Caspase-8 acts as key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

(1)

Caspase-8 acts as a key upstream executor of mitochondria during

justicidin A-induced apoptosis in human hepatoma cells

Chun-Li Su

a,1

, Lynn L.H. Huang

b,1

, Li-Min Huang

b

, Jenq-Chang Lee

c

,

Chun-Nan Lin

d

, Shen-Jeu Won

e,*

a

Department of Nursing, Chang Jung Christian University, Tainan 711, Taiwan

b_{Institute of Biotechnology, College of Science, National Cheng Kung University, Tainan 701, Taiwan} c

Department of Surgery, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan

d_{School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan} e

Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan Received 14 February 2006; revised 25 April 2006; accepted 27 April 2006

Available online 4 May 2006 Edited by Vladimir Skulachev

Abstract

Justicia procumbens is a traditional Taiwanese herbal

remedy used to treat fever, pain, and cancer. Justicidin A,

iso-lated from Justicia procumbens, has been reported to suppress

in vitro growth of several tumor cell lines as well as hepatoma

cells. In this study, justicidin A activated caspase-8 to increase

tBid, disrupted mitochondrial membrane potential (Dw

m

), and

caused the release of cytochrome c and Smac/DIABLO in Hep

3B and Hep G2 cells. Justicidin A also reduced Bcl-x

L

and

in-creased Bax and Bak in mitochondria. Caspase-8 inhibitor

(Z-IETD) attenuated the justicidin A-induced disruption of Dw

m

.

Growth of Hep 3B implanted in NOD-SCID mice was

sup-pressed signiﬁcantly by oral justicidin A (20 mg/kg/day). These

results indicate that justicidin A-induced apoptosis in these cells

proceeds via caspase-8 and is followed by mitochondrial

disrup-tion.

Supplementary materials

are available at

http://myweb.

ncku.edu.tw/~a725/

.

2006 Federation of European Biochemical Societies. Published

by Elsevier B.V. All rights reserved.

Keywords: Justicidin A; Hepatoma; Apoptosis; Caspase;

Mitochondria; Tumor growth

1. Introduction

Human hepatocellular carcinoma (HCC) is the ﬁfth most

frequent cancer and is the third most common cause of

can-cer-related death worldwide

[1,2]

. In Taiwan, HCC is also a

leading malignant neoplasm

[3]

. Unfortunately, it does not

re-spond well to chemotherapy and has a poor prognosis

[4,5]

. To

develop a more eﬀective chemotherapeutic agent for this

dis-ease, we concentrated our eﬀorts on natural compounds

tradi-tionally used to treat the disease. Justicia procumbens (J.

procumbens) is a traditional herbal remedy in Taiwan for fever,

pain, and cancer

[6,7]

. Justicidin A, puriﬁed from a methanolic

extract of J. procumbens, has various biological activities

including suppression of tumor cell growth

[8,9]

and release

of TNF-a

[10]

. The purpose of present study was to determine

whether apoptosis is stimulated by justicidin A-treatment of

human HCC. Two major apoptotic pathways (intrinsic and

extrinsic) have been concluded. The intrinsic apoptotic

path-way operates via mitochondria

[11]

. The extrinsic apoptotic

pathway activates caspase-8 and its downstream regulators

[12]

. The results of this study reveal that justicidin A induces

both intrinsic and extrinsic apoptotic pathways in HCC cells

since both caspase-8 and mitochondria are aﬀected. Induction

of apoptosis is characterized by phosphatidylserine

external-ization, accumulation of sub-G

1

cells, and DNA

fragmenta-tion. Activation of caspase-8 increases the amount of tBid to

change mitochondrial membrane potential (Dw

m

), which in

turn causes the release of cytochrome c and second

mitochon-dria-derived activator of caspase/direct IAP binding protein

with low pI (Smac/DIABLO) from mitochondria to activate

caspase-9 and caspase-3. The increase of Bax and Bak and

de-crease of Bcl-x

L

in mitochondria further promote the process

of apoptosis. The cytotoxicity of justicidin A is also eﬀective

in vivo.

2. Materials and methods

2.1. Materials

Justicidin A was isolated from J. procumbens plants[9]. DiOC6(3)

was obtained from Molecular Probes (Eugene, OR). Anti-cytochrome c mouse monoclonal antibody (mAB) and Annexin V–FITC were pur-chased from BD Pharmingen (San Diego, CA). Anti-caspase-3 mouse mAB and anti-Smac/DIABLO rabbit polyclonal antibody (pAB) were purchased from IMGENEX (San Diego, CA). Anti-caspase-8 mouse mAB and anti-poly(ADP-ribose) polymerase (PARP) rabbit pAB were purchased from Cell Signaling Technology (Beverly, MA). Anti-caspase-9 mouse mAB was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-receptors for activated C-kinase (RACK1) mouse mAB was purchased from BD Transduction Laboratories (Lex-ington, KY). Z-IETD, Z-LEHD, anti-Bax and anti-Bcl-xL mouse

mAB, Bak, DNA fragmentation factor (DFF) 45 and anti-DFF40 rabbit pAB, and goat anti-mouse conjugated HRP secondary antibody were purchased from Santa Cruz Biotech (Santa Cruz, CA). Abbreviations: HCC, hepatocellular carcinoma; PBMC, peripheral

blood mononuclear cells; Smac/DIABLO, second mitochondria-derived activator of caspase/direct IAP binding protein with low pI; PARP, poly(ADP-ribose) polymerase; DFF, DNA fragmentation factor; Dwm, mitochondrial membrane potential; XIAP, X-linked

apoptosis-inhibiting protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mAB, monoclonal antibody; pAB, polyclonal antibody

*_{Corresponding author. Fax: +886 6 2082705.}

E-mail address:[email protected](S.-J. Won).

1

Chun-Li Su and Lynn L.H. Huang contributed equally to this work.

(2)

Goat anti-rabbit conjugated HRP secondary antibody was purchased from Amersham Pharmacia Biotech (Quebec, Canada). Other reagents were purchased from Sigma (St. Louis, MO).

2.2. Cell cultures

Human HCC (Hep 3B and Hep G2 cells), and Chang liver cells from American Type Culture Collection (ATCC, Rockville, MD), were maintained in complete Dulbecco’s modiﬁed Eagle medium (DMEM; GIBCO BRL, Grand Island, NY). Human peripheral blood mononu-clear cells (PBMC) were isolated from healthy donors’ whole blood (Tainan Blood Bank Center, Tainan, Taiwan) by centrifugation over a Ficoll-Paque (Amersham Pharmacia, Uppsala, Sweden) density gra-dient at 400· g for 30 min in a Sorvall RT6000B (Du Pont, Wilming-ton, DE)[13]. The cells collected at the interface were washed thrice with serum-free RPMI-1640 (GIBCO BRL, Grand Island, NY) and subsequently resuspended in complete DMEM.

2.3. Cell viability assay

Cytotoxicity was determined using a modified 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [13]. Cells on 96-well plates (Nunc, Roskilde, Denmark) were treated with different concentrations of the test agent for 6 days. After addi-tion of 10 ll MTT to a final concentraaddi-tion of 0.5 mg/ml, cells were incubated at 37C for 4 h. After adding 100 ll of 10% SDS/0.01 N HCl, cells were left overnight at 37C. The absorbance of each well was measured at 590 nm in a Multiscan photometer (MRX II, Dyna-tech, McLean, VA).

2.4. Flow cytometric detection of phosphatidylserine exposure and cell cycle distribution

Cells were trypsinized, and resuspended in HEPES buffer solution (HBS) containing 1.25% (v/v) of Annexin V–FITC to stain phosphati-dylserine on the cell surface[14]. Stained cells were analyzed in a FAC-Scan flow cytometer (Becton Dickinson, Mountain View, CA)[15]. For cell cycle distribution analysis, cells were washed with HBS and resuspended in 70% ethanol at 4C. After centrifugation at 800 · g for 10 min, cells were resuspended in HBS containing 40 lg/ml propi-dium iodide (PI) and 0.1% NP-40 for flow cytometric analysis[16,17]. 2.5. Analysis of DNA fragmentation

Extraction and electrophoresis of DNA were performed as described [13]. Cells were incubated with lysis buffer (10 mM Tris–HCI [pH 7.6], 20 mM EDTA, and 1% NP-40) for 20 min at 37C. After centrifuga-tion, the supernatants were incubated with 50 ll of RNase A (20 mg/ ml) and 20 ll of SDS (10%) at 56C for 2 h. Proteinase K (35 ll of 20 mg/ml) was mixed with the cell lysates and incubated for another 2 h at 37C. Precipitated DNA fragments were resuspended in 15 ll of Tris–EDTA buffer, and separated by electrophoresis on a 1% (w/ v) agarose gel in TBE buffer. The patterns of DNA ladders were exam-ined after staining with ethidium bromide and under UV light. 2.6. Measurement of Dwm

Change in Dwm was determined by flow cytometry using the mito-chondria-sensitive dye rhodamine 123 in the dark[16,18]. After treat-ment, cells were stained with 5 lM rhodamine 123 for 30 min. After mixing with 2.5 lg/ml of PI, the stained cells were subjected to flow cytometry, and the data were analyzed using CellQuest software. Change of Dwm was also determined by laser scanning confocal microscopy (Leica TCS-SP2, Germany)[16,19]. Cells were seeded in 6-well plates containing methanol-sterilized glass cover slips. After treatment, the cells were stained with 5 lM rhodamine 123 at 37C, washed twice with PBS at room temperature, fixed with 4% parafor-maldehyde for 15 min, and then examined using a Leica TCSNT laser scanning confocal imaging system coupled to a Leica DMRBE micro-scope with a Leica 630 fluotar objective.

2.7. Subcellular fractionation and Western blot analysis Whole cells (1· 106

) were mixed with 200 ll of lysis buffer and then centrifuged at 15 000· g for 10 min[16]. The supernatant was used as total protein for immunoblotting. The cytosolic, mitochondrial, and nuclear proteins were prepared as previously reported [16,20,21]. Briefly, harvested cells (1· 106_{) were resuspended in TSE buffer}

(10 mM Tris, 0.25 M sucrose, and 0.1 mM EDTA [pH 7.4]), and homogenized with 10 strokes in a Dounce homogenizer (Glas-Col, Terre Haute, IN) using a Teﬂon pestle. After removing the cell debris by centrifuging the homogenates at 750· g at 4 C for 30 min, the supernatants were centrifuged at 12 000· g at 4 C for 30 min. The supernatants were centrifuged again at 100 000· g for another 1 h. The resulting supernatants were used as cytosolic fractions, and the resulting pellets were lysed with lysis buﬀer and used as mitochondrial fractions. For nuclear extract preparation, cells (1· 107_{) were lysed in}

400 ll of buﬀer A (10 mM HEPES [pH 7.9], 5 mM MgCl2, 10 mM

KCl, 3 mM Na3VO4, 10 mM NaF, 0.5 mM dithiothreitol, 0.5 mM

phenylmethylsulfonyl ﬂuoride, and 2 lg/ml of leupeptin, antipain, aprotinin, and pepstatin A) on ice for 20 min. After centrifugation at 11 000· g for 20 s at 4 C, the pellets were resuspended in 60 ll of buf-fer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM

EDTA, 25% glycerol, 1 mM Na3VO4, 10 mM NaF, 0.5 mM

dithio-threitol, 0.5 mM phenylmethylsulfonyl ﬂuoride, and 1 lg/ml each of leupeptin, antipain, aprotinin, and pepstatin A) for 15 min on ice with occasional mixing. Nuclear debris was removed by centrifugation again at 12 000· g for 15 min at 4 C. All isolated proteins were stored at70 C before immunoblotting analysis[22].

2.8. Animal study

NOD.CB17-PRKDCÆSCIDæ/J (NOD-SCID) mice were bred and maintained at the Animal Center of National Cheng Kung University (NCKU, Tainan, Taiwan) in a speciﬁc pathogen-free environment. Mice at 6–7 weeks of age were used in the experiments as described previously[16]. Food and water were provided ad libitum. Tumor vol-ume was measured using calipers (2–3 times/week)[23].

2.9. Statistical analysis

All of the experimental data are expressed as means ± S.E.M. Diﬀer-ences in tumor volumes were determined by Student’s t test using the Minitab (version 10.2) software package. We assigned statistical signif-icance if P < 0.05.

3. Results

3.1. Growth inhibition and apoptosis of justicidin A-treated cells

Low dosages of justicidin A suppressed the viability of HCC

Hep 3B and Hep G2 cells, and the IC

50

at day 6 was

0.048 ± 0.020 and 0.052 ± 0.050 lM, respectively. The IC

50

of justicidin A for non-malignant Chang liver cells was

0.95 ± 0.12 lM, which is at least 10-fold higher than the IC

50

of justicidin A for HCC cells. Human PBMC were much more

resistant to justicidin A treatment with an IC

50

of 23 ± 1 lM.

To examine the possible mechanism of justicidin A on cell

via-bility, three parameters of apoptosis (exposure of

phosphati-dylserine, cell cycle redistribution and DNA fragmentation)

were analyzed. As shown in

Fig. 1

A, the percentage of

Annex-in V–FITC positive cells were Annex-increased signiﬁcantly Annex-in a

dos-age- and time-related manner. Dose-related elevations in the

sub-G

1

fraction of both tumor cells were also observed (

Sup-plementary Fig. 1

). Justicidin A also caused time- and

dose-related enhancement of apoptotic DNA fragmentation in these

cells (data not shown).

3.2. Activation of caspases and involvement of mitochondria in

justicidin A-induced apoptosis

The process of apoptosis involves a cascade of proteolytic

activity, much of it carried out by caspases

[21]

. In this study,

both procaspase-8 (

Fig. 1

B) and procaspase-9 (

Supplementary

Fig. 2

) were cleaved into their active forms in a time-related

manner. Cleavage of their downstream molecule

procaspase-3 was also revealed (

Supplementary Fig. 2

). Since PARP and

DFF are substrates of activated caspase-3

[24–26]

, increase

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Fig. 1. Induction of apoptosis and expression of caspase-8, cytochrome c and Smac/DIABLO in hepatoma cells in response to justicidin A. Externalization of phosphatidylserine in Hep 3B and Hep G2 cells (A). After treatment with the indicated concentrations of justicidin A for 48 h or with 1 lM of justicidin A for the indicated time periods, cells (2· 105

) stained with Annexin V–FITC were analyzed by ﬂow cytometry. The percentages in the ﬁgure indicate the proportion of apoptotic cells with externalization of phosphatidylserine. Activation of caspase-8, and release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells (B). Total proteins from justicidin A-treated cells were subjected to Western blot analysis to determine the activation of procaspase-8 by using anti-caspase-8 mouse mAB. RACK1 was served as a loading control. Blots of the cytosol- and mitochondria-enriched fractions were used to demonstrate the translocation of cytochrome c and Smac/DIABLO using anti-cytochrome c mouse mAB or anti-Smac/DIABLO rabbit pAB. Relative protein expression is shown at the bottom of each panel, with control levels arbitrarily set to 1. JA, justicidin A.

(4)

in the cleavage PARP and nuclear DFF40 (

Supplementary

Fig. 2

) displayed the enzymatic activity of caspase-3. The

involvement of caspase-8 and caspase-9 in the process of

apop-tosis was conﬁrmed by showing that the presence of caspase-8

inhibitor (Z-IETD) or caspase-9 inhibitor (Z-LEHD) reduced

the number of justicidin A treated cells in the sub-G

1

phase

(

Supplementary Fig. 3

).

Activation of caspase-9 (

Supplementary Fig. 2

) implied the

involvement of mitochondria in justicidin A-induced

apopto-sis. Maintenance of Dw

m

is important for normal function

and survival of cells

[27]

. Changes of Dw

m

can cause the

re-lease of apoptogenic proteins. In this study, the time-related

release of cytochrome c and Smac/DIABLO from

mitochon-dria to cytosol (

Fig. 1

B) further demonstrated the pivotal role

of mitochondria in justicidin A-induced apoptosis.

Con-versely, mitochondrial cytochrome c decreased gradually in

both cells (

Fig. 1

B). Similar patterns of increased cytosolic

and decreased mitochondrial Smac/DIABLO were observed

(

Fig. 1

B). Anti-X-linked apoptosis-inhibiting protein (XIAP),

an antagonist of caspase-3 and caspase-9

[28]

, was reported

to be antagonized by cytosolic Smac/DIABLO

[29]

. Decrease

in XIAP expression (data not shown) may therefore favor

the increase in caspase-9 and -3 activities (

Supplementary

Fig. 2

). Involvement of mitochondria was also conﬁrmed by

changes in Dwm upon justicidin A stimulation. A dose- and

time-related decrease in the intensity of rhodamine 123

ﬂuorescence was detected in the mitochondria of justicidin

A-treated Hep 3B cells (

Supplementary Fig. 4

). A similar

time-related decrease in ﬂuorescence intensity was observed

in Hep G2 cells (data not shown). Confocal microscopy also

showed that justicidin A induced Dwm. Decrease in

ﬂuores-cence emission was found in both tumor cells after justicidin

A treatment (

Fig. 2

A).

Fig. 2

A reveals the relationship between mitochondria and

caspase-8. In both cells, Z-IETD (an inhibitor of caspase-8)

alleviated the justicidin A-induced decrease in mitochondrial

ﬂuorescence signals (

Fig. 2

A) and justicidin A-induced

in-crease in the number of apoptotic cells in the sub-G

1

fraction

(

Supplementary Fig. 3

). Similar results were observed when

cells were treated with justicidin A plus cyclosporin A, a

per-meability transition pore inhibitor that blocks the release of

cytochrome c from mitochondria (

Fig. 2

A).

3.3. Involvement of Bcl-2 family in justicidin A-induced

apoptosis

Members of Bcl-2 family regulate apoptosis by interacting

with mitochondria

[28]

. Bcl-2 and Bcl-x

L

protect against

mito-chondrial dysfunction and therefore inhibit apoptosis. In

con-trast, Bid, Bax, and Bak induce Dwm and thus promote

apoptosis. To test the involvement of the proteins of the

Bcl-2 family in justicidin A-induced apoptosis, total cell lysates,

and cytosolic and mitochondrial fractions of justicidin

A-treated tumor cells were prepared. In

Fig. 2

B, justicidin A

signiﬁcantly decreased total and mitochondrial Bcl-x

L

.

Trans-location of Bax was also observed in these two cells after

treatment of justicidin A (

Fig. 2

B). The expression of cytosolic

Bax decreased and the mitochondrial Bax increased (

Fig. 2

B).

In Hep 3B cells, mitochondrial Bak expression increased a

small amount, whereas, in Hep G2 cells it increased markedly

(

Fig. 2

B). Cytosolic Bid was decreased after treatment with

justicidin A (

Fig. 2

B). The translocation of the tBid fragment

to mitochondria began at 6 h of justicidin A treatment, kept

increasing between 12 and 24 h, and peaked at 72 h in Hep

3B cells or at 48 h in Hep G2 cells (

Fig. 2

B).

3.4. Tumor growth in mice

To examine the antitumor eﬀect of justicidin A in vivo,

Hep 3B cells were implanted in mice before they were fed

jus-ticidin A (20 mg/kg/day) for 60 consecutive days. As shown

in

Fig. 3

, the tumors were sensitive to justicidin A, and their

growth was halted throughout the period of justicidin A

administration.

4. Discussion

Our experimental ﬁndings suggest the following signaling

cascades in justicidin A-treated HCC. At 6 h of justicidin A

treatment, activation of caspase-8 (

Fig. 1

B) triggers the

cleav-age of Bid into tBid and causes the translocation of tBid to

mitochondria (

Fig. 2

B). Mitochondrial tBid may oligomerize

with the mitochondrial Bax (

Fig. 2

B) and Bak (

Fig. 2

B) (both

proteins ﬁrst increase in the mitochondria at 6 h of justicidin A

treatment) to damage Dwm (

Fig. 2

A and

Supplementary

Fig. 4

) and result in releasing cytochrome c and

Smac/DIA-BLO into the cytosol at a later time point (at 24 h in Hep 3B

and 48 h in Hep G2 cells) (

Fig. 1

B). Since Bcl-x

L

can bind to

Bax and prevent Bax insertion into the outer membrane of

mitochondria

[30]

, the decrease in total and mitochondrial

Bcl-x

L

(

Fig. 2

B) promotes the changes of Dwm. The released

cytochrome c may contribute to the formation of apoptosomes

in the cytosol to activate caspase-9 (

Supplementary Fig. 2

).

Decrease in XIAP (data not shown), because of interaction

with the cytosolic Smac/DIABLO, may further increase

apop-tosome formation and therefore facilitate caspase-9 activation

[28]

. The activation of caspase-8 or caspase-9 subsequently

activates caspase-3 (

Supplementary Fig. 2

). The activated

cas-pase-3 then cleaves PARP (

Supplementary Fig. 2

). Since

PARP participates in DNA repair mechanism

[31,32]

, the

in-crease in cleaved PARP disables the function of DNA repair

in both cells. The activated caspase-3 also cleaves DFF45

(

Supplementary Fig. 2

). Since DFF45 can bind DFF40 to

pre-vent DFF40-mediated DNA fragmentation

[33]

, the decrease

in DFF45 allows release of DFF40 into the nucleus. The

in-crease in nuclear DFF40 (

Supplementary Fig. 2

) may result

in the formation of DNA ladders in justicidin A-treated

HCC (data not shown). Greater increase in the level of cleaved

PARP than nuclear DFF40 (

Supplementary Fig. 2

) in Hep 3B

cells suggests that PARP is the more important determinant of

DNA fragmentation in these cells. The small increase in

pro-apoptotic Bak (

Fig. 2

B) in Hep 3B cells suggests that caspase

activation and Bax-dependent release of mitochondrial

apop-togenic proteins are suﬃcient to increase the level of cleaved

PARP and nuclear DFF40 (

Supplementary Fig. 2

) and thereby

to provoke Hep 3B cell death. Our results indicate that Hep

G2 but not Hep 3B cells express Bcl-2 (data not shown). Since

Bcl-2 protein has been reported to inhibit caspase-8 activity

[34,35]

, the greater increase in cleaved caspase-8 in Hep 3B

cells (

Fig. 1

B) may be explained. Greater cleavage of

procas-pase-8 (

Fig. 1

B) may result in more predominant increase in

mitochondrial tBid and tBid-induced translocation of Bax

(

Fig. 2

B), and thereby lead to the greater increase in cleaved

caspase-3 (

Supplementary Fig. 2

) in Hep 3B cells. Recently,

Bak has been reported to be a mitochondrial membrane

(5)

Fig. 2. Changes of Dwm and expressions of proteins involved in mitochondria-dependent apoptosis induced by justicidin A treatment. Inhibitory eﬀects of Z-IETD or cyclosporin A on Dwm and on the percentage of cells in the sub-G1fraction were evaluated respectively by confocal microscopy

and ﬂow cytometry (A). In the experiment, cells were pretreated with 20 lM of Z-IETD or 10 lM of cyclosporin A for 4 h prior to the addition of justicidin A (1 lM) for 30 h. Cells were either stained with rhodamine 123 for confocal microscopy or stained with PI for ﬂow cytometry. Expression of death related proteins in justicidin A-treated hepatoma cells (B). Blots were developed with anti-Bcl-xLor anti-Bax mouse mAB, or with anti-Bak

(6)

tein. In this study, mitochondrial Bak expression increases

upon justicidin A treatment in HCC cells. Similar result was

also reported in Hep G2 cells

[36]

. The predominant increase

in mitochondrial Bak in Hep G2 than in Hep 3B cells may

be due to the greater increase in total Bak protein expression

in Hep G2 cells upon justicidin A stimulation (data not

shown). However, the mitochondrial Bak in Hep G2 cells

might not all be in oligomerized form for cytochrome c release

(

Fig. 1

B). Of note, the control of caspase-8 inhibitor (Z-IETD)

on Dwm (

Fig. 2

A) indicates that caspase-8 is an upstream

reg-ulator of mitochondria in justicidin A-induced apoptosis. The

total blockage of justicidin A-induced apoptosis by either

cyclosporine A (

Fig. 2

A) or caspase-9 inhibitor (Z-LEHD)

(

Supplementary Fig. 3

) demonstrates that this apoptotic

pro-cess is mitochondria- and caspase-9-dependent, and the direct

activation of caspase-3 by caspase-8 only plays a minor role in

justicidin A-induced apoptosis.

Both the intrinsic and extrinsic pathways were induced in

the HCC cells since both caspase-8 (

Fig. 1

B) and

mitochon-dria (

Fig. 2

A and

Supplementary Fig. 4

) were aﬀected by

jus-ticidin A. Our previous experiments indicated that only the

intrinsic pathway is stimulated by justicidin A in colorectal

carcinoma cells

[16]

, in which, caspase-8 was not activated

in either HT-29 or HCT 116 cells. The justicidin A-induced

apoptotic pathway in colorectal carcinoma cells begins with

the suppression of Ku70, which causes the translocation of

Bax to mitochondria. The change of Dwm causes the release

of apoptogens (cytochrome c and Smac/DIABLO) to further

activate their downstream regulators. In contrast, in these

HCC cells, alteration of Ku70 expression was not detected

(data not shown).

In conclusion, justicidin A inhibits the growth of HCC cells

in vitro and in vivo. Induction of apoptosis is the result of

ticidin A cytotoxicity. The lower sensitivity of PBMC to

jus-ticidin A illustrates that this natural compound, jusjus-ticidin A,

is selective against malignant cells.

Acknowledgement: This work was supported by a grant from National Science Council, Taipei, Taiwan, ROC (NSC 91-2320-B-006-075).

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at

doi:10.1016/j.febslet.2006.

04.085 .

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