An angiogenesis inhibitor isolated from a marine-derived actinomycete, Nocardiopsis sp. 03N67

Article abstract of DOI:10.1016/j.phytol.2010.07.005

Cyclo-(l-Pro-l-Met) was isolated from the fermentation broth of a marine-derived actinomycete Nocardiopsis sp. 03N67 by chromatographic analysis and showed anti-angiogenesis activity against human umbilical vein endothelial cells (HUVECs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic data analysis, and Marfey's method, respectively.

Full text of DOI:10.1016/j.phytol.2010.07.005

Phytochemistry Letters 3 (2010) 194–197
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
An angiogenesis inhibitor isolated from a marine-derived actinomycete,
Nocardiopsis sp. 03N67
a,b,
a,b
a
a
a
c
Hee Jae Shin *, M.A. Mojid Mondol , Tae Kyung Yu , Hyi-Seung Lee , Yeon-Ju Lee , Hye Jin Jung ,
c
c
Jong Hyeon Kim , Ho Jeong Kwon
a
Marine Natural Products Chemistry Laboratory, Korea Ocean Research & Development Institute, 1270 Sadong, Ansan 426-744, Republic of Korea
University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon, Republic of Korea
b
c
Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Cyclo-( -Pro- -Met) was isolated from the fermentation broth of a marine-derived actinomycete
Article history:
L
L
Received 9 April 2010
Nocardiopsis sp. 03N67 by chromatographic analysis and showed anti-angiogenesis activity against
human umbilical vein endothelial cells (HUVECs). The structure and absolute stereochemistry of this
compound were determined based on extensive spectroscopic data analysis, and Marfey’s method,
respectively.
Received in revised form 14 July 2010
Accepted 18 July 2010
Available online 30 July 2010
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Marine actinomycetes
Angiogenesis inhibitors
HUVECs
Diketopiperazine
Cyclo-(L-Pro-L-Met)
1
. Introduction
Angiogenesis is an essential step in tumor cells proliferation,
isolated a rare bioactive diketopiperazine, cyclo-(L-Pro-L-Met),
from a marine actinomycete Nocardiopsis sp. 03N67. In this paper,
we report the isolation, absolute stereochemistry and anti-
development, and metastasis (Risau, 1997). It is generally accepted
that there are two stages of tumor progression regarding its
vasculature (Folkman, 1995; Reynolds et al., 1992). In initial
vascular stage of tumor growth (tumor mass < 0.5 mm), it receives
nutrition and oxygen from blood by diffusion. When tumor mass
grows larger than 0.5 mm, nutrition through diffusion is not
sufficient for further growth (vascular stage) and therefore, the
formation of new blood vessels (angiogenesis) is necessary to draw
nutrition directly from the systemic circulation (Folkman, 1990;
Gimbrone et al., 1972). The tumor remains in a dormant state until
it can stimulate blood vessel growth from nearby pre-existing
capillaries (Folkman, 1972; Horak et al., 1992; Weidner et al.,
angiogenesis activity of cyclo-(L-Pro-L-Met, 1, Fig. 1).
2. Results and discussion
Compound 1 was readily identified as cyclo-(L-Pro-L-Met) by
comparison of its NMR (Table 1) and MS data with those reported
(Jayatilake et al., 1996). The molecular formula of compound 1 was
1
13
16 2 2
assigned as C10H N O S based on its H and C NMR and MS data.
This compound was first reported from an Antarctic sponge-
associated bacterium, Pseudomonas aeruginosa. Marfey’s method
was applied to assign the absolute configuration of the Pro and Met
residues resulting from acid hydrolysis of compound 1. HPLC
1
993). Inhibiting angiogenesis has been considered as an impor-
analysis of the 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide
tant anticancer strategy to suppress tumor growth and metastasis.
As marine environmental conditions are extremely different
from terrestrial ones, so that marine microorganisms have
different characteristics from those of terrestrial counterparts
and, therefore, might produce different types of bioactive
compounds in challenging living conditions (Lam, 2006). As a
part of our on going search for bioactive secondary metabolites, we
(FDAA, Marfey’s reagent) derivatives of the acid hydrolysate of
compound 1 gave the almost same -retention times as those
prepared from samples of authentic
that both amino acids have -configuration. The specific rotation
value of [ -82.28 reported for this known compound (Jayatilake
et al., 1996) and a similar specific rotation value of [ -80.0
L-Pro and L-Met, suggesting
L
a]
D
a]
D
observed for compound 1 suggested the same configuration for
both isolates. To the best of our knowledge, we first isolated
compound 1 from an Arctic seaweed-associated actinomycete
Nocardiopsis sp. 03N67. Additionally, the bioactivities of compound
1 have never been reported. Recent reports have shown that
*
Corresponding author.
E-mail address: shinhj@kordi.re.kr (H.J. Shin).
1
874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.07.005

[
(
F
i
g
.
_
1
)
T
D
$
F
I
G
]
H.J. Shin et al. / Phytochemistry Letters 3 (2010) 194–197
195
Fig. 1. Structure and key HMBC correlations of cyclo-(L-Pro-L-Met).
several diketopiperazines inhibit angiogenesis, thereby suppres-
sing tumor growth (Brooks et al., 2004; Maloney et al., 2009). To
explore the anti-angiogenic activity of compound 1, we investi-
gated the effect of compound 1 on angiogenic phenotypes of
endothelial cells. In our HUVECs based capillary tube formation
and invasion assays, compound 1 significantly blocked the
capillary tube formation induced by tumor necrosis factor alpha
anti-angiogenic agent. Cyclo-(
duced tube formation and invasion at 10
which no cytotoxicity was observed. However, it is unclear why
L
-Pro-
L
-Met) inhibited TNF-
a
-in-
m
M, a concentration at
[F(ig._2)TD$FIG]
(
a
TNF-
, cultured HUVECs sprouted incompletely on the Matrigel to form
narrow tube-like capillary whereas endothelial cell sprouting was
stimulated by TNF- resulting in extensive network like thick
a) in a dose dependent manner (Fig. 2). In the absence of TNF-
a
capillary tube formation. In case of invasion assay, migration/
invasion of HUVECs was reduced with increasing concentration of
compound 1 induced by TNF-a. Compound 1 showed concentra-
tion dependent angiogenesis inhibition in the both assays. Trypan
blue staining was performed in parallel with these angiogenesis
assays and cytotoxicity was not observed at concentrations used in
this study.
Diketopiperazines possess diverse biological activities such as
antitumor (Nicholson et al., 2006; van der Merwe et al., 2008),
antifungal (Houston et al., 2004), antibacterial (Fdhila et al., 2003),
and antihyperglycemic (Song et al., 2003) activities. Due to their
chiral, rigid, and functionalized structures, they bind to a large
variety of receptors with high affinity, giving a broad range of
biological activities (Martins and Carvalho, 2007). Therefore,
diketopiperazines are attractive structures for the discovery of
new lead compounds for the rational development of new
therapeutic agents.
In conclusion, cyclo-(L-Pro-L-Met) and the like diketopipera-
zines are the smallest cyclic peptides, commonly biosynthesized
from amino acids by different organisms, including mammals, and
are considered to be secondary functional metabolites or side
products of terminal peptide cleavage and mainly act as cell
signaling molecules. The bioactivities of cyclo-(L-Pro-L-Met) have
never been known. Here, we identified cyclo-(L-Pro-L-Met) as a new
Table 1
NMR data of cyclo-(
L
-Pro-
L
3
-Met) in CD OD.
1
13
Position
H (mult., J = Hz)
C
COSY
HMBC
Pro
1
172.7
60.4
29.4
23.6
46.5
2
4.24, t (1H, 7.8)
2.31, m (2H)
2.02, m (2H)
3.52, m (2H)
H-3
C-1, C-3
C-4, C-5
C-5
3
H-2, H-4
H-3, H-5
H-4
4
5
C-4
Fig. 2. Inhibition of angiogenic activities by cyclo-(
cyclo-( -Pro- -Met) on TNF- -induced capillary tube formation in HUVECs: (a) non-
treated cells. (b) TNF- (50 ng/mL) alone. (c) TNF- and cyclo-( -Pro- -Met) (10 M).
d) Quantitative analysis of tube formation. Arrows indicate narrow or broken tubes
L-Pro-L-Met) in vitro. (A) Effect of
L
L
a
Met
a
a
L
L
m
0
1
168.0
55.4
30.3
(
0
0
0
0
0
0
2
4.27, t (1H, 5.3)
2.20, m (1H)
H-3
C-1 , C-4
formed by TNF-
a
-induced HUVECs after cyclo-(
-induced invasion of HUVECs: (a) non-treated cells. (b)
(50 ng/mL) alone. (c) TNF- and cyclo-( -Pro- -Met) (10 M). (d) Quantitative
L-Pro-L-Met) treatment. (B) Effect of
0
0
3
H-2 , H-4
C-1, C-2
cyclo-(L-Pro-L-Met) on TNF-a
2.07, m (1H)
TNF-a
a
L
L
m
0
0
0
0
4
2.61, m (2H)
2.10, s (3H)
30.5
15.2
H-3
C-2 , S-CH
3
analysis of invaded cells. Each value represents mean ꢀ SE from three independent
S-CH
3
C-4
experiments. *p < 0.01 versus TNF-a control.

1
96
H.J. Shin et al. / Phytochemistry Letters 3 (2010) 194–197
this strain produces cyclo-(
genesis activity against HUVECs. The reason behind this may be the
deterrence of predation from eukaryotic organisms. Anti-angio-
L
-Pro-
L
-Met) which shows anti-angio-
(MPLC) and fraction 5 (33.1 mg) containing the compound cyclo-(
Pro- -Met) was purified by reversed-phase HPLC (YMC ODS-A
column, 250 ꢂ 10 mm i.d.: 40% MeOH; flow rate: 1.5 mL/min;
detector: RI, t : 26 min) to yield pure compound 1 (3.6 mg).
L-
L
genesis activity of cyclo-(
L
-Pro-
L-Met) is an encouraging bioprobe
R
to develop new anticancer therapeutics from such type of small
molecules in near future.
3.5. Absolute configuration of compound 1
3
. Experimental section
Marfey’s method was performed to determine the absolute
configuration of methionine and proline moiety in compound 1 as
3
.1. General experimental procedures
described previously (Marfey, 1984). In short, 100
was taken in 500 L glass vial and hydrolyzed by heating at
110 8C for 12 h with 200 L 6 N HCl. Upon removal of excess HCl
under reduced pressure, 0.1% solution (100 L) of 1-fluoro-2,4-
dinitrophenyl-5- -alanine amide (FDAA, Marfey’s reagent) in
acetone, followed by 1.0N NaHCO (20 L) was added to derivatise
the hydrolysate and heated at 40 8C for 1 h, cooled to room
temperature, and then neutralized with 1N HCl (20 L). In a
similar fashion, standard -Pro, -Pro, -Met, and -Met were
derivatized separately with Marfey’s reagent. The derivatives of
the acid hydrolysate and the standard amino acids were subjected
to RP-HPLC analysis (YMC ODS-A column, 50 mm ꢂ 2.0 mm i.d.) at
25 8C using the following gradient program: solvent A, MeOH with
mg of compound
1
m
Specific rotations were measured on a JASCO digital polarimeter
m
(DIP-1000) with a 0.1 dm cell at 23 8C. ESI-MS experiments were
m
performed on a Thermo Finnigan mass spectrometer. 1D and 2D
NMR spectra were obtained on a Varian Unity 500 spectrometer
L
3
m
1
13
(
H at 500 MHz, 20 8C and C at 125 MHz, 24 8C). The resonances
of residual CD OD-d at 3.31 and 49.15 were used as internal
references for H and C NMR spectra, respectively. HPLC was
performed using a PrimeLine Binary pump with a Shodex detector
3
4
d
H
13
d
C
m
1
L
D
L
D
(RI-101). IR spectra were recorded on a JASCO FT/IR-4100. UV
spectra were measured in methanol using a Shimadzu spectro-
photometer (UV-1650PC). All solvents used were either spectral
grade or were distilled prior to use.
0.1% TFA; solvent B, CH
of A for 40 min with UV detection at 340 nm; flow rate: 0.2 mL/
min. The retention times for the FDAA derivatives of -Pro, -Pro,
Met, and -Met were 22.30, 24.87 30.41, and 37.10 min,
3
CN with 0.1% TFA; linear gradient, 10–40%
3.2. Isolation and taxonomy of strain 03N67
L
D
L-
D
The strain 03N67 was isolated from a seaweed (Undaria
pinnatifida) sample collected from the Arctic expedition in 2003
respectively, whereas those for the FDAA derivatives of Pro and
Met in the hydrolysate of compound 1 were 22.80 and 31.13 min,
respectively.
by serial dilution technique. In brief, one gram of the seaweed
ꢁ
1
ꢁ2
sample was blended, diluted in sterilized sea water (10 , 10 ,
ꢁ
3
ꢁ4
1
0
and 10 ) in aseptic conditions and 100
m
L from each dilution
3.6. Characteristics of compound 1
were spread on modified Bennett’s agar medium (0.1% yeast
extract, 0.1% beef extract, 0.2% tryptone, 1% dextrose, 100% natural
sea water, 1.8% agar and pH 7.2 before sterilization). The plates
were incubated for 14 days at 30 8C, and the resulting colony of
strain 03N67 was isolated and maintained on the modified
Bennett’s agar. The strain 03N67, which formed well-developed
white substrate mycelium and aerial mycelium on modified
Bennett’s agar medium. The strain was identified as Nocardiopsis
sp. on the basis of 16S rDNA sequence analysis. The strain is
currently deposited in the Microbial Culture Collection, KORDI,
with the accession number Nocardiopsis sp. 03N67 under the
curatorship of H.J.S.
1
13
D
White amorphous solid; [a] -80.0 (c 0.05, MeOH); H, C and
2D NMR data in CD
3
OD given in Table 1. ESI-MS: at m/z 251
+
ꢁ
(M + Na) and at m/z 227 (M ꢁ H) , calcd. 228 for C10
max
16 2 2
H N O S;
ꢁ
1
l
(
e
) 210 (11255); IR (neat)
n
max at 3709 and 1705 cm
.
3.7. Tube formation assay
Matrigel (10 mg/mL) was added to a 48-well plate and allowed
4
to polymerize for 1 h at 37 8C. HUVECs (6 ꢂ 10 cells) were seeded
onto the surface of the Matrigel in a 48-well plate. Compound 1
was then added for 6–18 h at 37 8C in the presence or absence of
TNF-a. The morphological changes of the cells and tubular
3
.3. Seed and mass cultures of strain 03N67
structure formed were observed under a microscope (IX71,
Olympus) and photographed at 100ꢂ magnification with a camera
(DP70, Olympus).). Tube formation was quantified by counting the
number of connected cells in randomly selected fields at 100ꢂ
magnification and dividing that number by the total number of
cells in the same field.
The composition and pH of the seed medium were same as
modified Bennett’s medium. The medium was dispensed at 50 mL
in 250 mL conical flask. A single colony from the agar plate was
inoculated into the flask and incubated at 30 8C at 120 rpm for 3
days. An aliquot (0.2%, v/v) from the seed culture was inoculated
into a 20 L fermenter containing 17 L of culture medium. The
composition of the mass culture medium was same to that of the
seed culture medium. The production culture was carried out at
3.8. Invasion assay
The invasiveness of HUVECs was examined in vitro using a
3
0 8C at 120 rpm for 7 days and then harvested.
Transwell chamber system with polycarbonate filter inserts with
8
.0-
with gelatin (10
Matrigel (10
L, 3 mg/mL). HUVECs (7 ꢂ 10 cells) were placed in
the upper part of the filter, and compound 1 was added to the
lower parts in the presence of TNF- (50 ng/mL). Then the
m
m-sized pores. Briefly, the lower side of the filter was coated
3.4. Extraction and isolation
m
L, 1 mg/mL), and the upper side was coated with
4
m
The fermentation broth (17 L) was centrifuged at 60,000 rpm by
using continuous centrifuge, and the supernatant was extracted
with EtOAc (17 L ꢂ 2). The EtOAc layer was concentrated to
dryness using a rotary evaporator under reduced pressure at 40 8C.
The residual suspension (1.2 g) was subjected to ODS open
chromatography with a stepwise gradient mixture of MeOH and
a
chamber was incubated at 37 8C for 18 h. The cells were fixed with
70% MeOH and stained with hematoxylin and eosin. The cell
invasiveness was measured by counting the whole-cell numbers
on the lower side of the filter using a microscope at 100ꢂ
magnification. Cells were also photographed at 100ꢂ magnifica-
tion with a camera.
H
2
O as eluant. The fraction eluted with MeOH: H
2
O (2:3, v/v) was
further fractionated by medium pressure liquid chromatography

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.9. Cell proliferation assay
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Maritime Affairs, Korea, Korea Ocean Research and Development
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