chelerythrine chloride from macleaya cordata induces growth inhibition and apoptosis ... · 2017....

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ORIGINAL ARTICLE

Chelerythrine chloride from Macleaya cordata inducesgrowth inhibition and apoptosis in human gastric cancerBGC-823 cells

Zhengfu Zhanga,b, Ying Guoc, Lingwei Zhanga, Jianbin Zhanga, Xionghui Weia,n

aDepartment of Applied Chemistry, College of Chemistry & Molecular Engineering, Peking University, Beijing 100871, ChinabCenter for Drug Certification, State Food and Drug Administration, Beijing 100061, ChinacDepartment of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China

Received 23 November 2011; revised 5 December 2011; accepted 23 December 2011

KEY WORDS

Macleaya cordata;

Chelerythrine chloride;

BGC-823 cells;

Apoptosis

stitute of Materia M

.V. All rights rese

016/j.apsb.2011.12

thor. Tel./fax: þ[email protected]

esponsibility of Inst

Abstract This study aimed to investigate the ability of chelerythrine chloride (CHE), the main

active ingredient of Macleaya cordata, to induce apoptosis in human gastric cancer BGC-823 cells.

The results demonstrate that CHE inhibits cell proliferation in a time- and dose-dependent manner

with accompanying S phase arrest. It also induces apoptosis by a mechanism involving a reduction

in the mitochondrial membrane potential, the release of cytochrome c, activation of caspase 3 and

cleavage of poly-ADP-ribose polymerase. In addition, CHE-induced apoptosis is accompanied by

down-regulation of Bcl-xl and Bcl-2 proteins with no change in the levels of Bax proteins. Taken

together, the results support the development of CHE as a potentially useful anticancer drug for the

treatment of gastric cancer.

& 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical

Association. Production and hosting by Elsevier B.V. All rights reserved.

edica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and

rved.

.013

10 62751529.

(Xionghui Wei).

itute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.

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Ability of chelerythrine chloride in human gastric cancer BGC-823 cells 465

1. Introduction

Cancer is a universal public health problem because of its high

incidence and mortality rate. Currently, gastric carcinoma is

the second leading cause of cancer-related deaths in the

world1, accounting for about 23.2% of all cancer deaths2.

This is despite recent advances in the development of new

diagnostic and therapeutic tools. Consequently, it remains of

some urgency to identify new and better therapeutic agents

and regimes for gastric cancer.

Natural products, including plants, microorganisms and the

halobios provide a rich resource for the discovery of cytotoxic

agents3. Recently, much effort has been directed towards the

search for new plant sources of anticancer drugs. One plant

receiving attention is Macleaya cordata, a wild plant growing

in Shanxi, Guizhou and Yunnan provinces of China. It is

extensively used in traditional Chinese medicine for the

treatment of wounds, arthritis, rheumatic arthralgia and

trichomonas vaginalis4. Of particular relevance is that the

roots of Macleaya cordata have been applied in folk medicine

for the treatment of cancer5.

CHE (Fig. 1), a benzophenanthridine alkaloid, is the major

active ingredient of Macleaya cordata. It is reported to

mediate diverse biological and pharmacological effects

through its antimicrobial, antifungal, and anti-inflammatory

properties6,7. It also significantly inhibits HaCaT keratinocytes

and exerts cytotoxic effects on various human normal and

cancer cell lines8–10. For instance CHE shows high cytotoxicity

against isolated rat hepatocytes and primary hepatocyte

cultures11. Induction of apoptosis has been mooted as a

possible mechanism of the anticancer activity of CHE12–15.

Recently, a differential induction of apoptosis was demon-

strated in cancer cells and normal cells in vitro, suggesting

CHE has potential as an effective anticancer drug10. To

confirm the ability of CHE to induce apoptosis and to

investigate the exact mechanism of apoptosis induction, we

investigated the effect of CHE on gastric carcinoma BGC-823

cells in vitro.

2. Materials and methods

2.1. Chemicals

CHE (purityZ99%) was provided by Yongfeng BoyuanIndustry Co. Ltd. (Jiangxi Province, China). RPMI-1640

medium were obtained from HyClone Laboratories Inc.

(Logan, UT, USA). A stock solution of CHE in DMSO was

diluted with RPMI-1640 medium to prepare standards with

concentrations of 1.25, 2.5, 5.0 and 10.0 mg/mL. The final

Figure 1 Structure of chelerythrine chloride.

concentration of DMSO was less than 0.1%. DNase free

RNase A and propidium iodide (PI) were purchased from

Sigma (St. Louis, MO, USA). Cytochrome c antibody and

goat anti-rabbit-HRP were obtained from Santa Cruz Bio-

technology, Inc. (Santa Cruz, CA). Bid, Bax, Bcl-2, Bcl-xl,

caspase-3, b-actin and poly-ADP-ribose polymerase (PARP)antibodies were purchased from Cell Signaling Technology

(Andover, MA, USA). All other chemicals were of the highest

grade available from commercial sources.

2.2. Cell culture

The human gastric carcinoma cell line BGC-823 was pur-

chased from the Institute of Basic Medical Sciences, Chinese

Academy of Medical Sciences, and cultured in complete

RPMI-1640 medium supplemented with 10% v/v fetal bovine

serum (FBS), 100 units/mL penicillin and 100 mg/mL strepto-mycin at 37 1C in a 5% CO2 incubator. Exponentially growingcultures at 80% confluence were used in all experiments.

2.3. MTT assay

The cytotoxicity of CHE on BGC-823 cells was determined

using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]

bromide (MTT) assay. The cells (2� 105/mL) were seeded in a96-well plate and, after 24 h, were treated with CHE solutions

and vehicle controls containing equivalent amounts of DMSO.

After incubation for 24, 36, and 48 h, 20 mL MTT solution(5 mg/mL in PBS) was added to each well and incubated for an

additional 4 h. The plate was then centrifuged at 1000 rpm for

5 min, the supernatants were aspirated and the formazan

crystals in each well were dissolved in 100 mL aliquots ofDMSO. Finally, the plate was shaken and the optical density

was determined at 570 nm using a Model 680 Microplate

Reader (Bio-Rad, Bath, UK). Cell survival was expressed as

the ratio of viable cells in treated samples to viable cells in

control samples expressed as a percentage. All doses were tested

in triplicate and experiments were repeated at least three times.

2.4. Annexin V-FITC/PI staining

Annexin V-FITC/PI double labeling was used for the detec-

tion of phosphatidylserine (PS) externalization, a hallmark of

the early phase of apoptosis. PS exposure on the outer layer of

cell membranes was measured using the annexin V-FITC

apoptosis detection kit (Biovision, USA) according to the

manufacturer’s instructions. Briefly, after treatment with

different concentrations of CHE for 24 h, cells were harvested,

washed twice with cold PBS and resuspended in 200 mLannexin V-FITC binding buffer. To this, 5 mL recombinantannexin V-FITC was added and the mixture was incubated at

room temperature for 10–15 min in the dark. Following this,

5 mL 20 mg/mL PI solution was added and a total of 2� 104cells was analyzed using a BD FACSCalibur flow cytometer

(Beckton–Dickinson, San Jose, CA, USA). Cells stained with

annexin V-FITC and PI were considered as late apoptotic or

necrotic cells while cells stained with annexin V-FITC alone

were considered as early apoptotic cells. All samples were

assayed in duplicate and experiments were repeated three

times.

Figure 2 Effect of CHE on BGC-823 cell viability as determined

by the MTT assay. Inhibition is expressed as cell viability relative

to control without CHE treatment. The cells (2� 104/well) weretreated with different concentrations of CHE for 24, 36 and 48 h.

Data are means7S.D. of three independent experiments.

Zhengfu Zhang et al.466

2.5. DNA fragmentation assay

After treatment with different concentrations of CHE for 24 h,

cells were harvested and washed twice with cold PBS. Total

DNA was isolated using a DNA extraction kit (Applygen,

China), analyzed by electrophoresis on 1% agarose gel

containing ethidium bromide and photographed by Bio-Rad

GD2000 (Bio-Rad, Hercules, CA, USA).

2.6. Cell cycle analysis

Cells in log-phase growth were treated with different concen-

trations of CHE for 24 h, trypsinized, resuspended, and fixed

with cold 70% ethanol at 4 1C overnight. Fixed cells weresubsequently washed in PBS, resuspended in 1.0 mL PBS

containing 50 mg/mL RNase and 50 mg/mL PI and incubatedfor 30 min at 37 1C. DNA content was then determined usinga BD FACSCalibur flow cytometer. The percentage of cells in

G0/G1, S and G2/M phases was determined using Cellquest

3.0 software measuring 10,000 events in each case.

2.7. Mitochondrial membrane potential (Dcm)

Dcm was determined by 5,50,6,60-tetrachloro-1,103,30-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining using the

mitochondrial membrane potential detection kit (Mountain

View, CA) according to the manufacturer’s instructions. Briefly,

after treatment with different concentrations of CHE for 24 h,

cells were harvested, washed, resuspended in 5 mg/mL JC-1solution (500 mL) and incubated at 37 1C for 20 min. Cells werewashed, resuspended in cold PBS (500 mL) and fluorescence of10,000 cells per sample was measured by flow cytometry using

excitation at 488 nm with emission at 525 nm for the green JC-1

monomers (FL1) and at 575 nm for the red JC-1 aggregates

(FL2). Data were analyzed using a FACSCalibur flow cyto-

meter equipped with Cellquest 3.0 software. All samples were

assayed in duplicate and experiments were repeated three times.

2.8. Protein and Western blot analyses


106 cells were harvested, washed, and lysed for 15 min on ice in

100 mL SDS lysis buffer (containing final concentrations of2% SDS, 10% glycerol and 62.5 mmol/L Tris, pH 6.8). The

protein concentration in the lysates was measured using the

BCA protein assay (Pierce Chemical Co., Rockford, USA)

according to the manufacturer’s protocol. For Western blot

analysis, protein samples (50 mg) from each cell lysate wereseparated on SDS–PAGE gels (10% for all proteins except

cytochrome c where a 15% gel was used) and then electro-

transferred onto immobilon membranes (Millipore, Waltham,

Massachusetts, USA). Blots were blocked using 5% nonfat

milk in TBST buffer (25 mM Tris HCl, 0.2 M NaCl, 0.1%

Tween 20) for 2 h at room temperature and then incubated

overnight at 4 1C with primary antibodies (200 mg/mL anddilutions of 1:1000 for Bax, Bad, Bcl-2, Bcl-XL, cytochrome c,

caspase-3 and PARP 1 mg/mL and dilution of 1:2500 for

b-actin). Blots were washed with TBST buffer (3� 5 min) andincubated with goat anti-rabbit antibodies conjugated with

horseradish peroxidase (1 mg/mL and dilution at 1:2000) for

1 h at room temperature. Blots were developed by enhanced

chemiluminescence (ECL Kits; Amersham Life Science) fol-

lowed by exposure to X-ray film using b-actin as loadingcontrol. Protein expression in treated cells was compared to

untreated controls.

2.9. Release of cytochrome c


106 cells were harvested, washed twice with ice-cold PBS and

then resuspended in 100 mL buffer (50 mM Tris–HCl pH 7.4,1 mM NaF, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1%

v/v Nonidet P 40 (NP-40) and 10 mg/mL leupeptin). Sampleswere centrifuged at 10,000� g for 30 min at 4 1C and thesupernatant (cytosolic fraction) was used to assess release of

cytochrome c. The remainder of the procedure was the same

as that for other Western blot analysis.

2.10. Statistical analysis

Results are expressed as means7S.D. of three independentexperiments. Differences in mean values were analyzed by the

Student’s t-test with a value of Po0.05 considered statisticallysignificant.

3. Results

3.1. Effect of CHE on cell viability

As shown in Fig. 2, cell growth inhibition by CHE was both

dose- and time-dependent. Cell growth inhibition increased

notably with increasing CHE concentration and decreased

with time with IC50 values of 2.42, 1.90, and 1.28 mg/mL for24, 36, and 48 h, respectively.

3.2. CHE-induced apoptosis and cell cycle arrest

As shown in Fig. 3A, treatment with CHE for 24 h caused

the percentage of early apoptotic cells (annexin V-FITC

positive, and PI negative) significantly increase from 4.7 to

27.7%. At the same time, the percentage of late apoptotic or

Figure 3 (A) Flow cytometric analysis of apoptosis with annexin-V/PI staining. Cells were treated with different concentrations of CHE

for 24 h and then labeled with a combination of annexin V-FITC/PI. Lower left quadrant (LL; annexin V and PI negative) represents

viable cells; Lower right quadrant (LR; annexin V positive and PI negative) represents apoptotic cells in early stage; Upper right quadrant

(UR; annexin V and PI positive) represents late apoptotic or necrotic cells; Upper left quadrant (UL; annexin V negative and PI positive)

represents necrotic cells or cellular debris. Numbers refer to the percentage of annexin-V and/or PI-labeled cells. (B) Agarose gel

electrophoresis of DNA ladders in BGC-823 cells. After treatment with CHE for 24 h, DNA in BGC-823 cells was extracted and loaded

on agarose gel. After electrophoresis, DNA ladders were photographed by BioRad GD2000 (bp). A: DNA marker; B: Control;

C: 1.25 mg/mL; D: 2.5 mg/mL; E: 5.0 mg/mL; F: 10.0 mg/mL.

Table 1 The relative proportion of cells (%) in the

G0/G1, S, and G2/M phases after treatment with CHE

for 24 h. Data are means7S.D. of three independentexperiments.

CHE

(mg/mL)Cell cycle distribution (%)

G0/G1 S G2/M

Control 67.5377.65 15.273.43 17.6772.081.25 62.0875.29 21.372.97 15.1573.162.5 53.1173.85 29.677.11 15.6172.985.0 52.9578.98 37.478.27 12.6070.7710.0 52.5277.34 39.6710.02 10.5272.63


necrotic cells (annexin V-FITC and PI positive) increased in a

dose-dependent manner. Degradation of DNA into a specific

fragmentation pattern (consisting of DNA ladders) is a

characteristic feature of apoptosis16. Agarose gel electrophor-

esis of the DNA from BGC-823 cells exposed to CHE revealed

formation of clear DNA fragmentation ladders in a dose-

dependent manner (Fig. 3B).

It has been previously demonstrated that CHE can induce

G1 phase arrest in cancer cells17. In the present study, the

effect of CHE on the cell cycle was determined by flow

cytometry of PI stained BGC-823 cells. As shown in Table 1

and Fig. 4, treatment with CHE for 24 h resulted in an

accumulation of cells in the S phase of the cell cycle from

15.2 to 39.6% while the number of cells in the G0/G1 and

G2/M phases decreased in a dose-dependent manner. The

Figure 4 Representative histograms of flow cytometric analysis of cell cycle phase distribution in BGC-823 cells after treatment with

CHE. Cells were treated for 24 h with 1.25, 2.5, 5.0 and 10.0 mg/mL CHE, harvested, fixed and stained for their DNA content with PI.Results are based on three independent experiments.


results demonstrate that CHE inhibits BGC-823 cell growth

through S phase arrest.

3.3. Mitochondrial membrane potential and cytochrome c

release

The results show that CHE treatment of BGC-823 cells leads

to a dramatic shift in fluorescence (Fig. 5A) and increase in

mitochondrial membrane depolarization from 34.4 to 98.4%

in a dose-dependent manner (Fig. 5B). In addition, cyto-

chrome c is released from mitochondria into the cytosol in a

dose-dependent manner (Fig. 6A). These observations suggest

CHE-induced apoptosis in BGC-823 cells involves cytochrome

c release from mitochondria and possibly mitochondrial

disruption.

3.4. Activation of caspase 3 and PARP cleavage

A role for caspase 3 in CHE-mediated induction of apoptosis

in BGC-823 cells is shown by formation of the cleaved forms

of caspase 3 (19 and 17 kDa fragments) in a dose-dependent

manner (Fig. 6A). Similarly, a role for the cleavage of PARP is

shown by the gradual increase in PARP fragments in the CHE

treated samples (Fig. 6A). These results confirm the involve-

ment of caspase 3 activation and PARP cleavage in CHE-

induced apoptosis of BGC-823 cells.

3.5. Expression of Bcl-2 family proteins

To further elucidate the molecular mechanism of CHE-

mediated apoptosis induction, the expression of several mem-

bers of the Bcl-2 family was examined by Western blot

analysis. As shown in Fig. 6B, the expressions of Bcl-xl and

Bcl-2 are down-regulated in a dose-dependent manner while

the expression of Bax is unchanged. The results reveal that

dysregulation of Bcl-2 family proteins is involved in CHE-

mediated apoptosis of BGC-823 cells.

4. Discussion

Apoptosis, also known as programmed cell death, is closely

related to the initiation, progression and metastasis of tumors18,19

and its induction is a basic strategy in the treatment of malignant

tumors. In fact, most of the available chemotherapeutic agents

work by destroying tumor cells through inducing apoptosis20.

Cellular growth and proliferation are closely related to apopto-

sis21,22 which is characterized by distinct morphological changes

including plasma membrane bleb, cell shrinkage, PS translocation

and DNA fragmentation23,24.

During a cellular screening of benzophenanthridine alka-

loids, we found that CHE, the major active ingredient in

Macleaya cordata, interacts with DNA bases25–27, inhibits

proliferation of human gastric carcinoma BGC-823 cells and

Figure 5 Induction of mitochondrial depolarization of BGC-823 cells treated with CHE at 1.25, 2.5, 5.0 and 10.0 mg/mL for 24 h. (A)representative histograms stained with the fluorescent probe JC-1; (B) percentage of cells with low mitochondrial potential. Data are

means7S.D. of three independent experiments.


induces apoptosis in vitro. As stated earlier, the mechanism of

apoptosis induction by CHE seems to be complex10,14,15, and

the molecular mechanism of its anticarcinogenic effect is not

fully understood. In this study, we evaluated the ability of

CHE to induce apoptosis in BGC-823 cells and investigated

how it occurs.

Figure 6 (A) Expression of cytochrome c, caspase-3 and PARP

in CHE-treated BGC-823 cells. Cells were treated with different

concentrations of CHE for 24 h followed by Western blot analysis

for detection of cytochrome c, caspase-3 and PARP proteins.

b-Actin was used as an equal loading control. Results are basedon three independent experiments. (B) Expression of Bcl-2 family

proteins including Bcl-2, Bcl-xl and Bax in CHE-treated BGC-823

cells. Cells were treated with different concentrations of CHE for

24 h followed by Western blot analysis for detection of Bcl-2, Bcl-

xl and Bax proteins. b-Actin was used as an equal loading control.Results are based on three independent experiments.


The MTT assay showed CHE inhibited the proliferation of

BGC-823 cells in a dose- and time-dependent manner (Fig. 2)

consistent with previous studies in various cell types9–11.

Contrary to previous studies14,17, we observed positive

annexin V-FITC/PI staining (Fig. 3A) and DNA ladder

formation (Fig. 3B) indicating that CHE efficiently inhibits

BGC-823 cell growth through apoptosis with no change in cell

viability leading to necrosis of BGC-823 cells. There are two

key checkpoints that regulate the cell cycle, the G1/S and

G2/M transitions28,29. Anticancer drugs can disturb cell cycle

progression, arrest cells in a particular phase and induce

apoptosis. Despite the fact that CHE was shown to induce

G1 cell cycle arrest in human leukemia HL-60 cells17, our

results suggest that CHE causes significant cell cycle arrest in

the S phase (Table 1 and Fig. 4).

As regards the mechanism of apoptosis induction, it is

known that apoptosis is regulated by two major signaling

pathways, namely the death receptor pathway and the mito-

chondrial pathway30,31. In the former, the binding of death

receptors with ligands such as Fas or cross-linking antibodies

results in initiation of the caspase cascade32. In the latter,

mitochondria are involved in a variety of key events leading to

apoptosis33,34. These include the collapse of Dcm and the

subsequent release of cytochrome c, both of which are

generally considered to play a central role in the early phase

of the mitochondrial pathway35,36. Cytochrome c is released

from mitochondria into the cytosol and forms the apoptosome

with Apaf-1 and procaspase 9, leading to activation of

executioner caspases including caspase 3. The activated cas-

pase 3 can cleave PARP which is an early and specific

indicator of apoptosis37.

As shown in Fig. 5A and 5B, CHE decreased Dcm in adose-dependent manner and CHE effectively induced cyto-

chrome c release from mitochondria into the cytosol. This

then activated caspase-3 to cleave PARP protein as shown in

Fig. 6A. These data are consistent with previous studies in

various cell types15. Bcl-2 family proteins have been reported

to regulate caspase activation by controlling mitochondrial

permeability and cytochrome c release36,38. The Bcl-2 family

includes anti-apoptotic proteins such as Bcl-2 and Bcl-xL and

pro-apoptotic proteins such as Bak and Bax39. It was reported

that changes in the ratio of pro- to anti-apoptotic members of

the Bcl-2 family, rather than the absolute expression level of

any single Bcl-2 member protein, determined apoptotic sensi-

tivity40. A study has shown that CHE can induce apoptosis by

influencing the balance of pro- and anti-apoptotic signaling

pathways41. In this investigation, the data show that down-

regulation of Bcl-xl and Bcl-2 proteins is involved in CHE-

induced apoptosis of BGC-823 cells (Fig. 6B).

Taken together, the results of this study clearly reveal that

induction of apoptosis by CHE involves multiple cellular and

molecular pathways. These include cell cycle arrest, reduction

in mitochondrial membrane potential, release of cytochrome c,

activation of caspase 3, PARP cleavage and dysregulation of

Bcl-2 family proteins. The present findings provide further

impetus to development of CHE as an effective agent for the

treatment of gastric cancer.

Acknowledgments

This work was supported by grant 20080430283 from the

China Postdoctoral Science Foundation and Jiangxi Yongfeng

Boyuan Industry Co., Ltd. (Jiangxi, China). We also thank the

following professors of Peking University: Wenting Hua and

Hongcheng Gao for suggestions and helpful discussions;

Ronghua Sun and Shouying Sun for experimental assistance.

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Chelerythrine chloride from Macleaya cordata induces growth inhibition and apoptosis in human gastric cancer BGC-823 cellsIntroductionMaterials and methodsChemicalsCell cultureMTT assayAnnexin V-FITC/PI stainingDNA fragmentation assayCell cycle analysisMitochondrial membrane potential (Deltapsim)Protein and Western blot analysesRelease of cytochrome cStatistical analysis

ResultsEffect of CHE on cell viabilityCHE-induced apoptosis and cell cycle arrestMitochondrial membrane potential and cytochrome c releaseActivation of caspase 3 and PARP cleavageExpression of Bcl-2 family proteins

DiscussionAcknowledgmentsReferences

chelerythrine chloride from macleaya cordata induces growth inhibition and apoptosis ... · 2017....

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