Targeting pro-invasive oncogenes with short chain fatty acid-hexosamine analogues inhibits the mobility of metastatic MDA-MB-231 breast cancer cells

Article abstract of DOI:10.1021/jm800873k

Per-butanoylated N-acetyl-D-mannosamine (Bu₄ManNAc), a SCFA-hexosamine cancer drug candidate with activity manifest through intact n-butyrate-carbohydrate linkages, reduced the invasion of metastatic MDAMB-231 breast cancer cells unlike per-butanoylated-D-mannose (Bu₅Man), a clinically tested compound that did not alter cell mobility. To gain molecular-level insight, therapeutic targets implicated in metastasis were investigated. The active compound Bu₄ManNAc reduced both MUC1 expression and MMP-9 activity (via down-regulation of CXCR4 transcription), whereas "inactive" Bu₅Man had counterbalancing effects on these oncogenes. This divergent impact on transcription was linked to interplay between HDACi activity (held by both Bu₄ManNAc and Bu₅Man) and NF-κB activity, which was selectively down-regulated by Bu ₄ManNAc. Overall, these results establish a new therapeutic end point (control of invasion) for SCFA-hexosamine hybrid molecules, define relative contributions of molecular players involved in cell mobility and demonstrate that Bu₄ManNAc breaks the confounding link between beneficial HDACi activity and the simultaneous deleterious activation of NF-κB often found in epigenetic drug candidates.

Full text of DOI:10.1021/jm800873k

J. Med. Chem. 2008, 51, 8135–8147
8135
Targeting Pro-Invasive Oncogenes with Short Chain Fatty Acid-Hexosamine Analogues Inhibits
the Mobility of Metastatic MDA-MB-231 Breast Cancer Cells
Christopher T. Campbell, Udayanath Aich, Christopher A. Weier, Jean J. Wang, Sean S. Choi, Mary M. Wen, Katharina Maisel,
Srinivasa-Gopalan Sampathkumar,† and Kevin J. Yarema*
Department of Biomedical Engineering, The Johns Hopkins UniVersity, Baltimore, Maryland 21218
ReceiVed July 15, 2008
Per-butanoylated N-acetyl-D-mannosamine (Bu4ManNAc), a SCFA-hexosamine cancer drug candidate with
activity manifest through intact n-butyrate-carbohydrate linkages, reduced the invasion of metastatic MDA-
MB-231 breast cancer cells unlike per-butanoylated-D-mannose (Bu5Man), a clinically tested compound
that did not alter cell mobility. To gain molecular-level insight, therapeutic targets implicated in metastasis
were investigated. The active compound Bu4ManNAc reduced both MUC1 expression and MMP-9 activity
(via down-regulation of CXCR4 transcription), whereas “inactive” Bu5Man had counterbalancing effects
on these oncogenes. This divergent impact on transcription was linked to interplay between HDACi activity
(held by both Bu4ManNAc and Bu5Man) and NF-κB activity, which was selectively down-regulated by
Bu4ManNAc. Overall, these results establish a new therapeutic end point (control of invasion) for SCFA-
hexosamine hybrid molecules, define relative contributions of molecular players involved in cell mobility
and demonstrate that Bu4ManNAc breaks the confounding link between beneficial HDACi activity and the
simultaneous deleterious activation of NF-κB often found in epigenetic drug candidates.
Introduction
checkpoint protein p21, halting cell cycle progression and
contributing to apoptosis. In studies dating from the 1980s,
clinical testing of Bu5Man (compd 2 in Figure 2) and similar
butanoylated carbohydrates showed moderate, but ultimately
disappointing, efficacy.5 SCFA-monosaccharides subsequently
have been superseded by small molecule HDACi such as
suberoylanilide hydroxamic acid (SAHA),6 a drug that was FDA
approved under the trade name Zolinza in 2006 for the treatment
of cutaneous T cell lymphoma (CTCL), a form of non-
Hodgkin’s lymphoma.7
Over the last 30 years, short chain fatty acid (SCFAa)-
monosaccharide hybrid molecules have been investigated by
two groups of researchers. By far, their most successful use
has been for metabolic glycoengineering of the cell surface1,2
where SCFA have been exploited to enhance the cellular
uptake of monosaccharide analogues.3,4 Earlier, in the reverse
strategy, carbohydrates were used as delivery vehicles for
n-butyrate, a histone deacetylase inhibitor (HDACi) used for
epigenetic cancer therapy.5 In both applications, the hybrid
molecules were assumed to be hydrolyzed by esterases or
other lipases upon entering a cell and any ensuing biological
activity was attributed to either the liberated sugar or SCFA
moieties. In this study, overlaid upon, and dominating, the
glycosylation-specific effects of the core sugar and the
HDACi activity of the accessory SCFA, we investigated the
therapeutic possibilities of a third source of biological activity
derived from molecular species with intact SCFA-hexosamine
ester linkages (Figure 1).
At the outset, a brief review of the first two uses of SCFA-
monosaccharide hybrid molecules is useful for understanding
why the therapeutic prospects of this class of compounds are
worth revisiting. Hybrid molecules consisting of a SCFA ester
linked to a carbohydrate were first employed by cancer biologists
who sought to exploit the sugar as a delivery vehicle for
n-butyrate. n-Butyrate is liberated by cytosolic esterases upon
cellular uptake of the prodrug, and the resulting histone
deacetylase inhibitory (HDACi) activity of this SCFA slows
the growth of cancer cells by up-regulating the cell cycle
During the past decade, as the fortunes of SCFA-monosac-
charides in cancer treatment waned, a subset of these molecules
found new life in metabolic glycoengineering and, in the process,
shed light on the shortcomings of the clinical tests. In early
metabolic glycoengineering experiments, a technique that
involves the introduction of non-natural monosaccharides into
biosynthetic glycosylation pathways for the display of chemi-
cally modified, i.e., engineered, glycans on the cell surface,2,8,9
acetate was linked to the sugar to increase membrane
permeability.3,4 As a consequence, monosaccharide concentra-
tions required to elicit robust cellular responses dropped from
the millimolar to the micromolar range.4,10,11 Because of the
success of this strategy with acetate, our group employed longer
chain SCFA of increased lipophilicity to further increase
membrane permeability and analogue uptake. Specifically, the
relative abilities of acetate- (as Ac4ManNAc), propionate- (as
Pr4ManNAc), and n-butyrate-modified (as Bu4ManNAc) N-
acetyl-D-mannosamine (ManNAc) to support flux into the sialic
acid pathway progressively increased from 600- to 1800- to
2100-fold.12
* To whom correspondence should be addressed. Phone: 410.516.4914.
Fax: 410.516.8152. E-mail: kyarema1@jhu.edu. Address: Clark Hall 106A,
3400 North Charles Street, Baltimore, Maryland 21218.
Coincident with its favorable impact on metabolic flux into
the sialic acid pathway, Bu4ManNAc proved to be moderately
toxic to cancer cells.12 This observation, reminiscent of the use
of Bu5Man for cancer treatment,5,13 raised the possibility that
the new compound also could be exploited therapeutically. As
a first step in evaluating whether Bu4ManNAc was a superior
drug candidate compared to Bu5Man, we tested its ability to
†
Current Address: Laboratory of Chemical Glycobiology, National
Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
a Abbreviations: SCFA, short chain fatty acids; HDACi, histone deac-
tylase inhibitor; MMP, matrix metalloproteinase; SAR, structure-activity
relationships; MUC1, mucin 1, NF-κB, nuclear factor kappa B; CXCR4,
chemokine (C-X-C motif) receptor 4.
10.1021/jm800873k CCC: $40.75
2008 American Chemical Society
Published on Web 11/20/2008

8136 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Campbell et al.
Figure 1. SCFA-ManNAc analogues have three modes of biological activity. The intracellular fate of short chain fatty acid (SCFA)-monosaccharide
hybrid molecules are illustrated with n-butanoylated ManNAc analogues (R1 modifications at the N-acyl position have been used extensively in
metabolic glycoengineering; this paper focuses on the natural core sugar where R1 is a methyl group as embodied in Bu4ManNAc, compd 1 in
Figure 2). In the past, complete hydrolysis of the analogue into the SCFA (in this case, n-butyrate (A)) and the carbohydrate (e.g., R1-modified
ManNAc (B)) moieties has been assumed to account for the biological activities of the parent molecule through HDACi-mediated chromatin
remodeling and glycosylation, respectively. (C) Recently, we reported that partial hydrolysis products of peracylated hexosamines, exemplified by
tributanoylated ManNAc analogues lacking the SCFA group at either the C1-OH or C6-OH position, have distinct biological activities that are not
primarily dependent on either the HDACi activity of n-butyrate or on the impact of the core sugar on glycosylation.15 In particular, C6-derivatized
hexosamine analogues (such as compds 1, 3, and 5 in Figure 2) support unique scaffold-dependent anticancer responses. (D) By contrast, “inactive”
analogues not based on the hexosamine scaffold or lacking an SCFA at the C6 position have negligible toxicity, do not suppress metastatic oncogenes
and introduce high metabolic flux into glycosylation pathways.15
Figure 2. Chemical structures of SCFA-derivatized monosaccharide analogues used in this study.
inhibit cell growth and induce apoptosis in several cancer lines
and found that it inhibited proliferation over a 3-5 day period
consistent with the HDACi activity of n-butyrate.14 Cells treated
with Bu5Man, by contrast, subsequently recovered and resumed
robust cell growth (thereby offering an explanation for the only
modest clinical efficacy of this compound), whereas cells treated
with Bu4ManNAc underwent apoptosis over a two week
period.14 The ability of Bu4ManNAc to augment growth
inhibition with apoptosis was a positive step in cancer drug
design, but an even more encouraging development was the
subsequent discovery that Bu4ManNAc, unlike Bu5Man, inhib-
ited the pro-metastatic oncogene MUC1.15 On the basis of its

Targeting Oncogenes with SCFA-Hexosamine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8137
prominent role in promoting metastatic breast cancer, MUC1
provides an attractive, but to date recalcitrant, therapeutic target.
Because of the difficulty of modulating MUC1 with current
small molecule drug candidates, new compounds such as
Bu4ManNAc that suppress this pro-invasive oncogene are
worthy of investigation; accordingly, in the first experiments
undertaken in this study, we tested whether Bu4ManNAc could
inhibit the mobility of metastatic breast cancer cells.
6-phosphofructose and preferentially shunted into energy pro-
duction rather than used for maximizing metabolic flux into
biosynthetic pathways. In this regard Bu5Man differed signifi-
cantly from Bu4ManNAc, which generates ManNAc, a sugar
dedicated to sialic acid biosynthesis. This point was important
because, at the outset of these experiments, the relative
contributions of the HDACi activity of n-butyrate, sugar-specific
responses, and the scaffold-dependent activity were unclear;
hence employing different core sugars helped clarify the
importance of glycosylation. Finally, because Bu5Man had
already undergone clinical testing, it was an established
benchmark for cancer therapy using SCFA-carbohydrate hybrid
molecules. Although it is difficult to extrapolate from cell culture
experiments to in vivo efficacy, the expectation was that
Bu4ManNAc needed to show clearly superior results in cell
culture tests compared to Bu5Man (which was only moderately
successful in vivo) to warrant further drug development interest.
The favorable outcome of the initial experiments reported in
this paper, where Bu4ManNAc inhibited invasion in MDA-MB-
231 cells (in contrast to Bu5Man that had no impact on cell
mobility), provided impetus to further probe the underlying
mechanism for two reasons. First, mechanisms beyond MUC1
influence the invasive behavior of metastatic cells making it
likely that additional factors were involved in modulating cell
mobility. Second, despite the favorable features of Bu4ManNAc
(i.e., the induction of apoptosis and suppression of MUC1), its
prospects in cancer drug development were clouded by potential
pitfalls arising from its hydrolysis products, n-butyrate and
ManNAc. For example, in some types of cancer, HDACi
suppress proliferation while also activating NF-κB,16 which leads
cells to become drug resistant, refractory to apoptosis, or
increasingly metastatic. Thus, n-butyrate generated by
Bu4ManNAc had the potential to negate the benefits of MUC1
suppression via NF-κB activation. Moreover, the pro-invasive
predisposition of n-butyrate threatened to be exacerbated by
ManNAc, the second hydrolysis product of Bu4ManNAc,
because ManNAc is the dedicated metabolic precursor for sialic
acid biosynthesis, a sugar that alters the adhesive properties of
cells and that has been associated with a highly metastatic
phenotype in cancer.
Because of the potential pitfalls introduced by the hydrolysis
products of Bu4ManNAc, the anti-invasive properties of this
compound, which ultimately proved to be an attribute of
structural features of the molecular species with intact SCFA-
hexosamine linkages (Figure 1), were evaluated throughout this
study against the backdrop of cellular responses emanating from
n-butyrate and ManNAc. The results reinforce the hypothesis
that the hexosamine core structure can serve as a scaffold for
pharmacophore development,15,17 overturning the previously
held premise that SCFA-carbohydrate molecules primarily
function as prodrugs. Importantly, we extend the newfound
“intact ester linkage” mode of activity from a single oncogene
(MUC1) to additional therapeutic candidates (e.g., MMP-9,
CXCR4, and NF-κB) implicated in metastasis and show that
the molecular-level changes translate into altered cell level
behavior with attributes that are attractive for confronting
malignant disease.
Bu4ManNAc Selectively Inhibited Invasion. Previously,
Bu4ManNAc had been shown to suppress MUC1, whereas
Bu5Man enhanced levels of this pro-invasive oncogene in the
highly metastatic MDA-MB-231 breast cancer line.15 Because
invasion is a multifactorial process, we were interested in testing
whether the modulation of MUC1 by these analogues translated
into corresponding changes to the invasive behavior of cells or
whether the changes to this oncogene were masked by com-
pensating or offsetting factors. In particular, because hydrolysis
of Bu4ManNAc results in the production of ManNAc that is
subsequently converted to sialic acid, any benefit gained from
MUC1 suppression could have been easily negated by the pro-
invasive tendencies of this sugar. Therefore, the first objective
of this study was to determine if Bu4ManNAc reduced invasion
more effectively than the MUC1-enhancing analogue Bu5Man.
Accordingly, to test cell mobility, MDA-MB-231 cells that
had been preincubated with Bu4ManNAc and Bu5Man was
evaluated in modified Boyden chamber assays. Two variations
of this assay were used to study either migration (measured by
passage of cells through a porous membrane) or invasion
(measured by penetration through Matrigel). Results obtained
at the minimally cytostatic concentration of 50 µM showed a
trend toward preferential inhibition of invasion compared to
migration after two days of exposure to Bu4ManNAc (Figure
3A). Increased Bu4ManNAc concentrations almost completely
inhibited invasion after two days (Figure 3B), and significant
inhibition of both invasion and migration were seen after three
days of exposure to 50 µM of this analogue (Figure 3C). By
contrast, Bu5Man did not change either end point at e125 µM
(data are shown for 50 µM in Figure 3D).
Reduced Mobility of Bu4ManNAc-Treated Cells Was
Not Due to Cytotoxicity. Upon demonstrating the therapeuti-
cally useful end point of reduced invasion in Bu4ManNAc-
treated metastatic breast cancer cells, we next sought to
understand the relative contributions of the recently discovered
third mode of scaffold-dependent activity of this SCFA-hex-
osamine hybrid molecule (e.g., through MUC1 suppression) in
comparison to the responses emanating from the hydrolysis
products n-butyrate and ManNAc. But first we eliminated the
trivial possibility that the toxicity of Bu4ManNAc contributed
substantially to reduced mobility of the analogue treated cells.
To do so, we carefully avoided thresholds previously associated
with cytotoxicity that included analogue concentration, cell
density, and duration of analogue exposure. For example, over
a 2 or 3 day period, Bu4ManNAc approaching 500 µM is
required for apoptosis;12 therefore the current experiments were
performed at less than a third of this level (i.e., at e150 µM).
Results and Discussion
Rationale for the General Experimental Design. The lead
compound Bu4ManNAc had both of the SAR features, an N-acyl
substitution at the C2 position and an ester-linked SCFA group
at the C6 position, hypothesized to provide the anticancer
activities uniquely held by “active” SCFA-hexosamine ana-
logues (Figure 1). For comparison purposes, Bu5Man (compd
2, Figure 2) was an attractive “negative” control for several
reasons (note that other compounds shown in Figure 2 were
also used as controls for specific experiments). First, being
hexose-based, Bu5Man did not have the critical N-acyl group
at the C2 position required for critical anticancer activities, such
as induction of apoptosis14 or MUC1 suppression,15 found in
hexosamine-based analogues. Another important feature of
Bu5Man is that its core sugar mannose can be converted to

8138 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Campbell et al.
analogue concentrations up to 125 µM.14 Moreover, the genes
that were influenced by the analogues were skewed toward
up-regulation but, because these genes were not involved in
the activation of apoptosis, these results were not consistent
with the transcriptional programs expected during cell death.
The robust sialic acid production in Bu4ManNAc-treated cells
(Figure 4D) was yet another indication that the cells were
not dying (we have previously found that sialic acid
production dramatically declines at analogue concentrations
that lead to cell death4).
Reduced Invasion Cannot be Explained by n-Butyrate
Generated by Bu4ManNAc. Once toxicity was discounted as
playing a significant role in the reduced mobility of Bu4ManNAc-
treated cells, we next investigated the relative impacts of
respective hydrolysis products of this analogue, specifically,
n-butyrate and ManNAc. We had previously extensively
investigated n-butyrate in other cancer lines, both as the sodium
salt and ester-linked to sugars, and found that while the expected
HDACi activity was manifest in changes to histone acetylation,
ancillary chromatin remodeling did not provide a convincing
explanation for the divergent activities of Bu5Man and
Bu4ManNAc on apoptosis,14 MUC1,15 or on cell mobility (as
described in this paper for the MDA-MB-231 line). This point
is illustrated by the data shown in Figure 5 for MDA-MB-231
cells, where modest changes to histone H3 acetylation occurred
(panels A and B) that were reflected in the expected up-
regulation of cell cycle checkpoint protein p21 (panel C).
Despite the “hallmark” HDACi activity exhibited by both
n-butanoylated analogues, the overall transcriptional changes
elicited by Bu4ManNAc and Bu5Man differed dramatically. The
different effects of the analogues were evident in previous
microarray analysis where each analogue regulated a completely
different set of genes14 and in this study where MUC1 (e.g.,
see Figure 5D) was up-regulated by one analogue (i.e., by
Bu5Man) and down-regulated by the other (i.e., by
Bu4ManNAc). These divergent responses indicated that HDACi
activity played only a partial role in regulating genes involved
in cell mobility because, if n-butyrate generated from
Bu4ManNAc and Bu5Man hydrolysis played a substantial role,
each compound should have led to similar responses.
Figure 3. Effects of Bu4ManNAc and Bu5Man on cell mobility. (A)
Two day pretreatment with the minimally cytostatic concentration of
50 µM Bu4ManNAc did not have a statistically significant effect on
migration or invasion in MDA-MB-231 cells. Higher concentrations
(e.g., up to 150 µM (B)) or a longer incubation period (e.g., three days,
(C)), however, did result in a preferential decrease in invasion compared
to migration. (D) In similar experiments, cells treated with Bu5Man
did not experience a change in migration or invasion. Error bars indicate
SEM for technical replicates (n ) 6 for (A), (C), and (D) and n ) 3
for B) using a double-sided t test.
A concentration of ∼ 150 µM can activate apoptosis and lead
to cell death, but only on time scales approaching 2 weeks;14
therefore in the current experiments, a maximum incubation
period of 3 days was used. A third parameter, cell density, was
also carefully controlled to avoid low seeding densities (e.g.,
6.25 × 104 cells/ml4) associated with toxicity.
A series of assays verified that Bu4ManNAc had minimal
cytotoxicity at the concentrations (i.e., e 150 µM) used to
evaluate mobility and associated genes. In these experiments,
proliferation was slower in cells incubated with Bu4ManNAc
than with Bu5Man; however, an increase in cell number
during incubation with the analogue indicated that the cells
nonetheless retained viability and were actively growing
(Figure 4). The small proportion of the cells that were dying,
indicated with the “rounded up” morphology in Figure 4A
(and evaluated more thoroughly as previously described by
DNA fragmentation analysis,12 PI/annexin staining,18 and
flow cytometry histogram analysis of DNA content and cell
cycle status,14 data not shown), were excluded from further
analysis. As an additional safeguard to separate the effects
of the analogues on cell mobility from toxicity, the WST-8
assay was employed to normalize the number of viable cells
used for subsequent analysis (e.g., for loading into the
migration or invasion chambers); in addition, aliquots of cells
were kept separate from the invasion assays and reevaluated
at end of the assay by the WST-8 method to ensure viability
was maintained throughout the entire experiment. Further
evidence that nonspecific toxicity did not explain the results
presented in this paper came from microarray analysis
previously done under similar conditions where >98% of
the gene probe sets did not show changes in expression over
Responses to Bu4ManNAc are Not
a Result of
Combined n-Butyrate and ManNAc Activity. Having ruled
out substantial contributions from either n-butyrate or ManNAc
in the anti-invasive activities of Bu4ManNAc, the possibility
remained that the divergent transcriptional activities of this
compound compared to Bu5Man resulted from a combination
of HDACi responses and glycosylation effects derived from
n-butyrate and the sugar acting as separate molecules. This
possibility was difficult to rigorously discount because the
simplest definitive experiment, the simultaneous addition of the
appropriate equimolar quantities of n-butyrate and ManNAc to
the culture medium, was not conclusive due to the poor cellular
uptake of these compounds as individual molecules (as described
previously14). Therefore a different approach was taken in this
study based on the premise that, if sialic acid produced from
Bu4ManNAc contributed to the anticancer activity of this
compound, peracylated sialic acid analogues should be highly
efficient at eliciting these responses because they directly
produce sialic acid (and n-butyrate) upon ester hydrolysis.
Accordingly, perbutanoylated Neu5Ac (as the C1-methyl ester
Bu5Neu5AcOMe, 7) was synthesized and tested in comparison
with peracetylated Neu5Ac (as the C1-methyl ester
Ac5Neu5AcOMe, 6). The results of proliferation assays (shown
in Figure 6A,B) followed the patterns associated with “inactive”

Targeting Oncogenes with SCFA-Hexosamine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8139
Figure 4. Effects of Bu4ManNAc and Bu5Man on cell viability, growth inhibition, and sialic acid production in MDA-MB-231 cells. Phase contrast
micrographs of cells incubated with Bu4ManNAc (A) and Bu5Man (B) for 3 days show the growth inhibitory properties of these analogues and the
“rounded-up” morphology indicative of dying cells. (C) Quantitatively, Bu4ManNAc inhibited proliferation more effectively than Bu5Man; at
concentrations used in the subsequent experiments, however, the cells remained viable and continued to proliferate (cell counts were determined
by trypsinization of viable cells followed by Coulter counting analysis). (D) Sialic acid levels increased in Bu4ManNAc-treated MDA-MB-231
cells in a dose-dependent manner while Bu5Man did not change levels of this sugar. Error bars indicate standard error of the mean (SEM) and in
each case n g 3.
hybrid analogues such as Bu5Man,14 where only HDACi
behavior indicative of n-butyrate was manifest. Specifically,
Ac5Neu5AcOMe, which delivered the weakly acting SCFA
acetate, had no measurable effect on proliferation (Panel A),
whereas Bu5Neu5AcOMe, which delivered the stronger acting
SCFA n-butyrate, inhibited proliferation over 3-5 days, after
which time the cells recovered and resumed robust growth
(Panel B).
The impact of Bu5Neu5AcOMe on proliferation closely
mimicked that of Bu5Man (as previously reported14). In more
detailed analysis, this compound also strongly increased MUC1
mRNA levels (Figure 6C), a distinctive feature of Bu5Man that
was consistent with reports that n-butyrate can up-regulate mucin
transcription.19,20 Finally, cells treated with Bu5Neu5AcOMe
experienced moderate enhancement of NFKB1 (NF-κB p105)
and down-regulation of CXCR-4 (Figure 6C), which are two
additional distinguishing features of “inactive” analogues (the
significance of which is discussed in more detail later in this
report). These results established that sialic acid did not
synergistically combine with n-butyrate to modulate the pro-
invasive oncogenes under investigation. They also indicated that
anticancer responses held by Bu4ManNAc cannot be reproduced
when n-butyrate is appended to sialic acid, thereby strengthening
the unique ability of the hexosamine core structure to support
scaffold-dependent responses by eliminating another monosac-
charide (i.e., sialic acid in addition to hexoses and glycerol that
were previously discounted5,14) from having the critical SAR
required for this new mode of bioactivity.
In a further experiment that conclusively ruled out a major
role for hydrolysis products in mediating the anti-invasive effects
of Bu4ManNAc, the impact of 3,4,6-O-Bu3ManNAc, 1,3,4-O-
Bu3ManNAc, and 3,4,6-O-Bu3GlcNAc, (compounds 3, 4, and
5 in Figure 2) on MUC1 expression were compared (Figure
7A). These three analogues generate similar molar equivalents
of n-butyrate, but 3,4,6-O-Bu3ManNAc and 1,3,4-O-
Bu3ManNAc, two compounds that support similar levels of sialic

8140 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Campbell et al.
Figure 5. Bu4ManNAc and Bu5Man have characteristic HDACi activity. (A) Western blots showed that the acetylation of histone H3 increased
in MDA-MB-231 cells exposed to either Bu4ManNAc, Bu5Man, or to n-butryate for 3 days, as quantified in (B) by densitometry. (C) Measurement
of mRNA levels by qRT-PCR showed that p21 was up-regulated by both analogues. (D) Bu4ManNAc decreased MUC1 mRNA levels while
Bu5Man had the opposite effect on this gene. In (A-B), representative data from one of three independent experiments are shown. In (C), p21 data
consistent with previous testing14 are shown, while errors bars represent SEM and n g 3 for (D).
acid production (Figure 7B), had opposing effects on MUC1
transcription, thereby unambiguously establishing that a simple
combination of n-butyrate and ManNAc does not suppress this
oncogene. By contrast, 3,4,6-O-Bu3GlcNAc, a compound that
does not generate ManNAc or by extension sialic acid, down-
regulated MUC1 as effectively as 3,4,6-O-Bu3ManNAc; together
these results eliminated the possibility that the core sugar as a
separate molecule (i.e., hydrolysis product) tunes the HDACi
activity of n-butyrate in a manner that could account for the
impact of Bu4ManNAc on MUC1 or invasion.
Bu4ManNAc and Bu5Man Both Reduced MMP-9
Activity in Zymogram Assays. The molecular basis for the
reduced invasion observed in Bu4ManNAc treated cells was
investigated in more detail by using various assays (described
in more detail below) that also provided insight into the lack of
a functional response seen in Bu5Man-treated cells. First, the
activities of matrix metalloproteinases (MMPs), important
players in extracellular matrix degradation during metastasis,21
were tested at both the functional and gene expression levels.
In the initial experiments, MMP activity was monitored in
conditioned medium and the involvement of the majority of
MMP family members was ruled out by a lack of bands
observed on casein gels (data not shown). By contrast, by
monitoring gelatinase activity, which is only characteristic of
two MMPs (MMP-2 and MMP-9), a band was identified that
was ∼92 kDa (Figure 9A) that corresponded to the mass of
MMP-9 (MMP-2, which is ∼72 kDa, was not observed).
Interestingly, unlike for MUC1 where the analogues had
divergent effects, both Bu4ManNAc and Bu5Man reduced the
activity of MMP-9 harvested from conditioned medium (Figure
9A,B). Taken together, the results for MUC1 and MMP-9
provide a plausible explanation for the impact of these analogues
on invasion at the cell level. Specifically, Bu4ManNAc sup-
pressed both pro-invasive proteins, which was consistent with
the decreased invasion observed at the cell level. By contrast,
Bu5Man up-regulated MUC1 while down-regulating MMP-9
activity, making it plausible that the two responses offset each
other accounting for the unchanged cell mobility.
Having concluded that the distinctive transcriptional responses
to Bu4ManNAc and Bu5Man could not be explained by the
HDACi activity of n-butyrate, either alone or in combination
with the core sugar, the formal possibility remained that the
core sugar contributed to the divergent invasive behavior of
Bu4ManNAc and Bu5Man strictly through changes to the cell
surface glycosylation patterns. More specifically, the altered
mobility of Bu4ManNAc-treated cells could have been a
consequence of robust sialic acid production (Figure 4D) that
changed their adhesive properties. Consequently, to test whether
ManNAc contributed to the reduced mobility of Bu4ManNAc-
treated cells in the absence of the HDACi influence of
n-butyrate, cells were incubated with sufficient ManNAc (up
to 50 mM) needed to replicate the levels of sialic acid (Figure
8A) produced in cells treated with the n-butanoylated analogues.
Subsequent testing of ManNAc-treated cells in mobility assays
revealed that there was a negligible impact on migration (data
not shown), while there was a biphasic response in the invasion
assays with a spike in activity at ∼10 mM that diminished at
higher levels (Figure 8B). In light of this pro-invasive response
to ManNAc, the ability of Bu4ManNAc to inhibit cell mobility
in the face of similar levels of sialic acid production (as shown
in Figure 4) was all the more remarkable and highlighted the
need for broadening the scope of the investigation to fully
appreciate the anti-invasive activity of this compound.
MMP-9 Activity was Correlated with CXCR-4
Transcription Rather Than with Pro-MMP-9 mRNA
Levels. Because of enticing therapeutic possibilities inherent
in the ability of Bu4ManNAc and Bu5Man to reduce MMP-9
activity, we sought to understand the molecular basis of this
finding more thoroughly. One explanation was that the HDACi
activity of these analogues (see Figure 5) reduced pro-MMP-9
transcription (although HDACi usually activate gene expression,

Targeting Oncogenes with SCFA-Hexosamine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8141
Figure 7. SAR implicated analogues with intact SCFA-hexosamine
structures as prerequisite for “active” responses. (A) The C6-OH
ManNAc analogue 1,3,4-O-Bu3ManNAc failed to suppress MUC1
transcription unlike the two C6-SCFA derivatized analogues 3,4,6-O-
Bu3ManNAc and 3,4,6-O-Bu3GlcNAc. Representative data from one
of three independent experiments are shown; error based on the
coefficient of variance (CV) for cycle number of quadruplicate replicates
was e2% (error bars were obscured by the symbols and therefore
omitted from the graph). (B) Both 1,3,4-O-Bu3ManNAc and 3,4,6-O-
Bu3ManNAc supported robust sialic acid production at low concentra-
tions (production from 3,4,6-O-Bu3ManNAc was inhibited by the
toxicity of this analogue at g150 µM), establishing further that the
hydrolyzed core sugar was not responsible for MUC1 suppression. Error
bars represent SEM, n ) 3.
Figure 6. SCFA-sialic acid analogues follow the pattern set by
“inactive” hexosamine analogues. (A) MDA-MB-231 cells exposed to
Ac5Neu5AcOMe did not experience growth inhibition at any time point,
whereas (B) the proliferation of cells incubated with Bu5Neu5AcOMe
was inhibited over the first 3-5 days. The cells subsequently recovered
and resumed robust growth by the 15th day. (C) Transcription patterns
of MUC1, CXCR-4, and NFKB1 in Bu5Neu5AcOMe treated MDA-
MB-231 cells follow the pattern set by the “inactive” compound
Bu5Man. The data shown in (A) and (B) are averaged from triplicate
experiments and in (C) from two independent experiments, each
measured by qRT-PCR in quadruplicate. Error bars were omitted for
clarity, in all cases SEM was e5%.
they are also capable of down-regulating transcription22,23).
Measuring mRNA levels by qRT-PCR, however, revealed that
Bu4ManNAc had no impact on pro-MMP-9 transcription while
Bu5Man actually increased mRNA levels (Figure 9C). Thus,
needing to probe deeper to explain the reduced MMP-9 activity
observed in the zymograms, we tested the expression of CXCR4.
This protein, an established therapeutic target for HIV as well
as cancer,24 regulates the secretion and activation of pro-MMP-
9.25 qRT-PCR revealed that CXCR4 mRNA levels were down-
regulated by both Bu4ManNAc and Bu5Man (Figure 9D).
Because CXCR4 plays a critical role in the secretion and
activation of pro-MMP-9,26 its down-regulation by both ana-
logues provided a compelling mechanism to explain the reduced
MMP-9 activity observed in culture medium of cells incubated
with either compound.
Figure 8. The suppression of invasion by Bu4ManNAc cannot be
explained by sialic acid production. (A) Millimolar concentrations of
ManNAc increased intracellular sialic acid in MDA-MB-231 cells to
levels comparable to cells treated with e125 µM of Bu4ManNAc (see
Figure 4D or 1,3,4-O-Bu3ManNAc and 3,4,6-O-Bu3ManNAc, in Figure
7B). (B) ManNAc increased invasion in MDA-MB-231 cells incubated
with low millimolar concentrations (e.g., at 10 mM shown) of this sugar,
but this potentiation was lost at higher concentrations. Errors bars
represent SEM and n g 3 for each data set shown.
mented the MUC1 data and helped explain the anti-invasive
attributes of Bu4ManNAc and the lack of a cell level response
to Bu5Man. These findings, however, left the important issue
of how the analogues engaged the underling mechanisms that
Transcriptional Control of Genes Regulated by Bu4ManNAc
Involves NF-KB. The evaluation of MMP-9 and CXCR4,
important therapeutic targets implicated in invasion, comple-

8142 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Campbell et al.
Figure 10. Analysis of NF-κB in HEK AD293 and MDA-MB-231
cells treated with Bu4ManNAc, Bu5Man, and controls. (A) HEK AD293
cells that were transfected with reporter gene plasmids and then
incubated with either Bu4ManNAc or Bu5Man; Bu5Man had no effect
on NF-κB activity in the reporter gene assay, while Bu4ManNAc
provided substantial inhibition in this cell line. (B) Similar results were
observed for Bu4ManNAc and Bu5Man in the reporter gene assay in
electroporated MDA-MB-231 cells. Error bars for the transcription
factor reporter assays in (A) and (B) represent SEM for independent
experiments done in triplicate. (C) Incubation with either analogue for
3 days did not alter proteasome activity in MDA-MB-231 cells, whereas
a 6 h treatment with epoxomicin produced potent inhibition; SEM was
e4% for all data points (and error bars were not included because they
were generally smaller than the data symbols). (D) Endogenous NFKB1
(p105) mRNA levels were reduced by Bu4ManNAc but not by Bu5Man
in MDA-MB-231 cells; error calculated based on the coefficient of
variance (CV) for cycle number of quadruplicate replicates was e2%
(not shown).
Figure 9. Control of MMP-9 activity in MDA-MB-231 cells by
Bu4ManNAc and Bu5Man. (A) Zymographic analysis of medium
conditioned by MDA-MB-231 cells incubated with Bu4ManNAc and
Bu5Man for 3 days showed a dose-dependent decrease in MMP-9
gelatinase activity for both compounds (bands from a representative
experiment were quantified by densitometry and shown in (B). Similar
results were obtained in two additional independent experiments. qRT-
PCR analysis of pro-MMP-9 (C) and CXCR4 (D) mRNA levels in
cells exposed to Bu4ManNAc and Bu5Man for 3 days. Error bars
indicate SEM for independent experiments done in triplicate.
controlled gene expression largely unresolved. Certain genes
(e.g., p21 involved in cell cycle arrest and CXCR4 implicated
in the control of MMP-9 gelatinase activity) were regulated in
a manner consistent with HDACi activity of n-butyrate generated
from the hydrolysis of the parent compound. By contrast,
HDACi could not explain the impact of these compounds on
other genes, exemplified by MUC1, that were regulated in a
dramatically different fashion by each compound. Clearly
HDACi, or most likely any single regulatory mechanism by
itself, did not adequately explain the impact of Bu4ManNAc
on gene expression.
Accordingly, we considered alternative possibilities to account
for the discordant effects of these analogues on gene regulation
and the NF-κB family of transcription factors that mediate
inflammation in normal cells and, among other functions in
cancer, help orchestrate invasion garnered interest for two
reasons. First, depending on the type of malignancy, HDACi
can activate NF-κB and offset anticancer benefits;16 at a
minimum, we wanted to eliminate this possibility for our
analogues. Second, and of specific relevance to this study,
MUC1 contains an NF-κB binding domain in its promoter
region27,28 and conversely, MUC1 can modulate constitutive
NF-κB signaling found in cancer cells.27 This crosstalk led us
to speculate that the link between reduced invasion (Figure
3B,C) and the transcription of MUC1 (Figure 5D) in cells
incubated with Bu4ManNAc plausibly involved NF-κB.
assay, a luciferase reporter plasmid was cotransfected with a
second plasmid encoding ꢀ-galactosidase driven by an SV
promoter and relative differences in luciferase to ꢀ-galactosidase
signaled changes in NF-κB transcriptional activity. The MDA-
MB-231 line used throughout this study proved difficult to
transfect by standard lipofectamine protocols; consequently,
these assays were first performed in the human HEK AD293
line where transfection efficiencies of >90% could be obtained
routinely. In these cells, the NF-κB activator TPA and inhibitor
EPO had the expected effects (see Figure S1 in the Supporting
Information), thereby validating the assay in our hands; it is
noteworthy that a relatively high level of NF-κB transcriptional
activity was found in unstimulated cells, likely reflecting high
basal levels of NF-κB activity associated with the cancerous
phenotype.27 In subsequent analogue testing, Bu4ManNAc
decreased NF-κB transcriptional activity in a dose-dependent
manner, while Bu5Man had negligible effect (Figure 10A).
Having found that NF-κB activity in analogue-treated HEK
cells reflected the impact of Bu4ManNAc and Bu5Man on
invasion in MDA-MB-231 cells (i.e., Bu4ManNAc was inhibi-
tory and Bu5Man hade no effect), we next tested NF-κB activity
in the breast cancer line by using electroporation (which was
more effective than lipid-based transfection in MDA-MB-231
cells). The reporter assay showed similar results in the MDA-
MB-231 cells (Figure 10B) as obtained in the HEK cells (Figure
NF-κB activity in cells exposed to Bu4ManNAc or Bu5Man
was evaluated in two ways. First, a reporter gene assay was
used to measure transcriptional activity driven by an NF-κB
consensus sequence upstream of the luciferase gene. In this

Targeting Oncogenes with SCFA-Hexosamine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8143
10A) with the exception that Bu5Man showed a trend toward
being slightly activating (instead of having an negligible impact
on activity). In both lines, however, it was unambiguous that
Bu4ManNAc dramatically inhibited NF-κB in a dose-dependent
manner, resulting in a ∼75% reduction at 125 µM. It is worth
noting that because NF-κB activity was normalized to an internal
control (a cotransfected plasmid with the ꢀ-galactosidase gene
driven by an SV promoter), the observed decrease in pathway
activity was not an artifact of nonspecific suppression of
transcription or translation due to nonspecific cytotoxicity.
Bu4ManNAc Inhibits the Endogeneous NF-KB Pathway.
Having demonstrated that Bu4ManNAc inhibited NF-κB activity
in reporter gene assays, we next verified that this compound
had a similar impact on the endogenous NF-κB pathway.
Reduction of proteasome activity, a common but by no means
ubiquitous characteristic of small molecule NF-κb inhibitors,
was tested as a potential mechanism underlying the suppressive
effects of Bu4ManNAc on NF-κB. The proteasome inhibitor
epoxomicin (EPO), which blocks degradation of ubiquinated
IκB by sequestering NF-κB in the cytoplasm, was used as a
positive control in this assay; unlike this compound, however,
neither Bu4ManNAc nor Bu5Man altered the degradation of a
luminescent proteasomal substrate (Figure 10C). Reduced NF-
κB signaling in the context of normal proteasome function
suggested Bu4ManNAc inhibited this pathway by regulating the
transcription of NF-κB proteins themselves. Therefore, as a
second method to assess the impact of the analogues on NF-κB
activity, we used qRT-PCR to monitor p105 (NFKB1) mRNA
levels. This gene encodes a subunit of the NF-κB transcription
complex (cleavage of p105 yields p50, one of five subunits that
assemble to form NF-κB) and is itself an NF-κB target gene.29
Figure 11. SAR of tributanoylated hexosamine analogues extend to
NF-κB. The effects of three tributanoylated hexosamine derivatives
1,3,4-O-Bu3ManNAc (3), 3,4,6-O-Bu3ManNAc (4), and 3,4,6-O-
Bu3GlcNAc (5) on NF-κB activity was measured by using the luciferase
reporter assay in HEK AD293 cells exposed to each of the analogues.
Error bars indicate SEM of independent experiments measured in
triplicate.
no inhibition of NF-κB (slight activation, in fact) while based
on a ManNAc core identical to 3 emphasized that the SAR of
the intact pharmacophore dominated any latent effects emanating
from the hydrolysis products. Another significant ramification
of these SAR is that simultaneous suppression of MUC1 and
NF-κB can be obtained with the GlcNAc scaffold, thereby
avoiding the potentially deleterious impact of sialic acid derived
from a ManNAc core on cancer progression. In other cases,
such as in the use of non-natural ManNAc analogues (i.e., with
R1 in Figure 1 not a methyl group) to install immunogenic sialic
acids in tumor-associated carbohydrate antigens for cancer
vaccine development,31,32 a ManNAc core may ultimately be
advantageous for cancer therapy. Either way, the results
described in this paper lay the foundation for a highly versatile
platform that exploits the emergence of carbohydrates as
scaffolds for drug discovery.33
Consistent with the reporter plasmid assays, Bu4ManNAc
suppressed NFKB1 transcription while Bu5Man did not signifi-
cantly alter its expression (Figure 10D), indicating that this
analogue directly altered, or interacted with, NF-κB proteins
rather than affecting this signaling pathway by a peripheral
mechanism such as proteasome activity. It is noteworthy that
current NF-κB inhibitors, while often proficient at knocking
down transiently elevated levels of NF-κB resulting from
pathway activating factors such TNFR, fail to reduce the
elevated basal NF-κB activity characteristic of certain types of
cancer. In one study, even small inhibitory RNAs targeted at
NF-κB proteins did not substantially inhibit the elevated basal
activity of NF-κB in MDA-MD-231 cells;30 consequently, the
discovery that Bu4ManNAc inhibited endogenous NF-κB rep-
resents a potentially important therapeutic advance.
Conclusions
Revisiting the therapeutic prospects of SCFA-monosaccharide
hybrid molecules was spurred by the discovery of therapeutically
relevant activities not derived from, as had previously been
presumed, the hydrolysis products of these compounds but
instead from structural features of the intact prodrug (see Figure
1 and references15,17). In particular, three specific factors
provided renewed interest in this class of drug candidates. First,
there is a growing appreciation that therapeutic compounds that
contain ester linkages, long exemplified by aspirin, are not
exclusively prodrugs; instead, the parent molecule can have
important biologic activity.17 Second, recent SAR have revealed
critical differences between the SCFA-carbohydrates previously
evaluated in clinical trials with only moderate success and the
current hexosamine-based compounds. Specifically, an N-acyl
group at the C2 position of a 6-carbon sugar is critical for
anticancer activity, and it is this C2 N-acyl group, found in
hexosamines but not in carbohydrates previously used for
n-butyrate delivery such as glycerol, glucose, or mannose, that
distinguishes the current report from earlier efforts. Finally, the
identification of important therapeutic targets involved in
invasion, including MUC1, MMP-9, CXCR4, and NF-κB,
suppressed by the lead compound Bu4ManNAc, provides a new
direction for drug discovery by expanding efforts previously
directed at killing cancer cells to now also reducing their
metastatic potential.
Anticancer SAR of SCFA-hexosamine Analogues
Extend to NF-KB. The close correlation between reduced
invasion and NF-κB in Bu4ManNAc-treated cells led us to
investigate whether the anticancer SAR previously observed for
apoptosis14 or MUC115 extended to NF-κB. Specifically, we
tested whether NF-κB was governed by the same controlling
mechanism as the other attractive anticancer attributes of
Bu4ManNAc (i.e., MUC1 suppression and apoptosis), where
both an N-acyl group at the C2 position and a SCFA substituent
at the C6 position were critical for activity (see Figures 11 and
12), by comparing 3,4,6-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAc,
and 3,4,6-O-Bu3GlcNAc, (compds 3, 4, and 5 in Figure 2). This
experiment verified the close link between the SAR responsible
for suppression of MUC1 and reduction of NF-κB activity as
the two C1-OH analogues (3 and 5) once again showed almost
identical inhibitory responses (Figure 11) despite having core
sugars that, as hydrolysis products, engaged different glycosy-
lation pathways. The fact that the C6-OH analogue 4 showed
The first important experimental result described in this paper,
verification that the lead compound Bu4ManNAc inhibited the

8144 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Campbell et al.
Figure 12. Summary of the anti-invasive effects of MDA-MB-231 cells to Bu4ManNAc and the muted response to Bu5Man. (A) Structure-activity
relationships (SAR) are summarized that are required for a unified molecular level response that reduces invasion through suppression of MUC1
and MMP-9 activity ((B), and dark arrows in (E) and (F)). By contrast, the SAR shown in (C) (bottom) resulted in offsetting molecular responses
((D), and gray arrows in (E) and (F)) that did not change the invasive behavior of cells. (E) The ability of Bu4ManNAc to inhibit invasion was a
consequence of coordinated down-regulation of MUC1 and (F) MMP-9 activity; in turn, MMP-9 activity was the aggregate result of the dominance
of CXCR-4 transcription over pro-MMP-9 mRNA expression. (G) A working hypothesis is that influence of Bu4ManNAc and Bu5Man on invasion
ultimately is a matter of balance between the differing impacts of these analogues on HDAC and NF-κB.
invasive behavior of metastatic breast cancer cells, established
a new therapeutic end point, control of pro-invasive oncogenes,
for SCFA-hexosamines. In addition, data presented in this paper
strongly confirmed the hypothesis that the unique biological
activity of this class of molecules is derived from intact ester-
linkages found in the parent molecules instead of merely
resulting from the hydrolysis products of the prodrug. Impor-
tantly for the control of pro-invasive oncogenes, this third mode
of scaffold-dependent activity dominated the potentially pro-
invasive attributes of the hydrolysis products n-butyrate and
ManNAc as well as illuminated shortcomings in previous
strategies that used broadly comparable molecules (i.e., SCFA-
carbohydrates in general) in cancer therapy: namely that the
wrong core sugars were utilized as delivery vehicles. Overall,
combined with the specific biological attributes discussed below
and the growing interest in carbohydate-based drug candidates,33
this work established SCFA-hexosamines as an attractive class
of molecules for the continuing development of antimetastatic
agents.
CXCR4) and NF-κB. Significantly, neither of two important
molecular players that regulate invasive behavior, MUC1 and
MMP-9, could by themselves explain the cell-level effects of
Bu4ManNAc and Bu5Man on mobility. Instead, the active
compound Bu4ManNAc evoked a unified molecular-level
response that led to reduced invasion whereas the “inactive”
analogue Bu5Man elicited counteracting molecular level re-
sponses that resulted in no change in cellular behavior (as
outlined in Figure 12).
The finding that Bu4ManNAc did not trigger the counterpro-
ductive relationship between HDACi and NF-κB found in some
cancer drug candidates invokes the principle described by
Minucci and Pelicci34 that epigenetic factors maintain a “matter
of balance” between genes that simultaneously promote malig-
nancy and those that suppress cancer progression. When this
balance is thrown out of kilter, as manifest in the highly invasive
phenotype of metastatic MDA-MB-231 cells, it can be extremely
difficult to remedy this aberrant behavior with a single thera-
peutic agent. This point is illustrated by unchanged mobility of
cells treated with Bu5Man, or other “inactive” analogues, that
modulate chromatin structure via the HDACi activity of their
n-butyrate groups. In essence, cancers cells exhibit an altered,
but multifaceted, equilibrium that is difficult to get “back in
balance” and was a feat that could only be achieved through
Although the impetus for this study originated in the ability
of Bu4ManNAc to suppress MUC1, we found that this com-
pound’s capacity to influence invasive behavior at the cell level
was critically dependent on suppression of additional therapeutic
targets including MMP-9 (via indirect regulation through

Targeting Oncogenes with SCFA-Hexosamine Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8145
mM CuSO4, 44% concentrated HCl, 36% dd H20), and heated
at 100 °C for 15 min. After cooling, 500 µL of tert-butyl alcohol
was added and optical density readings at 630 nm were promptly
recorded (the signal fades by ∼25% over the course of an hour;
typically readings were completed within five minutes, a time
frame where no measurable changes occur). The cellular content
of sialic acid, typically expressed as the number of molecules
of sialic acid per cell, was calculated based on the number of
cells harvested before lysis and on a calibration curve derived
from standards of sialic acid (Pfanstiehl, Waukegan, IL)
simultaneously assayed alongside the samples derived from the
cells.
Analysis of Histone Acetylation by Immunoblotting. MDA-
MB-231 cells (5.0 × 105) were plated into 100 mm dishes
containing complete medium and allowed to form monolayers
for 24 h prior to treatment with the specified compounds. After
incubation for two days with analogue or sodium n-butyrate,
cells were detached by scraping and were collected together with
floating cells and medium, pelleted by centrifugation, and washed
twice with PBS. Histones were extracted from nuclei following
a published procedure.39 Following the determination of protein
concentration by the micro BCA assay (Pierce, Rockford, IL),
histone extracts (4.2 µg protein/well) were separated by SDS-
PAGE using 18% polyacrylamide gel electrophoresis (Bio-Rad,
Hercules, CA). Samples then were blotted onto nitrocellulose
and immunostained with antibodies against histone H3 (total)
and acetyl-histone H3 (Upstate, Lake Placid, NY). Densitometry
was performed using NIH ImageJ software.
Analysis of Gene Expression. mRNA for gene expression
analysis was extracted from cells using Trizol (Invitrogen,
Carlsbad, CA), treated with DNase (Ambion, Austin, TX) to
remove contaminating genomic DNA, and purified using RNeasy
spin columns (Qiagen, Valencia, CA). RNA quality was assessed
with UV spectroscopy and agarose gel electrophoresis. For
quantitative real-time PCR (qRT-PCR), cDNA was obtained
using the SuperScript III First Stand synthesis kit (Invitrogen)
and amplified in an ABI 7700 thermocycler using a SYBR green
master mix (Applied Biosystems, Foster City, CA). Primer
sequences are given in Table S2 in the Supporting Information.
All Ct values were measured in quadruplicate (typically the SEM
was smaller than the symbols used in the figures and has not
been shown). Relative gene expression was calculated using
average Ct values according to the 2-∆∆Ct method.40,41 Correct
amplification was verified by agarose gel electrophoresis of the
PCR products.
Zymogram Analysis of MMP Activity. To measure the activity
of secreted matrix metalloproteinases (MMPs), MDA-MB-231
cells were stimulated with 100 ng/mL TPA (Sigma, St. Louis,
MO), incubated for three days, washed twice with serum-free
medium to remove serum proteases, and incubated with serum-
free medium for an additional day. The culture medium was
harvested, and the volumes of conditioned media were adjusted
according to cell number before measuring MMP activity using
SDS-PAGE gels containing either gelatin or casein (Biorad,
Hercules, CA). Following electrophoresis to separate the MMPs
according to molecular weight, the zymograms were incubated
in renaturation and development buffers according to the
manufacturer’s protocol (Biorad, Hercules, CA). Counterstaining
with Coomassie Blue identified protease activity as regions of
degraded substrate (either gelatin or casein). Densitometry was
performed using the NIH ImageJ software.
the combined HDACi activity and NF-κB suppression held by
active analogues such as Bu4ManNAc. Ultimately, the emerging
strategy of coupling HDACi activity with NF-κB inhibition in
a single molecule, described in this paper for Bu4ManNAc (and
other analogues with the appropriate SAR shown in Figure 12A),
promises to be an important step in intensifying efforts to
develop epigenetic therapies for invasive breast cancer.
Experimental Procedures
Synthesis of SCFA-monosaccharide Hybrid Molecules. The
structures of the n-butyrate monosaccharide hybrid molecules
used in this study are shown in Figure 2. These compounds, 2-
acetamido-2-deoxy-1,3,4,6-tetra-O-butanoyl-R,ꢀ-D-mannopyra-
nose (Bu4ManNAc, 112), 1,2,3,4,6-penta-O-butanoyl-R,ꢀ-D-man-
nopyranose (Bu5Man, 214), 2-acetamido-3,4,6-tri-O-butanoyl-2-
deoxy-R,ꢀ-D-mannopyranose (3,4,6-O-Bu3ManNAc, 315), 2-
acetamido-1,3,4-tri-O-butanoyl-2-deoxy-R,ꢀ-D-mannopyranose (1,3,4-
O-Bu3ManNAc, 415), 2-acetamido-3,4,6-tri-O-butanoyl-2-deoxy-
R,ꢀ-D-glucopyranose (3,4,6-O-Bu3GlcNAc, 515), and 5-actetamido-
2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-1-methyl ester-D-glycero-D-
galacto-2-nonulopyranosonic acid (Ac5Neu5AcOMe, 635) were
synthesized, purified, and characterized as described in the refer-
ences provided. The previously unreported compound 5-actetamido-
2,4,7,8,9-penta-O-butanoyl-3,5-dideoxy-1-methyl ester-D-glycero-
D-galacto-2-nonulopyranosonic acid (Bu5Neu5AcOMe, 7) was
synthesized and characterized as described in the Supporting
Information. For long-term storage, compounds were maintained
at -20 °C after lyophilization and stock solutions used for cell
culture assays were made periodically as needed by dissolving the
analogues in ethyl alcohol (EtOH) and storing at 4 °C; in both cases,
compounds were stable over several months of storage.
Cell Culture. Cell lines were obtained from the ATCC (Ma-
nassas, VA) and stored in aliquots in liquid nitrogen. MDA-MB-
231 cells were cultured in RPMI-1640 supplemented with 10% fetal
bovine serum (FBS). HEK AD293 cells were maintained in DMEM
supplemented with 10% FBS. Unless noted otherwise, cells were
plated at a density of 27000 cells/cm2 in 6-well plates for analogue
testing and, at the time of plating, stock solutions of analogues in
EtOH, or n-butyrate in PBS, were added to cell culture medium
such that the final EtOH concentration was 0.4% (v/v) in all samples
(control experiments established that this level of ethanol had no
effect on the biological parameters tested in this report). Cells were
cultured at 37 °C in a water saturated, 5.0% CO2 incubator.
In Vitro Invasion and Migration Assays. Modified Boyden
chambers (BD Biosciences, San Jose, CA) were used to measure
the migration and invasion of MDA-MB-231 cells as described by
Albini and co-workers.36 Briefly, migration chambers tested chemo-
taxis through 8.0 µm pores in response to a serum gradient. Invasion
chambers contained a layer of Matrigel on top of the porous
membrane. After pretreatment with analogue for two or three days,
cells were trypsinized, resuspended in serum-free RPMI-1640
medium, counted, tested for viability, and seeded at a density of
50000 viable cells per chamber (equal seeding of viable cells was
confirmed using the WST-8 assay (Dojindo, Gaithersburg, MD).
Following a 24 h incubation period at 37 °C, migrating or invading
cells were stained with Diff-Quik (Andwin Scientific, Addison, IL)
and counted using MetaMorph software (Molecular Devices
Corporation, Downingtown, PA); aliquots of cells were also
reevaluated at this 24 h time point by the WST assay to ensure
continued viability.
Reporter Gene Assays for Measurement of NF-KB. Trans-
activation activity of NF-κB was assessed with luciferase reporter
plasmids (Stratagene, La Jolla, CA). To normalize for transfec-
tion efficiency, reporter plasmids were cotransfected with an
equal mass of plasmid encoding ꢀ-galactosidase under the control
of a SV promoter (Promega, Madison, WI). MDA-MB-231 and
HEK 293 cells were transfected using a BTX electroporator and
Lipofectamine 2000 (Invitrogen, Carlsbad, CA), respectively,
according to the manufacturer’s directions. Following a 24 h
recovery period after transfection, the cells were replated and
Determination of the Total Sialic Acid Content of Cells. A
modification of the periodate-resorcinol assay originally de-
scribed by Jourdian and co-workers,37 adapted for smaller
volumes,4,38 was used to measure sialic acid levels in analogue-
treated cells. Briefly, trypsinized cells were washed twice and
resuspended in PBS, counted using a Coulter model Z2 cell
counter, and lysed by three freeze-thaw cycles. Lysates (300
µL) were oxidized by the addition of 5.0 µL of 0.4 M periodic
acid, cooled on ice for 10 min, reacted with 500 µL of freshly
prepared resorcinol mixture (10% 0.5 M resorcinol, 10% 2.5

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of luciferase and ꢀ-galactosidase were measured via chemilu-
minescent and colorimetric assays (Promega, Madison, WI),
respectively. Normalized transactivation activity of NF-κB was
calculated from ratios of luciferase and ꢀ-galactosidase activities.
Proteasome Activity. Proteasome activity was assayed using
a succinyl luminogenic proteasome substrate (Promega, Madison,
WI). Viable MDA-MB-231 cells (100000 cells in 100 µL of
RPMI) were incubated with the luminogenic substrate according
to the manufacturer’s instructions. Proteasome activity was
calculated from luminescent activity normalized to that of
untreated controls. The proteasome inhibitor epoxomicin (Sigma,
St. Louis, MO) provided a positive control.
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Acknowledgment. Funding was provided by the National
Institutes of Health (CA112314-01A1 for ManNAc analogue
synthesis and analysis of pro-invasive oncogenes and
AR054005-01 for GlcNAc analogue synthesis) and by the
Susan G. Komen Foundation (BCTR0503768) for the inva-
sion assays.
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Supporting Information Available: Synthesis and character-
ization of analogues; supporting information for cell-based
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
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