Glycobiology Advance Access originally published online on February 22, 2007
Glycobiology 2007 17(6):568-577; doi:10.1093/glycob/cwm020
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Cell growth arrest by sialic acid clusters in ganglioside GM3 mimetic polymers
2 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan
3 Core Research for Evolutional Science and Technology Program (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho Kawaguchi, Saitama, 332-0012, Japan
4 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences
5 Laboratory of Bio-Macromolecular Chemistry, Division of Biological Science, Graduate School of Science, Frontier Research Center for Post-Genomic Science and Technology
6 Research Center of Glycoscience, National Institute of Advanced Industrial Science and Technology, Hokkaido University, Kita 21-jo, Nishi 11-choume, Kita-ku, Sapporo 062-8517, Japan
1 To whom correspondence should be addressed; Tel: +81 22 727 0116; Fax: +81 22 727 0076; e-mail: jin{at}tohoku-pharm.ac.jp
Received on August 2, 2006; revised on February 8, 2007; accepted on February 19, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Ganglioside GM3, one of the sialic acid containing glycosphingolipids, is known to form clusters in lipid microdomains, which serve as platforms for effective signal transduction. In an attempt to clarify the GM3 cluster effect, we enzymatically synthesized GM3 mimetic polymer (GM3-p), with an acrylamide backbone from LacCer mimetic polymer (LacCer-p). Interestingly, GM3-p, but not LacCer-p, reversibly inhibited proliferation of NIH3T3 cells, which are normally resistant to exogenously added GM3. Moreover, we found that the introduction of carbonic acid into the acrylamide chain aided well-oriented cluster formation and enhanced the inhibitory effect of GM3-p. Since sialyllactosyl polymer and GM4 mimetic polymer, but not GM2 mimetic polymer, also inhibited cell proliferation, sialic acid-galactose units must be essential for the biological activity of GM3-p. These results suggest that the formation of sialic acid-galactose clusters is necessary for the suppressive effect of GM3-p. GM3-p treatment did not affect the serum-dependent activation of ERK1/2 or c-fos expression, but caused a reduction in the gene and/or protein expression of cyclin D1, cyclin E, cyclin-dependent kinase (cdk)4, and cdk2, which are involved in the cell cycle. Therefore, GM3-p inhibits cell proliferation by reducing cyclin D1-cdk4 and cyclin E-cdk2 complexes without affecting growth factor signaling from serum to c-fos.
Key words: ganglioside GM3 / cluster effect / cell cycle / growth arrest
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Gangliosides, sialic acid-containing glycosphingolipids, are an important class of biomolecules known to exhibit regulatory roles on cell growth, adhesion, cell–cell interactions and signal transduction (Hakomori 2004
). Gangliosides form lipid microdomains (rafts), which function as platforms for effective signal transduction, together with cholesterol and sphingomyelin in the plasma membrane (Simons and Toomre 2000). Since ganglioside molecules have both hydrogen bond acceptors and donors, gangliosides interact with each other in the microdomains, leading to cluster formation (Hakomori 2004).
Of over 200 species of ganglio-series ganglioside, GM3 is the first that is synthesized from the precursor lactosylceramide (LacCer). Interestingly, the effects of exogenously added GM3 differ in cell types (Bremer and Hakomori 1982; Bremer et al. 1984; Nakatsuji and Miller 2001; Noll et al. 2001; Mirkin et al. 2002). In the Swiss3T3 fibroblast cells, oral epidermoid carcinoma KB cells, ovarial epidermoid carcinoma A431 cells, baby hamster kidney (BHK) fibroblast cells, HCT116 colon cancer cells, primary cultures of astrocytes and glioblastoma multiforme tumors, and exogenously added GM3 inhibits cell proliferation remarkably. In contrast, NIH3T3 fibroblasts, A10 smooth muscle cells, and the primary cultures of skin and normal brain are resistant to exogenous GM3 effects. The mechanism of the cell growth inhibition by GM3 in each cell is reported as follows. (i) In Swiss 3T3 cells, GM3 inhibits the autophosphorylation of both platelet-derived growth factor and epidermal growth factor (EGF) receptors (Bremer et al. 1984). (ii) In A431 cells and KB cells, GM3 inhibits autophosphorylation of EGF receptor (Bremer et al. 1986). (iii) In BHK cells, GM3 suppresses the internalization of fibroblast growth factor (Bremer and Hakomori 1982). (iv) In HCT116 cells, GM3 causes the cell growth arrest through the up-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and reduction of cyclin E and cyclin-dependent kinase (cdk) 2 (Choi et al. 2006). (v) In Astrocyte, GM3 induces apoptosis through the up-regulation of cdk inhibitor p27Kip (Nakatsuji and Miller 2001). Therefore, the common mechanism of cell growth inhibition by exogenously added GM3 remains unclear. However, these studies suggest that GM3 may possess a potential of an inhibitor of cancer cell growth.
We have reported some methods for synthesizing sugar-containing polymers (Nishimura et al. 1990, 1991; Nishimura and Yamada 1997; Yamada et al. 1997). In the present study, we synthesized GM3 mimetic polymer (GM3-p) using sialyltransferase and LacCer mimeric polymer (LacCer-p) as primer to enhance the GM3 effects by clustering. Interestingly, GM3-p, but not LacCer-p, exhibited the cell growth inhibitory activity against GM3 resistant cells such as NIH3T3 cells, suggesting the distinctive effect of GM3-p on cell proliferation. In order to explore the applicability of sialic-acid containing glycopolymers as a candidate of new anticancer drug, we examined their structure–activity relationship, subcellular localization, and effects on proteins involving in cell cycle.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Exogenously added LacCer and GM3 do not affect cell proliferation in NIH3T3 cells
To examine the effect of exogenously added LacCer and GM3 on cell proliferation, NIH3T3 cells were treated with increasing concentrations (5, 15, 30, 50, 100, 150, or 300 µM) of LacCer or GM3, and the cell number was measured 24 h later. No effect on cell proliferation was observed with either LacCer or GM3 at any concentration tested (Figure 1). This result indicates that NIH3T3 cells are resistant to exogenously added GM3 monomer.
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Cell proliferation is inhibited by GM3-p
To enhance the GM3 effects by clustering, we synthesized GM3-p using sialyltransferase and LacCer-p as primer. LacCer-p were obtained by radical copolymerization of acrylamide with monomeric lactose precursors, which have lactose residues attached through a specific-ceramide mimetic linker (Nishimura and Yamada 1997
, Yamada et al. 1997). Our strategy for constructing GM3-p with different sugar densities (1:30–1:5, sugar:acrylamide) was to perform enzymatic elongation on the respective LacCer-p using 
-2,3-sialyltransferase and the sugar nucleotide cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-NeuAc) as the glycosyl donor (Figure 2A). The density of the sugar moieties on each glycopolymer was analyzed by the integration of all signals of 1H nuclear magnetic resonance (NMR) spectra and were estimated to be at the molar ratio of 1:5, 1:10, 1:15, 1:25, and 1:30, sugar to acrylamide (Figure 2A), although broadening of the signals due to the high molecular weights was observed. The ratio in each glycopolymer was determined to be governed by the feed ratio of the polymerizable glycomonomer and acrylamide, and could be manipulated as needed.
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To examine the effect of the glycopolymers on cell proliferation, NIH3T3 were treated with five kinds of LacCer-p or GM3-p (1:30, 1:25, 1:15, 1:10, or 1:5 sugar:acrylamide). After 24 h, the number of cells in each treatment was compared with that of untreated cells. Normally, the proliferation rate of NIH3T3 cells is around 2.2 to 2.5-fold per day, so the number of cells after 24 h in a treated culture, if complete growth inhibition is achieved, would be
40–45% compared with the untreated cells. Treating cells for 24 h with 30 µM GM3-p of varying sugar to acrylamide chain ratios (1:30, 1:25, 1:15, 1:10, and 1:5) resulted in the inhibition of cell growth, with culture cell numbers at 83, 54, 42, 63, and 64%, respectively, compared with untreated cultures (Figure 2B). In contrast, none of the LacCer-p of similar sugar to acrylamide chain ratios affected cell proliferation. Thus, every GM3-p, except the 1:30 polymer, significantly inhibited cell growth, with 1:15 GM3-p exhibiting the most potent inhibition. Moreover, GM3-p exhibited the distinctive effect of GM3-p on cell proliferation activity against GM3 resistant cells such as NIH3T3 cells.
Next, we compared the cell growth inhibitory effect of 1:15 GM3-p and 1:5 GM3-p over a range of concentrations. As shown in Figure 2C, 1:15 GM3-p exhibited higher inhibitory activity than 1:5 GM3-p, which had the higher density GM3 sugar unit, at all concentrations. A similar result has been reported in which a sharp optimum for sialic acid density was found at 10 mol% for a synthetic glycopolymer that inhibited influenza virus-receptor binding (Matrosovich et al. 1990). These results suggest that effective GM3 cluster formation would be decreased in the 1:5 GM3-p due to stronger electrostatic repulsion between the negative sialic acid units of trisaccharide on the side chains.
1:3:3 GM3-p strongly inhibits cell proliferation
In order to improve the inhibitory activity of 1:5 GM3-p, a carbonic acid unit was introduced into the main-chain of the acrylamide polymer, and 1:3:3 and 1:8:8 (sugar:acrylamide chain:carbonic acid) GM3-p were constructed (Figure 3A). We aimed to expand a mean square radius of gyration (S2)1/2 of the polymer and to enhance the GM3 cluster formation by taking advantage of the repulsion between the GM3 sugar unit and the carbonic acid unit on the main chain. The essential physicochemical properties of the constructed glycopolymers, for example, the weight-average molecular weights Mw and the square roots of mean-square radius of gyration (S2)1/2, were determined using static laser light-scattering (Flory 1953). The measurements yielded approximate values for the weight-average molecular weights Mw as 78870 for the composition of 1:5, 73170 for 1:15, 167309 for 1:3:3, and 100400 for 1:8:8 (Table I). The radii (S2)1/2 of glycopolymers in water were found to be 33.9 nm for the composition of 1:5, 26.9 nm for 1:15, 88.2 nm for 1:3:3, and 14.2 nm for 1:8:8 (Table I), under the presupposition that the glycopolymer in dilute water solution exists as a randomly kinked chain polymer, which is a flexibly polydisperse Gaussian chain coil (Holtzer et al. 1954). The square roots of mean-square radius of gyration (S2)1/2 of 1:3:3 GM3-p was 2.6 to 3.3-fold larger than that of 1:5 GM3-p or 1:15 GM3-p. We expected that the negative carbonic acid group introduced on the polymer main-chain would interact with the sialic acid unit on the side-chain by an electrostatic repulsion and thus causing the expansion of the polymer main-chain indicated by the larger radius in solution (Table I). That is to say that polymer main-chain changed from a compacted form to an expanded coil. Furthermore, this expansion would make the trisaccharide unit on the side chain exposure to the outer side of the polymer coil than that of 1:5 GM3-p. On the other hand, since the square roots of mean-square radius of gyration (S2)1/2 of 1:8:8 GM3-p was similar to that of 1:5 and 1:15 GM3-p, it suggests that the electrostatic repulsion between main chain and side sugar chain is more necessary to increase the main-chain expansion of polymer than that between main chains.
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We investigated the effects of 1:3:3 and 1:8:8 GM3-p on cell proliferation. 1:3:3 GM3-p strongly inhibited cell proliferation compared with 1:5 GM3-p and 1:15 GM3-p (Figure 3B). However, the inhibitory effect of 1:8:8 GM3-p was similar to that of 1:15 GM3-p (Figure 3C). These results indicate that the introduction of carbonic acid into the main acrylamide polymer chain is a useful strategy for improving the inhibitory effect of high density sugar chains fixed on the acrylamide chain like 1:5 GM3-p by enhancing expanded orientation and cluster formation.
Structure–activity relationship
In order to investigate the structure–activity relationship of GM3-p required for the inhibition of cell proliferation, we constructed a series of glycopolymers (Figure 4A). These included 1:15 lactosyl polymer (1:15 Lac-p), 1:15 sialyllactosyl polymer (1:15 Sia-p) with only one alkyl chain, 1:15 galactosyl ceramide (GalCer) mimetic polymer (1:15 GalCer-p), 1:15 GM4 mimetic polymer (1:15 GM4-p) having a sugar unit with sialic acid and galactose, and 1:7 GM2 mimetic polymer (1:7 GM2-p) with an N-acetylgalactosamine (GalNAc) linked to galactose in GM3-p as a ß 1-4. The degree of cell growth inhibition by 1:15 Sia-p was slightly weaker than that of 1:15 GM3-p (Figure 4B), suggesting that the hydrophobic interaction of the two alkyl chains in GM3-p formed a more effective cluster than that of the n-hexyl group in 1:15 Sia-p. In addition, the inhibitory effect of GM4-p was similar to that of 1:15 GM3-p (Figure 4C), demonstrating that a sialic acid-galactose unit is sufficient to inhibit cell proliferation. Most interestingly, 1:7 GM2-p did not affect cell proliferation at all (Figure 4D), suggesting that an additional GalNAc linked to galactose in GM3-p may perturb the interaction between the sialic acid clusters and its target molecule(s). Based on the chemical structures, the steric hindrance between the GalNAc and sialic acid may contribute to weakening the effective sialic cluster formation. At least, we can say that the exposure of the sialic acid cluster is necessary for the inhibitory activity.
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The inhibitory effect of GM3-p is reversible
We examined the effects of polymers on cell morphology and growth potential as a function of treatment time (Figure 5). Following a 24-h treatment with GM3-p, morphological changes, including the appearance of a spindle-like structure, and the decrease of cell number were observed. After culturing the cells in fresh media without any polymer for an additional 24 h, the growth inhibition was completely reversed (Figure 5B), however, the cell morphology did not recover completely (Figure 5A). As expected, LacCer-p had no effect on cell morphology, and the cell number increased
2.2 to 2.4-fold, similar to nontreated control cells. These results suggest that GM3-p treatment for 24 h causes cell growth arrest. Prolonged continuous treatment (i.e. 48 h) with GM3-p caused cell aggregation and irreversible growth inhibition leading to cell death. The nuclear condensation by the treatment of GM3-p for 48 h was not observed (data not shown). Moreover, the treatment of Z-aspartyl-2,6-dichlorobenzoyloxymethylketone, which is caspase inhibitor, did not suppress the cell death by GM3-p (data not shown). Thus, the cell death by GM3-p is nonapoptotic cell death.
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Cyclin D1, cyclin E, cdk4, and cdk2 are down-regulated by GM3-p
Since GM3-p causes cell growth arrest reversibly, we considered the possibility that GM3-p might inhibit any cell growth signal from serum in the medium. Therefore, we investigated serum-dependent ERK1/2 activation 24 h after the addition of GM3-p. Serum-dependent ERK1/2 activation was observed even in cells undergoing complete cell growth arrest following GM3-p treatment (Figure 6A), clearly indicating that growth signals from serum stimulation to ERK1/2 activation function normally (Figure 6A).
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Next, gene expressions of c-fos, cyclin D1, and cyclin E, which are induced by ERK1/2 activation, were quantified using real-time polymerase chain reaction (RT-PCR). Although the amount of c-fos mRNA was the same in the cells treated with LacCer-p or GM3-p, the amounts of cyclin D1 and cyclin E in GM3-p-treated cells decreased to half that found in untreated cells (Figure 6B). Furthermore, we examined the protein levels of cyclin D1, cyclin E, cdk4, cdk2, and Rb by immunoblotting with each specific antibody. Rb is phosphorylated by cyclin D1-cdk4 complex and cyclin E-cdk2 complex, leading to the S-phase transition. The protein levels of cyclin D1, cyclin E, cdk4, cdk2, and phosphorylated Rb remarkably decrease by GM3-p treatment (Figure 6C). Since GM3-p had little effect on the mRNA levels of cdk4 and cdk2, the decrease of cdk4 and cdk2 might be caused by some other reason, such as the protein instability leading to proteosomal degradation. These results suggest that GM3-p causes cell growth arrest by the decrease of cyclin D1-cdk4 and cyclin E-cdk2 complexes.
GM3-p and LacCer-p accumulate in lysosome
For investigations into the cellular localization of GM3-p, we constructed fluorescent polymers each containing dansyl group, including 1:15:0.1 Dansyl-LacCer mimetic polymer (1:15:0.1 D-LacCer-p), 1:15:0.1 Dansyl-GM3 mimetic polymer (1:15:0.1 D-GM3-p), and 15:0.1 Dansyl-acrylamide polymer (15:0.1 D-Acry-p) (Figure 7A). These fluorescent polymers inhibited cell growth similarly to the respective nonfluorescent polymers (data not shown). Using a laser-scanning microscope, we observed that the dansyl polymers were taken up by cells. As shown in Figure 7B, D-Acry-p, D-LacCer-p, and D-GM3-p were accumulated in the spherical organelles around the nucleus. These patterns were consistent with the staining of Lysotracker (lysosome marker), but not Mitotracker (mitochondria marker) and giantin (Golgi marker) (Figure 7B). These results suggest that all of these glycopolymers are incorporated into the cells by endocytosis and transported to lysosome.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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To our knowledge, this is the first report of artificially constructed GM3 mimetic polymers having inhibitory activities on cell proliferation, although studies have been performed on the bioactivities of related glycopolymers (Cao and Roy 1996
, Kamitakahara et al. 1998). For example, lysoganglioside GM3-poly-L-glutamic acid was demonstrated to act as a multivalent inhibitor on influenza A/PR/8/34 (H1N1) binding at picomolar concentrations (Kamitakahara et al. 1998). Another report discussed the chemical synthesis of a GM3-trisaccharide-containing glycopolymer (with molar ratios of 1:5, 1:10, 1:24, and 1:48 on a polyacrylamide chain) that exhibited high binding affinities for the plant lectin wheat germ agglutinin (Cao and Roy 1996). However, the structures of the glycopolymers in that included only the GM3-trisaccharide unit and not the two-alkyl chains of the ganglioside GM3 ceramide unit.
In our study, the most potent inhibitory activities were those of GM3-p with a sugar residue content of 3.8 mol% (1:25 GM3-p) to 9.1 mol% (1:10 GM3-p), rather than the GM3-p with the highest sugar content of 16.7 mol% (1:5 GM3-p) (Figure 2). A similar result has been reported, in which a sharp optimum (10 mol%) was observed for the sialic acid unit density in a synthetic glycopolymer that inhibited influenza virus receptor-binding (Matrosovich et al. 1990). Thus, an enhanced biological effect for GM3-p would be expected as the ratio of sugar chain in the polymer increased, however, the effect could be weakened by inefficient cluster formation due to electrostatic repulsion between the sugar units if the sugar content exceeds 9.1 mol%. In order to overcome this problem, we tried to introduce carbonic acid into the acrylamide backbone to facilitate cluster formation by electrostatic repulsion between the carbonic acids and sugar units. A marked increase in the radii (S2)1/2 in 1:3:3 GM3-p, as indicated in Table I, may imply an increase in well-oriented sugar units due to this substitution. In fact, 1:3:3 GM3-p completely inhibited cell proliferation, and its effect was stronger than 1:15 GM3-p. These observations suggest that introducing carbonic acid into an acrylamide backbone is a useful strategy for enhancing the biological effects of GM3-p (Figure 3). Moreover, the treatment of 1:3:3 GM3-p at 30 µM for 24 h caused the decrease of cell number up to 20%, suggesting that 1:3:3 GM3-p induced cell death. The cell morphological change by the treatment of 1:3:3 GM3-p for 24 h (data not shown) is quite similar to that of 1:15 GM3-p for 48 h (Figure 5A), suggesting that the induction of cell death by 1:3:3 GM3-p is earlier than that by 1:15 GM3-p.
The production of cyclin D and E is induced at the G1 phase in response to extracellular stimuli, and these proteins function as a complex with cdk4 and cdk2, respectively (Matsushime et al. 1991; Won et al. 1992; Ajchenbaum et al. 1993; Winston and Pledger 1993; Ohtsubo et al. 1995 ). The cyclin D-cdk4 and cyclin E-cdk2 complexes promote G1 to S phase transition by releasing E2F transcription factor from the E2F-Rb complex through phosphorylation of the Rb protein (Dowdy et al. 1993; Ewen et al. 1993; Kato et al. 1993). GM3-p significantly reduced the expression of cyclin D1, cyclin E, cdk4, and cdk2 at the gene and/or protein levels without affecting growth factor signaling from serum to c-fos, leading to a reduction in Rb phosphorylation and cell proliferation (Figure 6). GM3-p incorporated into cells by endocytosis and transported to lysosome (Figure 7). However, since Acry-p and LacCer-p also transported to lysosome, sialic acids clusters are not involved in this process. Moreover, after the incorporation of GM3-p into cells, the direct interaction between GM3-p and molecules involving the regulation of cell cycle will scarcely happen because GM3-p always exists in the lumen side of membranes. For this reason, the accumulation of GM3-p in lysosome may not be important for the cell growth suppression. Thus, we assume that GM3-p, but not LacCer-p, could interact with cell surface proteins that recognize sialic acid clusters, modify the signal transduction cascade or down-regulate the target protein through endocytosis, leading to the reduction of cyclin D-cdk4 and cyclin E-cdk2 complexes. But the interaction of sialic acid cluster of GM3-p to cell surface proteins may be independent event of the following internalization process, because GM3-p and LacCer-p exhibited the similar endocytotic pathway, resulting in lysosome accumulation. Currently, we are searching for GM3-p associated proteins using biotinylated GM3-p and hope to obtain a more definitive picture of the biological action of GM3-p.
The cyclin D1/pRb pathway is deeply linked to malignant transformation, and abnormalities have been observed in almost all tumor cell types. In particular, cyclin D1, the initial molecule of this pathway, is often overexpressed in esophageal, breast, and other cancers due to chromosomal inversion (Motokura et al. 1991), translocation (Withers et al. 1991; Seto et al. 1992), and amplification (Lammie et al. 1991; Jiang et al. 1992; Buckley et al. 1993; Tsuruta et al. 1993). A number of anticancer agents targeting cdk have been developed, but none possess selectivity for a particular cdk species such as cdk 4 or 6. There are also drug that exhibit anticancer activity through cell cycle arrest at G1. Especially, certain anticancer drug candidates are expected to promise efficacy against large cell lung cancer and colon cancer, many of which are resistant to currently available anticancer agents. Unlike anticancer agents that induce apoptosis, these drug candidates are expected to target many tumor cells possessing abnormal cyclin D1. In fact, GM3-p was able to induce potent cell growth arrest against many types of cancer cells such as 3LL Lewis lung carcinoma, B16 melanoma, HEK293T, and KB cells (data not shown). Thus, it is encouraged to develop GM3-p as a new anticancer drug that affects on the expression of molecules involved in the cell cycle, such as cyclin D1 cyclin E, cdk4, and cdk2.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Construction of mimetic glycopolymers
Chemical reactions (Nishimura and Yamada 1997
, Yamada et al. 1997) were monitored by thin-layer chromatography on precoated plates of Merck 60F254 Silica Gel (layer thickness, 0.25 mm), and compounds were detected by spraying the plates with 10% H2SO4 in ethanol, then heating them. Enzymatic reactions were performed using commercially available materials, including -2,3-sialyltransferase (Calbiochem, San Diego, CA), sodium cacodylate buffer (Sigma, St Louis, MO), and CMP-NeuAc (Sigma). Enzymatic reactions introducing sialic acid unit to LacCer-p were carried out at 37 °C for 72 h. Sugar compositions of glycopolymers were determined from the integration data of 1H NMR, according to a method described previously (Nishimura and Yamada 1997; Yamada et al. 1997).
For the synthesis of the GM2-p, GalNAc was transferred to the GM3-p in Tris–HCl buffer (pH 7.5) using ß-1,4-N-acetylgalactosaminyl transferase (Campylobacter jejuni) (Yamasa Corporation, Chiba, Japan) and UDP-GalNAc (Yamasa Corporation), and incubating at 30 °C for 72 h. The GM4-p was synthesized by introducing a sialic acid to the GalCer-p using the same method as for the GM3-p synthesis, i.e. using -2,3-sialyltransferase and CMP-NeuAc in sodium cacodylate buffer at 37 °C for 72 h (Yamada et al. 1997). The GalCer-p was synthesized by radical copolymerization of a GalCer monomer and an acrylamide monomer as described before (Yamada et al. 1997). The glycopolymers obtained were purified from the reactants by Sephadex G-25 (GE Healthcare Bio-Science, Piscataway, NJ) gel filtration or by dialysis (Zehavi and Herchman 1984; Zehavi et al. 1990).
Physicochemical properties
Static laser light scattering measurements (Flory 1953) were taken at 25 °C using a computer operated light-scattering spectrophotometer (DLS-7000) (Photal Otsuka Electronics CO., Osaka, Japan) with a 10 mV He–Ne laser-emitting vertically polarized light at a wavelength of 
= 632.8 nm as the light source (Flory 1953). MilliQ water (18.3 M
cm electric conductivity) was used as the solvent. Refractive index increments (dn/dc) of the glycopolymer solutions were measured using a double-beam differential refractometer (DRM-1020) (Photal Otsuka Electronics) at 633 nm and were estimated from the equation for a simple Cauchy dispersion (Huglin 1972). Intensities were calibrated against a well purified and clarified toluene (excess Rayleigh scattering ratio: 1.41 x 10–5 cm–1). No mechanical stirring was applied to the solution, which was gently shaken for several hours to promote complete dissolution into a perfectly homogeneous solution. Fractionations were extensively purified by repeatedly passing the solution over a 0.1 mm pore size MILLEX-VV Millipore filter (Millipore S.A. Molsheim, France). To maintain dust-free conditions, the solutions were directly pipetted into a polished 2 cm diameter precision glass cylindrical light-scattering cell. Intensities of scattered light were measured at angles of 20°, 30°, 40°, 60°, 90°, 120°, and 150° We used a standard Zimm plot (Zimm 1948) to analyze the data according to the well-known equation:
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2n2(dn/dc)2/NA4 for vertically polarized incident light], c is the glycopolymer concentration (g/ml), l is the wavelength of incident light, 
is the scattering angle (the angle between the incident and the scattered beams), n is the refractive index of solvent (water, 1.33), NA is Avogadro's number and R() is the excess Rayleigh ratio at the scattering angle q with a vertically polarized incident beam. The wave vector q = (4n)Sin(/2)/l. A2 is the second osmotic virial coefficient. S is the square root of the mean-square radius of gyration.
Cell culture conditions
NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 10% (v/v) fetal bovine serum (FBS) with 100 units/mL penicillin and 100 µg/mL streptomycin.
Cell proliferation assays
Cell viability and proliferation were determined using cell countig kit-8 (Dojindo, Kumamoto, Japan), which are based on a 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide method. Briefly, NIH3T3 cells (1 x 105 cells/ml) in DMEM with 10% FBS were plated in 96-well microtiter plates at 100 µL/well, then 24 or 48 h later the cells were treated with GM3, LacCer or a glycopolymer. After a specific culture time, 10 µL of cell counting kit-8 were added to each well, and the cultures were incubated for 2 h at 37 °C. The light absorbance at 450 nm of the formazan generated in the wells was measured with a dual-wavelength flying spot scanner (CS9300-PC, Shimadzu, Kyoto, Japan).
Confocal laser-scanning microscopy
Cells were cultured on coverslips and treated with 150 µM dansyl polymers for 24 h. For the staining with Mitotracker Red CMXRos (Invitrogen, Carlsbad, CA) or Lysotracker Red DND-99 (Invitrogen), the cells were incubated with 100 nM Mitotracker or 75 nM Lysotracker in DMEM for 30 min at 37 °C, washed twice with phosphate buffered saline (PBS), and fixed for 15 min with 3.7% formaldehyde in PBS at room temperature. For the staining with anti-giantin antibodies (CRP Inc., Berkeley, CA), cells were fixed for 15 min with 3.7% formaldehyde in PBS at room temperature, permeabilized with 0.5% Triton X-100 in PBS, blocked with blocking solution (10 mg/mL bovine serum albumin in PBS), and incubated for 1 h with anti-giantin antibodies (1:1000 dilution) in blocking solution. After washing three times with PBS, the cells were incubated for 30 min with Alexa 594-conjugated anti-rabbit antibodies (Invitrogen) diluted in blocking solution to 5 µg/mL. Coverslips were washed in PBS three times and were mounted onto glass slides using Mowiol 488 (Calbiochem) and analyzed by fluorescence microscopy FV1000 (Olympus, Tokyo, Japan).
Preparation of cell lysates
Cells were washed twice with ice-cold PBS and suspended in buffer A [Tris–HCl (pH 7.5)], 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (CompleteTM, ethylenediaminetetraacetic acid-free; Roche Molecular Biochemicals, Mannheim, Germany). Cells were sonicated for 3 min at 4 °C and centrifuged at 500g for 5 min at 4 °C. The supernatants were further centrifuged at 100 000 g for 1 h at 4 °C. The final supernatants (soluble fractions) were treated with 100 µL of a 0.2% deoxycholate solution (Sigma) for 10 min at 0 °C, then an equal volume of 10% (w/v) trichloroacetic acid was added, and the mixture was incubated for another 20 min at 0 °C. Protein precipitates were washed with acetone, suspended in 1 x sodium dodecyl sulfate (SDS) sample buffer [62.5 mM Tris–HCl (pH 6.8)], 2% SDS, 10% glycerol, 0.001% bromophenol blue, and 5% 2-mercaptoethanol), and boiled for 5 min.
Immunoblotting
Immunoblotting was performed as described previously (Uemura et al. 2006). Anti-cyclin D1 (Cell Signaling Technology, Berverly, MA), anti-cyclin E (Upstate, Charlottesville, VA), anti-cdk4 (Cell Signaling Technology), anti-cdk2 (BD Bioscience, Franklin Lakes, NJ), anti-Rb (BD Bioscience), and anti-actin (Sigma) antibodies were used as primary antibodies. Horseradish peroxidase conjugated donkey anti-mouse IgG F(ab')2 fragment (GE Healthcare Bio-Science) was used as the secondary antibody. The antigen was detected using an ECL plus kit (GE Healthcare Bio-Science).
Real-time polymerase chain reaction
First strand cDNA synthesis was performed using a first strand cDNA Synthesis kit for RT-PCR (AMV) (Roche Diagnostics Corporation, Penzberg, Germany) and total RNA isolated from NIH3T3 cells. Primers and probes (Taq-man probe) specific for c-fos, cyclin D1, cyclin E, cdk4, cdk2, and actin were purchased from Applied Biosystems (Foster, CA). A 2 x PCR universal master mix (Applied Biosystems), containing PCR buffer, MgCl2, deoxynucleotide tri-phosphates, and the thermally stable AmpliTaq Gold DNA polymerase, was used in the PCR reactions. In addition, the PCR reaction mixture contained forward and reverse primers at 0.9 µM, 0.25 µM Taq-man probe, and 0.05 µg first strand cDNA. RNase- and DNase-free water was added for a final volume of 10 µL. The PCR reaction mixture was incubated at 50 °C for 2 min and at 95 °C for 10 min, then run for 40 cycles at 95 °C for 15 s and 60 °C for 60 s on an Applied Biosystems 7500 Real Time PCR System.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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We thank Dr Eliabeth A. Sweeney for scientific editing and preparation of the manuscript.
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Footnotes |
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* These two authors contributed equally to this work.
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Abbreviations |
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BHK, baby hamster kidney fibroblasts; cdk, cyclin-dependent kinase; CMP-NeuAc, cytidine-5'-monophosphate-N-acetylneuraminic acid; D-Acry-p, dansyl-acrylamide polymer; D-GM3-p, dansyl-GM3-p; D-LacCer-p, dansyl-LacCer-p; DMEM, Dulbecco's modified Eagle's medium; GalCer-p, galactosylceramide mimetic polymer; GalNAc, N-acetylgalactosamine; GM3-p, GM3 mimetic polymer; GM4-p, GM4 mimetic polymer; LacCer, lactosylceramide; LacCer-p, LacCer mimetic polymer; Lac-p, lactosyl polymer; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SDS, sodium dodecyl sulfate; Sia-p, sialyllactosyl polymer.
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References |
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