Serotonin Analogues as Inhibitors of Breast Cancer Cell Growth

Article abstract of DOI:10.1021/acsmedchemlett.7b00282

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

Full text of DOI:10.1021/acsmedchemlett.7b00282

Letter
pubs.acs.org/acsmedchemlett
Serotonin Analogues as Inhibitors of Breast Cancer Cell Growth
Jiney Jose,○,◆,†,‡ Clint D. J. Tavares,○,□,†,§ Nancy D. Ebelt,○,¶,†,§ Alessia Lodi,∥
Ramakrishna Edupuganti,†,‡ Xuemei Xie,⊥,# Ashwini K. Devkota,†,§ Tamer S. Kaoud,†
Carla L. Van Den Berg,§,∇ Eric V. Anslyn,‡ Stefano Tiziani,∥ Chandra Bartholomeusz,⊥,#
and Kevin N. Dalby*,†,§
†Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712,
United States
‡Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States
§Graduate Program in Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, United States
∥Department of Nutritional Sciences, Dell Pediatric Research Institute, University of Texas at Austin, Austin, Texas 78723, United
States
⊥Section of Translational Breast Cancer Research, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United
States
#Department of Breast Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
∇Division of Pharmacology & Toxicology, College of Pharmacy, University of Texas at Austin, Dell Pediatric Research Institute,
Austin, Texas 78723, United States
S
* Supporting Information
ABSTRACT: Serotonin (5-hydroxytryptamine, 5-HT) is a
critical local regulator of epithelial homeostasis in the breast
and exerts its actions through a number of receptors.
Dysregulation of serotonin signaling is reported to contribute
to breast cancer pathophysiology by enhancing cell prolifer-
ation and promoting resistance to apoptosis. Preliminary
analyses indicated that the potent 5-HT1B/1D serotonin
receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like
molecule, induced cell death in breast cancer cell lines. Thus,
we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer
cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with
signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer
cells.
KEYWORDS: Breast cancer, tryptamine, serotonin receptor
progression, and the expression patterns of the serotonin
receptors are often severely altered.7 Interestingly, the
expression of some isoforms increase, while others decrease,
suggestive of significant alterations in downstream signaling,
which may underlie, resulting resistance to 5-HT-induced
apoptosis and to the promotion of proliferation.7
Here, we report the synthesis and characterization of a series
of 5-alkoxy tryptamine analogues, which inhibit proliferation of
breast cancer cell lines. Two compounds, 6 and 10, were
studied in detail and were shown to induce apoptosis in human
breast TNBC cell lines in a dose-dependent manner. Both
compounds inhibit the mTOR signaling pathway, as well as
ribosomal S6 phosphorylation, suggesting that they inhibit
erotonin (5-hydroxytryptamine, 5-HT), a monoamine
Sneurotransmitter, is a critical local regulator of epithelial
homeostasis in the breast.1 Serotonin exerts its actions through
a repertoire of receptor proteins belonging to seven discrete
families, six of which are G-protein-coupled receptors, including
Gi, 5-HT1; Gs, 5-HT4,6,7; and Gq/11, 5-HT2,5. Studies have
suggested that serotonin plays a mitogenic role in cancer cells2
and functions within the autocrine loops of growth factors
thereby contributing to cell proliferation in aggressive cancers,
such as small cell lung carcinoma,3 prostate cancer,4 bladder
cancer,5 and breast cancer.6 Recently, it was demonstrated that
in the normal mammary gland, serotonin acts as a physiological
regulator of lactation and involution, in part by favoring growth
arrest and cell death.7 This tightly regulated system becomes
dysregulated in human breast cancers. For example, tyrosine
hydroxylase (the rate-limiting enzyme in the serotonin
biosynthetic pathway) is overexpressed during malignant
Received: July 12, 2017
Accepted: September 14, 2017
Published: September 14, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.7b00282
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Letter
protein synthesis, a notion supported by an observed elevation
of free amino acids induced in MDA-MB-231 cells treated with
6.
The 5-alkoxy tryptamine analogues were synthesized from
serotonin (5-hydroxytryptamine) following a reported literature
procedure.8 The serotonin primary amine was protected using a
t-BOC group to afford intermediate 18, which was alkylated
with alkyl bromides of varying chain length to obtain 19. Facile
deprotection of the t-BOC group using HCl afforded
compounds 1−9 in 59−90% yield (Scheme 1A).
Table 1. Median Effects of Compounds on Viability in
Cancer Cell Lines
IC50 (μM)
MDA-MB-231 (breast)
MCF-7 (breast)
>50
tryptamine
>50
1
48 2.5
7.3 0.6
4.7 0.4
4.4 0.2
4.4 0.0
4.6 0.1
8.2 0.4
4.8 0.1
7.5 0.2
4.2 0.5
4.1 0.4
8.0 0.3
4.6 0.2
10 0.3
>50
45
5
2
13 1.0
8.8 0.1
9.0 0.6
6.1 1.1
5.1 0.9
15 1.3
8.4 0.6
13 1.5
4.9 0.1
5.0 0.2
15 1.0
8.6 0.7
17 0.8
>50
3
4
5
Scheme 1. General Schemes for the Syntheses of 3-(2-
Aminoethyl)-5-(alkoxy)indole Hydrochloride (Compound
6, n = 11) and 4/7-Alkoxy Tryptamine (Compound 10, n =
6
7
8
a
9
8)
10
11
12
13
14
15
16
17
20 0.0
18 2.1
24 3.2
22 0.8
in the aforementioned cell lines using full serum media.
Structures of all synthesized compounds are shown in Table S1
in the Supporting Information. Trends are immediately
apparent where those compounds containing a tryptamine
with a long linear alkoxy chain (chain length 8−14) at the 4, 5,
or 7 position exhibit the best activity (compounds 2−6, 8, 10−
13). Tryptamine itself does not reduce cell viability, and even
the addition of a short n-butyl group (compound 1) has little
effect. A nonlinear alkoxy analogue of 5-hydroxytryptamine
(compound 7) showed a 2-fold drop in activity. Compound 9,
which has a linear chain length of 16 carbon atoms, also showed
a reduction in activity similar to compound 7. The addition of a
bromide at the end of the linear alkyl chain seems to retain
some activity (compound 14), whereas substituting nitrile for a
bromide resulted in significant loss of activity (compound 15).
Various serotonin dimers, which act as bivalent ligands, have
been reported as selective 5-HT1B/1D agonists.9 Therefore, we
synthesized two homodimeric molecules, 16 and 17, which
showed similar but reduced activity compared to other
compounds. Since we observe a plateau in potency for
compounds with increasing chain length, it suggests that the
tryptamine scaffold may be the mediator of compound activity
and that the lipophilic hydrocarbon chain enables more efficient
entry into the cell (possibly bypassing SERT-mediated entry),
or it may target the compounds to receptors wholly different
from the serotonin receptors.
Compounds 6 and 10 were then chosen for further analysis.
Interestingly, both 10 (Figure 1a) and 6 (not shown) were
found to exhibit more potency following incubation with cells
for 48 or 72 h compared to 24 h, suggesting a cumulative effect
of the treatment with time. However, the efficacy of 10, but not
6, was dramatically increased in serum-free media, possibly
indicating a greater binding of 10 to serum compared to 6
(Figure 1b). This assertion is supported by the observation that
the addition of BSA to serum-free media suppresses the
inhibitory activity of 10 (Figure 1c). Compounds 10 (Figure
1d) and 6 (not shown) induce a dose-dependent increase in
apoptosis after treatment, as evidenced by a luciferase-based
caspase 3/7 assay, as well as Western blots showing poly-ADP
a
(A) Reagents and conditions: (a) (Boc)2O, K2CO3, H2O, 25 °C, 24
h, 90%; (b) alkyl bromide, K2CO3, CH3CN, 80 °C, 24 h, 52−74%; (c)
3 M HCl/EtOAc, 25 °C, 2 h, 59−90%. (B) Reagents and conditions:
(a) alkyl bromide, K2CO3, CH3CN, 25 °C, 24 h, 87−88% n = 8; 56−
59%, n = 11; (b) N,N-dimethyl-2-nitroethyleneamine, TFA, CH2Cl2,
25 °C, 1 h, 32−37%, n = 8; 59−60%, n = 11; (c) (i) LiAlH4, THF,
reflux, 6 h; (ii) 3 M HCl/EtOAc, 25 °C, 48−58%, n = 8; 46−55%, n =
11.
The 4- and 7-alkoxy tryptamine compounds 10−13 were
synthesized as shown in Scheme 1B. Alkylation of the 4- or 7-
hydroxy indole with the appropriate alkyl bromide afforded the
alkoxy indole intermediates 20. Michael addition with N,N-
dimethyl-2-nitroethyleneamine gave the corresponding nitro
vinyl intermediates 21, which was subjected to LAH reduction
to obtain the desired analogues in (46−58%) yields. Synthetic
details of the less potent compounds 14−17 are provided in the
Supporting Information.
Breast cancer cell lines MDA-MB-231 and MCF-7 were
treated with increasing concentrations of the various serotonin
derivatives. Viability was assayed using MTS, and IC50 values
were generated. Table 1 shows IC50 values for each compound
B
DOI: 10.1021/acsmedchemlett.7b00282
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Compound 10 represses activation of translation associated
proteins.
compound 6. NMR was performed to analyze levels of amino
acids and other metabolites in treated versus untreated cells.
Data sets were analyzed using unsupervised multivariate
statistical analysis (principal component analysis, PCA). The
score plots (Figure S3) obtained from the PCA analysis clearly
indicate very good grouping and separation of untreated and
treated cells. Notably, the separation between untreated and
compound 6-treated groups increased with the longer treat-
ment period (24 h versus 7 h). Direct metabolic modulation by
a compound is expected to occur shortly after treatment, and
those changes should intensify as well as occur with further
modulations at later time-points. These additional late time-
point modulations may be due to secondary or tertiary effects
as well as cellular adaptive response to the treatment. After the
initial PCA, we performed an in-depth analysis of the specific
metabolic changes induced by treatment at 24 h, indicating the
overall metabolic pathways affected by 6 and providing key
mechanistic information.
Notably, all the detected amino acids, including the branched
amino acids, alanine, aspartate, glutamine, proline, glycine,
phenylalanine, and tyrosine accumulated following treatment
(Figure 3). This observation could indicate that treatment with
6 hinders protein synthesis rates, possibly due to mTORC1
inhibition, or increases protein degradation. The activation of
autophagy after mTORC1 inhibition may also explain the
increased availability of amino acids.14 Similarly, glycans, such
as uridine diphosphate (UDP) glucose and UDP N-
acetylglucosamine, accumulated intracellularly at much higher
levels in the MDA-MB-231 cells treated with 6 for 24 h. These
results could be linked to a compound 6-induced endoplasmic
reticulum (ER) stress and unfolded protein response (UPR). In
fact, O-linked β-N-acetylglucosamine (O-GlcNAc) signaling is
upregulated in response to ER stress to prevent cardiomyocyte
death.15 In addition, the intracellular levels of both myo-inositol
and lactate were reduced, which may indicate a switch from
glycolysis followed by lactic acid fermentation to glycolysis
followed by the TCA cycle. As cancer cells rely heavily on the
former method of metabolism to maintain their rate of
proliferation (termed the Warburg effect16), these data suggest
that 6 may exert an antiproliferative effect through interruption
of this important mode of cancer cell survival.
Figure 1. Compounds 6 and 10 decrease viability and increase
apoptosis of TNBC cell lines.
ribose polymerase (PARP) cleavage. A similar effect is observed
in the MDA-MB-157 triple negative cell line (Figure S1).
To explore possible signaling mechanisms by which 6 and 10
might inhibit breast cancer cell growth, MDA-MB-231 (triple-
negative breast cancer) and MCF-7 (luminal A breast cancer)
cells were treated with vehicle, 6, or 10 (20 μM) for 24 h, and
samples were subjected to a reverse phase protein array
(RPPA) analysis, which revealed similar overall changes in
signaling the following treatment with either 6 or 10. Select
targets are represented as a heatmap in Figure S2 in the
Supporting Information. Differences between cell lines
including increased phosphorylation of AMPK and ACC in
MCF7 cells over MDA-MB-231 cells may indicate signaling
differences between the two breast cancer subtypes as they
relate to treatment with these compounds. Notably, phosphor-
ylation of proteins involved in downstream signaling of the
mTOR pathway, but not total protein, are significantly
decreased in MDA-MB-231 cells treated with 6 or 10. Targets
include p70S6K (threonine 389) and its substrate ribosomal
protein S6 (serine 235, 236, 240, 244). To explore whether
these effects are specific to the MDA-MB-231 cells or whether
they might represent a mechanism effective against triple
negative breast cancer in general, we confirmed these targets by
Western blotting in both the MDA-MB-231 and MDA-MB-157
triple negative cell lines (Figure 2). While phosphorylation of
p70S6K on T389 is strongly inhibited by compound 6 and 10,
the activation of the kinase responsible for this phosphor-
ylation, mTOR Signaling Complex 1 (mTORC1),10,11 as
judged by the PI3K/AKT-mediated phosphorylation of serine
2448,12 is unchanged (Figures 2 and S2). Thus, both 6 and 10
potentially inhibit the activation of p70S6K/S6 by mTORC1.
Nutrient sensing and therefore control of metabolism occurs
largely through mTOR signaling.13 Therefore, as 6 and 10
suppressed downstream signaling of mTOR to p70S6K/
ribosomal protein S6, it was of interest to assess the metabolic
changes induced by the treatment of MDA-MB-231 cells with
A recent study of “metabolic landscapes” from human breast
tumors revealed that a high degree of serotonin production was
a prominent feature in tumors with poor prognosis, many of
which are triple-negative.17 Here, we report that novel 5-HT
analogues induce apoptosis in human breast cancer cell lines by
diminishing signaling to nutrient sensing pathways resulting in
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Figure 3. Compound 6 decreases amino acid abundance and increases glycans.
reduced protein synthesis, increased autophagy, and cell death.
These effects may be due to the abrogation of cancer cell-
supervised by C.L.V., S.T., E.V.A., C.B., and K.N.D. All authors
have given approval to the final version of the manuscript.
specific functions of 5-HT receptors or distinct targeting of
these compounds to other molecules, possibly kinases involved
in the PI3K/Akt/mTOR signaling pathway.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00282.
■
The authors are grateful to NIH R21 CA167505, CPRIT
RP110532 and RP160657, the Welch Foundation (F-1390),
United States, and startup from The University of Texas MD
Anderson Cancer Center (111411), United States, to C.B. for
financial support of this study.
S
Materials and methods, and supplemental Tables and
Figures (PDF)
ABBREVIATIONS
■
mTOR, mammalian target of rapamycin; TCA, tricarboxylic
acid cycle; AMPK, 5′ AMP-activated protein kinase; ACC,
acetyl-CoA carboxylase; MAPK, mitogen activated protein
kinase
AUTHOR INFORMATION
Corresponding Author
*Tel: 512-471-9267. Fax: 512-232-2606. E-mail: dalby@austin.
utexas.edu.
■
REFERENCES
■
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Eric V. Anslyn: 0000-0002-5137-8797
Kevin N. Dalby: 0000-0001-9272-5129
Present Addresses
◆Auckland Cancer Society Research Centre, Faculty of
Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland, New Zealand.
□Department of Cancer Biology, Dana-Farber Cancer Institute,
and Department of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115, United States.
¶Department of Experimental Therapeutics, Beckman Research
Institute, City of Hope National Medical Center, Duarte,
California 91010, United States.
Author Contributions
○These authors contributed equally to this work. J.J., C.D.J.T.,
N.D.E. and K.N.D. conceived the project and designed the
research strategy and experimental approach; J.J. synthesized
the compounds, with assistance from R.E.; C.D.J.T. and N.D.E
performed the experiments, with assistance from X.X., A.K.D.
and T.S.K.; A.L. performed the metabolomic analysis; The data
was analyzed by J.J., C.D.J.T., N.D.E., A.L. and K.N.D.; The
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