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
4Department
of Nutrition, China Medical University, Taichung 404, Taiwan
of Chinese Medicine, Taichung Hospital, Department of Health, Executive Yuan,
Taichung 403, Taiwan
5School
of Pharmacy, China Medical University, Taichung 404, Taiwan
6Departments
7School
of Pharmacology, China Medical University, Taichung 404, Taiwan
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
INTRODUCTION
regarding effects of RL on the growth of human colon cancer
Cancer is one of the major causes of death worldwide in
cells. In this study, we investigated the effects of the
human populations (Huyghe et al., 2003; Molassiotis et al.,
water extract of RL (WERL) on cytotoxicity and apoptosis
2006). In the United States, the third most common cause
in human colon cancer cells. Results demonstrated that
of cancer-related deaths is colorectal cancer and in Europe,
WERL induced cell death through cell cycle arrest and apoptosis
it is the second most common cause of death (1999; Samuel
in LS1304 human colon cancer cells.
et al., 2009; Ross, 2010). In males and females, colon cancer
MATERIALS AND METHODS
is the third leading cause of cancer-related death in Taiwan
Chemicals and Reagents
with 19.6 individuals per 100,000 dying annually from
Dimethyl sulfoxide (DMSO), potassium phosphates, propidium
colorectal cancer based on the reports in 2009 from the
iodide (PI), ribonuclease-A (RNase A), trypan blue
Department of Health, ROC (Taiwan). Currently, treatments
and Tris-HCl were purchased from Sigma Chemical Co.
for colorectal cancer including surgery, radiotherapy,
RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine,
chemotherapy, and combinations of radio- and chemotherapy
penicillin-streptomycin, and trypsin-EDTA were
are not satisfactory (Kampfenkel et al., 2011; Shitara
obtained from Gibco BRL (15). CaspaLux-L1D2 for caspase-
et al., 2011; Brandi et al., 2012; Hompes et al., 2012).
8, CaspaLux-M1D2 for caspase-9 and PhiPhiLux-
Therefore, discovering new chemotherapeutic agents is
G1D1 for caspase-3 determinations were purchased from
urgent and agents with inducing apoptosis may be most
OncoImmunin (Gaithersburg, MD). Crude extract of
effective in cancer cells (McDermott et al., 2005; Taghizadeh
Rheum palmatum L. (CERP) was provided kindly by Dr.
et al., 2007; Zhong et al., 2008; Zhang et al., 2012).
Chien-Chih Yu (School of Pharmacy, China Medical University,
Apoptosis can be divided into extrinsic and intrinsic
Taichung 404, Taiwan).
pathways with the intrinsic pathway also referred to as the
Cell Culture of LS1034 cell
endoplasmic reticulum (ER) stress pathway (Landgraeber
The human colon adenocarcinoma cell line (LS1034) was
et al., 2008; Lan et al., 2012; Yang et al., 2012). The extrinsic
purchased from the Food Industry Research and Development
pathway involves death receptors (Fas and Fas ligand)
Institute (Hsinchu, Taiwan). Cells were cultured in
that activate caspse-9 caspase-3 causing apoptosis via a
RPMI 1640 medium with 2 mM L-glutamine, 10% FBS,
protease cascade without direct involvement of mitochondria
100 Units/mL penicillin and 100 mg/mL streptomycin in a
(Li et al., 2002; Putcha et al., 2002; Kim et al., 2007;
humid atmosphere of 5% CO2 (Lu et al., 2010).
Sayers, 2011). The intrinsic or ER stress pathway when
Cell Viability
induced alters calcium homeostasis and ER protein-folding
LS1034 cells (2 3 105 cells/well) were placed in 12-well
leading to ER dysfunction (Chu et al., 2012; Lu et al.,
plates and were incubated with 0, 250, 500, 750, 1000,
2012b; Wu et al., 2012; Xu et al., 2012).
1500, 2000, and 2500 lg/mL CERP or 0.5% DMSO used
Rheum palmatum L. (RL), has been used widely in traditional
as a vehicle control for 24 h then cells were harvested for
Chinese medicine for hundreds of years (Zhang
determination of viability as described previously (Lu
et al., 1993; Cheng et al., 1994; Zhang et al., 1997, 2001;
et al., 2012a). Harvested cells were stained with PI (5 lg/
Yang et al., 2006; Lin et al., 2008; Wang et al., 2011). In
mL) and then analyzed using a PI exclusion method by
East Asia, the root of Rheum undulatum L. was often used
flow cytometry (BD Biosciences, FACSCalibur, San Jose,
as a purgative and anti-inflammatory agent (Zhang et al.,
CA) as previously described (Lu et al., 2012a).
1993). Major components of RL such as emodin, aloe-emodin,
Cell Cycle Distribution by Flow
chrysonal and rhein induce apoptosis in many different
Cytometric Assay
types of human cancer cells (Cai et al., 2008; Yu et al.,
LS1034 (2 3 105 cells/) in 12-well plates were treated with
2008; Chiu et al., 2009; He et al., 2012; Ma et al., 2012; Ni
CERP at 0, 250, 500, 750, 1000, 1500, and 2000 lg/mL for
et al., in press; Suboj et al., 2012a, b). There are no reports
24 h. Cells were harvested, washed in cold phosphate-buffered
for Examining Protein Translocation
saline (PBS), fixed in 70% ethanol, and stored at 48C
in LS1034 Cells
overnight. Cells were washed with PBS and then were
LS1034 (2 3 105 cells/well) were maintained on 4-well
treated with RNase A (200 lg/mL) at 378C for 15 min and
chamber slides and treated with then 0 and 10 lg/mL of
stained by PI (20 lg/mL) with 0.1% Triton X-100 in PBS
CERP incubated for 24 h. Cells were fixed in 4% formaldehyde
in a dark room for 30 min. Cell distribution in sub-G1, G0/
in PBS for 15 min and permeabilized using 0.3% Triton-
G1, S, G2/M phases were analyzed using a FACScan flow
X 100 in PBS for 1 h. Nonspecific binding sites were
cytometer as described previously (Chiang et al., 2011).
blocked by using 2% BSA as described previously. Primary
Comet Assay and DAPI Staining
antibodies to AIF, Endo G, cytochrome c, and GADD153
LS1034 cells (2 3 105 cells/well) on 12-well plates were
(green fluorescence) were added and incubated overnight.
treated with 0, 500, 750, 1000, and 2000 lg/mL CERP for
Following incubation, cells were washed twice with PBS
24 h or 4 lM hydrogen peroxide (H2O2) positive control.
and then were stained with secondary antibody (FITC-conjugated
Cells were harvested for comet staining by PI stain (DNA
goat anti-mouse IgG), mitotracker (red fluorescence)
damage) or by 40-6-diamidino-2-phenylindole (DAPI)
for nuclein examination. Samples on slides were
staining (nuclear condensation and fragmentation) then all
examined and photo-micrographed using a Leica TCS SP2
samples were photographed using fluorescence microscopy
Confocal Spectral Microscope as described previously (Ma
as described elsewhere (Chiang et al., 2011).
et al., 2012).
Determinations of ROS, Intracellular Ca21
Western Blotting Assay for Cell Cycle and
Levels and Mitochondrial Membrane
Apoptosis Associated Proteins in LS1034
Potential (DCm) in LS1034 Cells
Cells
Cells (2 3 105 cells/well) were placed on 12-well plates for
Cells (5 3 106 cells) were placed in six-well plate then
24 h and then were incubated with 0 and 750 lg/mL of
were treated with 750 lg/mL CERP for 0, 6, 12, 24, and 48
CERP for various time periods. Following incubation cells
h then cells from each treatment were harvested with lysis
were trypsinized collected by centrifugation and washed
buffer containing 40 mM Tris-HCl (pH 7.4), 10 mM
twice with PBS, resuspended in 500 lL of H2DCF-DA (10
EDTA, 120 mM NaCl, 1 mM dithiothreitol, 0.1% Nonide
lM) for reactive oxygen species (ROS), 500 lL of Fluo-3/
P-40 A 30 lg protein from each sample was loaded on a gel
AM (2.5 lg/mL) for intracellular Ca21 and 500 lL of
(10% Tris-glycine-SDS-polyacrylamide) for Western blot
DiOC6 (1 lmol/L) for DCm, respectively, at 378C for 30
analysis then were transferred to a nitrocellulose membrane
min. ROS, Ca21, and DCm levels were analyzed by flow
by electro-blotting. Primary antibodies for the different proteins
cytometry as described previously (Wu et al., 2010).
were individually used to stain each sample followed
Caspase-3, Caspase-8, and Caspase-9
by staining with secondary antibody for enhanced
Activities
chemiluminescence
LS1034 cells (2 3 105 cells/well) were cultured on 12-well
(NEN Life Science Products, Boston, MA) as
plates for 24 h and were pretreated with inhibitors of caspase-
described previously. Anti-b-actin (a mouse monoclonal
3, caspase-8, and caspase-9 then were incubated with
antibody) was used as a loading control as described previously
0 and 750 lg/mL of CERP for 0, 24 and 48 h. Cells were
(Ma et al., 2012).
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 CaspaLuxM1D2 for caspase-9 and incubated at 378C for 60 min.
Samples were then washed again with PBS and were caspase8, -9, and -3 activity was determined by flow cytometry
as described previously (Wu et al., 2010; Ma et al.,
2012).
Confocal laser Scanning Microscopy
Statistical Analysis
Results are shown as mean 6 SD and data were analyzed
in LS1034 cells. After the treatment of LS1034 cells
for statistical significance using Student’s t-test. Significance
with CERP for 24 h then cells were harvest and were
was defined as p\0.05. All studies were done with
stained by primary antibodies then were stained with secondary
three independent experiments in duplicate.
antibody and then were photographed by confocal
RESULTS
laser microscopic systems. The results are showing in Figure
Effects of CERP on Cell Viability Of LS1034
6, which indicated that CERP promoted the AIF [Fig.
Cells
6(A)], Endo G [Fig. 6(B)] GADD153 [Fig. 6(C)] cytochrome
LS1034 cells were treated with to 0, 250, 500, 750, 1000,
c [Fig. 6(D)] in LS1034 cells when compared to
1500, 2000, and 2500 lg/mL of CERP for 48 hours then
control groups.
the percentage of viable cells were determined and results
Effects of CERP on the Activities Of
are shown in Figure 1. CERP induced cell death in a doseand
Caspase-3, -8, and -9 in LS1034 Cells
time-dependent manner in LS1034 cells.
LS1034 cells were pretreated with inhibitors of caspase-3, -
Effects of CERP on Cell Cycle Distribution of
8, and -9, incubated with CERP for various time periods
LS1034 Cells
and act vities of caspase-3, -8, and -9 or percentage of viable cells
LS1034 cells were treated with to 0, 250, 500, 750, 1000,
were determined. Results are shown in Figure
1500, and 2000 lg/mL of CERP for 24 or 48 h. Percent distribution
7, which indicate that CERP stimulated activities of caspase-
of each cell cycle can be seen in Figure 2(A,B).
3 [Fig. 7(A)], caspase-8 [Fig. 7(B)] and caspase-9
CERP induced G0/G1 phase arrest and sub-G1 phase was
[Fig. 7(C)] between 12 and 72 h. Inhibitors of caspase-8, -
present which indicated that CERP induced apoptosis in
9, and -3 reduced activities of caspase-3, -8, and -9 and promoted
LS1034 cells.
the percentage of viable cells. CERP induced apoptosis
Effects of CERP on DNA Damage and
involves activation of caspase-3, -8, and -9 in
Condensation in LS1034 Cells
LS1034 cells.8
Comet assay and DAPI staining were used to investigate
Effects of CERP on Cell Cycle and Apoptosis
the effects of CERP on DNA damage and nuclei condensations,
Protein Expression in LS1034 Cells
respectively. Results shown in Figure 3 demonstrated
LS1034 cells were treated with 750 lg/mL of CERP for 0,
that CERP induced DNA damage in a dose-response manner.
6, 12, 24, and 48 h and cell cycle and apoptosis associated
It can be seen in Figure 4 that CERP also induced
proteins were examined by Western blotting and results are
DNA condensation and fragmentation in a dose-dependent
shown in Figure 9. CERP significantly promoted the
manner.
expression of p27, p16, and p21 but inhibited the expression of
Effects of CERP on ROS, Ca21 and DCm
cyclin D1, E and CDK4 [Fig. 9(A,B)]. These
findings demonstrated that CERP induced G0/G1 phase
Levels in LS1034 Cells
arrest via inhibition of check point enzymes of cell cycle in
LS1034 cells were treated with 750 lg/mL of CERP for different
LS1034 cells. Figure 9(C,D) also demonstrated that CERP
time periods and levels of ROS, Ca21, and DCm
decreased the levels of anti-apoptotic proteins Bcl-2 and
were determined by flow cytometry. CERP increased the
Bid; however, it increased the pro-apoptotic protein BAX.
ROS levels (data not shown) and Ca21 [Fig. 5(A)] but
Results also showed that CERP promoted the expression of
reduced DCm levels (data not shown) compared with the
caspase-8, -9, and -3, GRP78, cytochrome c, AIF, and endo
vehicle treated (control) group. Effects were time-dependent.
G, and PARP cleavage in LS1034 cells [Fig. 6(C,D)].
CERP may induce apoptosis in LS1034 cells by
DISCUSSION
increasing ROS and Ca21, levels, and perturbation of
The purpose of this study was to determine effects of
mitochondria.
CERP on cytotoxicity and apoptosis in human colon cancer
Effects of CERP on Translocation of Apoptotic
cells. The results showed that: (1) CERP decreased the
Associated Proteins in LS1034 Cells
percentage of viable cells in a dose-dependent manner
We examined effects of CERP on AIF, Endo G, cytochrome
(Fig. 1); (2) CERP induced DNA damage and nuclei condensation
c, and GADD153 involved in CERP induced apoptosis
dose-dependent (Figs. 3 and 4); (3) CERP
induced G0/G0 phase arrest and induced sub-G1 phase
levels (Fig. 9). Our results suggest that AIF translocation
(apoptosis) (Fig. 3); (4) CERP ROS and Ca21 levels but
into the nucleus is required for CERP-induced apoptosis
decreased DCm levels (Fig. 5); (5) CERP stimulated activity
in LS1034 cells.
of caspase-8, -9, and -3 (Fig. 7); (6) CERP inhibited
A proposed mechanism of CERP-induced apoptosis is
cyclin E and CDK2 which was associated with cell cycle
presented in Figure 10. In conclusion, these results show
arrest (Fig. 9), promoted Bax expression but reduced Bcl-2
that CERP is cytotoxic in LS1034 human colon cancer
levels [Fig. 9(D)]; and 7) CERP increased levels of AIF,
cells. Cytotoxicity is due to stimulation of apoptosis which
cytochrome c, and GADD153 and cytochrome c in
was associated with the production of ROS and the activation
LS1034 cells.
of caspase-dependent and -independent mitochondrial
It is well documented that many natural plant extracts
pathways.
from plants have been proposed to be excellent candidates
REFERENCES
for cancer therapeutics (Shigemura et al., 2007). Induction
[No authors listed]. 1999. Primary Prevention of Colorectal Cancer
of apoptosis by such extracts is thought to be a mechanism
and Polyps: Does Fiber have a Role? Proceedings of a symposium.
of cell death. Rheum undulatum L. have been screened for
New York City, New York, USA. December 2, 1997.
anti-cancer activity in vitro in human breast, ovary, cervix and lung
Am J Med. 25;106:1S–51S.
and oral cancer cell lines. It is well known that apoptosis
Brandi G, Corbelli J, de Rosa F, Di Girolamo S, Longobardi C,
can be triggered through: (1) the extrinsic (death receptor)
Agostini V, Garajova I, La Rovere S, Ercolani G, Grazi GL,
based on FasL or tumor necrosis factor which binds
Pinna AD, Biasco G. 2012. Second surgery or chemotherapy
to its cognate receptor activating the Fas-associated death
for relapse after radical resection of colorectal cancer metastases.
domain and caspase-8 cleavage (Luschen et al., 2005); (2)
Langenbecks Arch Surg 397:1069–1077.
the intrinsic (mitochondrial) pathway by release of cytochrome
Cai J, Razzak A, Hering J, Saed A, Babcock TA, Helton S, Espat
c from mitochondria (Lee et al., 2008) and downstream
NJ. 2008. Feasibility evaluation of emodin (rhubarb extract) as
activation of caspase-3. Our results showed that
an inhibitor of pancreatic cancer cell proliferation in vitro.
CERP decreased DCm levels Bcl-2 protein abundance but
JPEN J Parenter Enteral Nutr 32:190–196.
increased Bax levels. The Bcl-2 family of sproteins regulate
Cheng DY, Wang JZ, Zhao XH. 1994. Analytical study on processing
mitochondria-dependent apoptosis through a balance of the
of Rheum palmatum L. by HPLC. Zhongguo Zhong Yao
ratio (anti- and pro-apoptotic members) such as Bcl-2 and
Za Zhi 19:538–539, 574.
Bax, respectively (Tsou et al., 2006; Wu et al., 2010) and
Chiang JH, Yang JS, Ma CY, Yang MD, Huang HY, Hsia TC,
changes in the ratio of Bcl-2 and Bax contribute to apoptosis.
Kuo HM, Wu PP, Lee TH, Chung JG. 2011. Danthron, an
We also showed that CERP-induced apoptosis was caspase-
anthraquinone derivative, induces DNA damage and caspase
dependent and involves the activation of the mitochondrial
cascades-mediated apoptosis in SNU-1 human gastric cancer
pathway.
cells through mitochondrial permeability transition pores and
CERP increased ROS and Ca21 and it also induced ER
Bax-triggered pathways. Chem Res Toxicol 24:20–29.
stress in LS1034 cells. Other studies have reported that
Chiu TH, Lai WW, Hsia TC, Yang JS, Lai TY, Wu PP, Ma CY,
persistent or intense ER stress can trigger apoptotic cell
Yeh CC, Ho CC, Lu HF, Wood WG, Chung JG. 2009. Aloeemodin
death (63, 64). Thus, in this study, CERP induced apoptosis
induces cell death through S-phase arrest and caspasedependent
in LS1034 cells involved ROS production and may
pathways in human tongue squamous cancer SCC-4
also be acted upon by ER stress. Results from confocal
cells. Anticancer Res 29:4503–4511.
laser microscope also demonstrated that CERP promoted
Chu W, Chai J, Feng Y, Ma L, Hu C. 2012. Role of endoplasmic
the expression of AIF (Fig. 6) in LS1034 cells. CERP
reticulum stress during myocardial apoptosis in rats with severe
induced DNA fragmentation and nuclear condensation in
burn injury. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi
LS1034 cells. It was reported that AIF is a mitochondrial
26:592–596.
protein and if apoptosis is caspase-independent pathway,
He L, Bi JJ, Guo Q, Yu Y, Ye XF. 2012. Effects of Emodin
AIF can be translocated into nuclei to mediate nuclear
Extracted from Chinese Herbs on Proliferation of Non-small
condensation and DNA fragmentation. CERP, AIF protein
Cell Lung Cancer and Underlying Mechanisms. Asian Pac J
Cancer Prev 13:1505–1510.
JP, Tang NY, Lee TH, Chung JG. 2012a. Inhibition of invasion
Hompes D, D’Hoore A, Van Cutsem E, Fieuws S, Ceelen
and migration by newly synthesized quinazolinone MJ-29 in
W, Peeters M, Van der Speeten K, Bertrand C, Legendre
human oral cancer CAL 27 cells through suppression of MMP-
H, Kerger J. 2012. The treatment of peritoneal carcinomatosis
2/9 expression and combined down-regulation of MAPK and
of colorectal cancer with complete cytoreductive surgery
AKT signaling. Anticancer Res 32:2895–2903.
and hyperthermic intraperitoneal peroperative chemotherapy
Lu CC, Yang JS, Chiang JH, Hour MJ, Lin KL, Lin JJ, Huang
(HIPEC) with oxaliplatin: A belgian multicentre
WW, Tsuzuki M, Lee TH, Chung JG. 2012b. Novel quinazolinone
prospective phase II clinical study. Ann Surg Oncol
MJ-29 triggers endoplasmic reticulum stress and intrinsic
19:2186–2194.
apoptosis in murine leukemia WEHI-3 cells and inhibits leukemic
Huyghe E, Matsuda T, Thonneau P. 2003. Increasing incidence of
mice. PLoS One 7:e36831.
testicular cancer worldwide: A review. J Urol 170:5–11.
Lu CC, Yang JS, Huang AC, Hsia TC, Chou ST, Kuo CL, Lu HF,
Kampfenkel T, Tischoff I, Bonhag H, Eckardt M, Schmiegel W,
Lee TH, Wood WG, Chung JG. 2010. Chrysophanol induces
Tannapfel A, Reinacher-Schick A, Viebahn R. 2011. Chemotherapy-
necrosis through the production of ROS and alteration of ATP
associated steatohepatitis in patients with colorectal
levels in J5 human liver cancer cells. Mol Nutr Food Res
cancer and surgery on hepatic metastasis: clinical validation of
54:967–976.
a histopathological scoring system and preoperative risk assessment.
Luschen S, Falk M, Scherer G, Ussat S, Paulsen M, Adam-Klages
Z Gastroenterol 49:1407–1411.
S. 2005. The Fas-associated death domain protein/caspase-8/c-
Kim HR, Chae HJ, Thomas M, Miyazaki T, Monosov A, Monosov
FLIP signaling pathway is involved in TNF-induced activation
E, Krajewska M, Krajewski S, Reed JC. 2007. Mammalian
of ERK. Exp Cell Res 310:33–42.
dap3 is an essential gene required for mitochondrial homeostasis
Ma YS, Weng SW, Lin MW, Lu CC, Chiang JH, Yang JS, Lai
in vivo and contributing to the extrinsic pathway for apoptosis.
KC, Lin JP, Tang NY, Lin JG, Chung JG. 2012. Antitumor
FASEB J 21:188–196.
effects of emodin on LS1034 human colon cancer cells in vitro
Lan YH, Chiang JH, Huang WW, Lu CC, Chung JG, Wu TS, Jhan
and in vivo: roles of apoptotic cell death and LS1034 tumor
JH, Lin KL, Pai SJ, Chiu YJ, Tsuzuki M, Yang JS. 2012. Activations
xenografts model. Food Chem Toxicol 50:1271–1278.
of both extrinsic and intrinsic pathways in HCT 116
McDermott U, Longley DB, Galligan L, Allen W, Wilson T, Johnston
human colorectal cancer cells contribute to apoptosis through
PG. 2005. Effect of p53 status and STAT1 on chemotherapy-
p53-mediated ATM/Fas signaling by Emilia sonchifolia extract,
induced, Fas-mediated apoptosis in colorectal cancer. Cancer
a folklore medicinal plant. Evid Based Complement Alternat
Res 65(19):8951–8960.
Med 2012:178178. Epub 2012 Feb 28.
Molassiotis A, Gibson F, Kelly D, Richardson A, Dabbour R,
Landgraeber S, von Knoch M, Loer F, Wegner A, Tsokos M,
Ahmad AM, Kearney N. 2006. A systematic review of worldwide
Hussmann B, Totsch M. 2008. Extrinsic and intrinsic pathways
cancer nursing research: 1994 to 2003. Cancer Nurs
of apoptosis in aseptic loosening after total hip replacement.
29:431–440.
Biomaterials 29:3444–3450.
Ni CH, Yu CS, Lu HF, Yang JS, Huang HY, Chen PY, Wu SH, Ip
Lee HJ, Lee EO, Ko SG, Bae HS, Kim CH, Ahn KS, Lu J, Kim
SW, Chiang SY, Lin JG, Chung JG. in press. Chrysophanolinduced
SH. 2008. Mitochondria-cytochrome C-caspase-9 cascade
cell death (necrosis) in human lung cancer A549 cells
mediates isorhamnetin-induced apoptosis. Cancer Lett 270:
is mediated through increasing reactive oxygen species and
342–353.
decreasing the level of mitochondrial membrane potential. Environ
Li S, Zhao Y, He X, Kim TH, Kuharsky DK, Rabinowich H, Chen
Toxicol. 2012. doi: 10.1002/tox.21801. [Epub ahead of
J, Du C, Yin XM. 2002. Relief of extrinsic pathway inhibition by
print]
the Bid-dependent mitochondrial release of Smac in Fas-mediated
Putcha GV, Harris CA, Moulder KL, Easton RM, Thompson CB,
hepatocyte apoptosis. J Biol Chem 277:26912–26920.
Johnson EM, Jr. 2002. Intrinsic and extrinsic pathway signaling
Lin YL, Wu CF, Huang YT. 2008. Phenols from the roots of
during neuronal apoptosis: Lessons from the analysis of mutant
Rheum palmatum attenuate chemotaxis in rat hepatic stellate
mice. J Cell Biol 157:441–453.
cells. Planta Med 74:1246–1252.
Ross WA. 2010. Colorectal cancer screening in evolution: Japan
Lu CC, Yang JS, Chiang JH, Hour MJ, Amagaya S, Lu KW, Lin
and the USA. J Gastroenterol Hepatol 25 (Suppl 1):S49–S56.
Samuel PS, Pringle JP, James NWt, Fielding SJ, Fairfield KM.
cells through ER stress and caspase cascade- and mitochondria-dependent
2009. Breast, cervical, and colorectal cancer screening rates
pathways. Anticancer Res 30:2125–
amongst female Cambodian, Somali, and Vietnamese immigrants
2133.
in the USA. Int J Equity Health 8:30.
Xu YY, You YW, Ren XH, Ding Y, Cao J, Zang WD, Feng R,
Sayers TJ. 2011. Targeting the extrinsic apoptosis signaling pathway
Zhang QX. 2012. Endoplasmic reticulum stress-mediated signaling
for cancer therapy. Cancer Immunol Immunother 60:
pathway of gastric cancer apoptosis. Hepatogastroenterology
1173–1180.
8:59, doi: 10.5754/hge12369. [Epub ahead of print]
Shigemura K, Arbiser JL, Sun SY, Zayzafoon M, Johnstone PA,
Yang JS, Wu CC, Kuo CL, Lan YH, Yeh CC, Yu CC, Lien JC, Hsu
Fujisawa M, Gotoh A, Weksler B, Zhau HE, Chung LW. 2007.
YM, Kuo WW, Wood WG, Tsuzuki M, Chung JG. 2012. Solanum
Honokiol, a natural plant product, inhibits the bone metastatic
lyratum extracts induce extrinsic and intrinsic pathways of
growth of human prostate cancer cells. Cancer 109:1279–1289.
apoptosis in WEHI-3 murine leukemia cells and inhibit allograft
Shitara K, Matsuo K, Kondo C, Takahari D, Ura T, Inaba Y,
tumor. Evid Based Complement Alternat Med 2012:254960.
Yamaura H, Sato Y, Kato M, Kanemitsu Y, Komori K, Ishiguro
Yang SH, Liu XF, Guo DA, Zhen JH. 2006. Induction of hairy
S, Sano T, Shimizu Y, Muro K. 2011. Prolonged survival of
roots and anthraquinone production in Rheum palmatum.
patients with metastatic colorectal cancer following first-line
Zhongguo Zhong Yao Za Zhi 31:1496–1499.
oxaliplatin-based chemotherapy with molecular targeting agents
Yu CX, Zhang XQ, Kang LD, Zhang PJ, Chen WW, Liu WW,
and curative surgery. Oncology 81:167–174.
Liu QW, Zhang JY. 2008. Emodin induces apoptosis in human
Suboj P, Babykutty S, Srinivas P, Gopala S. 2012a. Aloe emodin
prostate cancer cell LNCaP. Asian J Androl 10:625–634.
induces G2/M cell cycle arrest and apoptosis via activation of
Zhang B, Chen H, Xu A, Chen J, Sheng S. 1997. Effects of volatile
caspase-6 in human colon cancer cells. Pharmacology 89:91–98.
oil from Rheum palmatum on immunologic function in
Suboj P, Babykutty S, Valiyaparambil Gopi DR, Nair RS, Srinivas
mice. Zhong Yao Cai 20:85–88.
P, Gopala S. 2012b. Aloe emodin inhibits colon cancer cell
Zhang Q, Zhang C, Wang J, Guan L, Yu H. 2001. Effect of
migration/angiogenesis by downregulating MMP-2/9, RhoB
Rheum palmatum decoction on increasing intelligence. Zhong
and VEGF via reduced DNA binding activity of NF-kappaB.
Yao Cai 24:728–730.
Eur J Pharm Sci 45:581–591.
Zhang SJ, Zhang SY, Wang L, Zhu B. 1993. Studies on polysaccharide
Taghizadeh F, Tang MJ, Tai IT. 2007. Synergism between vitamin
of Rheum palmatum L. Zhongguo Zhong Yao Za Zhi
D and secreted protein acidic and rich in cysteine-induced apoptosis
18:679–681, 703.
and growth inhibition results in increased susceptibility
Zhang Y, Yuan J, Zhang HY, Simayi D, Li PD, Wang YH, Li F,
of therapy-resistant colorectal cancer cells to chemotherapy.
Zhang WJ. 2012. Natural resistance to apoptosis correlates with
Mol Cancer Ther 6:309–317.
resistance to chemotherapy in colorectal cancer cells. Clin Exp
Tsou MF, Lu HF, Chen SC, Wu LT, Chen YS, Kuo HM, Lin SS,
Med 12:97–103.
Chung JG. 2006. Involvement of Bax, Bcl-2, Ca21 and caspase-
Zhong YS, Lu SX, Xu JM. 2008. Tumor proliferation and apoptosis
3 in capsaicin-induced apoptosis of human leukemia HL-
after preoperative hepatic and regional arterial infusion
60 cells. Anticancer Res 26:1965–1971.
chemotherapy in prevention of liver metastasis after colorectal
Wang JB, Kong WJ, Wang HJ, Zhao HP, Xiao HY, Dai CM, Xiao
cancer surgery]. Zhonghua Wai Ke Za Zhi 46:1229–1233.
XH, Zhao YL, Jin C, Zhang L, Fang F, Li RS. 2011. Toxic
effects caused by rhubarb (Rheum palmatum L.) are reversed
on immature and aged rats. J Ethnopharmacol 134:216–220.
Wu M, Yang S, Elliott MH, Fu D, Wilson K, Zhang J, Du M,
Chen J, Lyons T. 2012. Oxidative and endoplasmic reticulum
stresses mediate apoptosis induced by modified LDL in human
retinal muller cells. Invest Ophthalmol Vis Sci 53:4595–4604.
Wu SH, Hang LW, Yang JS, Chen HY, Lin HY, Chiang JH, Lu
CC, Yang JL, Lai TY, Ko YC, Chung JG. 2010. Curcumin
induces apoptosis in human non-small cell lung cancer NCIH460