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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 significantly 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 significant 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 classified

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-hydroxyursolicacid, 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-inflammatory (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-cancermechanisms 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 identified 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 inFig. 1A. Z-vad-fmk, N-acetyl-L-cysteine (NAC), valinomy-

cin, and H2O2 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-

ified 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

Contents lists available at SciVerse ScienceDirect

Food and Chemical Toxicology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x

http://dx.doi.org/10.1016/j.fct.2013.02.002mailto:hkkim@kiom.re.krhttp://dx.doi.org/10.1016/j.fct.2013.02.002http://www.sciencedirect.com/science/journal/02786915http://www.elsevier.com/locate/foodchemtoxhttp://www.elsevier.com/locate/foodchemtoxhttp://www.sciencedirect.com/science/journal/02786915http://dx.doi.org/10.1016/j.fct.2013.02.002mailto:hkkim@kiom.re.krhttp://dx.doi.org/10.1016/j.fct.2013.02.002

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100 lg/ml streptomycin (Gibco BRL). All cultured cells were incubated at 37 C in ahumidified atmosphere containing 5% carbon dioxide. Cells were fed with fresh cul-

ture medium two to three times per week and subcultured when 80% confluent.

2.3. Cell viability assay

Cells were seeded in 96-well culture plates at a density of 2 104 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 (10ll) was added to each welland incubated for 1 h at 37C. 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, 3 105 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 (1040lM) for 24 h. Next, the cells were collected and fixedwith 3.7% formaldehyde in phosphate buffered saline (PBS) for 15 min and stained

with Hoechst 33342 (10lg/ml) at room temperature for 10 min. Finally, after thecells were washed with PBS, morphological changes, including a reduction in vol-

ume and nuclear chromatin condensation, were observed by fluorescence micros-

copy (Olympus Optical, Tokyo, Japan) and photographed at a 200 magnification.

2.5. Flow cytometric analysis for measurement of sub-G1 phase

Cells were seeded in six-well plates at 3 105 cells/well and allowed to attach

overnight. After exposure to CRA, cells were collected, washed twice with ice-coldPBS (pH 7.4), fixed 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 flow 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 flow 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 3 105 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, pH8.0, 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 specific 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. Briefly, cells were seeded at a den-

sity of 1 104 per well in triplicate wells onto 96-well plates and incubatedfor 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 (C30H48O4, FW: 472.70). (B) Concentration response and time course.

Cells were incubated with CRA (1040lM) over time (648 h). Cell viability was assessed by CCK-8 assay. The data are expressed as the means SD of triplicate samples.P

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(1040lM) for 24 h or incubated with 28 lM of CRA for 648 h. After exposure toCRA, culture supernatant (100 ll) was transferred into a white-walled 96-wellplate. 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 MitoCaptureapoptosis 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 (asa 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

fluorescence. 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,

fluorescing green. After labeling, cells were observed using a Fluoview FV10i confo-

cal laser-scanning microscope (Olympus Corporation, Tokyo, Japan) and fluores-

cence was measured using an EnVision 2103 Multilabel Reader (PerkinElmer,

Wellesley, MA, USA).

2.10. Preparation of mitochondrial and c ytosolic fractions

To detect the release of cytochrome cfrom mitochondria into the cytosol, a

Mitochondria/Cytosol fractionation kit (Abcam, Cambridge, MA, USA) was used.

Cells (1 107) were cultured in 75T-flasks 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 80C (cytosolic fraction). The pellet was resuspended in

mitochondrial extraction buffer and stored at 80C (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 H2DCFDA probe (Molecular Probes, Europe BV, Leiden, The

Netherlands) during the last 30 min of treatment. Then, cells were harvested bytrypsinization and washed twice with PBS before being analyzed by flow cytometry.

Flow cytometric analysis was performed on at least 1 104 cells using a FACS Cal-

ibur flow 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

ofP < 0.05 was considered to be statistically significant.

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 wasmeasured using PI staining and flow 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. Thedata are expressed as the means SD of triplicate samples. P< 0.05, P< 0.01 and P

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in formazan accumulation in the cells. The IC50 was 27.86 lM at24 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 progressionwas analyzed by flow 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 t he cell cycle analysis, CRA-induced

apoptosis was further confirmed 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 flow 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 cellpopulation 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 confirm 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

inFig. 3, the expression of bcl-xl and survivin was reduced after

treatment and the cleavage of bid was increased. These results con-

firm 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

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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 orincubated 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 confirm 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, andthen subsequently exposed to 28 lM CRA for 24 h. The activity of

caspase-3/-7, -8, and -9 was increased by CRA and completelydiminished 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

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using the fluorescent probe MitoCapture, a unique cationic dye.

Valinomycin, used here as a positive control, disrupts the Dwmand thus MitoCapture translocates to the cytoplasm and reverts

to its monomeric form, which is indicated by more diffuse fluores-

cence when viewed under a fluorescein filter. Similar effects were

observed in cells exposed to various concentrations of CRA; at 24 h,

control cells emitted a bright red fluorescence, viewed using a rho-damine filter, while in the CRA-treated cells, the majority of the

cytoplasm fluoresced green when a FITC filter was used (Fig. 6A).

Then the Dwm was analyzed in CRA-treated A549 cells using anEnvision 2103 Multilabel Reader. Exposure to CRA caused the loss

ofDwmin a concentration-dependent manner (Fig. 6B), as shownby the shift in the cell population from low to high green

fluorescence.

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 theformation 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

from both 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 thecytosolic fraction. These results show that CRA induces the release

of cytochrome c to the cytosol, supporting the fluorescence 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 confirmed. Cells were loadedwith H2DCFDA and stimulated with H2O2 (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 flow cytometry using annexin V/PIstaining. The data are expressed as the means SD of triplicate samples. P

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and survivin was reduced, and the cleavage of bid increased after

treatment, confirming 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 specific and

sensitive fluorescent 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 involvedin 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 efficacy of CRA in vivo and to demonstrate its

safety and efficacy 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 H2O2 and NAC for 6 and 1 h before being exposed to CRA. The cells were then exposed to H2DCFDA (10 lM) for an additional 20 min prior to flowcytometry 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

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Conflict of Interest Statement

The authors have no conflicts 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 Classification and helpful discussions.

References

Ahn, K.S., Hahm, M.S., Park, E.J., Lee, H.K., Kim, I.H., 1998. Corosolic acid isolatedfrom the fruit of Crataegus pinnatifida var. psilosa is a protein kinase C inhibitoras well as a cytotoxic agent. Planta. Med. 64, 468470.

Banno, N., Akihisa, T., Tokuda, H., Yasukawa, K., Higashihara, H., Ukiya, M.,Watanabe, K., Kimura, Y., Hasegawa, J., Nishino, H., 2004. Triterpene acidsfrom the leaves of Perilla frutescens and their anti-inflammatory andantitumor-promoting effects. Biosci. Biotechnol. Biochem. 68, 8590.

Budihardjo, I.,Oliver, H., Lutter, M., Luo, X., Wang, X., 1999. Biochemical pathways ofcaspase activation during apoptosis. Ann. Rev. Cell Dev. Biol. 15, 269290.

Cai, J., Yang, J., Jones, D.P., 1998. Mitochondrial control of apoptosis: the role ofcytochrome c. Biochim. Biophys. Acta 1366, 139149.

Cai, X., Zhang, H., Tong, D., Tan, Z., Han, D., Ji, F., Hu, W., 2011. Corosolic acid triggersmitochondria andcaspase-dependent apoptotic cell death in osteosarcoma MG-63 cells. Phytother. Res. February 21. doi: 10.1002/ptr.3422 (Epub ahead ofprint).

Cotter, T.G., 2009. Apoptosis and cancer: the genesis of a research field. Nat. Rev.Cancer 9, 501507.

Cragg, G.M., Newman, D.J., 2005. Plants as a source of anti-cancer agents. J.Ethnopharmacol. 100, 7279.

Curtin, J.F., Donovan, M., Cotter, T.G., 2002. Regulation and measurement ofoxidative stress in apoptosis. J. Immunol. Methods 265, 4972.

Fernandes, J., Weinlich, R., Castilho, R.O., Kaplan, M.A., Amarante-Mendes, G.P.,Gattass, C.R., 2005. Pomolic acid triggers mitochondria-dependent apoptoticcell death in leukemia cell line. Cancer Lett. 219, 4955.

Fujiwara, Y., Komohara, Y., Ikeda, T., Takeya, M., 2011. Corosolic acid inhibitsglioblastoma cell proliferation by suppressing the activation of signaltransducer and activator of transcription-3 and nuclear factor-kappa B intumor cells and tumor-associated macrophages. Cancer Sci. 102, 206211.

Fukushima, M., Matsuyama, F., Ueda, N., Egawa, K., Takemoto, J., Kajimoto, Y.,Yonaha, N., Miura, T., Kaneko, T., Nishi, Y., Mitsui, R., Fujita, Y., Yamada, Y., Seino,Y., 2006. Effect of corosolic acid on postchallenge plasma glucose levels.Diabetes Res. Clin. Pract. 73, 174177.

Harmand, P.O., Duval, R., Delage, C., Simon, A., 2005. Ursolic acid induces apoptosisthrough mitochondrial intrinsic pathway and caspase-3 activation in M4Beumelanoma cells. Int. J. Cancer 114, 111.

Hsu, H.F., Houng, J.Y., Kuo, C.F., Tsao, N., Wu, Y.C., 2008. Glossogin, a novel

phenylpropanoid from Glossogyne tenuifolia, induced apoptosis in A549 lungcancer cells. Food Chem. Toxicol. 46, 37853791.Jemal, A., Siegel, R., Xu, J., Ward, E., 2010. Cancer statistics. CA. Cancer J. Clin. 60,

277300.Kim, J.B., Lee, K.M., Ko, E., Han, W., Lee, J.E., Shin, I., Bae, J.Y., Kim, S., Noh, D.Y., 2008.

Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231. Planta. Med. 74, 3942.

Lee, M.S., Cha, E.Y., Thuong, P.T., Kim, J.Y., Ahn, M.S., Sul, J.Y., 2010a. Down-regulation of human epidermal growth factor receptor 2/neu oncogene bycorosolic acid induces cell cycle arrest and apoptosis in NCI-N87 human gastriccancer cells. Biol. Pharm. Bull. 33, 931937.

Lee, M.S., Lee, C.M., Cha, E.Y., Thuong, P.T., Bae, K., Song, I.S., Noh, S.M., Sul, J.Y.,2010b. Activation of AMP-activated protein kinase on human gastric cancercells by apoptosis induced by corosolic acid isolated from Weigela subsessilis.Phytother. Res. 24, 18571861.

Liby, K.T., Yore, M.M., Sporn, M.B., 2007. Triterpenoids and rexinoids asmultifunctional agents for the prevention and treatment of cancer. Nat. Rev.Cancer 7, 357369.

Martin, R., Carvalho, J., Ibeas, E., Hernandez, M., Ruiz-Gutierrez, V., Nieto, M.L., 2007.

Acidic triterpenes compromise growth and survival of astrocytoma cell lines byregulating reactive oxygen species accumulation. Cancer Res. 67, 37413751.

(D)

Fig. 7. (continued)

16 K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817

8/11/2019 1-s2.0-S0278691513001105-main

10/10

Miura, T., Ueda, N., Yamada, K., Fukushima, M., Ishida, T., Kaneko, T., Matsuyama, F.,Seino, Y., 2006. Antidiabetic effects of corosolic acid in KK-Ay diabetic mice.Biol. Pharm. Bull. 29, 585587.

Park, S.H., Oh, S.R., Ahn, K.S., Kim, J.G., Lee, H.K., 2002. Structure determination of anew lupane-type triterpene, tiarellic acid, isolated fromTiarella polyphylla. Arch.Pharm. Res. 25, 5760.

Reyes-Zurita, F.J., Rufino-Palomares, E.E., Lupianez, J.A., Cascante, M., 2009. Maslinicacid a natural triterpene from Olea europaea L induces apoptosis in HT29human colon-cancer cells via the mitochondrial apoptotic pathway. Cancer Lett.273, 4454.

Su, Y.T., Chang, H.L., Shyue, S.K., Hsu, S.L., 2005. Emodin induces apoptosis in humanlung adenocarcinoma cells through a reactive oxygen species-dependentmitochondrial signaling pathway. Biochem. Pharmacol. 70, 229241.

Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of disease.Science 267, 14561462.

Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 28, 13121316.

Wyllie, A.H., 1993. Apoptosis (the 1992 frank rose memorial lecture). Br. J. Cancer67, 205208.

Xian, M., Ito, K., Nakazato, T., Shimizu, T., Chen, C.K., Yamato, K., Murakami, A.,Ohigashi, H., Ikeda, Y., Kizaki, M., 2007. Zerumbone, a bioactive sesquiterpene,induces G2/M cell cycle arrest and apoptosis in leukemia cells via a Fas- andmitochondria-mediated pathway. Cancer Sci. 98, 118126.

Xu, Y., Ge, R., Du, J., Xin, H., Yi, T., Sheng, J., Wang, Y., Ling, C., 2009. Corosolic acidinduces apoptosis through mitochondrial pathway and caspase activation inhuman cervix adenocarcinoma HeLa cells. Cancer Lett. 284, 229237.

Yamaguchi, Y., Yamada, K., Yoshikawa, N., Nakamura, K., Haginaka, J., Kunitomo, M.,2006. Corosolic acid prevents oxidative stress, inflammation and hypertensionin SHR/NDmcr-cp rats, a model of metabolic syndrome. Life Sci. 79, 24742479.

Yoshida, M., Fuchigami, M., Nagao, T., Okabe, H., Matsunaga, K., Takata, J., Karube, Y.,Tsuchihashi, R., Kinjo, J., Mihashi, K., Fujioka, T., 2005. Antiproliferativeconstituents from Umbelliferae plants VII. Active triterpenes and rosmarinicacid from Centella asiatica. Biol. Pharm. Bull. 28, 173175.

Zong, W., Zhao, G., 2007. Corosolic acid isolation from the leaves ofEriobotrtajaponicashowing the effects on carbohydrate metabolism and differentiation of3T3-L1 adipocytes. Asia Pac. J. Clin. Nutr. 1, 346352.

K.J. Nho et al. / Food and Chemical Toxicology 56 (2013) 817 17