Antitumor Effects of Photodynamic Therapy Are Potentiated by 2-Methoxyestradiol

A SUPEROXIDE DISMUTASE INHIBITOR*

Jakub GołabDagger §, Dominika NowisDagger , Michał Skrzycki, Hanna Czeczot, Anna Baranczyk-Kuzma, Grzegorz M. Wilczynski||, Marcin MakowskiDagger , Paweł MrózDagger , Katarzyna KozarDagger , Rafał KaminskiDagger , Ahmad JaliliDagger , Maciej Kopec'Dagger , Tomasz Grzela**Dagger Dagger , and Marek JakóbisiakDagger

From the Departments of Dagger  Immunology,  Biochemistry, || Pathology, and ** Histology and Embryology, Center of Biostructure Research, and the Dagger Dagger  Department of General and Vascular Surgery and Transplantation, The Medical University of Warsaw, 02-004 Warsaw, Poland

Received for publication, September 6, 2002, and in revised form, October 28, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Photodynamic therapy (PDT), a promising therapeutic modality for the management of solid tumors, is a two-phase treatment consisting of a photosensitizer and visible light. Increasing evidence indicates that tumor cells in regions exposed to sublethal doses of PDT can respond by rescue responses that lead to insufficient cell death. We decided to examine the role of superoxide dismutases (SODs) in the effectiveness of PDT and to investigate whether 2-methoxyestradiol (2-MeOE2), an inhibitor of SODs, is capable of potentiating the antitumor effects of this treatment regimen. In the initial experiment we observed that PDT induced the expression of MnSOD but not Cu,Zn-SOD in cancer cells. Pretreatment of cancer cells with a cell-permeable SOD mimetic, Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride, and transient transfection with the MnSOD gene resulted in a decreased effectiveness of PDT. Inhibition of SOD activity in tumor cells by preincubation with 2-MeOE2 produced synergistic antitumor effects when combined with PDT in 3 murine and 5 human tumor cell lines. The combination treatment was also effective in vivo producing retardation of the tumor growth and prolongation of the survival of tumor-bearing mice. We conclude that inhibition of MnSOD activity by 2-MeOE2 is an effective treatment modality capable of potentiating the antitumor effectiveness of PDT.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>), 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> exerts multiple deleterious effects such as lipid peroxidation, DNA cross-linking, and formation of disulfide bonds in proteins (15). Intracellular O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production correlates with cell death in 5-aminolevulinic acid- and zinc(II) phthalocyanine-mediated PDT (16, 17). Cells scavenge O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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, beta -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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) divide  (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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

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.

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.

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.

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. black-square, 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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 gamma -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<UP><SUB>3</SUB><SUP>−</SUP></UP>) 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, 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.

    ACKNOWLEDGEMENTS

We thank Adam Gołab (Erco Leuchten GmBH, Warsaw) for the construction of the sodium lamp for in vitro experiments and Anna Czerepinska and Elzbieta Gutowska for excellent technical assistance.

    FOOTNOTES

* This work was supported by Grant 1M19/M2/2000 from the Medical University of Warsaw, Poland, Grant 4 P05A 025 18 from the State Committee for Scientific Research (to K. B. N.), and a grant from the Foundation for Polish Science (to F. N. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Immunology, Center of Biostructure Research, The Medical University of Warsaw, Chałubinskiego 5, 02-004 Warsaw, Poland. Tel./Fax: 48-22-622-6306; E-mail: jgolab@ib.amwaw.edu.pl.

Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M209125200

    ABBREVIATIONS

The abbreviations used are: PDT, photodynamic therapy; MnTBAP, Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride; LLC, Lewis lung carcinoma; SOD, superoxide dismutase; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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