Androsamide, a Cyclic Tetrapeptide from a Marine Nocardiopsis sp., Suppresses Motility of Colorectal Cancer Cells

Article abstract of DOI:10.1021/acs.jnatprod.0c00815

A cyclic tetrapeptide, androsamide (1), was isolated from a marine actinomycete of the genus Nocardiopsis, strain CNT-189. The planar structure of 1 was assigned by the interpretation of 1D and 2D NMR spectroscopic data. The absolute configurations of constituent amino acids of 1 were determined by application of the Marfey's and advanced Marfey's methods. Androsamide (1) strongly suppressed the motility of Caco2 cells caused by epithelial-mesenchymal transition.

Full text of DOI:10.1021/acs.jnatprod.0c00815

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Article
Androsamide, a Cyclic Tetrapeptide from a Marine Nocardiopsis sp.,
Suppresses Motility of Colorectal Cancer Cells
Jihye Lee,# Chathurika. D. B. Gamage,# Geum Jin Kim, Prima F. Hillman, Chaeyoung Lee,
Eun Young Lee, Hyukjae Choi, Hangun Kim,* Sang-Jip Nam,* and William Fenical*
Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00815
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ABSTRACT: A cyclic tetrapeptide, androsamide (1), was isolated
from a marine actinomycete of the genus Nocardiopsis, strain
CNT-189. The planar structure of 1 was assigned by the
interpretation of 1D and 2D NMR spectroscopic data. The
absolute configurations of constituent amino acids of 1 were
determined by application of the Marfey’s and advanced Marfey’s
methods. Androsamide (1) strongly suppressed the motility of
Caco2 cells caused by epithelial−mesenchymal transition.
has become one of the most important aims for the development
olorectal cancer (CRC) is the third most deadly diagnosed
Ccancer type and the fourth leading cause of cancer of anticancer therapeutics.
As part of our efforts to discover anticancer agents from
mortality in the world. According to global patterns and trends
in CRC incidence and mortality, it is expected to increase by
60% by 2030. More than two-thirds of all CRC incidents and
about 60% of all deaths have been recorded in countries with a
high or very high human development index (HDI).1 Metastases
are the main cause of mortality in CRC, similar to other
malignancies. About 25% of CRC patients were found to have
distant metastasis at their initial diagnosis.2 Human CRC
commonly metastasizes to the liver and lungs; more than 50% of
CRC patients tend to have liver metastases during their
lifespan.3 Given the severity of this disease, the discovery and
development of new diagnostic strategies and new CRC
treatments are of urgent need.
Human CRC has many underlying causes. Genetic mutations
such as inactivation of tumor suppressor genes or activation of
oncogenes, as well as chronic inflammation caused by Crohn’s
disease or ulcerative colitis, can ultimately lead to CRC
occurrence.4 Epithelial−mesenchymal transition (EMT) is a
complicated cellular mechanism in which cells change their
epithelial characteristic to acquire migratory and invasive
properties. EMT is considered a primary mechanism for CRC
progression and metastasis.5 That is, this phenomenon supports
tumor cells’ ability to survive against constant changes in the
tumor microenvironment and to invade other organs success-
fully.6 Some extracellular stimuli, such as inflammatory
cytokines, growth factors, or hypoxic conditions generated in
the tumor microenvironment, regulate EMT abnormally in
cancer cells. Given this situation, inhibition of the EMT process
natural products, we focused on screening the secondary
metabolites produced by the genus Nocardiopsis. The genus
Nocardiopsis has been a prominent marine bacterial source of
bioactive metabolites, along with members of the genus
Streptomyces.7 Nocardiopsis strains have been reported to
produce many unique bioactive metabolites, such as cyclic
peptides, polyketides, macrolides, chlorinated aromatic com-
pounds, and terpenoids, including lucentamycins A−D,8
lucentides A and B,9 borrelidins C−E,10 fijiolides A and B,11
and nocarimidazoles A and B.12 Nocardiopsis strain CNT-189,
isolated from a Bahamas shore sediment, was investigated to
discover new anticancer agents. This strain was first identified as
a Streptomyces sp. by analysis of a limited range of NCBI Blast
data. On re-examination, analysis of 16S rRNA sequence data
clearly showed this strain to be a member of the genus
Nocardiopsis. This bacterial strain has been reported to produce
the kromycin aglycones of pikromycin, 4Z-, 4E-12-deoxydihy-
drokromycins.13 In this work, following extensive investigation
of the chemical components from the culture broth of strain
CNT-189, a new cyclic peptide, androsamide (1), was isolated.
Received: July 21, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Herein, we report the isolation, structure elucidation, and
bioactivities of 1.
Figure 1. COSY, key HMBC, and ROESY NMR correlations for
androsamide (1).
Thr and Ile also supported this linkage. The HMBC correlations
from H-2 of Thr to C-5 of N-Me-4-O-Me-Trp permitted that the
modified Trp to be attached next to Thr. The connectivity of N-
Me-4-O-Me-Trp to N-Me-Met was identified based on HMBC
correlations from H3-17 to C-18 (δC 170.2). This linkage was
also confirmed by the ROESY correlation between N-methyl
protons of the modified Trp and H2-20 (δH 2.08, m, 1.56, m) of
N-Me-Met. Lastly, HMBC correlation from H3-23 to C-24 (δC
171.6) allowed the connection between N-Me-Met and Ile.
The absolute configurations of both Ile and Thr were
determined to be L by Marfey’s analysis with 1-fluoro-2,4-
dinitrophenyl-5-D-leucine amide (D-FDLA) by the comparison
of the retention time with authentic standards (Table S1 and
Figure S7). The absolute configurations of modified amino acids
(N-Me-4-O-Me-Trp and N-Me-Met) were analyzed by the
advanced Marfey’s method (Table S2 and Figure S8). The
reaction products of N-Me-4-O-Me-Trp with L-FDLA and D-
FDLA were detected at the retention times of 7.6 and 11.5 min,
respectively. The derivatives of N-Me-Met with L-FDLA and D-
FDLA were found at the retention times of 13.3 and 18.7 min,
respectively. Therefore, the absolute configurations of both N-
Me-4-O-Me-Trp and N-Me-Met were assigned as L config-
urations.14
Modified amino acids in peptides, and particularly those that
are N-methylated, are extremely important secondary metabo-
lites from ascidians and sponges.15 The occurrence of an N-Me-
Met and an N-Me-4-O-Me-Trp in marine natural products is
rare. With regard to N-Me-Met, it was found in microginin 674
and microginin 511 produced by the cyanobacterium Microcystis
aeruginosa.16 Interestingly, this is the first report of an N-Me-4-
O-Me-tryptophan residue in marine natural products. In the
terrestrial environment, the heptadepsipeptide cycloheptamycin
A produced by a terrestrial Streptomyces sp. is the only example in
the literature.17 Consequently, 1 is unique in containing this rare
amino acid.
Compound 1 was tested for its cytotoxicity against a variety of
cancer cell lines. Relative cell viability of human gastric
adenocarcinoma (AGS), human colorectal adenocarcinoma
(Caco2), and human colorectal carcinoma (HCT116) was
measured by MTT assay after treatment with various
concentrations of 1 for 48 h. Treatment with 1 decreased the
viability of AGS, Caco2, and HCT116 cells in a dose-dependent
fashion (Figure 2). Compound 1 had IC50 values of 13 μM
(Caco2), 18 μM (AGS), and 21 μM (HCT116) against the
three cell lines, indicating that 1 would not be considered
cytotoxic (cytotoxicity = IC50 < 10 μM). Treatment with high
concentrations of 1 (Caco2: 13 μM, 16 μM and HCT116: 21
μM, 26 μM) induces apoptosis in Caco2 and HCT116 cells
(Figure S9).
RESULTS AND DISCUSSION
■
Androsamide (1) was isolated as a yellowish powder, and its
molecular formula was determined to be C29H43N5O6S on the
basis of (+)-HRESIMS, coupled with analysis of NMR data. The
1H NMR spectrum of 1 showed two exchangeable amide
protons (δH 6.71 and 7.45), an amine proton (δH 10.85), and
four α-amino protons (δH 3.42, 3.80, 4.29, and 4.64), typical
features for a peptide, as well as an oxygenated methine proton
(δH 3.96), two methyl doublets (δH 0.90 and 0.97), two N-
methyl groups (δH 2.29 and 2.93), and one S-methyl group (δH
1.95). The 13C NMR spectrum displayed 29 carbons, including
four amide carbonyls (δC 170.2, 170.4, 171.1, and 171.6) and
four α-amino carbons (δC 52.7, 54.9, 55.2, and 61.8).
Analysis of COSY, HSQC, and HMBC spectra allowed the
establishment of four distinct amino acids: a threonine (Thr), an
N-methyl-4-methoxy-tryptophan (N-Me-4-O-Me-Trp), an N-
methyl-methionine (N-Me-Met), and an isoleucine (Ile). A Thr
unit was defined by COSY NMR data that illustrated cross-peaks
from H3-4 (δH 0.97, d, J = 5.0 Hz) to 2-NH (δH 7.45, d, J = 8.0
Hz) and by HMBC correlations from 2-NH to C-1 (δC 170.4).
The COSY correlations from H3-28 (δH 0.82, t, J = 5.8 Hz) to
25-NH (δH 6.71, d, J = 7.6 Hz) and from H3-29 (δH 0.90, d, J =
5.4 Hz) to H-26 (δH 1.65, m) and HMBC correlations from 25-
NH to C-24 (δC 171.6) indicated an Ile unit. A Met moiety was
assigned by interpretation of COSY cross-peaks from H-19 (δH
4.29, m) to H2-21 (δH 2.47, m; 2.35, m) and by HMBC
correlations from H3-22 (δH 1.95, s) to C-21 (δC 30.1) and from
H-19 to C-18 (δC 170.2). The HMBC correlation from H3-23
(δH 2.93, s) to C-19 (δC 55.2) indicated that an amide proton of
the Met residue was replaced with an N-methyl group. Lastly, a
Trp moiety was identified by the COSY NMR cross-peaks
between H-6 (δH 3.80, m) and H-7 (δH 3.26, d, J = 7.6 Hz) and
by HMBC correlations of H-6 and H-7 to C-5 (δC 171.1), of H-7
to C-8 (δC 110.9) and C-9 (δC 116.9), of H-15 (δH 6.88, s) to C-
8, C-9, and of 14-NH (δH 10.85, s) to C-14 (δC 138.0) and C-15,
along with the 1H−1H coupling of the aromatic protons, H-11
(δH 6.44, d, J = 5.6 Hz), H-12 (δH 6.94, t, J = 5.6 Hz), and H-13
(δH 6.91, d, J = 5.6 Hz), and their HMBC correlations. In
addition, HMBC correlations from methoxy protons H3-16 (δH
3.79, s) to C-10 (δC 153.9) and from N-methyl protons H3-17
(δH 2.29, s) to C-6 (δC 64.3) revealed that a Trp moiety was
modified as an N-Me-4-O-Me-Trp (Figure 1).
The sequence of amino acids was established by analyses of
key long-range HMBC and ROESY correlations. A Thr was
positioned next to Ile by HMBC correlations from H-25 of Ile to
C-1 of Thr. The ROESY correlation between amide protons of
B
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Figure 2. Cytotoxic effects of 1 in Caco2, AGS, and HCT116 cells. Relative cell viabilities of Caco2, AGS, and HCT116 cells were measured by MTT
assay, upon treatment with 1 for 48 h at concentrations from 3.125 to 100 μM. Data represent means SEM; n = 3. *p < 0.05; **p < 0.01; ***p <
0.001; NS, no significant difference compared with the DMSO-treated group.
In order to examine the effect of 1 on Caco2 cell motility at
nontoxic concentrations, Transwell migration and invasion
assays were performed.18 Cells were treated with nontoxic
concentrations of 1 for 6 h. Compound 1 inhibited Caco2 cell
migration in a dose-dependent fashion (Figure 3a). Treatment
with 1.3 μM (1/10 of IC50) inhibited the migration by ∼50%
(Figure 3b). Inhibition of Caco2 cell invasion was assessed after
treatment with nontoxic concentrations of 1 for 24 h. A dose-
dependent decrement of the number of invaded Caco2 cells was
observed upon treatment with 1 (Figure 3c). Approximately
45% of Caco2 cell invasion was inhibited by treatment with 1.3
μM (1/10 of IC50) of 1 (Figure 3d). Taken together, these
results indicate that nontoxic concentrations of 1 significantly
suppress the motility of Caco2 cells.
Caco2 cells treated with nontoxic concentrations of 1
modulated the expression of EMT markers and downregulated
EMT transcription factors. Quantitative real-time PCR (qRT-
PCR) assays measured the relative mRNA levels of EMT
markers such as E-cadherin, N-cadherin, and vimentin. Treat-
ment with 1 increased E-cadherin level slightly, whereas it
decreased N-cadherin and vimentin levels in a dose-dependent
fashion (Figure 4a). The levels of EMT transcription factors,
Snail, Slug, Twist, ZEB1, and ZEB2, were analyzed after
treatment with 1 at nontoxic concentrations for 24 h to
determine whether the suppression of Caco2 cell migration and
invasion was caused by EMT. mRNA expression of these EMT
transcription factors showed significant decrements after
treatment (Figure 4b). Moreover, modulation patterns of
protein expression of E-cadherin, N-cadherin, Snail, and Twist
by Western blotting confirmed the suppression of EMT (Figure
4c). Quantitative analysis of the relative protein level of E-
cadherin showed increasing patterns, and N-cadherin, Snail, and
Twist showed decreasing patterns similar to relative mRNA
levels (Figure 4d). These results suggested that 1 inhibited
Caco2 cell motility by regulating EMT. Next, the Human Cell
Motility RT2 Profiler PCR Array was used to examine the
expression of cell motility-related genes. Two genes related to
human cell motility were screened: calpain 1 (CAPN1) and Ras-
related C3 botulinum toxin substrate 2 (RAC2). CAPN1
destabilizes peripheral focal adhesions and increases the rate of
membrane detachment, leading to cell migration through a two-
dimensional substrate. Also, CAPN1 can cleave numerous
substrates to promote cellular motility, such as focal adhesion
kinase (FAK), talin paxillin, fodrin, ezrin, vinculin, and α-
actinin.19 RAC2 is responsible for encoding GTP-metabolizing
proteins. RAC2 is known to be important in macrophage and
endothelial cell migration by regulating vitronectin or
fibronectin via αvβ3 or α4β1 integrins.20 Modulation of RAC2
expression has been found in different kinds of human cancers
such as brain tumors, head and neck squamous cell cancer, and
leukemias.21 Our results indicate that treatment with 1
decreased the expression of cell motility-related genes CAPN1
and RAC2 in Caco2 cells (Figure 4e). Together, all these
findings confirm that 1 is a potential inhibitor against colorectal
cancer motility.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotation was
measured using an Autopol III polarimeter with a 5 cm cell. UV spectra
were recorded on a Scinco UVS-2100 spectrophotometer. IR spectra
were obtained using a Scimitar 800 FT-IR spectrometer. 1D and 2D
NMR spectra were recorded by a Varian Unity-Inova at 400 MHz, using
the signals of the residual solvent as internal references (δH 2.50 ppm
and δC 39.52 ppm for DMSO-d6). High-resolution ESIMS spectra were
obtained using a JEOL JMS-AX505WA mass spectrometer. Low-
resolution LC-MS data were obtained using an Agilent Technologies
6120 quadrupole LC/MS system with a reversed-phase C18 column
(Phenomenex Luna C18 (2), 50 mm × 4.6 mm, 5 μm) at a flow rate of
1.0 mL/min. Column chromatography separation was performed using
a C18 column eluting with a gradient of MeOH and H2O. The fractions
were purified using a Waters 1525 binary HPLC pump with a reversed-
phase C18 column (Phenomenex Luna C18 (2), 250 mm × 10 mm, 5
μm) eluting with 30% CH3CN in H2O at flow rate of 2.0 mL/min.
Strain Isolation. Actinomycete strain CNT-189 was isolated from
Bahamas shore sediment from the surf zone. Strain CNT-189 was
designated as a Nocardiopsis sp. with 99.6% identity by NCBI blast
analysis of 16S rRNA gene sequence. The strain CNT-189 DNA
sequence was deposited in GenBank (accession number KY111725.1).
Fermentation, Extraction, and Purifircation. Strain CNT-189
was cultured in 40 × 2.5 L Ultra Yield flasks, each containing 1 L of
seawater-based medium (10 g starch, 2 g yeast extract, 4 g peptone,
34.75 g artificial sea salt dissolved in distilled H2O), and shaken at 27 °C
at 150 rpm. After 7 days of cultivation, the broth was extracted using
EtOAc (40 L overall), and the solvent was removed in vacuo to yield 1.7
g of an EtOAc crude extract. The extract was fractionated using open
column chromatography on C-18 resin with a step gradient elution of
H2O and MeOH to yield eight fractions. Fraction 6, eluted with 30% of
MeOH in H2O (226.5 mg), was further separated using an isocratic
solvent system of C-18 reversed-phase column chromatography with
50% aqueous CH3CN to afford six subfractions. The second subfraction
(70.3 mg) was purified by reversed-phase HPLC under isocratic
conditions with 45% aqueous CH3CN to yield 1 (13.5 mg).
Androsamide (1): amorphous, yellowish powder; [α]25 −10 (c
D
0.75, MeOH); UV (MeOH) λmax (log ε) 200 (2.15), 281 (2.09), and
349 (1.24) nm; IR (KBr) νmax 3295, 2964, 2933, 1668, 1374, 1255,
1081, and 754 cm−1
;
1H and 13C NMR data, see Table 1;
C
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Figure 3. Suppression of Caco2 cell motility by 1. (a) Representative images of migrated Caco2 cells through the noncoated Transwell after treatment
with nontoxic concentrations (0.15, 0.65, 1.3 μM) of 1 for 6 h. (b) Quantitation of relative migrated cells compared to the DMSO-treated control. (c)
Representative images of invaded Caco2 cells through the Transwell coated with 1% gelatin after treatment with nontoxic concentrations (0.15, 0.65,
1.3 μM) of 1 for 24 h. (d) Quantitation of relative invaded cells in each treatment group. Data represent means SEM; n = 3. **p < 0.01; ***p < 0.001;
NS, no significant difference compared with the DMSO-treated group.
(+)-HRESIMS, m/z 590.3011 [M + H]+ (calcd for C29H44N5O6S,
590.3012).
reaction products were analyzed by LC-ESIMS with the following
chromatographic conditions: Agilent Zorbax SB-C3 column, 5 μm, 150
× 4.6 mm, 50 °C, 1 mL/min, solvent A (0.005% formic acid in H2O),
solvent B (MeOH), A:B = 48:52 → 48:52 (8 min) → 47.5:52.5 (35
min) → 38:62 (55 min) → 0:100 (60 min) → 0:100 (70 min) → 48:52
(72 min) → 48:52 (82 min). The standard amino acids were also
prepared and analyzed with the same protocol. All of the reaction
products were monitored under UV (340 nm) with a negative ESIMS
mode.13,22
Cell Culture. The human gastric cancer cell line AGS and the
human colorectal cancer cell lines Caco2 and HCT116 were used in
these experiments.23,24 AGS cells were cultured in RPMI, and Caco2
and HCT116 cells were cultured in Dulbecco’s modified Eagle medium
Acid Hydrolysis and Advanced Marfey’s Analysis. Compound
1 (100 μg) was hydrolyzed with 100 μL of 6 N HCl at 110 °C for 30 min
with stirring. The acid hydrolysate was evaporated under a N2 gas
stream. The dried hydrolysate was resuspended with distilled H2O and
divided into two individual vials. Each reaction vial was dried
completely under a N2 gas stream and in a vacuum freeze dryer. The
hydrolysate was dissolved in 100 μL of 1 M NaHCO3. In one vial, the
hydrolysate was treated with 25 μL of 1% L-FDLA in acetone. In the
second vial, the hydrolysate was treated with 1% D-FDLA in acetone.
The reaction mixtures were heated to 50 °C for 30 min with stirring,
and the reactions were quenched by addition of 100 μL of 1 N HCl. The
D
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Figure 4. Compound 1 suppressed Caco2 cell motility by suppressing epithelial−mesenchymal transition (EMT) and other motility-related genes. (a)
Relative mRNA levels of EMT effectors (N-cadherin, E-cadherin, and vimentin) of Caco2 cells treated with nontoxic concentrations (0.65, 1.3 μM) of
1. (b) Relative mRNA levels of EMT transcription factors (Slug, Snail, Twist, ZEB1, and ZEB2) of Caco2 cells treated with nontoxic concentrations
(0.65, 1.3 μM) of 1. mRNA levels were normalized against that of GAPDH. (c) Western blotting of E-cadherin, N-cadherin, Snail, Twist, and α-tubulin
in cells treated with nontoxic concentrations of 1. (d) Quantification of relative protein levels of E-cadherin, N-cadherin, Snail, and Twist normalized to
the level of α-tubulin, which served as the loading control. (e) Quantitative analysis of the mRNA levels of CAPN1 and RAC2 in Caco2 cells treated
with nontoxic concentrations of 1. Data represent means SEM; n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; NS, no significant difference compared
with the DMSO-treated group.
supplemented with 10% FBS and 1% penicillin and streptomycin. Cells
were maintained in an incubator at 37 °C with 5% CO2. All cell lines
were purchased from the Korean Cell Line Bank.
Cell Viability Assay. Viability of AGS, Caco2, and HCT116 cells
were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) colorimetric assay.25 Cells were seeded in 96-
well plates at a density of (2−3) × 103 cells/well and incubated for 12 h.
Then cells either were left untreated or were treated with 1 at
concentrations ranging from 3.125 to 100 μM and incubated for 48 h
followed by incubating with MTT for 4 h. DMSO was added to cells
E
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Table 1. 1H and 13C NMR Spectroscopic Data for
Androsamide (1) in DMSO-d6
bovine serum albumin (BSA). The lower chambers were filled with 400
μL of cultural medium with 0.2% BSA and 10 μg/mL fibronectin (EMD
Millipore Corp.) as a chemoattractant. Cells were treated with 0.15,
0.65, and 1.3 μM of 1 and incubated at 37 °C for 6 h. Migrated cells
were fixed and stained with a Diff Quick kit (Sysmex) and observed
under a K1-Fluo RT fluorescence confocal microscope (Nanoscope
Systems). Migrated cells were counted in five random places in each
Boyden chamber using iSolution FLAuto software (IMT i-solution
Inc.). Experiments were performed in triplicate.
Invasion Assay. Boyden chambers coated with 1% gelatin were
used for the invasion assay.28 Caco2 cells suspended in 100 μL of
cultural medium with 0.2% BSA were seeded in each Boyden chamber
at a density of 2.5 × 106 cells/mL. Lower chambers were filled with 600
μL of cultural medium with 0.2% BSA; 10 μg/mL fibronectin was used
as the chemoattractant. Cells were treated with 0.15, 0.65, and 1.3 μM 1
and incubated at 37 °C for 24 h. Invaded cells were fixed and stained
with a Diff Quick kit and observed using a Nikon Eclipse 400
fluorescence microscope (Nikon Instech, Co., Ltd.). Invaded cells were
counted in five random fields of each Boyden chamber and analyzed by
iSolution FLAutosoftware. Three replicates were performed.
Quantitative Real-Time PCR. Briefly, total RNA was isolated from
Caco2 cells using RNAiso Plus (TaKaRa) according to the
manufacturer’s instructions after treatment with nontoxic concen-
trations of 1 for 24 h.29 Total RNA (1 μg) from each treated group was
converted to cDNA using an M-MLV reverse transcriptase kit
(Invitrogen) and SYBR green (Enzynomics). E-Cadherin (forward)
5′-cagaaagttttccaccaaag-3′ and (reverse) 5′-aaatgtgagcaattctgctt-3′;
vimentin (forward) 5′-tgccctaaaggaaccaatg-3′ and (reverse) 5′-
tccagcagcttcctgtaggt-3′; N-cadherin (forward) 5′-ctcctat gagtggaacag-
gaacg-3′ and (reverse) 5′-ttggatcaatgtcataatcaagtgctgta3′; Snail (for-
ward) 5′-tcccgggcaatttaacaatg-3′ and (reverse) 5′-tgggagacacatcggtcga-
3′; Slug (forward) 5′-cgaactggacacacatacagtg-3′ and (reverse) 5′-
ctgaggatctctggttgtggt-3′; Twist (forward) 5′-cgggagtccgcagtctta-3′ and
(reverse) 5′-tgaatcttgctcagcttgtc-3′; ZEB1 (forward) 5′-atgacacagga-
aaggaagg-3′ and (reverse) 5′-agcagtgtcttgttgtag-3′; ZEB2 (forward) 5′-
caagaggcgcaaacaagcc-3′ and (reverse) 5′-ggttggcaataccgtcatcc-3′;
CAPN1 (forward) 5′-gaaggcaacgagttctggag-3′ and (reverse) 5′-
ggtccacgttgttccactct-3′; RAC2 (forward) 5′-aagaagctggctcccatcacctac-
3′ and (reverse) 5′-aacacggttttcaggcctctctg-3′; GAPDH (forward) 5′-
atcaccatcttccagga gcga-3′ and (reverse) 5′-agttgtcatggatgaccttggc-3′
were the primers used in RT-PCR. qRT-PCR reaction and analysis
were performed using CFX (Bio-Rad).
δH, (J in
a
b
unit
position δC, type
Hz)
COSY
HMBC
Thr
1
2
170.4, C
52.7, CH
4.64, dd
(8.4, 1.7)
3, 2-NH
5
3
4
66.1, CH
19.8, CH3 0.97, d (5.0)
7.45, d (8.0)
3.96, m
2, 4
3
2
4
2, 3
1, 2
2-NH
5
N-Me-4-O-
Me-Trp
171.1, C
6
7
64.3, CH
25.4, CH2 3.26, d (7.6)
3.80, m
7
6
5, 7
5, 6, 8, 9,
15
8
9
110.9, C
116.9, C
153.9, C
10
11
12
13
14
15
16
14-NH
11
98.8, CH 6.46, d (5.6) 10, 12
121.8, CH 6.95, t (5.6) 10, 13
105.0, CH 6.92, d (5.6) 12
138.0, C
122.9, CH
54.9, CH3
9, 10, 13
10
9, 14
6.88, s
3.79, s
10.85, s
2.29, s
8, 9, 13
10
14, 15
6, 18
17-
38.6, CH3
NCH3
N-Me-Met
18
19
170.2, C
55.2, CH
25.7 CH2
4.29, m
2.08, m
1.56, m
2.47, m
2.35, m
1.95, s
20a
19, 20b
21a, 21b
7
7
18, 21
21
21
20a
20b
21a
21b
22
30.1 CH2
14.6, CH3
30.6, CH3
21
19, 24
23-
NCH3
2.93, s
Ile
24
25
26
171.6, C
61.8, CH 3.42, t (6.6) 26
37.2, CH
1, 24
1.65, m
25, 27a,
27b, 29
27a
27b
24.7 CH2
1.48, m
1.17, m
26, 27b, 28
26, 27a, 28 26, 28,
29
Western Blotting. In total, 2 × 105 cells per well were seeded in six-
well plates and treated with nontoxic concentrations of 1 and incubated
for 24 h.29 Cells were lysed, and the extracted protein was separated
using SDS-PAGE. Proteins were detected against primary antibodies E-
cadherin, N-cadherin (BD Bioscience), Snail/Slug, Twist (Abcam),
and α-tubulin (Cell Signaling Technology). Primary antibodies were
detected with horseradish peroxidase-conjugated secondary antibodies
(Thermo Fisher Scientific). Band densities were detected under
luminescence imaging (iBright FL1000 Imaging System, Thermo
Fisher Sciences) and measured by Multi Gauge 3.0. Relative density
was calculated against the density of the α-tubulin bands. Values were
expressed as arbitrary densitometric units corresponding to signal
intensity.
28
10.8, CH3 0.82, t (5.8) 27a, 27b
25, 26,
27
26, 27
24, 26
29
15.6, CH3 0.90, d (5.4) 26
25-NH
6.71, d (7.6) 25
a
b
Measured at 100 MHz. Measured at 400 MHz.
after removing medium from the wells. Absorbance was measured at
540 nm using a microplate reader and analyzed with Gen 5 (2.03.1)
software. Percent relative cell viability was calculated by normalizing the
absorbance of cells treated with 1 by that of cells treated with DMSO.
IC50 was calculated using SPSS statistical software 23.
Flow Cytometric Analysis. Caco2 and HCT116 cells were seeded
in six-well plates at a density of (2.0−2.5) × 105 cells/well and treated
with 1 at cytotoxic concentrations (IC50 and 1.25 × IC50) for 6 h.26
Cells were harvested and washed with ice-cold phosphate-buffered
saline followed by adding 100 μL of 1× binding buffer, 6 μL of 50 μg/
mL PI, and 3 μL of annexin V−FITC. Cells were incubated in the dark
at room temperature for 15 min, and the apoptotic cell population was
measured by using a CytoFLEX flow cytometer.
RT2 Profiler PCR Array. The Human Cell Motility RT2 Profiler
PCR Array (Qiagen, SA Biosciences) was used to examine the
expression of cell motility-related genes from Caco2 cells treated with
1.27,28 The expression of 84 key genes related to cell motility was
profiled according to the manufacturer’s instructions. The fold change
of mRNA expression was calculated on the basis of the cycle threshold
values obtained from the RT-PCR experiment. The scatter plot of test
versus control samples indicated the validity of the experiment.
Statistical Analysis. All experiments were performed in triplicates.
All statistical analyses were performed using IBM Statistical Package for
Social Science (SPSS) version 22. Treatment effects were assessed by
one-way ANOVA posthoc analysis. Unless indicated otherwise, a p-
value of <0.05 was considered significant.
Transwell Migration Assay. Migration of Caco2 cells in response
to a chemoattractant was checked using noncoated Boyden chambers
(Corning Costar).27 In total, (2.0−2.5) × 105 cells were seeded in each
Boyden chamber containing 120 μL of cultural medium with 0.2%
F
https://dx.doi.org/10.1021/acs.jnatprod.0c00815
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
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* Supporting Information
The Supporting Information is available free of charge at
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AUTHOR INFORMATION
Corresponding Authors
Hangun Kim − College of Pharmacy, Sunchon National
University, Sunchon, Jeonnam 57922, Korea;
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William Fenical − Center for Marine Biotechnology and
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Chathurika. D. B. Gamage − College of Pharmacy, Sunchon
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Geum Jin Kim − College of Pharmacy and Research Institute of
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Prima F. Hillman − Department of Chemistry and Nanoscience,
Ewha Womans University, Seoul 03760, Korea
Chaeyoung Lee − College of Pharmacy, Ewha Womans
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Eun Young Lee − Department of Chemistry and Nanoscience,
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Hyukjae Choi − College of Pharmacy and Research Institute of
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Gyeongsangbukdo 38541, Korea; orcid.org/0000-0002-
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Complete contact information is available at:
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(25) Gamage, C. D. B.; Park, S. Y.; Yang, Y.; Zhou, R.; Tas, I.; Bae, W.
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Author Contributions
#J. Lee and C. D. B. Gamage contributed equally.
Notes
(26) Yang, Y.; Zhou, R.; Park, S. Y.; Back, K.; Bae, W. K.; Kim, K. K.;
Kim, H. Molecules 2017, 22, 453−460.
The authors declare no competing financial interest.
(27) Zhou, R.; Yang, Y.; Park, S. Y.; Nguyen, T. T.; Seo, Y. W.; Lee, K.
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ACKNOWLEDGMENTS
■
This research was funded by Basic Science Research Program
through the National Research Foundation of Korea (NRF) of
the Ministry of Science, ICT & Future Planning (NRF-
2017R1D1A1B03028172) and also supported by a Korea
Basic Science Institute (National Research Facilities and
Equipment Center) grant funded by the Ministry of Education
(2020R 1A 6C 101B194).
(28) Tas, I.; Han, J.; Park, S. Y.; Yang, Y.; Zhou, R.; Gamage, C. D. B.;
Van Nguyen, T.; Lee, J. Y.; Choi, Y. J.; Yu, Y. H.; Moon, K. S.; Kim, K.
K.; Ha, H. H.; Kim, S. K.; Hur, J. S.; Kim, H. Phytomedicine 2019, 56,
10−20.
(29) Yang, Y.; Nguyen, T. T.; Pereira, I.; Hur, J. S.; Kim, H.
Biomolecules 2019, 9, 797−809.
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https://dx.doi.org/10.1021/acs.jnatprod.0c00815
J. Nat. Prod. XXXX, XXX, XXX−XXX