Novel D-erythro N-octanoyl sphingosine analogs as chemo-and endocrine-resistant breast cancer therapeutics

Article abstract of DOI:10.1007/s00280-009-1233-0

Purpose Resistance to endocrine and chemotherapies remains the primary cause of breast cancer treatment failure. We have synthesized four novel D-erythro N-octanoyl sphingosine analogs and catalogued their activity in drugsensitive (MCF-7), endocrine-resistant (MDA-MB-231) and chemoresistant (MCF-7TN-R) breast cancer cells. Methods 3-(4,5-Dimethylthiazol-2-Yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to determine cell viability; colony assay was performed to determine effects on clonogenic survival and ¹H NMR, ¹³C NMR, HPLC spectra and elemental analytical data analyses were used to determine analog identity and purity. Results All four analogs inhibited both viability and clonogenic survival, with analog C exhibiting a log-fold improvement in anti-survival activity compared to the parent compound. Conclusion With resistance to current breast cancer chemotherapies on the rise, the development of novel therapeutic targets is of growing importance. Our results show that lipid analogs have therapeutic potential in treating chemo-and endocrine-resistant breast cancer. Springer-Verlag 2010.

Full text of DOI:10.1007/s00280-009-1233-0

Cancer Chemother Pharmacol (2010) 65:1191–1195
DOI 10.1007/s00280-009-1233-0
SHORT COMMUNICATION
Novel D-erythro N-octanoyl sphingosine analogs
as chemo- and endocrine-resistant breast cancer therapeutics
•
James W. Antoon Jiawang Liu
•
•
Adharsh P. Ponnapakkam Matthew M. Gestaut
•
•
Maryam Foroozesh Barbara S. Beckman
Received: 15 September 2009 / Accepted: 24 December 2009 / Published online: 14 February 2010
Ó Springer-Verlag 2010
Abstract
Keywords Novel D-erythro N-octanoyl sphingosine ꢀ
Purpose Resistance to endocrine and chemotherapies
remains the primary cause of breast cancer treatment fail-
ure. We have synthesized four novel D-erythro N-octanoyl
sphingosine analogs and catalogued their activity in drug-
sensitive (MCF-7), endocrine-resistant (MDA-MB-231)
and chemoresistant (MCF-7TN-R) breast cancer cells.
Methods 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltet-
razolium bromide (MTT) assay was used to determine cell
viability; colony assay was performed to determine effects
Sphingolipids ꢀ Breast cancer ꢀ Chemoresistance ꢀ
Endocrine resistance ꢀ Ceramide
Introduction
Breast cancer remains the most commonly diagnosed
cancer in women resulting in 192,370 estimated new cases
and 40,610 deaths in 2009 [1]. Resistance to endocrine and
chemotherapy is the major cause of treatment failure in the
clinic. Dysregulation of sphingolipid metabolism has been
shown to be an important mechanism of drug resistance in
breast cancer, specifically decreased endogenous and che-
motherapy-induced levels of the lipid ceramide [2–5].
Ceramide is known to mediate a wide range of cellular
activities including proliferation, apoptosis, differentiation,
senescence and survival in many human cancers, including
breast and prostate cancer [6–10]. From a clinical per-
spective, development of therapeutic agents aimed at the
sphingolipid pathway may improve patient response rates
in chemoresistant and endocrine therapy-resistant breast
cancer. Common breast cancer chemotherapies, such as
paclitaxel, doxorubicin and etoposide, all increase cera-
mide as a mechanism of inducing apoptosis and inhibiting
cancer growth. As a result, pharmacological targeting of
the active sphingolipid ceramide is of growing interest.
Recently, our laboratory showed that specific alterations
in the backbone of D-erythro N-octanoyl sphingosine ana-
logs, also known as C8-ceramide, increased the potency of
C8-ceramide and increased its effectiveness as a breast
cancer therapeutic [11, 12]. In this study, we used the short-
chain ceramide D-erythro N-octanoyl sphingosine as the
lead compound in developing novel breast cancer thera-
peutics. Endogenous ceramide is metabolized into
on clonogenic survival and H NMR, 13C NMR, HPLC
1
spectra and elemental analytical data analyses were used to
determine analog identity and purity.
Results All four analogs inhibited both viability and
clonogenic survival, with analog C exhibiting a log-fold
improvement in anti-survival activity compared to the
parent compound.
Conclusion With resistance to current breast cancer
chemotherapies on the rise, the development of novel
therapeutic targets is of growing importance. Our results
show that lipid analogs have therapeutic potential in
treating chemo- and endocrine-resistant breast cancer.
Electronic supplementary material The online version of this
article (doi:10.1007/s00280-009-1233-0) contains supplementary
material, which is available to authorized users.
J. W. Antoon ꢀ A. P. Ponnapakkam ꢀ M. M. Gestaut ꢀ
B. S. Beckman (&)
Department of Pharmacology,
Tulane University School of Medicine,
1430 Tulane Avenue, New Orleans, LA 70112, USA
e-mail: bbeckman@tulane.edu
J. Liu ꢀ M. Foroozesh
Department of Chemistry, Xavier University of Louisiana,
1 Drexel Drive, New Orleans, LA 70125, USA
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Cancer Chemother Pharmacol (2010) 65:1191–1195
sphingosine, which is then rapidly phosphorylated into
sphingosine-1-phosphate, a prosurvival lipid [11]. To pre-
vent ceramide from being degraded into sphingosine,
modifications have been carried out at the amide group
where the ceramidase-catalyzed hydrolysis reaction occurs.
Two strategies were used for the modification of the cer-
amide structure. Strategy I: Cycloalkyl group (analog A) or
branched alkyl group (analog D) were introduced as sub-
stituents on the amide group replacing the flexible heptyl
chain to enhance the rigidity and steric hindrance and
shield this hydrolysis site. Strategy II: The amide func-
tional group was replaced with an amine functional group
(analog B) or an imine functional group (analog C) to
prevent amide hydrolysis by the enzyme ceramidase.
later, cells were treated with varying concentrations of
ceramide analog and then monitored microscopically for
colony growth. Ten days later, the cells were fixed with 3%
glutaraldehyde. Following fixation for 15 min, the plates
were washed and stained with a 0.4% solution of crystal
violet in 20% methanol for 30 min, washed with PBS and
air-dried. Colonies of C30 cells were counted as positive.
Results are normalized to percent clonogenic survival from
untreated control cells. Statistical analysis of IC50 values
was calculated from concentration–response curves using
GraphPad Prism 5.0 (Graphpad Software, San Diego, CA,
USA) and the equation:
Y ¼ bottom þ ðtop ꢁ bottomÞ=1 þ 10 logEC50 ꢁ X:
Statistical analysis
Materials and methods
Statistical analysis of IC50 values was calculated from
concentration–response curves using GraphPad Prism 5.0
(Graphpad Software, San Diego, CA, USA), using the
equation:
Cell culture
Human cancer cell lines derived from breast, MCF-7,
MDA-MB-231 and MCF-7TN-R were cultured in 75-cm2
culture flasks in DMEM (Invitrogen, Co.) supplemented
with 10% FBS (Life Technologies, Inc., Gaithersburg, MD,
USA), basic minimum MEM essential (509, Invitrogen
Co.) and MEM non-essential (1009, Invitrogen, Co.)
amino acids, sodium pyruvate (1009 Invitrogen Co.), anti-
mycotic–antibiotic (10,000 U/ml penicillin G sodium;
10,000 lg/ml streptomycin sulfate; 25 lg/ml amphotericin
B as FungizoneÒ) and human recombinant insulin (4 mg/ml,
Invitrogen Co). Culture flasks were maintained in a tissue
culture incubator in a humidified atmosphere of 5% CO2 and
95% air at 37°C.
Y ¼ bottom þ ðtop ꢁ bottomÞ=1 þ 10logEC50 ꢁ X:
Assuming a standard slope, where the response goes
from 10 to 90% of maximal as X increases over two log
units. Differences in IC50 were compared using Student’s
unpaired t test with P \ 0.05 as the limit of statistical
significance. Experiments comparing multiple concentrations
to the control were tested with one-way ANOVA with
Bonferroni post-test to compare individual concentrations.
All statistical analysis were performed using GraphPad
Prism 5.0.
Results
MTT viability assay
The synthesis of analogs A–D is depicted in Fig. 1. At
0°C, coupling reactions of D-sphingosine/HCl with cyclo-
hexanecarboxylic acid and pivalic acid gave N-((2S,3R,E)-
1,3-dihydroxyoctadec-4-en-2yl)cyclohexanecarboxamide
(analog A) and N-((2S,3R,E)-1,3-dihydroxyoctadec-4-en-
2-yl)pivalamide (analog D) in 51 and 45% yields, respec-
tively. In the presence of sodium hydroxide, D-sphingosine/
HCl went through a condensation reaction with benzaldehyde
in methanol giving (2S,3R,E)-2-((E)-benzylideneamino)
octadec-4-ene-1,3-diol (analog C) in 71% yield, which
was then reduced by sodium cyanoborohydride to give
(2S,3R,E)-2-(benzylamino)octadec-4-ene-1,3-diol (analog B)
in 36% yield. These results show that all of these reactions
occurred in mild conditions, and ceramide analogs were
produced with acceptable yields.
The effect of ceramide and ceramide analogs on cell growth
was determined using MTT viability assays. MCF-7, MDA-
MB-231 or MCF-7TN-R cells were plated at 7.5 9 105 cells
per well in a 96-well plate in phenol-free DMEM supple-
mented with 5% FBS and allowed to adhere overnight. Cells
were then treated with analogs A, B, C or D (ranging from 0.1
to 100 lM) for 24 h. Following treatment, 20 ll of MTT
(5 mg/ml) reagent was added to each well prior to incubation
for 4 h. Cells were then lysed with 20% SDS in 50%
dimethylformamide. The pH and absorbances were read on
an ELx808 Microtek plate reader (Winooski, VT, USA) at
550 nm, with a reference wavelength of 630 nm.
Clonogenic survival assay
The four analogs were screened for their effects on
viability of drug-sensitive (MCF-7), endocrine therapy-
resistant (MDA-MB-231) and chemotherapy-resistant
MCF-7, MDA-MB-231 or MCF-7TN-R cells were plated
in 6-well plates at 1,000 cells per well. Twenty-four hours
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Cancer Chemother Pharmacol (2010) 65:1191–1195
1193
Fig. 1 a Structures of D-erythro
a
OH
OH
OH
N-octanoyl sphingosine analogs.
The IC50 values (lM) for
b viability assays and
c clonogenic survival assays
were calculated from
b
NaBH3CN
MeOH
1. NaOH/MeOH
2. Benzaldehyde
C13H27
C13H27
HO
HO
C13H27
HO
NH3+ Cl-
N
NH
D-Sphingosine/HCl
experiments shown in Fig. 2.
All values were statistically
significant with P values \0.05
DCC, HOBt, NMM
THF
C (71%)
B (36%)
OH
C13H27
HO
A R =
D R =
O
NH
R
A (51%), D (45%)
b
Analog
MDA-MB-231
MCF-7TN-R
MCF-7
22.6
6.3
A
B
C
D
5.4
3.7
4.8
8.0
2.4
3.0
3.6
4.2
9.0
13.5
c
Analog
MDA-MB-231
MCF-7TN-R
MCF-7
1.5
A
B
C
D
1.6
1.1
1.4
0.5
0.7
0.6
0.1
0.6
1.5
1.7
1.5
(MCF-7TN-R) breast cancer cells (Fig. 2a). Cells were
treated with various concentrations of an analog (0.1–100
lM) for 24 h, treated with MTT for 4 h and then solubi-
lized with SDS in DMF. As seen in Fig. 1b, all four ana-
logs displayed biological activity in both drug-resistant and
drug-sensitive breast cancers. Interestingly, no analog had
the lowest IC50 in all three cell lines. Analog A was the
most effective in MCF-7TN-R with an IC50 value of
2.4 lM. Analog C was the most potent of the analogs in
MCF-7 and MDA-MB-231 cells, with IC50 values of 6.3
and 3.7 lM, respectively.
The low micromolar efficacy of these analogs on via-
bility led us to examine the effects of the analogs on long-
term clonogenic survival (Fig. 2b). Cells were plated and
treated with varying concentrations of analogs (0.1–
100 lM) for 10 days and then monitored microscopically
for colony growth. Cells were fixed with 3% glutaralde-
hyde and stained with a 0.4% solution of crystal violet in
20% methanol before colonies were counted. The four
analogs displayed increased efficacy in blocking survival
compared to viability in all three breast cancer cell lines.
Interestingly, the analogs were most effective in the
chemoresistant MCF-7TN-R cell line, with analog C dis-
playing an IC50 of 0.1 lM.
In summary, we found that the four novel ceramide
analogs were effective in diminishing chemoresistant and
endocrine-resistant breast cancer cell viability and survival,
with analogs A and C displaying the lowest IC50 values for
these assays, respectively. Analog C is substantially more
effective than the other analogs in decreasing chemoresis-
tant breast cancer cell survival. This increased efficacy may
be due to the presence of the imine functional group in
analog C. This functional group is believed not to be
hydrolyzed by the enzyme ceramidase, effectively blocking
the production of sphingosine and preventing ceramide
analog depletion. Compared to previously published cera-
mide analogs in our laboratory with varying backbones,
these analogs display increased efficacy compared to their
counterparts [11]. The clonogenic survival IC50 values of
analogs A, B, C and D were significantly lower than their
counterparts with similar substitutions but amide back-
bones, suggesting that the structural changes to the short-
chain ceramide backbone in these analogs confer an
increased efficacy in drug-resistant breast cancer cell lines
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Cancer Chemother Pharmacol (2010) 65:1191–1195
a
100
75
50
25
0
100
75
50
25
0
MCF-7
MCF-7
MDA-MB-231
MCF-7TN-R
MDA-MB-231
MCF-7TN-R
10-3
10-2
10-1
100
101
102
10-3
10-2
10-1
100
101
102
[Analog A]µM
[Analog B]µM
100
75
50
25
0
100
75
50
25
0
MCF-7
MCF7
MDA-MB-231
MCF-7TN-R
MDA-MB-231
MCF-7TN-R
10-3
10-2
10-1
100
101
102
10-3
10-2
10-1
100
101
102
[Analog C] µM
[Analog D]µM
b
100
80
60
40
20
0
100
80
60
40
20
0
MCF-7
MCF-7
MDA-MB-231
MCF-7TN-R
MDA-MB-231
MCF-7TN-R
10-4
10-2
100
102
10-4
10-2
100
102
[Analog A] µM
[Analog B] µM
100
80
60
40
20
0
100
80
60
40
20
0
MCF-7
MCF-7
MDA-MB-231
MDA-MB-231
MCF-7TN-R
MCF-7TN-R
10-4
10-2
100
102
-4
10
10-2
100
102
[Analog D] µM
[Analog C] µM
Fig. 2 a Effect of analogs on breast cancer viability. MCF-7, MDA-
MB-231 and MCF-7TN-R cells were treated with varying concen-
trations of analog (0.1–100 lM) for 24 h. The values are the mean of
SE of four independent experiments. b Effect of ceramide analogs
on breast cancer clonogenic survival. MCF-7, MDA-MB-231 and
MCF-7TN-R cells were treated with varying concentrations of analog
(0.1–100 lM) for 10 days. The values are the mean of SE of three
independent experiments
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Cancer Chemother Pharmacol (2010) 65:1191–1195
1195
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Acknowledgments We thank Dr. James Robinson’s laboratory at
Tulane for help in quantifying viability results. This research was
supported by a grant from the Louisiana Cancer Research Consortium
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