, cells were seeded at 8 105 cells per well in 6-well plates overnight. After serum-starvation, cells were exposed to different concentrations of glucosamine for 24 or 48 h. The cells were then trypsinized, harvested using cold PBS, fixed in 70% ethanol and incubated with fluoro-chrome DNA staining solution containing 50 mg/ml propidium iodide (PI) and 100 mg/ml RNAse A for 40 min at 37 C in the dark. To determine the apoptosis rate, an annexin V-fluorescein isothiocyanate (annexin V-FITC) and PI apoptosis detection kit (Sigma) was used to determine the early and late apoptotic activities according to the manufacturer’s instructions. Overnight plated cells in 6-well plates were treated with different concentrations of glucosamine for 24 or 48 h, and the cells were harvested and resuspended in 100 ml of binding buffer. A total of 10 ml of annexin V-FITC and 10 ml of PI were added and the mixture was incubated for 30 min in the dark. Finally, 400 ml of binding buffer was added to the cells and the mixture was analyzed. The distribution of cells across the cell cycle and apoptotic analysis were determined by flow cytometry (Becton Dickinson FACSCalibur, Franklin Lakes, NJ,
Fig. 1. Inhibition of cell proliferation by glucosamine in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) to count alive cell numbers by trypan blue-exclusion assay. Each value represents means S.E.M. from four independent experiments *p < 0.05 compared with the control group.
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centrifugation at 6000 rpm for 7 min at 4 C. Protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). The total protein concentration was adjusted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and heated to 100 C for 10 min. Samples were then subject to regular immunoblotting to determine the protein expression profile of various proteins.
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b-actin. The final cDNA yields were then determined from the amplified DNA signals by comparing them against the internal standard house-keeping gene b-actin after amplification for 35 PCR cycles using appropriate parameters. The primer sequences are listed in Table 1. The PCR products were subject to electrophoresis on a 2% agarose gel with 1 mg/ml ethidium bromide. The DNA signals were captured and analyzed by ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).
2.6. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)
2.7. Statistical analysis
Overnight plated cells were treated with different concentrations of glucosamine for 12 or 24 h, and total cellular RNAs were then extracted using Tri-Reagent (Sigma) according to the manufacturer’s instructions. The isolated RNA samples were resuspended in RNase-free diethylpyrocarbonate (DEPC)-treated water and kept at 80 C. A two-step semi-quantitative RT-PCR method was used to measure the level of mRNAs encoding p21, p27, p53, HO-1, and
Experimental data were analyzed by SigmaStat v3.5 software (Baltimore, MD, USA) and were expressed as the means plus/minus their standard errors of the means (mean S.E.M.) and were analyzed by one-way analysis of variance (ANOVA) followed by least-significant difference (LSD) test to compare the differences between each treatment groups and the control group. Differences with a value of p < 0.05 are to be considered statistically significant.
Fig. 2. Alterations in cell cycle distribution in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) to determine the G0/G1 (black boxes), S (white boxes) and G2/M (gray boxes) cell cycle distribution by flow cytometric analysis. Representative graphs of the flow cytometric analysis are showed. Each value represents means S.E.M. from five independent experiments *, #, & p < 0.05 compared with control group within the same stage.
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3. Results 3.1. Impacts of glucosamine on cell proliferation, cell cycle distribution and cell apoptosis After serum starvation for 24 h to eliminate the contribution from the serum, A549 cells or HBECs were treated with glucosamine (0, 0.1, 10, 20, 50 mM) for 24 or 48 h. Cell viability was determined by classical trypan blue-exclusion assay. Glucosamine treatment of A549 cells at 0.1, 10, 20 or 50 mM for 24 h reduced viable cell numbers by 30e45% (Fig. 1A). Furthermore, an about 40% reduction in viable cell numbers after treatment with 50 mM glucosamine was noted at 48 h (Fig.1B). When HBECs were treated in a similar manner with glucosamine at 10, 20 or 50 mM, a more profound and longer effect was noted with a 20e40% reduction at 24 h and a 40e70% reduction at 48 h (Fig. 1C and D). To determine whether the inhibitory effect of glucosamine on cell proliferation was accompanied by an alteration in cell cycle progression, we next examined the cycle distribution profile after glucosamine treatment. In A549 cells,
glucosamine at 10, 20 or 50 mM increased the proportion of cells in G0/G1 phase, and decreased the proportion of cells in S and G2/M phases at 24 h (Fig. 2A); however this effect was not as obvious at 48 h (Fig. 2B). Similarly, when HBECs were treated with glucosamine at 20 or 50 mM, there was an increase in the proportion of cells in G0/ G1 phase at 24 h (Fig. 2C). Furthermore, glucosamine at 10, 20 or 50 mM reduced the proportion of cells in G2/M phase at 24 h (Fig. 2C). Interestingly, glucosamine at 20 or 50 mM for 48 h, seemed to cause an elevation in the proportion of cells in S phase and a reduction in the proportion of cells in G0/G1 phase (Fig. 2D). These results suggest that glucosamine inhibits the proliferation of A549 cells and HBECs by arresting the cell cycle progression. On the other hand, we also examined on whether the inhibition of cell proliferation under glucosamine exposure in A549 and HBECs was caused by apoptosis. At 24 h, glucosamine resulted in an increase in the late apoptosis rate from 8% (control) to 19% (10 mM), 20% (20 mM) and 21% (50 mM) in A549 cells (Fig. 3A), and from 8% (control) to 10% (10 mM), 11% (20 mM) and 12% (50 mM) in HBECs (Fig. 3C). Meanwhile, glucosamine (10, 20, 50 mM) for 24 h also mediated
Fig. 3. Induction of apoptosis by glucosamine in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) and using annexin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) staining to determine the viable (Annexin/PI, lower left, white boxes), early apoptotic (Annexinþ/PI, lower right, gray boxes) and late apoptotic (Annexinþ/PIþ, upper right, black boxes) cells. A representative result for each flow cytometric analysis was shown in upper panels. Each value represents means S.E.M. from four independent experiments *, #p < 0.05 compared with control group within the viable stage (*) or the late apoptotic stage (#).
a reduction in the viable rate in both A549 cells and HBECs (Fig. 3A and C). At 48 h, only 50 mM glucosamine remained to affect and to further decrease the viable rate from 86% (control) to 47%, and to increase the late apoptosis rate from 9% (control) to 45% in A549 cells (Fig. 3B). In HBECs, glucosamine resulted in a further reduction in the viable rate from 75% (control) to 55% (10 mM), 38% (20 mM), and 25% (50 mM), as well as a further elevation in the late apoptosis rate from 22% (control) to 40% (10 mM), 56% (20 mM), and 66% (50 mM) (Fig. 3D). In addition, no apparent change in the early apoptosis or the necrosis rate in either cell type was noted at 24 or 48 h (Fig. 3). 3.2. Decrease in Rb protein phosphorylation by glucosamine At the transition from G1 to S phase, Rb protein is phosphorylated in order to release E2 promoter-binding factor (E2F), which then initiates S phase progression [15]. In this context, the effect of glucosamine on Rb phosphorylation was examined and it was found that glucosamine at 20 and 50 mM appears to reduce Rb phosphorylation at 24 h in both cell types (Fig. 4).
Fig. 4. Inhibition of Rb protein phosphorylation by glucosamine in A549 cells and HBECs. A549 (A) and HBECs (B) were treated with various doses of glucosamine for 24 h and the nuclear proteins were collected to examine the expression of phosphorylated Rb by Western blotting with histone H1 as an internal control. Each value represents mean S.E.M. from three independent experiments. *p < 0.05 compared with control group.
3.3. Modulation of glucosamine on p21, p53 and HO-1, but not p27 protein expression It is well known that the Rb phosphorylation is regulated by CDKIs via the functional inhibition complexes of cyclins and CDKs which are pivotal to the G1 to S phase transition [10]. Therefore
Fig. 5. Regulation of protein expression of p21, p27, p53 and HO-1 by glucosamine in A549 cells. A549 cells were treated with various doses of glucosamine for 24 h (A, C, E, G) or 48 h (B, D, F, H), and total cell lysates were collected to determine p21, p27, p53, and HO-1 protein expression by Western blotting with human a-tubulin as an internal control. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
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the effect of glucosamine on the expression of two CDKIs, p21 and p27, both of which belong to kip family of CDKIs, was examined. In addition, it is also well known that p53, a tumor suppressor, is able to regulate cell cycle process [16]; therefore we also examined the effect of glucosamine on p53 expression. We also examined the inflammatory-related protein HO-1, which has been reported to participate in the inhibition of cell proliferation [11e13]. In A549 cells, after treatment with glucosamine for 24 and 48 h, glucosamine at 0.1 or 10 mM seemed to induce p21 protein expression at 24 h (Fig. 5A); however at the higher concentrations of 10, 20 or 50 mM, glucosamine seemed to reduce p21 protein expression at 48 h (Fig. 5B). Meanwhile, glucosamine at all doses had no effect on p27 protein expression at either 24 or 48 h (Fig. 5C and D). Glucosamine was only able to increase p53 expression at 48 h at 50 mM (Fig. 5E and F). Similarly, glucosamine at 50 mM was also able to elevate HO-1 protein expression at 24 and 48 h (Fig. 5G and H). In HBECs, glucosamine at 10, 20 or 50 mM was able to attenuate p21 protein expression at 24 h and at 20 or 50 mM this effect continued at 48 h (Fig. 6A and B). However, glucosamine at all doses had no effect on p27 or p53 protein expression at 24 or 48 h (Fig. 6CeF). In addition, glucosamine at 50 mM induced HO-1 protein expression at 24 h (Fig. 6G) and at 20 or 50 mM increased HO-1 expression at 48 h (Fig. 6H).
3.4. Induction of p21, p53 and HO-1 mRNA in A549 cells, but only HO-1 mRNA in HBECs by glucosamine To further elucidate the glucosamine effect at the mRNA level, both A549 cells and HBECs were treated with glucosamine for 12 or 24 h and then subject to RT-PCR. In A549 cells, glucosamine at 20 or 50 mM increased the mRNA levels of p21 at 12 and 24 h (Fig. 7A and B) and of p53 at 24 h (Fig. 7F); however, glucosamine at all doses had no effect on p27 mRNA expression (Fig. 7C and D). Furthermore, glucosamine at 50 mM elevated HO-1 mRNA level at 12 and 24 h (Fig. 7G and H). In HBECs, glucosamine at 20 and 50 mM increased HO-1 mRNA expression at 12 and 24 h (Fig. 8G and H). However, glucosamine at all doses did not seem to affect mRNA expression of p21, p27 or p53 (Fig. 8AeF). 3.5. Reduction of cellular p21 protein stability but promotion of nuclear p21 translocation by glucosamine In order to address the inconsistent effects of glucosamine whereby there was an inhibition of p21 protein expression (Figs. 5 and 6), but a promotion of p21 mRNA expression (Figs. 7 and 8), we examined whether glucosamine may directly affect the degradation of p21 protein. Glucosamine at 20 mM was able to promote the degradation of p21 protein at 6, 12, and 24 h (Fig. 9A). In addition, to further elucidate whether the lysosomal or proteasomal pathway is
Fig. 6. Regulation of protein expression of p21, p27, p53 and HO-1 by glucosamine in HBECs. HBECs were treated with various doses of glucosamine for 24 h (A, C, E, G) or 48 h (B, D, F, H), and total cell lysates were collected to determine p21, p27, p53, and HO-1 protein expression by Western blotting. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
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Fig. 7. Effect of glucosamine on p21, p27, p53 and HO-1 mRNA expression in A549 cells. A549 cells were treated with various doses of glucosamine for 12 h (A, C, E, G) or 24 h (B, D, F, H), and total cell lysates were collected to measure p21, p27, p53, and HO-1 mRNA concentration by semi-quantitative RT-PCR assay with human b-actin as an internal control. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
the method by which glucosamine enhances p21 protein degradation, the lysosome inhibitor chloroquine and the proteasome inhibitor MG132 were used separately on the above system. It was found that glucosamine-induced p21 protein degradation was reversed by MG132 but not by chloroquine (Fig. 9B), suggesting that glucosamine may reduce p21 protein expression by targeting the proteasomal proteolytic pathway. Consequently, we also examined the distribution of cytosolic and nuclear p21 expression in response to glucosamine and found that cytosolic p21 decreased at 12 and 24 h, while nuclear p21 appeared to increase at 12 and 24 h, particularly at 24 h (Fig. 9C). 4. Discussion This study investigates the influence of glucosamine at different concentrations on the cell growth and apoptosis of pulmonary epithelial cells and explores the molecular mechanisms associated with its effects. Our results demonstrate that glucosamine is able to mediate cell cycle arrest and apoptosis in both A549 cells and HBECs, resulting in inhibition of cell proliferation. Retardation of the cell cycle by glucosamine seems to be related to a reduction in Rb phosphorylation and the induction of p53 and HO-1 expression, as well as the promoting effect on nuclear p21 translocation. The lungs are often in contact with dust, pollen, chemicals, and microbial pathogens from the outside environment [17]. In
response to an exposure to such dangerous entities, an inflammatory response may be initiated in the lung epithelium [18]. Glucosamine has been demonstrated to modulate various inflammatory cascades [19], and an anti-inflammatory function of glucosamine at 10 and 1 mM in human airway epithelium was characterized in our previous study [14]. In fact, several studies have revealed effective doses of glucosamine with the antiinflammatory features varies in a great scale from mM to mM range, such as 500 mM [20] or 1e10 mM [21] in human chondrocytes; 0.5e5 mM in human chondrosarcoma cells [22]; 20 mM in rat chondrocytes [23]; 50 mM in equine chondrocytes [24]; 1e 4 mM in human retinal pigment epithelial cells [25]. Thus, the concentrations at 10, 20, and 50 mM of glucosamine in A549 cells and HBECs from our current study appear very similar with most studies. The difference in glucosamine dosage across different literature could be due to the differences in glucosamine manufacturers, the form of glucosamine (glucosamine hydrochloride or glucosamine sulfate), cell types (transformed cell line or primary cell), and species (rat or human). Whether the high dose glucosamine would impact cell proliferation or apoptosis in respiratory system remained unknown. Thus, in this study, we first characterized the effects of glucosamine from low to high concentrations (0.1, 10, 20, 50 mM) on the cell growth and apoptosis in primary HBECs and A549 alveolar epithelial cell line. Besides, so far there is no report of the glucosamine effect on cell proliferation in human
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Fig. 8. Effect of glucosamine on p21, p27, p53 and HO-1 mRNA expression in HBECs. HBECs were treated with various doses of glucosamine for 12 h (A, C, E, G) or 24 h (B, D, F, H), and total cell lysates were collected to measure p21, p27, p53, and HO-1 mRNA concentration by semi-quantitative RT-PCR assay. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
primary cells, but only in human cell lines, such as hepatoma cell line [7], leukemia cell line [8] and retinal pigment epithelial cell line [26]. Therefore, our findings of the glucosamine cytotoxicity in HBECs appeared to be the first examination in human primary cells. We first determined the effects of glucosamine on cell viability, cell cycle and apoptosis. It was found that glucosamine (10, 20 and 50 mM) significantly inhibited cell proliferation in both A549 cells and HBECs at 24 h, and this inhibition remained present only in HBECs at 48 h (Fig. 1). In addition, glucosamine caused accumulation in the G0/G1 phase at 24 h in both A549 cells and HBECs; however at 48 h, only accumulation in S phase for HBECs could be seen (Fig. 2). From the apoptosis assay, the glucosamine effect in A549 cells was noted at 24 h, but except the dose at 50 mM, the impact appeared to diminish at 48 h; however, the effect persisted from 24 to 48 h and indeed appeared to be more profound at 48 h in HBECs (Fig. 3). Thus, from the three lines of evidences on cell proliferation (Fig. 1), cell cycle progression (Fig. 2) and cell apoptosis (Fig. 3), HBECs seemed more sensitive than A549 cells to the glucosamine treatment and A549 cells appeared to be able to reverse at 48 h. The discrepancy between the two cell types in response to glucosamine may be the result of a different sensitivity to glucosamine between the two different cell types, as A549 were from an alveolar epithelium origin whereas HBECs were from a bronchial epithelium origin or as A549 is a cell line while HBECS are primary cells. Alternatively, the faster recovery rate and
stronger resistance to glucosamine in A549 cells may be due to its cancer cell features, such as chromosomal abnormalities in A549 cells. In addition, because the G1/S and S/G2 transitions of the cell cycle are controlled by different complexes of cyclins and CDKs [27], therefore glucosamine may also affect different target molecules among the cell cycle regulators in these two different cell types. It is well known that Rb has to be phosphorylated for progression from G1 into S phase [28]; this is because Rb is hypophosphorylated in quiescent cells and hyperphosphorylated during cell cycle progression, thus Rb phosphorylation promotes cell proliferation [29]. In our study, we observed that glucosamine markedly decreased the level of Rb phosphorylation (Fig. 4), suggesting that glucosamine-mediated G0/G1 arrest is possibly due to an inhibition of Rb phosphorylation. Similar to our findings, glucose-mediated stress, which results in a cell cycle arrest in G1 phase, has been reported to be accompanied by Rb hypophosphorylation in various human cancer cells [30]. In our study, it was observed that glucosamine appears to induce HO-1 protein (Figs. 5 and 6) and mRNA expression (Figs. 7 and 8), suggesting that HO-1 may have an important role in pulmonary epithelial cell proliferation. In addition, according to previous studies, high levels of various CDK inhibitors, such as p21 and p27, are able to interact with CDKs and thus inhibit their catalytic activities; this prevents the formation of active complexes
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Fig. 9. Effect of glucosamine on cellular p21 protein stability and translocation in HBECs. (A) After pre-treatment with cycloheximide (CHX) (10 mg/ml) for 1 h, HBECs were exposed or not exposed to glucosamine (20 mM) for indicated times and cell lysates were harvested to detect p21 protein expression by Western blotting. (B) HBECs were not treated (control) or treated with glucosamine (20 mM) alone or in combination with chloroquine (50 mM) or MG132 (1 mM) for 24 h. Total cell lysates were harvested and p21 protein expression was analyzed by Western blotting. (C) HBECs were not treated (control) or treated with glucosamine (20 mM) for indicated times and cytosolic and nuclear extracts were harvested and subject to Western blotting. Each value represents mean S.E.M. from three independent experiments. *p < 0.05 compared with control group at the same time point. &p < 0.05 compared at 1 h time point within the control group among the different time points. #p < 0.05 compared at 1 h time point within the glucosamine treatment group among the different time points.
of cyclins and CDKs, thereby blocking G1 phase progression and the G1/S transition [31]. A previous study has reported that glucosamine was able to induce p21 protein expression and mediated cell cycle arrest [32]. Similarly, our study also revealed that glucosamine is able to affect p21 expression in both A549 and HBECs (Figs. 5, 6 and 9). Our study demonstrated that high concentrations of glucosamine appeared to reduce p21 expression in both A549 cells and HBECs (Figs. 5 and 6); however, we also observed that glucosamine was able to promote p21 degradation in a proteasome-dependent pathway (Fig. 9). Similar to our finding, previous studies have also shown that low dose of ultraviolet (UV) irradiation is able to trigger p21 degradation by reducing its half-life [33]. Furthermore, another study has reported that p21 is a short-lived protein that is regulated post-translationally by a proteasomemediated pathway [34]. It is noteworthy that glucosamine promotes cellular p21 protein degradation, but also mediates a dramatic translocation of p21 into the nucleus (Fig. 9). These results suggest that even when glucosamine decreases total cellular p21 protein, there is overall a novel increase in nuclear p21, which presumably retards cell cycle progression. In fact, similar to our findings, previous studies have demonstrated UV-induced p21 proteolysis in the cytosol is accompanied by p21 protein
translocation and accumulation in the nucleus and only the nuclear form of p21 owns the ability to inhibit cell proliferation, while cytosolic p21 is regarded as a positive modulator of cell survival [35e37]. Therefore, it is likely that the accumulation of p21 in the nucleus as well as the reduction of p21 in the cytosol (Fig. 9) in lung epithelial cells would contribute significantly to the cell cycle arrest. At the same time, our results are inconsistent with respect to p21 protein expression, which appeared to be inhibited by glucosamine (Figs. 5 and 6) and p21 mRNA expression, which seemed to be induced by glucosamine (Fig. 7). Such an inconsistency between mRNA and protein regulation profiles is novel and deserves further investigation. Furthermore, p21 expression has been demonstrated to be regulated at the transcriptional stage by both p53-dependent and -independent mechanisms [16,38]. In our study, there seems to be no correlation between p21 and p53 expression (Figs. 5 and 6), suggesting that p21 regulation in both A549 cells and HBECs is likely to be mediated in a p53-independent manner. In addition to being an inhibitor of proliferation, some studies have pointed out that p21 may act as a positive regulator in proapoptotic response in mammary tumor cells and ovarian carcinoma cells [39,40]. Besides, p21 in cytoplasm is also regarded as an inhibitor of initiator caspase cleavage and the interaction with
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procaspase 3 leads to resistance to Fas-mediated cell death, and stabilization of the apoptotic inhibitor protein c-IAP1 [41,42]. These discoveries suggest that glucosamine may promote the cell apoptosis by accelerating p21 protein degradation as well as p21 nuclear accumulation. In summary, glucosamine-mediated anti-proliferation in A549 cells and HBECs may act, at least in part, via G0/G1 arrest, which is accompanied by a decrease in Rb phosphorylation and an induction of HO-1 expression, and via cell apoptosis in conjunction with a p21 accumulation in the nucleus. These findings provide new insights into the potential cellular toxicity effects of glucosamine on human lung epithelial cells. Acknowledgments The authors thank Dr. Ralph Kirby, Department of Life Sciences, National Yang-Ming University, for his help with language editing. The authors also gratefully acknowledge the funding support from the Taiwan National Science Council (NSC 100-2320-B-010-004) and the grant from the Ministry of Education, Aim for the Top University Plan. References [1] Janssen-Heininger YM, Poynter ME, Aesif SW, Pantano C, Ather JL, Reynaert NL, et al. Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control. Proc Am Thorac 2009;6:249e55. [2] Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66:1403e8. [3] Liao W, Goh FY, Betts RJ, Kemeny DM, Tam J, Bay BH, et al. A novel antiapoptotic role for apolipoprotein L2 in IFN-gamma-induced cytotoxicity in human bronchial epithelial cells. J Cell Physiol 2011;226:397e406. [4] Wang SX, Laverty S, Dumitriu M, Plaas A, Grynpas MD. The effects of glucosamine hydrochloride on subchondral bone changes in an animal model of osteoarthritis. Arthritis Rheum 2007;56:1537e48. [5] Largo R, Martinez-Calatrava MJ, Sanchez-Pernaute O, Marcos ME, MorenoRubio J, Aparicio C, et al. Effect of a high dose of glucosamine on systemic and tissue inflammation in an experimental model of atherosclerosis aggravated by chronic arthritis. Am J Physiol Heart Circ Physiol 2009;297:H268e76. [6] Kim YK, Park JH, Park SH, Lim B, Baek WK, Suh SI. Protective role of glucagonlike peptide-1 against glucosamine-induced cytotoxicity in pancreatic beta cells. Cell Physiol Biochem 2010;25:211e20. [7] Zhang L, Liu WS, Han BQ, Peng YF, Wang DF. Antitumor activities of Dglucosamine and its derivatives. J Zhejiang Univ Sci B 2006;7:608e14. [8] Wang Z, Liang R, Huang GS, Piao Y, Zhang YQ, Wang AQ, et al. Glucosamine sulfate-induced apoptosis in chronic myelogenous leukemia K562 cells is associated with translocation of cathepsin D and downregulation of Bcl-xL. Apoptosis 2006;11:1851e60. [9] Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1phase progression. Genes Dev 1999;13:1501e12. [10] Zarkowska T, Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 1997;272:12738e46. [11] Choi HC, Lee KY, Lee DH, Kang YJ. Heme oxygenase-1 induced by aprotinin inhibits vascular smooth muscle cell proliferation through cell cycle arrest in hypertensive rats. Korean J Physiol Pharmacol 2009;13:309e13. [12] Lee PJ, Alam J, Wiegand GW, Choi AM. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc Natl Acad Sci U S A 1996;93:10393e8. [13] Gonzalez-Michaca L, Farrugia G, Croatt AJ, Alam J, Nath KA. Heme: a determinant of life and death in renal tubular epithelial cells. Am J Physiol Renal Physiol 2004;286:F370e7. [14] Wu YL, Kou YR, Ou HL, Chien HY, Chuang KH, Liu HH, et al. Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells. Eur J Pharmacol 2010;635:219e26. [15] Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet 2001;10:699e703. [16] Liu PY, Chan JY, Lin HC, Wang SL, Liu ST, Ho CL, et al. Modulation of the cyclindependent kinase inhibitor p21(WAF1/Cip1) gene by Zac1 through the antagonistic regulators p53 and histone deacetylase 1 in HeLa Cells. Mol Cancer Res 2008;6:1204e14.
[17] Du Bois RM. Strategies for treating idiopathic pulmonary fibrosis. Nat Rev Drug Discov 2010;9:129e40. [18] Chen H, Cowan MJ, Hasday JD, Vogel SN, Medvedev AE. Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1Rassociated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists. J Immunol 2007;179:6097e106. [19] Runkel DR, Cupp MJ. Glucosamine sulfate use in osteoarthritis. Am J Health Syst Pharm 1999;56:267e9. [20] Nakamura H, Shibakawa A, Tanaka M, Kato T, Nishioka K. Effects of glucosamine hydrochloride on the production of prostaglandin E2, nitric oxide and metalloproteases by chondrocytes and synoviocytes in osteoarthritis. Clin Exp Rheumatol 2004;22:293e9. [21] Valvason C, Musacchio E, Pozzuoli A, Ramonda R, Aldegheri R, Punzi L. Influence of glucosamine sulphate on oxidative stress in human osteoarthritic chondrocytes: effects on HO-1, p22Phox and iNOS expression. Rheumatology 2008;47:31e5. [22] Lin YC, Liang YC, Sheu MT, Lin YC, Hsieh MS, Chen TF, et al. Chondroprotective effects of glucosamine involving the p38 MAPK and Akt signaling pathways. Rheumatol Int 2008;28:1009e16. [23] Gouze JN, Gouze E, Popp MP, Bush ML, Dacanay EA, Kay JD, et al. Exogenous glucosamine globally protects chondrocytes from the arthritogenic effects of IL-1b. Arthritis Res Ther 2006;8:R173. [24] Byron CR, Orth MW, Venta PJ, Lloyd JW, Caron JP. Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharidestimulated equine chondrocytes. Am J Vet Res 2003;64:666e71. [25] Chen JT, Chen PL, Chang YH, Chien MW, Chen YH, Lu DW, et al. Glucosamine sulfate inhibits leukocyte adhesion in response to cytokine stimulation of retinal pigment epithelial cells in vitro. Exp Eye Res 2006;83: 1052e62. [26] Liang CM, Tai MC, Chang YH, Chen YH, Chen CL, Chien MW, et al. Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells. Mol Vis 2010;16:2559e71. [27] Driscoll B, Wu L, Buckley S, Hall FL, Anderson KD, Warburton D. Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth. Am J Physiol 1997;273:L941e9. [28] Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev 1993;7:331e42. [29] Ferguson KL, Slack RS. The Rb pathway in neurogenesis. Neuroreport 2001;12: A55e62. [30] Tomida A, Suzuki H, Kim HD, Tsuruo T. Glucose-regulated stresses cause decreased expression of cyclin D1 and hypophosphorylation of retinoblastoma protein in human cancer cells. Oncogene 1996;13:2699e705. [31] Hunter T, Pines J. Cyclins and cancer. II: cyclin D and CDK inhibitors come of age. Cell 1994;79:573e82. [32] Masson E, Wiernsperger N, Lagarde M, El BS. Glucosamine induces cell-cycle arrest and hypertrophy of mesangial cells: implication of gangliosides. Biochem J 2005;388:537e44. [33] Bendjennat M, Boulaire J, Jascur T, Brickner H, Barbier V, Sarasin A, et al. UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair. Cell 2003;114:599e610. [34] Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol Cell 2000;5:403e10. [35] Lee JY, Kim HS, Kim JY, Sohn J. Nuclear translocation of p21(WAF1/CIP1) protein prior to its cytosolic degradation by UV enhances DNA repair and survival. Biochem Biophys Res Commun 2009;390:1361e6. [36] Rodríguez-Vilarrupla A, Díaz C, Canela N, Rahn HP, Bachs O, Agell N. Identification of the nuclear localization signal of p21(cip1) and consequences of its mutation on cell proliferation. FEBS Lett 2002;531:319e23. [37] Coqueret O. New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol 2003;13:65e70. [38] el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817e25. [39] Shibata MA, Yoshidome K, Shibata E, Jorcyk CL, Green JE. Suppression of mammary carcinoma growth in vitro and in vivo by inducible expression of the Cdk inhibitor p21. Cancer Gene Ther 2001;8:23e35. [40] Lincet H, Poulain L, Remy JS, Deslandes E, Duigou F, Gauduchon P, et al. The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells. Cancer Lett 2000;161: 17e26. [41] Xu SQ, El-Deiry WS. p21(WAF1/CIP1) inhibits initiator caspase cleavage by TRAIL death receptor DR4. Biochem Biophys Res Commun 2000;269: 179e90. [42] Suzuki A, Tsutomi Y, Akahane K, Araki T, Miura M. Resistance to Fas-mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene 1998;17:931e9.
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Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt
Cell cycle regulation by glucosamine in human pulmonary epithelial cells Kun-Han Chuang a, Chih-Shen Lu b, Yu Ru Kou a, Yuh-Lin Wu a, * a b
Department of Physiology, School of Medicine, National Yang-Ming University, No. 155, Sec. 2, Linong Street, Beitou District, Taipei 112, Taiwan Department of Neurosurgery, Cheng Hsin General Hospital, No. 45, Cheng Hsin Street, Beitou District, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2012 Received in revised form 4 October 2012 Accepted 24 October 2012
Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G0/G1 phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G0/G1 phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Apoptosis Cell cycle Cell proliferation Epithelial cells Glucosamine
1. Introduction Airway epithelial cells are in direct contact with inhaled materials, including pollutants, allergens and microbes as well as other factors, and these factors are important to the development of inflammation in the respiratory system [1]. Inflammation may initiate genetic and epigenetic changes that lead to regulation of a variety of cellular functions, such as proliferation, survival and apoptosis [2]. In fact, inducible inflammatory mediators have been demonstrated to play significant roles in cell proliferation and cell cycle progression via the modulation of anti-apoptotic proteins and cell cycle regulators; these changes involve multiple signaling pathways in both human lung adenocarcinoma and bronchial epithelial cells [3]. Glucosamine is a natural amino monosaccharide involved in the formation of cartilage and it has been widely used to treat human osteoarthritis-related symptoms [4]. Glucosamine has been reported to have anti-inflammatory and immune-regulatory functions that are related to a reduced expression of inflammatory
* Corresponding author. Tel.: þ886 2 2826 7081; fax: þ886 2 2826 4049. E-mail address: [email protected] (Y.-L. Wu). 1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.10.007
mediators [5]. At the same time, glucosamine also has been demonstrated to induce cytotoxicity in pancreatic beta cells [6]. In addition, recent studies have noted that a suppression of proliferation occurs when various cancer cell types are treated with glucosamine [7,8]. However, the effects of glucosamine in cell proliferation of airway epithelial cells and the molecular mechanisms by which glucosamine acts remain unknown. The cell cycle is controlled by several cell cycle regulators, including various kinds of cyclin-dependent kinase inhibitors (CDKIs), such as p21 and p27 being well-recognized to inhibit the functions of most complexes of cyclins and cyclin-dependent kinases (CDKs), those are required for cell cycle progression [9,10]. In addition, induction of heme oxygenase isoform-1 (HO-1), a major kind of heme-degrading enzyme, has also been shown to be associated with the inhibition of the proliferation in several cell types, including human pulmonary epithelial cells [11e13]. Based on our previous studies, it is known that glucosamine is able to effectively suppress the LPS-induced expression and subsequent secretion of inflammatory mediators in primary human bronchial epithelial cells (HBECs) [14]. However, the impact of glucosamine on the cell growth, cell cycle and apoptosis of human primary cells or human airway epithelial cells has not been characterized. In this study, the aim was to investigate the potential
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impacts of glucosamine on cell proliferation, cell cycle progression as well as cell apoptosis in human respiratory epithelial cells at the molecular level and to determine how this occurs.
Table 1 Primers used and the sizes of PCR products. Gene
Sequence
Size (bp)
p21
Forward-50 GCC GCG ACT GTG ATG CGC TAA TG -30 Reverse-50 CCG GCG TTT GGA GTG GTA GA -30 Forward-50 AGA GGC GAG CCA GCG CAA -30 Reverse-50 CTG CTC CAC AGA ACC GGC A -30 Forward-50 CGG TTT CCG TCT GGG CTT CT -30 Reverse-50 GCA CCT CAA AGC TGT TCC GTC CC -30 Forward-50 CAG GCA GAG AAT GCT GAG TTC -30 Reverse-50 GAT GTT GAG CAG GAA CGC AGT -30 Forward-50 GGC ACC ACA CCT TCT ACA AT -30 Reverse-50 CGT CAT ACT CCT GCT TGC TG -30
377
2. Materials and methods p27
2.1. Chemicals and reagents Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT, USA). Glucosamine hydrochloride, MG132 and chloroquine were from Sigma (St. Louis, MO, USA). Reverse transcriptase and Taq polymerase were from Promega (Madison, WI, USA). Antibodies against p21, histone H1, Rb and p-Rb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against p27 was from Cell Signaling Technology (Danvers, MA, USA). Antibody against p53 was from NeoMarkers (Fremont, CA, USA). Antibody against HO-1 was purchased from Enzo Life Sciences International Inc. (Plymouth Meeting, PA, USA). Donkey anti-rabbit IgG secondary antibody was purchased from Amersham Life Science Inc. (Arlington Heights, Illinois, USA). Unless otherwise specified, all other chemicals and reagents used in this project were from Sigma. 2.2. Cell culture A human alveolar epithelial cell line (A549) was from the American Type Culture Collection (Rockville, MD, USA) and used as a model of human alveolar epithelial cells. Primary human bronchial epithelial cells (HBECs) were from Cell Application Inc. (San Diego, CA, USA). Cells were maintained at 37 C in a humidified 5% CO2 atmosphere in F12K medium (Sigma) containing 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were plated on the previous day and grown for 16e18 h prior to various treatments, which were carried out on the following day.
p53 HO-1
b-actin
400 497 555 833
USA). Data were processed using CXP FC500 software (Beckman Coulter, CA, USA). 2.5. Western blotting Overnight plated cells were treated with different concentrations of glucosamine for 24 or 48 h and harvested with lysis buffer (50 mM Tris, 5 mM EDTA, 300 mM NaCl, 1% Triton X-100, 1 mM PMSF, 100 ng/ml Aprotinin and 100 ng/ml Leupeptin-Hemisulfate). The harvested cell lysates were centrifuged at 12,000 rpm for 15 min at 4 C and the supernatant was collected as the total cellular protein sample. Nuclear proteins were obtained by Nonidet P-40 lysis buffer (10 mm HEPES, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 0.02% sodium azide, 0.5% Nonidet P-40, 100 mM PMSF, 0.1% Aprotinin and 0.1% Leupeptin-Hemisulfat), followed by
2.3. Determination of cell viability by trypan blue exclusion assay A549 cells and HBECs were seeded in 6-well plates at a density of 8 105 cells per well overnight. After serum-starvation for 24 h, cells were treated with different concentrations of glucosamine for 24 or 48 h. Cells were then trypsinized and mixed (1:1 vol/vol) with 0.4% trypan blue solution for staining and the suspension was added to a hemocytometer. Viable (unstained) and nonviable (blue) cells from each well were counted over eight microscopic fields. 2.4. Cell cycle and apoptosis analyses To determine the cell cycle distribution profiles, cells were seeded at 8 105 cells per well in 6-well plates overnight. After serum-starvation, cells were exposed to different concentrations of glucosamine for 24 or 48 h. The cells were then trypsinized, harvested using cold PBS, fixed in 70% ethanol and incubated with fluoro-chrome DNA staining solution containing 50 mg/ml propidium iodide (PI) and 100 mg/ml RNAse A for 40 min at 37 C in the dark. To determine the apoptosis rate, an annexin V-fluorescein isothiocyanate (annexin V-FITC) and PI apoptosis detection kit (Sigma) was used to determine the early and late apoptotic activities according to the manufacturer’s instructions. Overnight plated cells in 6-well plates were treated with different concentrations of glucosamine for 24 or 48 h, and the cells were harvested and resuspended in 100 ml of binding buffer. A total of 10 ml of annexin V-FITC and 10 ml of PI were added and the mixture was incubated for 30 min in the dark. Finally, 400 ml of binding buffer was added to the cells and the mixture was analyzed. The distribution of cells across the cell cycle and apoptotic analysis were determined by flow cytometry (Becton Dickinson FACSCalibur, Franklin Lakes, NJ,
Fig. 1. Inhibition of cell proliferation by glucosamine in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) to count alive cell numbers by trypan blue-exclusion assay. Each value represents means S.E.M. from four independent experiments *p < 0.05 compared with the control group.
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centrifugation at 6000 rpm for 7 min at 4 C. Protein concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). The total protein concentration was adjusted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and heated to 100 C for 10 min. Samples were then subject to regular immunoblotting to determine the protein expression profile of various proteins.
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b-actin. The final cDNA yields were then determined from the amplified DNA signals by comparing them against the internal standard house-keeping gene b-actin after amplification for 35 PCR cycles using appropriate parameters. The primer sequences are listed in Table 1. The PCR products were subject to electrophoresis on a 2% agarose gel with 1 mg/ml ethidium bromide. The DNA signals were captured and analyzed by ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).
2.6. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)
2.7. Statistical analysis
Overnight plated cells were treated with different concentrations of glucosamine for 12 or 24 h, and total cellular RNAs were then extracted using Tri-Reagent (Sigma) according to the manufacturer’s instructions. The isolated RNA samples were resuspended in RNase-free diethylpyrocarbonate (DEPC)-treated water and kept at 80 C. A two-step semi-quantitative RT-PCR method was used to measure the level of mRNAs encoding p21, p27, p53, HO-1, and
Experimental data were analyzed by SigmaStat v3.5 software (Baltimore, MD, USA) and were expressed as the means plus/minus their standard errors of the means (mean S.E.M.) and were analyzed by one-way analysis of variance (ANOVA) followed by least-significant difference (LSD) test to compare the differences between each treatment groups and the control group. Differences with a value of p < 0.05 are to be considered statistically significant.
Fig. 2. Alterations in cell cycle distribution in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) to determine the G0/G1 (black boxes), S (white boxes) and G2/M (gray boxes) cell cycle distribution by flow cytometric analysis. Representative graphs of the flow cytometric analysis are showed. Each value represents means S.E.M. from five independent experiments *, #, & p < 0.05 compared with control group within the same stage.
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3. Results 3.1. Impacts of glucosamine on cell proliferation, cell cycle distribution and cell apoptosis After serum starvation for 24 h to eliminate the contribution from the serum, A549 cells or HBECs were treated with glucosamine (0, 0.1, 10, 20, 50 mM) for 24 or 48 h. Cell viability was determined by classical trypan blue-exclusion assay. Glucosamine treatment of A549 cells at 0.1, 10, 20 or 50 mM for 24 h reduced viable cell numbers by 30e45% (Fig. 1A). Furthermore, an about 40% reduction in viable cell numbers after treatment with 50 mM glucosamine was noted at 48 h (Fig.1B). When HBECs were treated in a similar manner with glucosamine at 10, 20 or 50 mM, a more profound and longer effect was noted with a 20e40% reduction at 24 h and a 40e70% reduction at 48 h (Fig. 1C and D). To determine whether the inhibitory effect of glucosamine on cell proliferation was accompanied by an alteration in cell cycle progression, we next examined the cycle distribution profile after glucosamine treatment. In A549 cells,
glucosamine at 10, 20 or 50 mM increased the proportion of cells in G0/G1 phase, and decreased the proportion of cells in S and G2/M phases at 24 h (Fig. 2A); however this effect was not as obvious at 48 h (Fig. 2B). Similarly, when HBECs were treated with glucosamine at 20 or 50 mM, there was an increase in the proportion of cells in G0/ G1 phase at 24 h (Fig. 2C). Furthermore, glucosamine at 10, 20 or 50 mM reduced the proportion of cells in G2/M phase at 24 h (Fig. 2C). Interestingly, glucosamine at 20 or 50 mM for 48 h, seemed to cause an elevation in the proportion of cells in S phase and a reduction in the proportion of cells in G0/G1 phase (Fig. 2D). These results suggest that glucosamine inhibits the proliferation of A549 cells and HBECs by arresting the cell cycle progression. On the other hand, we also examined on whether the inhibition of cell proliferation under glucosamine exposure in A549 and HBECs was caused by apoptosis. At 24 h, glucosamine resulted in an increase in the late apoptosis rate from 8% (control) to 19% (10 mM), 20% (20 mM) and 21% (50 mM) in A549 cells (Fig. 3A), and from 8% (control) to 10% (10 mM), 11% (20 mM) and 12% (50 mM) in HBECs (Fig. 3C). Meanwhile, glucosamine (10, 20, 50 mM) for 24 h also mediated
Fig. 3. Induction of apoptosis by glucosamine in A549 cells and HBECs. Cells were serum starved for 24 h, followed by treatment with various doses of glucosamine for 24 h (A, C) or 48 h (B, D) and using annexin V-fluorescein isothiocyanate (annexin V-FITC) and propidium iodide (PI) staining to determine the viable (Annexin/PI, lower left, white boxes), early apoptotic (Annexinþ/PI, lower right, gray boxes) and late apoptotic (Annexinþ/PIþ, upper right, black boxes) cells. A representative result for each flow cytometric analysis was shown in upper panels. Each value represents means S.E.M. from four independent experiments *, #p < 0.05 compared with control group within the viable stage (*) or the late apoptotic stage (#).
a reduction in the viable rate in both A549 cells and HBECs (Fig. 3A and C). At 48 h, only 50 mM glucosamine remained to affect and to further decrease the viable rate from 86% (control) to 47%, and to increase the late apoptosis rate from 9% (control) to 45% in A549 cells (Fig. 3B). In HBECs, glucosamine resulted in a further reduction in the viable rate from 75% (control) to 55% (10 mM), 38% (20 mM), and 25% (50 mM), as well as a further elevation in the late apoptosis rate from 22% (control) to 40% (10 mM), 56% (20 mM), and 66% (50 mM) (Fig. 3D). In addition, no apparent change in the early apoptosis or the necrosis rate in either cell type was noted at 24 or 48 h (Fig. 3). 3.2. Decrease in Rb protein phosphorylation by glucosamine At the transition from G1 to S phase, Rb protein is phosphorylated in order to release E2 promoter-binding factor (E2F), which then initiates S phase progression [15]. In this context, the effect of glucosamine on Rb phosphorylation was examined and it was found that glucosamine at 20 and 50 mM appears to reduce Rb phosphorylation at 24 h in both cell types (Fig. 4).
Fig. 4. Inhibition of Rb protein phosphorylation by glucosamine in A549 cells and HBECs. A549 (A) and HBECs (B) were treated with various doses of glucosamine for 24 h and the nuclear proteins were collected to examine the expression of phosphorylated Rb by Western blotting with histone H1 as an internal control. Each value represents mean S.E.M. from three independent experiments. *p < 0.05 compared with control group.
3.3. Modulation of glucosamine on p21, p53 and HO-1, but not p27 protein expression It is well known that the Rb phosphorylation is regulated by CDKIs via the functional inhibition complexes of cyclins and CDKs which are pivotal to the G1 to S phase transition [10]. Therefore
Fig. 5. Regulation of protein expression of p21, p27, p53 and HO-1 by glucosamine in A549 cells. A549 cells were treated with various doses of glucosamine for 24 h (A, C, E, G) or 48 h (B, D, F, H), and total cell lysates were collected to determine p21, p27, p53, and HO-1 protein expression by Western blotting with human a-tubulin as an internal control. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
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the effect of glucosamine on the expression of two CDKIs, p21 and p27, both of which belong to kip family of CDKIs, was examined. In addition, it is also well known that p53, a tumor suppressor, is able to regulate cell cycle process [16]; therefore we also examined the effect of glucosamine on p53 expression. We also examined the inflammatory-related protein HO-1, which has been reported to participate in the inhibition of cell proliferation [11e13]. In A549 cells, after treatment with glucosamine for 24 and 48 h, glucosamine at 0.1 or 10 mM seemed to induce p21 protein expression at 24 h (Fig. 5A); however at the higher concentrations of 10, 20 or 50 mM, glucosamine seemed to reduce p21 protein expression at 48 h (Fig. 5B). Meanwhile, glucosamine at all doses had no effect on p27 protein expression at either 24 or 48 h (Fig. 5C and D). Glucosamine was only able to increase p53 expression at 48 h at 50 mM (Fig. 5E and F). Similarly, glucosamine at 50 mM was also able to elevate HO-1 protein expression at 24 and 48 h (Fig. 5G and H). In HBECs, glucosamine at 10, 20 or 50 mM was able to attenuate p21 protein expression at 24 h and at 20 or 50 mM this effect continued at 48 h (Fig. 6A and B). However, glucosamine at all doses had no effect on p27 or p53 protein expression at 24 or 48 h (Fig. 6CeF). In addition, glucosamine at 50 mM induced HO-1 protein expression at 24 h (Fig. 6G) and at 20 or 50 mM increased HO-1 expression at 48 h (Fig. 6H).
3.4. Induction of p21, p53 and HO-1 mRNA in A549 cells, but only HO-1 mRNA in HBECs by glucosamine To further elucidate the glucosamine effect at the mRNA level, both A549 cells and HBECs were treated with glucosamine for 12 or 24 h and then subject to RT-PCR. In A549 cells, glucosamine at 20 or 50 mM increased the mRNA levels of p21 at 12 and 24 h (Fig. 7A and B) and of p53 at 24 h (Fig. 7F); however, glucosamine at all doses had no effect on p27 mRNA expression (Fig. 7C and D). Furthermore, glucosamine at 50 mM elevated HO-1 mRNA level at 12 and 24 h (Fig. 7G and H). In HBECs, glucosamine at 20 and 50 mM increased HO-1 mRNA expression at 12 and 24 h (Fig. 8G and H). However, glucosamine at all doses did not seem to affect mRNA expression of p21, p27 or p53 (Fig. 8AeF). 3.5. Reduction of cellular p21 protein stability but promotion of nuclear p21 translocation by glucosamine In order to address the inconsistent effects of glucosamine whereby there was an inhibition of p21 protein expression (Figs. 5 and 6), but a promotion of p21 mRNA expression (Figs. 7 and 8), we examined whether glucosamine may directly affect the degradation of p21 protein. Glucosamine at 20 mM was able to promote the degradation of p21 protein at 6, 12, and 24 h (Fig. 9A). In addition, to further elucidate whether the lysosomal or proteasomal pathway is
Fig. 6. Regulation of protein expression of p21, p27, p53 and HO-1 by glucosamine in HBECs. HBECs were treated with various doses of glucosamine for 24 h (A, C, E, G) or 48 h (B, D, F, H), and total cell lysates were collected to determine p21, p27, p53, and HO-1 protein expression by Western blotting. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
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Fig. 7. Effect of glucosamine on p21, p27, p53 and HO-1 mRNA expression in A549 cells. A549 cells were treated with various doses of glucosamine for 12 h (A, C, E, G) or 24 h (B, D, F, H), and total cell lysates were collected to measure p21, p27, p53, and HO-1 mRNA concentration by semi-quantitative RT-PCR assay with human b-actin as an internal control. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
the method by which glucosamine enhances p21 protein degradation, the lysosome inhibitor chloroquine and the proteasome inhibitor MG132 were used separately on the above system. It was found that glucosamine-induced p21 protein degradation was reversed by MG132 but not by chloroquine (Fig. 9B), suggesting that glucosamine may reduce p21 protein expression by targeting the proteasomal proteolytic pathway. Consequently, we also examined the distribution of cytosolic and nuclear p21 expression in response to glucosamine and found that cytosolic p21 decreased at 12 and 24 h, while nuclear p21 appeared to increase at 12 and 24 h, particularly at 24 h (Fig. 9C). 4. Discussion This study investigates the influence of glucosamine at different concentrations on the cell growth and apoptosis of pulmonary epithelial cells and explores the molecular mechanisms associated with its effects. Our results demonstrate that glucosamine is able to mediate cell cycle arrest and apoptosis in both A549 cells and HBECs, resulting in inhibition of cell proliferation. Retardation of the cell cycle by glucosamine seems to be related to a reduction in Rb phosphorylation and the induction of p53 and HO-1 expression, as well as the promoting effect on nuclear p21 translocation. The lungs are often in contact with dust, pollen, chemicals, and microbial pathogens from the outside environment [17]. In
response to an exposure to such dangerous entities, an inflammatory response may be initiated in the lung epithelium [18]. Glucosamine has been demonstrated to modulate various inflammatory cascades [19], and an anti-inflammatory function of glucosamine at 10 and 1 mM in human airway epithelium was characterized in our previous study [14]. In fact, several studies have revealed effective doses of glucosamine with the antiinflammatory features varies in a great scale from mM to mM range, such as 500 mM [20] or 1e10 mM [21] in human chondrocytes; 0.5e5 mM in human chondrosarcoma cells [22]; 20 mM in rat chondrocytes [23]; 50 mM in equine chondrocytes [24]; 1e 4 mM in human retinal pigment epithelial cells [25]. Thus, the concentrations at 10, 20, and 50 mM of glucosamine in A549 cells and HBECs from our current study appear very similar with most studies. The difference in glucosamine dosage across different literature could be due to the differences in glucosamine manufacturers, the form of glucosamine (glucosamine hydrochloride or glucosamine sulfate), cell types (transformed cell line or primary cell), and species (rat or human). Whether the high dose glucosamine would impact cell proliferation or apoptosis in respiratory system remained unknown. Thus, in this study, we first characterized the effects of glucosamine from low to high concentrations (0.1, 10, 20, 50 mM) on the cell growth and apoptosis in primary HBECs and A549 alveolar epithelial cell line. Besides, so far there is no report of the glucosamine effect on cell proliferation in human
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Fig. 8. Effect of glucosamine on p21, p27, p53 and HO-1 mRNA expression in HBECs. HBECs were treated with various doses of glucosamine for 12 h (A, C, E, G) or 24 h (B, D, F, H), and total cell lysates were collected to measure p21, p27, p53, and HO-1 mRNA concentration by semi-quantitative RT-PCR assay. Each value represents mean S.E.M. from four independent experiments. *p < 0.05 compared with control group.
primary cells, but only in human cell lines, such as hepatoma cell line [7], leukemia cell line [8] and retinal pigment epithelial cell line [26]. Therefore, our findings of the glucosamine cytotoxicity in HBECs appeared to be the first examination in human primary cells. We first determined the effects of glucosamine on cell viability, cell cycle and apoptosis. It was found that glucosamine (10, 20 and 50 mM) significantly inhibited cell proliferation in both A549 cells and HBECs at 24 h, and this inhibition remained present only in HBECs at 48 h (Fig. 1). In addition, glucosamine caused accumulation in the G0/G1 phase at 24 h in both A549 cells and HBECs; however at 48 h, only accumulation in S phase for HBECs could be seen (Fig. 2). From the apoptosis assay, the glucosamine effect in A549 cells was noted at 24 h, but except the dose at 50 mM, the impact appeared to diminish at 48 h; however, the effect persisted from 24 to 48 h and indeed appeared to be more profound at 48 h in HBECs (Fig. 3). Thus, from the three lines of evidences on cell proliferation (Fig. 1), cell cycle progression (Fig. 2) and cell apoptosis (Fig. 3), HBECs seemed more sensitive than A549 cells to the glucosamine treatment and A549 cells appeared to be able to reverse at 48 h. The discrepancy between the two cell types in response to glucosamine may be the result of a different sensitivity to glucosamine between the two different cell types, as A549 were from an alveolar epithelium origin whereas HBECs were from a bronchial epithelium origin or as A549 is a cell line while HBECS are primary cells. Alternatively, the faster recovery rate and
stronger resistance to glucosamine in A549 cells may be due to its cancer cell features, such as chromosomal abnormalities in A549 cells. In addition, because the G1/S and S/G2 transitions of the cell cycle are controlled by different complexes of cyclins and CDKs [27], therefore glucosamine may also affect different target molecules among the cell cycle regulators in these two different cell types. It is well known that Rb has to be phosphorylated for progression from G1 into S phase [28]; this is because Rb is hypophosphorylated in quiescent cells and hyperphosphorylated during cell cycle progression, thus Rb phosphorylation promotes cell proliferation [29]. In our study, we observed that glucosamine markedly decreased the level of Rb phosphorylation (Fig. 4), suggesting that glucosamine-mediated G0/G1 arrest is possibly due to an inhibition of Rb phosphorylation. Similar to our findings, glucose-mediated stress, which results in a cell cycle arrest in G1 phase, has been reported to be accompanied by Rb hypophosphorylation in various human cancer cells [30]. In our study, it was observed that glucosamine appears to induce HO-1 protein (Figs. 5 and 6) and mRNA expression (Figs. 7 and 8), suggesting that HO-1 may have an important role in pulmonary epithelial cell proliferation. In addition, according to previous studies, high levels of various CDK inhibitors, such as p21 and p27, are able to interact with CDKs and thus inhibit their catalytic activities; this prevents the formation of active complexes
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Fig. 9. Effect of glucosamine on cellular p21 protein stability and translocation in HBECs. (A) After pre-treatment with cycloheximide (CHX) (10 mg/ml) for 1 h, HBECs were exposed or not exposed to glucosamine (20 mM) for indicated times and cell lysates were harvested to detect p21 protein expression by Western blotting. (B) HBECs were not treated (control) or treated with glucosamine (20 mM) alone or in combination with chloroquine (50 mM) or MG132 (1 mM) for 24 h. Total cell lysates were harvested and p21 protein expression was analyzed by Western blotting. (C) HBECs were not treated (control) or treated with glucosamine (20 mM) for indicated times and cytosolic and nuclear extracts were harvested and subject to Western blotting. Each value represents mean S.E.M. from three independent experiments. *p < 0.05 compared with control group at the same time point. &p < 0.05 compared at 1 h time point within the control group among the different time points. #p < 0.05 compared at 1 h time point within the glucosamine treatment group among the different time points.
of cyclins and CDKs, thereby blocking G1 phase progression and the G1/S transition [31]. A previous study has reported that glucosamine was able to induce p21 protein expression and mediated cell cycle arrest [32]. Similarly, our study also revealed that glucosamine is able to affect p21 expression in both A549 and HBECs (Figs. 5, 6 and 9). Our study demonstrated that high concentrations of glucosamine appeared to reduce p21 expression in both A549 cells and HBECs (Figs. 5 and 6); however, we also observed that glucosamine was able to promote p21 degradation in a proteasome-dependent pathway (Fig. 9). Similar to our finding, previous studies have also shown that low dose of ultraviolet (UV) irradiation is able to trigger p21 degradation by reducing its half-life [33]. Furthermore, another study has reported that p21 is a short-lived protein that is regulated post-translationally by a proteasomemediated pathway [34]. It is noteworthy that glucosamine promotes cellular p21 protein degradation, but also mediates a dramatic translocation of p21 into the nucleus (Fig. 9). These results suggest that even when glucosamine decreases total cellular p21 protein, there is overall a novel increase in nuclear p21, which presumably retards cell cycle progression. In fact, similar to our findings, previous studies have demonstrated UV-induced p21 proteolysis in the cytosol is accompanied by p21 protein
translocation and accumulation in the nucleus and only the nuclear form of p21 owns the ability to inhibit cell proliferation, while cytosolic p21 is regarded as a positive modulator of cell survival [35e37]. Therefore, it is likely that the accumulation of p21 in the nucleus as well as the reduction of p21 in the cytosol (Fig. 9) in lung epithelial cells would contribute significantly to the cell cycle arrest. At the same time, our results are inconsistent with respect to p21 protein expression, which appeared to be inhibited by glucosamine (Figs. 5 and 6) and p21 mRNA expression, which seemed to be induced by glucosamine (Fig. 7). Such an inconsistency between mRNA and protein regulation profiles is novel and deserves further investigation. Furthermore, p21 expression has been demonstrated to be regulated at the transcriptional stage by both p53-dependent and -independent mechanisms [16,38]. In our study, there seems to be no correlation between p21 and p53 expression (Figs. 5 and 6), suggesting that p21 regulation in both A549 cells and HBECs is likely to be mediated in a p53-independent manner. In addition to being an inhibitor of proliferation, some studies have pointed out that p21 may act as a positive regulator in proapoptotic response in mammary tumor cells and ovarian carcinoma cells [39,40]. Besides, p21 in cytoplasm is also regarded as an inhibitor of initiator caspase cleavage and the interaction with
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procaspase 3 leads to resistance to Fas-mediated cell death, and stabilization of the apoptotic inhibitor protein c-IAP1 [41,42]. These discoveries suggest that glucosamine may promote the cell apoptosis by accelerating p21 protein degradation as well as p21 nuclear accumulation. In summary, glucosamine-mediated anti-proliferation in A549 cells and HBECs may act, at least in part, via G0/G1 arrest, which is accompanied by a decrease in Rb phosphorylation and an induction of HO-1 expression, and via cell apoptosis in conjunction with a p21 accumulation in the nucleus. These findings provide new insights into the potential cellular toxicity effects of glucosamine on human lung epithelial cells. Acknowledgments The authors thank Dr. Ralph Kirby, Department of Life Sciences, National Yang-Ming University, for his help with language editing. The authors also gratefully acknowledge the funding support from the Taiwan National Science Council (NSC 100-2320-B-010-004) and the grant from the Ministry of Education, Aim for the Top University Plan. References [1] Janssen-Heininger YM, Poynter ME, Aesif SW, Pantano C, Ather JL, Reynaert NL, et al. Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control. Proc Am Thorac 2009;6:249e55. [2] Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66:1403e8. [3] Liao W, Goh FY, Betts RJ, Kemeny DM, Tam J, Bay BH, et al. A novel antiapoptotic role for apolipoprotein L2 in IFN-gamma-induced cytotoxicity in human bronchial epithelial cells. J Cell Physiol 2011;226:397e406. [4] Wang SX, Laverty S, Dumitriu M, Plaas A, Grynpas MD. The effects of glucosamine hydrochloride on subchondral bone changes in an animal model of osteoarthritis. Arthritis Rheum 2007;56:1537e48. [5] Largo R, Martinez-Calatrava MJ, Sanchez-Pernaute O, Marcos ME, MorenoRubio J, Aparicio C, et al. Effect of a high dose of glucosamine on systemic and tissue inflammation in an experimental model of atherosclerosis aggravated by chronic arthritis. Am J Physiol Heart Circ Physiol 2009;297:H268e76. [6] Kim YK, Park JH, Park SH, Lim B, Baek WK, Suh SI. Protective role of glucagonlike peptide-1 against glucosamine-induced cytotoxicity in pancreatic beta cells. Cell Physiol Biochem 2010;25:211e20. [7] Zhang L, Liu WS, Han BQ, Peng YF, Wang DF. Antitumor activities of Dglucosamine and its derivatives. J Zhejiang Univ Sci B 2006;7:608e14. [8] Wang Z, Liang R, Huang GS, Piao Y, Zhang YQ, Wang AQ, et al. Glucosamine sulfate-induced apoptosis in chronic myelogenous leukemia K562 cells is associated with translocation of cathepsin D and downregulation of Bcl-xL. Apoptosis 2006;11:1851e60. [9] Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1phase progression. Genes Dev 1999;13:1501e12. [10] Zarkowska T, Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 1997;272:12738e46. [11] Choi HC, Lee KY, Lee DH, Kang YJ. Heme oxygenase-1 induced by aprotinin inhibits vascular smooth muscle cell proliferation through cell cycle arrest in hypertensive rats. Korean J Physiol Pharmacol 2009;13:309e13. [12] Lee PJ, Alam J, Wiegand GW, Choi AM. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc Natl Acad Sci U S A 1996;93:10393e8. [13] Gonzalez-Michaca L, Farrugia G, Croatt AJ, Alam J, Nath KA. Heme: a determinant of life and death in renal tubular epithelial cells. Am J Physiol Renal Physiol 2004;286:F370e7. [14] Wu YL, Kou YR, Ou HL, Chien HY, Chuang KH, Liu HH, et al. Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells. Eur J Pharmacol 2010;635:219e26. [15] Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet 2001;10:699e703. [16] Liu PY, Chan JY, Lin HC, Wang SL, Liu ST, Ho CL, et al. Modulation of the cyclindependent kinase inhibitor p21(WAF1/Cip1) gene by Zac1 through the antagonistic regulators p53 and histone deacetylase 1 in HeLa Cells. Mol Cancer Res 2008;6:1204e14.
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