ag4, a compound isolated from radix ardisiae gigantifoliae...

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AG4, a compound isolated from Radix Ardisiae Gigantifoliae,induces apoptosis in human nasopharyngeal cancer CNE cellsthrough intrinsic and extrinsic apoptosis pathwaysXian-Zhe Donga,*, Ting-Ting Xieb,*, Xiao-Jiang Zhoua,*, Li-Hua Mua,Xiao-Li Zhenga,c, Dai-Hong Guoa,b, Ping Liua and Xiao-Yue Gea,d

3β-O-{α-L-Pyran rhamnose-(1→3)-[β-D-xylopyranose-(1→2)]-β-D-glucopyranose-(1→4)-[β-D-lucopyranose-(1→2)]-α-L-pyran arabinose}-cyclamiretin A (AG4) is asaponin component obtained from the Giantleaf ArdisiaRhizome (Rhizoma Ardisiae Gigantifoliae). The presentstudy aimed to investigate the antitumor potential of AG4and its possible mechanisms in human nasopharyngealcarcinoma cells (CNE). We exposed tumor cells to AG4 toinvestigate which cell line was the most sensitive to AG4.Cell viability was assessed using the MTT reduction assay,and the effects of AG4 on apoptosis, reactive oxygenspecies (ROS) content, mitochondrial membranepotential (MMP), and cell cycle were detected using a flowcytometer; the glutathione, superoxide dismutase andmalondialdehyde activities were measured usingcolorimetric methods. The relative expressions of Bax, Bad,Bid, Bcl-2, and Fas mRNA were calculated using the 2�DDCt

comparative method by real-time PCR studies and proteinwas detected by western blotting. AG4 markedly inhibitedthe growth of CNE cells by decreasing cell proliferation,inducing apoptosis, and blocking the cell cycle in the Sphase. The release of caspase-3, caspase-8, and caspase-9was stimulated by AG4 in CNE, and the decreasedproliferation induced by AG4 was blocked by the inhibitor ofpan caspase (Z-VAD-FMK). Moreover, the MMP wasdecreased in AG4-treated cells, and AG4-induced cellapoptosis was accompanied by a rapid and lasting increasein ROS, which was abolished by N-acetyl-L-cysteine (NAC);glutathione, superoxide dismutase, and malondialdehyde

were regulated by AG4. AG4 inhibited Bcl-2 mRNA andprotein expression and stimulated Bax, Bad, Bid, Fas mRNA,and protein expression in CNE cultures, suggesting aneffect at the transcriptional and protein level. In addition,both the FasL inhibitor (AF-016) and the Bcl-2 familyinhibitor (GX15-070) could prevent the cell apoptosisinduced by AG4. The findings suggested that AG4-inducedapoptosis in CNE cells involved a death receptor pathwayand a Bcl-2 family-mediated mitochondrial signalingpathway by decreasing the MMPs in an ROS-dependentmanner and regulating genes and proteins relative toapoptosis; also, regulation of cell cycles may also play a rolein the antitumor mechanism of AG4. Anti-Cancer Drugs26:331–342 Copyright © 2015 Wolters Kluwer Health, Inc.All rights reserved.

Anti-Cancer Drugs 2015, 26:331–342

Keywords: apoptosis, caspases, CNE cells, Radix Ardisiae Gigantifoliae,redox system, signal pathway

Departments of aClinical Pharmacology, bPharmaceutical Care, Chinese PLAGeneral Hospital, Beijing, cCollege of Pharmacy, Tianjin University of TraditionalChinese Medicine, Tianjin and dCollege of Pharmacy, Bengbu Medical College,Bengbu, China

Correspondence to Ping Liu, MD, #28 Fuxing Road, Haidian District, Beijing100853, ChinaTel: + 86 010 66936676; fax: + 86 010 66936678; e-mail: [email protected]

*Xian-Zhe Dong, Ting-Ting Xie, and Xiao-Jiang Zhou contributed equally to thewriting of this article.

Received 19 November 2013 Revised form accepted 9 November 2014

IntroductionRhizoma Ardisiae Gigantifoliae is a traditional herbal

remedy that is produced in the eastern and southern

provinces and the long-river basin of China, and is used to

treat rheumatism, activating blood circulation to dissipate

blood stasis and inflammation. 3β-O-{α-L-Pyran rhamnose-

(1→3)-[β-D-xylopyranose-(1→2)]-β-D-glucopyranose-(1→4)-

[β-D-lucopyranose-(1→2)]-α-L-pyran arabinose}-cyclamir-

etin A (AG4) is a saponin component obtained from the

Giantleaf Ardisia rhizome (Ardisia densipidotula genera),

with the chemical constitution as in Fig. 1a, and has

beneficial effects on pain of rheumatism, wound, blood

stasis of postpartum, carbuncle-abscess, and ulcer dis-

orders. Previous reports have shown that AG4 also has

anticancer properties. Extensive work over the past

decade has shown that AG4 can induce obvious

cytotoxicity in many tumor cell lines, such as BGC

(human gastric cancer), HeLa (human cervical), HepG2

(human hepatocellular carcinoma), and EJ (human blad-

der cancer), and the method of isolation was as described

previously [1,2].

Ardisia pusilla A. (DC) is a whole grass plant of

Myrsinaceae, with a detoxifying and anti-inflammatory

analgesic effect, and is effective for rheumatism and

various inflammatory disorders, and for the treatment of

bruise and snake bites. In recent years, Ard ipusilloside-I

(ADS-I) and-II isolated from A. pusilla have been repor-

ted. ADS-I is a saponin that has a structure similar to that

of AG4; it can inhibit tumor growth in mice transplanted

models, promote spleen T lymphocyte proliferation in

S180, H22, EAC, and L1210 tumor-bearing mouse,

Preclinical report 331

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enhance macrophage phagocytic capability, and improve

immune function in tumor-bearing mice. It can sig-

nificantly improve immune function in CTX suppression

mice, and inhibit metastasis of Lewis lung carcinoma and

hepatocellular carcinoma in nude mice. On the basis of

the antitumor effects of saponin compounds, the aim of

the present study was to clarify the related mechanisms

of AG4 in human nasopharyngeal carcinoma CNE cells

[3–5].

Materials and methodsChemicals and drugsAG4 was isolated from Rhizoma Ardisiae Gigantifoliae,identified by a combination of spectra methods (ultraviolet,

infrared, mass spectrometry, and nuclear magnetic reso-

nance) and purified by high-performance liquid chroma-

tography (purity> 95%) (Fig. 1b). 3-(4,5-Dimethylthiazol-

2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Hoechst

33 258, propidium iodide (PI), fura-2 acetoxymethyl ester

(Fura-2AM), rhodamine 123 (Rh123), inhibitors of caspase-

3 (Ac-DEVD-CHO), caspase-8 (Z-IETD-FMK), caspase-9

(Z-LEHD-EMK·TFA), and pan caspase inhibitor Z-VAD-

FMK were purchased from Sigma Chemical Co. (St Louis,

Missouri, USA). Dulbecco’s modified Eagle’s medium

(DMEM), glucose-free DMEM, and fetal bovine serum

were obtained from Gibco (Carlsbad, California, USA).

The Annexin V–fluorescein isothioncyanate (FITC)

apoptosis detection kit was obtained from Beckmann

Coulter Inc. (Indianapolis, Indiana, USA). Caspase-3,

caspase-8, and caspase-9 activity kits were obtained from

Promega (Madison, Wisconsin, USA). Malondialdehyde

(MDA), superoxide dismutase (SOD), and glutathione

(GSH) test kits were purchased from the Nanjing

Jiancheng Bioengineering Institute (Nanjing, China).

Fig. 1

OH

CH3

O

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OHOH

O

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Time (min)

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Chemical structure of AG4 and its purity by high-performance liquid chromatography.

332 Anti-Cancer Drugs 2015, Vol 26 No 3


All other chemicals were of analytic grade and commer-

cially available. The antibodies were purchased from

(Abcam, California, USA). Obatoclax mesylate (GX15-070)

was purchased from Selleck Chemicals LLC (Houston,

Texas, USA), and dissolved in DMSO and stored at − 80°C, as recommended by the supplier; FasL inhibitor

(AF-016) was purchased from Kamiya Biomedical

Company (Seattle, Washington, USA).

Cell culture and treatmentThe human nasopharyngeal cancer cells CNE were

obtained from the Cancer Institute and Hospital of the

Chinese Academy of Medical Science and maintained in

DMEM supplemented with 10% fetal bovine serum

(heat inactivated at 56° for 30 min), and 100 U/ml of

penicillin and 100 U/ml streptomycin, in a 37°C incubator

under a humidified, 5% CO2 atmosphere. Confluent

CNE cells were seeded into 96-well plates at a density of

1× 104 cells/well. After 24 h, the cells were exposed to

various concentrations of AG4 and incubated for 24 h.

AG4 was dissolved in dimethyl sulfoxide (DMSO) before

they were added in cells. The final concentrations of

DMSO were always less than 0.01%, which was found to

exert no effects on cell viability. The absorbance was

read at 570 nm with DMSO as the blank.

Cell viabilityAfter the treatment of various concentrations of AG4

(2.03, 4.06, 8.13 μmol/l) for 24 h, or in the presence of

substrate peptides Ac-DEVD-CHO (1 μmol/l), Z-IETD-

FMK (20 μmol/l), Z-LEHD-FMK·TFA (20 μmol/l) [6],

Z-VAD-FMK (20 μmol/l), or FasL inhibitor AF-016 (50

μg/ml) and Bcl-2 family inhibitor GX15-070 (500 nmol/l)

[7,8], cell viability was evaluated using the MTT assay.

Briefly, 20 μl of MTT solution (2 mg/ml in PBS) was

added to the culture medium at a final concentration of

0.5 mg/ml and incubated at 37°C for 4 h. Then, the

supernatants were aspirated carefully, 150 μl of DMSO

was added to each well to dissolve the reaction product of

MTT, and the OD was measured spectrophotometrically

at 570 nm, with DMSO as a blank [9]. Viability was

expressed as the percentage of the values in vehicle-

treated (basal) cultures, set to 100%. Another method was

used to evaluate the cell viability: reversed microscopy.

Following the above cell treatment protocol, cells were

stained with Hoechst; the morphology of the cells was

observed and recorded as a photo under an Olympus

Microscope (Tokyo, Japan).

Cells apoptotic assayCell apoptosis was measured using an Annexin-

V–FITC/PI apoptosis detection kit. 5× 106 cells/well

were plated in six-well plates. After treatment with AG4

for 24 h, cells were harvested and washed in PBS, and

then centrifuged at 1000g for 5 min The cell pellet was

resuspended in the Annexin-V–FITC/PI labeling solu-

tion, mixed gently, and incubated for 15 min at room

temperature in the dark. Cells were then analyzed in a

Becton Dickinson flow cytometer (Franklin Lakes, New

Jersey, USA) equipped with the analysis software, and

each sample collected 10 000 cells [10].

Hoechst 33 258 staining was used to further analyze cell

apoptosis. Cells were cultured in 12-well culture plates

and treated with AG4 for 24 h. Afterwards, the cells were

washed with PBS and incubated with 500 μl 4% methanol

for 10 min, followed by staining with Hoechst 33 258 at

room temperature for 10 min, and then observed with

filters for blue fluorescence under fluorescence

microscopy.

Measurement of cell cycleThe cell cycle was measured using a PI according to the

manufacturer’s instructions. Briefly, cells were plated in

12-well plates at a density of 1× 106 cells/well. After

treatment with AG4 for 24 h, cells were harvested and

washed with 500 μl PBS and then fixed in ice-cold 70%

ethanol for 12 h at − 20°C. Then, the cells were collected

and resuspended in 100 μl of RNaseA (1 mg/ml) and

stained with 400 μl PI (50 μg/ml) at 4°C for 30 min in the

dark. Cells were analyzed by FACS Becton Dickinson

flow cytometry immediately.

Intracellular ROS quantificationThe level of intracellular reactive oxygen species (ROS)

was determined by the change in the fluorescent probe

dichlorofluorescein diacetate (DCFH-DA). Briefly,

6× 104 CNE cells were cultured into a six-well plate and

were treated with indicated concentrations of AG4 for

24 h. Cells were trypsinized and washed with PBS, and

then incubated with 10 mmol/l DCFH-DA for 30 min at

37°C. Subsequently, cells were washed twice with PBS

and analyzed using a flow cytometer. The protective

effect of N-acetyl-L-cysteine (NAC) was determined by

adding NAC with AG4 to the CNE cells before the ROS

measurement.

Measurement of SOD, MDA, and GSH activityCNE cells were plated in 96-well plates with

7× 104 cells/ml allowed to attach for 24 h. AG4 was added

at different final concentrations; after 48 h of incubation,

cells were thawed three times at a temperature of − 80°C.The supernatant was collected to measure the intracel-

lular SOD, MDA, and GSH activity according to the

instructions provided with the kits using an automatic

microplate reader.

Measurement of mitochondrial membrane potentialDiversities of mitochondrial membrane potential (MMP)

were assessed using the fluorescent cationic dye Rh123,

which accumulates in the mitochondria as a direct func-

tion of the membrane potential and is released upon

membrane depolarization. The CNE cells (5× 106 cells/

well) were plated in six-well plates; after treatment with

AG4 induces apoptosis in CNE cells Dong et al. 333


AG4 for 24 h, cells were harvested and washed with PBS,

and then incubated with 2 μmol/l Rh123 for 30 min at 37°C. Then, the Rh123 fluorescence intensity was measured

by flow cytometry, with the excitation filter wave set at

488 nm and the emission filter wave set at 535 nm. The

mean fluorescence intensity in the cells represented the

state of depolarization of MMP.

Measurement of caspase-3, caspase-8, and caspase-9activitiesCaspase-3, caspase-8, and caspase-9 activities were

measured using the Caspase Activity Kit according to the

manufacturer’s instructions. We exposed CNE cells to

various concentrations of AG4 (2.03, 4.06, 8.13 μmol/l) for

0, 6, 12, and 24 h, respectively. Briefly, cells were lysed,

and then the supernatant was mixed with buffer con-

taining the substrate peptides for caspase attached to

p-nitroanilide. The release of p-nitroanilide was qualified

by determining the absorbance using an ELISA reader

(Bio-Tek, Shoreline, Washington, USA) at 405 nm. The

caspase activities were expressed as percentages com-

pared with the controls.

Reverse transcriptase-polymerase chain reactionTotal RNA was extracted from cultured cells with Trizol

(Gibco BRL) according to the manufacturer’s instruc-

tions. cDNA was synthesized from 1 μg of purified RNA

with random primer using the First Strand Synthesis Kit

(ReverTra Ace-a-, Toyobo Co., Japan). Human β-actinand Bax, Bad, Bid, Bcl-2, and Fas primer pairs used were

synthesized by Biomed (Beijing, China) and described as

follows: β-actin: forward 5-GGACATCCGCAAAGA

CCTGTA-3, reverse 5-ACATCTGCTGGAAGGTGG

ACA-3; Bcl-2: forward 5-TCTGTGGATGACTGAGT

ACCTGAAC-3, reverse 5-AGAGACAGCCAGGAGAAA

TCAAAC-3; Bad: forward 5-CGGAGGATGAGTGAC

GAGTTT-3, reverse 5-GGGTGGAGTTTCGGGATG

TG-3; Bax: forward 5-CAGCAAACTGGTGCTCAA

GG-3, reverse 5-GCCACAAAGATGGTCACGGT-3;

Fas: forward 5-CAGAACTTGGAAGGCCTGCATC-3,

reverse 5-TCTGTTCTGCTGTGTCTTGGAC-3; Bid:

forward 5-ATGGACTGTGAGGTCAACAACGG-3,

reverse 5-CACGTAGGTGCGTAGGTTCTGGTT-3.

The PCR reaction was performed using the SYBR Green

real-time PCR Master Mix (Toyobo Co.) to detect the

abundance of PCR products among samples. The cycling

conditions were as follows: 95°C for 2 min, followed by 40

cycles of 95°C for 15 s, 62°C for 20 min, and 72°C for 20 s.

β-Actin was chosen as an internal control to normalize the

genes. Relative expressions of mRNA were determined

using the 2�DDCt comparative method. All quantities

were expressed as n-fold relative to the calibrator (control

value, which was defined as a value of ‘1.0’).

Western blotting analysisFor western blot analysis, total protein was extracted first,

cells were washed twice with cold PBS, and lysed in the

radioimmunoprecipitation assay buffer (20 mmol/l Tris-

HCl, pH 7.5, 150 mmol/l sodium chloride, 1% Triton,

1 mmol/l EGTA, 1 mmol/l EDTA, 2.5 mmol/l sodium

pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l

sodium orthovanadate, 1 μg/ml leupeptin, 1 mmol/l

PMSF) for 20 min on the ice. After centrifugation at

12 000g for 10 min, protein was then quantified using a

bicinchoninic acid Protein Assay Kit (Bioworld

Technology, St Louis, Missouri, USA). Samples were

heated at 95°C for 10 min, and then loaded on a SDS-

polyacrylamide gel for electrophoresis and transferred to

PVDF membranes (Millipore, Billerica, Massachusetts,

USA). Membranes were blocked 2 h in TBST [10 mmol/l

Tris base (pH 8.0), 150 mmol/l NaCl, and 0.1% Tween-

20] containing 5% (w/v) BSA (Sigma-Aldrich, St Louis,

Missouri, USA) at room temperature, and then probed

with each primary antibody at 4°C overnight. After three

washes with TBST, the membranes were incubated with

the appropriate horseradish peroxidase-labeled secondary

antibody for 2 h at room temperature. For quantitative

analysis of the immunoblot bands, the densities of the

bands were measured by scanning densitometry (Bio-

Rad, Hercules, California, USA). All the results of wes-

tern blot include three independent experiments, and

each independent experiment has three replicates.

Statistical analysisAll the data obtained in the experiments were repre-

sented as mean ± SD. Statistical analysis was carried out

using the software package SPSS, v. 10.0 (IBM Corp.,

Armonk, New York, USA). Differences were determined

using one-way analysis of variance. P values less than 0.05

and 0.01 were considered statistically significant.

ResultsEffect of AG4 on cell proliferation in tumor cell linesWe determined the antitumor effect of AG4 on a variety

of tumor cells using the MTT assay to investigate which

cancer cell line was the most sensitive to AG4 under the

same condition. The cell lines Bel-7402 (human liver

cancer), A549 (human lung cancer), HCT-8 and HT-29

(human coloncarcinoma), BGC, LS180 (human color-

ectal), HeLa, HepG2, EJ, SY5Y (human neuroblastoma),

CNE (human nasopharyngeal carcinoma), and MCF-7

(human breast cancer) were included. The results

showed that the CNE cell line was the most sensitive cell

line to the antitumor effect of AG4, the IC50 (50% inhi-

bition concentration) of AG4 was only 3.8 μmol/l, and

AG4 inhibited the proliferation of CNE cells in a time-

dependent manner (Fig. 2).

Effect of AG4 on cell apoptosis and cell cycleThe effects of AG4 could also be confirmed by mor-

phological observation. Distinct chromatin condensation

and nuclear fragmentation were identified in the AG4

treatment groups, while the nuclei of the control cells

stained a weak homogeneous blue. There was a



significant injury in CNE cells after treatment with AG4,

including the disappearance of cellular processes and

falling to pieces; cell nuclei were stained as high light

blue by Hoechst 33 258 (Fig. 3a). The Annexin-

V–FITC/PI staining assay was used to evaluate the

apoptosis in CNE cells. As shown in Fig. 3b, control cells

without the treatment with AG4 showed uniformly dis-

persed chromatin and an intact cell membrane. In the

cells treated with AG4 (4.06 and 8.13 μmol/l), the per-

centage of apoptotic cells was increased (P< 0.01). The

nuclear staining assay by PI was used to evaluate the

effect of AG4 on the cell cycle in CNE cells. As shown in

Fig. 3c, 4.06 and 8.13 μmol/l AG4 could decrease the

G1-phase cells and increase the percentage of S phase

cells from 20 to 34 and 68%, respectively.

ROS are responsible for AG4-induced cell apoptosis inCNE cellsTo investigate whether AG4 could increase ROS levels,

we examined the intracellular ROS production using a

fluorescent probe, DCFH-DA. As shown in Fig. 4a, after

AG4 treatment, the fluorescence intensity increased

markedly compared with the control group (P< 0.01). In

addition, the antioxidants, 5, 10, and 20 mmol/l of NAC,

were added together with AG4, respectively; the fluor-

escence intensity reduced markedly compared with

groups treated with AG4 alone. NAC (20 mmol/l)

induced total blockage, which showed that the NAC can

block the effect of AG4 in enhancing intracellular ROS

levels. Also, we found that AG4 increased the ROS

content in a time-dependent manner within 12 h. The

SOD and GSH activities were suppressed by AG4, and

the MDA content was increased after AG4 treatment in a

dose-dependent manner (Fig. 4b). The level of MMP

was determined using a flow cytometer after staining

with Rh123. As shown in Fig. 4c, the fluorescence

intensity of the cells treated with AG4 was significantly

decreased compared with the control group in a

concentration-dependent manner and inversed by

10 mmol/l NAC.

To further verify whether AG4-induced apoptosis was

correlated with ROS, we used NAC, the inhibitors of

ROS. As shown in Fig. 4d, AG4, either alone or in

combination with NAC, exerted quite different effects in

inducing cell apoptosis in CNE cells. The cell viability

rate decreased significantly when the CNE cells were

treated with 4.06 and 8.13 μmol/l AG4, and blocked by

10 mmol/l NAC.

AG4 increased the activities of caspase-3, caspase-8,and caspase-9The process of apoptosis involves a cascade of proteolytic

activity, much of it carried out by caspases; thus, we

investigated whether AG4 can activate the caspases. As

shown in Fig. 5a and b, after treatment with AG4 for

24 h, the activities of caspase-3, caspase-8, and caspase-9

all obviously increased in a dose-dependent manner

(2.03–8.13 μmol/l) and time-dependent manner (within

12 h) compared with the control group. Activation of

caspase-8, caspase-9, and the subsequent activation of

caspase-3 indicated that AG4 may be inducing intrinsic

and extrinsic apoptosis pathways.

To further verify whether the effect of apoptosis was

correlated with caspases, we used the inhibitors of

caspase-3 (Ac-DEVD-CHO), caspase-8 (Z-IETD-FMK),

caspase-9 (Z-LEHD-EMK·TFA), and pan caspase

inhibitor (Z-VAD-FMK). As shown in Fig. 5c, the

cell viability rate decreased significantly when the CNE

cells were treated with AG4 and reversed by

Z-VAD-FMK. However, Ac-DEVD-CHO, Z-IETD-

FMK, and Z-LEHD-EMK·TFA did not exert effects on

cell viability.

AG4 regulated the mRNA and protein levels related toapoptosisCNE cell cultures were exposed to AG4 for 24 h. Total

RNAs were extracted and the relative mRNA amounts

Fig. 2

15

10

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00 0.5 1 2 4 5 6 8 10 12

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LS180 EJ

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MCF-7 CNEHT-2

9BGC

HepG2

A549HeL

aSY5Y

12 h24 h48 h

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Concentration (μmol/l)

Growth-inhibitory effect of AG4 on the viability of tumor cells evaluatedby an MTTassay. (a) Effect of AG4 on the proliferation of tumor cells. (b)Effect of AG4 on the proliferation of CNE cells for different time points.Each data represented the mean±SD from three independentexperiments, each at least in triplicate. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.



were measured by real-time PCR and normalized to β-actin mRNA. The effects of AG4 on Bax, Bad, Bid, Bcl-2,

and Fas mRNA are shown in Fig. 6a, Bax, Bad, Bid, and

Fas mRNA were increased after AG4 treatment in a

dose-dependent manner (2.03–8.13 μmol/l), and Bcl-2

mRNA was suppressed by AG4. The same effects were

found for protein levels (Fig. 6b). To determine whether

the Fas or the Bcl-2 family was functional after AG4

treatment and whether the intrinsic and extrinsic path-

way was required for AG4-induced apoptosis, the FasL

inhibitor and the Bcl-2 family inhibitor were used.

Figure 6c shows that the FasL inhibitor (AF-016) and the

Bcl-2 family inhibitor (GX15-070) protected CNE cells

from apoptosis induced by AG4 under the conditions of

the experiments.

DiscussionHuman nasopharyngeal carcinoma cell CNE is a classical

cell line obtained from nasopharyngeal carcinoma

patients, and it is used in various kinds of studies of

nasopharyngeal carcinoma [11]. However, the CNE cell

line was more sensitive than other tumor cell lines to the

antitumor effect of AG4 in this experiment; thus, we

selected the CNE cells and expected that AG4 may be

effective for the treatment of nasopharyngeal carcinoma

someday clinically.

Apoptosis (or programmed cell death) is a physiological

mechanism that is crucial for the normal development of

organisms during embryogenesis, maintenance of tissue

homeostasis in adults, and elimination of diseased or

otherwise harmful cells during pathogenesis [12].

Dysregulated apoptosis has been implicated in many

human diseases, including neurodegenerative diseases

such as Alzheimer’s disease and Huntington disease,

ischemic damage, autoimmune disorders, and several

forms of cancer [13]. In this study, AG4 was shown to

enhance apoptosis, cell-cycle arrest, decrease MMPs, and

Fig. 3

104(a)(A) (B)

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Annexin V FITCAnnexin V FITC Annexin V FITC Annexin V FITC

PI (

%)

PI

PI

PI

Channels FL2-H

Effects of AG4 on the morphology, apoptotic rates, and cell cycle in CNE cells. (a) Cells nuclei were stained by Hoechst 33 258; A, B: control; C, D:treated with 2.03 μmol/l AG4; E, F: treated with 4.06 μmol/l AG4; G, H: treated with 8.13 μmol/l AG4. (b) Cell apoptosis was measured using aAnnexin-V-FITC/PI apoptosis detection kit by flow cytometry. (c) Cell cycle was measured using a propidium iodide (PI) by flow cytometry. **P<0.01versus the control.



Fig. 4

200(a)

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FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

8000 250

150

50

AG4 0 2.03 4.06 8.13Concentration (μmol/l)

8.13

Concentration (μmol/l) Concentration (μmol/l)

200

100

SO

D a

ctiv

ity (k

U/g

Pro

)

0RO

S c

onte

nt (D

CF

fluor

esce

nce)

6000

4000

2000

∗∗

∗∗

∗∗∗∗

10

15

5

MD

A c

onte

nt (μ

mol

/l/g

Pro

)

AG4 0 2.03 4.06 8.13AG4

GS

H c

onte

nt (μ

mol

/l/10

0m

g P

ro)

0 2.03 4.060

15

10

5

0

00 2 4 8 10 12 16 18 22 24

Time (h)

AG4 (μmol/l) 0 2.03 4.06 8.13

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120C

ount

s80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

200

160

120

Cou

nts

80

40

0

∗∗

∗∗

∗∗

ROS are responsible for AG4-induced cell death in CNE cells. (a, b) Effect of AG4 on levels of ROS, SOD, GSH, and MDA content on CNE cells.(c) Effect of different concentrations of AG4 on MMP of CNE cells for 24 h. (d) Proliferation of CNE cells treated with NAC for 24 h.**P<0.01 versuscontrol, compared with AG4 treatment-alone group, #P<0.05, ##P<0.01. GSH, glutathione; MDA, malondialdehyde; MMP, mitochondrialmembrane potential; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species; SOD, superoxide dismutase.



Fig. 4 (continued )

200

150

100

50

0

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

200

(c)

(d)

150

100

50

0

200

150

100

50

0100 101 102 103 104 100 101 102 103 104100 101 102 103 104100 101 102 103 104

200

150

100

50

0

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

100

NA

C (0

mm

ol/l)

NA

C (1

0m

mol

/l)

AG4 0 2.03

Concentration (μmol/l)

4.06 8.13

0.521%

M1M1 M1 M1

5.48% 25.3% 39.4%

M1 M1 M10.172% 2.40% 10.3%

M118.8%

∗∗

∗∗

#

##

##

150

100

50

0

AG4-(μmol/l)

NAC-(10 mmol/l)

0 2.03 4.06 8.13 0 2.03 4.06 8.13

++++−−−−

Cel

l via

bilit

y (%

of c

ontr

ol)



Fig. 5

60

40

20

0

70

60

50

40

30

20

10

0

150

100

50

0

kU/g

Pro

kUg−1

Pro

0 2.03 4.06 8.13 0 2.03 4.06 8.13 0 2.03 4.06 8.13

Caspase-8 Caspase-9 Caspase-3

0 6 12 18 24Different time points (h)

Cel

l via

bilit

y (%

of c

ontr

ol)

02.03

4.068.13 0

2.034.06

8.13 02.03

4.068.13 0

2.034.06

8.13 02.03

4.068.13

AC-DEVD-CHO Z-IETD-FMK Z-LEHD-EMK-TFA Z-VAD-FMK

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗

∗

∗∗∗∗

∗∗

∗∗

∗∗

Caspase-8Caspase-9Caspase-3

∗∗

∗∗

#

#

(a)

(b)

(c)

μmol/l

Influence of AG4 to the enzyme activities of caspase-3, caspase-9, and caspase-8 in CNE cells. (a) Effect of AG4 on levels of caspase-3, caspase-9,and caspase-8 for 24 h in CNE cells. (b) Effect of AG4 on caspase-3, caspase-9, and caspase-8 activities at different time points. (c) Proliferation ofCNE cells treated with AG4 and caspase inhibitors for 24 h. **P<0.01 versus control, compared with the AG4 treatment-alone group, #P<0.05.



Fig. 6

600400

300

150

50

0

100

200

100

0

400

∗∗

∗∗

∗∗

∗∗

∗∗

∗

∗∗

∗∗

∗∗∗

200

200

150

50

0

100

200

150

50

0

100

Bcl

-2 m

RN

A (%

of c

ontr

ol)

Fas

mR

NA

(% o

f con

trol

)

Bad

mR

NA

(% o

f con

trol

)

Bax

mR

NA

(% o

f con

trol

)B

id m

RN

A (%

of c

ontr

ol)

0Control Control

Control

Control

Bad

GAPDH

0 8.13 8.13 8.13 8.13 8.130 0 0 0

Bax Bid Bcl-2 Fas

AG4-2.03 AG4-2.03

AG4-2.03

AG4-2.03

AG4-4.06 AG4-4.06

AG4-4.06

AG4-4.06

AG4-8.13 AG4-8.13

Control AG4-2.03 AG4-4.06 AG4-8.13AG4-8.13

AG4-8.13

4

3∗∗

∗∗

∗∗

##

##

###

∗∗

∗∗

∗∗

∗2

Rel

ativ

e pr

otei

n le

vel (

/GA

PD

H)

1

0

150

(a)

(b)

(c)

100

Cel

l via

bilit

y (%

of c

ontr

ol)

50

AF-016 GX15-070

00

2.034.06

8.13 02.03

4.068.13 0

2.034.06

8.13

GAPDH Bad Bax Bid Bcl-2 Fas

AG4 regulated oncogene and anti-oncogene mRNA and protein expression. (a) Effect of AG4 on mRNA expression; the mRNA levels are normalizedto β-actin. (b) Effect of AG4 on protein expression. (c) FasL inhibitor (AF-016) and Bcl-2 family inhibitor (GX15-070) protected CNE cells fromapoptosis induced by AG4. Each bar is the mean ±SD of three independent experiments, statistical difference compared with the normal group(vehicle-treated) was calculated using Dunnett’s test after analysis of variance, **P<0.01.



increase ROS content and caspase activities in CNE

cells; the inhibitor of ROS (NAC), the FasL inhibitor

(AF-016), and the Bcl-2 family inhibitor (GX15-070)

could protect CNE cells from apoptosis.

ROS have recently been proposed to be involved in

tumor development and metastasis, which is a compli-

cated processes including epithelial–mesenchymal tran-

sition, migration, invasion of the tumor cells, and

angiogenesis around the tumor lesion [14]. Our results

suggested that the activity of ROS was increased sig-

nificantly in CNE cells after the treatment of AG4, and

showed that AG4 could mediate ROS production. The

apoptosis rate was increased following an increase in the

content of ROS and could be protected by NAC. We

found that the SOD and GSH activities were inhibited

and the MDA content was increased, which might be

involved in the ROS production [15].

Moreover, our results showed that AG4 could activate the

activities of caspase-3, caspase-8, and caspase-9 sig-

nificantly in a dose-dependent and time-dependent

manner. Caspases, a family of cysteine–aspartic pro-

teases, are key regulators of apoptosis; caspase-3 is a key

effecter molecule in the caspase-dependent cell apopto-

sis pathway that cleaves a number of cellular proteins,

leading to apoptotic changes. Cleavage of caspase-3,

caspase-8, and caspase-9 in this study suggested that the

extrinsic and intrinsic apoptotic pathways were activated.

The pathways were also found to crosstalk through the

truncation of members of the Bcl-2 protein family, which

transduced apoptotic signals from the cell surface to the

mitochondria [16–21]. Although caspases were activated

in CNE cells exposed to AG4, the addition of the caspase

inhibitors, Ac-DEVD-DHO, Z-IETD-FMK, and

Z-LEHD-EMK·TFA, failed to attenuate the apoptosis

induction effect of AG4; only Z-VAD-FMK could reverse

the decreased cell viability rate caused by treatment with

AG4. These results suggest that another caspase-

independent apoptotic pathway was also activated.

The mitochondria serve a critical function at the core of

the apoptotic pathways, and mitochondrial depolarization

is a major event in mitochondrial dysfunction; the loss of

MMPs is crucial for the balance between the caspase-

dependent and caspase-independent apoptotic pathways

[22–25]. In the present study, we found that treatment

with AG4 resulted in a decrease in MMPs and NAC

Fig. 7

Fas

FADD

Caspase8/10

GSK-3β

PI3K

RasRaf MEK1/2

ERK1/2P90 RSK

Akt

Bid

BidCa2+

Caspase 12

Caspase3/6/7 Apaf-1

Caspase-9

Cyt C

AIFApoptosis

BaxBak Bcl-xl

Bcl-2Mitochondria

PUMA NOXA

Bad

RTK

TRK

The signal pathway of cell apoptosis.



could reverse this decrease, suggesting that ROS gen-

eration was crucial for the decrease in MMPs induced

by AG4. Members of the Bcl-2 protein family, which

are categorized as prosurvival (antiapoptotic) and pro-

apoptotic (Fig. 7), act as gatekeepers of the mitochondria

by controlling the permeability of the mitochondrial

outer membrane [26–31]. In this study, the mRNA level

of Bcl-2 was downregulated in the presence of AG4;

moreover, AG4 could increase the relative expression of

Bad, Bid, and Bax mRNA, the proapoptotic genes. Two

distinct pathways of apoptosis have been identified as

mitochondria-initiated apoptosis occurs through caspase-

9 and the death receptor-mediated pathway requires

caspase-8 [32]. Therefore, the Fas mRNA level was

detected as a proapoptotic gene in the death receptor-

mediated pathway; we found that AG4 increased the

relative expression of Fas mRNA. The same effects were

found in protein level detection. The FasL inhibitor and

the Bcl-2 family inhibitor could protect CNE cells from

apoptosis induced by AG4. Cell-cycle analysis showed

that AG4 could arrest cell cycle in the S phase, and this

might be attributed to the inhibition of cell growth and

induction of cell apoptosis.

ConclusionAG4-induced apoptosis in CNE cells occurs by the

decrease in MMPs in a ROS-dependent manner and

regulation of genes and protein relative to apoptosis.

AG4-induced apoptosis in CNE cells was linked to a

death receptor pathway and Bcl-2 family-mediated

mitochondrial signaling pathway. Accordingly, the

results of this study suggest that AG4 may be a potential

candidate for nasopharyngeal carcinoma treatment.

AcknowledgementsThis work was supported by grants from the National

Natural Science Foundation (No. 31370006).

Conflicts of interestThere are no conflicts of interest.

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ag4, a compound isolated from radix ardisiae gigantifoliae...

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