A direct comparison of the anticancer activities of digitoxin MeON-Neoglycosides and O-Glycosides

Article abstract of DOI:10.1021/ml1000933

Digitoxin is a cardiac glycoside currently being investigated for potential use in oncology; however, an investigation of anticancer activity as a function of oligosaccharide chain length has not yet been performed. We generated mono-, di-, and tri-O-digitoxoside derivatives of digitoxin and compared their activities to the corresponding MeON-neoglycosides. Both classes of cardenolide derivatives display comparable oligosaccharide chain length-dependent cytotoxicity toward human cancer cell lines. Further investigation revealed that both classes of compounds induce caspase-9-mediated apoptosis in non-small cell lung cancer cells (NCI-H460). Because O-glycosides and MeON-neoglycosides share a similar mode of action, the convenience of MeON-neoglycosylation could be exploited in future SAR work to rapidly survey large numbers of carbohydrates to prioritize selected O-glycoside candidates for traditional synthesis.

Full text of DOI:10.1021/ml1000933

pubs.acs.org/acsmedchemlett
A Direct Comparison of the Anticancer Activities
of Digitoxin MeON-Neoglycosides and O-Glycosides
Anand Krishnan V. Iyer,† Maoquan Zhou,‡ Neelam Azad,† Hosam Elbaz,§ Leo Wang,‡
Derek K. Rogalsky, Yon Rojanasakul,*,§ George A. O'Doherty,*,‡ and
Joseph M. Langenhan*,
†Department of Pharmaceutical Sciences, Hampton University, Hampton, Virginia 23668, ‡Department of Chemistry, and
§Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia, 26506, and
Department of Chemistry, Seattle University, Seattle, Washington 98122
ABSTRACT Digitoxin is a cardiac glycoside currently being investigated for
potential use in oncology; however, an investigation of anticancer activity as a
function of oligosaccharide chain length has not yet been performed. We gene-
rated mono-, di-, and tri-O-digitoxoside derivatives of digitoxin and compared their
activities to the corresponding MeON-neoglycosides. Both classes of cardenolide
derivatives display comparable oligosaccharide chain length-dependent cyto-
toxicity toward human cancer cell lines. Further investigation revealed that
both classes of compounds induce caspase-9-mediated apoptosis in non-small
cell lung cancer cells (NCI-H460). Because O-glycosides and MeON-neoglyco-
sides share a similar mode of action, the convenience of MeON-neoglycosylation
could be exploited in future SAR work to rapidly survey large numbers
of carbohydrates to prioritize selected O-glycoside candidates for traditional synthesis.
KEYWORDS Apoptosis, caspase, digitoxin, cancer, cardiac glycoside
igitoxin (Figure 1, 3), a naturally occurring cardiac
glycoside isolated from digitalis, has been used to
treat congestive heart failure and cardiac arrhythmias
Naþ/Kþ-ATPase inhibitors than the corresponding aglycons.14,17
Because Naþ/Kþ-ATPase represents a promising anticancer
target, groups have begun to determine how cardiac glyco-
side carbohydrates influence anticancer properties.5,14,17,18
The difficulties inherent to chemical glycosylation have
limited the progress made at elucidating the relationship bet-
ween carbohydrate structure and anticancer activity in natural
digitoxin O-glycosides. MeON-neoglycosylation, a chemo-
selective glycosylation methodology that employs unpro-
tected, unactivated reducing sugars,19 has enabled the rapid
glycorandomization of digitoxin analogues to survey the
influence of differential glycosylation.14 Subtle sugar modi-
fications dramatically influenced the cytotoxicities of digi-
toxin MeON-neoglycosides, and several analogues displayed
enhanced potency and tumor selectivity relative to the parent
natural product.14 A combination of NMR experiments, ab
initio calculations, and crystallographic studies have revealed
that non-natural MeON-neoglycosides display similar confor-
mational properties to their natural O-glycoside counterparts.14,20
It remains unclear, however, whether or not MeON-neogly-
cosides and O-glycosides display comparable biological
activities toward human cancer cells. Also, while a number
of structure-activity relationship (SAR) studies of digitoxin
have been conducted, to our knowledge, anticancer activity
D
since the late 1700s. The canonically recognized target of
digitoxin is Naþ/Kþ-ATPase, a heterodimeric membrane
protein responsible for maintaining the Naþ/Kþ balance
across the plasma membranes of most eukaryotic cells.1
Partial Naþ/Kþ-ATPase inhibition leads to an increase in
intracellular Ca2þ concentration within myocardial cells,
resulting in an increase in heart contractility. More recently,
digitoxin has been shown to display significant anticancer
effects, and some researchers have begun to investigate its
potential use in oncology.2-4 Digitoxin inhibits cancer cell
growth in vitro,5-6 and for many cell lines, it does so at
concentrations commonly observed in the plasma of cardiac
patients.5 Cardiac glycosides have been shown to reduce
metastasis by inducing anoikis in tumor cell lines7 and to
induce apoptosis in a number of malignant cell lines, often at
nontoxic doses.6,8 Digitoxin's anticancer effects may in part
be related to the drug's cardiotonic activity (i.e., by increasing
Ca2þ concentrations).Forexample, sustainedcardiacglycoside-
induced increases in Ca2þ can trigger apoptosis in cancer
cells through caspase activation.9 However, cardiac glyco-
sides can also activate apoptosis pathways before ion cur-
rents are detected,10 consistent with other studies that
indicate that cardenolide-mediated signaling and cardio-
tonic activity are independent.10-16
Received Date: April 30, 2010
Accepted Date: June 9, 2010
Published on Web Date: July 12, 2010
The carbohydrate moiety of digitoxin is critical to Naþ/Kþ-
ATPase inhibition; cardiac glycosides are invariably better
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Scheme 1. Synthesis of Digitoxin Mono-, Di-, and Tri-O-digitoxo-
sides 1-3a
Figure 1. Digitoxin mono-, di-, and tri-O-digitoxosides 1-3 and
mono-, di-, and tri-MeON-digitoxosides 4-6.
as a function of oligosaccharide chain length has not been
investigated.
Thus, we initiated a study to compare directly the biological
effect of O-glycosidic versus MeON-neoglycosidic linkages
in mono-, di-, and tridigitoxoside analogues of digitoxin
(Figure 1). Our results suggest that digitoxin O-glycosides
and MeON-neoglycosides may share a common mechanism
of action in human ovarian adenocarcinoma cells (SK-OV-3),
human non-small cell lung carcinoma cells (NCI-H460), and
human intestinal epithelial cancer cells (HT-29). While MeON-
neoglycosides are less active, both classes of compounds
induce apoptosis in NCI-H460 cells via the caspase-9-mediated
intrinsic pathway, and for both classes of compounds, pro-
apoptotic activity is inversely correlated with oligosaccharide
chain length. On the basis of these findings, future carbohydrate
SAR work might be expedited by conducting rapid MeON-
neoglycosylation-based screens of carbohydrate structures
to provide a means for identifying corresponding O-glyco-
side structures worthy of synthesis and further investigation
as anticancer agents.
Previously, we have demonstrated the utility of our “de novo”
carbohydrate synthetic methodology for the synthesis of
natural and unnatural oligosaccharides.21-24 We synthesized
digitoxin (3) and its mono- (1) and didigitoxoside (2) ana-
logues as shown in Scheme 1.25-28 Briefly, palladium-catalyzed
glycosylation of pyranone 7 with benzyl alcohol or digitox-
igenin provided the pyranone 8 or 9, which then underwent
Luche reduction, reductive rearrangement of allylic alcohols,
and dihydroxylation to provide exclusively the diol 10 or
digitoxin monosaccharide 1. A regioselective protection of
diols 10 or 1 was achieved by the formation of an orthoester
and regioselective ring opening to give alcohol 11 or 12,
respectively. By applying the same reaction sequence above
to alcohol 11 or 12, the disaccharide 13 or 14 was prepared.
The regioselective protectionofdiols 13 or14gave the alcohol
15 or 16, which then underwent palladium-catalyzed glyco-
sylation, Luche reduction, 1,3-allylic rearrangement, dihy-
droxylation, and acetate hydrolysis to provide the digitoxin
trisaccharide 17 or 3. The digitoxin disaccharide 18 or 2 was
prepared by basic hydrolysis of the disaccharide 13 or 14.
To generate digitoxin MeON-neoglycosides, we synthesized
free, reducing digitoxose mono-, di-, and tridigitoxosides
a NBSH, o-nitrobenzenesulfonylhydrazide; DEAD, diethyl azodicarboxy-
late; and NMO, N-methylmorpholine-N-oxide.
(19, 20, and 21, respectively, Scheme 2) as well as secondary
oxyamine 22.14 Secondary oxyamines can be chemoselec-
tively glycosylated to provide hydrolytically stable glycosides
without sugar activation or protection.19 While our initial
attempts to generate 4-6 using published conditions14
failed, in the presence of AcOH and MeOH, aglycon 22 pro-
vided the corresponding MeON-neoglycosides 4-6 in mode-
rate yields when treated with 19-21. MeON-neoglycosides
4-6 exist predominantly in the β-pyranose form in solution.29
Because to our knowledge an investigation of anticancer
activity as a function of oligosaccharide chain length has not
yet been performed in digitoxin O-glycosides, we began by
assessing the growth inhibitory effects of 1-3 in the National
Cancer Institute's (NCI) panel of 60 human cancer cell lines
(Figure 2). GI50 values ranged from 2 to 223 nM; such a range
in susceptibilities is not unexpected since cancer cell lines
express different levels of Naþ/Kþ-ATPase isozymes.30,31 In
most cases, for a given cell line, mono-O-digitoxoside 1 was a
more potent inhibitor of tumor cell growth by a factor of 2-7
than the corresponding di- or tri-O-digitoxosides (2 and 3).
Interestingly, these findings are consistent with a compara-
tive molecular similarity index analysis for Naþ/Kþ-ATPase
inhibition, which predicted that for digitoxin the digitoxose
residue closest to the aglycon (the R-sugar) was essential for
Naþ/Kþ-ATPase inhibition, whereas further sugar substitution
was detrimental.17 These findings are also consistent with
the fact that Thorson and co-workers have identified a num-
ber of digitoxin MeON-neoglycosidic monodigitoxoside deri-
vatives that display improved cytotoxic properties relative to
the parent trisaccharide natural product.14 Since MeON-
neoglycosides display similar conformational properties to
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Figure 2. Summary of GI50 data from growth inhibition assays. Reciprocal GI50 values are displayed for clarity.
Scheme 2. Synthesis of Digitoxin Mono-, Di-, and Tri-MeON-
digitoxosides 1-3
that O-glycosides and MeON-neoglycosides share a similar
mechanism of action.
While the specific mechanism for cardenolide-induced
cytotoxicity remains controversial, a growing body of litera-
ture demonstrates that digitoxin can induce apoptosis in
malignant cell lines. However, few studies have been per-
formed in non-small cell lung cancer cells, despite the fact
that lung cancers have among the lowest 5 year survival
rates. Moreover, the ability of digitoxin MeON-neoglycoside
derivatives to induce apoptosis has not been assessed in
any cancer cell line. Thus, to investigate further the possibi-
lity of a shared mechanism of action, we measured the
ability of O-digitoxosides 1-3 and MeON-digitoxosides
4-6 to induce apoptosis in NCI-H460 (non-small cell lung
cancer) cells.
Both classes of compounds induced apoptosis in NCI-H460
cells (Figure 4). This is the first report of MeON-neoglycoside-
induced apoptosis. Consistent with our cytotoxicity results
(Figure 3), we observed that for both compound classes the
monodigitoxosides were more potent inducers of apoptosis
than the corresponding di- or tri-digitoxosides. We also obser-
ved a higher percentage of apoptotic cells with O-glycosides
1-3 than with MeON-neoglycosides 4-6, again mirroring
the trend observed in cytotoxicity assays. Because apoptosis
is mediated primarily through the action of the caspase
family of proteins,32 we assessed whether inhibiting these
pathways using pharmacological inhibitors would protect
NCI-H460 cells from undergoing apoptosis. We used the pan-
caspase inhibitor ZVAD-FMK to inhibit both intrinsic and
extrinsic pathways of apoptosis. Indeed, preincubation with
ZVAD-FMK completely abrogated apoptosis induced by 1-6
even at the highest dose tested (Figure 4, 100 nM þ ZV).
These results reveal that for both classes of compounds the
mode of drug action is dependent upon caspases.
their O-glycoside counterparts,14,20 we became interested in
learning whether the activity of MeON-neoglycosides 4-6
would mirror the oligosaccharide chain-length dependent
activity of O-glycosides 1-3.
First, we compared the activity of O-glycosides 1-3 and
corresponding MeON-neoglycosides 4-6 in cytotoxicity as-
says on a small subset of human cancer cell lines from the
NCI panel (Figure 3). Consistent with the growth inhibition
assay results shown in Figure 2, mono-O-digitoxoside 1 was at
least as potent a cytotoxin as the di- or tri-O-digitoxosides (2
and 3) in all cell lines. Interestingly, the corresponding neogly-
cosides 4-6 displayed a qualitatively similar potency trend
(mono- > or ≈ di- and tridigitoxoside) for three of the four cell
lines examined (SK-OV-3, NCI-H460, and HT-29). Despite the
fact that neoglycosides 4-6 were less potent than the corre-
sponding O-glycosides 1-3, the commonalities in oligo-
saccharide chain length-dependent potency trends suggest
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Figure 5. O-Digitoxoside 1 and MeON-digitoxoside 4 activate cas-
pase-9, not caspase-8, in NCI-H460 non-small cell lung cancer cells.
Cell lysates were incubated in fluorometric substrates for either
caspase-8 (IETD-AFC) or caspase-9 (LEHD-AFC); samples were then
read using a fluorescence plate reader. Ntx = no treatment. ZV =
pan-caspase inhibitor ZVAD-FMK.
caspase-9, with very little change in caspase-8 activity. These
results clearly implicate the intrinsic pathway of apoptosis in
the mechanism of action for these compounds. However, the
specific mechanism by which 1 and 4 activate the intrinsic
pathway of apoptosis remains to be elucidated. It is possible
that other proteins, such as Bcl-2, may be involved.34-35
In summary, we have investigated for the first time the anti-
cancer activity of digitoxin derivatives as a function of oligosac-
charide chain length; we have also directly compared the bio-
logical activities of O-glycoside and MeON-neoglycoside drugs.
O-Digitoxosides 1-3 and MeON-digitoxosides 4-6 both
display comparable oligosaccharide chain length-dependent
cytotoxicity toward human cancer cells. Further investiga-
tion revealed that both classes of compounds induce apop-
tosis in NCI-H460 cells; for both classes, apoptosis is driven
primarily through the caspase-9-mediated intrinsic pathway
ofcell death. This marks the first study where a MeON-neoglyco-
side has been observed to induce apoptosis. Our results pave
the way for the development of novel digitoxin analogues with
greater antitumor potential than naturally occurring derivatives;
more broadly, our results suggest a general strategy for carbo-
hydrate SAR work. Although MeON-digitoxosides 4-6 were
less potent than the corresponding O-digitoxosides in our
study, MeON-neoglycosides can be made extremely effici-
ently.14 Given the structural similarities between O-glyco-
sides and MeON-neoglycosides,14,20 as well as the parallel
biological activities indicated above, the convenience of
MeON-neoglycosylation could be exploited to rapidly sur-
vey large numbers of carbohydrates to prioritize selected
O-glycoside candidates for traditional synthesis.
Figure 3. Summary of IC50 data from cytotoxicity assays. Reci-
procal IC50 values are displayed for clarity; standard errors are
depicted with error bars.
Figure 4. O-Digitoxosides 1-3 and MeON-digitoxosides 4-6 in-
duce apoptosis in NCI-H460 non-small cell lung cancer cells.
Apoptotic cells were quantified by Hoechst dye staining and
microscopy. ZV = pan-caspase inhibitor ZVAD-FMK.
In the extrinsic pathway of apoptosis, the activation of pro-
apoptotic receptors on the cell surface leads to caspase-8
activation and cell death. In the intrinsic pathway of apop-
tosis, intracellular signals lead to the release of pro-apoptotic
proteins that activate caspase-9, eventually causing apoptosis.33
Because both O-digitoxosides 1-3 and MeON-digitoxosides
4-6 induced apoptotic cell death, we investigated the role of
specific caspases involved in the process. Using species-
specific fluorophore substrates, we assayed for both cas-
pase-8 (extrinsic pathway)and caspase-9 (intrinsic pathway)
to delineate the predominant mechanism of action of drugs
1 and 4 (Figure 5). Our results indicate that both O-digitoxo-
side 1 and MeON-digitoxoside 4 predominantly activated
SUPPORTING INFORMATION AVAILABLE Assay protocols
and synthetic procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. E-mail: yrojan@hsc.wvu.edu (Y.R.), george.odoherty@
mail.wvu.edu (G.A.O.), and langenha@seattleu.edu (J.M.L.).
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Funding Sources: J.M.L. was supported by the Camille and Henry
Dreyfus Foundation, the Research Corporation, the MJ Murdock Chari-
table Trust, and Seattle University. G.A.O. was supported by the NIH
(GM088839 and 090259) and the NSF (CHE-0749451).
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