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Involvement of Bcl-xL degradation and mitochondrial-mediated apoptoticpathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Lili Ji, Ying Chen, Tianyu Liu, Zhengtao Wang

PII: S0041-008X(08)00238-XDOI: doi: 10.1016/j.taap.2008.05.015Reference: YTAAP 11211

To appear in: Toxicology and Applied Pharmacology

Received date: 19 February 2008Revised date: 12 May 2008Accepted date: 20 May 2008

Please cite this article as: Ji, Lili, Chen, Ying, Liu, Tianyu, Wang, Zhengtao, Involvementof Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidinealkaloids-induced apoptosis in hepatocytes, Toxicology and Applied Pharmacology (2008),doi: 10.1016/j.taap.2008.05.015

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Involvement of Bcl-xL degradation and mitochondrial-mediated

apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in

hepatocytes

Lili Jia,b

, Ying Chena,c

, Tianyu Liua, Zhengtao Wang

a,b,c,*

a Key Laboratory of Standardization of Chinese Medicines of Ministry of Education,

Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese

Medicine, Shanghai 201203, PR China

b Shanghai R&D Centre for Standardization of Traditional Chinese Medicines,

Shanghai 201203, PR China

c Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210038,

Jiangsu, PR China

*Corresponding author:

Prof. Dr. Zhengtao Wang, Key Laboratory of Standardization of Chinese Medicines of

Ministry of Education, Institute of Chinese Materia Medica, Shanghai University of

Traditional Chinese Medicine, 1200 Cailun Road, shanghai 201203, PR China

E-mail: wangzht@hotmail.com; wangzht@shutcm.edu.cn

Phone: 86-21-51322507 Fax: 86-21-51322519

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Abstract

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide

distribution in more than 6,000 high plants including medicinal herbs or teas. The aim

of this study is to investigate the signal pathway involved in PAs induced

hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii

Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse

hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9

activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL

in a time (8-48 h)- and concentration (1-100 µM)-dependent manner. Furthermore,

inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited

clivorine-induced apoptosis and rescued clivorine-decreased cell viability.

Polyubiquitination of Bcl-xL was detected after incubation with 100µM clivorine for

40 h in the presence of proteasome specific inhibitor MG132, indicating possible

degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain

inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell

viability. All the other tested PAs such as senecionine, isoline and monocrotaline

decreased mouse hepatocytes viability in a concentration-dependent manner.

Clivorine (10µM) induced caspase-3 activation and decreased Bcl-xL was also

confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from

Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL

in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same

hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus

leading to the activation of mitochondrial-mediated apoptotic pathway.

Keywords: Pyrrolizidine alkaloid; Apoptosis; Hepatocytes; Caspase; Cytochrome c;

Bcl-xL; Protein degradation

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Introduction

Apoptosis is an important mode of cell death, and the characteristic changes

associated with apoptosis are due to the activation of a family of intracellular cysteine

proteases known as caspases (Fraser and Evan, 1996; Boyce et al., 2004).

Mitochondrial-mediated apoptotic pathway is one of major pathways involved in

apoptosis, which requires the release of mitochondrial cytochrome c and activation of

caspase-3/-9 signaling cascade, leading to the apoptotic destruction of the cell (Green

and Reed, 1998). Bcl-2 family members function as regulators of almost all known

forms of apoptosis and comprise three subfamilies, the anti-apoptotic subfamily (e.g.

Bcl-xL, Bcl-2), the multi-domain pro-apoptotic subfamily (e.g. Bax, Bak) and the

pro-apoptotic BH3-only subfamily (e.g. Bim, Bad) (Cory and Adams, 2002). The

anti-apoptotic Bcl-2 proteins protect the mitochondria during apoptosis, while the

pro-apoptotic members disrupt the mitochondria (Gross et al., 1999).

Pyrrolizidine alkaloids (PAs) are the most potential natural hepatotoxins found in

a wide variety of plant species of different families worldwide (Roeder, 1995, 2000).

Consumption of PAs-containing plants leads to high risk to humans and live stocks

(Coulombe, 2003). Based on its possible hazards to human health, the U.S. Food and

Drug Administration nominated riddelliine for genotoxicity and carcinogenicity

testing conducted by the National Toxicology Program (NTP), and British Medicines

Healthcare Products Regulatory Agency (MHRA) alert people to pay attention to the

toxicity of senecionine-containing herbs.

There are two mainly types of toxic PAs, retronecine- and otonecine-type, of

which characteristically the 8-membered heterocyclic necine base is bicyclic and

monocyclic respectively (Lin et al., 1998). There are already many reports about

retronecine-type PAs, which are bioactivated in the liver to pyrrolic derivatives via the

P450 system, and further react with DNA to form DNA adducting products (Mattocks,

1989; Huan et al., 1998; Kim et al., 1999). However there are few reports about the

signal molecules involved in their hepatotoxicity, and the research on otonecine PAs,

which is abundant in many plants, is fewer.

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Otonecine-type PA clivorine is abundant in Ligularia hodgsonii Hook and

Ligularia dentat Hara, which have been used for cough, hepatitis and inflammation

traditionally in Chinese medicine (Ji et al., 2002; Kuhara et al., 1980).

Retronecine-type PA senecionine is abundant in the genus of Senecio (Compositae)

plants in Asia, Europe, North American and other regions (Li et al., 2008; Wiedenfeld

et al., 2006; Walsh and Dingwell, 2007). In the present study, we have investigated

clivorine and senecionine induced apoptosis in hepatocytes and the involved apoptotic

signal pathways.

Materials and methods

Cells and reagents

The L-02 cell line was derived from adult human normal liver (Yeh et al., 1980;

Zhang et al., 2007) (Cell Bank, Type Culture Collection of Chinese Academy of

Sciences), and cells were cultured in RPMI1640 supplemented with 10% [v/v] fetal

bovine serum. Clivorine (Fig.1A) isolated from Ligularia hodgsonii Hook with the

purity ≥99.5%. Senecionine and Isoline (Fig.1A) isolated from Senecio vulgaris L.

and Ligularia duciformis respectively both with the purity≥95.0%. Monocrotaline was

from Sigma Chemical Co. (St. Louis, MO). Ac-IETD-pNA, z-VAD-fmk,

z-LEHD-fmk, z-DEVD-fmk, MG132 and calpain inhibitor I were from ALEXIS

Biochemicals (San Diego, CA). Caspase-3/CPP32, Caspase-9 Colorimetric Assay

Kits and Protein A-Agarose were from BioVision (Mountain View, CA).

The following antibodies for caspase-3, caspase-9, cytochrome c, Bcl-xL and

Ubiquitin were from Cell Signaling Technology (Danvers, MA). The antibody for

actin was from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody for Bcl-2

was from BioLegend (San Diego, CA). Peroxidase-conjugated goat anti-Rabbit

IgG(H+L) and peroxidase-conjugated goat anti-mouse IgG(H+L) were from Jackson

ImmunoResearch (West Grove, PA). Nitrocellulose membranes and prestained protein

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marker were from Bio-Rad (Hercules, CA) and enhanced chemiluminescence

detection system was from Amersham Life Science (Buckinghamshire, UK).

RevertAid first strand cDNA synthesis kit was from Fermentas International Inc.

(Ontario, Canada). All other reagents unless indicated were from Sigma Chemical Co.

DNA fragmentation assay

DNA fragmentation assay was performed as previously described method with

minor revision (Zhang et al., 2001). Briefly, cells were lysed with buffer containing

10mM Tris-HCl (pH8.0), 10mM EDTA, 150mM NaCl, 0.4% SDS and 100µg/ml

proteinase K and left in 37°C overnight. The fragmented DNA in the lysate was

extracted with phenol/chloroform/isopropyl alchol (25:24:1, v/v), and then

precipitated for 5 min at liquid nitrogen with chilled 100% ethanol and 3M sodium

acetate. The DNA pellets were saved by centrifuging at 15,000g for 15 min and then

washed with 70% ethanol and resuspended in Tris-HCl (pH8.0), with 100µg/ml

RNaseA at 37°C for 1 h. The DNA fragments were separated by 2% agarose gel

electrophoresis, stained with ethidium bromide and photographed in UV light.

Caspase colorimetric assay

Activities of caspase-3 and caspase-9 were determined according to the caspase

colorimetric assay kits. The cell lysates containing 80µg of protein were incubated

with 200µM of caspase-3 and caspase-9 specific labeled substrates Ac-DEVD-pNA

and Ac-LEHD-pNA at 37°C for 2 h. Caspase-3 and caspase-9 activities were

determined as the cleavage of colorimetric substrate by measuring the absorbance at

405nM. Results are expressed as the percent change of the activity compared to the

untreated control.

Preparation of mitochondrial and cytosolic fractions

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Cells were lysed in lysis buffer containing 10mM Tris (pH7.5), 10mM KCl, 0.15

mM MgCl2, 1mM dithiothreitol, 1mM phenylmethylsulfonyl fluoride, 10µg/ml

aprotinin, 10µg/ml leupeptin, 10µg/ml pepstatin A, 150mM sucrose for 15 min on ice,

then cells were broken with 10 passages through a 26-gauge needle, and the

homogenate was centrifuged at 800g for 10 min to remove nuclei and unbroken cells.

Supernatant was centrifuged at 12,000g for 15 min to collect supernatant as cytosolic

and pellet as mitochondrial fraction.

Cell viability assay

After various treatments, cells were incubated with 500µg/ml

3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetra-zolium bromide (MTT) for 4 h. The

functional mitochondrial succinate dehydrogenases in survival cells can convert MTT

to formazan that generates a blue color (Hansen et al., 1989). At last the formazan was

dissolved in 10%SDS-5% iso-butanol-0.01 M HCl. The optical density was measured

at 570nm with 630nm as a reference and cell viability was normalized as a percentage

of control.

Western-blot analysis and immunoprecipitation

Following the treatments, cells were lysed in lysis buffer containing 50mM Tris

(pH7.5), 1mM EDTA, 150mM NaCl, 20mM NaF, 0.5% NP-40, 10% glycerol, 1mM

phenylmethylsulfonyl fluoride, 10µg/ml aprotinin, 10µg/ml leupeptin, 10µg/ml

pepstatin A. Protein concentrations were assayed and normalized to equal protein

concentration. Proteins were separated by SDS-PAGE and blots were probed with

appropriate combination of primary and HRP-conjugated secondary antibodies. For

repeated immunoblotting, membranes were stripped in 62.5 mM Tris (pH 6.7), 20%

SDS and 0.1 M 2-mercaptoethanol for 30 min at 50°C.

To identify polyubiquitinated Bcl-xL, cells were pretreated with the proteasome

specific inhibitor MG132 (20µM) for 2 h and then incubated with clivorine (100µM)

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for 40 h. Cells were lysed and protein concentrations were determined, then equal

amounts of protein were subjected to immunoprecipitation with anti-Bcl-xL antibody.

The immunoprecipitate was separated via SDS-12% PAGE and the conjugates were

detected with anti-ubiquitin antibody. As a control for expression of Bcl-xL protein,

Western-blot analysis was also performed with an anti-Bcl-xL antibody.

Mouse hepatocyte isolation

Five or 7-week-old ICR male mice were purchased from Shanghai Laboratory

Animal Center (Shanghai, China). They were housed under standard laboratory

conditions with free access to standard laboratory chow and water. Experiments were

performed following the guidelines of the local Committee for Care and Use of

laboratory animals. Hepatocytes were isolated from ICR mice by enzymatic

dissociation (Guguen-Guillouzo et al., 1982). Hepatocytes were isolated by

decantation and centrifugation (500 rpm, 1.5 min). Freshly isolated hepatocytes were

seeded in RPMI1640 medium with 10% Fetal calf serum, 1µg/ml dexamethasone,

1µg/ml insulin, 2ng/ml epithelial growth factor. The medium was renewed every day.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from cells that had been treated with clivorine (100µM)

or vehicle for various times using a TRIZOL (Life Technologies, USA) reagent

according to the manufacturer’s protocol. To synthesize single strand cDNA, reverse

transcription of 2µg total RNA was carried out using 200 unit MMLV-RT and reaction

mixture (2.5 mM dNTP, 100 pmol oligo-dT primer, RT buffer, 50 unit RNasin)

according to the manufacturer’s protocol. Transcripts of the gene for

glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were used as an internal

control. The primer sequences: Bcl-xL forward: 5’-TTGGACAATGGACTGGTTG-3’,

Reverse: 5’-GTAGAGTGGATGGTCAGTG-3’(765bp product) (Weinmann et al.,

1999), GAPDH forward: 5’-CACCACCATGGAGAAGGCTGG-3’, Reverse:

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5’-CCAAAGTTGTCATGGATGACC-3’ (200bp product) (Hougardy et al., 2005).

The PCR protocol for Bcl-xL consisted of denaturation at 94°C for 5 min, 30 cycles

of denaturation at 94°C for 30s, annealing at 55°C for 30s and extension at 72°C for 2

min, final extension at 72°C for 15 min. The protocol for GAPDH was the same

except for annealing at 65°C for 1 min and amplification for 25 cycles. The PCR

products were electrophoresed in a 2% agarose gel and stained with ethidium bromide.

The quantity of Bcl-xL and GAPDH products were automatically analyzed by GIS gel

image processing system (Version 3.10, Tanon Technology Limited Company,

Shanghai, China).

Statistical analysis

The results were expressed as means ± SD. Differences between groups were

assessed by one-way ANOVA using the SPSS software package for windows. P≤0.05

was considered as statistically significant.

Results

Clivorine induced human normal liver L-02 cell apoptosis

Clivorine exposure (10-100µM) of L-02 cells for 48 h resulted in a dramatic

decrease of cell viability in the concentration-dependent manner (Fig.1B), and the

IC50 is about 40.8µM. As shown in Fig.1B that other PAs such as senecionine, isoline

and monocrotaline all have no significant toxicity on L-02 cells from 10-100µM. The

results are consistent with our previous studies that the toxicity of clivorine may not

need metabolic activation in liver (Ji et al., 2002). Our previous study has showed that

clivorine induced L-02 cells apoptosis as detected by FACS analysis of sub-G1 peak

and Hochest 33258 staining (Ji et al., 2005). L-02 cells were treated with 100µM

clivorine for 24, 36 and 48 h, as shown in Fig.1C, clivorine with 48 h treatment

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induced apoptotic DNA ladder in L-02 cells, which is considered a biochemical

hallmark for apoptosis (De Maria et al., 1997). The results further confirmed

clivorine-induced apoptosis in hepatocytes.

Clivorine induced caspase-3 and caspase-9 activation

Our previous study showed that clivorine induced PARP [Poly (ADP-ribose)

polymerase] cleavage (Ji et al., 2005), which is a specific physiological substrate of

executioner caspases (caspase3,6,7), indicating that clivorine may induce apoptosis

through caspase signaling cascade. Caspases are synthesized as inactive zymogens,

whose cleavage represents its activation (Boyce et al., 2004). To further investigate

whether caspase signal cascade is involved in clivorine-induced apoptosis, L-02 cells

are treated with 100µM clivorine for indicated times or treated with 1-100µM

clivorine for 48 h. As shown in Fig.2A,B,C, clivorine decreased pro-caspase-3/-9

expression and increased the expression of cleaved caspase-3/-9 in a time and

concentration-dependent manner, which indicate that clivorine can induce

caspase-3/-9 activation. The caspase colorimetric assay showed that 100µM clivorine

induced the cleavage of caspase-3/-9 substrates, which confirmed clivorine-induced

caspase-3/-9 activation (Fig.2D).

Effects of caspase inhibitors on the toxicity of clivorine

To determine whether the activation of the caspases is required for the induction

of apoptosis, L-02 cells are pretreated with various caspase inhibitors for 2 h prior to

the addition of 100µM clivorine. The results of DNA fragmentation and MTT assays

showed that pretreatment with specific caspase-3 inhibitor z-DEVD-fmk (20µM),

specific caspase-9 inhibitor z-LEHD-fmk (20µM) and pan-caspase inhibitor

z-VAD-fmk (20µM) significantly inhibited clivorine-induced cell apoptosis and

rescued clivorine-decreased cell viability (Fig.3), but specific caspase-8 inhibitor

Ac-IETD-pNA (20µM) had no such protective effect (Fig.3).

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Clivorine induced release of cytochrome c from mitochondria

The release of cytochrome c from mitochondria is believed to be an initiator of

the caspase cascade in mitochondrial-mediated apoptotic pathway. To investigate

whether the caspase-9 activation is due to the release of cytochrome c from

mitochondria, cellular cytosolic and mitochondrial fractions were prepared after

treated with 100µM clivorine for indicated times or various concentrations of

clivorine for 48 h. The Western-blot result of Fig.4 showed that clivorine caused a

time and concentration-dependent decrease in mitochondrial cytochrome c and a

concomitant increase in cytosolic cytochrome c.

Clivorine decreased the level of cellular anti-apoptotic Bcl-xL protein

Anti-apoptotic members of the Bcl-2 family (Bcl-xL, Bcl-2 etc.) play important

roles in regulating the release of cytochrome c from mitochondria. After treated with

100µM clivorine for indicated times or various concentrations of clivorine for 48 h,

the expression of Bcl-xL and Bcl-2 was detected by Western-blot. Fig.5 showed that

clivorine decreased Bcl-xL amount with a time and concentration-dependent manner,

while had no effect on another anti-apoptotic Bcl-2 protein.

Effects of clivorine on Bcl-xL protein biosynthesis and degradation

To further observe how clivorine decreased the level of Bcl-xL protein, we

analyzed the expression of Bcl-xL mRNAs in L-02 cells. After treated with 100µM

clivorine for indicated times, the results of RT-PCR showed that clivorine had no

effect on Bcl-xL gene expression in mRNA level (Fig.6A, B). Cycloheximide is a

well-known inhibitor of protein biosynthesis. We further used cycloheximide to

inhibit the biosynthesis of protein and observed the expression of Bcl-xL in

clivorine-treated cells. We found that in the presence of 200µM cycloheximide

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clivorine (100µM) still significantly decreased Bcl-xL level and cycloheximide did

not inhibit the decrease of Bcl-xL (Fig.6C), indicating that clivorine decreased Bcl-xL

expression not via inhibiting protein biosynthesis.

Ubiquitin-dependent proteolysis mediated by the 26S proteasome and calpain

(calcium-activated neutral protease) are implicated in proteolysis of a number of

proteins not only under normal conditions, but also during apoptosis (Relnstein and

Clechanover, 2006; Sorimachi et al., 1997). To observe whether clivorine-decreased

Bcl-xL is due to protein degradation, L-02 cells were pretreated with 20µM

proteasome inhibitor MG132 and 50µM calpain inhibitor I for 2 h, and then incubated

with 100µM clivorine for 40 h. As shown in Fig.7A MG132 and calpain inhibitor I

significantly rescued clivorine-decreased Bcl-xL protein.

If Bcl-xL is the target of the ubiquitin/proteasome pathway in clivorine treated

hepatocytes, inhibition of the proteasome activity by specific inhibitor MG132 shall

accumulate polyubiquitinated Bcl-xL. L-02 cells were pretreated with MG132 (20µM)

for 2 h, and then incubated with 100µM clivorine for 40 h. As shown in Fig.7B,

Bcl-xL immunoprecipitates from cells treated with clivorine in the presence of

MG132 displayed increases in high molecular weight ubiquitin immunoreative

materials as compared with cells not treated with clivorine.

Effect of proteasome and calpain inhibitors on cell viability and caspase-3/-9

activation induced by clivorine

To further observe the roles of ubiquitin/proteasome and calpain in

clivorine-induced hepatotoxicity, L-02 cells were pretreated with 20µM MG132 and

50µM calpain inhibitor I for 2 h prior to the addition of 100µM clivorine. MG132 and

calpain inhibitor I significantly reversed clivorine-decreased cell viability (Fig.8A).

Western-blot results showed that MG132 and calpain inhibitor I also inhibited

caspase-3/-9 activation induced by clivorine (Fig.8B).

Effects of various PAs on fresh isolated mouse hepatocytes

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We further observed the toxicity of other retronecine-type PAs and clivorine on

mouse hepatocytes. As shown in Fig.9A, all the tested PAs decreased mouse

hepatocytes viability in a concentration-dependent manner after 48 h treatment, and

the IC50 value of clivorine, senecionine, isoline and monocrotaline is about 2.1µM,

7.3µM, 39.6µM and 44.1µM respectively. Further results showed that clivorine

(10µM) and senecionine (100µM) induced apoptotic DNA ladder, caspase-3

activation and decreased anti-apoptotic Bcl-xL in mouse hepatocytes after 48 h

treatment (Fig.9B, C). All the results suggest that clivorine and senecionine induce

apoptosis in mouse hepatocytes, of which the involved toxic mechanisms are same as

clivorine in L-02 cells.

Discussion

In our previous work clivorine induced apoptosis in L-02 cells was reported (Ji et

al., 2005). In this study, our results showed that clivorine induced apoptotic DNA

ladder in L-02 and mouse hepatocytes (Fig.1C, Fig.9B), which further confirmed that

the hepatotoxicity of clivorine was due to inducing apoptosis. Our results also showed

that the toxicity of clivorine on mouse hepatocytes was stronger than L-02 cells

(Fig.1B, Fig.9B), which may be due to the species difference or the enhanced toxicity

of metabolic products in mouse hepatocytes.

In mammalian cells caspase-3 is an important downstream executioner in

apoptosis, and several substrates such as PARP, lamin are cleaved by activated

caspase-3 (Cohen, 1997; Lee et al., 2002). Our results showed that clivorine induced

caspase-3 activation in L-02 and isolated mouse hepatocytes (Fig.2, Fig.9C). The

caspase-3 inhibitor, z-DEVD-fmk significantly inhibited clivorine-induced cell

apoptosis and rescued clivorine-reduced cell viability (Fig.3). These results suggest

that caspase-3 plays an important role in clivorine-induced apoptosis on hepatocytes.

During apoptosis, caspase-3 can be activated by caspase-9 via

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mitochondrial-mediated pathway or by caspase-8 via death-receptor mediated

pathway (Kaufmann and Earnshaw, 2000). Our results in Fig.2 showed that clivorine

induced caspase-9 activation and further results in Fig.3 showed that caspase-9

inhibitor, z-LEHD-fmk, significantly inhibited clivorine-induced cell apoptosis and

rescued clivorine-decreased cell viability, indicating that clivorine may induce

caspase-3 cleavage via activation of caspase-9. To further investigate whether

death-receptor mediated apoptotic pathway is also involved in clivorine-induced

apoptosis, we observed the activation of caspase-8 after 48 h treatment, whose

activation is the hallmark of the death-receptor meditated apoptosis. The caspase

colorimetric assay showed no activation of caspase-8 (data not shown), and the

caspase-8 inhibitor, Ac-IETD-pNA, also had no effect on clivorine-induced cell

apoptosis and clivorine-reduced cell viability (Fig.3). All the results indicate that

death-receptor mediated apoptotic signal pathway may not be involved in

clivorine-induced apoptosis.

The release of cytochrome c from mitochondria plays an important role in

mitochondria triggered apoptosis (Newmeyer and Ferguson-Miller, 2003; Wang,

2001), which promotes the activation of caspase-9 by forming a complex with Apaf-1

in the presence of ATP (Li et al., 1997). In the present study, both cytosolic and

mitochondrial fractions were prepared and the cytosolic translocation of cytochrome c

was detected by Western-blot. Our results demonstrated that clivorine induced

cytochrome c release from mitochondria to cytosol in L-02 cells (Fig.4). These results

further confirmed that mitochondrial-mediated apoptotic pathway was involved in

clivorine-induced apoptosis.

The anti-apoptotic Bcl-xL protein is localized on outer mitochondrial membrane,

and functions to prevent cytochrome c release from mitochondria (Green and Reed,

1998; Terui et al., 1998). In our results, clivorine decreased the level of Bcl-xL protein

in L-02 and isolated mouse hepatocytes (Fig.5, Fig.9C). These results suggest that

clivorine may lead to the release of cytochrome c from mitochondria via decreasing

the level of anti-apoptotic Bcl-xL.

Clivorine decreased the level of Bcl-xL may be due to the inhibition of the gene

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expression or the biosynthesis of Bcl-xL protein. In our results we found that clivorine

had no effect on Bcl-xL gene expression and cycloheximide pretreatment did not

affect clivorine-induced decrease of Bcl-xL protein (Fig.6). These results indicate that

clivorine decreased Bcl-xL protein may not via inhibiting gene expression or protein

biosynthesis and probably through posttranslational modifications.

Ubiquitin is a ubiquitously expressed 76 amino acid protein that can be

covalently attached to target proteins, leading to their ubiquitination, and then the

ubiquitinated proteins can be degraded by the proteasome. Numerous studies have

demonstrated that the ubiquitin/proteasome system plays an important role in

controlling the levels of various cellular proteins involved in cell cycle progression,

apoptosis and cell transformation including Bcl-2 family member such as Bcl-2, Bim,

Bax and Bid (Ciechanover, 2004; Breitschopf et al., 2000; Li and Dou, 2000; Ley et

al., 2003; Chanvorachote et al., 2006), however there is no related report about Bcl-xL.

In our results, we showed that clivorine induced Bcl-xL polyubquitination in the

presence of proteasome specific inhibitor MG132 and clivorine-decreased Bcl-xL

level was enhanced by MG132 (Fig.7A). Further results showed that MG132 also

partially rescued clivorine-reduced cell viability and inhibited clivorine-induced

activation of caspase-3/-9 (Fig.8). Our results suggest that ubiquitin/proteasome plays

important roles in clivorine-induced degradation of Bcl-xL, and which is involved in

clivorine-induced apoptosis. All these results suggest that the ubiquitin/proteasome

system may change the ratio of Bcl-2 family members and therefore alter the

susceptibility of cells to various apoptotic stimuli. It is hoped that in the near future to

identify the enzymes responsible for ubiquitination of Bcl-xL, which could also

provide valuable targets for modulating apoptosis of normal or tumor cells.

Calpain is a family of Ca2+

-dependent cysteine proteases, which has been

implicated in proteolysis of a number of proteins including some Bcl-2 family

members (Gil-Parrado et al., 2002; Goll et al., 2003). Our results showed that

clivorine-decreased Bcl-xL level was enhanced by calpain inhibitor I (Fig.7A),

meanwhile calpain inhibitor I also partially improved clivorine-decreased cell

viability and inhibited clivorine-induced caspase-3/-9 activation (Fig.8). All the

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results indicate that calpain may play some roles in clivorine-induced degradation of

Bcl-xL and hepatocytes apoptosis, however which calpain is involved in the

degradation of Bcl-xL and whether Bcl-xL is the direct substrate of calpain in

clivorine-treated hepatocytes needs further investigation.

It has been reported that PAs like monocrotaline has no toxic effects on cells

lacking of hepatic enzymes (Thomas et al., 1998), and cultured L-02 cells may lose

the hepatic enzymes that's why the other tested PAs have no significant toxicity on

L-02 cells (Fig.1B). Next we observed that other PAs senecionine, monocrotaline and

isoline also decreased mouse hepatocytes viability in a concentration-dependent

manner (Fig.9A). The further study showed that clivorine and senecionine both

induced apoptotic DNA ladder, caspase-3 activation and decreased Bcl-xL (Fig.9B,C),

which indicating that senecionine induced hepatocytes apoptosis and the involved

apoptotic signal pathway is the same as clivorine.

Up to now, more than 400 PAs have been reported for structures elucidation, but

only few of them have been assayed for hepatotoxicity. In the present study, our

results suggest that clivorine induced anti-apoptotic Bcl-xL degradation via

ubiquitin/proteasome and calpain systems, thus regulating the release of cytochrome c,

leading to the activation of caspase-9/caspase-3 signaling cascade and finally

apoptosis, and further results indicate that the involved hepatotoxic mechanisms of

senecionine may be the same as clivorine. Our results define a novel-signaling

pathway for PAs-induced hepatocytes apoptosis and may also provide further

approach for the detoxification of PAs.

Acknowledgements

This work was financially supported by Shanghai Rising-Star program

(07QA14049) and National Natural Science Foundation of China (NSFC) for Key

Project (30530840). We gratefully acknowledge Murad Research Center for

Mordernized Chinese Medicine at Shanghai University of Traditional Chinese

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Medicine for kindly supplying some experimental materials, Ph.D. Beibei Wang and

Zhu Wang for helpful discussion.

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Footnotes

1To whom correspondence should be addressed:

E-mail address: wangzht@hotmail.com, wangzht@shutcm.edu.cn

Telephone: 86-21-51322507 Fax: 86-21-51322519

2Abbreviations used: NTP, National Toxicology Program; PARP, Poly(ADP-ribose)

polymerase; MHRA, British Medicines Healthcare Products Regulatory Agency; PAs,

Pyrrolizidine alkaloids; PBS, phosphate-buffered saline; RPMI1640, Roswell Park

Memorial Institute 1640; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)

2,5-diphenyltetrazolium bromide; DTT, dithiothreitol; Apaf-1, apoptosis protease

activating factor-1; RT-PCR, Reverse transcription-polymerase chain reaction;

GAPDH, glyceraldehydes-3-phosphate dehydrogenase; FACS, fluorescence-activated

cell sorting;

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Figure Legends

Fig.1 (A) The chemical structures of clivorine, senecionine, isoline and monocrotaline.

(B) Effects of various PAs on L-02 cell viability. Cells were treated with various PAs

(10-100µM) for 48 h, and the survival cells were determined by MTT assay. The

results are expressed in percent of control and presented as the means ± SD (n=6),

***p<0.001 versus control. (C) Clivorine induced DNA fragmentation in L-02 cells.

Cells were incubated with or without 100µM clivorine for the indicated times, and

L-02 cells with 1mM H2O2 treatment of 24 h is used as the positive control. After

treatment DNA was extracted (described in Materials and methods) and

electrophoresed on 2% agarose gel containing ethidium bromide.

Fig.2 Clivorine induced caspase-3 and caspase-9 activation in L-02 cells. (A) Cells

were incubated with 100µM clivorine for the indicated times, Pro-caspase-3 and

cleaved caspase-3 were assayed. (B) Cells were incubated with 100µM clivorine for

the indicated times, Pro-caspase-9 and cleaved caspase-9 were assayed. (C) Cells

were incubated with various concentrations of clivorine for 48 h. Pro-caspase-3/-9 and

cleaved caspase-3/-9 were determined by Western-blot analysis, and actin was used

for loading control. Each blot represents one of three independent experiments. (D)

Cells were incubated with clivorine (100µM) for 48 h, caspase-3/-9 activities were

measured as described in Materials and Methods. All values are means ± SD (n=5),

*p<0.05, ***p<0.001 versus control.

Fig.3 Effects of various caspase inhibitors on clivorine induced cell apoptosis and

decreased cell viability. L-02 cells were pretreated with 20µM various caspase

inhibitors for 2 h, and then incubated with clivorine (100µM) for 48 h. (A) Cell

apoptosis was determined by DNA fragmentation. The figure represents one of three

successive experiments. (B) The survival cells were determined by MTT assay. The

results are expressed in percent of control and presented as the means ± SD (n=6),

***p<0.001 versus control, ###

p<0.001 versus clivorine.

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Fig.4 Clivorine induced cytochrome c release. (A) L-02 cells were incubated with

100µM clivorine for the indicated times. (B) L-02 cells were incubated with various

concentrations of clivorine for 48 h. Mitochondrial and cytosolic cytochrome c were

determined by Western-blot analysis. Each blot represents one of three independent

experiments.

Fig.5 Clivorine decreased anti-apoptotic Bcl-xL level. (A) L-02 cells were incubated

with 100µM clivorine for the indicated times. (B) L-02 cells were incubated with

various concentrations of clivorine for 48 h. The expression of Bcl-xL, Bcl-2 and

actin were determined by Western-blot analysis. Each blot represents one of three

independent experiments.

Fig.6 Effects of clivorine on the expression of Bcl-xL mRNA and the biosynthesis of

Bcl-xL protein. (A) L-02 cells were incubated with clivorine (100µM) for the

indicated times, and then the expression of Bcl-xL mRNA was determined by

RT-PCR analysis. (B) Histograms indicate the ratio of Bcl-xL/GAPDH ± SD in

RT-PCR analysis (n=3), and GAPDH is used as a housekeeping gene. (C) L-02 cells

were pretreated with cycloheximide (200µM) for 2 h, and then incubated with 100µM

clivorine for 40 h. The expression of Bcl-xL and actin was determined by

Western-blot analysis. Each blot represents one of two independent experiments.

Fig.7 Effects of clivorine on the degradation of Bcl-xL protein. (A) L-02 cells were

pretreated with proteasome specific inhibitor MG132 (20µM) and Calpain Inhibitor I

(50µM) for 2 h, and then were incubated with 100µM clivorine for 40 h. The

expression of Bcl-xL and actin was determined by Western-blot analysis. (B) L-02

cells were pretreated with MG132 (20µM) for 2 h, and then were incubated with

100µM clivorine for 40 h. Cell extracts were subjected to immunoprecipitation with

anti-Bcl-xL antibody and the conjugates were detected with anti-ubiquitin and

anti-Bcl-xL antibodies. Each blot represents one of three independent experiments.

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Fig.8 Effects of proteasome specific inhibitor MG132 and Calpain inhibitor I on

clivorine-induced L-02 cells apoptosis. (A) Cells were pretreated with MG132 (20µM)

and Calpain Inhibitor I (50µM) for 2 h, and then incubated with clivorine (100µM)

for 48 h. The survival cells were determined by MTT assay. The results are expressed

in percent of control and presented as the means ± SD (n=6), ***p<0.001 versus

control, ###

p<0.001 versus clivorine. (B) L-02 cells were pretreated with MG132

(20µM) and Calpain Inhibitor I (50µM) for 2 h, and then were incubated with 100µM

clivorine for 40 h. Pro-caspase-3/-9 and cleaved caspase-3/-9 were determined by

Western-blot analysis, and actin was used as a loading control. Each blot represents

one of three independent experiments.

Fig.9 Effects of PAs on fresh isolated mouse hepatocytes. (A) Mouse hepatoyctes

were incubated with various concentrations of PAs for 48 h, and the survival cells

were determined by MTT assay. The results are expressed in percent of control and

presented as the means ± SD (n=5), **p<0.01, ***p<0.001 versus control. (B) Mouse

hepatocytes were incubated with clivorine (10µM) or senecionine (100µM) for 48 h,

and cell apoptosis was determined by DNA fragmentation. (C) Mouse hepatocytes

were incubated with clivorine (10µM) or senecionine (100µM) for 48 h, and then the

expression of pro-caspase-3, cleaved caspase-3, Bcl-xL and actin were determined by

Western-blot analysis. Each blot represents one of two independent experiments.

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Fig.1

A.

B.

0 10 25 50 100

Concentration, µM

Cell Via

bility (%

of Control)

0

20

40

60

80

100

120

Clivorine

Senecionine

Isoline

Monocrotaline

******

C.

Clivorine Senecionine Isoline Monocrotaline

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Fig.2

A.

B.

C.

D.

36 KDa

27 KDa

21 KDa

Pro-caspase-3 (35KDa)

Cleaved caspase-3 (19KDa)

Cleaved caspase-3 (17KDa)

0 8 12 16 24 32 40 48 Times/h

36 KDa Actin (43KDa)

47 KDa

36 KDa

Pro-caspase-9 (47KDa)

Cleaved caspase-9 (35KDa)

Cleaved caspase-9 (37KDa)

Actin (43KDa)

0 16 24 32 40 48 Times/h

36 KDa

Caspase-3 Caspase-9

Caspase A

ctivity (%

of Control)

0

50

100

150

200

250

300

Control

Clivorine

***

*

36 KDa

27 KDa

21 KDa

Pro-caspase-3 (35KDa)

Cleaved caspase-3 (19KDa)

Cleaved caspase-3 (17KDa)

36 KDa Actin (43KDa)

0 100 30 10 3 1 µM

47 KDa

36 KDa Cleaved caspase-9 (37KDa)

Pro-caspase-9 (47KDa)

Cleaved caspase-9 (35KDa)

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Fig.3

A.

B.

Clivorine + + + + + -

z-VAD - - - + - -

Ac-IETD - - + - - -

z-LEHD - + - - - -

z-DEVD + - - - - - DNA Marker

1000bp

900bp

500bp

300bp

200bp

100bp

Clivorine - + + + + + z-VAD - - + - - - Ac-IETD - - - + - - z-DEVD - - - - + - z-LEHD - - - - - +

Cell Viability (%

of Control)

0.0

20.0

40.0

60.0

80.0

100.0

120.0

***

###

######

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Fig.4

A.

B.

21KDa

Cyto C (cytosolic, 14KDa)

21KDa Cyto C (mitochondrial, 14KDa)

21KDa Cyto C (cytosolic, 14KDa)

21KDa

Cyto C (mitochondrial, 14KDa)

0 8 12 16 24 32 40 48 Times/h

0 100 30 10 3 µM

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Fig.5

A.

36KDa

27KDa

Bcl-xL (30KDa)

27KDa Bcl-2α/β(25/22KDa)

36KDa Actin (43KDa)

0 8 12 16 24 32 40 48 Times/h

B.

36KDa Bcl-xL (30KDa)

0 100 30 10 3 1 µM

36KDa Actin (43KDa)

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Fig.6

A.

B.

C.

0 6 12 24 36 48 Times/h

Bcl-xL

GAPDH

0 6 12 24 36 48 Times/h

The ratio o

f Bcl-xL/G

APDH

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Bcl-xL (30KDa)

Actin (43KDa)

36KDa

36KDa

Clivorine - + + -

Cycloheximide - - + +

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Fig.7

A.

B.

117KDa

85KDa

49KDa

IP: anti-Bcl-xL

WB: anti-Ubiquitin

IP: anti-Bcl-xL

WB: anti-Bcl-xL

85KDa

49KDa

34KDa

IgG

Bcl-xL (30KDa)

Poly-Ub

Bcl-xL

Clivorine - +

MG132 + +

IgG

Bcl-xL (30KDa) 36KDa

36KDa Actin (43KDa)

Clivorine - + + + - -

MG132 - - + - + -

Calpain Inhibitor I - - - + - +

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Fig.8

A.

B.

Pro-caspase-3 (35KDa)

Cleaved caspase-3 (19KDa) 27KDa

36KDa

21KDa Cleaved caspase-3 (17KDa)

Pro-caspase-9 (47KDa)

Cleaved caspase-9 (35KDa) 36KDa

47KDa Cleaved caspase-9 (37KDa)

Actin (43KDa)

Clivorine - + + +

MG132 - - + -

Calpain Inhibitor I - - - +

36KDa

Clivorine - + + +MG132 - - - +Calpain Inhibitor I - - + -

Cell Via

bility (%

of Control)

0

20

40

60

80

100

120

***

###

###

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Fig.9

A.

B.

C.

Marker Cont Cliv

0 1 10 25 50 100

Concentration, µM

Cell Via

bility

(%

of Control)

0

20

40

60

80

100

120

Clivorine

Senecionine

Isoline

Monocrotaline

**

*** *** *** ***

*** ****** ***

******

***

***

***

***

Pro-caspase-3 (35KDa)

Cleaved caspase-3 (19KDa)

36KDa

27KDa

21KDa

36KDa Bcl-xL (30KDa)

49KDa Actin (43KDa)

Cont Cliv Cont Sene