Anti-proliferative and pro-apoptotic effect of Smilax glabra Roxb. extract on hepatoma cell lines

Article (PDF Available)inChemico-Biological Interactions 171(1):1-14 · February 2008with117 Reads
DOI: 10.1016/j.cbi.2007.08.012 · Source: PubMed
Abstract
Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS. These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further development of SGR for liver disease therapy.
Chemico-Biological Interactions 171 (2008) 1–14
A
vailable online at www.sciencedirect.com
Anti-proliferative and pro-apoptotic effect of Smilax
glabra Roxb. extract on hepatoma cell lines
Fei Sa
a
, Jian-Li Gao
a
, Kwok-Pui Fung
b,c
, Ying Zheng
a,
,
Simon Ming-Yuen Lee
a,c,∗∗
, Yi-Tao Wang
a
a
Institute of Chinese Medical Sciences, University of Macau, Av. Padre Tom´as Pereira S.J., Taipa, Macao, China
b
Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China
c
Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China
Received 26 May 2007; received in revised form 28 August 2007; accepted 30 August 2007
Available online 12 September 2007
Abstract
Smilax glabra Roxb. (SGR) is the root of a traditional Chinese herb, referred to as tu fu ling in Chinese medicine. It is an
inexpensive traditional Chinese medicine commonly used for the treatment of liver diseases, and a few studies have indicated
that SGR has anti-hepatocarcinogenic and anti-cancer growth activities. In the current study, raw SGR plant was extracted with
Accelerate Solvent Extractor, and the molecular mechanism by which S. glabra Roxb. extract (SGRE) has an anti-proliferative effect
on the human hepatoma cell lines, HepG2 and Hep3B, was determined. We showed that SGRE inhibited HepG2 and Hep3B cell
growth by causing cell-cycle arrest at either S phase or S/G2 transition and induced apoptosis, as evidenced by a DNA fragmentation
assay. SGRE-induced apoptosis by alternation of mitochondrial transmembrane depolarization, release of mitochondrial cytochrome
c, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. The SGRE-mediated mitochondria-caspase dependent
apoptotic pathway also involved activation of p38, JNK, and ERK mitogen-activated protein kinase signaling. Isometric compounds
of astilbin (flavonoids) and smilagenin (saponin) have been identified as the main chemical constituents in SGRE by HPLC-MS/MS.
These results have identified, for the first time, the biological activity of SGRE in HepG2 and Hep3B cells and should lead to further
development of SGR for liver disease therapy.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Smilax glabra Roxb.; Apoptosis; HepG2; Hep3B; Caspase; Mitochondrial; Cytochrome c; p38; ERK; JNK; Liver cancer
Abbreviations: CBA, cytometric bead array; m, change of mito-
chondrial transmembrane potential; DMSO, dimethyl sulfoxide; PBS,
phosphate-buffered saline; ERK, extracellular signal-regulated kinase;
FBS, fetal bovine serum; NMR, nuclear magnetic resonance; MAPK,
mitogen-activated protein kinase; MTT, 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide; MS, mass spectrometry; IC
50
, 50%
of inhibitory concentration; JNK, c-Jun N-terminal kinase; PARP,
poly(ADP-ribose) polymerase; UV, ultraviolet.
Corresponding author. Tel.: +86 853 397 4687;
fax: +86 853 28841358.
∗∗
Corresponding author at: Institute of Chinese Medical Sciences,
University of Macau, Av. Padre Tom
´
as Pereira S.J., Taipa, Macao,
China. Tel.: +86 853 397 4695; fax: +86 853 28841 358.
E-mail addresses: yzheng@umac.mo (Y. Zheng),
simonlee@umac.mo (S.M.-Y. Lee).
1. Introduction
Hepatocellular carcinoma is the fifth most commonly
diagnosed cancer, with more than 1 million deaths
reported annually worldwide [1]. Apoptosis has been
characterized as a fundamental cellular activity that
maintains the physiological balance of organisms. It
also plays a critical role as a protective mechanism
against carcinogenesis by eliminating damaged or abnor-
mally excessive cells induced by various carcinogens
[2]. Emerging evidence has demonstrated that the anti-
0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2007.08.012
2 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
cancer activities of certain chemotherapeutic agents are
involved in the induction of apoptosis, which is regarded
as the preferred way to manage cancer [2,3]. In order
to develop an effective means for the prevention and
treatment of hepatocellular carcinoma and related liver
diseases, we have isolated and identified several chemi-
cal extracts and pure compounds from Chinese medicine
with anti-hepatoma and anti-liver disease effects [4–7].
SGR, a member of the Smilacaceae family and a
rhizome of the Liliaceae plant, is referred to as tu fu
ling in Chinese medicine. It is a crude drug used in
many traditional prescriptions in Asia. It is inexpen-
sive with a low toxicity to organs, but may be highly
advantageous in its long-term use for chronic inflamma-
tory diseases such as rheumatoid arthritis and hepatitis
[8]. Traditional Chinese medicinal uses of SGR have
included the dissipation of heat, resolution of toxins,
elimination of moisture, and promotion of mobility to
the joints. It is commonly used clinically to prevent
leptospirosis, and to treat syphilis, acute bacterial dysen-
tery, acute and chronic nephritis, mercury poisoning, and
rheumatoid arthritis [9]. Moreover, it has been used in the
preparation of traditional medications administered to
cancer patients in Thailand [10]. Previous investigators
have demonstrated that SGR can inhibit cell prolifera-
tion of the squamous cell carcinoma cell line, JTC226,
and the inhibitory rate was approximately 90% [11].
Moreover, the saponins of SGR can inhibit the growth
of EAC, S
180
, and H22 cells [12]. Recent in vivo investi-
gations have demonstrated that extract prepared from
SGR, Nigella sativa seeds, and Hemidesmus indicus
roots, as used in Sri Lanka, can offer significant pro-
tection against diethylnitrosoamine-induced hepatocar-
cinogenic changes in rats [13,14]. Furthermore, the effect
of this preparation on HepG2 cells, a human hepatoma
cell line, has been investigated in vitro, and the extract
possesses direct cytotoxic activity that contributes to
the anti-hepatocarcinogenic effects [15]. These stud-
ies indicate that SGR has potential protective effects
against hepatocarcinogenesis. In this study, we isolated
chemical extracts from SGR (SGRE) and attempted to
determine the molecular mechanism of action of the
inhibitory effect in human hepatoma HepG2 and Hep3B
cells.
2. Materials and methods
2.1. Chemicals and cell culture
SGR was purchased from the Zhuhai Medicine Com-
pany (Zhuhai, China). HPLC grade-methanol, -ethanol,
and -acetonitrile were purchased from Merck (Darm-
stadt, Germany). Deionized Milli-Q water was used in
all experiments (Millipore, Milli-Q & Rios Systems,
Bedford, MA).
The human hepatoma cell lines, HepG2 and Hep3B
(containing hepatitis B virus genome), were purchased
from the American Type Culture Collection (ATCC,
VA). Cell culture medium was purchased from Invitro-
gen (Guangzhou, China). Hep3B cells were cultured in
DMEM supplemented with 10% (v/v) fetal bovine serum
(FBS), 100 g/mL of streptomycin, and 100 unit/mL of
penicillin in 75 cm
2
tissue culture flasks in a humidified
incubator at 37
C with 5% CO
2
. HepG2 cells were cul-
tured in RPMI medium supplemented with 10% (v/v)
FBS, 100 g/mL of streptomycin, and 100 unit/mL of
penicillin in 75 cm
2
tissue culture flasks in a humidified
incubator at 37
C with 5% CO
2
.
2.2. Preparation of plant extracts
Two hundred grams of SGR were prepared from
a methanol extract with Accelerate Solvent Extractor
(ASE200, Dionex, CA) at 1500 psi and 60
C. The
resulting extract was concentrated by rotary evapora-
tion, dissolved in Milli-Q water, and then filtered with
a 0.45 m Millipore filter unit. The same volume of
petroleum ether was added to the extract for double phase
extraction for 12 h. The petroleum ether layer was dis-
carded, and five times volume of 99% alcohol (EtOH)
was added. After 2 h, the sediment was discarded and
the final extract (i.e., SGRE) was freeze-dried to a pow-
der form, 6.442 g in weight. The freeze-dried extract
was used in both chemical analysis and pharmacological
studies. The extract was dissolved in PBS containing 1%
DMSO to give stock solutions of 100 mg/mL.
2.3. UV–vis spectral methods for qualitative and
quantitative analyses of SGRE
The methanol solution of the SGRE was scanned; the
UV spectrum exhibited an absorption maxima at 280 nm
(band II) and 300–330 nm (band I); characteristic absorp-
tion bands of a dihydroflavonol skeleton. To investigate
the contents of the total flavonoids in the extract, AlCl
3
colorimetry [16,17] was used and rutin (Sigma, MO) was
used as a reference chemical standard. As a result, the
total flavonoid content in the extract was found to be
39.37 mg/g.
2.4. HPLC instrumentation and conditions
An Agilent 1100 series LC/MSD VL trap system
(Agilent Technologies, Palo Alto, CA) was used for sam-
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 3
ple analysis. An Agilent ZORBAX Eclipse XDB-C18
(150 mm × 4.6 mm, i.d.; 3.5 m, particle size) column
was used (Agilent). The separation was achieved using
a gradient elution with Milli-Q water (a) and acetonitrile
(b) as follows: 83–82% (a) for 0–10 min, 82–78% (a) for
10–15 min, 78–75% (a) for 15–16 min, 75–55% (a) for
16–35 min, 55–25% (a) for 35–45 min, 25–0% (a) for
45–50 min, and 100–100% (b) for the final 10 min. The
flow rate was 0.6 mL/min, the column temperature was
set at 35
C, and the detection wavelength was 330 nm.
2.5. Mass spectrometry
An Agilent trap mass spectrometer was interfaced to
the HPLC system with an electrospray ionization (ESI)
source. The sample was detected in both the positive and
negative ion modes, and scanned from m/z 150–1000.
The capillary voltage was set at 4 kV, the gas used for
drying and spraying was nitrogen, the nebulizer pressure
was 50 psi, the flow rate of dry gas was 11 L/min, the
dry gas temperature was 350
C, the collision energy
was set at 1.5 V, and the compound stability and trap
drive level were set at 80%. The HPLC-MS data were
acquired using the program, data analysis for LC/MSD
Trap, version 3.2 (Build 121), which corresponded with
LC/MSD Trap software 5.2 (Build 382).
2.6. Cell viability assay
Cells were seeded in 96-well microplates
(2 × 10
4
cells/well in 100 L of medium). SGRE
was added to the cells in serial concentrations and incu-
bated for 24, 48, and 72 h. Medium was discarded before
30 L of tetrazolium dye (MTT) solution (5 mg/mL
in PBS) was added to each well and incubated for an
additional 4 h. DMSO (100 L) was added to dissolve
the formed formazan crystals. The plate was then read
in a microplate reader (1420 Multilabel counter victor
3
,
Perkin-Elmer, MA) at 570 nm. MTT solution with
DMSO (without cells and medium) was used as a blank
control.
2.7. Cell-cycle analysis
Cells were seeded in six-well plates and incubated
with 0.6 mg/mL of SGRE in a humidified incubator
(37
C with 5% CO
2
) for 24, 48, and 72 h, respectively.
The adherent cells were washed with PBS, and then
300 L of trypsin was incubated with cells for 5 min
at room temperature to collect the cells. After centrifu-
gation at 350 × g for 5 min at 4
C, the cell pellet was
obtained. The cell pellet was then resuspended with 1 mL
of cold 70% EtOH at 4
C for 12 h. The cell pellet was
collected again by centrifugation at 350 × g for 5 min at
4
C. Finally, 1 mL of propidium iodide (PI) stain solu-
tion (20 g/mL of PI and 8 g/mL of DNase-free RNase)
was added to the samples, and the samples were then ana-
lyzed by flow cytometry (BD FACS Canto Trade Mark,
Franklin Lakes, NJ). The results were analyzed with Mod
Fit LT 3.0 software.
2.8. Terminal deoxynucleotidyl transferase-
mediated dUTP nick end labeling (TUNEL) assay
The TUNEL assay was performed according to
the manufacturer’s instructions (Apo-BrdUTM TUNEL
Assay Kit, Molecular Probes, Leiden, The Netherlands).
Cells were fixed with 1% paraformaldehyde (PFA) in
PBS on ice for 15 min. For a further fixation step, 70%
EtOH was added and cells were kept on ice for 10 min.
After 3 h of labeling at 37
C with the DNA-labeling
solution, cells were incubated with Alexa Fluor 488
conjugated anti-BrdU antibodies for 30 min at room tem-
perature. Cells were analyzed by flow cytometry and
were mounted on slides. The morphology of the cells was
observed with a fluorescent microscope (Axiovert 200,
Carl Zeiss, Thornwood, NY) mounted with a camera
(Carl Zeiss AxioCam HRc, Carl Zeiss).
2.9. Analysis of mitochondrial membrane potential
Mitochondrial injury was assessed by JC-1 dye, a
Mitochondrial Potential Sensors (Molecular Probes).
Red fluorescence of the J-aggregate form of JC-1 indi-
cates intact mitochondria, whereas green fluorescence
shows a monomeric form of JC-1 that is due to the
breakdown of the mitochondrial membrane potential.
Cells were seeded in six-well plates for 6 h. The medium
of each well was discarded and treated with 1 mL of
medium (5 mg/mL JC-1) for 15 min at 37
C and 5% CO
2
in the dark, then washed twice in PBS and serial con-
centrations of SGRE, and re-incubated in a humidified
incubator (37
C with 5% CO
2
) for 24 h. The cells were
collected and centrifuged, the cell pellet re-suspended
in 1 mL of medium, measured by flow cytometry and
mounted on slides. The morphology of the cells was
observed under a fluorescent microscope.
2.10. Cytochrome c labeling
Cytochrome c release was assessed by a SelectFX
Alexa Fluor 488 Cytochrome C Apoptosis Detection
Kit (Molecular Probes). Cells were seeded in 24-well
plates and treated with SGRE for 8 h. The media was dis-
4 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
carded and the cells were washed with warm PBS, fixed
with fresh 4% formaldehyde in PBS for 15 min at 37
C,
and permeabilized with 0.2% Triton X-100 for 5 min
at room temperature. The cells were washed and incu-
bated in a blocking buffer (10% heat-inactivated normal
goat serum) for 30 min at room temperature. The cells
were then incubated for 1 h with 1 g/mL primary anti-
body (anti-cytochrome c mouse IgG, Molecular Probes)
at room temperature. Green fluorescence was observed
with a fluorescent microscope.
2.11. Active caspase-3 protein and
poly(ADP-ribose) polymerase (PARP) levels
The BD Trade Mark CBA Human Apoptosis Kit (BD,
NJ) was used to quantify the active caspase-3 and PARP
protein levels. Cytometric bead array (CBA) employs a
particle with a discrete fluorescence intensity to detect
a soluble analyte. This kit provides two types of bead
populations with distinct fluorescence intensities that
have been coated with capture antibodies specific for
cleaved caspase-3 and PARP. Cells were seeded in a six-
well plate and incubated with desired concentrations of
SGRE in a humidified incubator (37
C with 5% CO
2
)
for 24 h. Cells were harvested and washed with PBS.
The cells of each sample were counted to 1.0 × 10
6
and
50 L of cell lysis buffer was added to each sample for
30 min on ice and vortexed at 10 min intervals. Cellu-
lar debris was pelleted by centrifugation at 12,500 rpm
for 10 min. The protein concentrations of all samples
were measured with a 2D Quant Kit (Amersham Bio-
sciences, Piscataway, NJ). Each sample was normalized
in a final concentration of 0.2 g/L. Thirteen standard
curves (standards ranging from 0 to 6000 unit/mL) were
obtained from one set of calibrators. For each sample and
the standard mixture of lysate standard (caspase-3 and
PARP beads), 50 L of a sample or a standard of beads
was added to the mixture of 50 L of two mixed capture
beads, incubated for 1 h, and then mixed with 50 mL
of PE detector beads for an additional 1 h. After that,
samples were washed before data acquisition by flow
cytometry. The results were analyzed by FCAP Array,
version 1.0.
2.12. Expression of phospho-ERK1/2, -JNK1/2, and
-p38
Expression of ERK1/2, JNK1/2, and p38 were mea-
sured by a Cell Signaling Master Buffer Kit (BD). Three
types of beads in a CBA provided a capture surface
for phospho-ERK1/2, -JNK1/2, and -p38 proteins. Cells
were seeded in a six-well plate and incubated with the
desired concentrations of SGRE in a humidified incu-
bator (37
C with 5% CO
2
) for the specific times, as
indicated. Cells were harvested and washed with PBS.
Cells of each sample were counted to 1.0 × 10
6
and
50 L of cell-denaturation buffer was added to each sam-
ple and immediately placed in a boiling water bath for
5 min. Cellular debris was pelleted by centrifugation at
10,000 × g for 5 min. The protein concentrations of all
samples were measured by a 2D Quant Kit (Amersham
Biosciences). Each sample was normalized to a final
concentration of 0.2 g/L. Standard curve (standards
ranging from 0 to 1000 pg/mL) was obtained from one set
of calibrators. For each sample and the standard mixture
of lysate standard (phospho-ERK1/2, -JNK1/2, and -p38
beads), 50 L of sample or standard beads was added
to the mixture of 50 mL of three mixed capture beads,
and 50 L of PE detector beads were incubated for 4 h.
After that, samples were washed before data acquisition
with flow cytometry. The results were analyzed by FCAP
Array, version 1.0.
2.13. Statistical analysis
The data are expressed as the mean ± S.D. from
at least three independent experiments. Differences
between groups were analyzed using a Student’s t-test.
3. Results
3.1. Chemical analysis
The typical HPLC chromatogram and total ion chro-
matogram (TIC) of SGRE are shown in Fig. 1A and
B. The MS/MS spectra of the main peaks in the chro-
matogram are shown in Fig. 1a–d. Compounds 1, 2, and
3, indicated in Fig. 1A and B, had the same molecu-
lar weight of approximately 450 m/z (Fig. 1 a and b)
and were predicted to be isomeric compounds of astil-
bin [18,19] (Fig. 1C) which structure was classified
as dihydroflavonol. In addition, compound 4 was pre-
dicted to be a glycoside of a steroid, namely smilagenin
(MW = 416.6), and the chemical structure of its aglycone
was shown in Fig. 1D.
3.2. Cell viability assay
The growth of the HepG2 and Hep3B cells in the
presence of various concentrations of SGRE was exam-
ined. Under the experimental conditions (24 and 48 h),
SGRE exhibited a marked growth inhibitory effect on
both HepG2 and Hep3B cells. The IC
50
for HepG2
and Hep3B cells ranged from approximately 1.8 mg/mL
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 5
Fig. 1. HPLC chromatogram, total ion chromatogram, and mass/mass spectra of the SGRE. (A) The HPLC chromatogram of SGRE at 330 nm. (B)
The total ion chromatogram of the SGRE in the negative mode (most of the major peaks were marked in the chromatograms): (a) the mass spectra of
compounds 1, 2, and 3 in the positive mode; (b) the mass spectra of compounds 1, 2, and 3 in the negative mode; (c) the mass spectrum of compound
4 in the positive mode; and (d) the mass spectrum of compound 4 in the negative mode. (C) Predicted chemical structure of compounds 1, 2 and 3,
belong to isomeric compounds of astilbin. (D) Predicted chemical structure of the aglycone of compound 4 (smilagenin).
6 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
Fig. 2. HepG2 and Hep3B cells were treated with drug-free medium
or medium containing different concentrations of SGRE for 24 or 48 h.
Cell growth was determined by MTT assay and was directly propor-
tional to the absorbance at a wavelength of 570 nm. Error bars represent
the mean ± S.E.M. (n = 16).
(24 h) to 1.1 mg/mL (48 h) and from 2.6 mg/mL (24 h) to
0.8 mg/mL (48 h), respectively (Fig. 2). Both inhibitory
curves of SGRE exposure to HepG2 and Hep3B cells
were similar, suggesting a parallel effect of cell death.
3.3. Cell-cycle analysis
The effect of different concentrations of SGRE on
cell-cycle progression of HepG2 and Hep3B cells was
studied after 24, 48 and 72 h of drug exposure. Treatment
of cells with 0.6 mg/mL SGRE led to profound changes
in the cell-cycle profiles after incubation of up to 72 h
(Fig. 3A and B). SGRE treatment resulted in a time-
dependent significant accumulation of cells in S phase
with concomitant losses from G1 phase (Fig. 3A and B).
These results could be explained by the hypothesis in
which S-phase accumulation is suggested to be caused
by a decrease in the progression through the cell cycle
or an inhibition of S/G2 phase transition. These results
suggested an anti-proliferative effect of SGRE on cells
and possible induction of cell-cycle arrest at the either S
phase or S/G2 transition.
3.4. TUNEL assay
Apoptosis of SGRE treated Hep3B cells was further
examined with the TUNEL assay at 24, 48, and 72 h of
treatment. The nuclei of the treated cells demonstrated
nuclear shrinkage and condensed chromatin, which was
consistent with the morphological hallmark of an apop-
totic nucleus (Fig. 4A). A significant number of cells
containing DNA strand breaks were found after treat-
ment with 1.0 mg/mL of SGRE for 48 h. The green
fluorescence intensity indicated that the quantity of
apoptotic cells of the 1.0 mg/mL treatment group was
approximately 10 times higher than the drug-free cells.
After treatment for 72 h, the green fluorescence inten-
sity was approximately 20 times higher than that of
the control cells (Fig. 4B). This result revealed a time-
dependent increase in the quantity of apoptotic Hep3B
cells.
3.5. Analysis of mitochondrial membrane potential
Some chemotherapeutic drugs induce apoptosis via
mitochondrial pathways by altering the mitochon-
drial transmembrane potential, m. We used a JC-1
probe to detect the m after cells were treated with
0.5 mg/mL of SGRE for 6 h. Mitochondria with nor-
mal m concentrates JC-1 into aggregates (red/orange
fluorescence), while in depolarized mitochondria, JC-
1 forms monomers (green fluorescence). As compared
to untreated (control) cells, the red/orange fluorescence
decreased by 89% in HepG2 cells (Fig. 5) and by 47%
in Hep3B cells (data not shown) after exposure to SGRE
under the same condition.
3.6. Cytochrome c labeling
Cytochrome c released from the mitochondria to the
cytosol is implicated in mitochondria-dependent apopto-
sis. Cytochrome c staining in the cytosol of both HepG2
and Hep3B cells showed markedly stronger levels of
green fluorescence than that of control cells after 6 h of
treatment with 1.0 mg/mL SGRE (Fig. 6). SGRE treated
cells showed obvious punctate green fluorescence stain-
ing or appeared to have green fluorescence accumulated
in large aggregates compared to control cells (Fig. 6).
3.7. Expression of active caspase-3 protein and
PARP levels
In drug-induced cell death via apoptosis, sig-
naling can generally be divided into receptor- and
mitochondrial-mediated pathways. These pathways con-
verge at several downstream points including the
mitochondria, caspase activation, and substrate cleav-
age. Active caspase-3 protein and PARP cleavage protein
levels were measured by the BD Trade Mark CBA
Human Apoptosis Kit. All the samples were counted
to 1.0 × 10
6
cells and normalized to a final protein con-
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 7
Fig. 3. Effect of SGRE on the cell-cycle distribution of HepG2 and Hep3B cells. (A) Flow cytometric analysis of PI-stained HepG2 and Hep3B cells
treated with 0.6 mg/mL SGRE for 24, 48, and 72 h. The x-axis represents fluorescent intensity on a logarithmic scale, whereas the y-axis represents
the number of events. (B) The results were analyzed by Mod Fit LT 3.0. Columns, mean of three independent plates; bars, S.D.; the results were
reproducible in three additional independent experiments. *P < 0.05;
$
P < 0.01;
#
P < 0.001; P value compared with the control group (0 h).
8 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
Fig. 3. (Continued ).
Fig. 4. (A) Morphological observation of TUNEL-stained Hep3B cells by fluorescence microscopy. Hep3B cells were treated with medium alone
(control) or 1 mg/mL of SGRE for 24, 48, and 72 h. Photographs were taken at a magnification of 20×. (B) The remaining cells were analyzed
by flow cytometry. The x-axis indicates green fluorescence intensity on a logarithmic scale and the y-axis indicates the number of events. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 9
Fig. 4. (Continued ).
centration of 0.2 g/L. The results were analyzed by
FCAP Array, version 1.0, and are shown in Fig. 7. Our
results demonstrated that there was a significant dose-
dependent increase in protein levels of cleaved PARP and
active caspase-3 in both HepG2 and Hep3B cells (Fig. 7).
Both results indicated that SGRE-induced apoptosis via
the caspase-dependent pathway.
3.8. Cell signaling cascades of mitogen-activated
protein kinases (MAPKs)
In order to evaluate if SGRE induced the HepG2
cell death through activation of the cell signaling cas-
cades of mitogen-activated protein kinases (MAPKs),
we assessed the kinetics of p38 MAPK, ERK1/2 and
JNK phosphorylation simultaneously by BD Trade Mark
CBA Cell Signaling Flex Set system. All samples were
counted to 1.0 × 10
6
cells and the protein concentra-
tions were normalized to 0.2 g/L. The results were
analyzed by FCAP Array, version 1.0, and are shown in
Fig. 8. Results showed that the expression levels of phos-
phorylated forms of p38, JNK1/2, and ERK1/2 increased
significantly and dose dependently in SGRE-induced
HepG2 and Hep3B cell death.
4. Discussion
The underlying mechanism of the pharmacologi-
cal action of SGR in cancer therapy is still largely
unclear. In the present study, SGRE has been identified
10 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
Fig. 5. Analysis of mitochondrial transmembrane potential in HepG2 cells. HepG2 cells were stained with JC-1 and treated with 0.5 mg/mL SGR
for 6 h. The cells were photographed at a magnification of 20× under bright field (A and B), red fluorescence (C and D) and green fluorescence (E
and F). The remaining cells were analyzed by flow cytometry and revealed a loss of intact mitochondria transmembrane potential in treated (H) vs.
untreated (G) cells. Representative density plots of green vs. red fluorescence are shown. Red fluorescence: intact mitochondrial potential; green
fluorescence: breakdown of mitochondrial transmembrane potential. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of the article.)
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 11
Fig. 6. Representative microscopic photos of cytochrome c immunostaining in HepG2 and Hep3B cells treated with 1.0 mg/mL SGRE. Cytochrome c
immunofluorescence was observed with Oregon green. HepG2 or Hep3B cells (B and D, respectively) exhibited higher cytochrome c immunostaining
in the cytosol than the control cells (A and C). Similar results were obtained from three separate experiments (original magnification, 20×). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
to inhibit HepG2 and Hep3B cell growth by inducing
apoptosis, as evidenced by activation of the depolar-
ization of the mitochondrial transmembrane potential,
mitochondrial cytochrome c release, caspase-3 and
PARP cleavage, S/G
2
cell-cycle arrest, and finally, DNA
fragmentation.
Cell-cycle progression was arrested in S/G
2
phase
in SGRE treated hepatoma cells. The results indicated
that there was a modulation of events at the S and
G
2
checkpoints, which may provide an opportunity
to enhance SGRE-induced cytotoxicity in hepatoma
cells. The results indicated that SGRE can be used
to sensitize malignant cells to drugs that destabilize
DNA during replication, such as methotrexate (MTX),
which is a cell-cycle-specific (S phase) chemother-
apeutic agent that is currently used to treat human
osteosarcoma [20].
It has been reported that the induction of cell detach-
ment is a prerequisite for the activation of caspase-3 in
an apoptosis execution process [21,22]. The quantity of
active caspase-3 protein (Fig. 7A and B) increased after
SGRE treatment. Also, our results showed that caspase-3
activity was dramatically increased after treatment with
1.0 mg/mL of SGRE for 24 h; the activity was 2.8 times
higher in HepG2 cells than drug-free (control) cells, and
4.5 times higher in Hep3B cells, as measured by the
EnzChek Caspase-3 Assay Kit (data not shown). We
demonstrated here that the caspase-3 protein is criti-
cally involved in SGRE-inducing apoptosis. PARP is a
116-kDa nuclear protein involved in DNA repair, and
a well-characterized substrate for caspase-3. Activated
caspase-3 cleaves PARP, generating 89- and 24-kDa
inactive fragments [23]. Fig. 7C and D indicated that
the amount of cleaved PARP increased depending on the
SGRE concentration and the quantity of active caspase-
3. These results suggested that the caspase-3 protein is
critically involved in SGRE-induced apoptosis. Taken
together, a mitochondrial-dependent caspase-3 pathway
may be involved in SGRE-induced apoptosis of hep-
atoma cells.
The MAPK superfamily consists of three ser-
ine/threonine kinase cascades [24]. ERKs respond
to growth factors or other external mitogenic sig-
nals by promoting cell proliferation and opposing cell
death signals. p38 and JNK are typically described
as stress-activated kinases that promote programmed
cell death [25], and it is now widely accepted as a
simplified scheme that p38 and JNK mediate apop-
totic signals. We thus wondered about the involvement
of these pathways in the SGRE-induced apoptosis
12 F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14
Fig. 7. Protein expression levels of active caspase-3 and cleaved PARP in SGRE-induced apoptosis. Both HepG2 and Hep3B cells were treated
with medium alone (control) or different concentrations of SGRE for 24 h. Cells of each sample were counted to 1.0 × 10
6
and all the samples were
normalized to a final protein concentration of 0.2 g/L. The results were analyzed by FCAP Array, version 1.0. Active caspase-3 expressions in
HepG2 and Hep3B cells are shown (A and B, respectively). CleavedPARP expression in HepG2 and Hep3B cells are presented (C and D, respectively).
The x-axis indicates the concentration of SGRE. The y-axis indicates unit of proteins per milliliter. Columns, mean of three experiments; bars, S.E.M.;
*P < 0.05;
#
P < 0.01;
$
P < 0.001; P value compared with a control group.
of hepatoma cells. Phospho-p38 and -JNK1/2 were
activated dose dependently after SGRE treatment
(Fig. 8C–F). Our results support the hypothesis that
p38 and JNK1/2 are activated in the process of SGRE-
induced apoptosis in human hepatoma cells (HepG2 and
Hep3B).
ERK promotes growth, differentiation and prolifer-
ation, and its activation alone does not fully reflect the
complex biology of cancer cells, especially in clinical
material [26]. Wang et al. showed that after treat-
ment, apoptosis was linked to ERK activation in the
HeLa cervical cancer cell line, whereas its resistant
variants, Hela-R1 and Hela-R2, established by a contin-
uous exposure to increasing concentrations of cisplatin,
showed a reduced activation of ERK [27]. However,
the pattern of ERK activation seems to be complex,
as it was reported to be biphasic in some kinetic
studies [28–30]. Therefore, substantial evidence sug-
gests that the activation of the ERK pathway increased
the cell death threshold in an unknown way. The
expression of phospho-ERK1/2 was up-regulated by
SGRE-induced apoptosis in our experiment. Interest-
ingly, in spite of a similar trend of up-regulated protein
expression levels of phospho-ERK1/2, -JNK1/2, and -
p38 in both cell lines, the activation profile of these
three kinases was shown to be different in response to
SGRE.
In this study, SGRE, a standardized chemical extract,
has been analyzed by UV spectrophotometry and
HPLC-MS/MS. Flavonoids (mainly isometric com-
pounds of astilbin) and saponins (e.g. smilagenin)
have been identified as the main chemical constituents.
Recent investigations have shown that the flavonoids
of SGR are the major active components which have
immunomodulating effects [8]. Previous reports have
shown that astilbin, isolated from SGR, possesses anti-
inflammatory and pain relief activities [31]. In addition,
the Smilax steroids, sarsasapogenin and smilagenin, have
been reported to be active components in the treat-
ment of senile dementia, cognitive dysfunction, and
Alzheimer’s disease. They improve memory by elevat-
ing the low muscarinic acetylcholine receptor density in
brains of memory-deficient rat models [32]. In addition,
the saponins of SGR have been reported to facilitate the
body’s absorption of other drugs and phytochemicals.
In short, our study suggested that the SGRE contain-
ing mainly flavonoids and saponins induced apoptosis
in HepG2 and Hep3B cells through activation of the
F. Sa et al. / Chemico-Biological Interactions 171 (2008) 1–14 13
Fig. 8. Protein expression levels of phospho-ERK1/2, -JNK1/2, and -p38 of SGRE-induced apoptosis. Both HepG2 and Hep3B cells were treated
with medium alone (control) or different concentrations of SGRE for 24 h. All the samples were normalized to a final protein concentration of
0.2 g/L. The results were analyzed by FCAP Array, version 1.0. The phospho-ERK1/2, -JNK1/2, and -p38 expression in HepG2 and Hep3B
cells are shown A–F, respectively. Columns, mean of three experiments; bars, S.E.M.; *P < 0.05;
#
P < 0.01;
$
P < 0.001; P value compared with the
control group.
mitochondrial and caspase-3 pathways, which involved
activation of p38, ERK1/2 and JNK1/2 MAPK signaling
cascades.
Acknowledgements
We thank Sandy Lao, Leon Lai, Chi Weng Leong,
Emilia Conceic¸
˜
ao Leong, Hio Wa Lam, and Xiao Yu for
their participation in the preliminary experiments. This
study was supported by grants from the Research Com-
mittee, University of Macau, Macao SAR (Ref. Nos.
RG054/05-06S and RG058/05-06S) and grants from
the Science and Technology Development Fund, Macao
SAR (Ref. Nos.: 078/2005/A2 and 012/2006/A).
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    • "It is also used in folk medicine alone or in combination with other herbal medicines for the treatment of a variety of diseases such as psoriasis and cancer in many other countries. Previous studies have demonstrated that the rhizome of S. glabra possesses a broad spectrum of bioactivities, including hypoglycemic [4], prevention of immunological hepatocyte damage [9], immunomodulatory [10], antiviral [11, 12], antiproliferative [11, 13, 14], and anti-inflammatory activity [13, 15, 16]. Phytochemical investigations on the rhizome of S. glabra led to the isolation and identification of more than 60 compounds, for example, flavonoids [12, 17– 20], phenylpropanoid derivatives [21], and phenolics [12, 22]. "
    [Show abstract] [Hide abstract] ABSTRACT: The rhizome of Smilax glabra has been used for a long time as both food and folk medicine in many countries. The present study focused on the active constituents from the rhizome of S. glabra, which possess potential anti-inflammatory activities. As a result, nine known compounds were isolated from the rhizome of S. glabra with the bioassay-guiding, and were identified as syringaresinol (1), lasiodiplodin (2), de-O-methyllasiodiplodin (3), syringic acid (4), 1,4-bis(4-hydroxy-3,5-dimethoxyphenyl)-2,3-bis(hydroxymethyl)-1,4-butanediol (5), lyoniresinol (6), trans-resveratrol (7), trans-caffeic acid methyl ester (8), and dihydrokaempferol (9). Among these compounds, 2 and 3 were isolated for the first time from S. glabra. In addition, the potential anti-inflammatory activities of the isolated compounds were evaluated in vitro in lipopolysaccharide- (LPS-) induced RAW264.7 cells. Results indicated that 4 and 7 showed significant inhibitory effects on NO production of RAW264.7 cells, and 1, 2, 3, and 5 showed moderate suppression effects on induced NO production. 1, 7, and 5 exhibited high inhibitory effects on TNF-α production, with the IC50 values less than 2.3, 4.4, and 16.6 μM, respectively. These findings strongly suggest that compounds 1, 2, 3, 4, 5, 7, and 9 were the potential anti-inflammatory active compositions of S. glabra.
    Full-text · Article · Mar 2015
    • "Smilax china (family Liliaceae) is a native plant grown in China, Japan, and Korea, and its root has been used as emergency foods in the past (Huang and others 2008, 2009; Kang and others 2011). It has also been used as a folk medicine for leptospirosis, syphilis, brucellosis , rheumatoid arthritis, nephritis, detoxification, and heavy metal poisoning (Chen and others 2000; Chu and Ng 2006; Sa and others 2008; Xia and others 2010; Shim 2012b). Although the Smilax china root contains important bioactive components including resveratrol and oxyresveratrol, nowadays most of them are thrown out because it refrains other trees from photosynthesis through spiraling. "
    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Resveratrol has a beneficial effect of lowering reactive oxygen species (ROS) and reduces cellular oxidative stress. We hypothesized that ethanol extract of Smilax china root (EESC) rich in resveratrol (RES) and oxyresveratrol (OXY) could reduce ROS caused by nicotine and promoting nicotine turnover by induction of CYP2A6. The amount of cotinine converted from nicotine was quantified by the direct barbiturate assay method. Expression of CYP2A6 was unregulated by RES, OXY, or EESC, respectively. Pretreatment of RES (50, 100, and 250 μM), OXY (50, 100, and 250 μM), and RES+OXY (50 and 100 μM) inhibited cytotoxicity and ROS production caused by nicotine in a dose-dependent manner. EESC pretreatment (1.8 mg/mL) increased cell viability by 1.5-fold higher than the control (nicotine only), and lowered cellular ROS levels. A significant amount of the conversion of nicotine to cotinine was observed in EESC pretreatment by CYP2A6 induction in HepG2 cells. These results suggested that hepatic induction of CYP2A6 and ROS reduction by EESC activate nicotine metabolism and reduce cellular oxidative stress. Practical application: Nicotine exposure due to smoking is very concerning because it is the major factor for lung diseases and cardiovascular disorders. It is necessary to examine natural ingredients that can detoxify from nicotine to cotinine as well as neutralize free radicals induced from nicotine. Results from the current study suggest potential applications of Smilax china root for detoxification of nicotine in the food industry.
    Full-text · Article · Oct 2014
    • "Smilax glabra Roxb. extractmediated mitochondria-caspase-dependent apoptotic pathway , however, involves p38, JNK, and ERK signalling activation (Sa et al., 2008). Our previous studies have demonstrated that p-PD induced apoptosis through p53, as well as intrinsic and extrinsic pathways in Mardin-Darby canine kidney (MDCK) cells (Chen et al., 2006; 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: para-phenylenediamine (p-PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p-PD-induced cell death in normal Chang liver cells was investigated. The results demonstrated that p-PD decreased cell viability in a dose-dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub-G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen-activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p-PD activated not only stress-activated protein kinase (SAPK)/c-Jun N-terminal kinases (JNK) and p38 MAPK but also extracellular signal-regulated kinase (ERK). Cytotoxicity and apoptosis induced by p-PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38-specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p-PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p-PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p-PD-induced apoptosis. Western blot analysis revealed that p-PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p-PD and that a decreased ERK signal contributes to growth inhibition or apoptosis. © 2012 Wiley Periodicals, Inc. Environ Toxicol, 2012.
    Full-text · Article · Sep 2014
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