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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Photodynamic therapy (PDT) is an increasingly popular anticancer treatment that uses photosensitizer, light and tissue oxygen to generate cytotoxic reactive oxygen species (ROS) within illuminated cells. Acting to counteract ROS-mediated damage are various cellular antioxidant pathways. In this study, we combined PDT with specific antioxidant inhibitors to potentiate PDT cytotoxicity in MCF-7 cancer cells. We used disulphonated aluminium phthalocyanine photosensitizer plus various combinations of the antioxidant inhibitors: diethyl-dithiocarbamate (DDC, a Cu/Zn-SOD inhibitor), 2-methoxyestradiol (2-ME, a Mn-SOD inhibitor), l-buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and 3-amino-1,2,4-triazole (3-AT, a catalase inhibitor). BSO, singly or in combination with other antioxidant inhibitors, significantly potentiated PDT cytotoxicity, corresponding with increased ROS levels and apoptosis. The greatest potentiation of cell death over PDT alone was seen when cells were preincubated for 24 h with 300 μm BSO plus 10 mm 3-AT (1.62-fold potentiation) or 300 μm BSO plus 1 μm 2-ME (1.52-fold), or with a combination of all four inhibitors (300 μm BSO, 10 mm 3-AT, 1 μm 2-ME and 10 μm DDC: 1.4-fold). As many of these inhibitors have already been clinically tested, this work facilitates future in vivo studies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Compared with their normal counterparts, many types of cancer cells have increased intracellular levels of reactive oxygen species (ROS), reflecting a disruption of redox homeostasis (1,2). The increase in ROS is attributed to intrinsic mechanisms (activation of oncogenes, aberrant metabolism, mitochondrial dysfunction and loss of functional P53) and extrinsic mechanisms (inflammatory cytokines, an imbalance of nutrients and hypoxic environment), thought to either elevate ROS production or impair the ROS-scavenging capacity of tumor cells (1–3).

Cancer cells adapt to this persistent oxidative stress by developing an enhanced endogenous antioxidant capacity, the extent of which correlates with the aggressiveness of the tumor, whereas at the same time making the malignant cells resistant to anticancer strategies that rely on inducing ROS stress (2). Several therapeutic approaches to killing cancer cells involve elevating cellular ROS levels: photodynamic therapy (PDT), chemotherapy, radiotherapy, immunotherapy, hormone therapy and hyperthermia (3). These anticancer therapeutic approaches are only successful in causing cytotoxicity if the increase in ROS exceeds a threshold level that is incompatible with cellular survival (2). The effective final concentrations of ROS in cancer cells are thus pivotal for pro-oxidant cancer therapies and depend on the balance of the intrinsic ROS levels, the increase in ROS caused by the therapy, and the competing antioxidant capacity of the tumor cells (3).

Several mechanisms are thought to be involved in the protective cellular responses to PDT. These include activation of redox sensitive transcription factors (causing an increase in detoxifying and antioxidant enzymes), activation of antiapoptotic pathways, and over expression of heat shock proteins (inhibiting the formation of an active apoptosome), as reviewed by Nowis et al. (4). Moreover, tumors upregulate antioxidant haeme oxygenase-1 (HO-1) and other cytoprotective molecules as an adaptive response against oxidative stress (5,6). In addition, PDT treatment is antagonized by three major cellular antioxidant defense mechanisms: superoxide dismutase enzymes (Cu/Zn-SOD and Mn-SOD), the glutathione (GSH) system and catalase (6–10).

Therefore, cellular antioxidant systems represent a useful target to improve the therapeutic efficacy of ROS-mediated anticancer therapies. For instance, both radiotherapy (11–13) and platinum-based chemotherapy (14,15) are augmented when combined with inhibitors of glutathione, or superoxide dismutases.

In this study, MCF-7 cancer cells were used to investigate whether combining PDT with inhibitors of the four main antioxidant defenses: diethyl-dithiocarbamate (DDC, an inhibitor of Cu/Zn-SOD), 2-methoxyestradiol (2-ME, an inhibitor of Mn-SOD), l-buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) and 3-amino-1,2,4-triazole (3-AT, an inhibitor of catalase), either singly or in combination, would augment PDT ROS-mediated cell death. In addition, we investigated whether: (1) there was any correlation between the inhibition of specific antioxidant pathway(s) and sensitivity to PDT-induced death; and (2) if there was any relationship between cellular ROS levels and cell death in the presence of the various antioxidant inhibitors. These approaches lead the way to the therapeutic use of antioxidant inhibition plus PDT to sustain a high intracellular level of ROS in cancer cells that would otherwise be resistant to oxidative stress, thereby improving existing PDT treatment and expanding its use to more aggressive tumor types.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

The various experimental conditions and subsequent assays are summarized in Fig. 1.

image

Figure 1.  Cartoon summary of the various experimental conditions. MCF-7 cells were pretreated with antioxidant inhibitors, loaded with photosensitizer, washed and illuminated, and then assayed immediately for ROS levels or after 24 h for cell viability.

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Cell cultures.  Human breast adenocarcinoma cell line MCF-7 was a kind gift from Dr. Marilena Loizidou, UCL Medical School, London. MCF-7 cells were maintained as monolayers in 25 mm glucose DMEM supplemented with: 100 μU mL−1 streptomycin, 100 μU mL−1 penicillin and 10% heat-inactivated fetal calf serum (FCS). For experiments, cells were grown in triplicate at a density of 1 × 105 cells per well in 500 μL growth medium in 24-well tissue culture plates and allowed to attach for 24 h to attain approximately 100% confluence.

PDT treatment.  All incubations and washes prior to PDT were carried out under subdued lighting. Thirty minutes prior to PDT, standard serum-containing DMEM was replaced with fresh medium without serum, containing 5 μg mL−1 AlPcS2 (a gift of Prof David Phillips, Imperial College. Stock 5 mg mL−1 in water [16]). Then cells were rinsed three times with warm PBS, followed by warm phenol red-free DMEM supplemented with 1% pen/strep and 10% FCS. Test samples were immediately exposed (for 15 min) to 28.6 J cm−2 water-filtered halogen white light from a 500 W bulb (or not in the case of dark cytotoxicity). Samples were then incubated under standard cell culture conditions for a further 24 h post PDT in the dark, and then assayed for viability.

Cell viability analysis.  Cells were washed three times with PBS and the collected culture medium and washes were combined to ensure that any detached cells were not lost. The remaining attached cells were removed with trypsin-EDTA and the cell suspension combined with the cells already collected and the total cell number was determined using a haemocytometer. The cells were incubated with 20 μg mL−1 propidium iodide (PI) on ice and analyzed using flow cytometry using a FACSCaliburTM cytometer (BD Biosciences, Oxford, UK). For each sample, 10 000 events were acquired on a logarithmic scale and the gating of single cells was achieved by analysis of forward and side scatter dot plots using BD CellQuest™ Pro software (BD Biosciences, Oxford, UK). PI fluorescence intensity was measured in FL-3 with an emission wavelength of 670 nm. For measurement of apoptosis 24 h after PDT, cells were incubated with 1:100 Annexin V-FITC (Sigma, A9210) for 15 min at room temperature in the dark and then analyzed using flow cytometry (as detailed above), but using FL-1 with an emission wavelength of 530 nm.

Measurement of ROS.  ROS levels were determined by flow cytometry using the fluorescent probes 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) or dihydroethidium (DHE). Cells were seeded in 24-well tissue culture plates at a density of 2 × 105 cells mL−1 and incubated at 37°C overnight and then treated with 5 μg mL−1 AlPcS2 and the various inhibitors for the indicated times. After incubation, the photosensitizer-containing medium was removed and the cells rinsed three times with warm PBS. Fresh phenol red-free culture medium with 10 μm DCFH-DA or 10 μm DHE was added under subdued light conditions and the test samples then exposed to 28.6 J cm−2 water-filtered white light (or not in the case of dark cytotoxicity). The cells were then washed twice with cold PBS, trypsinized and centrifuged for 5 min at 550 g and at 4°C. The cell pellet was resuspended in 200 μL cold PBS and probe fluorescence was measured using FACSCaliburTM cytometer by collecting 10 000 events for each sample. ROS levels were expressed as mean fluorescence intensity (MFI) as calculated by BD CellQuest™ Pro software. ROS was also measured in a cell-free system in 96-well plates (Corning: black, clear-bottom, flat) comprising fresh phenol red-free culture medium containing 0.2 μm DCFH-DA, 5 μg mL−1 AlPcS2 and the various inhibitors. Each plate was illuminated, as detailed above (or maintained in the dark) and the DCF fluorescence was measured immediately using a plate reader (BMG labtech, FLUOstar optima; Ex 485 nm, Em 520 nm, gain 300).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Initially, we identified a range of doses for each antioxidant inhibitor, in the absence of photosensitizer, that did not cause significant MCF-7 cell death or morphological change during 24 h, but were nevertheless within the accepted working range for inhibition (17–21). In half of these initial experiments, cells were illuminated 30 min after adding the antioxidant inhibitor to determine if any inhibitor had an intrinsic photosensitizing activity (Figs 2 and S1). Neither 3-AT nor BSO were found to be toxic or phototoxic at any of the doses used (Fig. 2c,d). By contrast, 2-ME led to the dose-dependent appearance of many rounded and floating cells, both in the dark and in photoirradiated samples (Fig. S1). However, cell viability analysis demonstrated this was not due to cell death (Fig. 2a), suggesting that 2-ME affected cell adhesion, as has been proposed before (22).

image

Figure 2.  Cell viability in the presence of antioxidant inhibitors, but without AlPcS2 photosensitizer. MCF-7 cells were treated with various concentrations of antioxidant inhibitors and exposed to 28.6 J cm−2 white light (dashed line) or kept in the dark (continuous line) and the percentage cell survival determined using propidium iodide exclusion assay. All conditions demonstrate minimal dark or phototoxicity, except (b) DDC at the highest concentrations.

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DDC demonstrated a concentration dependent increase in cell death that, at the highest dose (30 μm), was more pronounced when samples were illuminated (Figs 2b and S1), suggesting that DDC may have some innate photosensitizing activity and/or it interferes with the antioxidant systems that normally counteract ROS produced during light exposure by endogenous chromophores, such as riboflavin and porphyrin.

As a result of these dose-toxicity tests, we chose three concentrations of each inhibitor that were minimally toxic (in the absence or presence of light) for further analysis in combination with 5 μg mL−1 AlPcS2 photosensitizer. These concentrations were: 2-ME (0.3, 1 and 3 μm), DDC (1, 3 and 10 μm), 3-AT (1, 3 and 10 mm) and BSO (30, 100 and 300 μm).

Dark and phototoxicity studies of single antioxidant inhibitors with photosensitizer

MCF-7 cells were coincubated with antioxidant inhibitor and AlPcS2 photosensitizer for 30 min prior to PDT and then maintained in the dark with antioxidant inhibitor for a further 24 h prior to analysis of cell viability and number per well (Table 1: 0.5 h and Fig. 3).

Table 1.   Individual inhibitors.
Inhibitor concentration and preincubation time (h)5 μg mL−1 AlPcS2DarkLightViability ratio
Cell viability (%)Cell number (105)Cell viability (%)Cell number (105)
  1. The dark and phototoxicity effects on MCF-7 cells of AlPcS2 with 2-ME, or DDC, or 3-AT, or BSO on percentage cell viability, cell number, and viability ratio after 0.5, 1 or 24 h preincubation with antioxidant inhibitors. Cell viability was assessed by PI exclusion assay. For each treatment condition, results represent the mean of four independent experiments for 0.5 and 24 h, and five independent experiments for 1 h (mean ± SEM). Within each time-point, data were analyzed by one way ANOVA with Tukey’s multiple comparison test. The values in bold show statistically significant decreases compared with AlPcS2 alone; *P < 0.05, ***P < 0.001.

00.592.5 ± 0.52.99 ± 0.1593.1 ± 0.72.87 ± 0.140.97 ± 0.02
00.5+92.4 ± 0.62.98 ± 0.1587.7 ± 0.92.40 ± 0.150.70 ± 0.02
2-ME 0.3 μm0.5+92.7 ± 0.23.08 ± 0.4386.5 ± 1.81.97 ± 0.280.60 ± 0.03
2-ME 1 μm0.5+92.5 ± 0.62.87 ± 0.3784.6 ± 2.11.89 ± 0.250.57 ± 0.02*
2-ME 3 μm0.5+88.8 ± 0.2***2.45 ± 0.3882.5 ± 1.51.54 ± 0.270.58 ± 0.05
DDC 1 μm0.5+91.6 ± 1.12.72 ± 0.1788.9 ± 0.62.01 ± 0.120.72 ± 0.06
DDC 3 μm0.5+92.0 ± 0.72.68 ± 0.1588.2 ± 0.61.91 ± 0.770.69 ± 0.05
DDC 10 μm0.5+91.6 ± 0.82.60 ± 0.1488.4 ± 1.41.79 ± 0.080.64 ± 0.03
3-AT 1 mm0.5+93.1 ± 1.22.77 ± 0.1187.7 ± 1.71.85 ± 0.120.63 ± 0.05
3-AT 3 mm0.5+93.7 ± 1.22.72 ± 0.0688.3 ± 1.71.85 ± 0.160.64 ± 0.05
3-AT 10 mm0.5+94.1 ± 0.72.65 ± 0.1188.9 ± 1.71.70 ± 0.110.59 ± 0.04
BSO 30 μm0.5+91.5 ± 1.973.00 ± 0.4787.0 ± 1.72.03 ± 0.250.66 ± 0.05
BSO 100 μm0.5+92.4 ± 1.643.06 ± 0.4385.8 ± 2.21.84 ± 0.260.56 ± 0.01*
BSO 300 μm0.5+93.6 ± 1.222.99 ± 0 .4986.3 ± 2.01.79 ± 0.280.57 ± 0.03*
0195.3 ± 0.22.31 ± 0.1295.6 ± 0.32.16 ± 0.070.94 ± 0.03
01+94.8 ± 0.42.22 ± 0.0993.6 ± 0.91.70 ± 0.180.79 ± 0.03
2-ME 1 μm1+93.9 ± 0.82.08 ± 0.1192.2 ± 0.61.47 ± 0.120.68 ± 0.04
DDC 3 μm1+94.2 ± 0.52.05 ± 0.1392.5 ± 0.31.44 ± 0.110.67 ± 0.04
3-AT 10 mm1+93.7 ± 0.81.76 ± 0.2191.2 ± 1.21.36 ± 0.190.72 ± 0.04
BSO 300 μm1+92.6 ± 1.31.95 ± 0.1892.7 ± 0.51.47 ± 0.170.70 ± 0.01
02494.6 ± 0.63.44 ± 0.1296.1 ± 0.43.46 ± 0.121.02 ± 0.02
024+95.0 ± 0.73.37 ± 0.1389.2 ± 0.82.57 ± 0.190.74 ± 0.03
2-ME 1 μm24+94.2 ± 0.53.10 ± 0.1589.9 ± 0.62.03 ± 0.220.61 ± 0.05
DDC 3 μm24+94.8 ± 0.83.17 ± 0.1689.9 ± 1.02.25 ± 0.190.67 ± 0.03
3-AT 10 mm24+93.3 ± 0.72.96 ± 0.1587.4 ± 0.82.04 ± 0.140.65 ± 0.05
BSO 300 μm24+95.3 ± 0.43.37 ± 0.1585.5 ± 0.8*2.10 ± 0.200.55 ± 0.03*
image

Figure 3.  Representative phase-contrast micrographs of MCF-7 cells in dark and phototoxicity studies. The cells were treated with AlPcS2 and the different concentrations of antioxidant inhibitors for 30 min and then exposed to light or kept in the dark. The samples were incubated for a further 24 h under standard cell culture conditions in the presence of inhibitors, and the phase-contrast micrographs acquired at the end of the incubation period.

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The combination of AlPcS2 with the different concentrations of DDC, or 3-AT or BSO for 30 min in the dark demonstrated no altered cytotoxicity after 24 h, although there was a nonsignificant trend toward fewer cells with increasing antioxidant inhibitor concentration (Table 1: dark 0.5 h and Fig. 3b–d). Morphologically, there were very few rounded and floating cells even at the highest concentrations of these three antioxidant inhibitors (Fig. 3b–d). By contrast, the combination of AlPcS2 and 2-ME in the dark showed a dose-dependent increase in the number of rounded floating cells (Fig. 3a), as had been seen in the 2-ME-only experiments (Fig. S1). However, unlike the 2-ME-only experiments, the combination of 2-ME and photosensitizer in the dark reduced the percentage cell viability in a dose-dependent manner, with 3 μm 2-ME achieving a small, but highly significant (P < 0.001) decrease in cell viability when compared with AlPcS2 only (Table 1: dark 0.5 h, cell viability).

Unlike the situation in the dark, the combination of AlPcS2 with any of the antioxidant inhibitors for 30 min, followed by PDT, caused an increase in the number of floating cells (Fig. 3), a dose-dependent trend of decreasing total cell number (Table 1: light 0.5 h, cell number) and, for 2-ME, a dose-dependent trend of decreasing cell viability (Table 1: light 0.5 h, cell viability).

The numbers of viable cells per well in the dark and after PDT treatment were calculated from their respective percentage cell survival and total cell number per well (Table 1). Dividing the values for the total viable cells after PDT treatment by total viable cells in the dark, a viability ratio was obtained (Table 1: viability ratio). This ratio normalized any differences as a result of antioxidant inhibitors alone and allowed any specific PDT potentiating effect to be determined.

Cells treated with AlPcS2 alone or in combination with the different antioxidant inhibitors always produced a significant (P < 0.001) reduction in viability ratio compared with the control samples without photosensitizer or inhibitor.

Importantly, however, three inhibitor treatment conditions significantly potentiated (P < 0.05) the phototoxicity of AlPcS2 following 30 min preincubation (Table 1: viability ratio 0.5 h). These were 1 μm 2-ME, 100 μm and 300 μm BSO. Both DDC and 3-AT demonstrated a nonsignificant trend to potentiate phototoxicity at the highest concentrations used (10 μm DDC or 10 mm 3-AT).

Optimizing the preincubation time with single antioxidant inhibitors

The short-term incubation (30 min) of cells with antioxidant inhibitors helped to establish a single concentration for each antioxidant inhibitor that was not significantly dark toxic, but produced a reduction in the viability ratio, compared with AlPcS2 alone. These concentrations were 1 μm 2-ME, 10 μm DDC, 10 mm 3-AT and 300 μm BSO and were chosen for studies of longer-term (1 and 24 h) preincubation before PDT treatment. AlPcS2 photosensitizer was pulse-loaded into cells 30 min prior to PDT and cells were then maintained in the dark for a further 24 h before assessing cell viability, cell number, and viability ratio (Table 1: 1 and 24 h, and Fig. 4).

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Figure 4.  Optimizing the preincubation time with antioxidant inhibitors. MCF-7 cells were treated with the specified concentrations of antioxidant inhibitors for (a) 1 h or (b) 24 h, loaded with AlPcS2 photosensitizer for 30 min, and then illuminated (or kept in the dark for the dark toxicity studies). The representative images were a snapshot of the center of the well 24 h later, before analysis of the percentage cell viability.

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In dark toxicity studies, the combination of AlPcS2 with any of the specified antioxidant inhibitors resulted in very few rounded floating cells and did not significantly affect the percentage cell viability compared with no-drug/no-photosensitizer control, after either 1 h (Fig. 4a and Table 1: dark 1 h, cell viability) or 24 h (Fig. 4b and Table 1: dark 24 h, cell viability) preincubation. However, there was a nonsignificant trend toward fewer cells compared with AlPcS2 alone after 1 h preincubation (Table 1: dark 1 h, cell number) or 24 h preincubation (Table 1: dark 24 h, cell number). For either preincubation time, the greatest decrease in total cell number per well in the dark was achieved using a combination of AlPcS2 and 10 mm 3-AT (Table 1: dark).

Upon photoillumination, the combination of AlPcS2 with any of the antioxidant inhibitors led to an increase in floating cells in 1 (Fig. 4a) and 24 h (Fig. 4b) preincubated samples, compared with corresponding dark controls.

The combination of AlPcS2 with almost all of the specified antioxidant inhibitors showed a nonsignificant trend of decreased cell number and percentage cell viability following PDT when compared with AlPcS2-PDT alone, both in 1 and 24 h (Table 1: light) preincubated samples. The exception was with 300 μm BSO, which showed a significant (P < 0.05) decrease in cell viability in 24 h preincubated samples (Table 1: light 24 h, cell viability).

For 1 and 24 h preincubated samples, each of the antioxidant inhibitors demonstrated a trend to potentiate AlPcS2-PDT, (Table 1: viability ratio). However, it was only 300 μm BSO after 24 h preincubation that achieved a statistically significant reduction (P < 0.05) in viability ratio compared with AlPcS2 alone (Table 1: viability ratio 24 h, and Fig. 5c left side graph).

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Figure 5.  The effect of preincubation time with antioxidant inhibitor(s) on the viability ratio of MCF-7 cells. Viability ratios were calculated from cell survival and number data from the dark and phototoxicity studies after (a) 0.5 h, (b) 1 h or (c) 24 h preincubation with single antioxidant inhibitors (left side) or mixtures (right side). Results represent the mean of at least three independent experiments for each treatment condition (mean ± SEM) and were analyzed by one way ANOVA with Tukey’s multiple comparison test. Statistically significant differences compared with the relevant AlPcS2-only control (second bar in each graph pair) are indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Line (ratio of 1) indicates no difference in cell viability between the dark and phototoxicity.

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Combinations of antioxidant inhibitors

We selected four combinations of inhibitors, motivated by the antioxidant systems they are known to target (Fig. 9): 1 μm 2-ME plus 10 μm DDC target Mn-SOD and Cu/Zn-SOD, respectively, thereby inhibiting the breakdown of singlet oxygen to hydroperoxides. Ten millimolar 3-AT plus 300 μm BSO target both catalase and glutathione synthesis, thereby preventing the breakdown of hydroperoxides. One micromolar 2-ME plus 300 μm BSO target both Mn-SOD and glutathione synthesis (and this particular pairing was chosen since, as individual inhibitors, they showed the greatest potentiation of cytotoxicity). Finally, a cocktail of all four inhibitors was used to target all the major antioxidant systems. Each combination of inhibitors was added to cells 30 min, or 1 h, or 24 h before PDT and then analyzed 24 h after PDT.

Dark toxicity studies showed that the combination of AlPcS2 plus 30 min or 1 h preincubation with the antioxidant inhibitor mixtures were not toxic to the cells in the dark (Table 2: dark 0.5 and 1 h). By contrast, with 24 h preincubation, the combination of AlPcS2 with either 10 mm 3-AT plus 300 μm BSO, or the four inhibitor cocktail showed a small, but significant decrease (P < 0.05) in percentage cell viability compared with AlPcS2 alone (Table 2: dark 24 h, cell viability).

Table 2.   Combinations of inhibitors.
Inhibitors and preincubation time (h)5 μg mL−1 AlPcS2DarkLightViability ratio
Cell viability (%)Cell number (105)Cell viability (%)Cell number (105)
  1. The dark and phototoxicity effects of AlPcS2 with combinations of inhibitors on the viability and cell number after 24 h preincubation. MCF-7 cells were treated with AlPcS2 in the presence of the indicated concentrations of antioxidant inhibitors for up to 24 h. Cell viability was assessed by PI exclusion assay. Results represent the mean of four independent experiments (mean ± SEM) for each treatment condition and were analyzed by one way ANOVA with Tukey’s multiple comparison test. The values in bold show statistically significant differences compared with AlPcS2 alone. *P < 0.05, **P < 0.01, ***P < 0.001.

00.593.7 ± 0.52.09 ± 0.0895.0 ± 0.62.08 ± 0.050.98 ± 0.01
00.5+94.0 ± 0.72.02 ± 0.0591.0 ± 1.11.64 ± 0.060.72 ± 0.02
1 μm 2-ME 3 μm DDC0.5+93.4 ± 0.62.04 ± 0.0790.0 ± 1.01.46 ± 0.090.69 ± 0.03
10 mm 3-AT 300 μm BSO0.5+93.5 ± 0.52.00 ± 0.0891.0 ± 0.81.40 ± 0.080.68 ± 0.03
1 μm 2-ME 300 μm BSO0.5+94.0 ± 0.52.03 ± 0.0790.2 ± 0.51.37 ± 0.110.65 ± 0.04
All0.5+93.6 ± 0.52.00 ± 0.0791.1 ± 0.41.43 ± 0.080.70 ± 0.04
0193.7 ± 1.52.16 ± 0.0489.2 ± 4.72.16 ± 0.060.96 ± 0.03
01+93.3 ± 1.42.11 ± 0.0588.8 ± 2.21.90 ± 0.060.81 ± 0.02
1 μm 2-ME 3 μm DDC1+91.2 ± 2.32.06 ± 0.0786.7 ± 4.11.53 ± 0.10*0.71 ± 0.04
10 mm 3-AT 300 μm BSO1+90.4 ± 2.61.93 ± 0.0884.1 ± 6.11.61 ± 0.070.77 ± 0.05
1 μm 2-ME 300 μm BSO1+90.6 ± 3.22.03 ± 0.0786.0 ± 3.51.54 ± 0.11*0.72 ± 0.04
All1+90.6 ± 4.22.0 ± 0.0982.1 ± 7.91.41 ± 0.04**0.64 ± 0.04*
02495.5 ± 0.23.39 ± 0.3296.5 ± 0.43.24 ± 0.241.01 ± 0.02
024+95.9 ± 0.43.32 ± 0.2888.9 ± 1.12.58 ± 0.200.73 ± 0.02
1 μm 2-ME 3 μm DDC24+94.5 ± 0.13.09 ± 0.1786.8 ± 2.41.80 ± 0.120.54 ± 0.05*
10 mm 3-AT 300 μm BSO24+92.7 ± 1.0*3.06 ± 0.0378.6 ± 5.21.57 ± 0.21**0.45 ± 0.09***
1 μm 2-ME 300 μm BSO24+95.0 ± 0.52.89 ± 0.1780.3 ± 2.81.63 ± 0.11*0.48 ± 0.04**
All24+92.9 ± 0.6*2.59 ± 0.1179.8 ± 3.41.57 ± 0.07**0.52 ± 0.03**

In phototoxicity studies, each of the different antioxidant inhibitor combinations, at every preincubation time, demonstrated a trend of decreased cell number, which in several cases was statistically significant (Table 2: light, cell number). For each inhibitor combination, longer preincubation times gave a greater decrease in cell number and decrease in cell viability (Table 2).

Similarly, for each antioxidant inhibitor combination, increases in preincubation time gave progressively significant decreases in viability ratios, compared with AlPcS2 alone (Table 2: viability ratio and Fig. 5 right side graphs). Thus, 30 min preincubation provided no statistically significant decrease in viability ratio. One hour preincubation yielded a significantly decreased (P < 0.05) viability ratio for the inhibitor cocktail (1.27-fold decrease compared with AlPcS2 alone) (Table 2: viability ratio and Fig. 5b right side graph). Finally, in samples preincubated for 24 h, any of the inhibitor combinations significantly decreased the viability ratio compared with AlPcS2 alone: by 1.35-fold for 1 μm 2-ME plus 10 μm DDC (P < 0.05), 1.62-fold for 10 mm 3-AT plus 300 μm BSO (P < 0.001), 1.52-fold 1 μm 2-ME plus 300 μm BSO (P < 0.01) and 1.4-fold for the cocktail (P < 0.01) (Table 2: viability ratio and Fig. 5c right side graph).

Understanding the mechanisms of antioxidant inhibitor potentiated PDT

Apoptosis.  Next, it was assessed whether AlPcS2 plus the antioxidant inhibitors, either singly or in combination, led to apoptosis as assessed by annexin V-FITC flow cytometry 24 h after PDT (or dark control).

In the dark, none of the treatment conditions significantly increased the proportion of annexin V-positive cells when compared with no photosensitizer control (Fig. 6 left side graphs).

image

Figure 6.  The percentage of apoptotic MCF-7 cells, as determined using annexin V-FITC staining, following various preincubation times with antioxidant inhibitors, either maintained in the dark (left side) or after PDT (right side). MCF-7 cells were preincubated for (a) 0.5 h, (b) 1 h or (c) 24 h with the specified antioxidant inhibitors and analyzed 24 h later. Results represent the mean of at least three independent experiments for each treatment condition (mean ± SEM) and were analyzed by one way ANOVA with Tukey’s multiple comparison test. Statistically significant differences compared with the relevant AlPcS2-only control (second bar in each graph pair) are indicated by asterisks; *P < 0.05.

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All AlPcS2-PDT treatments produced an increase in annexin V-positive cells over light-exposed no photosensitizer controls (Fig. 6 right side graphs). However, only two antioxidant inhibitor combinations significantly increased the proportion of annexin V-positive cells compared with AlPcS2-PDT alone, and both of these occurred following 24 h preincubation. They were: 10 mm 3-AT plus 300 μm BSO (P < 0.05) and the inhibitor cocktail (P < 0.05; Fig. 6c right side graph). In future experiments, it will be interesting to examine both earlier and later patterns of apoptosis to better understand the mechanisms and kinetics of cell death with each of the antioxidant inhibitors.

Analysis of ROS levels.  At the end of each preincubation period, the intracellular ROS levels were assayed during illumination (or in the dark) using dichlorofluorescein (DCF) to detect general ROS (Fig. 7), or DHE to detect superoxide anions (Fig. 8). For each type of analysis, ROS values were expressed relative to the light-treated AlPcS2 sample of that preincubation time.

image

Figure 7.  The relative ROS levels in MCF-7 cells, determined by DCF fluorescence, as a function of preincubation time with various antioxidant inhibitors. For each preincubation time the ROS levels are reported as a ratio, obtained by dividing the mean of each sample DCF fluorescence by the mean DCF fluorescence of the light-treated AlPcS2 sample (second bar in each light-treated graph, right side). Results represent the mean of three independent experiments for each condition (mean ± SEM), analyzed by one way ANOVA with Dunnett’s test. Statistically significant differences compared with the relevant AlPcS2-only control (second bar in each graph pair) are indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Dotted line (ratio of 1) indicates no difference in DCF fluorescence compared with light-treated AlPcS2-only samples.

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image

Figure 8.  The relative superoxide levels in MCF-7 cells, determined by DHE fluorescence, as a function of preincubation time with various antioxidant inhibitors. For each preincubation time the ROS levels are reported as a ratio, obtained by dividing the mean of each sample DHE fluorescence by the mean DHE fluorescence of the light-treated AlPcS2 sample (second bar in each light-treated graph, right side). Results represent the mean of three independent experiments for each condition (mean ± SEM), analyzed by one way ANOVA compared with the relevant AlPcS2-only control (second bar in each graph pair). Dotted line (ratio of 1) indicates no difference in DHE fluorescence compared with light-treated AlPcS2-only samples.

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In the dark, none of the antioxidant inhibitors, singly or in combination, significantly increased ROS (Fig. 7 left side graphs) or superoxide levels (Fig. 8 left side graphs) compared with AlPcS2 alone.

During illumination, the presence of BSO either singly (Fig. 7c right side graph) or in combination with 3-AT or 2-ME, but not the cocktail (Fig. 7a,c right side graphs), produced higher levels of ROS (using DCF) compared with the other inhibitors analyzed. Conversely, the presence of 2-ME, or especially DDC, resulted in photoinduced ROS levels that were often lower than in samples treated with AlPcS2 alone (Fig. 7 right side graphs). This was unexpected as the SOD inhibitors 2-ME or DDC would be predicted to raise intracellular ROS, notably superoxides.

Although DCF is commonly used as a general indicator of ROS, and is thought to reflect the overall oxidative status of the cell (23,24), some studies have suggested that it is relatively insensitive to superoxides and hence not the appropriate probe for detecting superoxide radicals, as reviewed by Gomes et al. (25). To address whether this limitation might explain the apparent reduction in ROS observed with the two SOD inhibitors, DHE, a fluorescent probe that has relative specificity for superoxide anion radicals (O2; 25) was used (Fig. 8).

The combination of AlPcS2 and 1 μm 2-ME increased the photoinduced O2 levels in all the preincubation times (Fig. 8 right side graphs), with the maximum increase of 1.41-fold above AlPcS2 alone after 24 h preincubation (Fig. 8c right side graph). Surprisingly, 10 μm DDC did not demonstrate any increase in O2 levels (Fig. 8).

In cell-free media, 3-AT (alone or in combination with other inhibitors) resulted in a light-dependent increase in ROS, as measured by DCF fluorescence (Fig. S2). There are many differences between the cellular and cell-free systems, making direct comparisons inappropriate. Nevertheless, this observation does imply that our DCF fluorescence results with 3-AT should be treated with caution.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

The success of PDT as an antitumor treatment is determined by the balance between photo-oxidative damage to cells by ROS (26), versus elimination of ROS by the scavenging activity of the cellular antioxidant systems (2,27). In addition, there is increasing evidence that tumor cells initiate rescue mechanisms following PDT damage that include upregulation of antioxidant systems (4,6,8,28,29). In this study, we demonstrate potentiation of AlPcS2 PDT in MCF-7 cancer cells by inhibiting cellular antioxidant defenses. This was achieved at antioxidant inhibitor concentrations that did not significantly increase cytotoxicity by themselves, making this work of interest for future preclinical studies.

The main cellular antioxidant defenses that act against PDT are summarized in Fig. 9 and can be divided into two pathways. Initially, short-lived superoxides are converted to hydroperoxides by the superoxide dismutases, Cu/Zn-SOD and Mn-SOD. Subsequently, these hydroperoxides are broken down by glutathione and catalase.

image

Figure 9.  Summary diagram of the main cellular antioxidant systems responsible for detoxifying PDT-produced superoxides (O2˙) and the inhibitors that affect them.

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Previously published results, using different cell lines, showed improved PDT cytotoxicity following single inhibition of glutathione (30) or catalase (31,32) or Mn-SOD (8) or Cu/Zn-SOD (32) or HO-1 (33), which can itself upregulate SOD and catalase (34). However, unlike these previous studies, which had focussed on the effect of one or two antioxidants on PDT, our study directly compared inhibition of all these ROS-scavenging systems, and then took this one step further by examining combinations of inhibitors.

Thus, using singe antioxidant inhibitors at their optimum concentrations, we found that, in terms of protecting MCF-7 cancer cells against PDT: glutathione > Mn-SOD > catalase > Cu/Zn-SOD (Table 3), consistent with data demonstrating that tumor cells up-regulate Mn-SOD (8) and glutathione (35,36) following PDT-induced damage.

Table 3.   Summary of experimental results with 24 h preincubation.
Inhibitor(s)Antioxidant(s) inhibitedΔ viability ratioΔ ROSΔ annexin V
  1. Antioxidant inhibitors are ranked by the greatest decrease in PDT-specific cell kill (Δ viability ratio), and then by greatest increase in ROS (Δ ROS), and by greatest increase in apoptotic cells (Δ annexin V). Each value represents the difference compared with the corresponding light-treated AlPcS2 only control. Where this difference was statistically significant, it is indicated by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001.

3-AT 10 mm BSO 300 μmCatalase, glutathione0.28***2.79**15.37*
2-ME 1 μm BSO 300 μmMn-SOD, glutathione0.25**2.11**13.53
Cocktail 2-ME 1 μm DDC 3 μm 3-AT 10 mm BSO 300 μmCu/Zn-SOD, Mn-SOD, catalase, glutathione0.21**0.3813.53*
2-ME 1 μm DDC 3 μmCu/Zn-SOD, Mn-SOD0.19*−0.317.50
BSO 100 μmGlutathione0.14*1.30**3.35
2-ME 1 μmMn-SOD−0.13−0.30 (1.41 DHE)2.86
3-AT 10 mmCatalase−0.110.184.50
DDC 10 μmCu/Zn-SOD−0.06−0.68−1.42

We summarize our data for the 24 h preincubation period in Table 3, ranking antioxidants from most effective to least effective, in terms of augmentation of AlPcS2-PDT cytotoxicity. Decreased viability ratio, increased ROS and increased annexin V staining all rank in the same order for the first three inhibitor combinations (BSO plus 3-AT, BSO plus 2-ME and cocktail). This not only suggests a causal relationship between increased ROS levels and cell death, but also indicates that hydroperoxide degradation, normally occurring jointly via catalase (inhibited by 3-AT) and glutathione (inhibited by BSO), is of greater importance in protecting MCF-7 cells against AlPcS2-PDT than superoxide degradation, occurring jointly via Cu/Zn-SOD and Mn-SOD.

However, a disparity occurs between cell kill and ROS levels for some single inhibitors. For instance (in Table 3), BSO alone gives the third highest ROS increase, but only the fifth highest PDT-specific cell kill. Conversely, inhibition of catalase with 3-AT gives the fifth highest ROS increase, but only the seventh highest PDT-specific cell kill. In cell-free experiments, 3-AT caused a light-dependent increase in DCF fluorescence. If this occurred via a nonROS-mediated photochemical reaction then this could explain the disparity between apparent ROS levels and PDT cytotoxicity as a result of 3-AT. Alternatively, it is likely that a threshold level of ROS needs to be crossed before cytotoxic effects are obtained, as suggested by Trachootham et al. (2). Thus, whereas we observed several significant increases in ROS, these may be below a level that is sufficient to cause cell death. In addition, the disruption of the redox balance by depletion of one antioxidant enzyme often results in compensatory changes in other enzyme activities, as well as in low-molecular weight antioxidants (37,38).

The assessment of ROS production using DCF demonstrated that BSO, either singly or in combination with other inhibitors, produced the largest increase in ROS levels compared with the other inhibitors. Glutathione is the major ROS-scavenging system in all cells (2) and its inhibition by BSO has previously been shown to be followed by an increase in ROS levels (39).

2-ME and 3-AT have previously been shown to increase the ROS levels in different cell lines (40,41) and our results demonstrated a slight increase in ROS levels in the presence of 3-AT (using DCF) and 2-ME (using DHE) when compared with AlPcS2 alone. DDC, on the other hand, consistently yielded reduced ROS levels compared with AlPcS2 alone with both ROS assays and this agreed with results obtained by Han et al. (20) and Kimoto-Kinoshita et al. (42) who also observed a decrease in ROS in the presence of DDC. DDC is known to have both antioxidant and pro-oxidant effects in different cell systems (42,43). As an antioxidant, it can act directly by inhibiting superoxide production or blocking oxidoreductase enzymes such as xanthine oxidase that are involved in free radical production (43,44) and this might explain the decreased ROS levels in the presence of DDC, either singly or in combination with other inhibitors. It may also explain why the cocktail of inhibitors only showed a slight increase in ROS, compared with other inhibitor combinations that did not include DDC.

In summary, the pretreatment of MCF-7 cancer cells with antioxidant inhibitors prior to PDT (especially inhibitors of hydroperoxide degradation), causes ROS accumulation in the cells and enhances PDT cytotoxicity. It will be interesting to determine whether the elevated antioxidant capacity of many types of cancer cell results in a cancer-selective cell kill, compared with normal cells, when using antioxidant inhibitors together with PDT.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Acknowledgements— SK was funded by an Open University PhD fellowship. We thank members of the OU Department of Mathematics and Statistics for expert advice on statistical methods.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Figure S1. Representative phase-contrast micrographs of MCF-7 cells in dark and phototoxicity studies, without AlPcS2 photosensitizer. The cells were treated with the different concentrations of antioxidant inhibitors for 30 min and then exposed to light or kept in the dark. The samples were incubated for a further 24 h under standard cell culture conditions in the presence of inhibitors, and the phase-contrast micrographs acquired at the end of the incubation period.

Figure S2. The relative ROS levels in cell-free medium, determined by DCF fluorescence with various antioxidant inhibitors. ROS levels are reported as a ratio, obtained by dividing the mean of each sample DCF fluorescence by the mean DCF fluorescence of the light treated AlPcS2 sample (second bar in each light-treated graph, right side). Results represent the mean of three independent experiments for each condition (mean ± SEM), analysed by one way ANOVA with Dunnett’s test. Statistically significant differences compared with the relevant AlPcS2-only control (second bar in each graph pair) are indicated by asterisks; **P < 0.01.

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