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International Immunopharmacology
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Preliminary report
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].
509
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).
510
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 (-).
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.
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).
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
512
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).
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.
513
References
[1] P.B. Fontanarose (ed.), Alternative Medicine, JAMA, AMA, Chicago: 2000.
[2] Ernst E. The risk-benet prole of commonly used herbal therapies: Gingo, St. John's
Wort, ginseng, echinacea, saw palmetto, and kava. Ann Intern Med 2002;136:4253.
[3] Yamada H, Nagai T. In vivo antiinuenza virus activity of Kampo medicine Shoseiryu-to through mucosal immune system. Methods Find Exp Clin Pharmacol
1998;20:18592.
[4] Japanese Oriental Medicine Association(ed.). Introducing Kampo Medicine,
Nankodo,Tokyo: 2002 (in Japanese).
[5] Lu PX, Jim YV. Trichosanthin in the treatment of hydatidiform mole. Clinical
analysis of 52 cases. Clin Med J 1990;103:1835.
[6] Chan SH, Hung FS, Chan DS, Shaw PC. Trichosanthin interacts with acidic ribosomal
proteins P0 and P1 and mitotic checkpoint protein MAD2B. Eur J Biochem
2001;268:210712.
[7] Wang P, Li JC. Trichosanthin-induced specic changes of cytoskeleton conguration were associated with the decreased expression level of actin and tubulin
genes in apoptotic Hela cells. Life Sci 2007;31:113040.
[8] Dou CM, Li JC. Effect of extracts of trichosanthes root tubers on HepA-H cells and
HeLa cells. World J Gastroenterol 2004;10:20914.
[9] Kitajima J, Tanaka Y. Studies on the constituents of trichosanthes root. I.
Constituents of roots of Trichosanthes kirilowii Maxim. var. japonicum Kitam.
Yakugaku Zasshi 1989;109:2505 (in Japanese).
[10] Nishihara M, Morii H, Matsuno K, Ohga M, Stetter KO, Koga Y. Structural analysis by
reductive cleavage with LiAIH4 of an allylethercholine-phospholipid, archaetidylcholine, from the hyperthermophilic methanarchaeon Methanopyrus kandleri.
Archaea 2002;1:12331.
[11] Kim EJ, Park KS, Chung SY, Sheen YY, Moon DC, Song YS, et al. Peroxisome
proliferator-activated receptor-gamma activator 15-deoxy-delta12,14-prostaglandin J2 inhibits neuroblastoma cell growth through induction of apoptosis:
association with extracellular signal-regulated kinase signal pathway. J Pharmacol
Exp Ther 2003;307:50517.
[12] Gekle M, Schwerdt G, Freudinger R, Mildenberger S, Wilingseder C, Pollack V, et al.
Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar
concentrations. J Pharmacol Exp Ther 2000;293:83744.
[13] Salvesen GS, Dixit VM. Caspase activation: the induced-proximity model. Proc Natl
Acad Sci U S A 1999;96:109647.
[14] Blaskovich MA, Sun J, Cantor A, Turkson J, Sebti SM. Discovery of JSI-124
(cucurbitacin I), a selective Janus kinases/signal transducer and activator of
transcription 3 signaling pathway inhibitor with potent antitumor activity against
human and murine cancer cells in mice. Cancer Res 2003;63:12709.
[15] Shi X, Franko B, Frantz C, Amin HM, Lai R. JSI-124 (cucurbitacin I) inhibits Janus kinase3/signal transducer and activator of transcription-3 signaling, down regulates
nucleophosmin-anaplastic lymphoma kinase(ALK), and induces apoptosis in ALKpositive anaplastic large cell lymphoma cells. Brit J Haematol 2006;135:2632.
[16] Sun J, Blaskovich MA, Jove R, Livingston SK, Coppola D, Sebti SM. Cucurbitacin Q: a
selective STAT3 activation inhibitor with potent antitumor activity. Oncogene
2005;24:323645.
[17] Duncan KL, Duncan MD, Alley MC, Sausville EA. Cucurbitacin E-induced disruption
of the actin and vimentin cytoskeleton in prostate carcinoma cells. Biochem
Pharmacol 1996;52:155360.
[18] Yin D, Wakimoto N, Xing H, Liu D, Huynh T, Wang X, et al. Cucurbitacin B markedly
inhibits growth and rapidly affects the cytoskeketon in glioblastoma multiforme.
Intern J Cancer 2008;123:136475.
[19] Duncan MD, Duncan KL. Cucurbitacin E targets proliferating endothelia. J Surg Res
1997;69:5560.
[20] Blum HE. Hepatocellular carcinoma: therapy and prevention. World J Gastroenterol 2005;11:7391400.
[21] Wilson JF. Liver cancer on the rise. Ann Intern Med 2005;142(12 Pt1):102932.
[22] Johnson PJ. Hepatocellular carcinoma: is current therapy really altering outcome?
Gut 2002;51:45962.