Corosolic acid induces apoptotic cell death in human lung adenocarcinoma

A549 cells in vitro

Kyoung Jin Nho, Jin Mi Chun, Ho Kyoung Kim

Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811, Republic of Korea
a r t i c l e i n f o
Article history:
Received 5 December 2012
Accepted 3 February 2013
Available online 20 February 2013
Keywords:
Corosolic acid
Apoptosis
Caspase
Mitochondria
Reactive oxygen species
A549 cells
a b s t r a c t
Corosolic acid (CRA), a triterpenoid from medicinal herbs, has been shown to induce apoptosis in several
cell lines, with the exception of A549 cells. In this report, we investigated the apoptotic effect and mech-
anism of CRA in A549 cells. The present study shows that CRA signicantly inhibits cell viability in a con-
centration- and time-dependent manner. Exposure to CRA induces sub-G1 cell cycle arrest and causes
apoptotic death in A549 cells. CRA also triggers the activation of caspases and poly(ADP-ribose) polymer-
ase, an effect antagonized by z-vad-fmk. In addition, exposure to CRA leads to a signicant increase in the
levels of reactive oxygen species (ROS) inA549 cells. Furthermore, exposure to the ROS scavenger N ace-
tylcysteine (NAC)prevents CRA-induced apoptosis, suggesting a role for ROS in CRA-induced apoptosis.
ROS are critical regulators of caspase-mediated apoptosis in A549 cells. These results indicate that CRA
induces mitochondria-mediated and caspase-dependent apoptosis inA549 cells by altering anti-apoptotic
proteins in a ROS-dependent manner.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Lung cancer is the most common cause of cancer mortality
worldwide. Approximately 8085% of all lung cancers are classied
as non-small-cell lung cancer (NSCLC), an aggressive tumor type
with a 5-year survival rate of only 16% that has improved little over
the last 35 years (Jemal et al., 2010). Even in patients with early
stage NSCLC, about half will relapse despite surgery, radiation,
and adjuvant chemotherapy. Therefore, the search for better thera-
peutic agents with enhanced activity against lung cancer continues.
Over the past few decades, a large number of plant-derived bioac-
tive compounds have been isolated that are now widely used to
treat cancers, including paclitaxel, vinblastine, and camptothecin.
Corosolic acid (CRA), a triterpenoid named 2a-hydroxyursolic
acid, has been discovered in many traditional Chinese medicinal
herbs, such as Lagerstroemia speciosa (Fukushima et al., 2006),
Eriobotrta japonica (Zong and Zhao, 2007), Tiarella polyphylla (Park
et al., 2002), etc. The triterpenoids have been used widely in Asian
medicine (Liby et al., 2007) and are reported to possess anti-
tumoral properties (Fernandes et al., 2005; Harmand et al., 2005;
Martin et al., 2007; Reyes-Zurita et al., 2009). Recent data suggest
that CRA may be of therapeutic value for its variety of biological
activities, such as its anti-diabetic (Fukushima et al., 2006; Miura
et al., 2006), anti-inammatory (Banno et al., 2004), and anti-
obesity activity (Yamaguchi et al., 2006; Zong and Zhao, 2007). In
addition, CRA displays cytotoxic activity against several human
cancer cell lines (Ahn et al., 1998; Yoshida et al., 2005; Lee et al.,
2010a,b) but the underlying anti-cancer mechanisms of CRAremain
unknown.
Apoptosis is a fundamental cellular event during development
and is critical for the cytotoxicity induced by anti-cancer drugs
(Cotter, 2009). Over the past two decades, more and more bioactive
compounds identied from traditional Chinese medicinal herbs
have been shown to kill NSCLC cells by apoptosis including, for
example, glossogin (Hsu et al., 2008) and emodin (Su et al.,
2005); however, to our knowledge, the apoptotic effect of CRA
has not been evaluated in lung cancer cells. In this study, we used
A549 cells to investigate the apoptotic effect and molecular mech-
anisms of CRA.
2. Materials and methods
2.1. Chemicals and reagents
CRA was obtained from ChromaDex Inc. (Irvine, CA, USA), and its molecular
structure is illustrated in Fig. 1A. Z-vad-fmk, N-acetyl-L-cysteine (NAC), valinomy-
cin, and H
2
O
2
were purchased from SigmaAldrich Co. (St. Louis, MO, USA).
2.2. Cell culture
A549 lung adenocarcinoma cells were obtained from the American Type Culture
Collection (Manassa, VA, USA). Cells were routinely maintained in Dulbeccos Mod-
ied Eagles Medium (DMEM, HyClone, Logan, UT, USA) with 10% heat-inactivated
FBS (Gibco BRL, Gaithersburg, MD, USA), 100 U/ml penicillin (Gibco BRL), and
0278-6915/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.02.002

Corresponding author. Tel.: +82 42 868 9502; fax: +82 42 863 9434.
E-mail address: hkkim@kiom.re.kr (H.K. Kim).
Food and Chemical Toxicology 56 (2013) 817
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100 lg/ml streptomycin (Gibco BRL). All cultured cells were incubated at 37 C in a
humidied atmosphere containing 5% carbon dioxide. Cells were fed with fresh cul-
ture medium two to three times per week and subcultured when 80% conuent.
2.3. Cell viability assay
Cells were seeded in 96-well culture plates at a density of 2 10
4
cells/well and
allowed to adhere at 37 C for 12 h. The following day, cells were exposed to several
concentrations of CRA and further incubated for 24 h. Finally, cell viability was
measured using the CCK-8 assay. The CCK-8 reagent (10 ll) was added to each well
and incubated for 1 h at 37 C. The assessment of cell viability by the CCK-8 assay is
based on the bioconversion of tetrazolium into formazan by intracellular dehydro-
genase. Absorbance was measured at 450 nm using a Benchmark Plus Microplate
Spectrophotometer (Bio-Rad, Hercules, CA, USA). Cytotoxicity was expressed as a
percentage of the absorbance measured in control untreated cells.
2.4. Hoechst 33342 staining
Hoechst 33342 (Invitrogen, Eugene, Oregon, USA) staining was used to observe
the apoptotic morphology of cells. First, 3 10
5
cells/ml were seeded in six-well
plates and incubated for 24 h, after which the cells were exposed to different con-
centrations of CRA (1040 lM) for 24 h. Next, the cells were collected and xed
with 3.7% formaldehyde in phosphate buffered saline (PBS) for 15 min and stained
with Hoechst 33342 (10 lg/ml) at room temperature for 10 min. Finally, after the
cells were washed with PBS, morphological changes, including a reduction in vol-
ume and nuclear chromatin condensation, were observed by uorescence micros-
copy (Olympus Optical, Tokyo, Japan) and photographed at a 200 magnication.
2.5. Flow cytometric analysis for measurement of sub-G1 phase
Cells were seeded in six-well plates at 3 10
5
cells/well and allowed to attach
overnight. After exposure to CRA, cells were collected, washed twice with ice-cold
PBS (pH 7.4), xed with 80% ethanol at 4 C for 2 h and then stained with PI/RNase
Staining Buffer (BD PharMingen, San Diego, CA, USA) for 20 min in the dark at room
temperature. Apoptotic cell analysis was conducted on a FACS Calibur ow cytom-
eter (BD Biosciences, San Jose, CA, USA) and the data were analyzed using the Cell-
Quest software.
2.6. Annexin V/propidium iodide (PI) staining
Double staining with annexin V and PI was conducted using the BD PharMingen
Annexin V-FITC Apoptosis Detection kit II (BD Biosciences, Schwechat, Austria)
according to the manufacturers instructions. Data were acquired using a FACS Cal-
ibur ow cytometer and analyzed using the CellQuest Pro data analysis software
provided by the manufacturer.
2.7. Protein extraction and Western blot analysis
Cells were seeded at 3 10
5
cells/well in six-well plates and incubated with
CRA, NAC, and z-vad-fmk for the times indicated and at the concentrations indi-
cated. Following treatment, cells were washed in PBS, and total cell lysates were
prepared by scraping the cells in 200 ll 1X RIPA lysis buffer (50 mM TrisHCl, pH
8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM Prote-
ase Inhibitor Cocktail). 30 lg of protein, measured by Bradford assay, was electro-
phoretically separated using 12% sodium dodecyl sulfatepolyacrylamide gel
electrophoresis (SDSPAGE), transferred to nitrocellulose membranes (Scheicher
& Schnell BioScience, Dassel, Germany) and then immunoblotted with specic anti-
bodies. Immunodetection was performed using the enhanced chemiluminescence
(ECL) detection kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
2.8. Detection of caspase catalytic activity
Caspase activity was assayed using the Caspase-Glo assay (Promega, Madison,
WI, USA) according to manufacturer protocols. Briey, cells were seeded at a den-
sity of 1 10
4
per well in triplicate wells onto 96-well plates and incubated
for 24 h. Afterwards, the cells were exposed to several concentrations of CRA
(A) (B)
(C)
Fig. 1. CRA inhibits the growth and alters the morphology of A549 cells. (A) Molecular structure of CRA (C
30
H
48
O
4
, FW: 472.70). (B) Concentration response and time course.
Cells were incubated with CRA (1040 lM) over time (648 h). Cell viability was assessed by CCK-8 assay. The data are expressed as the means SD of triplicate samples.

P < 0.01 and

P < 0.001 vs. untreated CRA. (C) Cells were incubated in the presence or absence of several concentrations of CRA for 24 h. Hoechst stain showed CRA-induced
chromatin condensation (arrow). Magnication 200.
K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817 9
(1040 lM) for 24 h or incubated with 28 lM of CRA for 648 h. After exposure to
CRA, culture supernatant (100 ll) was transferred into a white-walled 96-well
plate. An equal volume of caspase substrate was added and samples were incubated
at room temperature for 1 h. Culture medium was used as a blank control sample
and luminescence was measured using an EnVision 2103 Multilabel Reader (Perk-
inElmer, Wellesley, MA, USA).
2.9. Detection of mitochondrial transmembrane potential (Dwm) disruption
Mitochondrial membrane potential (Dwm) was assessed using MitoCapture
apoptosis detection kit (Trevigen for R&D Systems Inc, Minneapolis, MN, USA). Cells
were cultured on glass chamber slides and incubated with 1 lM of valinomycin (as
a positive control), and CRA for 24 h. Subsequently, the cells were stained with
MitoCapture according to the manufacturers instructions. In healthy cells, Mito-
Capture accumulates and aggregates in the mitochondria, giving off a bright red
uorescence. In apoptotic cells, MitoCapture cannot aggregate in the mitochondria
due to the altered Dwm, and thus it remains in the cytoplasm in its monomer form,
uorescing green. After labeling, cells were observed using a Fluoview FV10i confo-
cal laser-scanning microscope (Olympus Corporation, Tokyo, Japan) and uores-
cence was measured using an EnVision 2103 Multilabel Reader (PerkinElmer,
Wellesley, MA, USA).
2.10. Preparation of mitochondrial and cytosolic fractions
To detect the release of cytochrome c from mitochondria into the cytosol, a
Mitochondria/Cytosol fractionation kit (Abcam, Cambridge, MA, USA) was used.
Cells (1 10
7
) were cultured in 75T-asks and exposed to CRA for the time indi-
cated and at the concentration indicated. Afterwards, the cells were washed with
ice-cold PBS and resuspended in cytosol extraction buffer. After incubation on ice,
the cells were homogenized and the homogenates were centrifuged at 700g for
10 min at 4 C. The supernatants were further centrifuged at 10,000g for 30 min
at 4 C and stored at 80 C (cytosolic fraction). The pellet was resuspended in
mitochondrial extraction buffer and stored at 80 C (mitochondrial fraction).
30 lg of protein were loaded onto a 12% SDSPAGE. The standard Western blot pro-
cedure described above was followed.
2.11. Detection of ROS
To measure intracellular ROS, cells treated with CRA and untreated cells were
loaded with 10 lM H
2
DCFDA probe (Molecular Probes, Europe BV, Leiden, The
Netherlands) during the last 30 min of treatment. Then, cells were harvested by
trypsinization and washed twice with PBS before being analyzed by ow cytometry.
Flow cytometric analysis was performed on at least 1 10
4
cells using a FACS Cal-
ibur ow cytometer (BD Biosciences, San Jose, CA, USA) and the data were analyzed
using the CellQuest software.
2.12. Statistical analysis
Statistical analyses were performed with the Prism 5 software (GraphPad, San
Diego, USA). Analysis of variance (ANOVA) was followed by Dunetts test. A value
of P < 0.05 was considered to be statistically signicant.
3. Results
3.1. CRA induces apoptosis in A549 cells
The effect of CRA on A549 cell growth was assessed using the
CCK-8 assay. Fig. 1B shows inhibition of A549 cell viability by sev-
eral concentrations (1040 lM) of CRA and over time (648 h).
CRA induced both a concentration- and time-dependent decrease
(A)
(C)
(D)
(B)
Fig. 2. CRA induces apoptosis in A549 cells. (A) Cells were exposed to several concentrations of CRA for 24 h or (B) exposed to CRA (28 lM) over time. Apoptosis was
measured using PI staining and ow cytometry. (C) Flow cytometry analysis of annexin V-FITC staining and PI accumulation after exposure of A549 cells to several
concentrations of CRA. (D) The number of early and late apoptotic cells (annexin V
+
/PI

and annexin V
+
/PI
+
, respectively) was calculated using CellQuest Pro software. The
data are expressed as the means SD of triplicate samples.

P < 0.05,

P < 0.01 and

P < 0.001 vs. untreated CRA.
10 K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817
in formazan accumulation in the cells. The IC
50
was 27.86 lM at
24 h. To investigate further the effect of CRA on the morphology
of apoptotic cells, Hoechst 33342 staining was conducted. Very
few apoptotic cells were observed in the control culture, while
the percentage of apoptotic cells in the presence of CRA increased
in a CRA concentration-dependent manner (Fig. 1C). The cytotoxic-
ity caused by CRA may be due in part to anti-proliferative and
proapoptotic effects. The effect of CRA on cell cycle progression
was analyzed by ow cytometry. Exposure of cells to CRA in-
creased the number of cells in the sub-G1 phase, possibly due to
DNA fragmentation, resulting in increased CRA-induced apoptotic
cell death (Fig. 2A and B). Since a concentration- and time-depen-
dent sub-G1 phase appeared in the cell cycle analysis, CRA-induced
apoptosis was further conrmed using annexin V-FITC and PI
staining to differentiate early apoptotic cells (annexin V
+
/PI

) from
late apoptotic cells (annexin V
+
/PI
+
). Fig. 2C shows a dot-plot dis-
play produced from annexin V-FITC/PI with ow cytometry of
A549 cells. Representative data from three independent experi-
ments are shown. The lower left (LL) quadrants of the cytograms
show viable cells, excluding PI and negative for annexin V-FITC
binding. The lower right (LR) quadrant represents the early apopto-
tic cells, which were annexin V-FITC positive and PI negative. The
upper right (UR) quadrant represents the late apoptotic cells,
which were positive for annexin V-FITC binding and PI uptake.
When cells were exposed to 40 lM CRA for 24 h, 63.5% of the cell
population emitted a strong FITC signal with a weak and/or strong
PI signal. Quantitative analysis showed that CRA markedly de-
creased the live cell population whereas apoptotic cell populations
were increased by CRA in a concentration-dependent manner
(Fig. 2D). A bar diagram of cumulative data from three independent
experiments is shown. These results indicate that the cell death in-
duced by CRA is mainly due to apoptosis.
3.2. CRA alters the expression of apoptosis-related proteins in A549
cells
Many proteins play important roles in apoptosis. Bcl-xl, survi-
vin, and bid are anti-apoptotic proteins, the degradation of which
is required for the induction of apoptosis. The expression level of
these proteins, which interact with mitochondria, was studied.
To conrm that the observed cell death is mediated by these
anti-apoptotic proteins, the protein level of bcl-xl, survivin, and
cleaved bid was assessed in A549 cells exposed to CRA. As shown
in Fig. 3, the expression of bcl-xl and survivin was reduced after
treatment and the cleavage of bid was increased. These results con-
rm that CRA induces apoptosis by regulating anti-apoptotic pro-
tein expression. Since proteins from the IAP family bind to
caspases, leading to caspase inactivation in eukaryotic cells, the
(A) (B)
Fig. 3. CRA alters the expression of bcl-xl and IAP family members in A549 cells. (A) Cells were exposed to several concentrations of CRA for 24 h or (B) exposed to CRA
(28 lM) over time. Cells were subjected to Western blot analysis using the antibodies indicated.
K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817 11
involvement of the IAP family in CRA-induced apoptosis was
examined. Results indicate that the levels of IAP family members,
such as cellular inhibitor of apoptosis protein (cIAP)-1 and cIAP-
2, remained virtually unchanged in response to CRA, whereas
X-linked inhibitor of apoptosis protein (XIAP) was inhibited by
exposure to CRA (Fig. 3A and B).
3.3. CRA induces caspase-3/-7, -8, and -9 activity in A549 cells
The activation of caspases, which are key mediators of apopto-
sis, was analyzed upon exposure of A549 cells to CRA. Caspase-3/
-7, -8, and -9 activity and expression was measured in cells
exposed to several concentrations of CRA (1040 lM) for 24 h or
incubated with 28 lM CRA for 648 h. The levels of caspase activa-
tion in A549 cells exposed to CRA were compared to those of con-
trol untreated cells arbitrarily set to 1.0. Results showed that CRA
markedly increased caspase-3/-7 and -9 activity in a concentra-
tion-dependent manner, while the activity of caspase-8 increased
only slightly (Fig. 4A). Results also showed that caspase activity
reached the maximum level at 24 h (Fig. 4B). Furthermore, CRA in-
duced the degradation of poly (ADP-ribose) polymerase (PARP,
116 kDa), a substrate of caspase-3, and PARP cleavage fragments
(89 kDa) increased over time (Fig. 4C). The results in Figs. 3 and
4 suggest that CRA causes apoptosis through both mitochondria-
mediated and caspase-dependent pathways.
3.4. CRA-induced apoptosis is inhibited by a caspase inhibitor in A549
cells
To conrm whether caspase cascade activation is involved in
CRA-mediated apoptosis, A549 cells were pretreated with z-vad-
fmk (100 lM), a broad-spectrum caspase inhibitor, for 1 h, and
then subsequently exposed to 28 lM CRA for 24 h. The activity of
caspase-3/-7, -8, and -9 was increased by CRA and completely
diminished in the presence of z-vad-fmk (Fig. 5A). As shown in
Fig. 5B, apoptosis was observed in about 57.7% of the cells at
24 h following exposure to CRA in the absence of z-vad-fmk, but
50% of the cells in the presence of z-vad-fmk. To understand fur-
ther the signal transduction pathways involved in CRA-induced
apoptosis, western blot analysis was conducted. The CRA-mediated
events, including the degradation of bcl-xl, XIAP, and survivin, the
increase in cleaved PARP proteins, and the activation of caspase-3
and -9, were apparently blocked in the presence of z-vad-fmk
(Fig. 5C). These results clearly indicate that CRA-induced apoptosis
is associated with caspase activation.
3.5. CRA alters the mitochondrial transmembrane potential (Dwm)
To explore the mechanisms of apoptosis mediated by CRA, we
focused initially on mitochondria-dependent pathways and as-
sessed alterations in mitochondrial membrane potential (Dwm)
(A) (B)
(C)
Fig. 4. CRA activates caspase activity and PARP protein degradation in A549 cells. (A) Concentration response. Cells were incubated in the presence or absence of several
concentrations of CRA for 24 h. (B) Time course. Cells were incubated in the presence or absence of 28 lM CRA for different lengths of time. Upon completion of each exposure
time, caspase activity was assessed using the Caspase-Glo assay. The data are expressed as the means SD of triplicate samples.

P < 0.05,

P < 0.01 and

P < 0.001 vs.
untreated CRA. (C) Cells were subjected to Western blot analysis using anti-PARP and anti-c-PARP antibodies.
12 K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817
using the uorescent probe MitoCapture, a unique cationic dye.
Valinomycin, used here as a positive control, disrupts the Dwm
and thus MitoCapture translocates to the cytoplasm and reverts
to its monomeric form, which is indicated by more diffuse uores-
cence when viewed under a uorescein lter. Similar effects were
observed in cells exposed to various concentrations of CRA; at 24 h,
control cells emitted a bright red uorescence, viewed using a rho-
damine lter, while in the CRA-treated cells, the majority of the
cytoplasm uoresced green when a FITC lter was used (Fig. 6A).
Then the Dwm was analyzed in CRA-treated A549 cells using an
Envision 2103 Multilabel Reader. Exposure to CRA caused the loss
of Dwm in a concentration-dependent manner (Fig. 6B), as shown
by the shift in the cell population from low to high green
uorescence.
3.6. CRA induces cytochrome c release from mitochondria
Mitochondria play an essential role in the apoptosis triggered
by chemical agents. The mitochondrial response includes the re-
lease of cytochrome c into the cytosol. In the cytosol, cytochrome
c binds to Apaf-1, allowing the recruitment of caspase-9 and the
formation of an apoptosome complex, resulting in caspase-3 acti-
vation and execution of cell death [19]. To analyze the involvement
of the mitochondrial release of cytochrome c in A549 cells, proteins
fromboth cytosolic and mitochondrial fractions were prepared and
analyzed by western blot. COX IV was used as internal control for
the mitochondrial fractions and b-actin for the cytosolic fractions
(Fig. 6C). Exposure of A549 cells to CRA caused a gradual decrease
in mitochondrial cytochrome c, with concomitant increase in the
cytosolic fraction. These results show that CRA induces the release
of cytochrome c to the cytosol, supporting the uorescence studies
and indicating that this agent alters mitochondrial membrane per-
meability. These data suggest that CRA induces apoptosis via alter-
ations in the mitochondrial membrane permeability of A549 cells.
3.7. CRA induces apoptosis via the generation of ROS in A549 cells
Mitochondria are the major sites of ROS production, and accu-
mulation of ROS may lead to the initiation of apoptosis. To investi-
gate further whether CRA-induced ROS are required for the
induction of apoptosis, A549 cells were exposed to CRA in the pres-
ence or absence of N-acetylcysteine (NAC). First, the generation of
ROS in A549 cells exposed to CRA was conrmed. Cells were loaded
with H
2
DCFDA and stimulated with H
2
O
2
(positive control).
(A)
(C)
(B)
Fig. 5. Caspase inhibition prevents CRA-induced apoptosis in A549 cells. Cells were incubated in the presence or absence of z-vad-fmk for 1 h before being exposed to CRA
(28 lM). (A) After 24 h of incubation with CRA, caspase activity was measured. (B) The percentage of apoptotic cells was detected by ow cytometry using annexin V/PI
staining. The data are expressed as the means SD of triplicate samples.

P < 0.05 and

P < 0.01 vs. CRA + z-vad-fmk. (C) Cells were subjected to Western blot analysis using
the antibodies indicated.
K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817 13
Exposure of the cells to CRA caused a substantial and concentra-
tion-dependent increase in ROS (Fig. 7A). Flow cytometry analysis
showed that the ROS scavenger NAC decreased CRA-induced ROS
production (Fig. 7A), indicating that CRA-induced apoptosis pro-
ceeds via the activation of ROS. Furthermore, NAC blocked caspase
activity and apoptotic cell death (Fig. 7B and C), providing further
evidence that ROS contribute to the apoptosis induced by CRA. To
evaluate further the role of ROS in the apoptosis induced by CRA,
the effect of NAC on the expression of bcl-xl, survivin, XIAP, bid,
caspase-3, -9, and PARP proteins was measured. As shown in
Fig. 7D, the CRA-mediated events, including the degradation of
bcl-xl, XIAP, survivin, and t-bid, were apparently blocked in the
presence of NAC. In addition, the cleavage of procaspase-3 and -9
was noted in the presence of CRA in A549 cells. By contrast, pre-
treatment with NAC completely protected the procaspases from
cleavage in the presence of CRA. The cleavage of PARP was also
protected by NAC. These results demonstrate that ROS production
mediates CRA-induced apoptotic cell death in A549 cells.
4. Discussion
Several anti-cancer agents were originally developed from nat-
ural sources (Cragg and Newman, 2005). Epidemiological investi-
gation and experimental studies indicate that bioactive natural
compounds play an important role in the treatment of many can-
cers (Xian et al., 2007; Kim et al., 2008). In the present study, we
demonstrate that CRA, a naturally-occurring triterpenoid from Chi-
nese medicinal herbs, is a potent inhibitor of human NSCLC
proliferation.
Apoptosis is essential for the development and maintenance of
tissue homeostasis and for the elimination of unwanted or dam-
aged cells form multicellular organisms (Wyllie, 1993; Thompson,
1995). Several genes have been identied as either inducers or
repressors of apoptosis. Among these, caspases, a growing family
of cysteine proteases that cleave specic substrates at aspartic acid
residues, have been identied as major components of apoptosis
(Thornberry and Lazebnik, 1998; Budihardjo et al., 1999). Caspase
activation is regulated by various proteins, including the inhibitory
proteins of the IAP (inhibitors of apoptosis) family and the bcl-2
family. Our data reveal that the caspase-dependent pathway is in-
volved in CRA-induced apoptosis; indeed, caspase cascade activa-
tion was apparent in cells exposed to CRA. The cleavage of PARP,
which is mediated by the activation of executive caspases, was also
observed in the cells. Furthermore, a pan-caspase inhibitor pro-
tected cells from CRA-mediated toxicity (Figs. 4 and 5). These data
conrmed that CRA induces growth inhibition in A549 cells via the
induction of apoptosis. In addition, the expression of bcl-xl, XIAP,
(A)
(B) (C)
Fig. 6. CRA reduces mitochondrial membrane potential in A549 cells. (A) Upper panels show images acquired with a rhodamine lter, middle panels with a FITC lter, and
lower panels show DIC images. (B) The graph shows quantication of uorescence measured using an EnVision 2103 Multilabel Reader. The data are expressed as the
means SD of triplicate samples. (C) Cytochrome c release from mitochondria to cytosol in A549 cells exposed to CRA. COX IV and b-actin were used as internal controls for
the mitochondrial fractions and the cytosolic fraction, respectively.
14 K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817
and survivin was reduced, and the cleavage of bid increased after
treatment, conrming that CRA induces apoptosis by regulating
anti-apoptotic protein expression. Production of c-bid could induce
mitochondrial stress, and also participate in the release of cyto-
chrome c into the cytosol. These results suggest that mitochondrial
stress mediated by caspase-8, bid and bcl-xl, and subsequent re-
lease of cytochrome c followed by caspase cascade activation, are
the executive mechanisms involved in CRA-mediated apoptosis.
ROS generation has been recognized as a mediator of apoptotic
signaling cascades (Cai et al., 1998; Curtin et al., 2002). Consistent
with this notion, we found that CRA caused cytochrome c release
from mitochondria, activation of caspase-3 and -9, and cleavage
of PARP. Importantly, the activation of the mitochondria-mediated
intrinsic death signaling pathway was completely blocked by an
antioxidants (NAC). These results suggest that CRA induces the
production of ROS, which causes the collapse of mitochondrial
membrane potential and triggers the activation of mitochondria-
mediated death signaling. It is likely that ROS are the critical medi-
ators of CRA-induced cell toxicity.
Since mitochondria play an important role in oxidative stress-
induced apoptosis, we focused our attention on the intrinsic
death pathway. Collapse of mitochondrial membrane potential
is a sensitive indicator of mitochondrial damage induced by sev-
eral toxins. A concentration assessment of mitochondrial mem-
brane potential (MMP) was performed using the specic and
sensitive uorescent dye MitoCapture. Our results showed that
CRA induced loss of MMP in a concentration-dependent manner
(Fig. 6). This result reveals that a CRA-induced ROS surge pre-
cedes the loss of MMP.
Many studies have examined the cellular mechanisms involved
in CRA-mediated toxicity (Xu et al., 2009; Lee et al., 2010a,b; Cai
et al., 2011; Fujiwara et al., 2011). Although ROS is thought to be
related to CRA-mediated cell death, the precise mechanisms by
which CRA induces apoptosis in A549 cells have not been eluci-
dated. Our data provide evidence that ROS play an important role
in CRA-induced apoptosis in A549 cells. Apoptosis induced by
CRA is mediated through the mitochondrial- and caspase-depen-
dent pathway, which are negatively regulated by the anti-apopto-
tic molecules. By showing that ROS is implicated in CRA-induced
cell death, we have revealed a novel mechanism of apoptosis
induction by CRA, which could be exploited for the treatment of
cancer and related apoptosis disorders. Further studies are needed
to determine the efcacy of CRA in vivo and to demonstrate its
safety and efcacy in clinical trials.
(A)
(B) (C)
Fig. 7. CRA induces cell death mainly through generation of ROS in A549 cells. (A) Cells were incubated with various concentrations of CRA for 24 h or incubated in the
presence or absence of H
2
O
2
and NAC for 6 and 1 h before being exposed to CRA. The cells were then exposed to H
2
DCFDA (10 lM) for an additional 20 min prior to ow
cytometry analysis. ROS levels are expressed as fold increase relative to control cells cultured in complete medium. The data are expressed as the means SD of triplicate
samples.

P < 0.05 and

P < 0.01 vs. untreated CRA. (B) After 24 h of incubation with CRA, caspase activity was measured. (C) The percentage of apoptotic cells was detected by
ow cytometry using annexin V/PI staining. The data are expressed as the means SD of triplicate samples.

P < 0.05 and

P < 0.01 vs. CRA + NAC. (D) Cells were subjected to
Western blot analysis using the antibodies indicated.
K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817 15
Conict of Interest Statement
The authors have no conicts of interest to declare.
Acknowledgements
This work was supported by the project Construction of the Ba-
sis for Practical Application of Herbal Resources funded by the
Ministry of Education, Science and Technology (MEST) of Korea
to the Korea Institute of Oriental Medicine (KIOM). We thank the
KIOM Classication and helpful discussions.
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