Cucurbitacin E as a new inhibitor of cofilin phosphorylation in human leukemia U937 cells

Article abstract of DOI:10.1016/j.bmcl.2010.02.062

Cucurbitane-type triterpenes, cucurbitacins B and E, were reported to exhibit cytotoxic effects in several cell lines mediated by JAK/STAT3 signaling. However, neither compound inhibited phosphorylation of STAT3 in human leukemia (U937) cells at low concentrations. We therefore synthesized a biotin-linked cucurbitacin E to isolate target proteins based on affinity for the molecule. As a result, cofilin, which regulates the depolymerization of actin, was isolated and suggested to be a target. Cucurbitacins E and I inhibited the phosphorylation of cofilin in a concentration-dependent manner, and their effective concentrations having the same range as the concentrations at which they had cytotoxic effects in U937 cells. In addition, the fibrous-/globular-actin ratio was decreased after treatment with cucurbitacin E in HT1080 cells. These findings suggested that the inhibition of cofilin's phosphorylation increased the severing activity of cofilin, and then the depolymerization of actin was enhanced after treatment with cucurbitacin E at lower concentrations.

Full text of DOI:10.1016/j.bmcl.2010.02.062

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2994–2997
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cucurbitacin E as a new inhibitor of cofilin phosphorylation in human
leukemia U937 cells
Souichi Nakashima a, Hisashi Matsuda a, Ai Kurume b, Yoshimi Oda a, Seikou Nakamura a,
Masayuki Yamashita b, Masayuki Yoshikawa a,
*
a Department of Pharmacognosy, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
b Department of Functional Molecular Chemistry, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cucurbitane-type triterpenes, cucurbitacins B and E, were reported to exhibit cytotoxic effects in several
cell lines mediated by JAK/STAT3 signaling. However, neither compound inhibited phosphorylation of
STAT3 in human leukemia (U937) cells at low concentrations. We therefore synthesized a biotin-linked
cucurbitacin E to isolate target proteins based on affinity for the molecule. As a result, cofilin, which reg-
ulates the depolymerization of actin, was isolated and suggested to be a target. Cucurbitacins E and I
inhibited the phosphorylation of cofilin in a concentration-dependent manner, and their effective con-
centrations having the same range as the concentrations at which they had cytotoxic effects in U937 cells.
In addition, the fibrous-/globular-actin ratio was decreased after treatment with cucurbitacin E in
HT1080 cells. These findings suggested that the inhibition of cofilin’s phosphorylation increased the
severing activity of cofilin, and then the depolymerization of actin was enhanced after treatment with
cucurbitacin E at lower concentrations.
Received 8 January 2010
Revised 12 February 2010
Accepted 16 February 2010
Available online 25 February 2010
Keywords:
Cucurbitane-type triterpene
Cucurbitacin E
Cytotoxic effect
Cofilin
Target protein
Biotin-linked cucurbitacin E
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis plays an important role in the maintenance of tissue
homeostasis through the selective elimination of excessive cells.
On the other hand, induction of apoptosis of cancer cells is also
useful for treatment of cancer, since the cytotoxic drugs (e.g., eto-
poside, cisplatin, and paclitaxel) used in chemotherapy cause
apoptosis in target cells.1–4
Previously, cucurbitacins B, E (1), and I (2) and several related
compounds were reported to show cytotoxic effects in several cell
lines such as A549, MDA-MB-468, HepG2, and KB, and their apop-
tosis-inducing activities mediated by the inhibition of Janus kinase
(JAK)/signal transducer and activator of transcription 3 (STAT3)
signaling.5–10 We have recently reported that cucurbitacins B and
E (1) isolated from the roots of Bryonia cretica showed strong cyto-
U937, HL60, and HT1080 cells, respectively, after incubation for
72 h. These findings confirm the importance of an acetoxy group
at the 25-position.11,17
With regard to effects on the cell cycle, inhibitors of JAK/STAT3
signaling are reported to induce arrest at G0/G1 in several human
and mouse cancer cell lines.18–20 However, few effects of cucurbi-
tan-type triterpenes on cell cycle arrest have been reported to date,
though a weaker active 23,24-dihydrocucurbitacin B was reported
to induce arrest at G2/M in human breast cancer cells (Bcap37).21
Therefore, we examined the effect of cucurbitacin E (1) on the cell
cycle in U937 cells by flow cytometry after staining with propidi-
um iodide (PI).22
Consistent with a previous report on 23,24-dihydrocucurbitacin
B,21 the population of cells in the G0/G1 and S phases was reduced
[G0/G1 (control: 45.1%, 1: 37.7%), S (control: 44.0%, 1: 28.1%)] and
that in G2/M was increased (control: 10.9%, 1: 34.2%) by treatment
with 1 (100 nM) for 24 h, indicating that 1 caused arrest at G2/M.
Furthermore, 1 did not inhibit the phosphorylation of STAT3 at
toxic effects in human leukemia (U937) cells, and an
a,b-conju-
gated ketone moiety at the 22–24-positions and an acetoxy
group at the 25-position are important for the greater activity.11
In the present study, we compared effects of the active triter-
pene, cucurbitacin E (1),12,13 and a moderate active triterpene,
cucurbitacin I (2),14,15 on proliferation of U937, human leukemia
(HL60), and human fibrosarcoma (HT1080) cells (Fig. 1).16 Com-
pound 1 showed greater activity than 2; IC50 values of 1 were 16,
concentration 10
lM, and inhibit it at a higher concentration
(10
l
M) in U937 cells (Fig. 2).23 Similar results were observed after
treatment with cucurbitacin B (data not shown). Since our results
are not consistent with reports about an inhibitor of STAT3 phos-
phorylation, other mechanisms of action of cucurbitane-type tri-
terpenes were considered to exist.
18, and 40 nM, and those of 2 were 0.30, 0.10 and 0.47 lM against
* Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595 4768.
E-mail addresses: shoyaku@mb.kyoto-phu.ac.jp, myoshika@mb.kyoto-phu.ac.jp
(M. Yoshikawa).
Next, we synthesized a biotin-linked cucurbitacin E (3)24,25 and
tried to isolate target proteins based on its affinity for the
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.062

S. Nakashima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2994–2997
2995
O
H
O
HO
HO
OH
H
OAc
O
H
OH
O
H
OH
HO
O
HO
O
cucurbitacin I (2)
cucurbitacin E (1)
O
HO
H
OAc
O
H
OH
O
S
H
N
O
O
H
N
H
H
NH
HN
O
O
O
3
Figure 1. Chemical structures of cucurbitacins E (1) and I (2) and biotin-linked cucurbitacin E (3).
Cucurbitacin E (1)
molecule. Briefly, cell lysate without SDS was treated with 3 and
passed through a streptavidin-column. After washing with a wash-
ing buffer, bound proteins were eluted with an elution buffer and
separated by SDS–PAGE (Fig. 3). A band at 20 kDa that was ex-
cluded by addition of a high concentration of 1, considered specific
to 1, was excised from the gel. After in-gel digestion, tryptic pep-
tides were subject to LC–MS/MS analysis.26 As a result, cofilin
was isolated and suggested to be a target protein. Cofilin is a mem-
ber of a family of essential conserved small actin-binding proteins
that play pivotal roles in cytokinesis, endocytosis, embryonic
development, stress response, and tissue regeneration.27 In re-
sponse to stimuli, cofilin promotes the regeneration of actin fila-
0
10
100 (µM)
p-STAT3
(Y705)
STAT3
Figure 2. Effects of cucurbitacin E (1) on phosphorylation of STAT3 in U937 cells.
U937 cells (5.0 ꢀ 104 cells/mL) were incubated with the test compound for 24 h.
Equivalent amount of protein (60 lg of protein/lane) of each lysate was electro-
phoresed in 10% SDS–polyacrylamide gels. The STAT3 and p-STAT3 proteins were
detected using Western blotting.23
A
B
C
D
Cucurbitacin E (1)
0
0.1
1
10 100 (nM)
p-cofilin
p-LIMK1/2
β-actin
cofilin
Cucurbitacin I (2)
0.1 10 100 1000 (nM)
0
1
p-cofilin
p-LIMK1/2
β-actin
Figure 3. Isolation of cucurbitacin E (1)-selective binding protein from U937 cells.
A: cucurbitacin E (1, 1 M), B: biotin-linked cucurbitacin E (3, 1 M), C: biotin-
linked cucurbitacin E (3, 1 M), D: total protein. U937
l
l
l
M) + cucurbitacin E (1, 100 l
cells were harvested from a 50 mL culture (about 5.0 ꢀ 107 cells), resuspended in
500 lL of a lysis buffer (pH 7.0, 50 mM imidazole, 50 mM NaCl, 5 mM 6-
aminohexanoicacid, 0.5% DDM, 1.1% triton-X100, proteinase inhibitor, and phos-
phatase inhibitor), and sonicated for 90 s three times on ice. Debris was removed by
centrifugation of the sample at 10000g for 10 min. Test compounds were incubated
with 500 lL of the cell lysates for 30 min on ice in binding buffer (50 mM
ammonium carbonate and 0.5 M NaCl), and the mixture was passed through HiTrap
Streptavidin HP (GE Healthcare). After washing with the binding buffer, the bound
proteins were eluted with the elution buffer (50 mM ammonium acetate and 0.5 M
NaCl) according to the manufacturer’s instructions and separated by 10% SDS–
PAGE. The 20 kDa band specific to the test compound was excised from the gel.
After in-gel digestion, tryptic peptides were subjected to LC–MS/MS analysis.26
Figure 4. Effects of cucurbitacins E (1) and I (2) on p-cofilin and p-LIMK1/2 levels in
U937 cells. U937 cells (5.0 ꢀ 104 cells/mL) were incubated with the test compounds
for 24 h. Equivalent amount of protein [60 lg of protein/lane for phospho-cofilin
(p-cofilin) and phospho-LIMK1/2 (p-LIMK1/2), 30 lg of protein/lane for b-actin] of
each lysate was electrophoresed in 10% SDS–polyacrylamide gels. The p-cofilin,
p-LIMK1/2, and b-actin proteins were detected using Western blotting.23

2996
S. Nakashima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2994–2997
F-/G-actin ratio (%)
125
F-/G-actin ratio (%)
125
100
75
100
75
**
**
50
25
0
50
25
0
0
0.1
1
10
100 1000 (nM)
0
0.1
1
10
100 1000 (nM)
Cucurbitacin E (1)
Cucurbitacin I (2)
Figure 5. Effects of cucurbitacins E (1) and I (2) on F-/G-actin ratio in HT1080 cells. HT1080 cells (1.0 ꢀ 104 cells/100
lL/well) were seeded in 96-well microplates and
incubated with test compounds for 24 h at 37 °C. The cells were then washed three times with PBS, and fixed with 4% formaldehyde in PBS for 15 min. Next they were
permeabilized with ice-cold methanol and blocked with Blocking One for 30 min. After washes with PBS, the cells were incubated for 15 min at rt in the dark with the Alexa
FluorÒ 350 phalloidin (Invitrogen) in PBS. The cells were then incubated for 15 min at rt in the dark with the deoxyribonuclease Alexa FluorÒ 488 conjugate (Invitrogen) in
PBS. After another washes with PBS, the fluorescence intensity of each well was measured with a microplate reader (ex: 355 nm, em: 405 nm and ex: 480 nm, em: 520 nm,
FLUOstar OPTIMA, BMG Labtechnologies). Each bar represents the mean with SEM (n = 4). Significantly different from the control group, **p <0.01 (Dunnett’s test).
ments by severing preexisting filaments.28 The severing activity of
Acknowledgments
cofilin is suppressed by its phosphorylation by LIM kinase (LIMK)
or TES kinase.29–31
This research was supported by the 21st COE Program, Aca-
Next, to confirm the involvement of cofilin in the cytotoxic ef-
fect of cucurbitane-type triterpenes, effects of cucurbitacins E (1)
and I (2) on the phosphorylation of cofilin were examined using
a SDS–PAGE and Western blotting.22 As shown in Figure 4, both
compounds inhibited the phosphorylation of cofilin, but not
LIMK1/2, in a concentration-dependent manner. In addition, the
range of concentrations at which they were effective was the same
as that at which they were cytotoxic in U937 cells.
Finally, we examined the fibrous (F)-/globular (G)-actin ratio to
examine the influence of cucurbitacins E (1) and I (2) on the struc-
ture of actin in an adherent cell line, HT1080 cells, using fluores-
cent probes [Alexa FluorÒ 350 phalloidin and deoxyribonuclease
Alexa FluorÒ 488 (Invitrogen)] for F- and G-actins. The F-/G-actin
ratio was decreased after treatment with cucurbitacin E (1) at
demic Frontier Project, and a Grant-in Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Tech-
nology of Japan.
References and notes
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100 nM. Cucurbitacin
I (2) tended to decrease the ratio at
10. Shi, X.; Franko, B.; Franz, C.; Amin, H. M.; Lai, R. Br. J. Haematol. 2006, 135, 26.
11. Matsuda, H.; Nakashima, S.; Abdel-Halim, O. B.; Morikawa, T.; Yoshikawa, M.
Chem. Pharm. Bull. 2010, in press (c091036).
1000 nM, but not significant. These findings suggested that cofilin’s
severing activity increased when its phosphorylation was inhibited
and the depolymerization of actin was enhanced after treatment
with 1 at low concentrations, with 1 eventually exhibiting anti-
proliferative effect. Compound 2 also tended to decrease the F-/
G-actin ratio at 1000 nM, though the decrease was small. This re-
sult implies that the inhibition of JAK/STAT3 signaling is also
important at higher concentrations as reported (Fig. 5).8–10 To the
best of our knowledge, this is the first report about a low molecular
inhibitor against cofilin’s phosphorylation at low concentrations.
In conclusion, we synthesized a biotin-linked cucurbitacin E (1)
and tried to identify target proteins based on its affinity. As a re-
sult, cofilin, which regulates the depolymerization of actin, was
isolated. Cucurbitacins E (1) and I (2) inhibited the phosphoryla-
tion of cofilin in a concentration-dependent manner, and the range
of concentration at which they were effective was the same as that
at which they were cytotoxic in U937 cells. In addition, F-/G-actin
ratio was decreased after treatment with cucurbitacin E (1) in
HT1080 cells. These findings suggested that the inhibition of cofi-
lin’s phosphorylation increased the severing activity of cofilin,
and then the depolymerization of actin was enhanced after treat-
ment with 1 at lower concentrations.
12. Cucurbitacin E (1) was obtained by an enzymatic hydrolysis of cucurbitacin E
2-O-b-D a solution of cucurbitacin E 2-O-b-D-
-glucopyranoside.13 Briefly,
glucopyranoside (50 mg) in 0.2 M acetate buffer (pH 3.8, 10 mL) was treated
with hesperidinase (75 mg, from Aspergillus niger, Sigma) and the solution was
stirred at 40 °C for 48 h. After EtOH was added to the reaction mixture, the
solvent was removed under reduced pressure and the residue was purified by
reversed-phase silica gel column chromatography [MeOH/H2O (50:50, v/v)] to
give 1 (31 mg, 80%).
13. Yoshikawa, M.; Morikawa, T.; Kobayashi, H.; Nakamura, A.; Matsuhira, K.;
Nakamura, S.; Matsuda, H. Chem. Pharm. Bull. 2007, 55, 428.
14. A solution of 1 (20 mg) in 50% aqueous 1,4-dioxane (1.5 mL) was treated with
5% aqueous KOH (0.5 mL) and the whole was stirred at room temperature for
2 h. The reaction mixture was neutralized with Dowex HCR W2 (H+ form).
After the resin was removed by filtration, the filtrate was extracted with EtOAc.
The EtOAc layer was evaporated and purified by normal-phase silica gel
column chromatography [n-hexane/EtOAc (1:1, v/v)] to give 215 (7 mg, 38%).
15. Velde, V. V.; Lavie, D. Tetrahedron 1983, 39, 317.
16. U937 cells (Cell No. JCRB9021), HL60 cells (Cell No. JCRB0163), and HT1080
cells (Cell No. JCRB9113) were obtained from Health Science Research
Resources Bank (Osaka, Japan), and the cells were grown in RPMI-1640 and
MEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin
(100 lg/mL) at 37 °C in 5% CO2/air. After 68 h incubation of U937 cells
(5.0 ꢀ 103 cells/100
l
L/well), HL60 cells cells (5.0 ꢀ 103 cells/100
lL/well), or
HT1080 cells (5.0 ꢀ 103 cells/100
lL/well), with test compounds in RPMI-1640
in 96-well microplates, 10 lL of WST-8 solution (Cell Counting Kit-8™, Dojin)

S. Nakashima et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2994–2997
2997
was added to each well. After a further 4 h in culture, the optical density of the
water-soluble formazan produced by the cells was measured with a microplate
reader (Model 550, Bio-Rad) at 450 nm (reference: 655 nm). Inhibition (%) was
calculated and IC50 value was determined graphically.
conjugated secondary antibody for 1 h. Immunoreactive proteins were
detected using an enhanced chemiluminescence kit (ECL plus, GE
Healthcare), according to the manufacturer’s instructions. The following
antibodies and dilutions were used for Western blotting: rabbit polyclonal
antibody against anti-phospho-STAT3, anti-STAT3, anti-phospho-cofilin, anti-
phospho-LIMK1/2, anti-b-actin (1:1000) (Cell Signaling Technology); anti-
rabbit IgG; and HRP-linked whole antibody from donkeys (1:5000) (GE
Healthcare).
17. The IC50 values of cucurbitacins B and D and 23,24-dihydrocucubitacins B and
E were 13 nM, 0.28
lM, 0.29 lM, and 3.1 lM in HL60 cells and 27 nM, 0.50 lM,
1.2 M, and 9.6 M in HT1080 cells, respectively. These results confirm the
l
l
importance of a double bond at the 23,24-positions as well as an acetoxy group
at the 25-position.
24.
A
solution of
D
-biotinylaminohexanoylaminohexanoic acid25 (24 mg,
18. Amin, H. M.; McDonnell, T. J.; Ma, Y.; Lin, Q.; Fujio, Y.; Kunisada, K.; Leventaki,
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22. After a 24 h incubation of U937 cells (5.0 ꢀ 104 cells/mL) with test compounds,
cells were harvested, transferred to test tubes and centrifuged at 1500 rpm for
5 min at 4 °C. The supernatant was discarded and the cell pellets were
0.05 mmol) in pyridine (0.5 mL) was treated with 1 (36 mg, 0.065 mmol) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCꢂHCl, 19 mg, 0.10 mmol) and 4-dimethylaminopyridine (4-DMAP, 12 mg,
0.10 mmol) and the solution was stirred under N2 at room temperature for 49 h.
The solvent was removed under reduced pressure and the residue was purified
by preparative TLC [PTLC, CHCl3/MeOH (10:1, v/v)] to give 3 (23 mg, 45%). The
methods of synthesis will be presented in detail in a full paper.
3:
a
white powder. ½ ꢃ ꢁ21.9 (c 0.20, MeOH). HR-FAB-MS m/z: Found,
a 1D7
1009.5566 [M+H]+ (Calcd for C54H81N4O12S: 1009.5572), Found, 1031.5396
[M+Na]+ (Calcd for C54H80N4O12SNa: 1031.5391). IR (KBr): 3440, 3300, 2999,
2936, 1697, 1655, 1259, 1232 cmꢁ1 1H NMR (270 MHz, CDCl3) d: 0.99, 1.03, 1.31,
.
1.31, 1.39, 1.44 (3H each, all s, 18, 30, 29, 21, 28, 19-H3), 1.55, 1.57 (3H each, all s,
26, 27-H3, reversible), 2.01 (3H, s, –OC(O)CH3), 3.57 (1H, br s, 10-H), 4.35 (1H, m,
16-H), 6.50 (1H, d, J = 15.6 Hz, 23-H), 7.05 (1H, d, J = 15.6 Hz, 24-H). 13C NMR
(68 MHz, CDCl3) dC: 18.2, 19.9, 20.1, 20.2, 21.9, 23.5, 24.0, 24.3, 25.1, 25.6, 25.9,
26.0, 26.3, 27.2, 27.9, 28.0, 28.8, 29.0, 33.4, 35.7, 35.8, 36.3, 39.0, 39.1, 40.5, 40.9,
41.4, 45.5, 48.0, 48.4, 48.9, 49.3, 50.5, 55.6, 58.2, 60.2, 61.7, 71.2, 78.2, 79.3, 120.4,
121.6, 132.1, 135.8, 143.1, 151.9, 163.8, 170.3, 171.7, 173.2, 173.2, 195.4, 202.5,
213.1. Positive-ion FAB-MS: m/z 1009 [M+H]+, 1031 [M+Na]+.
resuspended in 300 lL of cold PBS and fixed by the addition of 700 lL of ice-
cold ethanol. Fixed cells were incubated overnight at ꢁ20 °C after which they
were centrifuged at 1500 rpm for 5 min. The cell pellets were resuspended in
500
at 37 °C for 30 min. After the incubation, propidium iodide (final concn: 50
mL) was added. The flow cytometric determination of DNA content
(20,000 cells/sample) was analyzed with FACSCalibur Flow Cytometer
l
L of PBS with DNase-free RNase (0.25 mg/mL) and incubated in the dark
lg/
a
(Becton Dickinson). The fractions of cells in G0/G1, S, and G2/M phase were
analyzed using cell cycle analysis software, ModFit LT ver. 3.0 (Verity Software
House).
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algorithm can be found in the technical information section of the website of
NextGen Sciences (www.nextgensciences.com).
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24 h. They were then lysed in a lysis buffer containing a phosphatase inhibitor
(Roche) and a proteinase inhibitor (Thermo Scientific). Protein concentrations
of cell lysate were determined using the Protein Assay Kit (Thermo Scientific).
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Equivalent amounts of protein (60 lg or 30 lg of protein/lane) were
electrophoresed in 10% SDS–polyacrylamide gels and transferred to
polyvinylidene difluoride membranes. The membranes were blocked for
30 min in Blocking One or Blocking One-P (Nacalai Tesque). The blots were
probed with primary antibody at the appropriate dilution in Tris-buffered
saline containing 0.1% Tween 20 (T-TBS) for 1 h. The membranes were then
washed three times with T-TBS and incubated with the appropriate HRP-
29. Arber, S.; Barbayannis, F. A.; Hanser, H.; Schneider, C.; Stanyon, C. A.; Bernard,
O.; Caroni, P. Nature 1998, 393, 805.
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