Cancer Biology & Therapy

ISSN: 1538-4047 (Print) 1555-8576 (Online) Journal homepage: http://www.tandfonline.com/loi/kcbt20

Furanonaphthoquinones cause apoptosis of

cancer cells by inducing the production of reactive
oxygen species by the mitochondrial voltage-
dependent anion channel

Eriko Simamura, Kei-Ichi Hirai, Hiroki Shimada, Junko Koyama, Yukie Niwa &
Shigeomi Shimizu

To cite this article: Eriko Simamura, Kei-Ichi Hirai, Hiroki Shimada, Junko Koyama, Yukie Niwa
& Shigeomi Shimizu (2006) Furanonaphthoquinones cause apoptosis of cancer cells by inducing
the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel,
Cancer Biology & Therapy, 5:11, 1523-1529, DOI: 10.4161/cbt.5.11.3302

To link to this article: http://dx.doi.org/10.4161/cbt.5.11.3302

Published online: 14 Nov 2006.

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 [Cancer Biology & Therapy 5:11, 1523-1529, November 2006]; 2006 Landes Bioscience

Furanonaphthoquinones Cause Apoptosis of Cancer Cells by Inducing

Research Paper

the Production of Reactive Oxygen Species by the Mitochondrial

Voltage-Dependent Anion Channel
Eriko Simamura1 ABSTRACT
Kei-Ichi Hirai1 The mitochondrial production of reactive oxygen species has been implicated in the

Hiroki Shimada1

.
anticancer activity of furanonaphthoquinone. However, the mechanism of the activation

E
Junko Koyama2
remains elusive. In the current study, we found that treatment of HeLa cells with

UT
2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial

Yukie Niwa3
swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical

RIB
Shigeomi Shimizu4
scavengers -tocopherol and trolox. Cytochemical experiments in isolated mitochondria
showed that a combination of FNQ13 and NADH induces the production of H2O2 at the
exterior mitochondrial membrane surface. This production of H2O2 was reduced by an
1Molecular and Cell Structural Science; Kanazawa Medical University; Uchinada,

IST
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antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC

Ishikawa, Japan by transfection with vdac1 cDNA increased the production of H2O2 by HeLa cells,
2Kobe Pharmaceutical University; Higashinada, Kobe, Japan whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced

D
3Niwa Institute for Immunology; Tosashimizu, Kochi, Japan
H2O2 production and cell death due to an almost complete knockdown of VDAC expression.

4Laboratoryof Molecular Genetics; Department of Post-Genomics and Diseases;
Osaka University Medical School; Suita, Osaka, Japan
*Correspondence to: Eriko Simamura; Molecular and Cell Structural Science;
Kanazawa Medical University; Uchinada, Ishikawa 920-0293 Japan; Tel.:
+81.76.286.9513; Fax: +81.76.286.9513; Email: simamura@kanazawa-med.ac.jp
Received 07/27/06; Accepted 08/19/06
OT
We also found significant correlations between the expression of VDAC and the induction
of H2O2 production and cell death by FNQ13 in 11 human cancer cell lines. These
results indicate that the anticancer activity of furanonaphthoquinones depends on the
ON
production of reactive oxygen species by mitochondrial permeability transition pores
including the VDAC.
.D

INTRODUCTION
Previously published online as a Cancer Biology & Therapy E-publication:
CE

http://www.landesbioscience.com/journals/cbt/abstract.php?id=3302

KEY WORDS
IEN

Several quinones are used as anticancer drugs, including adriamycin (anthraquinones)

and mitomycin C (benzoquinones); however, these agents frequently cause severe
VDAC (voltage-dependent anion channel), side-effects such as nausea and fatal hemopoietic failure. Furanonaphthoquinones (FNQs)
SC

furanonaphthoquinone, reactive oxygen are effective anticancer agents found in the inner bark of the tropical trees Tabebuia
species, mitochondrial permeability transition (Tecoma) impetiginosa and T. heptaphylla, which are members of the Bignoniaceae family.
BIO

pore, cancer cells, apoptosis, molecular target We have synthesized several FNQ derivatives, including 2-methyl-furanonaphthoquinone

ACKNOWLEDGEMENTS
(FNQ3) or 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13). FNQ3 is
10-fold more toxic to human cancer cell lines than to normal epithelial cells and effectively
ES

We are grateful to Ms. Hiroko Ikeda for inhibits the growth of human cancer xenografts in nude mice.1 FNQ13 was more toxic
technical assistance, Ms. Mayumi Mitani for than FNQ3 and approximately 14-fold more toxic to cancer cells than non-cancerous cells.1
ND

secretarial assistance. This work was supported Such selectivity seems to be important for avoiding side-effects of anticancer treatments.
in part by a Grant-in-aid for scientific FNQs have been reported to be effective for treating leukemia and carcinomas. FNQ3
research from the Japan Society for the induces the differentiation of HL-60 myeloid cells in the presence of 1, 25(OH)2-
LA

Promotion of Science (15591664 and dihydroxyvitamin D3 or all-trans-retinoic acid, and it reduces clonogenic growth of primary
17591899) and Grant for Promoted Research acute myeloid leukemia cells.2 On the other hand, Rieber et al. reported that 2-acetyl-fura-
06

(S2003-12, S2004-12) from Kanazawa nonaphthoquinone preferentially induces apoptosis in p53 mutant breast carcinoma cells3
Medical University. Production of FNQ13 and becomes more active under conditions of glucose depletion, which reduces cell
was sponsored by the Japan Science and
20

proliferation and decreases the efficacy of some genotoxic drugs.4

Technology Corporation (Tokyo, Japan).
We have previously studied the ability of FNQ3 to damage the structure of mitochondria

via the production of reactive oxygen species (ROS) in HeLa cervical cancer cells,5 HuO9
osteosarcoma cells,6 and A549 lung adenocarcinoma cells.7 We found that, unlike FNQs,
adriamycin and mitomycin C do not cause mitochondrial rupture in cancer cells8 but are
equally harmful to normal cells.1 Treatment of cancer cells with 6 mM FNQ3 caused
collapse of the mitochondrial membrane potential, leakage of cytochrome c from the
mitochondria into the cytosol, and activation of caspase-9, leading to apoptosis.7 We thus
concluded that the toxicity of FNQ3 depends on ROS production in mitochondria, but
the target of FNQs in the mitochondria remains unclear.

www.landesbioscience.com Cancer Biology & Therapy 1523

 Production of Ros by FNQ

It was reported that voltage-dependent anion channel (VDAC),

which a component of a mitochondrial permeability transition pore
(MPTP),9 promoted the release of O2- and H2O2 from mitochondria
to the cytosol.10 In the present study, we tested the hypothesis that
VDAC promotes the ROS production and the cell survival by FNQ13,
using VDAC-overexpressed or knockdown HeLa cells.

MATERIALS AND METHODS

Cells. HeLa and A549 cells were provided by RIKEN Cell Bank
(Tsukuba, Japan) and cultured in Dulbeccos modified Eagles medium
(DMEM) (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented
with 10% fetal bovine serum (FBS) at 37C in a humidified CO2
incubator. Other cell lines used were also obtained from RIKEN
Cell Bank and cultured in the following media: T24 bladder
carcinoma cells in McCoy5A + 10% FBS; 5637 bladder carcinoma
cells, RPMI1640 + 10% FBS; WI-38 fibroblasts, DMEM + 10%
FBS; GCIY gastric cancer cells, DMEM + 15% FBS; MKN1
(lymph node metastatic), MKN7, and MKN74 (liver metastatic)
gastric cancer cells, DMEM + 10% FBS; T98G glioblastoma cells,
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DMEM + 10% FBS and 1% non essential amino acids; and

RCC10RGB kidney cancer cells, DMEM + 10% FBS.
Production of 2-methyl-5(or -8)-hydroxy-furanonaphtho-
quinone (FNQ13). FNQ13 (C13H8O4; molecular weight = 228.2)
was chemically synthesized in our laboratory in collaboration with
Dr. Yoshinobu Nakagawa (Tatsumi Kagaku Co., Ltd., Kanazawa,
Japan).
Effects of radical scavengers on cell survival. -Tocopherol
acetate (Sigma-Aldrich) and trolox (Sigma-Aldrich), a water-soluble
analog of vitamin E were used. HeLa cells (4 x 103 cells per well)
were precultured on 96 well-plates for 24 h and treated with 0.22 to
22 mM FNQ13 for 1 h. The cells were washed with medium and
cultured in the presence of 5 mM -tocopherol or 1 mM trolox for
72 h.11 Finally, the cells were fixed with 2% glutaraldehyde and
stained with 0.5% crystal violet to measure the number of cells.12
Ultrastructural observations. Monolayers of HeLa cells were
cultured in the presence and absence of FNQ13, fixed for 1 h in
cold 0.1 M phosphate-buffered saline, pH 7.4 (PBS) containing
2.5% glutaraldehyde, post-fixed for 1 h in Michaelis Veronal buffer
(pH 7.4) containing 1% OsO4, and block-stained with 0.5% uranyl
acetate for 20 min at 4C.6,8 The cells were then dehydrated and
embedded in Quetol 653 resin. Ultrathin sections were stained with
uranyl acetate and lead citrate for transmission electron microscopy
(JEM-1200EX, JEOL Co., Ltd, Tokyo, Japan).
Isolation of mitochondria from cultured cells. Monolayers of
subconfluent HeLa cells and A549 cells were harvested and slowly
homogenized in a cold mannitol solution containing 0.1% bovine
serum albumin using a Teflon-glass homogenizer.13,14 Heavy
mitochondrial fractions were resuspended in Tris solution (0.05 M
Tris-HCl buffer, pH 7.4, 0.25 M sucrose, 20 mM KCl, 2 mM
MgCl2, and 1 mM Na2HPO4) with 0.1% bovine serum albumin
and starved for 30 min at 37C to deplete endogenous substrates.13
Cytochemical detection of H2O2. Cytochemical detection of
H2O2 was performed essentially as described for the determination
of mitochondrial NADH-quinone oxidoreductase activity except
that paraquat was replaced by FNQ13.15 Briefly, mitochondria
isolated from A549 cells were incubated for 20 min at 37C in a
reaction mixture containing 10 M FNQ13, 2 mM -NADH, 1 mM
KCN, 1 mM CeCl3, 0.25 M sucrose, and 0.05 M Tris-maleate
buffer (pH 7.5) in the presence of 5 M rotenone and 1 M
Figure 1. Cytotoxicity of FNQ13 (A) and inhibition by radical scavengers
(B). (A) Cells were cultured for 72 h after exposure to FNQ13 for 1 h, and
cell survival was measured. The IC50 value was calculated from approxi-
mate curve. (B) Protective effects of radical scavengers. Cells were treated
for 1 h with FNQ13 and cultured for 72 h in the presence of -tocopherol
or trolox. Each point is the average of triplicate. Students t test was per-
formed on the different sets of data. *Differences were considered signifi-
cant when the p value was <0.01 vs. control (no radical scavenger).
p-hydroxymercuribenzoic acid (pHMB; inhibitor of NADH-
cytochrome b5 reductase) without or with 6 g/mL mouse
anti-human VDAC monoclonal antibody (anti-Porin 31HL;
Calbiochem, San Diego, CA, USA). The cells were fixed with 2.5%
glutaraldehyde to prepare them for electron microscopy. This
method has been previously shown to produce specific staining.16
Fluorescent Detection of H2O2. Isolated HeLa cell mitochondria
were attached to glass-bottomed dishes coated using BD CELL-TAK
cell adhesive (Becton Dickinson, Franklin Lakes, NJ, USA) and
incubated in a mixture of 10 M FNQ13, 2 mM -NADH, 10 M
2',7'-dichlorofluorescin diacetate (DCFH-DA; Molecular probes,
Eugene, OR, USA), 5 M rotenone, 1 M pHMB, and 1 mM KCN
in Tris buffer without or with 6 g/mL mouse anti-human VDAC
monoclonal antibody.17 The production of H2O2 was detected using
an inverted light microscope (IX70-FLA; Olympus Corp., Tokyo,
Japan) equipped with an IB cube filter (excitation at 460-490 nm;
emission at 515 nm). The fluorescence of 2,7-dichlorofluorescein
was detected using a Pixera Penguin 140CL CCD camera and
analyzed with a Lumina Vision visual bioimaging analytical system
(Mitani Corp., Fukui, Japan). For fluorescent detection of H2O2 in
cells, cells were cultured on 35-mm glass-bottomed dishes for 24 h,

1524 Cancer Biology & Therapy 2006; Vol. 5 Issue 11

 Production of Ros by FNQ
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Figure 2. Ultrastructural changes in HeLa cells caused by 3 M FNQ13 as
determined by electron microscopy after 12 or 24 h. 0 h indicates cells
prior to treatment with FNQ13. FNQ13 caused changes in the ultrastructure
of mitochondria. Mt, mitochondria; rER, rough endoplasmic reticulum. At 12
h, swollen mitochondria are visible (arrows), and at 24 h, apoptotic cells
are observed. Bars, 1 m
treated for 30 min in the dark at 37C with 10 M DCFH-DA in
DMEM, and then incubated with 1 M FNQ13.
Transfection of Cells with DNA. HeLa cells were cultured in
60-mm dishes and transfected with human vdac1 cDNA (2.5 g)
using EffectenTM (Qiagen GmbH, Hilden, Germany) as described
by the manual. The vdac1 cDNA, which was subcloned into
pUC-CAGGS, was prepared as described by Narita.18 Cells were
cotransfected with pDSred2 vector (Clontech, Mountain View,
CA, USA) and cultured in G418 disulfate solution (Nacalai Tesque,
Inc., Kyoto, Japan). Western blot analysis was performed as
described in our previous report.7 Whole- cell lysate (10 g protein)
from transfected cells were subjected to electrophoresis on a
SuperSep gel (WAKO, Wako Pure Chemical Industries, Ltd., Osaka,
www.landesbioscience.com Cancer Biology & Therapy
Figure 3. Cytochemical detection of FNQ13-induced H2O2 production in
isolated, unfixed mitochondria from A549 cells. The cells were incubated for
20 min at 37C in the presence of FNQ13, NADH, both NADH and
FNQ13, or NADH, FNQ13 and an anti-VDAC antibody (Ab). H2O 2
production was determined by cytochemical detection, followed by electron
microscopy. Arrows indicate cerium perhydroxide deposits. Bars, 200 nm
Japan) and then electrophoretically transferred to a polyvinylidene
difluoride membrane (ATTO, Tokyo Japan). The membranes were
reacted with an anti-VDAC antibody (1:500; Santa Cruz
Biotechnology, Inc., CA, USA) as the primary antibody and subse-
quently reacted with peroxidase-conjugated secondary anti-goat
antibody (1:10,000; Rockland Immunochemicals, Inc., PA, USA).
Transfection of Cells with Small Interfering RNA (siRNA).
HeLa cells (2.3 105 per 35-mm dish) were transfected for 48 h
with 5 nM Control siRNA or Hs_VDAC1_1HP siRNA (Qiagen)
using HiPerFect Transfection Reagent (Qiagen).
Cell Survival. HeLa cells stably overexpressing VDAC (4 103
per well) in 96 well-plates were cultured with FNQ13 for 48 h.
HeLa cells (1.5104 per well) were transfected with 5 nM siRNA
using Hiperfect Transfection Reagent and incubated for 48 h.
Thereafter, the cells were treated with FNQ13, and the number of
cells were measured after 4872 h. FNQ13 doses were set after several
preexperiments.
Analysis of the correlation between VDAC expression, H2O2
production, and the concentration of FNQ13 for 50% inhibition
of cell survival (IC50). The IC50 of cancer cell lines for FNQ13 was
determined based on the effects of a 72-h treatment with 0.01 to
10 M FNQ13. VDAC contents were analyzed by Western blotting
in cancer cell lines, and their relative amounts per mitochondria were
calculated based on the level of cytochrome oxidase subunit I determined
using a mouse monoclonal antibody to OxPhos Complex IV subunit
I (Invitrogen Corp., Carlsbad, CA, USA). To determine the level of
H2O2, confluent cells treated with 1 M FNQ13 were reacted with
10 M DCFH-DA in 96-well plates, and H2O2 production was
1525
 Production of Ros by FNQ
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Figure 4. H2O2 production in isolated mitochondria. Mitochondria from
HeLa and A549 cells were incubated with no addition (blank), FNQ13,
NADH, both NADH and FNQ13, or NADH, FNQ13, and an anti-VDAC
antibody (Ab). H2O2 production by mitochondria was determined using
DCFH-DA, followed by observation with a fluorescence microscope. The
fluorescence intensity is expressed as the number of positive counts per
1000 mitochondria. Each point is the average of triplicate. Students t test
was performed on the different sets of data. *Differences were considered
significant when the p value was <0.01.
determined based on the fluorescence detected with a Labsystems
Fluoroscan II (Dainippon Pharmaceutical Co., Ltd. Osaka, Japan).
RESULTS
Cytotoxicity of FNQ13 and the effects of radical scavengers.
Treatment of HeLa cells with FNQ13 for 1 h inhibited their survival
with an IC50 of 0.6 M. A 72-h treatment with 5 M FNQ13
caused the death of all of the cells (Fig. 1). Cell survival following
exposure to 0.6 M FNQ13 was 40.7 8.5%, but it was increased
up to 72.5 8.7% and 67.1 5.2% by treatment with the antioxidants
-tocopherol and trolox (Fig. 1B).
Effect of FNQ13 on cellular ultrastructure. As shown in Figure 2,
treatment with 3 M FNQ13 caused ultrastructural alterations in
HeLa monolayers. A 12-h treatment caused swelling of mitochondria
and a loss of electron density in their matrices. These damaged cells
appeared to be undergoing apoptosis after 24 h, as indicated by the
1526 Cancer Biology & Therapy
Figure 5. Effect of VDAC overexpression in HeLa cells. (A) Western blot of
control and vdac1-transfected cell lysates using an anti-human VDAC antibody.
Wild, wild type HeLa cells. (B) Effect of 4 M FNQ13 on H2O2 production
as determined using DCFH-DA, followed by observation with a fluorescence
microscope. (C) H2O2 fluorescence in monolayer cells in the presence of
4 M FNQ13. The total fluorescence per cell was integrated. (D) Survival of
HeLa cells treated with 0.6 M FNQ13 for 48 h. Each point is the average
of triplicate. Students t test was performed on the different sets of data.
*Differences were considered significant when the p value was <0.01 vs.
control (Cont; cells transfected with a control vector; Overexpressed, cells
overexpressing pUC-CAGGS-human vdac1).
presence of cytoplasmic blebs, abnormal nuclei with heavily pyknotic
chromatin, and spherical apoptotic bodies.
Localization of H2O2 production. Figure 3 shows the effects of
FNQ13 on the localization of H2O2 production as determined by
cytochemistry in mitochondria isolated from A549 cells. When the
cells were incubated with a combination of FNQ13 and NADH,
H2O2 was detected as cerium perhydroxide deposits on the mito-
chondrial outer membrane. In contrast, neither FNQ13 nor NADH
alone caused formation of the deposits. The formation of deposits
was strongly inhibited by an antibody to VDAC.
Fluorescent detection of H2O2 in isolated mitochondria using
DCFH-DA indicated that treatment of HeLa and A549 cells with a
2006; Vol. 5 Issue 11
 Production of Ros by FNQ
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Figure 6. Effect of VDAC knockdown in HeLa cells. (A) Western blot of HeLa
cells expressing control (Cont) and Hs_VDAC1_1HP (Knockdown) siRNAs
by Western blotting using an anti-human VDAC antibody. Wild, wild type
HeLa cells. (B) Effect of 4 M FNQ13 on H2O2 production as determined
using DCFH-DA, followed by observation with a fluorescence microscope.
(C) H2O2 fluorescence in monolayer cells in the presence of 4 M FNQ13.
The total fluorescence per cell was integrated. (D) Survival of HeLa cells
treated with 0.6 M FNQ13 for 48 h. Each point is the average of triplicate.
Students t test was performed on the different sets of data. *Differences
were considered significant when the p value was <0.01 vs. control (cells
transfected with a control siRNA).
combination of FNQ13 and NADH induced H2O2 production
(Fig. 4). The fluorescence in the presence of FNQ13 and NADH
was significantly reduced by treatment with an anti-VDAC antibody
but not with normal serum (data not shown). These results agreed
with those showing destruction of the mitochondrial ultrastructure
by FNQ13 in the presence of NADH (Fig. 2).
Effect of VDAC overexpression. When treated with 4 M
FNQ13, HeLa cells transfected with vdac1 exhibited 1.9-fold higher
levels of VDAC contents as indicated by Western blotting (Fig. 5A)
and produced 1.6-fold higher levels of H2O2 (Fig. 5B and C). After
a 48-h treatment with 0.6 M FNQ13, the survival of the VDAC-
overexpressing cells (52.7 10.7%) decreased from that of control
cells transfected with plasmid alone (91.1 11.6%) (Fig. 5D).
www.landesbioscience.com Cancer Biology & Therapy
Figure 7. Induction of H2O2 production by 1 M FNQ13 as determined by
fluorescent staining of mitochondria using DCFH-DA. FNQ13 potently
induced H2O2 production in A549, HeLa, T24, MKN1 and 5637 cells but
only weakly in RCC10RGB and MKN74 cells and WI-38 fibroblasts.
Effect of VDAC knockdown. Transfection of HeLa cells with
Hs_VDAC1_1HP siRNA, eliminated the production of VDAC
protein as indicated by Western blotting (Fig. 6A). In addition, the
cells did not produce H2O2 in response to FNQ13 (Fig. 6B and C).
After a 48-h treatment with 4 M FNQ13, 10.8 1.2% of the cells
survived, whereas 47.6 12.7% of the cells transfected with the
VDAC siRNA survived (Fig. 6D).
Induction of H2O2 and cell death in various cancer cell lines by
FNQ13. Fluorescent staining with DCFH-DA revealed that a
30-min treatment with 1 M FNQ13 potently induced mitochon-
drial H2O2 production in A549, HeLa, T24, MKN1 and 5637 cancer
cells; however, FNQ13 caused only weak production of H2O2 in
RCC10RGB and MKN74 cancer cells and WI-38 fibroblasts (Fig. 7).
Figure 8A shows that a significant correlation also existed between
the mitochondrial VDAC content and the IC50 values for FNQ13
(r = -0.763; p < 0.01). There was a significant correlation between
the mitochondrial VDAC contents of the cells and the FNQ13-
induced H2O2 production (r = 0.642; p < 0.05; Fig. 8B). There was
no significant correlation, however, between the VDAC protein
contents and the IC50 values (Fig. 8C).
DISCUSSION
The present study demonstrated that, like FNQ3,5,7 FNQ13 is
selectively toxic to the mitochondria of cancer cells. Cytochemical
experiments revealed that treatment of mitochondria with FNQ13
in the presence of rotenone and KCN, which are inhibitors of the
mitochondrial respiratory chain, caused the release of H2O2 from
the mitochondrial outer membrane. The induction of H2O2 produc-
tion required the presence of NADH as an electron donor; however,
pHMB had no effect, indicating that H2O2 production was inde-
pendent of NADH-cytochrome b5 reductase, an enzyme localized
on the mitochondrial outer membrane.19
HeLa cells overexpressing VDAC, which is localized in the
mitochondrial outer membrane, showed enhanced FNQ13-induced
H2O2 production and cell death, whereas knockdown of VDAC by
1527
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Figure 8. Correlation between VDAC protein per mitochondria, VDAC pro-
tein per cell, IC50 for FNQ13, and H2O2 production. (A) Correlation
between VDAC protein per mitochondrion and IC50 for FNQ13. (B)
Correlation between VDAC protein per mitochondrion and H2O2 produc-
tion following treatment with 1 M FNQ13 for 30 min. (C) Correlation
between VDAC protein per cell and IC50 for FNQ13. Each point is the aver-
age of triplicate. r, Pearsons correlation coefficient. VDAC/COX, ratio of
VDAC vs. cytochrome oxidase levels.
a siRNA reduced the production of H2O2 and induction of cell
death in response to FNQ13. The induction of H2O2 production
and cell death by FNQ13 correlated with the amount VDAC per
mitochondrion (VDAC/cytochrome oxidase ratio) rather than the
amount of VDAC per cell. Therefore, it is likely that the mitochon-
drial VDAC participates in FNQ-induced cell death by participating
in the production of ROS.
The VDAC may act as a MPTP on the outer membrane. Madesh
et al. reported that O2- but not H2O2 induces a rapid and massive
release of cytochrome c from mitochondria,20 which is a central
event in apoptosis. Reduced cytotoxicity of FNQ13 in VDAC
knockdown cells might be due to lower cytochrome c release and,
therefore, reduced induction of apoptosis. On the other hand,
VDAC1 in the plasma membrane has recently been shown to possess
1528 Cancer Biology & Therapy
Figure 9. Schematic diagram of the mechanism of FNQ-induced H2O2 pro-
duction in cancer cells.
NADH-ferricyanide reductase activity, which could directly catalyze
the reduction of ferricyanide in the presence of NADH.21 In the
present study, we showed that FNQs induce the NADH-dependent
production of ROS on the mitochondrial outer membrane. We
propose that VDAC has a similar function as NAD(P)H-quinone
oxidoreducase1 and therefore, mitochondrial VDAC may catalyze
the reduction of FNQs and leading to mitochondrial production of
ROS (Fig. 9); however NAD(P)H-quinone oxidoreductase1, which
activates mitomycin C, is localized in the cytosol but not on the
mitochondrial outer membrane and mediates the two-electron
reduction of substrates,22 suggesting that VDAC and NAD(P)H-
quinone oxidoreductase1 have different functions.
MPTP proteins are often the molecular targets of chemical
reagents. The peripheral benzodiazepine receptor, which is a critical
component of the MPTP, may be a molecular target of anticancer
reagents because some ligands of the peripheral benzodiazepine
receptor have anticancer activity.23,24 Thus, it is very likely that the
mitochondrial VDAC has multiple functions and is a molecular
target of anticancer reagents. The most important discovery in the
current study was that cancer cells expressed higher levels of the
VDAC than normal cells such as WI-38 fibroblasts. The peripheral
benzodiazepine receptor was also reported to be overexpressed in
several cancers.25 Therefore, the expression of MPTPs may be higher
in cancer tissues, which may explain why FNQs are preferentially
toxic to cancer cells.
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