- Major reagents
Pyropheophorbide α methyl ester (MPPa, C34H36N4O3) was obtained from Sigma-Aldrich (St. Louis, MO). The laser (630 nm) was purchased from Chongqing Jingyu Laser Technology Co., Ltd. (Chongqing, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone (Logan, UT). The Cell Counting Kit-8 (CCK-8) was procured from Dojindo Molecular Technologies (Kumamoto, Japan). Akt (catalogue number: 4691, dilution: 1:1000), phospho-Akt (catalogue number: 4060, Ser473, dilution: 1:2000), phospho-NF-κB p65 (catalogue number: 3033, Ser536, dilution: 1:1000) and NF-κB p65 (catalogue number: 8242, dilution: 1:1000) were obtained from Cell Signaling Technology (Danvers, MA). GAPDH was purchased from Sungene Biotech (Tianjin, China). Trypsin and Actin-tracker Green were procured from Beyotime (Shanghai, China).
- Cell culture
The human breast cell line MCF-7 (Shanghai Institute of Cell Biology China, catalogue number: SCSP-531) was cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Beyotime, Shanghai, China). MCF-7 cells were routinely cultured in 5% CO2 at 37°C.
- PDT protocol
Four groups were designed in the present study—A: control group; B: MPPa-only group; C: laser-only group; D: MPPa-PDT group. After cell attachment, the media of groups A and C were replaced with fresh media, while the media of groups B and D were replaced with media containing MPPa (2 μmol/L). Cells of groups B and D were washed with PBS to remove the MPPa after 12 h, and then groups C and D were exposed to LED light (630 nm, 30 mW/cm2) for 30, 60, 90, 120, or 180 sec to obtain an energy density of 0.9, 1.8, 2.7, 3.6, or 5.4 J/cm2, respectively. Energy density (J/cm2) = power (mW/cm2) × irradiation time (s). The cells were cultured in the incubator after irradiation. All the operations were performed in the dark.
- Detection of intracellular MPPa
MPPa sparkles with red fluorescence upon corresponding excitation. To observe the uptake of MPPa in MCF-7 cells, cells were seeded into 6-well plates and incubated with 2 μmol/L MPPa for different times (0 h, 3 h, 6 h, 12 h, and 24 h). The intracellular accumulation of MPPa was detected by fluorescence microscopy (Leica DMRE Fluorescence Microscope, Germany).
- Cell viability and cytotoxicity tests
Cells were seeded in 96-well plates at a density of 5 x 103 cells/well. After 24 h of different treatments, the medium of each well was discarded, 100 μl serum-free medium containing 10 μl CCK-8 solution was added to each well, and the cells were further incubated for some time in 5% CO2 at 37°C. Optical densities (ODs) were measured by a microplate reader. Cell viability (%) = (Average OD of experiment group – Average OD of blank group)/ (Average OD of control group – Average OD of blank group) × 100%. The wells of the blank group contained only DMEM with CCK-8 solution.
- ROS detection
MCF-7 cells were seeded in 24-well plates. After the abovementioned treatments, the medium of each group was replaced by DCFH-DA (Invitrogen, Paisley, UK) at a concentration of 10 μmol/L. After 30 min, the cells were washed with PBS three times to wash away the extracellular DCFH-DA. Fluorescence microscopy was used to detect the production of ROS (Zeiss Fluorescence Microscope, Germany).
- Wound healing assay
MCF-7 cells were seeded in 6-well plates at a density of 1 × 105 cells/well. The MPPa-only group and MPPa-PDT group were subjected to MPPa-medium (2 μmol/ml) and incubated for 12 h. Then the medium was replaced with PBS, and a scratch was made with a sterile pipet tip (200 μl) in all groups. The detached cells were removed. The corresponding irradiation was performed in the laser-only group and MPPa-PDT group. Images were taken immediately after irradiation and 24 h later using a ﬂuorescence microscope. The area of the scratch was analyzed using ImageJ software.
- Matrigel invasion assay
A Matrigel invasion assay was utilized to evaluate the invasiveness of cells pre- and post-PDT. Cells were divided into 4 groups and received the corresponding treatment. Matrigel (Becton Dickinson, Bedford, MA) was added into Transwell inserts (Corning Costar, Tokyo, Japan) for solidification in a 24-well plate. Cells were collected and resuspended in serum-free medium and then transferred into the upper chambers, 5 × 104 cells for each Transwell insert. The lower wells were supplemented with 750 μl of complete medium. After 48 h, the cells that did not invade the lower surface of the transwell inserts were cleaned with a cotton swab, while the invaded cells of the inserts were fixed with 4% paraformaldehyde, washed with PBS three times and subjected to crystal violet staining (Beyotime, Shanghai, China). Transwell inserts were visualized by light microscopy (Zeiss Fluorescence Microscope, Germany).
- Real-time PCR Analysis
RNA was extracted by RNAiso Plus reagent (Takara) after 24 h. The compounds were centrifuged for 15 min at 12000 rpm, and the supernatant was collected in new EP tubes to obtain the sediment of RNA by isopropyl alcohol precipitation. Extracted RNA was purified with 75% ethyl alcohol and suspended in 20 μl diethyl pyrocarbonate (DEPC water). The cDNAs were synthesized through reverse transcription reactions with a mixture following the instructions (Takara, Japan), and real-time polymerase-chain reaction (PCR) was performed with CFX96™ Bio-Rad. Primers were synthesized by Qingke (Chongqing, China): ATGGGGAAGGTGAAGGTCGG (Forward) and GACGGTGCCATGGAATTTGC (Reverse) for GAPDH; (Forward) CCGCTCACCTTCACTCG and CTCCGCGACACCAAACT (Reverse) for MMP9; and (Forward) CCCACTGCGGTTTTCTCGAAT and CAAAGGGGTATCCATCGCCAT (Reverse) for MMP2.
- Western blotting
To collect the cell protein at the indicated time points (times are shown in the figure) or after 24 h, cells were lysed in RIPA buffer containing PMSF (RIPA:PMSF= 100:1) and centrifuged at 12000 g at 4 °C. Protein concentrations were quantified by BCA assay and prepared with RIPA and loading buffer to make the final concentrations the same. Target proteins were separated by gel electrophoresis and then transferred to PVDF membranes (Millipore). Five percent nonfat dry milk was used to block membranes at room temperature, and Tris-buffered saline plus tween (TBST) was used to wash the membranes. Then, the membranes were exposed to homologous primary antibodies and shaken tardily at 4 °C overnight. The next day, the membranes were washed with TBST to remove antibodies and then exposed to secondary antibodies of the corresponding species. The images were obtained by Fusion with the ECL coreaction (Advansta, USA). All experimental results were analyzed by Fusion (Fusion, Vilber Lourmat, France).
- F-actin cytoskeleton analysis
MCF-7 cells were seeded onto glass coverslips in a 12-well plate. After treatment, the coverslips were collected and fixed with 4% paraformaldehyde (PFA). Cells were permeabilized using 0.1% Triton X and then blocked in 5% BSA. Cells were stained with Actin-tracker Green (Beyotime Biotechnology, China) for 1 h at room temperature. Then the surface of slides was smeared with Antifade mounting medium, and all coverslips were transferred onto slides. The image series were captured using a ZEISS LSM800 confocal microscope (Carl Zeiss AG, Germany).
- Tumor models
All female nude mice (4–6 w) were obtained from the Experimental Animal Center of Chongqing Medical University. All animal studies abided by the ethics guidelines of the Animal Ethics Committee of Chongqing Medical University. The nude mice were inoculated subcutaneously with 1×106 MCF-7 cells in 100 μL PBS to form MCF-7 xenograft breast cancer. These mice were randomly divided into four groups when the tumor grew to approximately 100 mm3: control group, MPPa group, laser group and MPPa-PDT group (n=3 in each group). MPPa was administered intravenously to the tumor-bearing mice at a concentration of 15 mg/kg, and the laser was applied 12 h after injection at 120 J/cm2 every 2 days for 10 days. Tumor sizes and body weights were recorded during treatment.
Tumor Volume (TV) = Length × Width2/2.
- Tissue histology and immunohistochemistry
Nude mice were euthanized by cervical dislocation, and the major organs were harvested on day 16. Lung tissues were formalin-fixed, paraffin-embedded, and visualized by hematoxylin and eosin staining. Tumor tissues were stained by Actin-tracker Green and DAPI as well as for collagen at room temperature for immunohistochemistry.
- Statistical analysis
Data are shown as mean ± SD. GraphPad Prism Software version 7.00 (San Diego, CA) was used for statistical analysis. The method of statistical analysis was one-way analysis of variance (ANOVA). P < 0.05 was regarded as statistically significant.