ARTICLE IN PRESS
Cancer Letters xxx (2009) xxx–xxx
Contents lists available at ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet
Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort
Marchantia emarginata subsp. tosana induces apoptosis in human
MCF-7 breast cancer cells
Wei-Jan Huang a, Chia-Li Wu b, Chia-Wei Lin c, Li-Ling Chi c, Pen-Yuan Chen c, Chun-Jung Chiu c,
Chung-Yang Huang c, Chia-Nan Chen c,*
a
b
c
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan
Department of Chemistry, Tamkang University, Tamsui 251, Taiwan
Naturewise Biotech and Medicals Corporation, Taipei 115, Taiwan
a r t i c l e
i n f o
Article history:
Received 30 June 2009
Received in revised form 3 October 2009
Accepted 9 October 2009
Available online xxxx
Keywords:
Marchantia tosana
Marchantin A
Breast cancer
Apoptosis
Cyclic bis(bibenzyl ether)
a b s t r a c t
Liverwort constituents have been reported to exert a broad spectrum of biological activities. In this study, we used a bioactivity-guided separation of an extract from the liverwort
species Marchantia emarginata subsp. tosana to determine its anticancer activity. A high
level of the active ingredient was isolated from this liverwort and its chemical structure
was identified and characterized by various spectra. It was found to be identical to a
well-known compound, marchantin A, a cyclic bisbibenzyl ether. However, no anticancer
activities of this compound have previously been reported. We found that marchantin A
efficiently induced cell growth inhibition in human MCF-7 breast cancer cells, with an
IC50 of 4.0 lg/mL. Fluorescence microscopy and a Western blot analysis indicated that
marchantin A actively induced apoptosis of MCF-7 cells. The levels of cleaved caspase-8,
cleaved caspase-3, cleaved caspase-9, and cleaved poly (ADP ribose) polymerase (PARP)
increased. However, the level of Bid markedly decreased in a dose- and time-dependent
manner. We also evaluated the anticancer activities of marchantin A on the regulation of
cell cycle regulators such as p21, p27, cyclin B1, and cyclin D1. The p21 and p27 gene
expressions increased markedly while cyclin B1 and D1 gene expression decreased markedly by treatment with marchantin A. Many report demonstrated that liverwort was suggested to possess potent antioxidant activity. Our results indicate that marchantin A
possesses free radical-scavenging activity (EC50 = 20 lg/mL). Taken together, for the first
time, the compound marchantin A from liverworts demonstrated to be a potent inducer
of apoptosis in MCF-7 cells.
Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
There are about 6000 species of liverwort identified in
the world. Liverworts contain oil bodies in their cells and
* Corresponding author. Address: Center of Research and Development,
NatureWise Biotech and Medicals Corporation, ER-13, 17 Fl., No. 3, YuanQu St., Nankang, Taipei 115, Taiwan. Tel.: +886 2 2655 9109; fax: +886 2
2655 9110.
E-mail address: [email protected] (C.-N. Chen).
elaborate mainly sesquiterpenes, diterpenes and lipophilic
aromatics as their major chemical constituents. Several
biological activities of liverworts are triggered from these
active ingredients. Plants of Marchantia species had been
used as traditional medicine in ancient China. Many bioactive components, particularly the bis(bibenzyl) ethers,
such as marchantin A–C, have been isolated from various
Marchantia species [1]. Marchantia emarginata subsp.
tosana (Marchantiaceae) is a common thallus liverwort
species. Humulane-type sesquiterpenes and bis(bibenzyl)
0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2009.10.006
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
compounds had been reported from the Japanese liverwort
M. tosana [1,2]. In Taiwan, this liverwort is widely distributed in moist and endolithic areas at low elevations, however, the chemical components as well as their
bioactivities have not been seriously investigated. In the
present study, we found that the Taiwanese species M.
tosana contains a fairly high amount of marchantin A,
which had been isolated from M. tosana previously, yet
never in such rich scale. Marchantin A has been reported
to possess diverse biological activities, such as antifungal
[3], antimicrobial [4], anti-inflammatory [5], antioxidative,
and skeletal muscle relaxing [6] activities. Askawa demonstrated that marchantin A was efficient against fungi of
several species [7] and inhibited LPS-induced iNOS in
RAW264.7 cells [5]. As far as we know, no anticancer activities of this compound have yet been reported. In the present study, we are interested in the anticancer activities of
marchantin A against human breast cancer MCF-7 cells.
Apoptosis, or programmed cell death, plays an essential
role in controlling cell numbers in many developmental
and physiological settings. Its morphological characteristics include cell shrinkage, nuclear condensation, plasma
membrane blebbing, chromosomal DNA fragmentation,
and the formation of apoptotic bodies [8]. Recent reports
have shown that many anticancer drugs or chemoprevention agents act by inducing apoptosis to inhibit tumor promotion and progression. Mitochondria are currently
regarded playing a central role in mediating ‘‘intrinsic
death signals” and can serve as a novel target for chemotherapies [9,10]. Cytochrome c (Cyt c) is a mitochondrial
protein that can activate caspase-9. However, the release
of Cyt c is regulated by anti- and proapoptotic members
of the Bcl-2 family (such as Bcl-2, Bcl-xL, Bax, Bak, Bad,
and Bid). Once in the cytoplasm, it binds Apaf-1 to procaspase-9, leading to activation of caspase-9 and triggering of
the caspase cascade [11]. Many parameters of mitochondrial physiology have been shown to be hallmarks of apoptosis. These include loss of the mitochondrial membrane
potential, generation of reactive oxygen species (ROS), termination of oxygen consumption, and the release of Cyt c
[12]. In the present study, we investigated the effect of
marchantin A on the induction of apoptosis in MCF-7 cells.
Breast cancer is the second most common type of cancer and the fifth most common cause of cancer death in
the world. The incidence of breast cancer had increased
4-fold in the past 25 years (1980–2005) according to the
statistics of Department of Health in Taiwan. In Taiwan,
incidence of female breast cancer was from 12.7 per
100,000 women in 1980 to 49 per 100,000 women in
2005. In 2008, calculated 182,460 new cases of invasive
breast cancer will be diagnosed in women in the United
States and breast cancer is expected to cause 40,480 deaths
in the US. In North America, women have the highest incidence of breast cancer. Although no one method can predict the development of breast cancer, several risk factors
have been found and allow clinicians to identify women
at highest risk [13]. Recent research focused on exploring
options such as chemoprevention to reduce the rate of
developing breast cancer among high risk women. Conventional therapeutic strategies include surgery, radiation, and
chemotherapy. Due to their side effects and limited effec-
tiveness, there is still a pressing need for the development
of anti-breast cancer drugs. Natural products exerting diverse bioactivities and possessing unique structural properties are an important source for the development of
novel anticancer drugs [14]. In this study, the anticancer
effects of marchantin A, a class of secondary metabolites
produced exclusively in liverworts (M. tosana), like the dimer of stilbenoids, were evaluated in human MCF-7 cells.
2. Materials and methods
2.1. Plant material
M. tosana (Fig. 1a) was collected from Nankang, Taipei,
and the species was identified by Prof. Shan-Hsiung Lin
(Department of Life Science, Tunghai University, Taiwan).
The specimen was stored at NatureWise Biotech and Medicals Corp. Samples were collected in the summer 2007.
2.2. Extraction and isolation of marchantin A
The air-dried and powdered material of M. tosana
(500 g) was extracted with 5 L of MeOH, sonicated for
1 h, then left to stand for 2 weeks at room temperature
(24–25 °C). The filtered methanol extract was evaporated
to dryness under reduced pressure to obtain a crude extract (11.7 g), which was suspended in water (500 mL)
and partitioned with n-hexane and EtOAc (3 500 mL). A
bioassay test showed that the EtOAc (ethyl acetate) extract
exhibited significant anticancer activity than n-hexane or
water extract against MCF-7 cells. The EtOAc extract was
evaporated under reduced pressure (1.8 g). A portion
(1.0 g) of the EtOAc extract was chromatographed over a
silica gel column and eluted with an n-hexane:EtOAc gradient system (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8,
1:9, 0:10, then pure MeOH) to obtain ten (F1–F10) fractions. These fractions were assayed for their effects on
MCF-7 cell proliferation. The most active fraction (F4)
was purified again by silica gel column and eluted with
an n-hexane: EtOAc gradient system (from 10% to 60%).
The inhibitory activities of these eluted fractions against
MCF-7 cells were screened in a proliferation platform assay. The active fractions were purified on a reversed-phase
preparative high-performance liquid chromatographic/
ultraviolet (HPLC/UV) unit. Conditions included a Phenomenex Luna C18 silica gel column (10.0 250 mm, USA), a
solvent system of methanol:water (80:20), a flow rate of
3.0 mL/min, detection at UV 210 nm, the temperature of
column at 25 ± 1 °C, and the elution conditions are used
the isocratic. Fractions with a retention time of 7.3 min
containing the active ingredient were collected. The active
ingredient was identified as a known compound, marchantin A (Fig. 1b). Marchantin A, a white amorphous solid, has
a molecular formula of C28H24O5, as deduced from 13C NMR
(CPD and DEPT) data and HREIMS (440.1632). By means of
1
H, 13C NMR analysis (Table 1) and 2D NMR (including
HMQC, HMBC, COSY, and NOESY methods), the structure
was elucidated as marchantin A [15]. The purity of marchantin A was estimated to be at least 97% by HPLC/UV, based
on the peak area.
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
ARTICLE IN PRESS
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
a
b
1
2
6
3
OH
O
1'
5
7
8
6'
3'
5'
7'
8'
9
9'
10
14
11
OH
2'
13
OH
O
14'
10'
13'
11'
c
120
140
d
e
Control
Marchantin A
120
100
Cell Viability (%)
Cell Viability (%)
100
80
60
40
80
60
40
20
20
0
0
0
2.5
5.0
7.5
MarchantinA ( g/mL)
10.0
0
24
48
72
Incubation Times (h)
Fig. 1. Structure of marchantin A and inhibition of cell growth. (a) Morphology of the liverwort, M. tosana, which was collected in summer 2007. (b) The
structure of the active ingredient, marchantin A. (c) Cell growth inhibition activity of marchantin A after treatment with a fixed concentration of 10 lg/mL
for 24 and 72 h or (d) treatment with various concentrations of 2.5–10.0 lg/mL for 48 h on human MCF-7 breast cancer cells. (e) MCF-7 cells were untreated
or treated with marchantin A at a fixed concentration of 7.5 lg/mL for 24, 48, and 72 h. Cells were counted and determined by a trypan blue exclusion assay.
Fig. 1a–c, data presented were from three independent experiments with similar results. Fig. 1d and e, data represent means ± SD (n = 3).
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
Table 1
1
H and 13C NMR data (d/ppm) and HMBC data of 1 in CDCl3 (500 MHz).
Position
1
2, 6
3, 5
4
7
8
9
10
11
12
13
14
10
20
30
40
50
60
70
80
90
100
110
120
130
140
dH mult. (J/Hz)a
6.58 d (8)
6.92 d (8)
2.98–3.20 m
2.81–2.96 m
7.01 d (7.8)
7.13 t (7.8)
6.85 d (7.8)
5.27 d (1.7)
6.46 d (1.7)
2.78–2.80 m
2.72–2.75 m
6.59 s
6.53 d (7.8)
6.96 t (7.8)
6.39 d (7.8)
dC (mult.)b
152.9 (s)
121.2 (d)
129.6 (d)
139.1 (s)
35.2 (t)
30.3 (t)
136.1 (s)
121.9 (d)
126.0 (d)
114.3 (d)
148.7 (s)
139.6 (s)
143.1 (s)
146.4 (s)
107.8 (d)
130.6 (s)
109.2 (d)
144.2 (s)
34.1 (t)
35.5 (t)
132.5 (s)
115.5 (d)
156.7 (s)
128.9 (d)
112.0 (d)
123.2 (d)
Cells were counted and determined by a trypan blue exclusion assay.
HMBC (H ? C)
2.5. Morphological analysis of apoptotic cells
1
1, 7
3, 5, 8
7, 9, 10, 14
8, 12, 14
9, 13
10, 13, 14
20 , 50 , 7
0
30 , 40 , 60 , 70
0
0
0
4,6,8
70 , 90 , 100 , 140
80 , 110 , 120
100 , 110 , 140
100 , 110
80 , 100 , 130
a
Data without multiplicities were obtained using the COSY-45 and
HMQC pulse sequences.
b
Multiplicities were obtained from DEPT experiments.
2.3. Analytical conditions for marchantin A
The M. tosana profile in the total methanol extract and
marchantin A were analyzed with a reversed-phase
HPLC/UV unit. The conditions included a Luna Phenomenex column (C18, 4.6 250 mm), a solvent system of methanol:water (75:25), a flow rate of 1.0 mL/min, detection at
UV 210 nm, the temperature of the column at 25 ± 1 °C, the
elution conditions are isocratic, and the volume of injection is 10 lL.
2.4. Cell culture
Human MCF-7 cells were purchased from the Food
Industry Research and Development Institute (Hsinchu,
Taiwan). MCF-7 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS), a 1% dilution of penicillin–streptomycin,
and 2 mM glutamine. Cells were maintained at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. Marchantin
A was dissolved in dimethyl sulfoxide (DMSO) and prepared at a fixed concentration of 10 mg/mL. Cells
(1 106 per dish) were cultured in a 100-mm dish and
incubated for 14 h before being treated with DMSO or with
various concentrations (2.5, 5.0, 7.5, and 10.0 lg/mL) of
marchantin A at different time points. Marchantin A is a
small compound (MW 440.16 Da). When these values
were converted to molarities, they were 5.7, 11.4, 17.0,
and 22.7 lM, respectively. All of these treatments with
various doses of marchantin A. The vehicle (DMSO) in the
cell culture medium was fixed concentration at 2 lL/mL.
MCF-7 cells were cultured on six-well culture plates
(3 105/well) and treated with marchantin A at a fixed
concentration of 10.0 lg/mL for 24 h. After treatment, cells
were fixed with methanol for 30 min, washed with phosphate-buffered saline (PBS), and then stained with 50 lg/
mL of propidium iodide (PI) in the presence of 50 lg/mL
RNase A. The morphology of nuclear chromatin was defined by a Confocal Imager of DNA-binding dye, PI, under
a confocal microscope (BDTM, USA).
2.6. Analysis of the cell cycle
MCF-7 cells (1 106) in a 100-mm dish were treated
with various concentrations (2.5–10.0 lg/mL) of marchantin A for 48 h or cells were treated with a fixed concentration of 10 lg/mL marchantin A for 2, 4, 6, and 8 h. Cells
were trypsinized and collected with ice-cold PBS. Cells
were resuspended in 200 lL PBS and fixed by adding
800 lL of iced 100% ethanol, then incubated overnight at
20 °C. The cell pellets were collected by centrifugation,
resuspended in 1 mL of hypotonic buffer (0.5% Triton X100 in PBS and 1 lg/mL RNase A), and incubated at 37 °C
for 30 min. Then, 1 mL of the PI solution (50 lg/mL) was
added, and the mixture was allowed to stand at 4 °C for
30 min. The cellular DNA content was then analyzed by
FACScan cytometry (Becton Dickinson).
2.7. Annexin V-staining
Double staining for Annexin-V-fluorescein isothiocyanate (FITC) binding and for cellular DNA using PI was performed. MCF-7 cells were plated at a density of 3 105
cells/well on 6-well plates. After treatment with or without
the marchantin A (10 lg/mL) for 2, 4, and 6 h, or treatment
with 2.5–10.0 lg/mL of marchantin A for 48 h, cells were
trysinized and collected in ice-cold PBS. The cells were
washed twice with cold PBS and resuspended in 1 mL
binding buffer (BD PharMingen, San Diego, CA). They were
then stained with Annexin-V-FITC and PI according to the
protocol from BD PharMingen. Briefly, 2 lL of Annexin-VFITC (50 lg/mL) and 1 lL of PI (500 lg/mL) were added
to a 100 lL solution of cells and the solution was incubated
for 15 min at room temperature in the dark. Binding buffer
was then added, and early and late apoptosis was visualized by constructing a dot-plot using a Becton Dickinson
FACScan. Green fluorescence from the Annexin-V-FITC
was determined using an FL1 detector having a bandpass
filter with specifications 530 ± 15 nm. Red fluorescence
from PI was determined using an FL2 detector having a
bandpass filter with specifications 585 ± 21 nm. A total of
10,000 events were recorded for each sample.
2.8. Western blot assay
MCF-7 cells (1 106) on 100-mm dishes were treated
with various concentrations (0, 2.5, 5, 7.5, and 10 lg/mL)
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
Total RNA from MCF-7 cell was treated with marchantin
A (0, 2.5, 5.0, 7.5, and 10 lg/mL) for 24 h. Cells were lysed,
and the total RNA was extracted with a Qiagen RNeasy
Mini kit (Qiagen, Valencia, CA, USA). All real-time quantitative PCR primers were designed with Primer Express Software (Applied Biosystems, Foster City, CA) based on
GenBank sequence data. Each real-time quantitative PCR
reaction contained 30 ng of cDNA, each primer at 100
nM, and 7.5 lL of SYBR Green PCR Master Mix (1-step
kit, Applied Biosystems) in a total volume of 15 lL. Each
sample was performed in triplicate on an Applied Biosystems Prism 7500 Fast Sequence Detection System and the
relative mRNA levels were calculated by the comparative
threshold cycle method with GAPDH as an internal control.
Multiple PCR was performed using the following primers
for humans: p21waf1 forward 50 -CAGACCAGCATGACAGATTTC-30 and reverse 50 -TTAGGGCTTCCTCTTGGAGA-30 ;
cyclin B1 forward 50 -AGCTGCTGCCTGGTGAAGAG-30 and
reverse 50 -GCCATGTTGATCTTCGCCTTA-30 ; cyclin D1 forward 50 -TATTGCGCTGCTACCGTTGA-30 and reverse 50 -CCAATAGCAGCAAACAATGTGAAA-30 ; P27 forward 50 -CTGCAACCGACGATTCTTCTACT-30 and reverse 50 -GGGCGTCTGCTCCACAGA-30 ; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 50 -ATGCTGGCGCTGAGTACGT-30
and reverse 50 -AGCCCCAGCCTTCTCCAT-30 . After reverse
transcriptase step at 48 °C for 30 min, then enzyme activation reaction at 95 °C for 10 min. Initial denaturation at
95 °C for 15 s, anneal/extend at 60 °C for 1 min, 40 cycles
were performed in the duration.
2.10. DPPH free radical-scavenging activity
The free radical-scavenging capacities of marchantin A
(5–30 lg/mL) and caffeic acid phenyl ester (CAPE (1–
16 lg/mL), a well-known antioxidant used as a positive
Scavenging effect ð%Þ
¼ ½1 ðA517 of sample=A517 of controlÞ 100%:
The EC50 value was defined the concentration of a test
compound which scavenged 50% of the free radicals.
2.11. Densitometric analyses
Bands were scanned with Adobe Photoshop 6.0 (Adobe
System Inc., San Jose, CA, USA). The mean density of each
band was analyzed by the Scion image program. In each
case, blots were subjected to multiple exposures on the
film to ensure that the band density was in the linear
range. The data are presented in terms of fold change over
internal control (b-actin) for each treatment. All data presented were from three independent experiments with
similar results.
a
800
mAU
2.9. Real-time quantitative PCR analysis
control) at various concentrations were measured with
1.0 mL of 0.3 mM 1,2-diphenyl-2-picryhydrazyl (DPPH) in
methanol. The DPPH radical has a deep violet color due
to its unpaired electron, and its radical-scavenging capability can be followed spectrophotometrically by an absorbance loss at 517 nm when the pale-yellow non-radical
form is produced. The mixtures were vigorously shaken
and left to stand at room temperature for 30 min in the
dark. The absorbance at 517 nm was measured against
methanol as a blank. The ability to scavenge DPPH radicals
was then calculated by the following equation:
400
b
1000
Total Methanol Extract
0
Marchantin A
DMSO
mAU
of marchantin A for 48 h, or cells were treated with a fixed
concentration of 10 lg/mL of marchantin A for 24 and 72 h.
After treatment, cells were collected and resuspended in
100 lL lysis buffer. Equal amounts of proteins (30 lg) were
mixed with 2 sample buffer and resolved by 12.5% sodium dodecylsulfate polymerase chain reaction (SDS–
PAGE) for b-actin, poly (ADP ribose) polymerase (PARP),
Bid, caspase-3, caspase-8, and caspase-9 detection. Proteins were electrotransferred to an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica,
MA, USA), and equivalent protein loading was verified by
staining the membrane with the reversible dye, amido
black (Sigma Chemical, St. Louis, MO, USA). This was followed by overnight blocking with a solution composed of
20 mM Tris–HCl (pH 7.4), 125 mM NaCl, 0.2% Tween 20,
and 3% bovine serum albumin (BSA). Specific antibodies
used were anti-human PARP and Bid (1:500 of a rabbit
polyclonal antibody; Cell Signaling Technology, Danvers,
MA, USA), anti-caspase-3, anti-caspase-8, and caspase-9
antibodies (1:800 of mouse monoclonal antibody; Cell Signaling Technology). These proteins were detected by
chemiluminescence (ECL, Amersham, GE Healthcare, Buckinghamshire, UK).
500
0
0
5
10
15
20
Retention Times (min)
Fig. 2. Separation of the methanol extract from liverwort and marchantin
A by HPLC/UV. (a) Separation of total methanol extracts from the
liverwort M. tosana. The conditions included a Luna Phenomenex column
(C18, 250 4.6 mm), a solvent system of methanol:water of 75:25, a flow
rate of 1.0 mL/min, and detection at UV 210 nm. (b) The retention time of
the active ingredient, marchantin A, at 6.5 min was identified by HPLC/
UV. Conditions for the separation and identification of marchantin A are
the same as those given in Fig. 2a.
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
ARTICLE IN PRESS
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
from Marchantia species. It exhibits antibacterial and antifungal activities
against several species [3]. However, no anticancer activities of this compound (marchantin A) had previously been reported. In this report, we
investigated the mechanism of apoptosis of human MCF-7 breast cancer
cells induced by marchantin A.
3. Results and discussion
3.1. Purification and identification of marchantin A
The active ingredient, called marchantin A (Fig. 1b), was isolated by
repeated passages of the total methanol extract of the liverwort through
chromatography. Final purification of the active ingredient was achieved
by HPLC/UV on a reversed-phase column. The total content marchantin A
was roughly 6.6% of the methanol extract of liverwort (M. tosana). Fig. 2
shows the HPLC profile of the total methanol extracts of liverwort, indicating liverwort is rich in marchantin A. Marchantin A was previously isolated from several different species of liverworts, and was first identified
3.2. Inhibition of cell growth by marchantin A
MCF-7 cells were treated with marchantin A at a fixed concentration
of 10 lg/mL for 24 and 72 h (Fig. 1c) or treated with various concentrations (2.5, 5.0, 7.5, and 10 lg/mL) of marchantin A for 48 h (Fig. 1d). Treatment with marchantin A at a fixed concentration of 7.5 lg/mL for 24, 48,
a
b
MarchantinA (
4
10
12.17%
10
FL3-H
FL3-H
3.19%
3
10
2
10
1
2
10
1
10
10
1.58%
0
10
0
10
1
10
2
10
3
10
0
10
10
1
10
10
6.91%
3
10
3
10
4
10
10.0
4
11.57%
2
FL1-H
84.35%
5.0
4
10
0.27%
0
10
4
FL1-H
82.54%
3.86%
11.90%
3
10
FL3-H
10
FL3-H
12.19%
3.71%
3
2
10
1
2
10
1
10
10
1.47%
0
10
2.5
4
0
10
g/mL)
0
10
1
10
80.05%
2
10
FL1-H
3
10
10.91%
0
4
10
10
0
10
73.33%
1
10
2
10
3
10
4
10
FL1-H
Fig. 3. Marchantin A induced apoptosis in MCF-7 cells. (a) Effect of marchantin A on the morphology of nuclear chromatin. MCF-7 cells were untreated or
treated with marchantin A at a concentration of 10 lg/mL for 24 h and stained as described in Section 2. Morphological changes of nuclear chromatin were
then viewed under a confocal microscope. (b) Flow cytometric analysis of marchantin A-treated MCF-7 cells after fluorescinated anti-Annexin V monoclonal
antibody binding (FL1-H) and propidium iodide uptake (FL2-H). Cells were treated with or without 10 lg/mL of marchantin A for 2, 4, and 6 h, or treated
with 2.5–10.0 lg/mL of marchantin A for 48 h, and subsequently measured by flow cytometry. The lower right quadrant represents early apoptosis; the
upper right quadrant represents late apoptosis. (c) Flow cytometric analysis of MCF-7 cells treated with a fixed concentration of marchantin A at 10 lg/mL
for 2, 4, 6, and 8 h or (d) treated with marchantin A at various concentrations of 2.5–10.0 lg/mL for 48 h and stained with propidium iodide as described in
Section 2. Following the flow cytometric analysis, the cellular DNA profile was analyzed by Cell Quest software.
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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c
UT
d
T
7
µ g/mL)
Marchantin A (µ
0
2h
2.5
4h
5.0
6h
7.5
10.0
8h
DNA content
DNA content
Fig. 3 (continued)
and 72 h significantly inhibited cell proliferation as shown in Fig. 1e. All
these result demonstrated that marchantin A possesses the ability to inhibit cell growth, with an IC50 of 4.0 lg/mL. We also evaluated marchantin A on several different cancer cell lines such as Rat c6 glioma cells,
human Hs683 glioma cells, human HT-29 colon cancer cells, and human
A549 lung cancer cells. All of these cell lines exhibited similar results to
those of MCF-7 cells (data not shown). A related compound, marchantin
C was reported to induce microtubule depolymerization and suppress tumor cell growth [16]. Our data demonstrated that suppression of tumor
cell growth capacity by marchantin A was similar to that of marchantin C.
3.3. Marchantin A induces apoptotic cell death in MCF-7 cells
Through a cell growth inhibition assay (Fig. 1c and d), we showed that
marchantin A induced significant cell death in MCF-7 cells. Inhibition of
cell proliferation was clearly present in marchantin A-treated cells. The
effect of cell death appeared to occur through the induction of apoptosis.
The cytotoxic effect of marchantin A was further evidenced by nuclear
condensation under a confocal microscope as shown in Fig. 3a. The morphological features of apoptosis, such as chromatin condensation and nuclear fragmentation, were seen in marchantin A-treated cells. We next
questioned whether marchantin A induced apoptosis in MCF-7 cells.
Here, we used annexin V-staining and flow cytometry to evaluate
whether marchantin A-caused cell death is via apoptosis (Fig. 3b). Cells
were treated with marchantin A (10 lg/mL) in 0, 2, 4, and 6 h for the
apoptosis analysis. Our result indicates no significantly increased in a
time-course dependent in apoptotic cell population following marchantin
A treatment (data not shown). Furthermore, MCF-7 cells were treated
with 2.5–10.0 lg/mL of marchantin A for 48 h. The result indicated a
dose-dependent increase (1.58–10.91%) in apoptotic cell population following marchantin A treatment. To investigate the induction of a subG1 cell population, the DNA content of MCF-7 cells was treated with
marchantin A at a fixed concentration of 10 lg/mL for 2, 4, 6, and 8 h
(Fig. 3c) or treated with various concentrations of marchantin A for
48 h and then analyzed by flow cytometry as shown in Fig. 3d. Marchantin A-treated cells for 8 h, markedly increased the G2/M cell population
from 20.5% to 33.3% and the G0/G1 cell population dropped markedly
from 60.0% to 42.7% as shown in Fig. 3c. The results of flow cytometric
analysis indicated that marchantin A increased the sub-G1 cell population
from 0.7% to 6.8%, the G0/G1 cell population dropped significantly from
62.3% to 36.1%, and the G2/M cell population cell increased markedly from
17.1% to 48.0% in a dose-dependent manner as shown in Fig. 3d. These results suggested that marchantin A efficiently influenced cell cycle progression in a period times (2–8 h) and induced apoptosis in MCF-7 cells
may be needed incubation over 24 h. Marchantin C was reported to significantly induce cell cycle arrest at the G2/M phase in A172 and HeLa
cells [16]. Our data also demonstrated that marchantin A suppressed tumor cell growth similarly to that of marchantin C.
3.4. Marchantin A induces apoptosis through the activation of caspases
Apoptosis is a programmed cell suicide in which caspases are the central components of this process. In mammalian cells, caspases involved in
apoptosis are classified into two groups according to their function and
structure [17]. In this present study, we examined on how marchantin
A induced apoptosis by activating caspases-3, -8, and -9 in MCF-7 cells.
First, we evaluated whether caspase-dependent signal pathways were involved. Treatment of cells with a fixed concentration (10 lg/mL) of
marchantin A for 24 and 72 h or with various concentrations (2.5, 5.0,
7.5, and 10.0 lg/mL) for 48 h resulted in a significant increase in the
cleaved form of PARP (85 kDa) as shown in Fig. 4a. Furthermore, our results also demonstrated that protein levels of the active forms of caspases-3 and -9 were markedly increased in dose- and time-dependent
manners as shown in Fig. 4b and c. Activation of caspase-9 is a very
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
a
24 h
72 h
Marchantin A (
0
10
g/mL)
0
10
PARP
116 kDa
Cleaved PARP
85 kDa
-actin
43 kDa
48 h
Marchantin A (
0
2.5
g/mL)
5.0
7.5
10.0
Cleaved PARP
85 kDa
-actin
43 kDa
b
24 h
72 h
Marchantin A (
0
10
g/mL)
0
10
Procaspase-3
32 kDa
Cleaved caspase-3
17 kDa
12 kDa
43 kDa
-actin
48 h
Marchantin A (
0
2.5
5.0
g/mL)
7.5
10.0
Procaspase-3
32 kDa
Cleaved caspase-3
17 kDa
12 kDa
-actin
43 kDa
Fig. 4. Marchantin A induces apoptosis through a caspase-dependent pathway. Apoptosis was induced via caspase-dependent pathways such as caspase8, -9, and -3, Bid, and PARP. MCF-7 cells were treated with a fixed concentration of 10 lg/mL for 24 and 72 h or treated with various concentrations of
marchantin A (2.5–10.0 lg/mL) for 48 h. Cell lysates were prepared and subjected to SDS–PAGE and immunoblotting using respective specific antibodies.
(a) Dose- and time-dependent changes in PARP and cleaved PARP, (b) dose- and time-dependent changes in procaspase-3 and cleaved caspase-3, (c)
dose- and time-dependent changes in Bid, and (d) dose-dependent changes in procaspase-8 and cleaved caspase-8. The Western blot assay is described
in Section 2.
important pathway, because it can induce apoptosis through the mitochondrion-dependent pathway. Therefore, evaluation of Bid was an
important issue. Our findings suggested that marchantin A decreased
Bid protein levels (Fig. 4d) in dose- and time-dependent manners. Furthermore, we were interested in evaluating caspase-8 levels following
marchantin A treatment. Because caspase-8 plays an important role in
both the mitochondrion-dependent and -independent pathways in apoptosis [18], we further evaluated whether caspase-8 might be activated
during the induction of apoptosis by marchantin A, as shown in Fig. 4e.
Our data demonstrated that marchantin A-caused a marked decrease in
the procaspase-8 protein level and a slight increase in the cleaved cas-
pase-8 protein level in a dose-dependent manner. These results suggested
that marchantin A increased protein levels of the cleaved forms of PARP,
and caspases-3, -8, and -9. In this study, we assessed the apoptotic effect
induced by marchantin A on MCF-7 cells and found that this effect might
occur through a mitochondrion-dependent pathway.
3.5. Effects of marchantin A on cell cycle regulators
Recently, a study demonstrated that cyclins D1 and E and the cyclindependent kinase inhibitor, p21(Waf1/Cip1), are important regulators of cell
cycle in breast cancer [19]. Many reports also demonstrated that cell
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
c
24 h
72 h
Marchantin A (
0
10
g/mL)
0
10
Procaspase-9
47 kDa
Cleaved caspase-9
35 kDa
-actin
43 kDa
48 h
Marchantin A (
0
2.5
g/mL)
5.0
7.5
10.0
Procaspase-9
47 kDa
Cleaved caspase-9
35 kDa
-actin
43 kDa
24 h
d
72 h
Marchantin A (
0
10
g/mL)
0
10
Bid
22 kDa
-actin
43 kDa
48 h
Marchantin A (
0
2.5
5.0
g/mL)
7.5
10.0
22 kDa
Bid
43 kDa
-actin
Relative intensity
1.0
0.82
e
0.75
0.55
0.66
48 h
Marchantin A (
0
2.5
5.0
g/mL)
7.5
10.0
Procaspase-8
57 kDa
Cleaved caspase-8
18 kDa
-actin
43 kDa
Relative intensity
1.0
1.1
1.1
1.1
1.2
Fig. 4 (continued)
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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W.-J. Huang et al. / Cancer Letters xxx (2009) xxx–xxx
Fig. 5. Regulation of cell cycle regulator genes in marchantin A-treated MCF-7 cells. Cell cycle regulator genes expression was used real-time Q-PCR
analysis. MCF-7 cells were treated marchantin A with various concentrations at 0, 2.5, 5.0, 7.5, and 10.0 lg/mL for 24 h. (a) p21, (b) p27, (c) cyclin B1, and (d)
cyclin D1 cell cycle regulator genes were determined by Q-PCR as described in Section 2. We have used the t-test for statistical analysis. The data represent
means ± SD (n = 3); *p < 0.05; **p < 0.02.
Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006
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1.4
DPPH absorption (517 nm)
1.2
1.0
0.8
EC50
0.6
0.4
0.2
0
0
1
2
4
8
16
0
5
10
15
20
Marchantin A (
CAPE ( g/mL)
25
30
g/mL)
Fig. 6. DPPH free radical-scavenging activity of marchantin A. The DPPH free radical-scavenging activity of marchantin A and CAPE were assessed as
described in Section 2. The data represent means ± SD (n = 3).
growth inhibition can be regulated by cell cycle regulators, including the
cyclin-dependent kinase inhibitor, p21waf1 [20], and cyclins B1 [21], D1
[22], and E [23]. We investigated the effect of inhibiting MCF-7 cell
growth by marchantin A. Using a real-time quantitative PCR (Q-PCR),
we studied gene expressions of those cell cycle regulators following treatment of MCF-7 cells with marchantin A at various concentrations (0, 2.5,
5.0, 7.5, and 10.0 lg/mL) for 24 h. The results are shown in Fig. 5. In each
case, multiple gene analyses with GAPDH were used to standardize mRNA
levels over the course of the experiment. The p21 and p27 gene expressions increased markedly by treated with marchantin A at concentrations
from 5.0 to 10.0 lg/mL as shown in Fig. 5a and b; while cyclin B1 and D1
gene expression decreased markedly after treatment with marchantin A
at concentrations from 2.5 to 7.5 lg/mL as shown in Fig. 5c and d. Many
reports have revealed an association of cancer and cell cycle regulation.
Cell cycle regulators are also frequently found to be mutated in many human cancers [24]. Therefore, cell cycle regulation has been a target for
controlling cancers. In this report, we evaluated the anticancer activities
of marchantin A not only by evaluating its apoptotic mechanism but also
by investigating its effects on cell cycle regulators. P21 is a very important
cell cycle regulator and tumor suppress gene [25]. P21 gene overexpression can increase the cell population at the G0/G1 phase after treatment
with marchantin A (0–5.0 lg/mL) as shown in Fig. 3c. Many histone
deacetylase (HDAC) inhibitors such as SAHA [26], trichostatin A [27], sodium butyrate [28], LAQ-824 [29], and MS-275 [30], were reported to
cause p21 gene overexpression. Cyclin B1 is another important cell cycle
regulator. Our data demonstrated that cyclin B1 gene expression was
markedly suppressed after cells were treated with marchantin A. However, marchantin C treated human A172 glioma cells markedly increased
cyclin B1 levels [16]. These results implied that the anticancer activities of
marchantin A and C may go through different regulatory pathways.
3.6. Free radical-scavenging activity
The liverwort has been reported to exert a broad spectrum of biological activities such as cytotoxic, antifungal, antimicrobial, and antioxidant
properties. We were interested in evaluating the antioxidant property of
marchantin A. The free radical-scavenging activities of various concentrations of marchantin A and CAPE (a potent antioxidant agent used as a positive control [31]) were evaluated with DPPH, and the results are
presented in Fig. 6. The respective EC50 values of marchantin A and CAPE
were 20.0 and 5.5 lg/mL indicating that marchantin A was not as strong
an antioxidant as CAPE. Previous studies had been reported that the
neighboring hydroxyl groups on the aromatic ring are essential for the
antioxidant activity of flavonoids [32]. The chemical structure of marchantin A clearly demonstrates that two hydroxyl groups attached at C10
and C60 are very important parameters for scavenging free radicals of
DPPH. For the same reason we believe that the antioxidant activity of
marchantin A is stronger than that of marchantin C, because marchantin
C has no hydroxyl group at the 60 position. Similarly, two adjacent hydroxyl groups at C10 and C60 are also very important for inducing cytotoxicity. Many reports suggested that phenolic compounds in DMEM cell
culture medium could produce hydrogen peroxide (H2O2) [33,34]. This
free radical producing agent may induce cytotoxicity or apoptosis in cultured cells. This leads us to think that marchantin A induces apoptosis in
MCF-7 cells may partly due to generating of H2O2.
In summary, we think that the marchantin A inhibited proliferation of
MCF-7 cells via apoptosis. Our data demonstrated that marchantin A possibly induced apoptosis through a caspase-dependent pathway. Cyclin B1,
cyclin D1, P27, and p21 were significantly changed by treating the cells
with marchantin A. These results clearly show that marchantin A has
strong capability to influence the cell cycle process. Flow cytometric data
demonstrated that low doses (<5 lg/mL) of marchantin A arrested cell
growth at the G0/G1 phase, while high doses (7.5–10.0 lg/mL) arrested
cell growth at the G2/M phase. The antioxidant and cytotoxic effects
may be related to the chemical structure of marchantin A. The neighboring hydroxyl groups attached at C10 and C60 on the aromatic ring are
important for the antioxidant and cytotoxic activities of marchantin A.
Both marchantin A and marchantin C have similar chemical structures
and anticancer activities and may have potential to be developed into
candidate drugs for chemotherapy.
Conflict of interest
None of the authors have financial relationship with a
commercial entity that has an interest in the content of
this study.
Acknowledgements
We gratefully acknowledge the National Science Council of Taiwan and National Science Foundation, Directorate
for Engineering in Taiwan for their financial support under
the Grants NSC97-2320-B-038-010-MY3 and SBIR (IZ950295), respectively.
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Please cite this article in press as: W.-J. Huang et al., Marchantin A, a cyclic bis(bibenzyl ether), isolated from the liverwort Marchantia
emarginata subsp. tosana induces apoptosis in human MCF-7 breast cancer cells, Cancer Lett. (2009), doi:10.1016/j.canlet.2009.10.006