Crude Extract of Rheum Palmatum L Induced
cell Death in LS1034 Human Colon Cancer
Cells acts through the Caspase-Dependent
and -Independent Pathways
Yi-Shih Ma,1,2 Shu-Chun Hsu,3 Shu-Wen Weng,1,4 Chien-Chih Yu,5 Jai-Sing Yang,6
Kuang-Chi Lai,7,8 Jing-Pin Lin,1 Jaung-Geng Lin,1 Jing-Gung Chung9,10
1Graduate Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan 2Department of Chinese Medicine, Changhua Hospital, Department of Health, Executive Yuan,
Changhua 513, Taiwan
3Departments of Nutrition, China Medical University, Taichung 404, Taiwan
4Department of Chinese Medicine, Taichung Hospital, Department of Health, Executive Yuan,
Taichung 403, Taiwan
5School of Pharmacy, China Medical University, Taichung 404, Taiwan
6Departments of Pharmacology, China Medical University, Taichung 404, Taiwan 7School of Medicine, China Medical University, Taichung 404, Taiwan
8Department of Surgery, China Medical University Beigang Hospital, Yunlin 651, Taiwan 9Departments of Biological Science and Technology, China Medical University,
Taichung 404, Taiwan
10Department of Biotechnology, Asia University, Taichung 413, Taiwan
ABSTRACT: Crude extract of Rheum palmatum L (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub-G1 phase (apoptosis), the activities of caspase-8, -9, and -3, reactive oxygen species (ROS) and Ca21 levels,
and mitochondrial membrane potential (DCm). DNA damage, nuclei condensation, protein expression,
and translocation were examined by Comet assay, 40-6-diamidino-2-phenylindole (DAPI) staining, Western
blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration- and time-dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl-2 protein ratio and CERP promoted the activities of
caspase-8, -9, and -3. Both ROS and Ca21 levels were increased by CERP but the compound decreased levels of
DCm in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G,
cytochrome c, and GADD153 to induce apoptosis through mitochondrial-dependent pathway. Keywords: crude extract of Rheum palmatum L; human colon cancer LS1034 cells; apoptosis; mitochondrial
in
human populations (Huyghe et al., 2003; Molassiotis et al.,
2006). In the United States, the third most common cause
of cancer-related deaths is colorectal cancer and in Europe,
it is the second most common cause of death (1999; Samuel
et al., 2009; Ross, 2010). In males and females, colon cancer
is the third leading cause of cancer-related death in Taiwan
with 19.6 individuals per 100,000 dying annually from colorectal cancer based on the reports in 2009 from the
Department of Health, ROC (Taiwan). Currently, treatments
for colorectal cancer including surgery, radiotherapy, chemotherapy, and combinations of radio- and chemotherapy
are not satisfactory (Kampfenkel et al., 2011; Shitara et al., 2011; Brandi et al., 2012; Hompes et al., 2012). Therefore, discovering new chemotherapeutic agents is
urgent and agents with inducing apoptosis may be most
effective in cancer cells (McDermott et al., 2005; Taghizadeh
et al., 2007; Zhong et al., 2008; Zhang et al., 2012). Apoptosis can be divided into extrinsic and intrinsic pathways with the intrinsic pathway also referred to as the
endoplasmic reticulum (ER) stress pathway (Landgraeber
et al., 2008; Lan et al., 2012; Yang et al., 2012). The extrinsic
pathway involves death receptors (Fas and Fas ligand) that activate caspse-9 caspase-3 causing apoptosis via a
protease cascade without direct involvement of mitochondria
(Li et al., 2002; Putcha et al., 2002; Kim et al., 2007; Sayers, 2011). The intrinsic or ER stress pathway when
induced alters calcium homeostasis and ER protein-folding
leading to ER dysfunction (Chu et al., 2012; Lu et al., 2012b; Wu et al., 2012; Xu et al., 2012).
Rheum palmatum L. (RL), has been used widely in traditional
Chinese medicine for hundreds of years (Zhang et al., 1993; Cheng et al., 1994; Zhang et al., 1997, 2001;
Yang et al., 2006; Lin et al., 2008; Wang et al., 2011). In
East Asia, the root of Rheum undulatum L. was often used
as a purgative and anti-inflammatory agent (Zhang et al.,
1993). Major components of RL such as emodin, aloe-emodin,
chrysonal and rhein induce apoptosis in many different
types of human cancer cells (Cai et al., 2008; Yu et al.,
2008; Chiu et al., 2009; He et al., 2012; Ma et al., 2012; Ni
et al., in press; Suboj et al., 2012a, b). There are no reports
regarding effects of RL on the growth of human colon cancer
cells. In this study, we investigated the effects of the water extract of RL (WERL) on cytotoxicity and apoptosis
in human colon cancer cells. Results demonstrated that
WERL induced cell death through cell cycle arrest and apoptosis
in LS1304 human colon cancer cells.
MATERIALS AND METHODS Chemicals and Reagents
Dimethyl sulfoxide (DMSO), potassium phosphates, propidium
iodide (PI), ribonuclease-A (RNase A), trypan blue and Tris-HCl were purchased from Sigma Chemical Co. RPMI 1640 medium, fetal bovine serum (FBS), L
-glutamine,
penicillin-streptomycin, and trypsin-EDTA were obtained from Gibco BRL (15). CaspaLux-L1D2 for
caspase-8, CaspaLux-M1D2 for caspase-9 and PhiPhiLux-G1D1 for caspase-3 determinations were purchased from
OncoImmunin (Gaithersburg, MD). Crude extract of Rheum palmatum L. (CERP) was provided kindly by Dr.
Chien-Chih Yu (School of Pharmacy, China Medical University,
Taichung 404, Taiwan).
Cell Culture of LS1034 cell
The human colon adenocarcinoma cell line (LS1034) was
purchased from the Food Industry Research and Development
Institute (Hsinchu, Taiwan). Cells were cultured in RPMI 1640 medium with 2 mM L-glutamine, 10% FBS,
100 Units/mL penicillin and 100 mg/mL streptomycin in a
humid atmosphere of 5% CO2 (Lu et al., 2010).
Cell Viability
LS1034 cells (2 3 105 cells/well) were placed in
12-well
plates and were incubated with 0, 250, 500, 750, 1000,
1500, 2000, and 2500 lg/mL CERP or 0.5% DMSO used
as a vehicle control for 24 h then cells were harvested for
determination of viability as described previously (Lu et al., 2012a). Harvested cells were stained with PI (5 lg/
mL) and then analyzed using a PI exclusion method by
flow cytometry (BD Biosciences, FACSCalibur, San Jose,
CA) as previously described (Lu et al., 2012a).
Cell Cycle Distribution by Flow Cytometric Assay
LS1034 (2 3 105 cells/) in 12-well plates were treated
with
CERP at 0, 250, 500, 750, 1000, 1500, and 2000 lg/mL for
24 h. Cells were harvested, washed in cold phosphate-buffered
saline (PBS), fixed in 70% ethanol, and stored at 48C overnight. Cells were washed with PBS and then were treated with RNase A (200 lg/mL) at 378C for 15 min and
stained by PI (20 lg/mL) with 0.1% Triton X-100 in PBS in a dark room for 30 min. Cell distribution in sub-G1, G0/
G1, S, G2/M phases were analyzed using a FACScan flow
cytometer as described previously (Chiang et al., 2011).
Comet Assay and DAPI Staining
LS1034 cells (2 3 105 cells/well) on 12-well plates
were
treated with 0, 500, 750, 1000, and 2000 lg/mL CERP for
24 h or 4 lM hydrogen peroxide (H2O2) positive
control.
Cells were harvested for comet staining by PI stain (DNA
damage) or by 40-6-diamidino-2-phenylindole (DAPI)
staining (nuclear condensation and fragmentation) then all
samples were photographed using fluorescence microscopy
as described elsewhere (Chiang et al., 2011).
Determinations of ROS, Intracellular Ca21 Levels and Mitochondrial Membrane Potential (DCm) in LS1034 Cells
cytometry as described previously (Wu et al., 2010).
Caspase-3, Caspase-8, and Caspase-9 Activities
LS1034 cells (2 3 105 cells/well) were cultured on
12-well
plates for 24 h and were pretreated with inhibitors of
caspase-3, caspase-8, and caspase-9 then were incubated with
0 and 750 lg/mL of CERP for 0, 24 and 48 h. Cells were
harvested, washed twice with PBS and resuspended in 50
lL of 10 lM the substrate solution PhiPhiLux-G1D1 for caspase-3, CaspaLux-L1D2 for caspase-8 and
CaspaLux-M1D2 for caspase-9 and incubated at 378C for 60 min.
Samples were then washed again with PBS and were
caspase-8, -9, and -3 activity was determined by flow cytometry
as described previously (Wu et al., 2010; Ma et al., 2012).
Confocal laser Scanning Microscopy for Examining Protein Translocation in LS1034 Cells
LS1034 (2 3 105 cells/well) were maintained on 4-well
chamber slides and treated with then 0 and 10 lg/mL of
CERP incubated for 24 h. Cells were fixed in 4% formaldehyde
in PBS for 15 min and permeabilized using 0.3%
Triton-X 100 in PBS for 1 h. Nonspecific binding sites were blocked by using 2% BSA as described previously. Primary
antibodies to AIF, Endo G, cytochrome c, and GADD153
(green fluorescence) were added and incubated overnight.
Following incubation, cells were washed twice with PBS
and then were stained with secondary antibody (FITC-conjugated
goat anti-mouse IgG), mitotracker (red fluorescence) for nuclein examination. Samples on slides were examined and photo-micrographed using a Leica TCS SP2
Confocal Spectral Microscope as described previously (Ma
et al., 2012).
Western Blotting Assay for Cell Cycle and Apoptosis Associated Proteins in LS1034 Cells
Cells (5 3 106 cells) were placed in six-well plate then
were treated with 750 lg/mL CERP for 0, 6, 12, 24, and 48
h then cells from each treatment were harvested with lysis
buffer containing 40 mM Tris-HCl (pH 7.4), 10 mM EDTA, 120 mM NaCl, 1 mM dithiothreitol, 0.1% Nonide P-40 A 30 lg protein from each sample was loaded on a gel
(10% Tris-glycine-SDS-polyacrylamide) for Western blot
analysis then were transferred to a nitrocellulose membrane
by electro-blotting. Primary antibodies for the different proteins
were individually used to stain each sample followed by staining with secondary antibody for enhanced chemiluminescence
(NEN Life Science Products, Boston, MA) as described previously. Anti-b-actin (a mouse monoclonal
antibody) was used as a loading control as described previously
(Ma et al., 2012).
Statistical Analysis
Results are shown as mean 6 SD and data were analyzed
for statistical significance using Student’s t-test. Significance
was defined as p\0.05. All studies were done with three independent experiments in duplicate.
RESULTS
Effects of CERP on Cell Viability Of LS1034 Cells
LS1034 cells were treated with to 0, 250, 500, 750, 1000,
1500, 2000, and 2500 lg/mL of CERP for 48 hours then
the percentage of viable cells were determined and results
are shown in Figure 1. CERP induced cell death in a doseand
time-dependent manner in LS1034 cells.
Effects of CERP on Cell Cycle Distribution of LS1034 Cells
LS1034 cells were treated with to 0, 250, 500, 750, 1000,
1500, and 2000 lg/mL of CERP for 24 or 48 h. Percent distribution
of each cell cycle can be seen in Figure 2(A,B).
CERP induced G0/G1 phase arrest and sub-G1 phase was
present which indicated that CERP induced apoptosis in
LS1034 cells.
Effects of CERP on DNA Damage and Condensation in LS1034 Cells
Comet assay and DAPI staining were used to investigate
the effects of CERP on DNA damage and nuclei condensations,
respectively. Results shown in Figure 3 demonstrated that CERP induced DNA damage in a dose-response manner.
It can be seen in Figure 4 that CERP also induced DNA condensation and fragmentation in a dose-dependent
manner.
Effects of CERP on ROS, Ca21 and DCm Levels in LS1034 Cells
LS1034 cells were treated with 750 lg/mL of CERP for different
time periods and levels of ROS, Ca21, and DCm
were determined by flow cytometry. CERP increased the
ROS levels (data not shown) and Ca21 [Fig. 5(A)] but
reduced DCm levels (data not shown) compared with
the
vehicle treated (control) group. Effects were time-dependent.
CERP may induce apoptosis in LS1034 cells by increasing ROS and Ca21, levels, and perturbation of
mitochondria.
Effects of CERP on Translocation of Apoptotic Associated Proteins in LS1034 Cells
We examined effects of CERP on AIF, Endo G, cytochrome
c, and GADD153 involved in CERP induced apoptosis in LS1034 cells. After the treatment of LS1034 cells with CERP for 24 h then cells were harvest and were stained by primary antibodies then were stained with secondary
antibody and then were photographed by confocal laser microscopic systems. The results are showing in Figure
6, which indicated that CERP promoted the AIF [Fig. 6(A)], Endo G [Fig. 6(B)] GADD153 [Fig. 6(C)] cytochrome
c [Fig. 6(D)] in LS1034 cells when compared to control groups.
Effects of CERP on the Activities Of Caspase-3, -8, and -9 in LS1034 Cells
LS1034 cells were pretreated with inhibitors of caspase3,
-8, and -9, incubated with CERP for various time periods
and act vities of caspase-3, -8, and -9 or percentage of viable cells were determined. Results are shown in Figure
7, which indicate that CERP stimulated activities of
caspase-3 [Fig. 7(A)], caspase-8 [Fig. 7(B)] and caspase-9 [Fig. 7(C)] between 12 and 72 h. Inhibitors of caspase8,
-9, and -3 reduced activities of caspase-3, -8, and -9 and promoted
the percentage of viable cells. CERP induced
apoptosis
involves activation of caspase-3, -8, and -9 in LS1034 cells.8
Effects of CERP on Cell Cycle and Apoptosis Protein Expression in LS1034 Cells
LS1034 cells were treated with 750 lg/mL of CERP for 0,
6, 12, 24, and 48 h and cell cycle and apoptosis associated
proteins were examined by Western blotting and results are
shown in Figure 9. CERP significantly promoted the expression of p27, p16, and p21 but inhibited the expression of cyclin D1, E and CDK4 [Fig. 9(A,B)]. These
findings demonstrated that CERP induced G0/G1 phase
arrest via inhibition of check point enzymes of cell cycle in
LS1034 cells. Figure 9(C,D) also demonstrated that CERP
decreased the levels of anti-apoptotic proteins Bcl-2 and
Bid; however, it increased the pro-apoptotic protein BAX.
Results also showed that CERP promoted the expression of
caspase-8, -9, and -3, GRP78, cytochrome c, AIF, and endo
G, and PARP cleavage in LS1034 cells [Fig. 6(C,D)].
DISCUSSION
The purpose of this study was to determine effects of CERP on cytotoxicity and apoptosis in human colon cancer
cells. The results showed that: (1) CERP decreased the
percentage of viable cells in a dose-dependent manner
(Fig. 1); (2) CERP induced DNA damage and nuclei condensation
dose-dependent (Figs. 3 and 4); (3) CERP
induced G0/G0 phase arrest and induced sub-G1 phase
(apoptosis) (Fig. 3); (4) CERP ROS and Ca21 levels but
activity
of caspase-8, -9, and -3 (Fig. 7); (6) CERP inhibited cyclin E and CDK2 which was associated with cell cycle
arrest (Fig. 9), promoted Bax expression but reduced Bcl-2
levels [Fig. 9(D)]; and 7) CERP increased levels of AIF, cytochrome c, and GADD153 and cytochrome c in LS1034 cells.
It is well documented that many natural plant extracts
from plants have been proposed to be excellent candidates
for cancer therapeutics (Shigemura et al., 2007). Induction
of apoptosis by such extracts is thought to be a mechanism
of cell death. Rheum undulatum L. have been screened for
anti-cancer activity in vitro in human breast, ovary, cervix and lung and oral cancer cell lines. It is well known that apoptosis
can be triggered through: (1) the extrinsic (death receptor)
based on FasL or tumor necrosis factor which binds to its cognate receptor activating the Fas-associated death
domain and caspase-8 cleavage (Luschen et al., 2005); (2)
the intrinsic (mitochondrial) pathway by release of cytochrome
c from mitochondria (Lee et al., 2008) and downstream
activation of caspase-3. Our results showed that CERP decreased DCm levels Bcl-2 protein abundance
but
increased Bax levels. The Bcl-2 family of sproteins regulate
mitochondria-dependent apoptosis through a balance of the
ratio (anti- and pro-apoptotic members) such as Bcl-2 and
Bax, respectively (Tsou et al., 2006; Wu et al., 2010) and
changes in the ratio of Bcl-2 and Bax contribute to
apoptosis.
We also showed that CERP-induced apoptosis was
caspase-dependent and involves the activation of the mitochondrial
pathway.
CERP increased ROS and Ca21 and it also induced ER
stress in LS1034 cells. Other studies have reported that
persistent or intense ER stress can trigger apoptotic cell
death (63, 64). Thus, in this study, CERP induced apoptosis
in LS1034 cells involved ROS production and may also be acted upon by ER stress. Results from confocal
laser microscope also demonstrated that CERP promoted
the expression of AIF (Fig. 6) in LS1034 cells. CERP induced DNA fragmentation and nuclear condensation in
LS1034 cells. It was reported that AIF is a mitochondrial
protein and if apoptosis is caspase-independent pathway,
AIF can be translocated into nuclei to mediate nuclear condensation and DNA fragmentation. CERP, AIF protein
levels (Fig. 9). Our results suggest that AIF translocation
into the nucleus is required for CERP-induced apoptosis
in LS1034 cells.
A proposed mechanism of CERP-induced apoptosis is presented in Figure 10. In conclusion, these results show
that CERP is cytotoxic in LS1034 human colon cancer cells. Cytotoxicity is due to stimulation of apoptosis which
was associated with the production of ROS and the activation
of caspase-dependent and -independent mitochondrial
pathways.
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