ORIGINAL PAPER

Danthron Triggers ROS and Mitochondria-Mediated ApoptoticDeath in C6 Rat Glioma Cells Through Caspase Cascades,Apoptosis-Inducing Factor and Endonuclease G MultipleSignaling

Shang-Ming Chiou • Chiz-Hao Chiu •

Su-Tso Yang • Jai-Sing Yang • Hui-Ying Huang •

Chao-Lin Kuo • Po-Yuan Chen • Jing-Gung Chung

Received: 26 January 2012 / Revised: 2 April 2012 / Accepted: 27 April 2012 / Published online: 17 May 2012

� Springer Science+Business Media, LLC 2012

Abstract This research focused on the induction of

cytotoxic effects by danthron, a natural anthraquinone

derivative on C6 rat glioma cells through exploring the

means of cell death and the effects on mitochondrial

function. We found that danthron decreased the percentage

of viable C6 cells and induced cell morphological changes

in a dose-and time-dependent manner. The morphological

and nuclei changes (DAPI staining) in C6 cells were

observed using a contrast-microscope and fluorescence

microscopy, respectively. The results suggest that cell

death of C6 cells which are induced by danthron is closely

related to apoptotic death. Danthron decreased the level of

mitochondrial membrane potential (DWm), stimulated the

release of cytochrome c from mitochondria to cytosol and

promoted the levels of caspase-9 and caspase-3, or induced

the release of AIF and Endo G from mitochondria. Based

on both observations, we suggest that the danthron-pro-

voked apoptotic death of C6 cells is mediated through the

mitochondria-dependent pathway. Furthermore, our results

also indicated that danthron triggered apoptosis through

reactive oxygen species (ROS) production which were

increased after 1 h exposure of danthron, which was

reversed by the ROS scavenger N-acetyl-L-cysteine (NAC).

As a consequence, danthron-mediated cell death of C6 cells

via ROS production, mitochondrial transmembrane poten-

tial collapse and releases of cytochrome c, AIF and Endo

G. Taken together, danthron was demonstrated to bePo-Yuan Chen and Jing-Gung Chung made equal contribution to this

work.

S.-M. Chiou

Department of Functional Neurosurgery, Gamma Knife Center,

China Medical University Hospital, Taichung 404, Taiwan

C.-H. Chiu � P.-Y. Chen � J.-G. Chung

Department of Biological Science and Technology,

China Medical University, Taichung 404, Taiwan

S.-T. Yang

Department of Radiology, China Medical University Hospital,

Taichung 404, Taiwan

S.-T. Yang

School of Chinese Medicine, China Medical University,

Taichung 404, Taiwan

J.-S. Yang

Department of Pharmacology,

China Medical University, Taichung 404, Taiwan

H.-Y. Huang

Department of Nutrition, China Medical University,

Taichung 404, Taiwan

C.-L. Kuo

School of Chinese Pharmaceutical Sciences and Chinese

Medicine Resources, China Medical University, Taichung 404,

Taiwan

P.-Y. Chen (&) � J.-G. Chung (&)

Department of Biological Science and Technology,

China Medical University, No 91, Hsueh-Shih Road,

Taichung 40402, Taiwan

e-mail: pychen@mail.cmu.edu.tw

J.-G. Chung

e-mail: jgchung@mail.cmu.edu.tw

J.-G. Chung

Department of Biotechnology, Asia University,

Taichung 413, Taiwan

123

Neurochem Res (2012) 37:1790–1800

DOI 10.1007/s11064-012-0792-3

effective in killing C6 rat glioma cells via the ROS-pro-

moted and mitochondria-dependent apoptotic pathways.

Keywords Danthron � C6 rat glioma cells � Apoptosis �ROS � Caspase cascades � Mitochondria

Abbreviations

DWm Mitochondrial membrane potential

AIF Apoptosis-inducing factor

DAPI 40,6-Diamidino-2-phenylindole

DMSO Dimethyl sulfoxide

Endo G Endonuclease G

FBS Fetal bovine serum

GRP78 Glucose-regulated proteins 78

PARP Poly ADP ribose polymerase

PBS Phosphate buffer saline

PI Propidium iodide

ROS Reactive oxygen species

SDS-PAGE Sodium dodecylsulfate polyacrylamide gel

electrophoresis

Introduction

Brain tumor is one of the most threatening malignant

cancers in humans today and notoriously hard to treat.

Glioma is a tumor of dismal prognosis and ultimately

progresses to form high-grade malignancies with local

infiltration of the brain [1, 2]. Unfortunately, high-grade

glioma is difficult and their prognosis is low. The treatment

of high-grade glioma is limited for resection followed by

radio/chemotherapy, resulting in a median survival of

14 months [3]. Currently, the treatments for glioma include

surgery, radiotherapy, chemotherapy or a combination of

multiple modalities but the results are still not satisfactory.

The new for development of new treatment strategies for

glioma patients is urgent. The current clinical anticancer

drugs have been demonstrated to cause cell death through

interfering the processes of the cell cycle and induction of

apoptosis [4]. Therefore, one of the best strategies for

chemoprevention or chemotherapy of agents is to induce

apoptosis in cancer cells [5–9].

Danthron (1,8-dihydroxyanthraquinone), an anthraqui-

none derivative, is a possible human carcinogen (IARC

2B), and has been withdrawn from the market as a laxative

by the US Food and Drug Administration (FDA) [10–13].

Recently, it was reported that the oxidative stress may be a

major contributing pathway in the genotoxicity of danthron

[14]. It was reported that danthron functions as a retinoic X

receptor antagonist by stabilizing tetramers of the receptor

[15]. In our laboratory, we have reported that danthron not

only induced DNA damage and inhibited DNA repair

genes expression [16] but triggered apoptosis [17] in

human brain glioblastoma multiforms GBM 8401 cancer

cells. Our report also found that danthron-induced DNA

damage and apoptotic cell death was involved in mito-

chondrial depolarization and caused the activation of cas-

pase cascades in SNU-1 human gastric cancer cells [5].

Until now, there is no information to address danthron-

induced cell death (apoptosis) in C6 rat glioma cells. In the

present study, we try to examine whether danthron inhibits

the growth and induces apoptosis in C6 cells. Our finding

provides insight into the mechanism of danthron-induced

mitochondria-mediated apoptosis via caspase cascades,

apoptosis-inducing factor (AIF) and endonuclease G (Endo

G)-dependent pathways in C6 cells in vitro.

Materials and Methods

Chemicals and Reagents

Danthron, dimethyl sulfoxide (DMSO), propidium iodide

(PI) and agarose were purchased from Sigma-Aldrich

Corp. (St. Louis, MO, USA). The primary antibodies were

obtained as follows: antibodies for BAX, BCL-2, cyto-

chrome c, APAF-1, AIF, Endo G, caspase-3, -8 and -9,

PARP, p53, GRP78, GADD153, caspase-12 and b-Actin

were purchased from Cell Signaling Technology (Beverly,

MA, USA) and the secondary antibodies, which were

horseradish peroxidase (HRP)-linked goat anti-mouse IgG

and goat anti-rabbit IgG were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA, USA). The protein assay

kit was purchased from Bio-Rad (Hercules, CA, USA).

Cell Culture

C6 rat glioma cell line was obtained from the Food

Industry Research and Development Institute (Hsinchu,

Taiwan). The culture medium consisted of Kaighn’s

modification of Ham’s F-12 (KMHF) medium, 10 %

fetal bovine serum (FBS), 1 % penicillin–streptomycin

(100 Units/ml penicillin and 100 lg/ml streptomycin) and

2 mM L-glutamine which were obtained from Invitrogen

Life Technologies (Carlsbad, CA, USA). C6 cells were

cultured in KMHF medium as monolayers and maintained

in an incubator with a humidified atmosphere of 95 % air

and 5 % CO2 at 37 �C [8].

Danthron Treatment

In all treatments of experiments, danthron was initially

dissolved in DMSO and made up to the maximum final

concentration of 0.2 % (v/v) in the complete cell culture

medium (KMHF). The sub-cultured cells from experiments

were treated with either varying concentrations of danthron

Neurochem Res (2012) 37:1790–1800 1791

123

or 0.2 % DMSO in media alone that served as control

[5, 17].

Assessment of Cell Morphology and Viability

Approximately 2 9 105 cells/well of C6 cells were cul-

tured onto 12-well plates in KMHF culture medium and

each well was then treated individually with 0, 10, 25, 50,

75 and 100 lM of danthron for 24, 48 and 72 h. For cell

morphological changes examination, cells onto the plate

were directly examined and were photographed under a

phase-contrast microscope [18]. For determination of the

percentage of viable cells, cells were collected from each

well and added PBS containing PI (4 lg/ml). Thereafter,

nonviable cells were stained by a PI dye exclusion

(indicative of an intact membrane) and displayed brighter

fluorescence than the unstained (viable cells). Cells were

immediately determined and counted by flow cytometric

analysis (BD Biosciences, FACSCalibur, Franklin Lakes,

NJ, USA) as previously described [19–21].

DAPI Staining for Apoptosis

Approximately 2 9 105 cells/well of C6 cells were placed

in 12-well plates with KMHF medium as described above

for 24 h. Cells were treated individually with 0, 10, 25, 50,

75 and 100 lM of danthron for a 48-h incubation. Cells

were stained by using 6-diamidino-2-phenylindole dihy-

drochloride (DAPI, Invitrogen Life Technologies) as

described previously [22, 23]. The cells were examined and

were photographed under a fluorescence microscope.

DNA Fragmentation by Agarose Gel Electrophoresis

A total 5 9 105 C6 cells/well on 6-well plates were cul-

tured with or without 50 lM of danthron for 48 h. DNA

was isolated using DNA Purification kit (Genemark tech-

nology Co., Ltd, Tainan, Taiwan) and ladder formation

assays were performed by DNA agarose gel electrophoresis

as described previously [24, 25].

Determinations for Reactive Oxygen Species (ROS),

Mitochondrial Membrane Potential (DWm) and Ca2?

Release

A density of 2 9 105 cells/well of C6 cells were placed

onto 12-well plates and treated with 50 lM danthron at

37 �C, 5 % CO2 and 95 % air for 0, 1, 3, 6, 12 and 24 h to

detect the changes of ROS, DWm and Ca2? release. Cells

were harvested from each treatment by centrifugation

(1,500 rpm) and then re-suspended in 500 ll of DCFH-DA

(10 lM) for ROS determination, in 500 ll of DiOC6

(100 nmol/l) for the levels of DWm and in 500 ll of Fluo-3

(2.5 lg/ml) for Ca2? release at 37 �C for 30 min and

immediately analyzed by flow cytometry [26–28]. Or cells

were pretreated with 20 mM NAC (a ROS scavenger,

Sigma-Aldrich Corp.) and then were treated with or with-

out danthron for 24 h before cells were harvested for

measuring the percentage of viable cells using a PI

exclusion method and flow cytometry as described above.

Apoptosis-Associated Proteins Levels Utilizing

Western Blotting Analysis

C6 cells at the density of 5 9 105 cells/well were placed

onto 6-well plates and treated individually with 50 lM

danthron for 0, 6, 12, 24 and 48 h. Cells were harvested

from each treatment for the total protein determination and

then for Western blotting analysis. The protein levels of

BAX, BCL-2, cytochrome c, APAF-1, AIF, Endo G, cas-

pase-3, -8 and -9, PARP, p53, GRP78, GADD153, caspase-

12 and b-Actin expressions were examined using sodium

dodecylsulfate polyacrylamide gel electrophoresis (SDS-

PAGE) and Western blotting as described previously

[5, 19, 20].

Caspase-3, -8 and -9 mRNA Gene Expressions

by Real-Time PCR

C6 cells (5 9 105 cells/well) were plated in 6-well plates

and placed with KMHF medium containing with or without

50 lM danthron for a 24-h treatment. Cells were then

collected and total RNA was isolated using the Qiagen

RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA

samples were reverse-transcribed with High Capacity

cDNA Reverse Transcription Kit according to the standard

protocol of the supplier (Applied Biosystems, Foster City,

CA, USA). Quantitative PCR was done, and 29 SYBR

Green PCR Master Mix (Applied Biosystems) and 200 nM

of forward and reverse primers (rat caspase-3: F-AG

GCCGACTTCCTGTATGCTT, R-AGCATGGCGCAAA

GTGACT; rat caspase-8: F-TCCTTAGTTCTCTCAGTT

GCCTTTC, R-CCGGGCTCACTTCCTGTTC; rat cas-

pase-9: F-CTGTCCCGTGAAGCAAGGAT, R-CCCACG

TCTCCTCCAACCT; rat GAPDH: F-GGTGGACCTCAT

GGCCTACA, R-CAGCAACTGAGGGCCTCTCT) were

applied as previously described [29, 30]. Each assay was

run on an Applied Biosystems 7300 Real-Time PCR sys-

tem in triplicate and expression fold-changes were derived

using the comparative CT method.

Confocal Laser Scanning Microscopy for Protein

Translocation

C6 cells at a density of 1 9 105 cells/well were cultured on

four-well chamber slides for treatment without or with

1792 Neurochem Res (2012) 37:1790–1800

123

50 lM danthron for 24 h. Then cells on the slides were

fixed in 4 % formaldehyde in PBS for 15 min, permeabi-

lized with 0.3 % Triton-X 100 in PBS for 1 h with

blocking of nonspecific binding sites using 2 % BSA as

described previously [31]. Primary antibodies against AIF,

Endo G and cytochrome c (1:100 dilution) (green fluores-

cence) were used to stain the fixed cells overnight, washed

twice with PBS before being stained with secondary anti-

body (FITC-conjugated goat anti-mouse IgG at 1:100

dilution), and followed by DNA and mitochondria staining

with PI (red fluorescence) and MitoTracker (Invitrogen

Life Technologies) (red fluorescence). All samples were

microphotographed using a Leica TCS SP2 Confocal

Spectral Microscope as described previously [18, 31].

Statistical Analysis

The statistical analyses of the data were represented as

means ± SD in triplicate (n = 3) and one-way ANOVA

followed by the Dunnett’s test was used to analyze dif-

ferences between danthron-treated and control groups. The

p value of less than 0.05 was considered a significant

difference.

Results

The Effects of Danthron on the Morphological Changes

and the Percentages of C6 Rat Glioma Cells

Cells were treated with 0, 10, 25, 50, 75 and 100 lM

danthron for 24, 48 and 72 h, and then cell morphological

changes and cell viability were examined as shown in

Fig. 1a, b. The results indicated that danthron induced cell

morphological changes (Fig. 1a) and cell death (decreased

the percentage of viable cells) at over 25 lM and this effect

is a dose- and time-dependent response (Fig. 1b). Based on

these observations, danthron induced cell morphological

changes (Fig. 1a) and cell death induction mostly in dose-

and time-dependent (Fig. 1b) situations in examined time

periods. We selected the 50 lM dose of danthron and next

assessed whether the growth-inhibitory and cell death

Fig. 1 The effects of danthron

on the morphological changes

and percentage of viable C6

cells. Cells were plated in

KMHF medium ? 10 % FBS

with various concentrations of

danthron for 24, 48 and 72 h,

and the morphological changes

were examined under phase-

contrast microscope (a). Total

percentage of viable cells

(b) was determined by flow

cytometer. Each point is

mean ± SD (n = 3)

Neurochem Res (2012) 37:1790–1800 1793

123

effects of danthron are accompanied by its effect on

apoptotic cell death.

Danthron Induced Apoptosis in C6 Rat Glioma Cells

C6 cells after exposure to danthron at various concentra-

tions (0, 10, 25, 50, 75 and 100 lM) of danthron for 48 h

were then stained by DAPI. Cells were examined and

photographed by fluorescence microscope and results can

be seen in Fig. 2. As shown in Fig. 2a, b, the photographs

from DAPI staining assays revealed that apoptotic cells

were observed in dantrhron-treated C6 cells compared with

intact control cells and this effect is a dose-dependent

course (Fig. 2b). We also found that danthron promoted

DNA fragmentation in C6 cells after treatment with or

without 50 lM of danthron for 48 h as illustrated in

Fig. 3c.

Danthron Affected the Levels of ROS, DWm and Ca2?

Release in C6 Cells

To confirm whether or not danthron-induced apoptosis in

C6 cells is involved in the effects of ROS, DWm and Ca2?

levels, cells were pre-treated with or without NAC (a ROS

Fig. 2 Danthron-induced DNA

damage (apoptosis) in C6 cells.

Cells were incubated with

various concentrations of

danthron and apoptotic cells

were determined by DAPI

staining. Cells were then

photographed by fluorescence

microscopy (a). The levels of

apoptosis were elevated (b).

MFI indicates mean

fluorescence intensity. Each

point is mean ± SD in

triplicate. *p \ 0.05 shows

significant difference from the

untreated control. (c) DNA

fragmentation was performed

by agarose gel electrophoresis

in C6 cells after treatment with

or without 50 lM of danthron

for 48 h

1794 Neurochem Res (2012) 37:1790–1800

123

scavenger) and exposed to 50 lM danthrone for various

time periods. The levels of ROS production were analyzed

and quantified by flow cytometry. The results from Fig. 3a

demonstrated that danthron induced ROS production quite

early and time-dependently and pretreatment with NAC led

to decrease the dead cells of C6 cells after treatment with

danthron (Fig. 3d). Figure 3b shows that danthron pro-

moted the loss of DWm in C6 cells and this effect also is a

time-dependent manner (Fig. 3b). The results from Fig. 3c

indicated that danthron induced Ca2? production was sig-

nificantly increased from 1 h treatment and up to 24 h to

produce high levels of Ca2? (Fig. 3c).

Danthron Altered the Apoptosis-Associated Proteins

and Gene Levels in C6 Rat Glioma Cells

Our data in Figs. 1 and 2 already indicated that danthron

induced cell death of C6 cells (apoptotic death). For

Fig. 3 Danthron affected the

levels of ROS, DWm and Ca2?

in C6 cells. Cells were

incubated with 50 lM danthron

for 0, 1, 3, 6, 12 and 24 h and/or

pre-treated with NAC before

stained by DCFH-DA for ROS

levels determination (a) after

treatment, stained with DiOC6

for the DWm level determination

(b) and stained by Fluo-3/AM

and the Ca2? level

(c) determined as described in

‘‘Materials and Methods’’. Cells

were harvested for the

determination of percentage of

viable cells (d). Each point is

mean ± SD of three

experiments. *Values are

significantly different from the

untreated control (p \ 0.05)

Neurochem Res (2012) 37:1790–1800 1795

123

determining the signaling pathways of danthron-induced

apoptosis of C6 cells via a mitochondria-dependent

pathway, cells were treated with 50 lM danthron for

various time periods, harvested and measured for the total

protein levels from each treatment using Western blotting

analysis. The results are shown in Fig. 4, which indicated

that danthron promoted the expressions of BAX (Fig. 4a),

cytochrome c, APAF-1, AIF and Endo G (Fig. 4b), cas-

pase-3, -8 and -9 and p53 (Fig. 4c), GRP78, GADD153

and caspase-12 (Fig. 4d), but it decreased the level of

BCL-2 (Fig. 4a). As shown in Fig. 4c, treatment of cells

with danthron resulted in a time-dependent increase in

caspase-3, caspase-8, and caspase-9 with strongest effects

at 12, 24 and 48 h, suggesting a possible involvement of

caspases activation in the apoptotic effect of danthron in

C6 cells in vitro. Alternatively, it can be seen in Fig. 4e

that danthron treatment for 24 h led to a significant

increase for caspase-3, -8 and -9 gene expressions in C6

cells.

Danthron Trafficked the Proteins Translocation in C6

Rat Glioma Cells

Cells were cultured on 4-well chamber slides and were then

treated with or without 50 lM danthron for 24 h and then

were individually stained by anti-AIF, Endo G and cyto-

chrome c. The results shown in Fig. 5 indicated that dan-

thron promoted the expression of AIF (Fig. 5a), Endo G

(Fig. 5b) and cytochrome c (Fig. 5c) and all samples also

translocated from mitochondria to nuclei or cytoplasma in

C6 cells.

Discussion

Although it has been shown that danthron induced geno-

toxicity and has been reported by the FDA [12, 13], our

previous studies do show that danthron can induce apop-

tosis in cancer cell lines [5, 16, 17, 32, 33]. Our earlier

Fig. 4 Danthron altered the

apoptosis-associated proteins

levels in C6 cells. A total of

5 9 105 C6 cells/ml cells were

treated with 50 lM danthron for

0, 6, 12, 24 and 48 h. Cells were

harvested from each sample and

associated proteins were

determined by Western blotting.

The levels of BAX and BCL-2

(a), cytochrome c, APAF-1, AIF

and Endo G (b), caspase-3, -8

and -9, PARP and p53 (c),

GRP78, GADD153 and

caspase-12 (d) expressions were

examined using sodium

dodecylsulfate polyacrylamide

gel electrophoresis (SDS-

PAGE) and Western blotting.

(e) Cells were individually

treated with 50 lM danthron for

24 h, and then cells were

harvested for real-time PCR to

determine the gene expression

of caspase-3, -8 and -9 as

described in ‘‘Materials and

Methods’’. *p \ 0.05 indicates

significant difference between

control and treated cells

1796 Neurochem Res (2012) 37:1790–1800

123

Fig. 5 Danthron trafficked AIF,

Endo G and cytochrome

c distributions in C6 cells. Cells

were incubated with 50 lM

danthron for 24 h then were

fixed and stained with primary

antibodies to AIF (a), Endo G

(b) and cytochrome c (c) which

were then stained by FITC-

labeled secondary antibodies

(green fluorescence) and the

proteins were detected by a

Confocal laser microscopic

system. The nuclei and

mitochondria were stained by PI

and MitoTracker (redfluorescence), respectively.

Areas of colocalization between

AIF, Endo G and cytochrome

c expressions as well as

cytoplasm and nuclei in the

merged panels are yellow.

Scale bar, 20 lm

Neurochem Res (2012) 37:1790–1800 1797

123

study has also demonstrated that danthron at 10–75 lM

had no significant effect on cell viability in rat embryo

aortic smooth muscle cell line (A10), normal fetal osteo-

blast cell line (hFOB) and normal hepatocyte cell line

(Chang liver). We found that no significantly adverse effect

levels of renal, hepatic and immune response in danthron-

treated nude mice at the lower dose [5].

The present study herein provides evidence that dan-

thron-induced apoptosis in C6 rat glioma cells is involved

in the mitochondria-dependent pathways. Furthermore, the

data suggest that induction of cytotoxicity is via apoptotic

death in C6 cells (Fig. 2). The results from flow cytometric

assay already showed that danthron promoted the ROS

production in C6 cells (Fig. 3a). The ROS generation is a

common hall mark for cancer cells due to cancer cells

response to oxidative stress which led to ROS production

[34, 35]. Furthermore, over production of ROS could affect

the levels of DWm accompanying with mitochondrial-

associated event including the apoptosis [36, 37]. Our

results as shown in Fig. 4b also demonstrated that danthron

decreased the level of DWm and these effects are time-

dependent. It was reported that mitochondria plays a crit-

ical role in apoptotic cell death [38]. Thus, mitochondrial

target is becoming the approach to screen therapeutic

agents against cancer [39–41].

It was reported that the accumulation of ROS could lead

to the collapse of mitochondrial membrane potential in

cancer cells, leading to the release of cytochrome c [42]

even AIF and Endo G releases from mitochondria and

finally cause cell apoptosis [39, 43]. To confirm if ROS is

one of the reasons for induction of cell death in C6 cells

after exposure to danthron, NAC was used to pretreat the

C6 cells and then followed by danthron treatment. Cells

were then harvested for the measurements of viable cells

in comparison to danthron treatment alone sample. The

results showed that NAC, a ROS scavenger, significantly

reduced danthron-induced cell death (Fig. 3d), and pro-

tected in vitro mitochondria from depolarization. Our data

showed that ROS was involved and may play a critical role

in danthron-induced apoptosis of C6 cells.

We also examined the apoptosis-associated proteins

levels in C6 cells after exposure to danthron. Results in

Fig. 4a indicated that danthron up-regulated the levels of

pro-apoptotic protein (BAX) but down-regulated the level

of anti-apoptotic protein (BCL-2). BAX and BCL-2 pro-

teins belong to the BCL-2 family and both proteins play an

important role in mitochondrial death cascade [5, 44] and

the ratio of BAX/BCL-2 proteins have been reported to

affect the levels of DWm in several cancer cell lines [6, 45].

BAX translocates to the mitochondria and integrates into

Fig. 6 Schematic diagram

showed danthron-induced

signaling on the activation of

apoptotic machinery in C6 rat

glioma cells

1798 Neurochem Res (2012) 37:1790–1800

123

the outer mitochondrial membrane for causing the disrup-

tion of DWm before leading to the release of cytochrome

c into the cytosol followed by the activation of caspase-9

and -3 [46, 47], but the release of AIF and Endo G from

mitochondria into the nuclei caused apoptosis [5, 48].

However, BCL-2 prevents the process of BAX function via

preserving mitochondrial integrity. Therefore, the ratio of

BAX/BCL-2 has been demonstrated to play a crucial role

for the sustenance of drug-induced apoptosis in the mito-

chondria-mediated apoptotic pathway [6, 45].

Furthermore, the results from Fig. 4b demonstrated that

danthron promoted the levels of cytochrome c, AIF and

Endo G in C6 cells. We also used confocal laser micro-

scope to examine the translocations of cytochrome c, AIF

and Endo G and results. Our data showed that danthron

promoted the releases of cytochrome c, AIF and Endo G in

C6 cells (Fig. 5). Based on these actions, danthron-induced

apoptosis of C6 cells is mediated through mitochondria-

dependent pathway. The possible signal pathways of dan-

thron-induced apoptosis in C6 rat glioma cells was sum-

marized in Fig. 6, which indicated that danthron induced

ROS production, dysfunction of mitochondria, and then led

to the releases of cytochrome c, AIF and Endo G during

cell apoptosis.

Acknowledgments This study was supported by the grant

CMU100-TC-05 from China Medical University and by a research

grant from the National Science Council of Republic of China (Tai-

wan) (NSC 97-2815-C-039-034-B).

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