Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells

Non-tumorigenic human MCF-10A (ATCC CRL-10317™) breast cell line was maintained in phenol red-free Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DMEM-F12; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5% heat-inactivated horse serum (Vitrocell Embriolife, Campinas, Sao Paulo, Brazil), insulin (10 μg/ml; Sigma-Aldrich), cortisol (500 ng/ml; Sigma-Aldrich), cholera enterotoxin (100 ng/ml; Sigma-Aldrich), and epidermal growth factor (20 ng/ml; Sigma-Aldrich). Human breast adenocarcinoma cell line MCF-7 (ATCC HTB-22™) was maintained in phenol red-free Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 Ham (DMEM-F12; Sigma-Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Vitrocell Embriolife). Human breast adenocarcinoma cell line MDA-MB-231 (ATCC HTB-26™) was cultured in phenol red-free Roswell Park Memorial Institute Medium Modified (RPMI 1640; Sigma-Aldrich) supplemented with 10% FBS (Vitrocell Embriolife). All cultures were maintained at 37 °C under water-saturated atmosphere containing 5% CO₂. For the 3D culture assays, (2x10⁴/cm²) cells were seeded on top of lamin-rich extracellular matrix gels (lrECM);commercially available as Matrigel (BD Biosciences, San Jose, CA, USA)- in phenol red-free medium supplemented with 2.5% serum and 5% lrECM and maintained for four days after treatments [28]. All 2D assays were also performed in 2.5% serum-supplemented medium.

Phenotiazonium salt, MB (Labsynth Products, São Paulo, Brazil) was used as Ps to perform the PDT treatment. Cells were incubated for 2 h with 0.2, 2 or 20 μM MB, in phenol red-free medium supplemented with 2.5% FBS and maintained in these conditions during both irradiation as well as post-treatment times (1, 3 and 24 h). The whole microplate was irradiated with a light emitting diode (LED) array, with maximum emission wavelength at 640 nm, corresponding to total light doses of 4.5 J/cm². Control experiments such as cells neither exposed to the Ps nor to light (control); cells not exposed to the Ps but washed and exposed to light (phototoxicity); and cells exposed to the Ps alone without irradiation (dark toxicity) were performed in all experiments.

4 × 10⁴ cells/cm² were plated and maintained in control conditions or exposed to MB-PDT and then stained with the DNA-binding dyes Propidium iodide (PI, Sigma-Aldrich) and Hoechst 33342 (HO, Sigma-Aldrich) for 10 min. Following incubation, the percentage of viable and dead cells was determined using an inverted fluorescence microscope (Nikon Eclipse Ti, Kyoto, Japan) with 20x of magnification. Tri-dimensional cell cultures were transferred to a glass slide and visualized using a confocal microscope (Axiovert 200 LSM 510 Laser and Confocor Modules, Carl Zeiss, Göttingen, Germany) equipped with water immersion objective (40X). Fluorescence of labelled cells was detected using laser 461 nm and 545 nm for excitation of HO and PI respectively. The cultures were evaluated according to: the total number of cells, determined by counting the nuclei stained with HO; and the number of dead cells determined by the number of nuclei stained with PI or by brightly HO (condensed chromatin) [29]. A minimum of 500 cells was counted in each experimental condition. Results were expressed as percentage of dead cells.

1 × 10⁵ cells/cm² were plated and incubated with 5 mL of medium containing MB (20 μM) and maintained for 1, 2, 4, 6 and 8 hours. At each time point, the supernatant was discarded and the cells were washed twice with PBS and then 1 mL of 50 mM SDS was added to promote lysis of the cell membrane. The supernatant was collected and absorbance was measured at the wavelength of maximum absorption of the MB solution used (655 nm). The incorporation of MB was determined by correcting the absorbance of MB by the number of cells remaining in each well after the incubation period.

Singlet oxygen measurements were performed in a specially designed Edinburgh F900 instrument (Edinburgh, UK) consisted of a Rainbow OPO (Quantel Laser-France) 10Hz, 2 mJ/pulse, which was pumped by a Brilliant Nd-YAG laser (Quantel Laser-France) and equipped with a cuvette holder, a silicon filter, monochoromator, a liquid-nitrogen-cooled NIR PMT (R5509) (Hamamatsu Co., Bridgewater, NJ, USA) and a fast multiscaler analyser card with 5 ns/channel (MSA-300; Becker & Hickl, Berlin, Germany). The cells were seeded in six-well plates (4x10⁵ cells/well) and after 24 h were incubated with MB for 2 h. The cells were washed in PBS, removed from the plates using trypsin solution, centrifuged and suspended in D₂O saline solution and were directly excited at 664 nm inside a fluorescence quartz cuvette. We obtained ¹O₂ emission spectra by measuring emission intensities from 1200 to 1348 nm with 1 to 5 nm steps. The intensities of the near infrared (NIR) emission peak (centred at 1275 nm) are correlated with the amount of ¹O₂ generated.

Reduced glutathione (GSH) was quantified as previously described by Kand’ár et al [30] with minor modifications. Cells were seeded in Petri dishes (100 mm) at an initial density of 2.6 × 10⁶ cells/Petri dish. After 48 h the cells were washed in PBS, removed from the plates using 0.1% trypsin, centrifuged and suspended in deionized water. An aliquot was separated for cell count before lysis with 0.15% polidocanol (Sigma-Aldrich). For protein precipitation, samples were incubated with cold 10% metaphosphoric acid (10 min, 4 °C), centrifuged (22,000 x g, 15 min, 4 °C) and supernatants were collected. The supernatant was diluted 20 folds in 100 mM phosphate buffer pH 8.9 containing 0.1 mM diethylene triamine pentaacetic acid (DTPA, Sigma-Aldrich). Twenty microliters of this sample were diluted to 320 μL in a 100 mM phosphate buffer pH 8.0 containing 0.1 mM DTPA. Derivatization was performed by adding 20 μL of 148 mM orthophthaldehyde (OPA, Sigma-Aldrich) to this solution. The reaction was incubated at 25 °C for 15 min in dark. The samples were then filtered through a 0.22 μm polyvinyl difluoride (PVDF) filter and injected onto high performance liquid chromatography (HPLC). The GS-OPA product was separated in a VP-ODS/C8/Phenyl column (250 mm × 4.6 mm × 4.6 μm, Shimadzu, Kyoto, Japan). The HPLC was equipped with two LC-20AT solvent delivery systems, SIL-20 AC HT autosampler, CTO-20HC column oven, RF-20A fluorescent detector and CBM-20A system controller (Shimadzu, Kyoto, Japan). The separation was achieved using an isocratic elution of 15% methanol in 85% 25 mM Na₂HPO₄ (Merck, Darmstadt, German) pH 6. The flow rate was constant at 0.5 mL/min 37 °C. Fluorescent GS-OPA was monitored with a ʎ_ex 350 nm and ʎ_em 420 nm. The peak area was plotted against an external GS-OPA standard curve previously derivatized with pure GSH (Sigma-Aldrich). Results were presented as pmol/cell.

We used confocal microscopy to characterize the subcellular localization of MB. To this end, we compared the fluorescence arising from cell cultures simultaneously incubated in the presence of MB and standard fluorescent markers of organelles. MitoTracker Green (Invitrogen, Paisley, UK) was used as a mitochondrial marker, LysoTracker Green (Invitrogen) as a lysosome marker and HO as a marker for the cell nucleus. Confocal images were taking using a laser scan microscope (LSM) - 510 from Zeiss using 1.2 N.A. 40x water immersion or 1.4 N.A. 63x oil immersion objective lenses. The laser and filter settings were: laser lines for MB = 633, Lyso = 488 and Hoechst 33342 = 364; beam splitter = HFT UV/488/543/633; emission filters for MB: 651-704, Lyso = 501-554 and Hoechst 33342 = 435-485. The imaging settings were: zoom = 1, dimensions = 512x512 pixels, image depth = 16 bit, averaging signal = 2 and optical section thickness = 2 μm. Images had their brightness and contrast adjusted for the figures and were analysed with ImageJ Software (National Institutes of Health).

Acridine orange (AO) is a weak base that can accumulate in acidic spaces and emits bright red fluorescence. The intensity of the red fluorescence is proportional to the pH of the cellular acidic compartments [31]. In order to detect and quantify acidic vesicle formation during the process of autophagy, 4x10⁴ cells/cm² were plated and then subjected or not to MB-PDT. Cells were then washed with PBS and stained with AO (Sigma Aldrich) at a final concentration of 5 μg/ml for 10 min at 37 °C in the dark. After a washing step, live cells were visualized using an inverted microscope for transmitted light and epifluorescence (Axiovert 200, Carl Zeiss) equipped with a C-APOCHROMAT 40×/1.20 M27 objective (Zeiss™). Fluorescence of AO-stained vesicles was detected by using a filter set 09 (Zeiss™) that provides an excitation band pass (BP) of 450-490 nm with emission long pass (LP) of 515 nm.

4.0 × 10⁴ cells/cm² were plated and incubated with each inhibitor in the presence or in the absence of MB for 2 h, in a 5% CO₂ humidified atmosphere at 37 °C. Cells were then subjected or not to light irradiation as described above. Apoptosis was inhibited by using a specific caspase-3 inhibitor (100 nM Caspase-3 Inhibitor VI; Calbiochem, La Jolla, CA, USA), a pan-caspase inhibitor (20 μM Z-VAD-FMK; Calbiochem) or a BAX inhibitor (10 μM; Tocris, Ellisville, MO, USA). Autophagy was activated using mTOR inhibitor rapamycin (20 nM; Cell Signaling Technology, Danvers, MA, USA) or inhibited by using either PI3-kinase inhibitor LY294002 (50 μM; Tocris), class III PI3K inhibitor 3-MA (5 mM; Calbiochem), chloroquine (5 μM; Sigma-Aldrich) or bafilomycin A1 (50 nM; Calbiochem). We analysed the dose-response and toxicity of each inhibitor and we used the highest concentration that presented no cytotoxicity in the control conditions for each cell line.

The siRNA used in this study was a Silencer pre-designed siRNA (Invitrogen) of sequence 5’GCUCUGCCUUGGAACAUCAtt 3’. “AllStars negative control siRNA” (Qiagen, Venlo, Netherlands) was used as a negative siRNA control of scrambled sequence (siCTR). Transfection of siRNA was done using the lipid carrier Lipofectamine RNAiMAX (Invitrogen). Lipid-RNA complexes were formed in Opti-MEM (Invitrogen) in a proportion of 0.6 μl of Lipofectamine to 0.45 μl of 20 μM siRNA, at room temperature for 20 min and were further added to cells in antibiotic-free medium to reach a final volume of 300 μl for overnight transfection. Cells were maintained in culture for a 24-h recovery period before experiments were carried out. The efficiency of transfection/silencing was validated by Western blot, with at least 60% of inhibition.

Total protein extracts were prepared from each culture subjected to the treatments described above. Equal amounts (100 μg) of protein from each extract were solubilized in sample buffer (60 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 0.01% bromophenol blue) and subjected to SDS-PAGE (16%). Proteins were transferred to PVDF membranes, incubated with Blocking Buffer solution (Thermo Fisher Scientific) and 5%BSA 1:1, and then incubated with the following antibodies: rabbit polyclonal anti-BAX (2772), rabbit polyclonal anti-BCL2 (2876) (all from Cell Signaling Technology), rabbit polyclonal anti-LC3 (L8918) and mouse monoclonal anti-alpha-tubulin clone B-5-1-2 antibody (T5168) (all from Sigma-Aldrich) as a loading control. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA). Enhanced chemiluminescence was performed according to the manufacturer’s instructions (Amersham Biosciences, Little Chalfont, UK). Quantitative densitometry was carried out using the ImageJ software (National Institute of Health [NIH]). The volume density of the chemiluminescent bands was calculated as integrated optical density × mm² after background correction.

4 × 10⁶ cells were collected and caspase activity was measured using a specific fluorimetric assay (BioVision Research Products, Mountain View, CA, USA). The reactions were started at 37 °C by incubating 50 μg of total protein extracts with a specific caspase substrate (50 μM DEVD-AFC, 50 μM IETD-AFC or 50 μM LEHD-AFC for caspase-3 and -7; -8 and -9, respectively; BioVision), following the manufacturer’s instructions. Protease activity was evaluated at an excitation wavelength of 400 nm and an emission wavelength of 505 nm using a 96-well plate spectrofluorometer (Spectra MAX M2; Molecular Devices, Sunnyvale, CA, USA). Total protein extracts from Min6 cells exposed to either vehicle or a combination of pro-inflammatory cytokines which induced a significant and well documented degree of apoptosis [32‐35], were always included as a positive control for caspase-3, -7, -8 and -9 activity assays, in each experiment performed. Caspase-3, -7, -8 and -9 activities were expressed as arbitrary fluorescence units per 50 μg of total protein (At least 3 independent experiments were performed in triplicate for each condition).