J Nat Med (2010) 64:423–429

DOI 10.1007/s11418-010-0425-6

ORIGINAL PAPER

Cytotoxic effect of artocarpin on T47D cells

Enos Tangke Arung • Britanto Dani Wicaksono •
Yohana Ayupriyanti Handoko • Irawan Wijaya Kusuma •

Kuniyoshi Shimizu • Dina Yulia • Ferry Sandra

Received: 14 January 2010 / Accepted: 19 April 2010 / Published online: 11 June 2010
Ó The Japanese Society of Pharmacognosy and Springer 2010

Abstract In our screening projects for anticancer agents together, these data indicated that artocarpin induced apop-
from natural resources, artocarpin [6-(3-methyl-1-butenyl)- tosis in T47D cells possibly via an extrinsic pathway.
5,20 ,40 -trihydroxy-3-isoprenyl-7-methoxyflavone] isolated
from wood of jack fruit (Artocarpus heterophyllus) showed Keywords Artocarpin 
potent cytotoxic activity on human T47D breast cancer cells. 6-(3-Methyl-1-butenyl)-5,20 ,40 -trihydroxy-3-isoprenyl-7-
The mode of action of artocarpin was evaluated by its effect methoxyflavone  T47D cells  Apoptosis 
on cell viability, nuclear morphology, cell cycle progression, Extrinsic pathway  Artocarpus heterophyllus
expression of protein markers for apoptosis, and mitochon-
drial membrane potential (Dwm). These results showed that
artocarpin caused a reduction of cell viability in a concentra- Introduction
tion-dependent manner and an alteration of cell and nuclear
morphology. Moreover, the percentage of the sub-G1 phase For centuries, people have been using herbs for healing. In
formation was elevated dose-dependently. Artocarpin induced fact, the use of herbal medicine can be traced back for at
activation of caspase 8 and 10 as indicated by stronger signal least 5,000 years. Nowadays, there are more than 85,000
intensity of cleaved-caspase 8 and weaker signal intensity of plant species that have been documented for medical use
caspase 10 markers detected after artocarpin treatment. In globally. The World Health Organization (WHO) estimates
addition, we also noticed the activation of caspase 3 by arto- that almost 75% of the world’s population has therapeutic
carpin. There were negligible changes in mitochondrial experience with herbal remedies [1]. The major categories
membrane potential (Dwm) due to artocarpin treatment. All of plant-derived compounds that have medicine properties
are terpenoids, flavonoids, and alkaloids [2].
A total of more than 6,500 different flavonoids have
E. T. Arung  B. D. Wicaksono  Y. A. Handoko  D. Yulia 
F. Sandra (&) been identified from plant sources of which at least 400
Stem Cell and Cancer Institute, appear to be prenylated. Flavonoids are known to exhibit a
Jalan Ahmad Yani no. 2, Pulomas, Jakarta 13210, Indonesia number of beneficial properties for human health, such as
e-mail: fsandra@sci-indonesia.org
antioxidant and free radical scavenging activities, and anti-
E. T. Arung  I. W. Kusuma inflammatory, antiviral, and anticancer properties [3, 4]. In
Wood Chemistry Laboratory, Forestry Faculty, their review of applications of prenylated flavonoids in
Mulawarman University, Jalan KH Dewantara, pharmacology and biotechnology, Botta et al. [5] pointed
Kampus Gn. Kelua, Samarinda, East Kalimantan 75123,
out that prenylated flavonoids display a series of interesting
Indonesia
e-mail: tangkearung@agr.kyushu-u.ac.jp; biological activities that have attracted interest from
tangkearung@yahoo.com chemists, biologists, pharmacologists, and physicians. This
activity may be due to the properties of prenylated flavo-
K. Shimizu
noids; the substitution of the flavonoid ring system with
Department of Forest and Forest Products Science,
Agriculture Faculty, Kyushu University, 6-10-1 Hakozaki, lipophilic prenyl groups increases their affinity to biologi-
Higashi-ku, Fukuoka 8128581, Japan cal membranes.

123
424 J Nat Med (2010) 64:423–429

Some prenylated flavonoids have been isolated from Cell culture

Moraceae plants and showed cytotoxicity in human cancer
cells. Artelasticin was cytotoxic to MCF-7, TK-10, and Human breast cancer T47D cells were grown and main-
UACC-62 cells with IC50 values of 8.9, 10.6, and 8.8 lM, tained in Dulbecco’s modified Eagle medium (DMEM)
respectively; and artelastin was with IC50 values of 2.2, 4.6, with L-glutamine supplemented with 10% (v/v) fetal bovine
and 2.2 lM, respectively. Artelastoxanthone exhibited serum, sodium bicarbonate, 100 lg/ml streptomycin, and
cytotoxicity in A549, Hep3B, HT-29, and MCF-7 cells 100 U/ml penicillin at 37°C in a humidified 5% CO2
with IC50 values of 10.0, 3.2, 3.9, and 3.1 lg/ml, while atmosphere.
artonol A had IC50 values of 1.1, 21.9, 3.1, and 2.7 lg/ml,
respectively [6, 7]. Moreover, Watjen et al. [8] reported Artocarpin
that prenylation of apigenin and liquiritigenin enhanced
their cytotoxicity in rat H4IIE hepatoma and C6 glioma The prenylated flavonoid artocarpin was purified from
cells. Wang et al. [9] reported that artocarpin [6-(3-methyl- Artocarpus heterophyllus, as described in previously pub-
1-butenyl)-5,20 ,40 -trihydroxy-3-isoprenyl-7-methoxyflavone] lished papers [10, 11], that was part of a collection
(Fig. 1) has a cytotoxic effect on some cancer cells such as belonging to the Wood Chemistry Laboratory, Forest
lung carcinoma (A549), breast adenocarcinoma (MCF-7 Product Department, Forestry Faculty, Mulawarman Uni-
and MDA-MB-231), ovarian carcinoma (1A9), ileocecal versity, Samarinda, East Kalimantan, Indonesia.
carcinoma (HCT-8), kidney carcinoma (CAKI-1), mela-
noma (SK-MEL-2), glioblastoma (U87-MG), prostate Cell viability assay
cancer (PC-3), and epidermoid carcinoma of the naso-
pharynx (KB). To determine cell viability, trypan blue dye exclusion assay
Although some prenylated flavonoids, including arto- was performed according to the method described by Jamil
carpin, have exhibited cytotoxic effects on human cancer et al. [12], with minor modifications. Trypan blue penetrates
cell lines [6–9], the mechanism underlying these activities dead cells through damaged membrane, thereby staining the
are not well understood. Understanding the mechanism of nucleus. Cells were counted by using a hemocytometer and
the prenylated flavonoids with potential anticancer effects, the numbers of viable and dead cells were scored by direct
such as artocarpin, is important for their future therapeutic observation under a bright field microscope. Briefly, cells
application. Therefore, in this present study we evaluated were seeded into 96-well plates (5 9 103 cells per well).
the cytotoxic property of artocarpin on human breast can- After 24 h incubation, the medium was replaced with fresh
cer (T47D) cells and examined its possible mode of action. medium containing different concentrations of artocarpin,
dimethyl sulfoxide (DMSO; 1%, v/v), hydrogen peroxide
(H2O2; 0.07%, v/v), or medium alone. The cells were then
Materials and methods incubated for a further 24 h. DMSO was used as a negative
control. Cells were trypsinized, then trypan blue (0.4% w/v
Chemicals in PBS) was added to the cell suspensions. The cell sus-
pension was then transferred (dropped) onto a hemocy-
Dimethyl sulfoxide (DMSO), ethanol, H2O2, and trichloro- tometer fitted with a coverslip and viewed at 910
acetic acid were purchased from Wako (Osaka, Japan). The magnification under a microscope. The data were analyzed
3,30 -dihexyloxacarbocyanine iodide (DiOC6), 40 ,6-diami- with Student’s t test against control.
dino-2-phenylindole (DAPI), streptomycin, penicillin, pro-
pidium iodide, sodium fluoride, sodium b-glycerophosphate, Apoptosis measurement and DAPI staining
sodium orthovanadate, sodium pyrophosphate, pepstatin A,
leupeptin, and p-amidinophenyl methanesulfonyl fluoride) DAPI staining was performed as described by Sandra et al.
were from Sigma (St. Louis, USA). [13, 14]. Briefly, the cells were seeded onto chamber slides
for 24 h, and treated with artocarpin for 24 h. Untreated
and treated cells were rinsed with phosphate-buffered sal-
ine (PBS), fixed with ice-cold 10% trichloroacetic acid, and
Fig. 1 Structure of artocarpin HO OH further washed with cold 70, 80, 90%, and absolute etha-
MeO O
nol. Cells were permeabilized with Triton-X and stained
with 1 lg/ml 40 ,6-diamidino-2-phenylindole (DAPI) for
3 min. After staining with DAPI, the cells were covers-
OH O lipped with 90% glycerol and observed under a fluores-
cence microscope.

123
J Nat Med (2010) 64:423–429 425

Sub-G1 apoptosis assay and flow cytometry by 50%. Values are given as geometric means. Differences
were considered to be statistically significant when
The cells were analyzed for their sub-G1 contents with the p \ 0.05 and p \ 0.01.
method described by Sandra et al. [13, 14]. Briefly, the
cells were seeded into 24-well plates (25 9 104 cells per
well) for 24 h. The medium was replaced with the fresh Results
medium containing artocarpin. After 24 h, cells were har-
vested and suspended in 1 ml of hypotonic fluorochrome Cytotoxic effects of artocarpin in T47D cells
solution (50 lg/ml propidium iodide in 0.1% sodium cit-
rate containing 0.1% Triton X-100). Cell suspensions were To evaluate the cytotoxicity of artocarpin, cell viability
placed at 4°C in a dark room 1–2 h before flow cytometric was measured following 24 h incubation in different con-
analysis. The propidium iodide fluorescence of individual centrations of artocarpin (5.7, 11.5, 20, and 28.7 lM)
nuclei was measured by using FACSCalibur (Benton dissolved in DMSO. Control is with media only. DMSO
Dickinson, San Jose, California). (vehicle for artocarpin) was used as the negative control;
H2O2 (0.07%) was used as the positive control (Fig. 2).
Immunoblotting Figure 2 shows that artocarpin exhibits cytotoxic activity
in a concentration-dependent manner. Comparing with
Western blot was performed as described by Sandra et al. viable cells in control (16.3 9 103 cells/well), the number
[13]. Briefly, the cells were lysed with buffer [20 mM of viable cells was decreased even at the lowest concen-
HEPES buffer pH 7.2, 150 mM NaCl, 2 mM EDTA, 1% tration, i.e., 5.7 lM (15.2 9 103 cells/well), and increasing
Triton X-100, 50 mM sodium fluoride, 40 mM sodium concentration of artocarpin concomitantly decreased the
b-glycerophosphate, 2 mM sodium orthovanadate, 30 mM viable cells, 11.5 lM (7.1 9 103 cells/well), 20 lM
sodium pyrophosphate, and a cocktail of protease inhibitors (5.1 9 103 cells/well), and 28.7 lM (4.45 9 103 cells/
(containing 10 lM pepstatin A, 10 lM leupeptin, and well), respectively (Fig. 2). Data analysis showed that the
1 mM p-amidinophenyl methanesulfonyl fluoride)] and IC50 value of artocarpin on T47D cells was 12.6 lM.
scraped. Proteins from cells were separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS- Cell and nuclear morphology
PAGE) and transferred to a polyvinylidene difluoride
(PVDF) membrane. After blocking with 5% skim milk in DAPI staining was conducted to investigate the effect of
Tris-buffered saline (150 mM NaCl and 20 mM Tris–HCl, artocarpin in inducing apoptosis. Treatment of cells with
pH 7.2), the membrane were incubated in 1:1,000 diluted artocarpin at 12.6 lM caused morphological changes
rabbit polyclonal anti-cleaved-caspase 3, 1:1,000 diluted (Fig. 3a, b) compared with normal cells (data not shown)
rabbit polyclonal anti-caspase 10, or 1:1,000 diluted mouse that might be indicative of apoptosis. Morphological
monoclonal anti-b-actin antibodies. The secondary anti- changes observed were cell shrinkage, nuclei broken into
body was 1:2,000 diluted horseradish peroxidase-conju- discrete fragments (indicated by arrows), and cell budding
gated donkey anti-rabbit or anti-mouse IgG antibodies
(Amersham, Buckinghamshire, UK). The bound antibodies
were visualized by G:Box Syngene system (Beacon House,
Cambridge, UK).

Dwm analysis and flow cytometry

Dwm analysis was conducted as described by Sandra et al.

[13]. Briefly, the cell pellet was suspended in 250 ll of
20 nM DiOC6(3) in PBS and incubated in an incubator for
15 min. In this experiment, 0.07% H2O2 was used as a
positive control. Dwm was measured by using flow
cytometry (FACSCalibur).

Statistical analysis
Fig. 2 Effect of artocarpin on T47D cell viability. The values are
averages per well. Values were expressed as mean ± SD, n = 3.
The IC50 (median inhibition concentration) is the concen- Treatments significantly different from control group (*p \ 0.05 and
tration of the compound that reduces the biological activity **p \ 0.01) according to Dunnett’s test

123
426 J Nat Med (2010) 64:423–429

that resulted in cells of various sizes. Higher concentrations Immunoblotting

of artocarpin appeared to cause more morphological
changes (data not shown), indicating that apoptosis Morphological and sub-G1 apoptosis assay clearly dem-
occurred in a concentration-dependent fashion. onstrated that artocarpin induced apoptosis in T47D cells.
To investigate the apoptotic signaling pathway of artocar-
Sub-G1 apoptosis pin, expression of proteins involved in apoptotic signaling
pathway was examined by using western blotting.
To understand the mechanism of artocarpin-induced Figure 5a shows that the expression of cleaved-caspase 3
apoptosis, the effect of artocarpin on cell cycle progression was increased with increasing concentration of artocarpin
was analyzed by flow cytometry. The results of this as indicated by stronger signal intensity observed. Similar
experiment are presented in Fig. 4. The percentages of to the expression of cleaved-caspase 3, the expression of
cells in the sub-G1 phase were 0.89, 25.71, and 82.86% as cleaved-caspase 8 appeared to gradually increase after
the concentration of artocarpin was increased to 5.7, 12.6, treatment with artocarpin at 5.7, 12.6, and 20 lM (Fig. 5b).
and 20 lM, respectively, whereas control was 1.85%. In Figure 5c shows that the signal of caspase 10, an initiator
addition, the percentages of total phase G1, S, and G2/M of apoptosis, was detected in control, DMSO, and H2O2.
were decreased from 99.48, 74.84, and 17.41% with The signal intensity of cells treated with artocarpin, how-
increasing concentration of artocarpin at 5.7, 12.6, and ever, was decreased with increasing concentration of
20 lM, respectively. artocarpin at 5.7 and 12.6 lM, and the signal disappeared
at 20 lM (Fig. 5c). We also examined caspase 9, but there
was no signal detected (data not shown). As a control for
protein loading, we also examined the expression of
b-actin. Figure 5d shows that the signal intensity was
similar between cells treated with artocarpin, DMSO, and
H2O2 and untreated cells indicating the expression of
b-actin was similar in all samples.

Mitochondrial membrane potential (Dwm)

Changes in the mitochondrial membrane potential (Dwm)

Fig. 3 Cell and nuclear morphology of T47D cells treated with
artocarpin. T47D cells (1 9 104 cells/well) were treated with arto-
have been linked to initiation and activation of the
carpin at 12.6 lM (a) and after DAPI staining (b). Arrows indicate apoptotic cascade via the intrinsic (mitochondria) pathway.
apoptotic cells. Magnification 9200 This then contributes to the activation of caspases and

Fig. 4 Effect of artocarpin on T47D cell cycle progression. Untreated T47D cells (25 9 104 cells/well, a) and cells treated with artocarpin at
5.7 lM (b), 12.6 lM (c), 20 lM (d)

123
J Nat Med (2010) 64:423–429 427

control. According to Stridh et al. [16], H2O2 in Jurkat T

lymphocytes showed Dwm dissipation and subsequently a
release of cytochrome c into the cytosol. Our experiment
showed that changed Dwm values were also found on
treatment with H2O2.

Discussion

In this experiment, the artocarpin was isolated from wood

of A. heterophyllus [11]. Previous studies reported that
isolated artocarpin from A. heterophyllus had some bio-
logical activities in vitro, such as inhibition of 5a-reductase
[17], antioxidant [18], antiplatelet [19], and antibacterial
activities [20], cytotoxicity against some human cancer
cells [21] and B16 melanoma cells [22], antiherpetic
activity [23], melanin inhibition [11], inhibition of lipo-
polysaccharide-induced nitric oxide production [24], and
Fig. 5 Immunoblotting analysis of artocarpin on T47D cells. skin whitening activity [25]. In this present study, we
Untreated T47D cells and cells treated with DMSO, 0.07% H2O2,
reported the potential anticancer properties of artocarpin in
and artocarpin at 5.7, 12.6, and 20 lM. Cell extracts were blotted by
using polyclonal antibody against cleaved-caspase 3 (a), cleaved- breast cancer cells (T47D cells) and its potential mecha-
caspase 8 (b), and caspase 10 (c) and using monoclonal antibody nism in inducing apoptosis.
against b-actin (d) The cytotoxic effect evaluated by trypan blue dye
exclusion assay showed that viable cells decreased with
increasing concentration of artocarpin; artocarpin has an
IC50 value of 12.6 lM (5.5 lg/ml). Wang et al. [9]
reported that artocarpin tested on other breast cancer cells,
MCF-7 and MDA-MB-231, had IC50 values of 3.3 and
3.8 lg/ml, respectively. The IC50 value of artocarpin in
T47D cells is, therefore, relatively similar to those in MCF-7
and MDA-MB-231. On the basis of these data, we carried
out cell and nuclear morphology analysis with DAPI to
examine whether the cells underwent apoptosis after
treatment with artocarpin. The 12.6 lM of artocarpin was
applied to the cells and compared with the untreated cells.
The results shows that exposure of the cells to 12.6 lg/ml
of artocarpin caused morphological changes of the nucleus
and demonstrated cells undergoing apoptosis (Fig. 3,
apoptotic cells are marked by the arrows). Fragmented
nuclei were more clearly observed with DAPI staining
(Fig. 3b). Our observations of altered cell and nuclear
morphology after treatment with the artocarpin (Fig. 3)
Fig. 6 Effect of artocarpin on T47D cells on mitochondrial were consistent with previous reports of cells undergoing
membrane potential (Dwm). Untreated T47D cells (2 9 105 cells/ apoptosis. Kerr [26] described that some characteristics of
well, a), 0.07% H2O2 (b), and cells treated with artocarpin at 12.6 (c)
cells undergoing apoptosis are the formation of sharply
and 20 lM (d)
delineated, uniformly finely granular masses adjacent to the
nuclear envelop and cytoplasmic condensation; breaking
subsequent induction of apoptotic cell death [15]. Since the up of the nucleus into discrete fragments surrounded by a
signal of caspase 9, a protein marker used to examine double layered enveloped; and cell budding to produce
apoptosis cascade via mitochondria, was not detected, we membrane-bound apoptotic bodies. In line with the mor-
investigated Dwm in cells treated with artocarpin and found phological analysis, a sub-G1 apoptosis assay also con-
that Dwm of cells treated were similar to the control firmed that artocarpin induced apoptosis in T47D cells
(Fig. 6). In this experiment, H2O2 was used as a positive (Fig. 4).

123
428 J Nat Med (2010) 64:423–429

Apoptosis is a regulated and ubiquitous cellular mech- These results also suggested that artocarpin may induce
anism in response to prodeath signaling. Apoptosis is apoptosis in T47D cells via the extrinsic pathway.
morphologically characterized by cell shrinkage and con- Previous studies reported that some other prenylated
densation, nuclear envelope disassembly, and DNA frag- flavonoids have induced apoptosis in some cancer cells.
mentation [26]. All these changes are due to activation of a For example, xanthohumol and xanthoaurenol induced
cascade of highly specific proteases, called caspases [27]. apoptosis via decreasing of NFjB activation in prostate
Within the cascade, caspases can be categorized into two cancer cells (BPH-1) [31]; licoflavone C and icobavachin
initiator caspase and downstream effectors of apoptosis caused apoptosis via activation of caspase 3/7 in H4IIE rat
[28]. Initiator caspases (caspase 2, 8, 9, 10, 11, and 12) are hepatoma cells [8], as did abyssinoflavanone VII [32];
closely coupled to pro-apoptotic signals. Once activated, propolin A and propolin B trigger apoptosis in melanoma
these caspases are cleaved and activate downstream cancer cells (A2058) through the mitochondria-dependent
effector caspases (caspase 3, 6, and 7), which in turn or intrinsic pathway [33]; and artonin B showed apoptotic
execute apoptosis by cleaving cellular proteins following effects in human acute lymphoblastic leukemia cells via the
specific Asp residues [27]. mitochondrial pathway [34]. This current study may add to
Nowadays, there are two well-understood caspase acti- this list of reports about apoptosis induction due to
vation pathways. The first is the assembly of the death- prenylated flavonoid compounds. In summary, on the basis
induced signaling complex (DISC) induced by the binding of the data obtained, we conclude that artocarpin induced
of the members of the death receptor family including Fas apoptosis in human breast cancer (T47D cells) possibly via
to its ligand [28]. This pathway is also called the extrinsic the extrinsic pathway.
pathway. The second is the mitochondrial (intrinsic)
pathway, which is initiated by the release of cytochrome c Acknowledgments T47D cells were generously donated by Tjan-
drawati Mozef, M.Sc., Indonesian Institute of Sciences Research
from mitochondria to the cytosol, leading to activation of Centre for Chemistry (Natural Products, Food and Pharmaceuticals
caspase 9 [28]. Division), Bandung, Indonesia.
To understand the mechanism of artocarpin-induced
apoptosis of T47D cells, we investigated the expression
level of some protein markers involved in the apoptosis References
cascade, such as caspase 8 and 10 as markers for the
extrinsic pathway, caspase 9 as a marker for the mito- 1. Liu Y, Wang MW (2008) Botanical drugs: challenges and
chondrial pathway, and caspase 3 as a marker for effector opportunities Contribution to Linnaeus Memorial Symposium
caspase. By immunoblotting analysis, we detected cleaved- 2007. Life Sci 82:445–449
2. Gupta R, Gabrielsen B, Ferguson SM (2005) Nature’s medicines:
caspase 3, an active form of caspase 3, in the cells traditional knowledge and intellectual property management.
(Fig. 5a). This caspase was observed and gradually Case studies from the National Institutes of Health (NIH), USA.
increased with increasing concentration of artocarpin. Curr Drug Discov Technol 2:203–219
Similarly, the detection of cleaved-caspase 8, a marker for 3. Barron D, Di Pietro A, Dumontet C, McIntosh DB (2002) Iso-
prenoid flavonoids are new leads in the modulation of chemo-
apoptosis via the extrinsic pathway, showed the increased resistance. Phytochem Rev 1:325–332
signaling of this protein due to artocarpin treatment 4. Di Pietro A, Conseil G, Peres-Victoria JM, Dayan G, Baubichon-
(Fig. 5b). Detection of another extrinsic marker protein, Cortay H, Trompier D, Steinfels E, Jault JM, de Wet H,
caspase 10, showed that this protein level is reduced due to Maitrejean M, Comte G, Boumendjel A, Mariotte AM, Dumontet
C, McIntosh DB, Goffeau A, Castanys S, Gamarro F, Barron D
artocarpin treatment (Fig. 5c). This might indicate that (2002) Modulation by flavonoids of cell multidrug resistance
artocarpin induced apoptosis in T47D cells via the extrinsic mediated by P-glycoprotein and related ABC transporters. Cell
pathway. Mol Life Sci 59:307–322
To investigate the possible involvement of the mito- 5. Botta B, Vitali A, Menendez P, Misiti D, Monache GD (2005)
Prenylated flavonoids: pharmacology and biotechnology. Curr
chondrial pathway of apoptosis due to artocarpin treatment, Med Chem 12:713–739
a marker for this pathway, caspase 9, was tested; however, 6. Cidade HM, Nacimento MS, Pinto MM, Kijjoa A, Silva AM,
the caspase 9 signal was not detected. We therefore Herz W (2001) Artelastocarpin and carpelastofuran, two new
investigated the mitochondrial membrane potential (Dwm) flavones, and cytotoxicities of prenyl flavonoids from Artocarp-
use elasticus against three cancer cell lines. Planta Med 67:867–
of cells treated with artocarpin. It was reported that mito- 870
chondrial transmembrane potential precedes the release of 7. Ko HH, Lu YH, Yang SZ, Won SJ, Lin CN (2005) Cytotoxic
cytochrome c which was important for Apaf-1-mediated prenylflavonoids from Artocarpus elasticus. J Nat Prod 68:1692–
caspase 9 activation [29, 30]. As shown in Fig. 6, similar 1695
8. Watjen W, Weber N, Lou YJ, Wang ZQ, Chovolou Y,
results of Dwm were observed in untreated cells and those KampkÖtter A, Kahl R, Proksch P (2007) Prenylation enhances
treated with artocarpin, except by H2O2. This Dwm result cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma
may support the absence of caspase 9 activation signal. and C6 glioma cells. Food Chem Toxicol 45:119–124

123
J Nat Med (2010) 64:423–429 429

9. Wang YH, Hou AJ, Chen L, Chen DF, Sun HD, Zhao QS, 22. Arung ET, Yoshikawa K, Shimizu K, Ryuichiro K (2010) Iso-
Bastow KF, Nakanish Y, Wang XH, Lee KH (2004) New prenoid-substituted flavonoids from wood of Artocarpus hetero-
isoprenylated flavones, artochamins A-E, and cytotoxic principles phyllus on B16 melanoma cells: cytotoxicity and structural
from Artocarpus chama. J Nat Prod 67:757–761 criteria. Fitoterapia 81:120–123
10. Arung ET, Shimizu K, Ryuichiro K (2006) Inhibitory effect of 23. Likhitwitayawuid K, Chaiwiriya S, Sritularak B, Lipipun V
artocarpanone from Artocarpus heterophyllus on melanin bio- (2006) Antiherpetic flavones from the heartwood of Artocarpus
synthesis. Biol Pharm Bull 29:1966–1969 gomezianus. Chem Biodivers 10:1138–1143
11. Arung ET, Shimizu K, Ryuichiro K (2006) Inhibitory effect of 24. Han AR, Kang YJ, Windono T, Lee SK, Seo EK (2006) Preny-
isoprenoid-substituted flavonoids isolated from Artocarpus het- lated flavonoids from the heartwood of Artocarpus communis
erophyllus on melanin biosynthesis. Planta Med 72:847–850 with inhibitory activity on lipopolysaccharideinduced nitric oxide
12. Jamil K, Shaik AP, Mahboob M, Krishna D (2004) Effect of production. J Nat Prod 69:719–721
organophosphorus and organochlorine pesticides (Monochroto- 25. Shimizu K, Kondo R, Sakai K, Takeda N, Nagahata T (2002) The
phos, chlorpyriphos, dimethoate, and endosulfan) on human skin-lightening effects of artocarpin on UVB-induced pigmenta-
lymphocytes in vitro. Drug Chem Toxol 27:133–144 tion. Planta Med 68:79–81
13. Sandra F, Matsuda M, Yoshida H, Hirata M (2002) Inositol 26. Kerr JFR (1994) Apoptosis. Its significance in cancer and cancer
hexakisphospahate blocks tumor cell growth by activating therapy. Cancer 73:2013–2026
apoptotic machinery as well as by inhibiting the Akt/NFkB- 27. Degterev A, Boyce M, Yuan J (2003) A decade of caspases.
mediated cell survival pathway. Carcinogenesis 23:2031–2041 Oncogene 22:8543–8567
14. Sandra F, Hendarmin L, Nakao Y, Nakao Y, Nakamura N, 28. Mor G, Montagna MK, Alvero AB (2008) Modulation of apop-
Nakamura S (2005) TRAIL cleaves caspase-8, -9 and -3 of AM-1 tosis to reverse chemoresistance. Apoptosis and cancer, methods
cells: a possible pathway for TRAIL to induce apoptosis in and protocols. Humana, Totowa
ameloblastoma. Tumor Biol 26:258–264 29. Zamzami N, Susin SA, Marchetti P, Hirsch T, Gómez-Monterrey
15. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science I, Castedo M, Kroemer G (1996) Mitochondrial control of nuclear
281:1309–1312 apoptosis. J Exp Med 183:1533–1544
16. Stridh H, Kimland M, Jones DP, Orrenius S, Hampton MB (1998) 30. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M,
Cytochrome c release and caspase activation in hydrogen per- Alnemri ES, Wang X (1997) Cytochrome c and dATP-dependent
oxide- and tributyltin-induced apoptosis. FEBS Lett 429:351–355 formation of Apaf-1/caspase-9 complex initiates an apoptotic
17. Shimizu K, Fukuda M, Kondo R, Sakai K (2000) The 5a- protease cascade. Cell 91:479–489
reductase inhibitory components from heartwood of Artocarpus 31. Colgate EC, Miranda CL, Stevens JF, Bray TM, Ho E (2007)
incisus: structure–activity investigations. Planta Med 66:16–19 Xanthohumol, a prenylatedflavonoid derived from hops induces
18. Rajendran M, Manisankar P, Gandhidasan R, Murugesan R apoptosis and inhibits NF-kappaB activation in prostate epithelial
(2004) Free radical scavenging efficiency of a few naturally cells. Cancer Lett 246:201–209
occurring flavonoids: a comparative study. J Agric Food Chem 32. Watjen W, Suckow-Schnitker AK, Rohrig R, Kulawik A, Addae-
52:7389–7394 Kyereme J, Wright CW, Passreiter CM (2008) Prenylation fla-
19. Lin CN, Lu CM (1996) Novel antiplatelet constituents from vonoid derivatives from the bark of Erythrina addisoniae. J Nat
formosan Moraceous plants. J Nat Prod 59:834–838 Prod 71:735–738
20. Sato M, Fujiwara S, Tsuchiya H, Fujii T, Iinuma M, Tosa H, 33. Chen CN, Wu CL, Lin JK (2007) Apoptosis of human melanoma
Ohkawa Y (1996) Flavones with antibacterial activity against cells induced by the novel compounds propolin A and propolin B
cariogenic bacteria. J Ethnopharmacol 54:171–176 from Taiwanese propolis. Cancer Lett 245:218–231
21. Itoigawa M, Ito C, Jun-Ichi M, Nobukuni T, Ichiishi E, Tokuda 34. Lee C, Lin CN, Jow GM (2006) Cytotoxic and apoptotic effects
H, Nishino H, Furukawa H (2002) Cancer chemopreventive of prenylflavonoid artonin B in human acute lymphoblastic leu-
activity of flavanones on Epstein–Barr virus activation and two- kemia cells. Acta Pharmacol Sin 27:1165–1174
stage mouse skin carcinogenesis. Cancer Lett 176:25–29

123