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References
Introduction
Natural products from plants or Chinese herbs have been used as traditional remedies in Asian countries for hundreds of
years. The development of compounds with antitumor effects from natural products has become a very important topic.
Flavonoids are commonly found in most plants and exert a remarkable spectrum of biological activities that affect basic cell
functions, and several beneficial biological activities of flavonoids including antioxidant, antitumor, and anti-inflammation
properties have been identified in several
studies[1,2]. Flavonoids are also dietary pharmacological agents, which may block
neoplastic inception or delay disease
progression[3,4]. These data indicate that certain flavonoids may be used as possible
chemopreventive or chemotherapeutic agents.
The great prevalence of flavonoids in the vegetal kingdom act, not only as the colored pigments of flowers, but also as
enzyme inhibitors, precursors of toxic substance, and a defense against ultraviolet radiation exposure. Flavonoids were
found to act on the growth of cancer cells, which means that it possesses potential anti-tumor activity. For example, baicalein,
epigallocatechin (EGC) gallate and green tea extract were reported to inhibit tumor
growth[5_7]. Furthermore, epidemiological
studies indicated that diets containing linseed and soy (rich in isoflavonoids and lignans) might protect against colon,
breast, and prostate cancer[8]. Flavonoids are benso-r-pirone derivatives that can be grouped according to the presence of
different substituents on the rings and to the degree of benso-r-pirone ring saturation. Artonin B is a prenylflavonoid that is
obtained from the root bark of Artocarpus
heterophyllus Lamk[9]. Artonin B is a derivative from heterophyllin and possesses
the structure in C-10 position of isoprenoid moiety. However, the effects of artonin B on cancer cell growth have rarely been
investigated in great detail.
Apoptosis is a cell suicide program, which is essential for the development and maintenance of tissue homeostasis and
the elimination of unwanted or damaged cells from multicellular
organisms[10,11]. Apoptosis is characterized by a series of
morphological changes involving cell shrinkage, chromatin condensation and the formation of apoptotic
bodies[12]. It can be triggered by various extracellular and intracellular stimuli that result in the coordinated activation of
family proteases called caspases. The activation of caspase
3 pathways is an important downstream executioner in
apopto-sis[13].
Human leukemia is a commonly diagnosed neoplasm and the major leading cause of human death. CCRF-CEM cells are
acute lymphoblastic leukemia (ALL) cells. ALL represents the clonal proliferation of malignantly transformed lymphoid
progenitors in the bone marrow. The treatment of patients with recurrent cancer is usually unsuccessful, and the
development of new potent treatments has become the focal point for cancer treatment. Therefore, we evaluated the effects and
action mechanism of artonin B on human acute lymphoblastic leukemia CCRF-CEM cells. In this present study, the cell
cytotoxicity of four prenylflavonoid compounds was examined. Furthermore, morphological nuclear fragmentation, apoptotic
body formation, the change of mitochondrial membrane potential, cytochrome c release, cell cycle change, Bcl-2, Bax, and Bak
protein expression, and caspase 3 activity in artonin B treated human CCRF-CEM leukemia cells were investigated.
Materials and methods
Materials RPMI-1640, fetal bovine serum (FBS), and antibiotics were purchased from Hyclone. Caspase 3 assays kits
and caspase 3 inhibitor (z-DEVD-fmk) were obtained from Biovision. The cytochrome c was purchased from R&D Systems.
Monoclonal antibodies of Bcl-2, Bax, Bak, and anti-rabbit IgGs were obtained from Cell Signaling. Hoechst 33258 and the rest
of chemicals were purchased from Sigma.
The four prenylflavonoid compounds that were extracted from Artocarpus species (Moraceae) were obtained from Dr
Chun-nan LIN (School of Pharmacy, Kaohsiung Medical University, Taiwan, China). Artocarpanone (Figure 1A) was an
isoprenoid-flavone[14]. Artonin A and artonin B (Figure 1B and 1C) were extracted from the root bark of Artocarpus heterophyllus Lamk (9). And, the new prenylflavonoid-Artocammunols CE (Figure 1D) was obtained from the Artocarpus communis[15]. These four prenylflavonoid compounds were dissolved in dimethyl sulfoxide (DMSO) as stock solution. The final
concentration of DMSO in each experiment was less than 0.1%.
Cell culture Human acute lymphoblastic leukemia cell line (CCRF-CEM) was purchased from the Culture Collection and
Research Center (Taiwan ). CCRF-CEM cells were maintained in RPMI-1640 medium (Hyclone) supplemented with 10%
heat-inactivated fetal bovine serum (Hyclone), 100 µg/mL penicillin, 100 µg/mL streptomycin and 100 µg/mL amphotericin B
(Hyclone). The cells were grown in a humidified incubator at 37 ºC under a 5%
CO2/95% air atmosphere. For each experiment,
3×105 cells were seeded in each well in a 24-well plate containing 1 mL of fresh medium and incubated with or without chemical
treatment for the indicated time. For toxicity study, the cells were treated with drugs during the exponential phase of cell
growth.
Cytotoxicity assay The cytotoxic effect of drugs was determined using the MTT
method[16]. In brief, 100 µL MTT solution
(0.5 mg/mL in phosphate-buffer saline or PBS) was added to each well at the end of each experiment. After 1_2 h incubation
at 37 ºC, 10 µL Triton X-100 (10%) was added and mixed
well. Once the cells were completely dissolved, the absorbance
difference at 550 nm was measured using a microplate reader, with the RPMI medium as a blank.
Microscopic observation of morphology and nuclear
fragmentation After artonin B treatment, cells were harvested by
centrifugation, washed with PBS, and fixed with 1% glutaraldehyde in 100 µL of PBS at room temperature for 1 h. Fixed cells
were washed with PBS and then stained with 200 µmol/L Hoechst 33258 in 20 µL PBS for 30 min at room temperature. Five
hundred stained cells from each treatment group were examined and counted under an Olympus fluorescence microscope.
DNA content and cell cycle analysis Human CCRF-CEM cells were collected and rinsed with PBS, after being cultured
with 0, 1, 5, or 10 µmol/L artonin B for 24 h, and suspended in 75% ethanol at -20 ºC overnight. Fixed cells were centrifuged
at 1200×g and washed with PBS twice. To detect DNA content, cells were contained in the dark with Propidium Iodide (PI) 50
mg/L and 0.1% RNase A in 400 µL PBS at 25 ºC for 30 min. Stained cells were analyzed on FACSort (Becton Dickinson). The
percentage of apoptotic cells was determined using the CellQuest software program.
Assay of mitochondrial membrane potential Initially, 1×106 human CCRF-CEM cells/mL were incubated with 2 mmol/L
rhodamine 123 for 10 min at 37 °C. After the incorporation of a fluorescent probe, the cells were incubated for up to 4 h with
or without 10 µmol/L artonin B. At the end of incubation, the cells were washed twice with PBS, harvested by centrifugation,
and then resuspended in 1.5 mL PBS. The fluorescent intensity of each cell suspension was measured at an excitation
wavelength 480 nm and an emission wavelength 530 nm in a Perkin-Elmer Victor 3 fluorescent microplate reader. The
fluorescence intensity was used as an arbitrary unit representing the mitochondrial transmembrane potential.
Cytochrome c release Human CCRF-CEM cells were seeded in 2 mL fresh medium at an initial density of
1×106 cells/mL and incubated for up to 4 h with or without 10
mmol/L artonin B. After the incubation, the cells were harvested by
centrifugation and washed twice with PBS. The cells were suspended in 200 mL lysis buffer (195 mmol/L mannitol; 65 mmol/L
sucrose; 2 mmol/L HEPES, pH 7.4; 0.05 mmol/L EGTA; 0.01 mmol/L
MgCl2; 0.5 g/mL BSA) and lysed by the addition of 0.01%
digitonin. The cytosolic fraction was
obtained from 10 000×g centrifugation for 10 min and was collected for cyt c assay in 1×RD5P calibrator diluent (cyto-chrome
c Immunoassay Kit; R&D Systems, MN, USA).
After reacting with cyt c antibody and substrate, the
absorbance was measured at 450 nm (reference wavelength is 540 nm).
Western blot analysis After being exposed to the indicated concentration of artonin B, human CCRF-CEM cells were
washed with cold PBS. Whole cell extracts were prepared
by incubating the cells with cold lysis buffer (20 mmol/L
Tris-HCl; pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1
mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1
mmol/L b-glycerophosphate, 1 mmol/L
Na3VO4, 1 mg/mL leupeptin, and 1 mmol/L PMSF). The protein content of the lysates was
determined using the DC protein assay kit (Bio-Rad). The cell lysates (25 µg protein/lane) were electro-phoresized on 12%
SDS-polyacrylamide gels. The cellular proteins were
then transferred to PVDF membranes by electroblotting for 2
h and Western blot analysis was carried out as previously
described[17]. The protein levels were visualized with an
enhanced chemiluminescence detection kit (Amersham).
Preparation of cytosolic extract and measurement of caspase 3 activity After the treatment with indicated agents, cells
were harvested and washed with PBS by centrifugation at
750× g for 5 min at 4 ºC. The cell pellets were resuspended in lysis
buffer (caspase colorimetric assay kits; Biovision) and left on ice for 30 min. The lysates were centrifuged at
10 000×g for 10 min and the supernatant (20 µL) was used for caspase-3 activity assay in the lysis buffer containing
DEVD-pNA, a specific substrate to caspase-3. The concentration of pNA, as the product from enzymatic converting of DEVD-pNA
by caspase-3, was measured at 405 nm and used as an indication of caspase-3 activity.
Assessment of cell necrosis The necrotic cell death was measured by the release of lactate dehydrogenase (LDH) into
the culture medium, which indicates the loss of membrane integrity and cell necrosis. LDH activity was measured using a
commercial assay kit (Cytotoxicity assay kit, Promega), where the released LDH in culture supernatants is measured with a
coupled enzymatic assay, which results in the conversion of a tetrazolium salt into a red formazan product. The necrotic
percentage was expressed as (sample value/maximal release)×100%, where the maximal release was obtained following the
treatment of control cells with 0.5% Triton X-100 for 10 min at room temperature.
Statistic analysis For each experiment involving assessment of cell survival, apoptotic cell, and caspase-3 activity are
presented as the mean and standard error (SEM) for four to five experiments. The statistical analysis of data was performed
by one-way ANOVA, followed by the Schefft test, and P-values less than 0.05 were considered significant.
Results
Effects of prenylflavonoid compounds on cytotoxicity in human CCRF-CEM leukemia
cells The cytotoxic effects of four prenylflavonoid compounds were examined in human CCRF-CEM leukemia cells. When CCRF-CEM leukemia cells were
incubated with 10 µmol/L of four prenylflavonoids, the data showed that artonin B had a more potent cytotoxicity than the
other compounds (Figure 2). In addition, the cell survival rate decreased in a dose-dependent manner (Figure 3A). Under the
same treatment, artonin B did not cause any cell loss in HaCa cells (Figure 3B). Artonin B elicited a significant decrease of cell
survival rate in human CCRF-CEM leukemia cells, which also exhibited a time-dependent manner (Figure 4). After an exposure
time of 6 h, the cytotoxic effects were noticed at a concentration of 5 µmol/L and 10 µmol/L of artonin B, with a the survival
rate decreasing to 64% at 5 µmol/L and 22% at 10 µmol/L. Accordingly, after
24 h exposure cytotoxic effects started to become apparent at a concentration of approximately 3_10 µmol/L, cell viabi-lity
declined considerably at 3 mmol/L and was approximately 10% at 10 µmol/L of artonin B (Figure 4). The
IC50 value of artonin B was 3.45±0.50 µmol/L.
Assessment of artonin B-induced cell apoptosis and
intracellular events To determine whether the artonin B-
induced cytotoxicity was to undergo the apoptotic cell pathway, human CCRF-CEM leukemia cells were incubated
in presence of 1_10 µmol/L artonin B for 24 h. The morphological examination reveled that artonin B-treated cells showed
typical apoptotic morphological changes, such as cell shrinkage, nuclear fragmentation, and apoptotic body formation
(Figure 5). Artonin B-treatment significantly
increased the number of cells with apoptotic body formation (Figure 5D,5E), while the control cells and 1_3 µmol/L of artonin
B-treated cells showed seldom apoptotic body formation (Figure 5A_5C). The number of apoptotic cells, which carry
fragmented nuclear particles, were significantly increased in artonin B-treated cells in a dose-dependent manner (Figure 5F).
The necrotic indication of cellular lactate dehydrogenase (LDH) release was also examined after artonin B treatment
(Figure 6). The data showed that 5 mmol/L and 10
mmol/L artonin B induced an increase of LDH release in only
20.59%±2.12% and 28.23%±0.81%, respectively,
while the cell survival rates were 35.33%±0.9% and 14.76%±3.56%, respectively.
These data indicated necrotic cytotoxicity was less involved in artonin B action. Thus, artonin B induces leukemia cell death
employing an apoptotic pathway.
Cell cycle analysis Figure 7 illustrates the changes of DNA content distribution treated with artonin B 0, 1, 5, or 10
µmol/L for 24 h. We examined these cells for DNA degradation characteristic of apoptosis, indicated by hypoploid DNA content
using hypotonic PI staining. Exposure of human CCRF-CEM leukemia cells to 1 µmol/L artonin B promoted approximately the
same percentage of hypoploid cells observed in the DMSO treated control. As the treatment dose increased the percentage
of cells in the hypoploid (sub-G1) phase increased accordingly. Treated with 5 or
10 µmol/L artonin B for 24 h, the rate of
sub-G1 phase cells were
increased by 11.43% or 18.56%, respectively.
Changes of mitochondrial membrane potential and
release of cytochrome c from mitochondria In the present study, the mitochondrial membrane potential and cytochrome c
release were analyzed spectrophotometrically. As shown in Figure 8, artonin B-induced a time-dependent mitochondrial
transmembrane depolarization, represented as the decrease of mitochondrial membrane potential (Figure 8A). Concomitantly,
a time-dependent artonin B-induced cytochrome c release was also observed in human leukemia CCRF-CEM cells,
representing a significant increase of cytosolic cytochrome c concentration (Figure 8B). These data suggest that loss of mitochondrial
membrane potential may be required for artonin B-induced cytochrome c release into cytosol, that later triggered the cleavage
and activation of mitochondrial downstream caspases and onset of apoptosis.
Regulation of Bcl-2 family proteins in artonin B-treated human leukemia CCRF-CEM cells To determine whether Bcl-2 family proteins were modulated in artonin B-induced apoptosis in human leukemia CCRF-CEM cells, the expression of
several members of Bcl-2 family proteins was examined by Western blot analysis. As shown in Figure 9, the exposure of
human leukemia CCRF-CEM cells to 1_10 µmol/L artonin B resulted in a marked decrease of Bcl-2 protein expression, but a
drastic increase of Bax and Bak protein expression.
Determination of the involvement of caspase 3
activation Artonin B-induced nuclear fragmentation may thus be an
apoptotic event provoked along with endonuclease activation
via caspase 3 protein. We also examined the caspase
3 activity under the 1_10 µmol/L artonin B treatment. The present study has demonstrated that artonin B treatment increased caspase
3 activities in human CCRF-CEM leukemia cells in a dose-dependent manner (Figure 10). Human leukemia CCRF-CEM cells
were pretreated with 50 µmol/L caspase 3 inhibitor (z-DEVD-fmk) for 2 h, and then induced to undergo apoptosis by treatment
with artonin B. The results clearly showed that the administration of caspase 3 inhibitor alone did not affect the caspase 3
activation, apoptotic cell formation, and cell viability (Figure 11). How-ever, z-DEVD-fmk (a specific caspase 3 inhibitor)
significantly inhibited artonin B-induced caspase 3 activation, apoptotic cells formation, and cell death in human acute
lymphoblastic leukemia cells.
Discussion
In the present work, we demonstrated that artonin B, one of prenylflavonoids, strongly inhibited the growth of human
acute lymphoblastic leukemia CCRF-CEM cells, whereas artonin A, artocarpanone, and artocammunols prenylfla-vonoid
compounds had no effect on the growth of CCRF-CEM cells. This is a pioneer study of artonin B-induced cell cytotoxicity
in human leukemia CCRF-CEM cells. Prenylfla-vonoids exist extensively in plants; however, the structure-activity
relationship of their effect is still unknown. These results indicate that heterophyllin structure would be needed in the growth
inhibitory effect of prenylflavonoid compounds. Importantly, we observed that artonin B had no effect on the growth of
HaCa cells; this is in agreement with data showing that artonin B had a growth inhibitory effect on cancer cells, but not on
normal cells. Therefore, artonin B could be a good candidate for acute lymphoblastic leukemia cells therapy without toxicity
for normal cells.
Our results revealed that human CCRF-CEM cells treated with artonin B exhibited characteristic morphological features of
apoptosis, such as membrane shrinkage chromosomal condensation. The notion that artonin-B treated cells undergo apoptosis
rather than necrosis is further supported by the results from cell cycle analysis. The proportion of hypoploid cells
(sub-G1) was dramatically increased after artonin-B treatment. These results support the finding that artonin-B induces cell death
through apoptotic pathway.
The several mechanisms of activation of apoptosis in different physiological or pathological conditions in cells have
been proposed and studied
intensively[18]. Numerous factors, such as cytosolic cytochrome c release, the expression of
Bcl-2 family proteins, and caspase 3 activation have been suggested to play an essential role in the apoptotic process in cancer
cells. Our study demonstrated that a progressive decrease of the mitochondrial membrane potential and release of
cytochrome c into the cytosol were observed in artonin-B treated human CCRF-CEM leukemia cells. It has been noticed in many in vitro systems that apoptosis was associated with a loss of mitochondrial membrane potential, which may correspond to
the opening of an outer membrane permeability transition pore. Thus, this event has been suggested to be responsible for
cytochrome c release into cytosol from
mitochondria[19]. In our present study, the cytosolic cytochrome c accumulation in
artonin B-induced human CCRF-CEM cells is probably the consequence of the loss of mitochondrial membrane potential,
which finally leads to cell death.
The Bcl-2 family is composed of a number of genes that play critical roles in the control of mitochondrial integrity. Several
studies have shown that overexpression of Bcl-2 prevents the mitochondrial release of cytochrome c, thereby
inhibiting the activation of caspases cascade and
apopto-
sis[20_23]. In the present study, artonin B-induced apoptosis in human CCRF-CEM leukemia cells was accompanied by
upregulation of Bax and Bak and downregulation of Bcl-2. Other studies have demonstrated that Bcl-2, Bax, and Bak can act
as channel proteins within the mitochondrial
membrane[21,24,25]. It is conceivable that the channel property of Bax and Bak may
control the mitochondrial permeability transition and other early mitochondrial perturbation. Thus, Bax and Bak may facilitate
the passage of some important proteins, such as cytochrome c or other apoptosis-inducing factors that trigger the activation
of caspases cascade and apoptosis. Previous reports have also documented that the ratio of pro-and anti-apoptotic proteins
determines, at least in part, the susceptibility of cells to a death
signal[21,26,27]. Our results showed that expression of Bcl-2
family proteins Bcl-2, Bax, and Bak can be regulated differently by artonin B, suggesting that the artonin B-induced apoptosis
is controlled by a balanced expression between those apoptosis-
inducing and apoptosis-suppressing molecules.
Apoptosis is a type of cell death, and agents with the ability to induce apoptosis in tumors have the potential to be used
for antitumor therapy. The apoptotic mechanism has been extensively studied, and activation of caspase 3
has been shown to occur in the common apoptotic
pathway[28]. The activation of caspases plays a pivotal role in the execution of cell
apoptosis[29]. Recent studies have demonstrated that the caspase 3 is a major caspase, which is activated in response to distinct stimuli [30_33]. Moreover, human acute lymphoblastic leukemia CCRF-CEM cells were preincubated with specific caspase 3 inhibitor
(z-DEVD-fmk) before treatment of artonin B, and the caspase 3 activity, apoptotic cells and cell viability were analyzed by
spectrophotometry analysis, Hoechst 33258 staining and MTT assay, respec-tively. Results showed that pre-incubation of
cells with z-DEVD-fmk effectively inhibited artonin B-induced caspase 3 activity, apoptotic cell formation and cell death. Our
data reveled that artonin B-induced nuclear fragmentation may be an apoptotic event provoked along with endonuclease
activation via a caspase protein[34,35]. We also examined the possibility that caspase 3 was involved in the morphological
changes in artonin B-treated cells by measuring the caspase 3 activity with or without its inhibitor, z-DEVD-fmk. Theses data
demonstrated that artonin B activated caspase 3 activity and consequent cell death.
However, the development of effective chemopreventive approaches must take into consideration the selective and
differential effects manifested by different bioactive substances. Target specific agents that are capable of
inducing selective apoptosis of cancer cells, but are harmless to normal cells are receiving considerable attention in the fields
of cancer prevention and therapy[36]. Artonin B-
induced cell cytotoxicity on human leukemia CCRF-CEM cells, but not on normal cells. Therefore, artonin B is a candidate for
development as a chemopreventive agent.
In summary, our results demonstrate that cell cytotoxicity induced by artonin B in human CCRF-CEM leukemia cells is
possibly mediated through apoptotic cell formation, mitochondrial pathways, Bcl-2 family protein expression, and the
activation of caspase 3. However, artonin A, artocarpanone, and artocammunols CE have no effect on the cell survival rate of
human CCRF-CEM leukemia cells. By analyzing the structure, more effective compounds might be reconstruc-tured and new
strategies for cancer therapy can be explored.
Acknowledgements
We would like to thank Dr C N LIN (School of Pharmacy, Kaohsiung Medical University, Taiwan, China) for his generosity
in providing the drugs.
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