Antiangiogenic activity of 3,4-seco-cycloartane triterpenes from Thai Gardenia spp. and their semi-synthetic analogs

Article abstract of DOI:10.1016/j.bmcl.2011.10.128

Twelve naturally occurring 3,4-seco-cycloartane triterpenes (1-12) isolated from Gardenia sootepensis and Gardenia obtusifolia, and eight semi-synthetic derivatives (13-20) were evaluated for their antiangiogenic activity on a rat aortic sprouting assay,

Full text of DOI:10.1016/j.bmcl.2011.10.128

Bioorganic & Medicinal Chemistry Letters 22 (2012) 512–517
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiangiogenic activity of 3,4-seco-cycloartane triterpenes from Thai Gardenia
spp. and their semi-synthetic analogs
Khanitha Pudhom , Thanesuan Nuanyai a,b, Kiminori Matsubara c, , Tirayut Vilaivan a
a,
⇑
⇑
a
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Center for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Bangkok 10330, Thailand
Department of Human Life Science Education, Graduate School of Education, Hiroshima University, Hiroshima 739-8524, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2011
Revised 7 October 2011
Accepted 27 October 2011
Available online 16 November 2011
Twelve naturally occurring 3,4-seco-cycloartane triterpenes (1–12) isolated from Gardenia sootepensis
and Gardenia obtusifolia, and eight semi-synthetic derivatives (13–20) were evaluated for their antiangio-
genic activity on a rat aortic sprouting assay, an ex vivo model of angiogenesis. Among these compounds,
sootepin B (1) displayed the most potent activity in terms of the inhibition of microvessel sprouting from
rat aortic rings in a dose-dependent manner with IC50 value of 4.46 lM. Its angiogenic effect was found to
occur via suppression of endothelial cell proliferation and tubular formation, and was likely mediated by
regulation (inhibition) of the Erk1/2 signaling pathway.
Keywords:
3
,4-seco-cycloartane
Ó 2011 Elsevier Ltd. All rights reserved.
Gardenia
Antiangiogenesis
Cancer is one of the important public health problems all over the
world and continues to cause a large number of present deaths,
including pre-reproductive deaths, and the per capita population le-
vel appears to be increasing in the affected populations. Despite
many anticancer drugs having been discovered and developed for
use in clinical treatments over the last few decades, most of available
drugs are non-selective cytotoxic molecules that provoke severe ad-
verse effects, as well as acquiring drug resistance. Therefore the
discovery and development of new effective chemotherapeutic
agents is still required in the fight against cancer.
the genetic stability of endothelial cells whose migration and pro-
liferation are involved in the angiogenesis process.
Ring-A 3,4-seco-cycloartane type triterpenes have been mainly
found in plants belonging to the genus Gardenia from family Rubi-
6
–11
ceae.
Many of them have been found to exhibit cytotoxic activ-
6
–10
ities towards various tumor cell lines.
Recently we reported the
isolation and characterization of a number of these triterpenoids
from the apical buds and their exudate of some Thai Gardenia spe-
cies that exhibit in vitro cytotoxicity against five human cancer
1
2–15
lines.
However, the effect of these triterpenes on angiogenesis
The use of antiangiogenic therapy as an alternative treatment
for cancer patients has attracted considerable attention from
researchers in recent years since the development and progression
of tumors is crucially dependent upon angiogenesis.1 Indeed,
angiogenesis, the formation of new blood vessels from the pre-
existing vasculature, is not only required for tumor growth and
survival, but also for the transplantation and metastasis of tu-
mors. The newly formed blood vessels promote tumor progres-
sion by supplying oxygen and nutrients in sufficient amounts
and by removing the waste metabolites. Thus the inhibition of tu-
mor angiogenesis is one of the most promising current approaches
in the cancer prevention and treatment.5 Furthermore, therapy
based upon targeting angiogenesis can be applied for a broad spec-
trum of tumors because angiogenesis is required for all of them,
yet it has a low potential for the evolution of resistance due to
is seldom investigated. In this Letter, we focus on the assessment of
the antiangiogenic activity of 3,4-seco-cycloartanes obtained from
Gardenia sootepensis and Gardenia obtusifolia and their semi-syn-
thetic analogs in order to gain more information about the struc-
ture–activity relationship (SAR).
,2
The twelve naturally occurring 3,4-seco-cycloartane triterpenes
used in this study were isolated from the apical buds of G. sootepen-
3
,4
12,13
sis and the exudate of G. obtusifolia as previously reported.
These
are sootepin B (1), coronalolide (2), turbiferolide methyl ester (3),
sootepin A (4), coronalolide methyl ester (5), gardenoins B and C
(6, 7), sootepins C and E (8, 9), secaubryenol (10), and dikamakiar-
tanes A and C (11, 12). As for the semi-synthetic derivatives, the first
compound series (13–17) were prepared from coronalolide (2) by
modification of the COOH at C-1 to ester and amide functions using
the combination of DCC (EDC for 17) and HOBt as the coupling
agent. The second series of compounds (18–20) were obtained by
modification of the primary alcohol of sootepin A (4). The benzoate
⇑
Corresponding authors. Tel.: +66 2 2187639; fax: +66 2 2541309.
E-mail addresses: Khanitha.P@chula.ac.th (K. Pudhom), kmatsuba@hiroshima-u.
1
8 was prepared by treating 4 with benzoyl chloride in the presence
ac.jp (K. Matsubara).
of Et N. Reactions of 4 with the corresponding acids, pretreated with
3
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.128

K. Pudhom et al. / Bioorg. Med. Chem. Lett. 22 (2012) 512–517
513
oxalyl chloride in the presence of a catalytic amount of DMF to
generate the acid chlorides, afforded 19 and 20. The structures of
the modified compounds, shown in Figure 1, were confirmed by
spectroscopic data (see Supplementary data).
and amidation of the C-1 carboxylic acid (3–5, 13–20), as well as
the presence of highly oxidized functionalities in place of the
methyl group at C-26 (2, 4, 5, 13–20) caused a significant loss of
activity, revealing from only 40–60% and 10–50% inhibition,
respectively (Fig. 2). Similar results were obtained from the deriv-
atives with a cleaved C-6–C-29 bond (8–12). The free COOH at C-1
is also required for activity as observed in 9 and 10, but they exhib-
ited a lower activity than 1, which suggests that the exomethylene
To investigate the SARs of both the natural and the semi-syn-
thetic derivatives, their effect on angiogenesis was evaluated by
monitoring the inhibition of microvessel sprouting from rat aortic
16
rings (an ex vivo model) at a single dose of 25
pounds possessing an exomethylene -lactone (1–7 and 13–20),
the naturally occurring sootepin B (1), bearing a free COOH at C-
and Me at C-26, showed the strongest antiangiogenic activity,
lM. Among com-
c
c-lactone (formed by lactonization of C-29 onto C-6) plays a crucial
role for the antiangiogenic effect. Furthermore, there was no
apparent significant or only a slight inhibition of angiogenesis by
the four derivatives having a furan ring in the side chain (6–7,
11–12).
Based on the results obtained from the screening using the
ex vivo microvessel sprouting model, only sootepin B (1) was cho-
sen for further evaluation, starting with its potency to suppress
microvessel outgrowth by treating aortic rings with various doses
1
completely suppressing the microvessel sprouting, followed by
coronalolide (2) at ꢀ85% inhibition, but their semi-synthetic deriv-
atives were all less active. For the five natural compounds without
an exomethylene c-lactone group, sootepin E (9) showed the high-
est anti-angiogenic activity, at ꢀ92% inhibition, which was the sec-
ond highest of all 20 compounds after that of 1. The esterification
Figure 1. Structures of the naturally occurring 3,4-seco-cycloartanes (1–12) and their semisynthetic derivatives (13–20).

5
14
K. Pudhom et al. / Bioorg. Med. Chem. Lett. 22 (2012) 512–517
Figure 2. Effect of each of 3,4-seco-cycloartanes (1–20) at 25
l
M on the ex vivo angiogenesis rat aortic sprouting assay as an angiogenesis model. Values are the mean ± SD
⁄
⁄⁄
(n = 6). Significantly different from the control: p <0.05 and p <0.01.
Figure 3. Inhibitory effect of sootepin B (1) at various concentrations on the ex vivo rat aortic sprouting assay. (a) Microscopic photograph of microvessel formation. (b) The
⁄
⁄
average microvessel length on the seventh day of culture. Values are the mean ± SD (n = 6). Significantly different from the control: p <0.01.
of the compound. As shown in Figure 3, the inhibitory effect of 1
of human umbilical vein endothelial cells (HUVECs), in terms of
was found to be dose-dependent manner with an IC50 value of
the in vitro cell proliferation, tubular formation and migration,
4
.46
To investigate how sootepin B (1) could exert the anti-angio-
genic effect, its in vitro inhibitory activity towards the functions
l
M.
were evaluated. The effect of 1 on the proliferation of HUVECs
was evaluated by using the WST-8 assay, following treatment
of HUVECs with various concentrations of 1 or vehicle (DMSO)
1
7

K. Pudhom et al. / Bioorg. Med. Chem. Lett. 22 (2012) 512–517
515
inhibition at 20
lM (Fig. 5). Finally, the effect of sootepin B (1) on
the VEGF-induced migration of HUVECs was examined using a
2
1
modified Boyden chamber assay. VEGF is known as a specific
and pivotal growth factor involved in endothelial cell proliferation,
migration and survival during blood vessel formation.2
2,23
HUVECs
were seeded on cell culture inserts with sootepin B (1) at concen-
trations ranging from 5 to 50 M and VEGF (10 ng/mL). After incu-
l
bation for 6 h, the membranes containing migrated cells were
stained and cut off from the inserts. The number of migrated cells
was then counted under a microscope. As seen in Supplementary
Figure S9, VEGF strongly stimulated HUVECs migration and soote-
pin B (1) did not display any detectable effect upon this, even at a
dose as high as 50 lM. Thus 1 might not be involved in the VEGF-
induced endothelial cell migration.
Based on these results from the above ex vivo and in vitro as-
says, sootepin B (1) would appear to be a potent angiogenic inhib-
itor, and that it mainly functions by suppression of endothelial cell
proliferation and tubule formation, but has no effect upon cell
migration.
Figure 4. Effect of sootepin B (1) on the HUVEC proliferation. Values are the
mean ± SD (n = 5). Significantly different from the control: p <0.05 and p <0.01.
⁄
⁄⁄
The mechanism by which sootepin B (1) regulates angiogenic
inhibition was then investigated by western blot analysis.24 In
angiogenesis there are two known important pathways responsi-
ble for VEGF stimulus on endothelial cells, the phosphoinositide
3-kinase/Akt and the extracellular signal-regulated kinase (Erk1/
2) pathways. Both of these pathways are reported to be involved
in endothelial cell proliferation, migration and survival.25 The
treatment of HUVECs with sootepin B (1) suppressed the phos-
phorylation of Erk1/2 in both a time- and dose-dependent manner
(Fig. 6), but did not affect the expression of phosphorylated Akt
(data not shown). This suggested that the antiangiogenic property
of sootepin B (1) is mediated by alteration of the Erk1/2 signaling
pathway. The strong suppressive effect of sootepin B (1) on the
alone for 72 h. The results (Fig. 4) indicated that sootepin B (1)
inhibited the proliferation of the endothelial cells in a dose-depen-
dent manner with an IC50 value of 1.20
B (1) could affect the HUVECs tubule formation was investigated,
by inoculating HUVECs onto reconstituted basement membranes
lM. Next, whether sootepin
1
8
(
Matrigel) and then allowing them to migrate, attach to each other
19,20
and then form capillary-like structures.
The effect of the com-
pound was assessed by measuring the length of tube-structured
cells compared to the vehicle control after incubation for 12 h.
Sootepin B (1) was found to suppress tubule formation in the endo-
thelial cells in a dose dependent manner with a significant inhibi-
tion being observed at 5 lM, an IC50 of 11.20 lM and the complete
Figure 5. Effect of sootepin B (1) on the HUVEC tubular formation. (a) Microscopic photograph of tubular formation on the reconstituted gel after incubation for 12 h. (b) The
⁄
⁄
average capillary length. Values are the mean ± SD (n = 5). Significantly different from the control: p <0.01.

5
16
K. Pudhom et al. / Bioorg. Med. Chem. Lett. 22 (2012) 512–517
Figure 6. Effect of sootepin B (1) concentration on the phosphorylation level of Erk1/2 in HUVECs. (a) HUVECs were pretreated with various doses of sootepin B (1) or vehicle
(
DMSO) alone for 1 h. (b) The level of phosphorylated (pErk1/2) and total Erk1/2 protein was determined by western blot analysis. Values are the mean ± SD (n = 3).
⁄
Significantly different from the control: p <0.05.
phosphorylation of Erk1/2 is in line with a previous report in which
inhibitors for Erk1/2 signaling pathway suppress endothelial cell
proliferation. Furthermore the inactive effect of sootepin B (1)
on migration is consistent with the absence of any detectable effect
on the phosphorylation of Akt since this pathway plays an impor-
tant role in endothelial cell migration.27
In summary, a series of 3,4-seco-cycloartane triterpenes, com-
prised of twelve naturally occurring and eight semi-synthetic
derivatives, were evaluated for their antiangiogenic activity. The
results indicated that the natural compound sootepin B (1) pos-
sesses a potent angiogenic activity, presumably mediated via sup-
pression of endothelial cell proliferation and tubular formation. Its
effect on angiogenesis might be mediated by regulation of the
Erk1/2 signaling pathway. Therefore sootepin B (1) might be
potentially useful in cancer therapy as an angiogenic inhibitor.
References and notes
2
6
1. Folkman, J.; Shing, Y. J. Biol. Chem. 1992, 267, 10931.
2
3
.
.
Folkman, J. Nat. Med. 1995, 1, 27.
Carmeliet, P.; Jain, R. K. Nature 2000, 407, 2249.
4. Bergers, G.; Benjamin, L. E. Nat. Rev. Cancer 2003, 3, 401.
5
6
.
.
Herbst, R. S. Expert Opin. Emerg. Drugs 2006, 11, 635.
Silva, G. L.; Gil, R. R.; Cui, B.; Chai, H.; Santisuk, T.; Srisook, E.; Reutrakul, V.;
Tuchinda, P.; Sophasan, S.; Sujarit, S.; Upatham, S.; Lynn, S. M.; Farthing, J. E.;
Yang, S.-L.; Lewis, J. A.; O’Neill, M. J.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J.
M.; Kinghorn, A. D. Tetrahedron 1997, 53, 529.
7.
Tuchinda, P.; Pompimon, W.; Reutrakul, V.; Pohmakotr, M.; Yoosook, C.;
Kongyai, S.; Sophapan, S.; Sujarit, K.; Upatham, S. E.; Santisuk, T. Tetrahedron
2002, 58, 8073.
8. Reutrakul, V.; Krachangchaeng, C.; Tuchinda, P.; Pohmakotr, M.; Jaipetch, T.;
Yoosook, C.; Kasisit, J.; Sophapan, S.; Sujarit, K.; Santisuk, T. Tetrahedron 2004,
6
0, 1517.
9.
Tuchinda, P.; Saiai, A.; Pohmakotr, M.; Yoosook, C.; Kasisit, J.; Napaswat, C.;
Santisuk, T.; Reutrakul, V. Planta Med. 2004, 70, 366.
1
1
0. Grougnet, R.; Magiatis, P.; Mitaku, S.; Loizou, S.; Moutsatsou, P.; Terzis, A.;
Cabalion, P.; Tillequin, F.; Michel, S. J. Nat. Prod. 2006, 69, 1711.
1. Kunert, O.; Sreekanth, G.; Babu, G. S.; Adavi Roa, B. V. R.; Radhakishan, M.;
Kumar, B. R.; Saf, R.; Appa Roa, A. V. N.; Schühly, W. Chem. Biodivers. 2009, 6,
Acknowledgments
1
185.
This work was supported by the Thailand Research Fund (Grant
no. DBG5380038), the Hitachi Scholarship Foundation, Research
Funds from the Faculty of Science (RES-A1B1-23), Chulalongkorn
University, and the 90th Anniversary of Chulalongkorn University
Fund (Ratchadaphisek Somphot Endowment Fund). The authors
are also grateful for the Thai Government Stimulus Package
1
2. Nuanyai, T.; Sappapan, R.; Teerawatananond, T.; Muangsin, N.; Pudhom, K. J.
Nat. Prod. 2009, 72, 1161.
13. Nuanyai, T.; Chokpaiboon, S.; Vilaivan, T.; Pudhom, K. J. Nat. Prod. 2010, 73, 51.
1
4. Nuanyai, T.; Sappapan, R.; Vilaivan, T.; Pudhom, K. Chem. Pharm. Bull. 2011, 59,
85.
5. Nuanyai, T.; Sappapan, R.; Vilaivan, T.; Pudhom, K. Phytochem. Lett. 2011, 4, 26.
3
1
16. Ex vivo angiogenesis assay. The rats were maintained according to the guide
for the care and use of laboratory animals established by Hiroshima University.
All experiments using animals were approved by Hiroshima University Animal
Research Committee. A six-week-old specific pathogen free male Wistar rat
was sacrificed by bleeding from the right femoral artery under anesthesia with
diethyl ether. The thoracic aorta was removed, washed with RPMI 1640
medium, turned inside out, and cut into 1-mm lengths. The aortic rings were
then placed on 6-well culture plate and covered with 0.5 mL of gel matrix
solution (8 volumes of porcine tendon collagen solution, 1 volume of 10Â
Eagle’s MEM and 1 volume of reconstitution buffer), and then allowed to gel at
(
TKK2555) under the Project for Establishment of Comprehensive
Center for Innovative Food, Health Products and Agriculture.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.128.

K. Pudhom et al. / Bioorg. Med. Chem. Lett. 22 (2012) 512–517
517
3
7 °C for 30 min. Two millilitre of RPMI 1640 medium containing 1% (v/v) of
TIS+ with the designated doses of the test compounds or vehicle (DMSO) were
added to the wells. After incubation for 7 days at 37 °C in 5% CO , the capillary
added with 400
human recombinant VEGF with or without sootepin B (1). The assembled
chamber was incubated for 6 h at 37 °C in 5% CO . Non-migrated cells on the
lL of Medium 199 containing 0.1% (w/v) BSA and 10 ng/mL of
2
2
length was estimated under phase-contrast microscopy by measuring the
distance from the cut end of the aortic segment to the approximate mid-point
of the capillary.
surface of the membrane were removed by scrubbing with a cotton swab. The
migrated cells were then fixed with methanol, stained with Diff-Quik stain
(Sysmex, Kobe, Japan), and then counted on three randomly selected fields of
view for each membrane under a light microscope at 200Â magnification.
22. Ferrara, N. Curr. Top. Microbiol. Immunol. 1999, 237, 1.
23. Shinkaruk, S.; Bayle, M.; Laïn, G.; Déléris, G. Curr. Med. Chem. – Anti-Cancer
Agents 2003, 3, 95.
24. Western blot analysis. HUVECs were grown to confluence and then starved for
16 h in RPMI 1640 containing 5%(v/v) FBS. Cells were pretreated for 60 min
1
1
7. HUVEC proliferation assay.
A
HUVEC suspension in HuMedia EG2
1.5 Â 10 cells/mL) was seeded into each well of 96-well plate (100 L) and
incubated for 24 h at 37 °C in 5% CO . The medium was then removed and
replaced with fresh HuMedia EG2 containing various doses of sootepin B (1) or
vehicle (DMSO) and incubated for 72 h at 37 °C in 5% CO . Cell proliferation
4
(
l
2
2
was detected using the WST-8 reagent, and the inhibition of proliferation was
measured at 450 nm using a microplate reader.
with sootepin B (0–10 lM) or vehicle (DMSO) alone and stimulated by the
8. HUVEC tube formation assay. HUVEC tube formation was assayed using a BD
addition of VEGF (10 ng/mL) for 10 min. After stimulation, cells were washed
twice with PBS and then lysed with mammalian protein extraction reagent (M-
PER; Pierce, Rockford, IL, USA). Lysates were clarified by centrifugation for
15 min at 12000 rpm at 4 °C. The protein concentration was determined using
the microBCA protein assay kit (Pierce, Rockford, IL, USA) with BSA as standard.
Matrigel.1
9,20
The solid gel was prepared in a 96-well tissue culture plate
5
according to the manufacture’s instruction. HUVECs (1 Â 10 cells/mL) in
HuMedia EG2 medium containing various doses of sootepin B (1) or vehicle
(
DMSO) alone were seeded onto the surface of the solid BD Matrigel and
incubated for 12 h at 37 °C in 5% CO . Tube formation was then observed under
2
For each sample, 50
gel electrophoresis under reducing conditions and then transferred to
lg of total protein was separated by SDS polyacrylamide
an inverted light microscope at 40Â magnification. Microscopic fields were
a
photographed with a digital camera (OLYMPUS DSE330-A system).
nitrocellulose membrane. After blocking, the membranes were incubated with
primary antibody for phopho-Erk1/2 or phospho-Akt (Cell Signaling
Technology, Tokyo, Japan) at 4 °C overnight and for Erk1/2 or Akt at room
temperature, respectively. Immunoreactive bands were visualized by
chemiluminescence using a WesternBreeze (Invitrogen) with a ChemiDoc
XRS (BIO-RAD).
1
2
9. Kayashima, T.; Mori, M.; Yoshida, H.; Mizushina, Y.; Matsubara, K. Cancer Lett.
2
009, 278, 34.
0. Kayashima, T.; Mori, M.; Mizutani, R.; Nishio, K.; Kuranochi, K.; Tsubaki, K.;
Yoshida, H.; Mizushina, Y.; Matsubara, K. Bioorg. Med. Chem. Lett. 2010, 18,
6
305.
2
1. HUVEC migration assay. HUVEC migration was assayed using a modified
25. Zachary, I. Biochem. Soc. Trans. 2003, 31, 1171.
Boyden chamber.1
9,20
A microporous membrane (8
l
m) as an insert inside each
26. Meadows, K. N.; Bryant, P.; Pumiglia, K. J. Biol. Chem. 2001, 276, 49289.
27. Goetze, S.; Eilers, F.; Bungenstock, A.; Kintscher, U.; Stawowy, P.; Blashchke, F.;
Graf, K.; Law, R. E.; Fleck, E.; Grafe, M. Biochem. Biophys. Res. Commun. 2002,
293, 1431.
well of 24-well cell culture plates was coated with 0.1%(w/v) gelatin. The
5
HUVEC suspension (2.5 Â 10 cells/mL) in Medium 199 with 0.1% (w/v) bovine
serum albumin was seeded into each chamber (400 lL) and to each well was