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INTRODUCTION |
Photodynamic therapy
(PDT)1 is an effective
treatment modality that has been approved for the palliation or
treatment of esophageal, lung, and bladder cancers in at least 10 countries (1). Moreover, numerous clinical trials investigate the
effectiveness of PDT in the treatment of gastric, colon, bile duct,
pancreatic, breast, and other cancers (2-9). The treatment consists of
a systemic administration of a photosensitizer followed by illumination
with a laser light. Neither of the PDT components alone can induce antitumor effects but when combined with oxygen they produce lethal cytotoxic agents that can either directly kill tumor cells or destroy
blood vessels within the tumor (1). The photochemical reaction produces
singlet oxygen (1O2) as well as numerous forms
of reactive oxygen, such as superoxide ion (O
), hydrogen
peroxide (H2O2), and hydroxyl radical
(OH·) (10). Increasing evidence indicates that tumor cells can
respond to photodynamic damage by either initiating a rescue response or by undergoing cell death by apoptosis or necrosis (11). Rescue response to sublethal changes allows tumor cells to cope with the
damage induced by the physicochemical stress. This effect is
particularly important in deeper layers of the tumor exposed to laser
illumination. Because the light penetration is limited by the
absorption, scattering, and reflection characteristics of the tissues
the effective dose of energy reaching the deeper layers of the tumor
might cause insufficient damage allowing the initiation of the rescue
response (12). The surviving cells might be the cause of relapse
rendering the treatment less effective. Therefore, elucidation of
molecular changes in the treated cells as well as identification of
drugs that might interfere with rescue responses becomes an important
area of investigation.
Although singlet oxygen has been demonstrated to play a dominant role
in the cytotoxic effects invoked by the photodynamic therapy (13) there
are also a number of observations implicating superoxide anion in
phototoxicity. Superoxide generation increases severalfold after
illumination of PhotofrinTM-bearing cells (14) and
O
exerts multiple deleterious effects such as lipid
peroxidation, DNA cross-linking, and formation of disulfide bonds in
proteins (15). Intracellular O
production correlates with
cell death in 5-aminolevulinic acid- and zinc(II)
phthalocyanine-mediated PDT (16, 17). Cells scavenge O
with
the help of a constitutive Cu,Zn-SOD (SOD-1) as well as an inducible
MnSOD (SOD-2) (18). The latter is associated with the mitochondrial
matrix and participates in dismutating superoxide anion from the site
of PhotofrinTM-mediated generation of reactive oxygen
species during photodynamic therapy (19). Mice treated with a
SOD mimetic,
-carotene, had considerably less ear swelling following
PhotofrinTM-mediated PDT (20), and intravenous
administration of bacterial SOD decreased, to a significant extent, the
antitumor efficacy of PhotofrinTM mediated PDT in three
different murine tumor models (21). Accordingly, sodium
diethyldithiocarbamate, a SOD inhibitor augments cutaneous photosensitization (20). Intuitively, combinations of SOD inhibitor with treatment modalities that produce free radicals might result in
synergistic anticancer activity. All these observations prompted us to evaluate the role of superoxide dismutases in the antitumor effectiveness of PDT and to investigate whether 2-MeOE2, an
estrogen metabolite that has recently been shown to inhibit SOD
activity (22), would be capable of potentiating the antitumor effects of this treatment regimen.
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EXPERIMENTAL PROCEDURES |
Mice--
(C57BL/6 × DBA/2)F1 mice, hereafter
called B6D2F1, and Balb/c mice, 8-12 weeks of age, were
used in the experiments. Breeding pairs were obtained from the Inbred
Mice Breeding Center of the Institute of Immunology and Experimental
Medicine (Wroclaw, Poland) and from the Institute of Oncology (Warsaw,
Poland). All experiments with animals were performed in accordance with
the guidelines approved by the Ethical Committee of the Medical
University of Warsaw.
Reagents--
PhotofrinTM was a generous gift of QLT
PhotoTherapeutics, Inc. (Vancouver, BC, Canada). Manganese-containing
SOD from Escherichia coli, 2-MeOE1, and
2-MeOE2 were purchased from Sigma.
Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was
purchased from Calbiochem (San Diego, CA).
Tumors--
Human pancreatic cancer (Panc-1, HPAC-I, and
HPAF-II), breast cancer (T47-D), and bladder cancer (T24) cell lines
were purchased from ATCC. Murine colon-26 (C-26), a poorly
differentiated colon adenocarcinoma cell line, and Lewis lung carcinoma
(LLC) cells were obtained from Prof. C. Radzikowski (Institute of
Immunology and Experimental Medicine, Wroclaw, Poland) and Prof.
W. W. Jedrzejczak (Department of Immunology, Central Clinical
Hospital, Military School of Medicine, Warsaw, Poland), respectively.
Cells were cultured in RPMI 1640 medium (Invitrogen)
supplemented with 10% heat-inactivated fetal calf serum, antibiotics,
2-mercaptoethanol (50 µM), and L-glutamine (2 mM) (all from Invitrogen), hereafter referred to as culture medium.
Measurements of SOD Activity--
The SOD activity was measured
using a Ransod assay kit purchased from Randox Laboratories Ltd.
(Antrim, United Kingdom) according to the manufacturer's protocol. The
SOD inhibitory activity of 2-MeOE1 or 2-MeOE2
was measured using a manganese-containing SOD. For the measurements of
SOD activity in tumor cells extracts, colon-26 cells were scraped using
a rubber policeman, washed twice in cold PBS, and resuspended in 0.01 M sodium phosphate buffer with 1% Triton X-100. After a
freeze-thaw cycle the cells were homogenized, the extracts were
preincubated with Me2SO (controls) or 2-MeOE2
for 15 min, and the enzymatic activity of SOD was measured using a
Ransod assay kit. In some experiments colon-26 cells were preincubated
with either Me2SO (controls) or 2-MeOE2 added
to the cell cultures for 3, 6, 12, 24, and 48 h. The SOD activity was measured directly from the cell homogenates.
Western Blotting--
For Western blotting C-26 cells were
cultured with 2.5 µg/ml PhotofrinTM for 24 h before
illumination. After washing with PBS, the cells were illuminated using
a 50-watt sodium lamp (Phillips) with light filtered through a red
filter to a final dose of 4.5 kJ/m2. After 1, 2, 4, 12, or
24 h of culture in the fresh medium the cells were washed with PBS
and lysed in a sample buffer containing 2% SDS with protease
inhibitors. Protein concentration was measured with the use of BCA
protein assay (Pierce). Equal amounts of proteins were separated on
15% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride
membranes, and blocked with TBST (Tris-buffered saline, pH 7.4, 0.05%
Tween 20) with 5% nonfat milk and 5% fetal bovine serum. The
following primary antibodies were used for the 6-h incubation: mouse
monoclonal anti-
-tubulin at 1:10000 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), rabbit polyclonal anti-SOD-1 at 1:1000 (Santa Cruz)
and rabbit polyclonal anti-MnSOD at 1:1000 (Research and Diagnostics,
Flanders, NJ). After extensive washing the membranes were incubated for
45 min with the corresponding alkaline phosphatase-coupled secondary
antibodies (Jackson ImmunoResearch Inc., West Grove, PA). The color
reaction was developed using p-nitro blue tetrazolium
chloride and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).
Immunocytochemistry--
Four hours after in vitro
PDT (2.5 µg/ml PhotofrinTM and 4.5 J/m2
light), colon-26 cells were fixed in situ in culture dishes, in a mixture of 4% paraformaldehyde plus 0.1% glutaraldehyde for 1 h at room temperature, scraped, suspended in 10% liquified
gelatin, and pelleted at 1000 rpm at 4 °C. The pellets were cut into
smaller fragments, washed in PBS, cryo-protected in 2.3 M
sucrose (overnight at 4 °C), snap frozen in liquid nitrogen, and
stored at
70 °C until use. Cryo sections (0.5 µm thick) were cut
in a cryo-ultramicrotome (MT-X model with CR-X cryosectioning system,
RMC Boeckeler Instruments, Tucson, AZ) at
60 °C, collected on
glass slides using a drop-of-sucrose technique (according to Ref. 48),
washed in distilled water, equilibrated in PBS, and processed
immediately for immunocytochemistry. The immunocytochemical procedure
included overnight incubation (at 4 °C) in a solution of anti-MnSOD
primary antibody (Research and Diagnostics) diluted 1:200 in 5% normal
goat serum, followed by a standard avidin-biotin immunoperoxidase
reaction, with diaminobenzidine (Sigma) as a chromogen. Omission of the
primary antibody or its replacement by an irrelevant antibody served as
controls of antibody specificity. Phase-contrast images of the
specimens were recorded using Axioplan microscope (Zeiss, Oberkochen,
Germany) coupled to a CCD camera, and image analysis software
(Analysis, Soft Imaging System, Münster, Germany).
Transient Transfection--
For the transfection experiments the
pcDNA3 (Invitrogen) plasmids containing Cu,Zn-SOD (from Dr. L. W. Oberley, Department of Radiation Oncology, University of Iowa) (23)
and MnSOD (from Dr. K. Scharffetter-Kochanek, Department of
Dermatology, University of Cologne, Germany) (24) were used.
T24 cells were seeded at 8 × 105 cells/35-mm dish and
incubated overnight at 37 °C in a 5% CO2 incubator.
Subconfluent proliferating cultures were incubated overnight with 5 µg of each vector in serum-free Dulbecco's modified Eagle's medium
containing LipofectAMINETM 2000 (Invitrogen). Cultures were
washed with PBS to remove the excess vector and
LipofectAMINETM and then incubated again for 24 h in
fresh complete culture medium containing PhotofrinTM (2.5 µg/ml). Immediately after illumination with 4.5 kJ/m2
light the cells were trypsinized and dispensed into the wells of the
96-well flat-bottomed microtiter plate (Nunc) at concentrations of 8, 16, or 32 × 104 cells/200 µl/well. Similarly, T24
cells transiently transfected with each of the plasmid vectors
(Cu,Zn-SOD, MnSOD, or pcDNA3) and not exposed to PDT were
trypsinized and dispensed into the wells at the same concentrations as
PDT-treated cells. Following 24 h incubation the cultures were
rinsed with PBS and stained with 0.5% crystal violet in 30% ethanol
for 10 min at room temperature. Plates were washed four times with tap
water. Cells were lysed with 1% SDS solution, and dye uptake was
measured at 550 nm using an enzyme-linked immunosorbent assay reader
(SLT Labinstrument GmbH, Salzburg, Austria), equipped with a 550-nm
filter. Cytotoxicity was expressed as relative viability of tumor cells
(% of control cultures transiently transfected with corresponding
plasmid vector) and was calculated as follows: cytotoxicity = (APDT
Ab) × 100/(Ac
Ab), where
Ab is background absorbance,
APDT is absorbance of cells exposed to PDT, and
Ac is the absorbance of transfected controls.
Cytotoxicity Assays--
The cytostatic and/or cytotoxic effects
of treatment on tumor cells were measured using crystal violet staining
as described above or a standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as
described previously (25). Briefly, cells were dispensed into a 96-well
flat-bottomed microtiter plate (Nunc) at a concentration of 5-10 × 104 cells/100 µl/well. Cells were treated with serial
dilutions of 2-MeOE1 or 2-MeOE2 or with a
control Me2SO-containing medium (to a final volume of 200 µl) 2 h after plating. After 24 h the medium was completely
removed and replaced with fresh 2-MeOE1 or
2-MeOE2 or a Me2SO-containing medium in a
volume of 100 µl. Then, 100 µl of culture medium with
PhotofrinTM (2.5 µg/ml final concentration) was added to
each well. Following 24 h of incubation with the photosensitizer
the cells in each well were exposed to a laser light delivered through
the fiber optic light delivery system. The illumination area was
exactly matching the size of the wells. In some experiments the cells were preincubated with MnTBAP (100 µM) for 2 h
before laser illumination. After another 24 h 25 µl of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added
to each well for the last 4 h of incubation. The plates were read
in an enzyme-linked immunosorbent assay reader (SLT Labinstrument GmbH,
Salzburg, Austria), using a 450-nm filter. In all experiments a
cytostatic/cytotoxic effect was expressed as relative viability of
tumor cells (% of control cultures incubated with medium only) and was
calculated as follows: relative viability = (Ae
Ab) × 100/(Ac
Ab), where
Ab is background absorbance, Ae is experimental absorbance, and
Ac is the absorbance of untreated controls.
Interaction Analysis--
For the examination of the
interactions between PDT and 2-MeOE2, an ISOBOLOGRAM
analysis was used as described in detail elsewhere (26). Briefly,
inhibition of cell proliferation was determined as described in the
above section. For analysis of the interactions equi-effective doses
were used. These were the doses of either treatment alone or used in a
combination that produced equivalent inhibition of cell growth in
comparison with controls (p < 0.005; Student's
t test). Combinations of the treatments had to induce growth
inhibition higher than those of the same doses used alone (p < 0.001; Student's t test). The
interaction index for two-treatment combinations was computed according
to the following formula: interaction index = (Pc/Pa) + (Mc/Ma), where
Pa and Ma are doses of PDT and
2-MeOE2, respectively, that produce a specified effect when
used alone; Pc and Mc are doses
of PDT and 2-MeOE2, respectively, that produce the same
effect when used in combination. Synergy is claimed when the
interaction index is <1.0.
Tumor Treatment and Monitoring--
For in vivo
experiments exponentially growing tumor cells were harvested,
resuspended in PBS medium to an appropriate concentration of cells, and
injected (1 × 105 C-26 cells or 5 × 105 3LL cells in 20 µl of PBS) into the footpad of the
right hind limb of experimental mice. Tumor cell viability measured by
trypan exclusion was always above 95%.
Tumor-bearing mice were treated orally with 2-MeOE2 at a
dose of 100 mg/kg dissolved in Me2SO and suspended in olive
oil. The treatment with 2-MeOE2 started on day 3 following
inoculation of tumor cells and continued for 6 consecutive days. Mice
in the control groups received oral Me2SO in olive oil in
the same regimen as the treated mice.
PhotofrinTM was administered intraperitoneally at a dose of
10 mg/kg 24 h before illumination with 630 nm light (day 5 of
2-MeOE2 administration, day 7 of the experiment). Control
mice received 5% dextrose. The light source was a He-Ne ion laser
(Amber, Warsaw, Poland). The light was delivered on day 8 of the
experiment using a fiber optic light delivery system. The power density
at the illumination area, which encompassed the tumor and 1-1.5 mm of the surrounding skin, was ~80 mW/cm2 (40 mW laser
output). The total light dose delivered to the tumors was 150 J/cm2. During light treatment mice were anesthetized with
ketamine (87 mg/kg) and xylazine (13 mg/kg) and restrained in a
specially designed holder. Local tumor growth was determined as
described (27) by the formula: tumor volume (mm3) = (longer diameter) × (shorter diameter)2. Relative
tumor volume was calculated as: relative tumor volume = [(tumor
volume)
(initial tumor volume)] × 100%.
Statistical Analysis--
Data were calculated using
MicrosoftTM Excel 98. Differences in in vitro
cytotoxicity assays and tumor volume were analyzed for significance by
Student's t test. Additionally, data were analyzed with the
nonparametric Mann-Whitney U test (InstatTM,
GraphPad Software, San Diego, CA). Kaplan-Meier plots were
generated using days of animal death (after inoculation of tumor cells) as a criterion, and survival time of animals was analyzed for significance by log-rank survival analysis. Significance was defined as
a two-sided p < 0.05.
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RESULTS |
Photodynamic Therapy Induces the Expression of MnSOD--
It has
previously been demonstrated using Northern blotting that PDT induces
the expression of the SOD gene (28). However, it was not investigated
whether increased RNA levels correlate with the expression of SOD at
the protein level. Therefore, we have initially investigated whether
and to what extent PDT influences the level of Cu,Zn-SOD and MnSOD
proteins using Western blotting. C-26 cells were exposed to 2.5 µg/ml
PhotofrinTM and after 24 h of incubation with the
photosensitizer the cells were washed with PBS and illuminated with a
sublethal light dose of 4.5 kJ/m2. Although, there was no
influence of PDT on the expression of Cu,Zn-SOD, we have observed a
time-dependent increase in the level of MnSOD (Fig.
1A). Immunocytochemical
staining of tumor cells exposed to PDT revealed that MnSOD expression
was detectable in surviving but not in lethally damaged cells (Fig. 1,
B1 and B2).

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Fig. 1.
PDT induces MnSOD in tumor cells.
A, C-26 cells were exposed to 2.5 µg/ml
PhotofrinTM for 24 h and then to 4.5 kJ/m2
light and incubated for the indicated times. Total cell lysates were
prepared, and Western blot analysis was performed using anti-Cu,Zn-SOD,
anti-MnSOD, or anti- -tubulin antibodies. B,
immunocytochemical detection of MnSOD in control (B1) C-26
cells and in cells exposed to PDT (B2). In B2,
the characteristic granular MnSOD immunoreactivity is present
exclusively in cells that appear to survive the treatment, whereas it
is virtually absent in severely damaged, presumably necrotic cells. The
images were recorded under phase-contrast optics. Bar
indicates 10 µm.
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The influence of MnSOD Overexpression on the Antitumor Effects of
PDT--
To determine the role of SOD in the antitumor effectiveness
of PDT, C-26 and T24 cells were preincubated with MnTBAP, a cell permeable SOD mimetic, and then exposed to laser illumination. Remarkably, the antitumor efficacy of the PDT was significantly reduced
(Fig. 2, A and B).
Because these studies did not reveal which of the SOD isoforms might be
responsible for the protective effects, T24 cells have been transiently
transfected with Cu,Zn-SOD, MnSOD, or an empty control (pcDNA3)
vector. Exposure of pcDNA3- or Cu,Zn-SOD-transfected cells to PDT
resulted in a comparable cytotoxicity (Fig. 2C). However,
T24 cells transiently transfected with a MnSOD containing plasmid were
significantly less susceptible to the antitumor effects of PDT (Fig.
2C).

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Fig. 2.
Superoxide dismutase protects tumor cells
from lethal damage induced by PDT. A, C-26, and
B, T24 cells were incubated with 2.5 µg/ml
PhotofrinTM for 24 h and then preincubated for 1 h prior to light exposure with 100 µM MnTBAP, a
cell-permeable SOD mimetic. Prior to light treatment the cells were
rinsed with PBS. Immediately after illumination the cells were refed
with fresh MnTBAP (100 µM). Following 24 h
incubation the cytotoxic effects were measured by crystal violet
staining. The bars represent percent cytotoxicity
versus nontreated controls. Black bars represent
cultures of MnTBAP-treated cells exposed to light illumination without
PhotofrinTM. Data refer to mean ± S.D. *,
p < 0.01 versus PDT alone group (Student's
t test). B and C, T24 cells were
transiently transfected with a control plasmid (pcDNA3) or plasmids
containing Cu,Zn-SOD or MnSOD as described under "Experimental
Procedures." After an overnight incubation with plasmids complexed
with LipofectAMINETM 2000 the cultures were postincubated
for 24 h in fresh complete culture medium with
PhotofrinTM (2.5 µg/ml). Immediately after illumination
with 4.5 kJ/m2 light the cells were trypsinized and
dispensed into the wells of the 96-well flat-bottomed microtiter plate
at concentrations of 8, 16, or 32 × 104 cells/200
µl/well. Similarly, T24 cells transiently transfected with each of
the plasmid vectors (Cu,Zn-SOD, MnSOD, or pcDNA3) were trypsinized
and dispensed into the wells at the indicated concentrations. The
bars represent the percent cytotoxicity of PDT-exposed cells
to vector-transfected cells seeded at the same concentration. Data
refer to mean ± S.D. *, p < 0.01 versus all other groups (Student's t test).
D, efficiency of transfection was verified by Western
blotting. Total lysates of T24 cells were prepared, and Western blot
analysis was performed using anti-Cu,Zn-SOD, anti-MnSOD, or
anti-tubulin antibodies.
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Inhibition of SOD Activity by 2-MeOE2--
We have
observed that 2-MeOE2, but not 2-MeOE1, another
estrogen metabolite, significantly suppresses the activity of
manganese-containing SOD from E. coli (Table
I). Additional experiments were done to
determine the potential suppressive effects of 2-MeOE2 on
the SOD activity in colon-26 cells. These studies demonstrated that 2-MeOE2 indeed effectively inhibits in a
dose-dependent manner the SOD activity in extracts from
colon-26 cells (Table II). Moreover, preincubation of colon-26 cells with 2-MeOE2 revealed a
time-dependent inhibition of SOD activity in tumor cells
(Table III). These studies confirmed that
2-MeOE2 can effectively enter the tumor cells and inhibit
SOD activity.
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Table I
The influence of 2-MeOE2 or 2-MeOE1 on the activity of
manganese containing SOD
Manganese containing SOD from E. coli (100 or 200 units/ml)
was preincubated with either 2-MeOE1 or 2-MeOE2 (at
concentrations of 1, 10, and 50 µM) for 15 min at
37 °C. Controls (SOD at 100 or 200 units/ml) were incubated for 15 min with a diluent (Me2SO). Then the SOD activity was measured
as described under "Experimental Procedures."
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Table II
The influence of 2-MeOE2 on the activity of SOD in extracts
from C-26 cells
C-26 cells (50 × 106) were rinsed with PBS, scraped,
pelleted, and resuspended in a sodium-phosphate buffer with 1% Triton
X-100. After one freeze-thaw cycle the cells were homogenized and the
SOD activity was measured in samples incubated for 15 min at 37 °C
with 2-MeOE2 (at 5, 10, or 50 µM) or with a
diluent (Me2SO).
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Table III
The influence of preincubation of C-26 cells with 2-MeOE2 on
the activity of SOD
C-26 cells (5 × 106) were incubated (for 3 to
48 h) with 2-MeOE2 (at 0.5 or 1.0 µM) or
with Me2SO (controls). The cells were then rinsed with PBS,
scraped, pelleted, and resuspended in a sodium-phosphate buffer with
1% Triton X-100. After one freeze-thaw cycle the cells were
homogenized and the SOD activity was measured in samples.
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Inhibition of SOD Activity Potentiates the Cytotoxic Effects of
PDT--
Because 2-MeOE2 has been shown to inhibit SOD
activity in tumor cells we decided to examine the influence of this
agent on the antitumor activity of PDT in vitro. The
experiments performed in three murine cell lines (C-26, LLC, and MDC)
revealed that 2-MeOE2 potentiates the cytotoxic effects of
sublethal doses of PDT (Fig. 3).
According to Berenbaum analysis the interactions between
2-MeOE2 and PDT in all cell lines were synergistic.
Moreover, the antitumor effects of the 2-MeOE2 + PDT
combination were examined in 5 human tumor cell lines of breast
(T47-D), pancreatic (PANC-1, HPAF-II, and HPAC), and bladder (T24)
origin. In all these cell lines there was also a synergistic
potentiation of the antitumor effects of PDT by 2-MeOE2
(Fig. 3). Of note, 2-MeOE1 another estrogen derivative
devoid of SOD inhibitory activity did not influence the antitumor
effects of PDT in two murine (C-26 and LLC) and one human (T47-D)
cancer cell lines (Fig. 4).

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Fig. 3.
Potentiation of in
vitro cytotoxic effects of PDT by 2-MeOE2.
Tumor cells of murine (C-26, LLC, and MDC) and human (T47-D, PANC-1,
HPAF-II, HPAC, and T24) origin were dispensed into a 96-well
flat-bottomed microtiter plate at a concentration of 5-10 × 104 cells/100 µl/well. Cells were pretreated with serial
dilutions of 2-MeOE2 or with a control
Me2SO-containing medium. After 24 h the medium was
completely removed and replaced with fresh 2-MeOE2 or a
control medium. Then, PhotofrinTM (2.5 µg/ml final
concentration) was added to each well and after 24 h of incubation
with the photosensitizer the cells in each well were exposed to a laser
light delivered through the fiber optic light delivery system.
Cytotoxic effects were tested in a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and
are expressed as mean ± S.D. An ISOBOLOGRAM analysis
presents an interaction between PDT and 2-MeOE2 at
inhibiting the growth of tumor cells. The solid line
represents concentrations of both drugs required to inhibit cell growth
to 50% (IC50). The broken lines represent doses
of each treatment required to produce the same growth inhibition as if
the interactions were additive. The numbers in boxes
represent interaction indexes.
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Fig. 4.
The antitumor effects of PDT are not
potentiated by 2-MeOE1. Tumor cells of murine (C-26,
LLC) and human (T47-D) origin were dispensed into a 96-well
flat-bottomed microtiter plate at a concentration of 5-10 × 104 cells/100 µl/well. Cells were pretreated with serial
dilutions of 2-MeOE1 or with a control
Me2SO-containing medium. After 24 h the medium was
completely removed and replaced with fresh 2-MeOE1 or a
control medium. Then, PhotofrinTM (2.5 µg/ml final
concentration) was added to each well and after 24 h of incubation
with the photosensitizer the cells in each well were exposed to a laser
light delivered through the fiber optic light delivery system.
Cytotoxic effects were tested in a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and
are expressed as mean ± S.D.
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Antitumor Effects of PDT in Combination with 2-MeOE2 in
Mice--
Next, we decided to evaluate the antitumor activity of the
combination treatment in vivo in two murine models of
syngenic tumors, namely in C-26 adenocarcinoma syngenic with Balb/c
mice and LLC growing in B6D2F1 mice. Treatment with
2-MeOE2 was started 5 days prior to laser illumination of
the tumors. Although 2-MeOE2 initially inhibited tumor
growth, it did not show significant antitumor effects as measured by
tumor volume or mouse survival time. PDT caused a typical edema that
disappeared 24-36 h after illumination and was followed by a
significant retardation of tumor growth as compared with controls.
Importantly, administration of 2-MeOE2 significantly
potentiated the antitumor effects of PDT in both tumor models, leading
not only to the retardation of tumor growth but also to the
prolongation of mouse survival time (Fig.
5). Remarkably, 60% of colon-26-bearing
mice were completely cured (no tumor for 150 days of observation).

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Fig. 5.
Antitumor effects of the combined treatment
with PhotofrinTM-based PDT and
2-MeOE2. Oral administration of 2-MeOE2
(100 mg/kg) was started on day 3 following inoculation of tumor cells
and continued for six consecutive days. PhotofrinTM was
administered intraperitoneal at a dose of 10 mg/kg, 24 h before
laser illumination (150 J/cm2 on day 7 after inoculation of
tumor cells). Measurements of tumor diameter were started on day 5 after inoculation of tumor cells. A, the influence of the
combined treatment on the growth of LLC tumors in B6D2F1
mice (n = 8). B, Kaplan-Mayer plot of the
survival of B6D2F1 mice bearing LLC tumors. C,
the influence of the combined treatment on the growth of C-26 tumors in
Balb/c mice (n = 6-8). D, Kaplan-Mayer plot
of the survival of Balb/c mice bearing C-26 tumors. ,
p < 0.03 (Mann-Whitney U test) in
comparison with all other groups. #, p = 0.038; ##,
p = 0.003 (log-rank survival analysis) in comparison
with all other groups.
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DISCUSSION |
The mechanisms of the tumoricidal effects of PDT have not yet been
completely elucidated. It has been well established that the presence
of molecular oxygen is an absolute requirement for effective cell
killing during PDT. Hypoxic or anoxic conditions almost completely
reduce the antitumor effectiveness of PDT in vitro (29).
Moreover, PDT was shown to be ineffective in a poorly vascularized
xenograft model (30) and chemotherapy-induced anemia leads to decreased
effectiveness of PDT in mice (31). During photodynamic therapy, a
photosensitizer absorbs light and crosses from its excited singlet
state to the reactive triplet state. It then either transfers its
energy to triplet-state molecular oxygen-generating singlet oxygen
(type II photochemical reactions) or by hydrogen atom extraction or
electron transfer reaction it generates radicals and radical ions (type
I reactions) (10). The latter, which include reactive oxygen species
such as superoxide anion, are molecules with a higher reactivity than
ground-state molecular oxygen. For the effective photodamage both type
I and type II reactions seem to be necessary and inhibition of the
enzymes scavenging reactive oxygen species might offer an attractive
way of potentiating the cytotoxic action of PDT.
Numerous previous studies demonstrated an increased generation of
O
in cancer cells exposed to PDT (14, 16, 17). Because normal
as well as malignant cells have the capacity to scavenge this reactive
oxygen species we decided to investigate the role of superoxide
dismutases in the antitumor effectiveness of PDT. We observed that
following PDT there is an induction of MnSOD expression in tumor cells. Interestingly, there was no influence of PDT on the expression of
Cu,Zn-SOD in tumor cells. These observations can be interpreted through
the intracellular localization of the PhotofrinTM. This
photosensitizer localizes initially in the plasma membrane and after
24 h incubation it can be found mainly in the mitochondria (32).
Although both short and long term incubations with
PhotofrinTM are used experimentally in in vitro
studies, we chose a 24-h preincubation because this is concordant with
clinical situations, where laser illumination is performed 24-48 h
following PhotofrinTM injection. The total light dose
delivered to tumor cells in our experiments was 4.5 kJ/m2
because previous studies with PhotofrinTM revealed that at
this dose superoxide generation increases by a factor of ~2.5
(14).
SODs appear to be important antioxidative enzymes that regulate the
sensitivity of cancer cells to various treatment modalities. Indeed,
the expression of SODs negatively correlates with the sensitivity of
cancer cells to anticancer drugs and radiation therapy (33, 34).
Overexpression of MnSOD suppresses apoptosis (35) and transfection of
tumor cells with antisense oligonucleotides that block SOD activity are
more susceptible to apoptosis induced by chemotherapeutics,
hyperthermia, and
-radiation (36, 37). Remarkably, pretreatment of
tumor cells with a cell-permeable SOD mimetic, MnTBAP, as well as
transient transfection of tumor cells with plasmids encoding MnSOD
renders tumor cells less susceptible to the cytotoxic effects of PDT
(Fig. 2, A and B).
Although several small molecule compounds, such as cyanide ion
(CN
), hydroxyl ion (OH
), and azide ion
(N
) were previously shown to inhibit
SOD at the catalytic site (38), these agents are highly toxic and
therefore of limited potential for cancer therapy. Recently,
2-MeOE2, an endogenous estrogen metabolite with antitumor
activity, was shown to selectively inhibit the activity of superoxide
dismutases (22). Although this finding was questioned in one study
(39), our observations (Tables I-III) as well as a recent report (40)
confirmed that 2-MeOE2 can effectively block superoxide
dismutase activity. In direct, cell-free inhibition assays 50 µM concentration of 2-MeOE2 was required to
inhibit SOD activity by less that 50% (Tables I and II). However, in cell cultures the effective inhibition of SOD was obtained at much
lower concentrations and in a time-dependent manner (Table III). One speculative explanation for this apparent discrepancy might
be that intracellular 2-MeOE2 undergoes a metabolic
activation that converts this mediator to a more potent SOD inhibitor.
Additionally, 2-MeOE2 demonstrates a number of antitumor
effects including antiproliferative and apoptotic effects (41), induction of p53 (42), tubulin depolymerization (43), and inhibition of
angiogenesis (44). This drug has already proved effective in
potentiating the antitumor effects of radiotherapy (45, 46). Moreover,
2-MeOE2 has proved effective in the treatment of
experimental tumors (43, 46, 47) and two clinical trials with
2-MeOE2 in patients with advanced solid tumors have
recently been initiated.
To verify whether cancer cells use MnSOD to avoid the action of
O
, we have evaluated the antitumor effectiveness of PDT in
cells treated with a SOD inhibitor, 2-MeOE2. In all
investigated cell lines (3 murine and 5 human) there was a significant
potentiation of the antitumor effects of PDT (Fig. 3). Berenbaum
analysis of the results revealed that in all instances the cytotoxic
effects of the combination treatment are synergistic. These
observations prompted the in vivo studies evaluating the antitumor efficacy of the combination treatment. Our studies revealed that 2-MeOE2 can sensitize tumors in two murine models to
more effective PDT (Fig. 5). The combined treatment led not only to retardation of tumor growth, but also to prolonged survival and in C-26
colon adenocarcinoma bearing mice to complete cures in 60% of animals.
Altogether, these studies indicate that interference with the
mechanisms used by tumor cells to resist PDT is a rational approach of
potentiating the antitumor effectiveness of this promising treatment
modality. Moreover, our studies suggest that treatment regimens
designed to selectively enhance free radical generation and suppress
antioxidant defenses in the tumor cells may form an effective approach
in the treatment of cancer although toxic side effects of increased
activity of reactive oxygen species should be taken into consideration
in such therapeutic modalities.