Glycomics for drug discovery: Metabolic perturbation in androgen-independent prostate cancer cells induced by unnatural hexosamine mimics

Article abstract of DOI:10.1002/anie.201108742

Inhibited: N-acetylglucosamine (GlcNAc) derivatives with a fluorine atom at the C4 position (2-4) were synthesized, and their ability to inhibit cancer-cell growth was investigated. The administration of these 4F-GlcNAc derivatives to cells led to the unn

Full text of DOI:10.1002/anie.201108742

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DOI: 10.1002/anie.201108742
Sugar Derivatives
Glycomics for Drug Discovery: Metabolic Perturbation in Androgen-
Independent Prostate Cancer Cells Induced by Unnatural Hexosamine
Mimics**
Shin-Ichiro Nishimura,* Megumi Hato, Satoshi Hyugaji, Fei Feng, and Maho Amano
Prostate cancer is the most frequently diagnosed malignancy
and the second leading cause of cancer-related deaths among
men. The incidence is very high in the United States followed
by Australia/New Zealand and Europe. The American
Cancer Society estimated that in the United States about
240890 new cases of prostate cancer will be diagnosed and in
2011 about 33720 cases will be fatal.[1] Androgens are critical
to the growth and differentiation of prostate epithelial cells.
Therefore an androgen ablation therapy is the first choice for
the treatment of advanced prostate cancer. However, meta-
static prostate cancer eventually becomes hormone-refrac-
tory and resistant to many treatments, while it is initially
responsive to androgen ablation. Although systemic chemo-
therapy has been evaluated in men with hormone-refractory
prostate cancer for many years, there is no current standard
treatment for such a metastatic androgen-independent pros-
tate cancer.[2,3] It is clear that novel therapeutic approaches
are strongly needed to improve the outlook for patients
suffering from advanced prostate cancer.
Cell-surface glycans regulate many different states of
cancer progression such as proliferation, invasion, angio-
genesis, and metastasis.[4] Cancer cells often express many
aberrant glycoforms when compared with normal cells. It is
documented that the expression of highly branched N-glycans
is distinctly enhanced during the proliferation of different
cancer cells and metastasis.[5,6] Recently, Novotny and co-
workers reported an increase of the expression of highly
branched N-glycans, such as tri- and tetra-antennary struc-
tures, in the serum of human metastatic prostate cancer
patients when compared with the expression level in the
serum of healthy individuals.[7] We also observed similar
alterations of N-glycan profiles in the human prostate cancer
cell line PC-3 in comparison with those of normal human
prostate epithelial cells.[8] Therefore, it is considered that
inhibitors and modifiers of the biosynthesis of such highly
branched glycans may become novel candidates for cancer
therapy.[9–13] The Golgi N-glycan biosynthetic pathway seems
to be ultrasensitive to the hexosamine flux that is required for
the production of tri- and tetra-antennary N-glycans, which
regulate topologies/distribution and functional roles of differ-
ent cell-surface glycoprotein receptors.[14] Increasing the
intracellular uridyl diphosphate N-acetyl-glucosamine
(UDP-GlcNAc) concentration leads to increased branching
of N-glycans of such glycoprotein receptors[15] and enhances
the affinity with the cell-surface galectin-3, a key lattice-
forming lectin.[16,17] In cancer cells, the interaction between
glycoprotein receptors having multiple hyperbranched N-
glycans and galectin-3 seems to be crucial for retaining
growth-promoting receptors at the cell surface by forming
galectin-mediated glycoprotein lattice. Reagents that can
control the intracellular UDP-GlcNAc levels and N-glycan
branching by perturbing a hexosamine biosynthetic pathway
may become promising leads to the discovery of a new class of
anti-prostate-cancer drugs. GlcNAc can normally be salvaged
from glycoprotein turnover by degradation in lysosomes and
be employed for further enzymatic modifications in the
cytoplasm to re-enter the UDP-GlcNAc pool (Scheme 1A).
We hypothesized that GlcNAc analogues bearing a fluorine
atom at the C4 position[18–20] might depress the expression
levels of such hyperbranched N-glycans in human prostate
cancer PC-3 cells[21] by perturbing the hexosamine biosyn-
thetic pathway.
Herein, we communicate for the first time evidence for
and the mechanism of a metabolic inhibitory effect on human
prostate cancer cell proliferation by a new class of 2-
acetamido-2,4-dideoxy-4-fluoro-d-glucopyranose (1, FGN-
2Ac) derivatives, notably the known 2-acetamido-2,4-
dideoxy-1,3,6-tri-O-acetyl-4-fluoro-d-glucopyranose
(2,
FGN-1236Ac)[18] and the novel 2-acetamido-2,4-dideoxy-3,6-
di-O-acetyl-4-fluoro-d-glucopyranose (3, FGN-236Ac; Sche-
me 1B,C). Synthetic 4F-GlcNAc-1-phosphate is converted
into UDP-4F-GlcNAc (5) efficiently in vitro by the recombi-
nant human UDP-GlcNAc pyrophophorylase in the presence
of uridine triphosphate (UTP);[22] thus per-O-acetate 2 and its
analogue may be accepted in the hexosamine biosynthetic
pathway leading to compound 5, an unnatural sugar nucleo-
tide (Scheme 1C). We thought that an interconversion
between closed and open forms at the anomeric carbon of
compound 3 (1-OH analogue) might influence a series of
metabolic reactions of enzymes comprising the GlcNAc
salvage pathway and result in a different biological activity
against cancer cell growth in comparison with that of per-O-
acetate 2. To obtain a set of the required derivatives 1–4, we
[*] Prof. S.-I. Nishimura, Dr. M. Hato, S. Hyugaji, Dr. F. Feng,
Dr. M. Amano
Graduate School of Advanced Life Science
Hokkaido University
N21, W11, Kita-ku, Sapporo, 001-0021 (Japan)
E-mail: shin@sci.hokudai.acjp
[**] This work was partly supported by a grant for “Innovation COE
Project for Future Medicine and Medical Research” from the
Ministry of Education, Culture, Science, and Technology (Japan). We
appreciate the assistance with the analysis of sugar nucleotides by
Dr. Y. Takegawa of Hokkaido University.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201108742.
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3
cell proliferation (IC50 = 277 mm; IC50 = concentration
required for 50% inhibition of target cells), whereas the
parent non-O-acetylated FGN-2Ac (1) did not have any
influence owing to the poor cell permeability. Surprisingly,
FGN-236Ac (3) exhibited the highest growth inhibition
against PC-3 cells (IC50 = 61 mm) among all 4F-GlcNAc
analogues tested herein. In contrast, FGN-123Ac (4) having
a 6-OH group exhibited little inhibitory effect, indicating that
the position of O-acetyl groups influences the metabolic
acceptability of these molecules on the cytoplasmic side.
Strikingly, a partially O-acetylated GlcNAc analogue having
a 1-OH group also showed a similar type of activity to that of
FGN-236Ac (3; Figure S1 in the Supporting Information),
thus suggesting that the free hemiacetal group liberated
predominantly during de-O-acetylation within cells may
specifically perturb further enzymatic modification processes.
Because similar results were obtained in a common human
lung cancer cell line, A549 (Figure S2 in the Supporting
Information), we may conclude that per-O-acetylated deriv-
ative FGN-1236Ac (2) is a precursor of the metabolic
inhibitor of human cancer cell proliferation, and spontaneous
liberation of the hemiacetal group during de-O-acetylation
within cells induces perturbation in the GlcNAc salvage
pathway. The inhibitory effect on PC-3 cell growth of FGN-
236Ac (3) seemed to be higher than that of 5-fluorouracil (5-
FU) or cisplatin (Figure 1B).
The significant inhibitory effects of FGN-236Ac (3) on
PC-3 cell growth prompted us to elicit the available UDP-
Scheme 1. Mammalian hexosamine biosynthetic pathway and com-
pounds used in this study. A) Possible pathway using Glc, GlcN, or
GlcNAc salvaged from endocytic glycoconjugates. Per-O-acetate of
GlcNAc exogenously added can penetrate into cells across the plasma
membrane owing to enhanced hydrophobicity. B) Equilibrium between
the open and closed forms of 3. C) Proposed synthesis of the
unnatural sugar nucleotide 5 from compounds 2–4 by hijacking
a biosynthetic pathway.
Scheme 2. A synthetic route for the synthesis of 4F-GlcNAc analogues
1–4 from GalNAc. Reagents and conditions: a) 1. PhCH(OMe)2, CSA,
CH3CN, RT, 8 h. 2. Ac2O, pyridine, RT, 12 h, 98%. b) H2, Pd/C, MeOH/
dioxane, RT, 2 h, 99%. c) Triphenylmethyl chloride, Et3N, DMAP,
CH2Cl2, RT, 13 h, 83%. d) DAST, pyridine, CH2Cl2, RT, 8 h, 85%.
e) 80% AcOH, 408C, 5 h, 67%. f) Ac2O, pyridine, RT, 23 h, 100%.
g) K2CO3, MeOH, À208C, 4 h, 94%. h) Benzylamine, THF, RT, 3 h,
68%. CSA=camphorsulfonic acid, DMAP=4-(dimethylamino)pyri-
dine, DAST=N,N-(diethylamino)sulfur trifluoride, Tr=triphenylmethyl.
designed a facile and efficient synthetic route from N-
acetylgalactosamine (GalNAc) as a starting material that
might be feasible for large-scale synthesis as indicated in
Scheme 2. The effect of compounds 1–4 on PC-3 cell viability
after 48 h treatment is shown in Figure 1A. As anticipated,
FGN-1236Ac (2) showed a significant inhibitory effect on PC-
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of FGN-236Ac (3) appears to be at a lower level than the
UDP-4F-GlcNAc levels observed in the presence of two
other analogues, FGN-1236Ac (2) and FGN-123Ac (4).
It is likely that the decrease of the UDP-GlcNAc level and
the accumulation of UDP-4F-GlcNAc (5) directly affect the
total N-glycan profiles of PC-3 cells either through changes in
the availability of UDP-GlcNAc or through an inhibitory
effect of UDP-4F-GlcNAc (5) against different GlcNAc
transferases.
We employed glycoblotting-based enrichment analysis to
characterize the whole N-glycome of PC-3 cells.[24,25] Advan-
tageous in this method is that the sensitivity of the Golgi N-
glycan biosynthetic pathway to the hexosamine flux can be
directly monitored as the up-regulation of hyperbranched N-
glycoforms of PC-3 cells that were treated with high concen-
trations (10–100 mm) of free GlcNAc (Figure S5 in the
Supporting Information). In other words, direct monitoring
of the total cellular glycome[8,26] is a facile and efficient assay
to estimate the available GlcNAc concentration within cells
during dynamic cancer progression. Surprisingly, treatment of
PC-3 cells with three fluorine-containing GlcNAc mimics (2–
4, 50 mm) down-regulated remarkably the expression levels of
most hyperbranched N-glycans such as tri- and tetra-antenn-
ary glycoforms, which were profiled as characteristic cellular
and serum biomarkers for human prostate cancer[7,8]
(Figure 3). As anticipated, treatment with 5-FU, cisplatin, or
UV light did not induce any notable changes in the N-glycan
profiles. Moreover, we could not see any significant ion peaks
originating from 4F-GlcNAc-terminated N-glycans in the
lower-molecular-weight region of MALDI-TOF-MS (Fig-
ure S6 in the Supporting Information), thus indicating that
Figure 1. Inhibition of PC-3 cell growth by exogenous addition of
unnatural GlcNAc mimics. A) Cell viability after 48 h incubation with 4-
fluorine-containing compounds FGN-2Ac (1, pink trace), FGN-1236Ac
(2, blue trace), FGN-236Ac (3, red trace), and FGN-123Ac (4, orange
trace) was determined by the method described in the Supporting
Information. B) Cell-growth arrest of PC-3 cells by FGN-236Ac (3) in
comparison with two representative chemotherapy agents, 5-FU and
cisplatin. Red squares correspond to measurements with PC-3 cells
and white circles to those with normal prostate epithelial cells.
% Control=the percentage of living cells.
GlcNAc concentration in the intracellular sugar-nucleotide
pools and the alteration of cell-surface whole N-glycan
profiles during cell cultivation in the presence of this class
of unusual GlcNAc mimics. To verify that UDP-4F-GlcNAc
(5) can be synthesized within PC-3 cells from fluorine-
containing unusual sugars, the relative levels of intracellular
UDP sugars were evaluated by using cells cultured in the
presence of compounds 2–4 (50 mm). Considering that FGN-
236Ac (3) completely arrested PC-3 cell growth at a dose of
about 100 mm (Figure 1A), administration of 50 mm of com-
pounds is an appropriate condition to test the effect on the
GlcNAc salvage biosynthetic pathway independent from cell-
growth inhibition. The HPLC and MS analysis using authentic
sugar nucleotides[23] clearly revealed that all three compounds
used herein can be efficiently converted within PC-3 cells into
UDP-4F-GlcNAc (5), and in contrast, the UDP-GlcNAc level
seemed to be dramatically decreased (Figure 2, and Fig-
ures S4 and S5 in the Supporting Information). The intra-
cellular accumulation of UDP-4F-GlcNAc (5) in the presence
Figure 2. Accumulation of UDP-4F-GlcNAc in sugar-nucleotide pools
of PC-3 cells. HPLC analysis of sugar nucleotides of PC-3 cells treated
with 50 mm of 4F-GlcNAc mimics 2–4 and 0.5% DMSO as a control
for 48 h. Sugar nucleotides were separated on a Develosil RPAQU-
EOUS column (250ꢀ4.6 mm) and detected by measuring the absorb-
ance at 260 nm (A260).
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non-natural UDP-4F-GlcNAc (5) cannot be incorporated
into the Golgi N-glycosylation pathways instead of the native
UDP-GlcNAc. Interestingly, it was reported that 2-acetam-
ido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-a-d-glucopyranose
(Ac-5S-GlcNAc) can be salvaged within COS-7 cells and be
converted into the unnatural UDP-5S-GlcNAc, and disturb
the synthesis of O-linked GlcNAc residues (O-GlcNAc),
whereas 5S-GlcNAc was not incorporated.[27] Furthermore,
no significant changes in the expression levels of major bi-
antennary N-glycans of PC-3 cells were observed in the
presence of 2 and 3 at a concentration of 50 mm (Figure S7 in
the Supporting Information). This finding suggests that an
intracellular UDP-GlcNAc level of at least approximately
1 mm is retained, which is needed for the N-acetylglucosami-
nyltranferases (GnTs), GnT-I and GnT-II, because the values
of the Michaelis constant Km obtained in vitro of GnT-I and
GnT-II for UDP-GlcNAc are 0.04 and 0.96 mm, respec-
tively.[15,16] These results imply that, within PC-3 cells, UDP-
4F-GlcNAc (5) is a highly specific and efficient metabolic
inhibitor of GlcNAc transferases that are responsible for the
synthesis of tri- and tetra-antennary N-glycans, notably GnT-
IV and GnT-V (Km values of GnT-IV and GnT-V are 5.0 and
11.0 mm, respectively).[15,16] Most importantly, the loss of the
hyperbranched N-glycans caused by a 50 mm dose of 2–4 did
not correlate with the results of cell-growth inhibition as
shown in Figure 1A. It means that 6-O-acetylated 4F-GlcNAc
analogues bearing a free hemiacetal group might efficiently
inhibit other enzymes and result in the perturbation of the
total hexosamine biosynthetic pathway. This conclusion was
supported by the lower accumulation of UDP-4F-GlcNAc (5)
in PC-3 cells after treatment with 3 when compared with the
Figure 4. G2/M cell-cycle arrest and apoptosis analyzed by flow
cytometry. A) A schematic presentation of the cell cycle is shown on
the left, with the mitotic phase (M) and the interphase, which consists
of G1, G0, S, and G2. Cell-cycle distribution in PC-3 cells treated with
DMSO (0.5%) as control, 5-FU (50 mm), cisplatin (50 mm), and FGN-
236Ac (50 mm) for 48 h (*P<0.01 compared with control). B) Cell-
cycle distribution in PC-3 cells treated with the shown related sugar
derivatives (G: Glc, GN: GlcNAc, numbers 1–6 indicate the positions
of acetylation). Above the histograms in (A) and (B), an indication of
the ploidy (2N, 4N) of the cells is given. During the G2/M phases,
cells are usually tetraploid (4N), that is, they have double the number
of chromosomes.
Figure 3. Perturbation of the Golgi N-glycosylation biosynthetic path-
way by 4F-GlcNAc analogues. Hyperbranched N-glycans of PC-3 cells
cultured in the presence of different GlcNAc mimics, 5-FU, cisplatin,
and UV light. MALDI-TOF-MS focused on the high-molecular-weight
region involving major tri-antennary and tetra-antennary N-glycans.
Structures were identified by using tandem MS and the GlycoMod Tool
and the GlycoSuite website. Yellow circle: galactose, blue square: N-
acetylglucosamine, green circle: mannose, red triangle: fucose, purple
diamond: sialic acid.
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UDP-4F-GlcNAc levels observed after treatment with 2 and 4
(Figure 2). However, notably the hyperbranched N-glyco-
forms are essential for the retention of cancer cell surface
glycoprotein receptors through the formation of galectin-
mediated lattice that greatly influences cancer malignancy
phenomena, such as invasion and metastasis.[5,6] It should be
emphasized that 4F-GlcNAc derivatives are potential
reagents that reduce expression levels of hyperbranched N-
glycans during cancer-cell progression, although confirmation
by using appropriate aggressive cancer cells or such disease
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Cell flow cytometry of PC-3 cells treated with 3 (50 mm)
for 48 h showed a marked increase in the cell population in
the G2/M phases from 19.2% (control) to 39.9% with
a concomitant decrease of the cells in the G1 phase from
51.2% (control) to 27.5% (Figure 4A). In contrast, treatment
with 50 mm 5-FU or cisplatin for 48 h showed no significant
differences in the populations of phases G1 (60.4% and
45.5%) and G2/M (16.8% and 22.5%), because 5-FU is
known to be one of the S-phase-specific anticancer agents.
The induction of G2/M cell-cycle arrest was most efficient
after treatment with 3 (50 mm) compared with related sugar
compounds (Figure 4B). This finding clearly indicates that
the hemiacetal group of GlcNAc is essential for the specific
cell-cycle arrest, when other hydroxy groups involving the C-6
position are acetylated. The estrogen metabolite 2-methox-
yestradiol[28] also induces G2/M cell-cycle arrest and apoptosis
in LNCaP, DU145, ALVA-31, and PC-3 cells in vitro, and
inhibits the growth of androgen-independent prostate cancer
in a transgenic mouse model in vivo.[29] To assess the role of 3
in the apoptosis, double staining with annexin V-FITC
(FITC = fluorescein isothiocyanate) and propidium iodide
(PI) was employed for PC-3 cells treated with 3 for 48 h at
concentrations of 50, 100, 200 mm to differentiate early
apoptotic cells and late apoptotic/necrotic cells, respectively
(Figure S8 in the Supporting Information). 66.6% of PC-3
cells treated with 3 (200 mm) underwent apoptosis (9.7% early
apoptosis and 56.9% late apoptosis/necrosis), whereas no
significant changes were observed at concentrations of 3
below 100 mm. These results may motivate us to target the late
cell-cycle stage for therapeutic intervention in androgen-
insensitive diseases,[29] and the novel class of non-natural
GlcNAc derivatives would allow for expanding the available
chemical space to discover promising anti-prostate cancer
drugs.
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Received: December 12, 2011
Revised: January 9, 2012
Published online: February 17, 2012
Keywords: cancer · carbohydrates · glycomics ·
.
hexosamine pathway · N-glycans
[1] American Cancer Society, Cancer Statistic 2011, available at
http://www.cancer.org.
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