International Immunopharmacology 9 (2009) 508513

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International Immunopharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p

Preliminary report

Cucurbitacin D isolated from Trichosanthes kirilowii induces apoptosis in human

hepatocellular carcinoma cells in vitro
Norito Takahashi a, Yasuhiro Yoshida b, Tsutomu Sugiura b, Koji Matsuno c, Akihiro Fujino a, Uki Yamashita b,
a
b
c

Department of Medical Humanities, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
Department of Immunology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan
Kyushu Health and Welfare University, Nobeoka 882-8508, Japan

a r t i c l e

i n f o

Article history:
Received 27 March 2008
Received in revised form 7 January 2009
Accepted 13 January 2009
Keywords:
Cucurbitacin D
Trichosanthes kirilowii
Hepatocellular carcinoma
Apoptosis
Caspase-3
JNK

a b s t r a c t
The aim of the present study is to examine the effects of the anti-tumor component isolated from Trichosanthes kirilowii on human hepatocellular carcinoma cells. Using Sephadex G-25 column chromatography,
Sep-Pak Plus C18 cartridge and high-performance liquid chromatography (HPLC), we isolated the active
component from trichosanthes extract. By fast atom bombardment mass spectrometric analysis, the
molecular mass of the active fraction was determined, the active components identied, and their
mechanisms of action were analyzed by cell growth assay, cell cycle analysis, TUNEL staining and Western
blot analysis. We found that the anti-tumor components isolated from the extract of trichosanthes (EOT) are
cucurbitacin D and dihydrocucurbitacin D, and suggest that cucurbitacin D induces apoptosis through
caspase-3 and phosphorylation of JNK in hepatocellular carcinoma cells. These results suggest that
cucurbitacin D isolated from Trichosanthes kirilowii could be a valuable candidate for anti-tumor drug.
2009 Elsevier B.V. All rights reserved.

1. Introduction
There has been a growing interest in the use of traditional
medicinal herbs (TMHs) as a potent source of new therapeutic drugs
for several diseases [1,2]. In Japan standardized crude extracts and
dried powder from TMHs are prepared, commercially supplied by
some pharmaceutical companies and the combination of each TMH
extracts has been clinically used for the therapy of many diseases
(Kampo formura), especially chronic diseases including diseases of the
respiratory, digestive and nervous systems [3,4]. Trichosanthes
kirilowii is a type of liana of the Cucurbitaceae family whose root
tuber has been used to reset menstruation and expel retained placenta
in China [5]. In the 1980s, Trichosanthin (TCS; 27 kD protein) was
isolated from the root tuber of trichosanthes and proved to be the
active component, a type I ribosome-inactivating protein (RIP). In
recent years, TCS has also been found to possess anti-tumor activity
[6,7]. The extract of trichosanthes (EOT) has been found to have a
better inhibitory effect on the growth of tumor cells, with the
induction of apoptosis, than TCS. Therefore it is suggested that the
better anti-tumor activity of EOT is probably due to other active
components in EOT [8].
In this study, we isolated anti-tumor components from EOT,
cucurbitacin D and dihydrocucurbitacin D, and it is suggested that the
active component from EOT and cucurbitacin D, induces apoptosis by

Correspondence author. Tel.: +81 93 691 7241; fax: +81 93 692 2479.
E-mail address: yama-uki@med.uoeh-u.ac.jp (U. Yamashita).
1567-5769/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2009.01.006

the activation of caspase-3 and phosphorylation of JNK in hepatocellular carcinoma, Hep3B, cell line in vitro.

2. Experimental procedures
2.1. Preparation of traditional medicinal herb (TMH) extracts
Dried powder of the extracts of 11 TMHs (Aconitum carmichaeli
(root), Asparagus cochinchinensis (root), Bupleurum falcatum(root), Coix
lacryma-jobi (semen), Cornus ofcinalis (fruit), Glycyrrhiza uralensis
(root), Ophiopogon iaponicus (root), Ostrea gigas (shell), Panax ginseng
(root), Poria cocos (sclerotia) and Trichosanthes kirilowii (root)) were
supplied by Tsumura & Co. (Tokyo, Japan). One gram of each TMH extract
was dissolved overnight in 100 ml of distilled water at room
temperature. The extracts were ltrated with Millipore lters (MilexGP, 0.22 m pore size; Nippon Millipore Ltd, Tokyo, Japan) after
centrifugation at 2000 rpm for 10 min and used as extracts for
subsequent experiments. Puried cucurbitacin D was donated by Dr. J.
Kitajima (Showa Pharmaceutical University, Tokyo, Japan) [9].

2.2. Cell line and cell culture

Human hepatocellular carcinoma cell line, Hep3B, was obtained
from the Cell Resource Center for Biomedical Research, Tohoku
University (Sendai, Japan). Cells were grown in DMEM medium
(Nissui Seiyaku Co., Yokohama, Japan) supplemented with 10% (v/v)
fetal calf serum (FCS) (Biowhittaker, Walkersville, MD), 50 g/ml

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509

penicillin, and 50 U/ml streptomycin (Gibco, Grand Island, NY). Cells

were maintained at 37 C in a humidied atmosphere of 5% CO2 in air.
2.3. Cell growth assay
Alamar Blue dye assay was used to analyze the cell growth. Hep3B
cells were plated at a density of 1 104/0.1 ml DMEM10% FCS in
microtiter culture plates (Falcon #3072, Becton Dickinson Labware,
Franklin Lakes, NJ). Each TMH extract (5 l; nal concentration is
0.05%) was added and the cells were cultured for 48 h. Finally, 5 l
Alamar Blue (Wako Junyaku Co., Osaka, Japan) was added to the cells.
After 4 h incubation, cell growth was evaluated at an absorbance of
560 nm using a microtiter plate reader (MPRA4, Toyo Soda Co., Tokyo,
Japan). In Fig. 4A, Hep3B cells were cultured at a density of 1 105/ml
DMEM10% FCS with or without the component of retention time of
30 min (CRT30) (5 g/ml) or cucurbitacin D (5 g/ml) in culture dishes
(Falcon #3046) and the viable cell number was counted using 0.1%
trypan blue after trypsination.
2.4. Isolation of active component from crude extract of root from
Trichosanthes kirilowii (EOT)
EOT (5 ml) was applied on a Sephadex G-25 column (2 50 cm,
Pharmacia Fine Chemicals, Uppsala, Sweden), equilibrated, eluted
(15 ml/h) with distilled water, and 5-ml fractions were aliquoted. The
protein content in each fraction was monitored at an absorbance of
280 nm. The activity of each fraction was assayed by the inhibition of
Hep3B cell growth. The active fractions (fraction # 30, 31, 32 in Fig. 2A)
were pooled, loaded on a Sep-Pak Plus C18 cartridge, (cartridge
volume: 1.4 ml, Waters, Milford, MA), eluted (1 ml/min) stepwise with
060% acetonitrile in 0.1% triuoroacetic acid, and 2-ml fractions were
aliquoted. The activity of each fraction was assayed by the inhibition of
Hep3B cell growth. The resulting active fractions were pooled
(fraction #7, 8, 9, 10 in Fig. 2B), freeze dried, rechromatographed on
high performance liquid chromatography (HPLC, Hitachi-L2130, Diode
Array Detector, Hitachi L-2455, Tokyo, Japan) using a column of
Wakopack: Wakosil-II5C18HG, 4 250 mm (Wako Junyaku Co.), and
eluted continuously with 2060% acetonitrile in 0.1% triuoroacetic
acid and 0.5 ml/min fractions were aliquoted. Each fraction was
monitored at an absorbance of 230 nm (Fig. 2C). The activity of each
fraction was assayed by the inhibition of Hep3B cell growth (Fig. 2D).
2.5. FAB-MS analysis
The molecular mass of the peak observed at the retention time of
30 min (CRT30) was determined by fast atom bombardment mass
spectrometry (FAB-MS) with a JMS SX102A double-focusing mass
spectrometer (JEOL, Tokyo, Japan), with nitrobenzyl alcohol + dithiothreitol (1:1) matrix in a positive ion mode [10].
2.6. Analysis of cell-cycle of Hep3B cells by ow cytometry with PI
staining
Hep3B (1.5 106) cells treated with the nal active component
(CRT30; 5 g/ml) extracted from trichosanthes or cucurbitacin D (5 g/
ml) at 37 C for 24 h were harvested by trypsinization and washed
with phosphate buffered saline, then xed with ethanol (70% nal
concentration) at 4 C for 0.5 h. Cells were treated with 0.1 mg/ml of
RNase A (Sigma Chemical Co., St Louis, MO) at 37 C for 1 h. After
washing, the cells were stained with propidium iodide (PI; Sigma
Chemical Co.) (50 mg/ml nal concentration) at room temperature for
10 min. Cell-cycle analysis was performed on a ow cytometer
(Beckman Coulter Co., Miami, FL). The apoptotic cells were assessed by
ow cytometric detection of sub-G1 DNA content.

Fig. 1. Effect of TMH extracts on Hep3B cells. Hep3B cells (1 104/0.1 ml) were cultured
with extracts of 11 kinds of medicinal herbs (5 l) for 48 h and the growth of cells was
detected by Alamar Blue assay. The results were expressed as mean SE of optical
density (OD) at 560 nm in triplicate cultures. Signicantly suppressed (p b 0.05).

2.7. TUNEL analysis

Hep3B (1 105/well) cells treated with CRT30 (5 g/ml) or
cucurbitacin D (5 g/ml) at 37 C for 48 h were stained with a
commercial TUNEL kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer's instructions. The cells were
observed under a light microscope.
2.8. Western blot analysis
Hep3B cells(1 106) were treated with CRT30 (5 g/ml) or
cucurbitacin D (5 g/ml) for the indicated period. Whole cell lysates
were prepared for western blotting of caspase-3 and phosphorylated
JNK. 15-deoxy-12,14-prostagladin J2 (15d-PGJ2) and lipopolysaccharide (LPS) served as the positive controls. Whole cell lysates were
suspended in RIPA buffer (10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl,
1% Triton X-100, 1% Deoxycholic acid, 0.1% SDS, 5 mmol/L EDTA (pH
8.0), 1 mmol/L PMSF). Protein concentration was determined by the
Bio-Rad protein assay system according to the manufacturer's
directions (Bio-Rad, Hercules, CA). Equivalent amounts of protein
(10 g) were resolved on SDS-PAGE gels, transferred, and immobilized
onto nitrocellulose membranes (Amersham, Little Chalfont, UK). The
membranes were probed with anti-caspase-3 primary antibody
(#9662, Cell Signaling Technology, Danvers, MA), anti-phosphoSARK/JNK primary antibody (#9251S, Cell Signaling Technology,
Danvers, MA), anti--actin primary antibody (Sigma Chemical Co.),
anti-rabbit-IgG-HRP antibody (sc-2030, Santa Cruz, CA), and antimouse-IgG-HRP antibody (NA 931, Amersham). Immunodetection
was accomplished by a chemiluminescence detection system (Alpha
Innotech, San Leandro, CA).
2.9. Statistical analysis
All experiments were repeated more than three times, and the
representative results are shown. In Figs. 1 and 4, the means and
standard errors were from triplicate cultures of one representative
experiment. Statistical analyses were performed by a student's t-test.
A condence level of b0.05 was considered signicant.
3. Results
3.1. Effect of TMHs on Hep3B cell growth
At rst, we studied the effect of EOT on the growth of Hep3B cells
along with 10 other kinds of TMH extracts, which have been popularly
used in clinical therapy of several diseases in Japan. As shown in Fig. 1,
the EOT markedly inhibited the growth of Hep3B cells, but the other
10 TMH extracts had no effect on Hep3B cell growth.

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N. Takahashi et al. / International Immunopharmacology 9 (2009) 508513

Fig. 2. Fractionation of trichosanthes extract by column chromatography. (A)The extract of trichosanthes (5 ml) was fractionated by a Sephadex G-25 column (2 50 cm), and 5-ml
fractions were aliquoted. The protein concentration was monitored by the OD at 280 nm (-). The activity of each fraction was assayed in Hep3B cells. The results were expressed as
OD at 560 nm; stained with Alamar Blue (-). The column size was calibrated by cytochrome c (M.W. 12300 D) and trypan blue (M.W. 960 D). (B)The active fractions from Sephadex
G-25 column were pooled, loaded on Sep-Pak Plus C18 cartridge (1.4 ml volume), eluted stepwise with 060% acetonitrile (-), and 2-ml fractions were aliquoted. The activity of
each fraction was assayed by Hep3B cells. The results were expressed as OD at 560 nm; stained with Alamar Blue (-). (C)The active fractions from Sep-Pak cartridge were pooled,
freeze-dried, loaded on HPLC column (4 250 mm) and eluted continuously with 2060% acetonitrile (), and 0.5-ml/min fractions were aliquoted. The abscissa is the retention time
(min) and the ordinate is the absorbance units at 230 nm. (D) The activity of each fraction of (C) was assayed by Hep3B cells. The results are expressed as OD at 560 nm; stained with
Alamar Blue (-).

3.2. Isolation of active component extracted from trichosanthes

EOT was chromatographed on a Sephadex G-25 column. The
activity of each fraction was assayed by the inhibition of Hep3B cell
growth. As shown in Fig. 2A, the active fractions (#30, 31 and 32) had a

molecular mass of less than 1 kD. The active fractions were pooled,
loaded on a Sep-Pak Plus C18 cartridge, and eluted in a stepwise
manner with 060% acetonitrile. As shown in Fig. 2B, the active
fractions on a Sep-Pak Plus C18 were observed from #7 to #10 (30
40% (v/v) acetonitrile). The resulting active fractions were pooled and

Fig. 3. (A) Fast atom bombardment mass spectrum (FAB-MS) of the active fraction isolated from trichosanthes extract. The fraction eluted at 30 min (of Fig. 2C) was analyzed by FAB-MS
with a JMS SX102A double-focusing mass spectrometer with nitrobenzyl alcohol + dithiothreitol (1:1) matrix in a positive ion mode. The abscissa is the molecular size and the ordinate is %
ion count. (B) shows the structural formula of cucurbitacin D.

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511

Fig. 4. (A) Dose response curve of the effect of CRT30 and cucurbitacin D on Hep3B cells. Hep3B (1104/0.1 ml) were cultured with CRT30 (5 g/ml) or cucurbitacin D (5 g/ml) for 48 h, and
stained with Alamar Blue. The results were expressed as the mean SE of OD at 560 nm; stained with Alamar Blue in triplicate cultures. Signicantly decreased from the control group
(p b 0.05). (B) Time course of the effect of CRT30 and cucurbitacin D on Hep3B cell growth. Hep3B cells (1 105/ml) were cultured without (-) or with CRT30 (5 g/ml) (-) or cucurbitacin
D (5 g/ml) (-), and the recovery of viable cells was counted after trypsin treatment on each day indicated in triplicate cultures. Signicantly decreased from the control group (pb 0.05).

rechromatographed on HPLC. The absorbance at a wavelength of

230 nm was monitored, because the active fraction of Fig. 2B showed
the maximum absorption at 230 nm (data not shown).
As shown in Fig. 2C, the highest peak was observed at the retention
time of 30 min, which showed strong inhibiting activity of Hep3B cell
growth (Fig. 2D). We performed fast atom bombardment mass
spectrometry (FAB-MS) of this peak. As shown in Fig. 3A, a large
peak was observed at the molecular mass of 501. This corresponds to
dihydrocucurbitacin D. A small peak was observed at the molecular
mass of 499. This corresponds to cucurbitacin D that was puried by
Dr. J. Kitajima (Showa Pharmaceutical University) from T. kirilowii
MAXIM. var. japonicum KITAM [9]. Taken together, the active
components with a retention time of 30 min on the HPLC (CRT30)
are thought to be dihydrocucurbitacin D and cucurbitacin D.
3.3. Dose and time course study of inhibitory effect of CRT30 and
cucurbitasin D on Hep3B cell growth
The doseresponse of the inhibitory effect of CRT30 and cucurbitacin D on Hep3B cell growth was examined. CRT30 and cucurbitacin

D showed similar dose response curves (Fig. 4A). The 50% inhibition of
Hep3B cell growth was observed at 0.320.63 g/ml. Fig. 4B shows the
time course study of the inhibitory effect of CRT30 and cucurbitacin D.
The Hep3B cell numbers showed a time-dependent increase, which
was inhibited similarly by CRT30 and cucurbitacin D.
3.4. CRT30 and cucurbitacin D induce apoptosis in Hep3B cells
In order to analyze the inhibitory mechanism of Hep3B cell growth
with CRT30 and cucurbitacin D, the cell cycle of treated Hep3B cells was
examined. As shown in Fig. 5B and C, the treatment with CRT30 and
cucurbitacin D increased sub-G1 populations (apoptosis) and decreased
G1 populations in Hep3B cells. Additionally, TUNEL assay showed that
Hep3B cells treated with CRT30 and cucurbitacin D for 48 h become
round, and stained intense dark-brown (Fig. 5E and F). These results
suggest that CRT30 and cucurbitacin D induce apoptosis in Hep3B cells.
3.5. CRT30 and cucurbitacin D activate caspase-3 and JNK
Western blot analysis was performed to investigate the mechanisms
of apoptosis induced by CRT30 and cucurbitacin D. Procaspase-3 was

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N. Takahashi et al. / International Immunopharmacology 9 (2009) 508513

Fig. 5. (A, B, C) Effect of CRT30 and cucurbitacin D on cell cycle of Hep3B cells. Hep3B cells (1.5 106/well were cultured without (A) or with CRT30 (5 g/ml: B) or cucurbitacin D (5 g/ml:
C) for 24 h, stained with propidium iodide (PI), then the DNA content was analyzed by ow cytometry. The percentages of cells in each phase of the cell cycle: sub G1 (apoptotic cells), G1, S,
and G2/M phases are shown under the graph. (D, E, F) TUNEL staining of Hep3B. Hep3B cells (1 105/well) were cultured without (D) or with CRT30 (5 g/ml: E) or cucurbitacin D (5 g/ml: F)
for 48 h, then stained with TUNEL method. The cells showing brown-dark nuclear staining were apoptotic cells (200).

expressed in Hep3B cells, which was cleaved by 15d-PGJ2 known to

activate caspase-3 [11] (Fig. 6A, lanes 8 and 12). CRT30 and cucurbitacin
D also activated caspase-3 at 12 and 24 h, but not at 6 h after treatment
(Fig. 6A, lanes 6 and 10). Fig. 6B shows that CRT30 and cucurbitacin D
phosphorylated JNK at 6 h (lanes 2 and 3) in Hep3B cells. These results
suggest that the ability of CRT30 and cucurbitacin D to induce apoptosis
is related to their ability to activate caspase-3 and phosphorylate JNK.

4. Discussion
TCS is the active protein component isolated from EOT. It is known
to be cytotoxic and induces apoptosis of gastric cancer cells, human
choriocarcinoma cells and leukemia cells [8]. However, EOT has a
better inhibitory effect on the growth of tumor cells with the
induction of apoptosis than TCS, suggesting that the more effective

Fig. 6. Apoptosis induced by CRT30 and cucurbitacin D depends on caspase-3 and p-JNK. Hep3B cells (1 106) were treated with CRT30 (5 g/ml) or cucurbitacin D (Cu.D, 5 g/ml) for
indicated period. Whole cell extract was prepared to Western blotting of caspase-3 (A) and phosphorylated JNK (B). Ethanol (0.05%) served as the negative control. 15d-PGJ2 and LPS
served as the positive control.

N. Takahashi et al. / International Immunopharmacology 9 (2009) 508513

anti-tumor activity of EOT is probably due to the presence of other

active components in EOT [8]. To nd the more effective anti-tumor
components than TCS, we studied the effect of EOT on the growth of
tumor cells along with 10 other kinds of TMH extracts, which have
been used for the clinical therapy of several diseases in Japan. Hep3B
cell growth assay identied two highly oxygenated tetracyclic
triterpenes, cucurbitacin D and dihydrocucurbitacin D, from EOT as
the active compounds, whose molecular masses are 499 and 501, their
molecular formulae are C30H43O6 and C30H45O6, respectively, and the
structural formula of cucurbitacin D is shown in Fig. 3B.
Apoptosis is considered as one of the ideal paths to tumor
suppression. The signaling pathways leading to tumor cell apoptosis
have been extensively investigated [1113]. Several caspases play an
important role in the regulation of apoptosis. They are broadly
grouped into initiator or effector caspases, due to the role that they
play in an apoptosis inducing system. In particular, activation of
caspase-3 plays a central role in the initiation of apoptosis [13].
Furthermore, it has been reported that JNK might play a role in tumor
suppression. The fact that many proapoptotic stimuli activate JNK
provides evidence of a role for the JNK signaling pathway in apoptosis
[12,14,15]. Therefore, we investigated whether cucurbitacin D induces
apoptosis in Hep3B cells through caspase-3 and phosphorylation of
JNK. Our results suggest that procaspase-3 is cleaved by cucurbitacin D
at 12 and 24 h after treatment. Furthermore, cucurbitacin D
phosphorylated JNK at 6 h after treatment. Together, these results
suggest that the ability of cucurbitacin D to induce apoptosis is related
with its ability to activate caspase-3 and phosphorylate JNK.
Several studies have been published to point out that different
cucurbitacin species exhibit various biological activities against tumor
expansion. Recent studies indicate that cucurbitacin I and Q effectively
block the growth of several tumor cells by disrupting STAT3 signaling
pathways [1416]. Cucurbitacin B and E were demonstrated to disrupt
the cytoskeleton in tumor cells [17,18]. It has been reported that
cucurbitacin B has anti-proliferative activity on glioma cells in part
through activation of JNK pathway [18]. However, the present results
are the rst to demonstrate that cucurbitacin D induces apoptosis via
activation of caspase-3 and phosphorylation of JNK in hepatocellular
carcinoma. Moreover, cucurbitacin D is not cytotoxic to peripheral
blood lymphocytes from healthy donors (data not shown). A previous
study also reported that 23, 24-dihydrocucurbitacin D was not toxic to
non-tumor cells, whereas it was toxic to tumor cells [19].
Hepatocellular carcinoma (HCC) is one of the most common
malignancies in Asia and its incidence is rapidly increasing in Europe
and the U.S.A. [20,21]. HCC is widely considered to be chemotherapyresistant[22]. Thus, cucurbitacin D isolated from EOT could be a
valuable candidate for an anti-HCC drug.
In conclusion we found that the anti-tumor components isolated
from EOT are cucurbitacin D and dihydrocucurbitacin D, and it is
suggested that the active components from EOT and cucurbitacin D
induce apoptosis through caspase-3 and phosphorylation of JNK in
HCC. Based on these ndings, we suggest that cucurbitacin D isolated
from EOT could be a valuable candidate for an anti-tumor drug.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientic
Research from the Ministry of Education, Science, Sports and Culture

513

of Japan (205921329) and a Grant-in-Aid for Advanced Research from

University of Occupational and Environmental Health, Japan. We
thank Dr. J. Kitajima and Tsumura & Co for providing us the puried
sample of cucurbitacin D and crude TMH extracts.

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