Chrysophanol-Induced Cell Death (Necrosis) in Human Lung Cancer A549 Cells Is Mediated
Through Increasing Reactive Oxygen Species and Decreasing the Level of Mitochondrial
Membrane Potential
Chien-Hang Ni,
1,2
3
4
5
6
7
Chun-Shu Yu, Hsu-Feng Lu, Jai-Sing Yang, Hui-Ying Huang, Po-Yuan Chen, Shin-Hwar Wu,
2
8,9
6
Siu-Wan Ip, Su-Yin Chiang,
2
7,10
Jaung-Geng Lin, Jing-Gung Chung
1
Department of Chinese Medicine, E-DA Hospital/I-Shou University, Kaohsiung 824, Taiwan
2
Graduate Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan
3
School of Pharmacy, China Medical University, Taichung 404, Taiwan
4
Department of Clinical Pathology, Cheng Hsin General Hospital, Taipei 112, Taiwan
5
Department of Pharmacology, China Medical University, Taichung 404, Taiwan
6
Department of Nutrition, China Medical University, Taichung 404, Taiwan
7
Department of Biological Science and Technology, China Medical University, Taichung 404,
Taiwan
8
Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan
9
Division of Critical Care Medicine, Department of Internal Medicine, Changhua
Christian Hospital, Changhua 500, Taiwan
10
Department of Biotechnology, Asia University, Taichung 413, Taiwan
Received 9 July 2011; revised 28 June 2012; accepted 30 June 2012
ABSTRACT: Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is one of the anthraquinone compounds, and it has been
shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have
not been well studied. The purpose of this study is to examine chrysophanol-induced cytotoxic effects and also to investigate such
influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol
decreased the viable A549 cells in a dose-and time-dependent manner. Chrysophanol also promoted the release of reactive
21
oxygen species (ROS) and Ca and decreased the levels of mitochondria membrane potential (DCm) and adenosine triphosphate
in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly,
chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis-associated protein levels including
caspase-3, caspase-8, Apaf-1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a
cellular pattern associated with necrotic cell death and not apoptosis in vitro. # 2012 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2012.
Keywords: chrysophanol; necrosis; human lung cancer A549 cells; reactive oxygen species; mitochondria membrane potential
Correspondence to: J.-G. Chung; e-mail: jgchung@mail.cmu.edu.tw Published online in Wiley Online Library (wileyonlinelibrary.com). Contract grant sponsor: China Medical University; Contract grant num-DOI 10.1002/tox.21801
ber: CMU100-ASIA-4.
。C 2012
Wiley Periodicals, Inc. 1
INTRODUCTION
USA), and the other primary and secondary antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The fl uorescent probes
Lung cancer is one of the major causes of death in human population. In
Taiwan, 36.3 individuals per 100,000 died of lung cancer in 2011, and it is the
first most frequent cause of cancer death among males in Taiwan (Department
of Health). Over the past decades, the clinical results are far from satisfactory
although the treatment options for lung cancer have undergone tremendous
change (Jassem, 1999; Magrini et al., 2006). Traditional herbs or natural
products are commonly used in cancer patients (Aravindaram and Yang, 2010);
however, the exact mechanisms of action from these compounds are not yet
understood. Numerous studies are focused on new compounds from traditional
herbs.
Many studies have been shown that anthraquinone compounds such as
emodin, aloe-emodin, and rhein induced apoptosis in many cancer cell lines (Su
et al., 2005; Lin et al., 2009, 2010; Aviello et al., 2010). Chrysophanol, a
2 ,7 -dichloro fl uorescin diacetate (DCFH-DA), Indo 1/AM, DiOC6, and
member of the anthraquinone family, is a component of Rheum officinale
(rhubarb) and Polygpnum cuspidatum (Huang et al., 2007; Chiang et al.,
2011). Chrysophanol has been shown to inhibit the growth of L1210 leukemic
cells and did not affect apoptosis in HL-60 human leukemia cells (Ueno et al.,
1995), but it also exist antiproliferative effects in MCF-7 and MDA-MB-231
human breast cancer cells (Kang et al., 2008). Recently, we found that
chrysophanol-induced cells death is necrosis not apoptosis in J5 human liver
cancer cells (Lu et al., 2010). Other investigator also showed that anticancer
activity for chrysophanol via EGFR/mTOR mediated signaling transduction
pathway in human colon cancer cells (Lee et al., 2011). Also, chrysophanol
exhibited anti-in fl ammatory activity through suppressing NF-jB/caspase-1
activation in vitro and in vivo (Kim et al., 2010).
Numerous reports have shown that reactive oxygen species (ROS) involved
the components of cell signaling in host defense (Fialkow et al., 2007;
Boncompain et al., 2010). After the cells were exposed to growth factors, the
ROS appear to be rapidly produced (Sundaresan et al., 1995; Ushio-Fukai et al.,
2001). In mammalian cells, they can rapidly respond to ligand stimulation with
a change in intracellular ROS, which can stimulate cell proliferation (Burdon,
1995; Wedgwood et al., 2001), and the excessive accumulation of ROS is
cytotoxic due to oxidative damage (Yan et al., 1997; Cadenas and Davies,
2000).
The effects of chrysophanol on cancer cell death are unclear, especially in
human lung cancer cells. Thus, the present study investigated the cytotoxic
effects of chrysophanol on human lung cancer A549 cells, and result indicated
that chrysophanol induced ROS production and then led to cell necrosis in
0
0
0
4 -6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life
Technologies (Carlsbad, CA). Adenosine triphosphate (ATP) detection kit was
purchased from Luminescence ATP Detection Assay by ATPliteTM kit
(PerkinElmer, Waltham, MA). RPMI-1640 medium, fetal bovine serum (FBS),
L-glutamine, penicillin, and streptomycin were obtained from Invitrogen Life
Technologies.
Cell Culture
The human lung cancer cell line (A549) was obtained from the Food Industry
Research and Development Institute (Hsinchu, Taiwan) and maintained in
RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/
2
mL penicillin, and 100 lg/mL streptomycin in 75-cm tissue-culture flasks at
378C under a humidified 5% CO2 and 95% air atmosphere as we have
previously reported (Lu et al., 2008).
Morphological Changes and Percentage of Viable Cell
Examination in A549 Cells
5
A549 cells at a density of 2 3 10 cells/well were placed on 12-well plates,
treated with 1, 5, 10, 25, 50, 75, or 100 lM chrysophanol or only with vehicle
(DMSO, 1% in culture media), and then incubated for 24 and 48 h. All cells in
each well were examined and photographed under a phase-contrast microscope
for morphological changes. Then, cells from each treatment were collected to
measure the percentage of viable cells. Cell viability was determined by a PI
exclusion method and flow cytometric assay as described elsewhere (Tan et al.,
2006; Hsu et al., 2007). In brief, cells were exposed to various concentrations of
chrysophanol for 24, 48, and 72 h. At the end of the experiment, cells were
harvested, resuspended in PI (4 lg/mL), and then immediately determined using
a BD FACSCalibur (Becton-Dickinson, San Jose, CA) and BD CellQuest Pro
software (Macintosh) as previously described (Su et al., 2006; Hsu et al., 2007).
Determination of Cell-Cycle Distribution of A549 Cells
5
vitro.
Cells were plated onto 12-well plates at a density of 2 3 10 cells/well, and
MATERIALS AND METHODS
chrysophanol was added to cells at final concentrations of 0, 12.5, 25, 50, 75,
100, 120, and 150 lM and incubated for 24 h. DMSO (solvent) was served as
the vehicle control. For DNA content analysis, cells were washed with PBS, fi
xed in 70% ethanol at -20C
Chemicals and Reagents
Chrysophanol, dimethyl sulfoxide (DMSO), propidium iodide (PI), RNase A,
and triton X-100 were obtained from Sigma-Aldrich Co. (St. Louis, MO).
Anti-caspase-8 (Cat. AB1879) was bought from Merck Millipore (Bedford, MA,
overnight, resuspended in PBS-containing 40 lg/mL PI, 0.1 mg/mL RNase A, and 0.1% Triton X-100 in a dark room for 30 min at
378C, and analyzed by flow cytometry as previously described (Su et al., 2006; Hsu et al., 2007).
21
Assays for ROS, Ca Release, and Mitochondrial Membrane Potential (DCm)
A549 cells/well at a density of 2 3 10 cells/well on a 1250 lM chrysophanol for 6, 12, 24, 48, or 72 h under 5% CO2 and 95% air at
378C. Then, all cells from each treatment were harvested, washed twice by PBS, and then were resuspended in 500 lL of DCFH-DA
5
(10 lM) for reactive oxygen species (ROS), Indo 1/AM (3 lg/mL) for Ca production, and DiOC6 (1 lmol/L) for the level of DCm under
dark room for 30 min at 378C. Then, all samples were analyzed immediately by flow cytometry as described previously (Lin et al.,
2007; Lu et al., 2008; Chiang et al.,
well plate were maintained and then were treated with 2011).
21
ATP Level Assay
then detected by ECL kit and autoradiography using X-ray film (Ji et al., 2009;
Lu et al., 2012).
4
A549 cells at the density of 1 3 10 cells/well were seeded in 100-lL phenol
red-free medium with various concentrations (0, 25, 50, 75, and 100 lM) of
chrysophanol for 6 h onto 96-well white microplates, and the intracellular ATP
content level was measured using Luminescence ATP Detection Assay by
ATPliteTM kit (PerkinElmer) as described previously (Huan et al., 2006; Lu et
al., 2010). The resulting luminescence was monitored by SynergyTM HT
Multi-Mode Microplate Reader (BioTek, Winooski, VT).
Comet Assay and DAPI Staining
Statistical Analysis
The quantitative data are shown as mean 6 SD. Results are representative of
three independent experiments. The statistical differences between the
chrysophanol-treated and control samples were calculated using Student’s t-test.
A p-value of \0.05 was considered significant.
RESULTS
5
A549 cells/well at a density of 2 3 10 cells/well on 12well plates were treated
with 0, 10, 25, 50, 75, 100, and 120 lM chrysophanol for 48 h. All treated cells
were divided into two parts for Comet staining by PI and DAPI staining. Then,
all samples from each treatment were examined and photographed using fl
uorescence microscopy as described elsewhere (Chiang et al., 2006; Yu et al.,
2011).
Western Blotting Analysis
6
A549 cells at a density of 5 3 10 cells/well on six-well plates were maintained
and then were incubated with 50 lM chrysophanol for 0, 6, 12, 24, 48, and 72 h.
Cells from each treatment were isolated, washed twice with PBS, and then
Chrysophanol Induced Cell Morphological Changes and
Decreased the Percentage of Viable A549 Cells
After the cells were treated with various concentrations of chrysophanol for
different time periods, cells were examined under a phase-contrast microscope
for morphological changes and collected for determining the percentage of
viable cells as can be seen from Figure 1. Results indicated that chrysophanol
induced morphological changes of A549 cells in a dose-dependent manner at
24-h [Fig. 1(A)] and 48-h [Fig. 1(B)] exposure. The total viable cells were also
examined, and results showed that chrysophanol decreased the percentage of
viable cells in a dose-dependent manner [Fig. 1(C)].
TM
lysed in the PRO-PREP protein extraction solution (iNtRON Biotechnology,
Seongnam-si, Gyeonggi-do, Korea). The total proteins from each lysed cells
were determined by using Bio-Rad protocol as described previously (Chiang et
al., 2011; Yu et al., 2011). The determined proteins levels [cyclin D, CDK2,
thymidylate synthase, GST, catalase, SOD (Mn), SOD (Cu), cytochrome c,
PARP, caspase-3, caspase-8, Apaf-1, and AIF] were associated with apoptotic
cell death by Western blotting. Each sample was stained with primary
antibodies, washed twice, followed for staining by secondary antibody, and
A549 cells. Results shown in Figure 2 indicated that chrysophanol induced
S-phase arrest [Fig. 2(A)]. The treatment of A549 cells with 50 lM for 24 h
resulted in a higher number of cells in the S phase (28%) compared to the
control (12%). Also, the decreases in the levels of (B) cyclin D, (C) CDK2, and
(C) thymidylate synthase occurred in chrysophanol-treated A549 cells for 6–72
h.
Chrysophanol Increased the Levels of ROS and Ca
21
Chrysophanol Induced S Phase Arrest and Inhibited
Associated Protein Levels in A549 Cells
Cells were treated with various concentrations of chrysophanol for 24 h and
analyzed cell-cycle distribution in
level [Fig. 3(C)] were observed in the chrysophanol-treated A549 cells when
compared with the control cells. At the treatment of chrysophanol at 12 h until
72 h, the ROS levels were significantly higher than those of the control [Fig.
3(A)]. At the earlier treatment of chrysophanol (6 h), A549 cells were initially
2
1
significantly increased the cytosolic Ca level in 72-h exposure as shown in
Figure 3(B). Furthermore, Figure 3(C) shows that chrysophanol significantly
decreased the level of DCm around 65% at 12-h treatment in A549 cells when
compared with the untreated groups [Fig. 3(C)].
Production and Decreased the Level of DCm in A549 Cells
Chrysophanol Alters ATP Level in A549 Cells
Cells were treated with 50 lM chrysophanol for different time periods, and then
A549 cells were treated with different concentrations of chrysophanol, and then
all cells were measured by the levels of ATP. Results are shown in Figure 3(D),
2
1
all the cells were measured by the levels of ROS, Ca , and DCm. The results
are shown in Figure 3(A–C). A significant increase in intracellular ROS [Fig.
2
1
3(A)] and cytosolic Ca levels [Fig. 3(B)] and a significant decrease DCm
which indicated that chrysophanol decreased ATP levels around 20% (p \ 0.05)
from the doses of chrysophanol about 25–75 lM in A549 cells.
Chrysophanol Induced DNA Damage in A549 Cells
Cells were isolated from chrysophaol treatment to determine DNA damage by
Comet assay and DAPI staining, and results are shown in Figure 4(A,B). Figure
4(A) indicates that chrysophanol induced DNA damage in a dose-dependent
manner by using the Comet assay. Figure 4(B) also shows that chrysophanol
induced DNA that is broken in a dose-dependent manner because of the higher
density of white color on the nuclei compared to the control by fluorescence
microscopy examination (DAPI staining examination). Based on these results,
chrysophanol induced DNA damage in A549 cells in vitro.
Chrysophanol Affected the Levels of Oxidative Stress and
Apoptosis-Associated Proteins in A549 Cells
Cells were treated with 50 lM chrysophanol for 0, 6, 12, 24, 48, and 72 h and
then were isolated to determine the levels of oxidative stress and
apoptosis-associated proteins by Western blotting as shown in Figures 5 and 6.
Our results indicated that chrysophanol decreased the levels of GST [Fig. 5(A)],
catalase [Fig. 5(B)], and SOD (Mn) [Fig. 5(D)], but did not affect the SOD (Mn)
level [Fig. 5(C)] in A549 cells. In Figure 6(A), we showed that chrysophanol
promoted the level of cytochrome c in A549 cells. However,
chrysophanol does not alter the levels of PARP [Fig. 6(B)],
caspase-3 [Fig. 6(C)], caspase-8 [Fig. 6(D)], Apaf-1 [Fig. 6(E)], and
AIF [Fig. 6(F)] in A549 cells. On the basis of these observations, we
suggest that chrysophanol triggered nonapoptotic cell death in A549
cells.
DISCUSSION
It is well documented that anticancer drug-induced apoptosis of cancer cells is
the best effective strategy for cancer therapy (Wu et al., 2010, 2011; Yu et al.,
2010, 2011). It was reported that extracts prepared from many plants and from
semisynthesized compounds have been demonstrated can induce apoptotic
processes (Calixto et al., 1998; Vergote et al., 2002). This study is the first to
show that chrysophanol inhibits the growth of human lung carcinoma
A549 cell-line in vitro. A549 cells treated with chrysophanol accumulated in
the S phase of the cell cycle and underwent necrosis not induced apoptosis in a
dose and time-dependent manner in vitro. It was reported that agents arrested
the cell-cycle progression by means of the downregulation the levels of cyclin
D, CDK2, and thymidylate synthase that are involved in DNA replication in S
phase (Yin et al., 1999). Thus, we suggest that chrysophanol arrested A549
cells in the S phase through modulation cyclin D, CDK2, and thymidylate
synthase pathway [Fig. 7(A)].
Apoptosis has also been reported to vial the mitochondria, and
mitochondrial function is regulated through Bcl2 family proteins comprising
both antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic members (Bax, Bak;
Hengartner, 2000; Adams and Cory, 2007). Herein, results from Western
blotting did not show cyrysophanol promoting the Bax or Bak, and it did not
inhibit the levels of Bcl-2 in A549 cells (data not shown). In the present study,
we found that chrysophanol decreased the levels of DCm [Fig. 3(C)] and ATP
[Fig. 3(D)], and Western blotting analysis showed that chrysophanol treatment
resulted in a no significant increase of caspase-3, caspase-8, Apaf-1, AIF, and
PARP expression. Thus, we suggest that chrysophanol-induced cell death may
not mediate apoptotic signaling in A549 cells.
Our results also showed that chrysophanol promoted the ROS production in
A549 cells [Fig. 3(A)]. It was reported that decreased ROS accumulation led to
tumor growth inhibition (Fruehauf and Meyskens, 2007), but ROS production
was associated with the apoptotic response induced by several anticancer agents
(Su et al., 2005; Antosiewicz et al., 2006). It is well known that high levels of
ROS can induce apoptosis through triggering mitochondrial permeability
transition pore opening, release of proapoptotic factors, and activation of
caspase-9 (Zu et al., 2005; Iwamaru et al., 2007). However, necrosis is
associated with a loss of DCm and ATP levels (Halestrap, 2005), and our study
indicated that chrysophanol decreased both the DCm [Fig. 3(C)] and ATP
levels [Fig. 3(D)] in A549 cells. Therefore, our finding is similar to other
investigations regarding the production of ROS, loss of DCm, and ATP
depletion in mammalian cells that lead to undergo necrosis because of failure in
the formation of apoptosome (Leist et al., 1997; Green and Reed, 1998). The
previous study demonstrated that ROS has been shown to activate autophagy
through autophagy-related 4 (Scherz-Shouval et al., 2007). However, we also
determined the protein expression of autophagic marker light chain 3, and we
found that there is no significant alteration in chrysophanol-treated cells (data
not shown). Therefore, we can rule out the possible involvement of autophagy
in chrysophanol-induced cell death.
In conclusion, our data indicated that human lung cancer A549 cells are
sensitive to growth inhibition but not through the induction of apoptosis by
chrysophanol in vitro. Chrysophanol induced cell death and growth inhibition
through the necrotic cell death, and it is associated with mitochondrial cell death pathways,
diallyl trisulfide-induced generation of reactive oxygen species and cell cycle arrest in
which are mediated by ROS generation and decreased the levels of ATP [Fig. 7(B)]. Thus,
human prostate cancer cells. Cancer Res 66:5379–5386.
these properties of chrysophanol could be further explored in vivo.
Aravindaram K, Yang NS. 2010. Anti-inflammatory plant natural products for cancer
therapy. Planta Med 76:1103– 1117.
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