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Centre National de la Recherche Scientifique, UMR 6022, Université de Technologie de Compiègne, 60205, Compiègne, France [A-S. B., D. M., C. B.]; Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, 94805 Villejuif, France [E. J., H. L. A. V., G. K.]; QLT PhotoTherapeutics, Inc., Vancouver, British Columbia, V5Z 4H5, Canada [D. J. G.]; and The Burnham Institute, La Jolla, California 92037 [Z. X., J. C. R.]
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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m). Light-activated
verteporfin-induced apoptosis was abolished by transfection with Bcl-2,
a procedure reported to inhibit the mitochondrial permeability
transition pore complex (PTPC). Verteporfin triggered the m loss
in isolated mitochondria in vitro, and this effect was
suppressed by bongrekic acid and cyclosporin A. Verteporfin plus light
also permeabilized proteoliposomes containing the semipurified PTPC or
the purified PTPC component adenine nucleotide translocator (ANT), yet
had no effect on protein-free control liposomes. Verteporfin
phototoxicity on ANT proteoliposomes was mediated by reactive oxygen
species and was prevented by recombinant Bcl-2 or the adenine
nucleotides ATP and ADP. In conclusion, verteporfin belongs to a class
of clinically used chemotherapeutic agents acting on PTPC and ANT. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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m, enhanced generation of ROS, and the
release of apoptogenic proteins from the intermembrane space to the
cytoplasm. Such mitochondrial apoptogenic effectors include the caspase
activator cytochrome c, AIF, and pro-caspases
(2, 3, 4, 5, 6)
. All of the signs of apoptosis induced by CD437,
lonidamine, betulinic acid, and arsenite are prevented by two agents
acting on specific PTPC proteins, namely CsA (a cyclophilin D ligand)
and BA (a ligand of the ANT). It thus appears that PTPC opening is a
critical event of apoptosis triggered by these agents. The PTPC is a dynamic protein complex located at the contact site between the two mitochondrial membranes, its opening allowing the free diffusion of solutes Mr <1500 on the inner membrane. Formation of PTPC involves the association of proteins from different compartments, hexokinase (cytosol), porin, also called VDAC (outer membrane), PBR (outer membrane), ANT (inner membrane), and cyclophilin D (matrix). PTPC has been implicated in many examples of apoptosis because of its capacity to integrate multiple proapoptotic signal transduction pathways and because of its control by proteins from Bcl-2/Bax family (8 , 9) . The Bcl-2 family comprises death-inhibitory (Bcl-2-like) and death-inducing (Bax-like) members (10 , 11) , which, respectively, prevent or facilitate PTPC opening. Bax and Bcl-2 reportedly interact with VDAC (12) and ANT within PTPC (9 , 13) . In physiological conditions, ANT is a specific antiporter for ADP and ATP. However, ANT can also form a lethal pore on interaction with different proapoptotic agents including Ca2+, Atr, the HIV-1 protein Vpr, and pro-oxidants (9 , 13, 14, 15) . Mitochondrial membrane permeabilization may also be regulated by the nonspecific VDAC pore modulated by Bcl-2/Bax-like proteins in the outer membrane (12 , 16) , and/or by changes in the metabolic ATP/ADP gradient between the mitochondrial matrix and the cytoplasm (17) .
PDT is based on the combined use of light-absorbing compounds and light irradiation (18) . PDT has been previously evaluated for treatment of tumors of bronchus, bladder, skin, head, neck, and cervix, and more recently for esophageal carcinoma or nonmalignant disorders such as age-related macular degeneration. As a consequence of their selective retention in cancer cells as compared with normal tissues, photosensitizers may kill cells by the local production of ROS on light irradiation. PDT has also been used as an ex vivo purging procedure for leukemia cells (19) . Constant interest in producing new photosensitizers allowed production of a second generation of compounds including benzoporphyrin derivatives, which exhibit superior pharmacological and photochemical properties with minimal dark toxicity. Verteporfin or benzoporphyrin derivative monoacid ring A, is a photosensitizer that induces apoptosis in cancer cells and tumor cell lines (reviewed in Ref. 20 ). Cancer cells treated with verteporfin plus red light exhibit a rapid cytochrome c release into the cytoplasm, followed by caspase activation (21, 22, 23, 24) . Conflicting results have been reported on Bcl-2, which, depending on the concentration and the cell line studied, would inhibit all, some, or none of the signs of verteporfin-induced cell death (Ref. 25 ; for review, see Ref. 20 ). Although the efficacy of verteporfin to elicit proapoptotic mitochondrial photodamage has been clearly demonstrated in various cell lines, its primary molecular target in mitochondria has been elusive.
These observations prompted us to examine the functional interaction between verteporfin, PTPC components, and the oncoprotein Bcl-2. Using a variety of different experimental set-ups, studies of intact cells, purified mitochondria, proteoliposomes containing PTPC, or defined PTPC components, we show here that ANT is a critical target of verteporfin-dependent photochemotherapy. We also provide evidence that Bcl-2 regulates verteporfin-mediated membrane permeabilization via an effect on ANT.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Photoactivation of Verteporfin.
Verteporfin was added to cells, mitochondria, or liposomes at the
indicated dose. After 1-h, 5-min, or 10-min incubations, respectively,
in the dark, photoactivation was performed with a tungsten-halogen lamp
(100W) with a 650-nm-long pass filter in front of it at a dose of
2J/cm2 as published previously (23)
.
Cell Lines, Immunoblot, and Cytofluorometric Analysis of
Apoptosis.
Jurkat cells (J-NEO) or Jurkat cells overexpressing Bcl-2
(J-Bcl-2) were kindly provided by N. Israel (Ref. 27
;
Pasteur Institute, Paris, France) and were cultured in DMEM
supplemented with 10% heat-inactivated FCS and antibiotics at 37°C
under 5% CO2. Bcl-2 and ANT expression levels in
J-NEO and J-Bcl-2 were analyzed by immunoblot (12.5% SDS-PAGE;
500-µg cell lysates proteins/lane) using a polyclonal rabbit
antiserum recognizing ANT and a monoclonal antibody recognizing Bcl-2
(4C11; Santa-Cruz, CA). One x 106 cells/ml were treated with various doses of
verteporfin for 1 h at 37°C. Cells were illuminated (or not) and
then labeled with 40 nM DiOC(6)
3
(Molecular Probes, Eugene, OR) and 2 µM
dihydroethidine (hydroethidine; Molecular Probes, Eugene,
OR) for 10 min at 37°C. Cells were then analyzed by cytofluorometry
(FACSVantage, Becton Dickinson) as described previously
(28)
.
Flow Cytometric Analysis of Purified Mitochondria.
Mouse liver purified mitochondria were resuspended in a buffer
containing 200 mM sucrose, 10 mM
Tris-3-[N-morpholino]propanesulfonic acid (pH 7.4),
5 mM Tris-succinate, 1 mM Tris-phosphate, 2
µM rotenone, and 10 µM EGTA.
Cytofluorometric (FACSVantage, Becton Dickinson) detection was
restricted to mitochondria by gating on the FSC/sideward light
scatters parameters and on the main peak of the FSC-W parameter.
Confirmation a posteriori of the validity of the double
gating was obtained by labeling of mitochondria with the
m-insensitive mitochondrial dye MitoTracker Green (75
nM; Molecular Probes; green fluorescence). To
determine the percentage of mitochondria having a low m, the
m-sensitive fluorochrome JC 1 (200 nM;
570–595 nm) was added 10 min before CCCP or verteporfin. Percentage of
mitochondria having a low m was determined in dot-plot FSC/FL-2
(red fluorescence) windows.
PTPC Purification and Reconstitution in Liposomes.
PTPC was purified and reconstituted in liposomes after previous
protocols (8)
. Briefly, four Wistar rat brains
(3-month-old males) were homogenized in 40 ml of 1 mM
-monothioglycerol-10 mM glucose (pH 8.0) and were
centrifuged twice (15 min, 12,000 x g,
4°C). The pellets were resuspended in the same buffer + 0.5% (v:v) Triton X-100 (Boehringer Mannheim, Mannheim, Germany) for
30 min at room temperature while stirring. The Triton-soluble
protein fraction obtained by ultracentrifugation (40 min,
50,000 x g, 4°C) was mixed with 17 g
of DE52 resin previously equilibrated with a buffer containing
1.5 mM
Na2HPO4, 1.5
mM
K2HPO4, 100
mM glucose, and 1 mM DTT
(pH 8.0). The resin was packed into an FPLC column (XK16/20;
Pharmacia) and eluted with previous buffer supplemented with 50
mM KCl or 400 mM KCl. After
equilibration (0.8 ml/min, 6 ml), elution was performed on a linear
gradient from 50 to 400 mM KCl, followed by
determination of hexokinase activity. Lipid vesicles were prepared by
mixing 100 mg of PC and 20 mg of cholesterol in 1 ml of chloroform,
evaporation of the chloroform under nitrogen, and resuspension in 1 ml
of liposomes buffer [125 mM sucrose + 10 mM HEPES (pH 7.4)] + 0.3%
n-octyl-ß-D-pyranoside by vortexing
(40 min, room temperature). One volume of liposomes was incubated with
one volume of PTPC-containing fraction during 20 min at room
temperature and dialyzed overnight against liposomes buffer at 4°C.
ANT Purification and Reconstitution in Liposomes.
ANT was purified from rat heart mitochondria as described
previously (9)
. After mechanical shearing, mitochondria
were suspended in 220 mM mannitol, 70 mM
sucrose, 10 mM HEPES, 200 µM EDTA, 10
mM DTT, and 0.5 mg/ml subtilisin (pH 7.4), kept 8 min on
ice, and sedimented twice by differential centrifugations (5 min,
500 x g, and 10 min, 10,000 x g). Mitochondrial proteins were solubilized by 6%
(v:v) Triton X-100 (Boehringer Mannheim) in 40 mM
K2HPO4, 40
mM KCl, and 2 mM EDTA (pH
6.0), for 6 min at room temperature, and solubilized proteins were
recovered by ultracentrifugation (30 min, 24,000 x g, 4°C). Then, 2 ml of this Triton X-100 extract were
applied to a column filled with 1 g of hydroxyapatite (BioGel HTP;
Bio-Rad), eluted with previous buffer. and diluted (v:v) with 20
mM MES, 200 µM
EDTA, and 0.5% Triton X-100 (pH 6.0). Subsequently, the sample was
separated with a Hitrap SP column using a FPLC system
(Pharmacia) and a linear NaCl gradient (0–1
M). Proteins concentration was determined using
microBCA-assay (Pierce, Rockford, IL). Purified ANT and/or recombinant
Bcl-2 were reconstituted in PC/cardiolipin liposomes. Briefly, to
prepare liposomes, 45 mg of PC and 1 mg of cardiolipin were mixed in 1
ml of chloroform, and the solvent was evaporated under nitrogen. Dry
lipids were resuspended in 1 ml of liposomes buffer containing 0.3%
n-octyl-ß-D-glucopyranoside and
mixed by continuous vortexing for 40 min at room temperature. ANT (0.1
mg/ml) and/or recombinant Bcl-2 (0.1 mg/ml) were then mixed with
liposomes (v:v) and incubated for 20 min at room temperature.
Proteoliposomes were finally dialyzed overnight at 4°C.
ADP/ATP Translocase Assay.
ANT liposomes were sonicated in the presence of 5 mM
ATP and 10 mM KCl (50 W, 22 s; Branson sonifier 250)
on ice. Then, liposomes were separated on Sephadex G-25 columns
(PD-10; Pharmacia) from unencapsulated products. 25-µl aliquots of
liposomes were mixed with various concentrations of ADP, verteporfin,
or diamide, illuminated (or not), and incubated for 1 h at room
temperature. Luciferase (25 µl, 0.5 units/ml; Boehringer Mannheim)
was added, and the emitted luminescence was determined using a
Perkin-Elmer spectrophotometer. Results are expressed as the percentage
of maximum translocase activity, as determined by the addition of 400
µM ADP.
Pore Opening Assay.
PTPC or ANT proteoliposomes were sonicated in the presence of 1
mM 4-MUP and 10 mM KCl (50W, 22 s; Branson
sonifier 250) on ice as described previously (15)
. Then,
liposomes were separated on Sephadex G-25 columns (PD-10; Pharmacia)
from unencapsulated products. Aliquots (25-µl) of liposomes were
diluted to 3 ml in 10 mM HEPES-125 mM
saccharose (pH 7.4), mixed with various concentrations of the
proapoptotic inducers Atr or verteporfin, illuminated (or not), and
incubated for 1 h at room temperature. Potential inhibitors of
mitochondrial membranes permeabilization such as histidine, SOD,
catalase, DTT, ATP, and ADP, were added to the liposomes 30 min prior
to verteporfin treatment. After addition of 10 µl of alkaline
phosphatase (5 units/ml; Boehringer Mannheim) diluted in liposomes
buffer + 0.5 mM MgCl2,
samples were incubated for 15 min at 37°C under agitation, and the
enzymatic conversion of 4-MUP in 4-MU was stopped by the addition of
150 µl of Stop buffer [10 mM HEPES-NaOH and 200
mM EDTA (pH 10)]. Fluorescence was subsequently determined
using a Perkin-Elmer spectrofluorometer. Atr, a proapoptotic
permeability transition inducer, was used in each experiment as a
standard to determine the 100% response. The percentage of 4-MUP
release induced by verteporfin was calculated as follows:
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, were cultured in the presence of verteporfin and
irradiated by red light. Then mitochondrial and nuclear parameters of
apoptosis were assessed by flow cytofluorometry as described in
Material and Methods. The combination of verteporfin plus light, but
not verteporfin alone, kill Jurkat cells in a dose-dependent manner
(Fig. 1)
. When used at a dose of 140 nM,
verteporfin induced a significant loss of the mitochondrial inner
membrane potential (m) 3 h after light irradiation, as shown
by a decrease in the DiOC(6)
3 fluorescence (Fig. 1B)
. This event was accompanied by Can enhanced generation
of ROS capable of oxidizing hydroethidine oxidation to ethidium
(Fig. 1B)
, as well as 8 h later by nuclear DNA loss, as
shown by the staining with the DNA intercalating dye propidium
iodide (Fig. 1C)
. No toxicity of verteporfin was observed in
the absence of light (Fig. 1, B and C)
. As
expected, preincubation of cells with zVAD, the caspase inhibitor,
failed to inhibit the verteporfin-induced m dissipation (Fig. 1B)
but prevented the appearance of hypoploid cells (Fig. 1C)
.
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m dissipation (Fig. 1B)
. However, Bcl-2
did not reduce the production of ROS induced by verteporfin plus light.
This latter observation suggests that ROS production may be stimulated
independently of the m loss, presumably by the photoactivation
process of verteporfin itself, which leads to the local generation of
ROS. Although Bcl-2 has been initially proposed to function as an
antioxidant (32)
, this result is in agreement with several
reports that indicate Bcl-2 protects from apoptosis via a mechanism
that does not rely on the inhibition of ROS production or activity
(33
, 34) . Collectively, these data indicate that light-activated verteporfin elicits a mitochondrion-dependent apoptosis under the control of Bcl-2. Because Bcl-2 generally inhibits cell death induced by chemotherapeutic agents acting on PTPC, namely CD437, lonidamine, betulinic acid, and arsenite (3, 4, 5, 6) , a hypothesis was that verteporfin may act on mitochondria through the PTPC.
Verteporfin Can Act Directly on Isolated Mitochondria.
To determine possible direct effects of verteporfin on
mitochondria, these organelles were purified from healthy mouse liver
and treated with verteporfin plus light; their m was determined
by flow cytofluoro-metry using the potential sensitive dye JC1.
When mitochondria were incubated with the potentially sensitive
fluorochrome JC1 (Fig. 2
, upper panels), they exhibited a fluorescence significantly
higher than unlabeled mitochondria (Fig. 2
, upper panel,
Co.). This JC1-dependent fluorescence was reduced by
depolarization of the m with the protonophore CCCP (Fig. 2
,
upper panel, JC1+CCCP). Verteporfin
triggered a rapid loss of the m measured with JC1, within less
than 5 min (Fig. 2
, lower panel,
JC1+verteporfin). This effect was complete, as compared with
that of the maximum m depolarizing agent CCCP (Fig. 2
,
upper panel, JC1+CCCP). The m loss
triggered by verteporfin was inhibited by pretreatment of mitochondria
with BA and CsA, two PT inhibitors (Fig. 2
, lower panel),
which suggests again that verteporfin mediates its proapoptotic
activity by acting on PTPC. In addition, this indicated that
verteporfin toxicity did not require any pre-mitochondrial activation
of an intermediate cytoplasmic compound which, in turn, would act on
mitochondria. We conclude that verteporfin can exert its toxicity
directly on isolated mitochondria.
|
. The verteporfin-induced 4-MUP release was dose-dependent
and attained the same magnitude as did the 4-MUP release induced by the
Atr, a proapoptotic ligand of ANT (Fig. 3A)
. To further
investigate the mechanism of action of verteporfin, we evaluated a
series of ROS-detoxificating agents that were added to PTPC
containing liposomes prior to treatment with verteporfin plus
light. Catalase, an
H2O2-scavenging enzyme, and
DTT, a thiol reducing agent, fully inhibited the membrane
permeabilization induced by verteporfin plus light. In contrast, SOD
and histidine, a singlet oxygen quencher, failed to inhibit the
permeabilization of liposomal membranes under the same conditions (Fig. 3B)
. These results suggest that the PTPC opening effect of
light-activated verteporfin are mediated, at least in part, by
H2O2 and/or thiol oxidation
of PTPC components. ATP and ADP, the two physiological ligands of ANT,
also inhibited the effect of verteporfin plus light on PTPC, thus
confirming that ANT constitutes (one) of the physical target(s) of the
photosensitizer within PTPC (Fig. 3B)
.
|
. As expected, verteporfin did not trigger the opening of
the ANT pore in the absence of light irradiation, nor did it induce
permeabilization of plain liposomes, underscoring the specificity of
the verteporfin effect for ANT and its absence of effects on lipids and
notably cardiolipin, a phospholipid that is reputed for its sensitivity
to ROS (35)
. In contrast to diamide, a
proapoptotic thiol-oxidating agent (15)
, verteporfin had
no inhibitory effect on the antiporter function of ANT (Fig. 4B)
. Again, the two ligands of ANT, ATP and ADP, prevented
the pore opening effect of verteporfin plus light (Fig. 4C)
.
Pretreatment of ANT liposomes with DTT and catalase, confirmed that the
light-activated verteporfin effect is mediated by an oxidative
modification of ANT by H2O2
and/or by thiol oxidation. In contrast, SOD did not inhibit the
light-activated verteporfin-induced permeabilization of ANT liposomes,
indicating an oxidative mechanism independent of the anion superoxide
generation or, alternatively, an incapacity of SOD to intercept the
formation of anion superoxide as an intermediate in the cascade of
reactive oxygen intermediates triggered by the photoactivation of
verteporfin (Fig. 4D)
.
|
. Because ANT appears (one of) the verteporfin targets in
mitochondria, we wondered whether Bcl-2 exerts its protective effect
via ANT. To clarify this issue, verteporfin was added to
proteoliposomes containing either ANT alone or ANT combined with
recombinant Bcl-2. In this chemically defined, synthetic set-up, Bcl-2
totally inhibited the ANT-dependent permeabilization of membranes
induced by verteporfin plus light (Fig. 4D)
. These data
constitute the first demonstration, to our knowledge, that a clinically
relevant chemotherapeutic agent may act on ANT to exert its cytocidal
function. Moreover, they establish for the first time that Bcl-2 may
intercept proapoptotic photochemotherapy at the level of ANT. In
conclusion, based on the present results, ANT becomes a plausible
target of the photosensitizer verteporfin. Of note, the PBR has been
proposed in the past to be the mitochondrial receptor for porphyrin
derivatives (25
, 36
, 37)
, and PBR has been shown to
physically interact with ANT (38)
. However, PBR may not be
the only target of verteporfin, given that Jurkat cells lack PBR
expression (39)
are efficiently killed by this
porphyrin derivative. It remains possible, however, that interaction of
porphyrins with PBR ameliorates their proapoptotic potential, by
increasing the local concentration of verteporfin, near to its true
physiological target, ANT. Irrespective of these theoretical
considerations, the present data suggest ANT to be a target of at least
one photosensitizing agent. Future studies will determine whether the
active search for agents acting on ANT will yield a new class of
anticancer agents.
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FOOTNOTES |
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1 This work has been supported by a special grant
from the French National League against Cancer as well as grants from
Agence Nationale pour la Recherche sur le SIDA and European
Commission, Fondation pour la Recherche Médicale (to
G. K. and C. B.), and Institut Gustave Roussy (to G. K.).
A-S. B. received an Association pour la Recherche sur le
Cancer fellowship, E. J. an Agence Nationale pur la Receherche sur le
SIDA and European Commission, fellowship, and H. L. A. V. a
fellowship from the Fundacao para a Ciencia e a Tecnologia PRAXIS XXI,
Portugal. 
2 G. K. and C. B. share senior coauthorship.
3 To whom requests for reprints should be
addressed, at Centre National de la Recherche Scientifique, UMR 6022,
Université de Technologie de Compiègne, Royallieu, BP20529,
60205 Compiègne, France. Phone: 33-3-44-23-44-16; Fax:
33-3-44-20-39-10; E-mail: catherine.brenner{at}utc.fr
4 The abbreviations used are: PTPC, permeability
transition pore complex; ANT, adenine nucleotide translocator; Atr,
atractyloside; BA, bongkrekic acid; CCCP, carbonylcyanide
m-chlorophenylhydrazone; CsA, cyclosporin A;
DiOC(6)3, 3–3'-dihexyloxacarbocyanineiodide; m,
mitochondrial transmembrane potential; PC, phosphatidylcholin; ROS,
reactive oxygen species; 4-MUP, 4-methylumbelliferyl phosphate; 4-MU,
4-methylumbelliferone; PDT, photodynamic therapy; JC1,
5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine
iodide; PBR, peripheral benzodiazepin receptor; SOD, superoxide
dismutase; VDAC, voltage-dependent anion channel.
Received 8/31/00. Accepted 12/28/00.
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104 events. Pretreatment of
mitochondria with the PTPC inhibitors CsA (5 µM; 5 min.)
or BA (50 µM; 5 min.) strongly reduce the 
) or were
preincubated with 1 mM ATP (
), 1 mM
ADP (
), 1 mM DTT, 500 units/ml catalase (
), and 500
units/ml SOD before verteporfin plus light was applied.
D, Bcl-2 inhibits the verteporfin pore-opening effect on
ANT. ANT liposomes (open symbols) and ANT-Bcl-2
liposomes (full symbols) were incubated with Atr
(circles) or verteporfin (squares) and
illuminated with red light. Experiments are representative of three
independent determinations.