MPPa-PDT suppresses breast tumor migration/invasion by inhibiting Akt-NF-κB-dependent MMP-9 expression via ROS
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Breast cancer is one of the most commonly diagnosed cancers in women, with high morbidity and mortality. Tumor metastasis is implicated in most breast cancer deaths; thus, inhibiting metastasis may provide a therapeutic direction for breast cancer. In the present study, pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) was used to inhibit metastasis in MCF-7 breast cancer cells.
Uptake of MPPa was detected by fluorescence microscopy. Cell viability was evaluated by the Cell Counting Kit-8 (CCK-8). ROS generation was detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The migration of cells was assessed by wound healing assay, and invasion ability was assessed by Matrigel invasion assay. Levels of MMP2 and MMP9 were measured by PCR. Akt, phospho-Akt (Ser473), phospho-NF-κB p65 (Ser536) and NF-κB p65 were measured by western blotting. The F-actin cytoskeleton was observed by immunofluorescence. Lung tissue was visualized by hematoxylin and eosin staining.
Following MPPa-PDT, migration and invasion were decreased in the MCF-7 cells. MPPa-PDT downregulated the expression of MMP2 and MMP9, which are responsible for the initiation of metastasis. MPPa-PDT reduced the phosphorylation of Akt and NF-κB. MPPa-PDT also reduced the expression of F-actin in cytoskeleton in MCF-7 cells. These effects were blocked by the reactive oxygen species scavenger NAC or the Akt activator SC79, while the PI3K inhibitor LY294002 or the Akt inhibitor triciribine enhanced these effects. Moreover, MPPa-PDT inhibited tumor metastasis and destroyed F-actin in vivo.
Taken together, these results demonstrate that MPPa-PDT inhibits the metastasis of MCF-7 cells both in vitro and in vivo and may be involved in the Akt/NF-κB-dependent MMP-9 signaling pathway. Thus, MPPa-PDT may be a promising treatment to inhibit metastasis.
KeywordsPhotodynamic therapy Reactive oxygen species Breast tumor Migration Invasion
Protein kinase B
Cell viability and cytotoxicity test kit
Dulbecco’s modified Eagle’s medium
Hematoxylin and Eosin
Pyropheophorbide-α methyl ester
Reactive oxygen species
Breast cancer is the second leading cause of cancer death in women around the world . Metastasis is the dominant cause of death in breast cancer patients . It is related to many elements, such as destruction of the extracellular matrix (ECM)  and generation of new metastatic tumors in secondary sites after transport in blood and lymph vessels . Matrix metalloproteinases (MMPs) play a crucial role in the degradation of the ECM and the subsequent invasion and metastasis of tumor cells [5, 6]. MMPs are zinc-dependent endopeptidases that include gelatinases, collagenases, stromelysins, and membrane-associated MMPs . The relationships of MMP-2 and MMP-9 to the degradation of the ECM and tumor metastasis  are significant; thus, they are regarded as progression markers in breast cancer.
Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is an important signaling pathway that is involved in tumor cell growth, proliferation, apoptosis, metabolism, angiogenesis, metastasis and immunity [8, 9, 10]. It also has a close connection with the NF-κB signaling pathway, in which the phosphorylation of Akt can activate NF-κB , triggering the regulation of downstream MMP-2 and MMP-9, regulating cancer cell proliferation, migration and invasion [12, 13]. Inhibition of MMP-2 and MMP-9  may be a suitable therapeutic option for cancer.
Photodynamic therapy (PDT), as a valid therapy modality for multiple solid tumors, is minimally invasive and innoxious and possesses selective cytotoxicity for targeted cells . It is based on photosensitizers and laser light with a specific wavelength, causing the production of reactive oxygen species (ROS) and inducing tumor cell apoptosis/necrosis . PDT has been clinically used for various cancers, including cervical , lung , bladder , skin  and head and neck cancers [5, 21].
Pyropheophorbide-α methyl ester (MPPa), a derivate of chlorophyll , provides more advantages due to its better absorbance of and stronger permeability to PDT compared with first-generation photosensitizers. Our previous studies demonstrated that MPPa-PDT can inhibit breast cancer cell growth ; however, the role of MPPa-PDT on invasion and migration in breast cancer is not clear. In the present study, we observed that MPPa-PDT inhibited breast cancer cell MCF-7 metastasis and the underlying molecular mechanisms, which may provide important implications for breast cancer treatment.
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). Loading control was obtained from Beyotime (Shanghai, China). Trypsin and Actin-tracker Green were procured from Beyotime (Shanghai, China).
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.
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 s 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 × 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 1 h 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.
MCF-7 cells were seeded in 24-well plates. After the above mentioned 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) at 24 h after irradiation. The compounds of cells and RNAiso Plus reagent 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.
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 (Radio Immunoprecipitation Assay) Lysis buffer containing PMSF (Phenylmethylsulfonyl fluoride) (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 in excess 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).
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 for 30 min at room temperature for immunohistochemistry.
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.
MPPa-PDT suppresses MCF-7 cell viability
MPPa-PDT suppresses MCF-7 cell migration, invasion and MMP-9 expression
MPPa-PDT downregulates the PI3K/Akt/NF-κB signaling pathway
MPPa-PDT suppresses MCF-7 cell metastasis by inhibiting PI3K/Akt/NF-κB-dependent MMP-9 expression
MPPa-PDT induces MCF-7 cell cytoskeleton destruction by inhibiting PI3K/Akt/NF-κB
MPPa-PDT decreases the tumor progression of MCF-7 cells in vivo
MPPa-PDT decreases the tumor metastasis of MCF-7 cells in vivo
Lung tissues were harvested to perform the hematoxylin and eosin (H&E) histopathological analysis. In the three control groups, there was apparent generation of micrometastatic foci; in contrast, there were few metastases in the MPPa-PDT group (Fig. 6d). Above mentioned results demonstrated that MPPa-PDT possessed successful therapeutic efficacy in inhibiting tumor metastasis. We analyzed the F-actin cytoskeleton and collagen of tumor tissues through immunofluorescence assay. The cytoskeleton in the MPPa-PDT group exhibited undoubted destruction, while the three control groups exhibited relatively complete structures. Collagen in the three control groups was destroyed, while in MPPa-PDT, it was apparent (Fig. 6f). There was no significant difference among the three control groups.
Due to the limitations of current clinical treatment of breast cancer and accompanying unsatisfied prognosis, safe and effective therapy methods are urgently needed . PDT exhibits unique advantages, such as minimal invasion to the human body, little system toxicity and no resistance . PS, as an important element of PDT, should be readily available, inexpensive, and stable and possess an absorption peak at longer wavelengths. MPPa is a desirable photosensitizer that possesses good stability and a 630 nm absorbance wavelength ; thus, it was chosen for the present study.
ROS play the most pivotal role in PDT , which is implicated in cellular homeostasis and possesses bidirectional effects on cellular processes. Chemotherapeutic drugs exert anticancer effects via the generation of ROS such as doxorubicin, cisplatin and mitomycin C in many cancer diseases. Moderate ROS levels influence inflammation of tissues or cells, cellular defense systems, and progression of cancer. Overproduction of ROS induces cancer cell apoptosis; nucleic acid, lipid, and protein damage; and suppression of pro-survival pathways . Therefore, appropriate levels of ROS may be an effective therapeutic method. Our previous study showed that ROS produced by PDT-induced breast cancer cell apoptosis, including MCF-7 and MDA-MB-231 cells. Considering that cancer metastasis is the most important cause of death in breast cancer patients , we wondered whether MPPa-PDT could influence breast cancer cell line metastasis and what the interrelated mechanisms would be. Our study found that ROS were significantly increased after MPPa-PDT treatment, which was compatible with previous studies.
High migration and invasion capability are major malignant characteristics of cancer cells, while ECM is the crucial barrier to cancer metastasis. Metastasis occurs when cancer cells spread to other organs from a primary tumor site and form new tumors [28, 29], including migration and invasion, intravasation, arrest of cancer cells and extravasation, as well as metastatic colonization . Metastases are a leading cause of breast cancer mortality , and suppression of metastasis would ameliorate patient prognosis. Our study found that MPPa-PDT alleviated the invasion and migration of MCF-7 cells, suppressed the expression of MMP-2 and MMP-9, and inhibited Akt and NF-κB phosphorylation.
MMP-9 is an irreplaceable player in the degradation of ECM  whose abnormal expression disequilibrates ECM and contributes to cancer cell metastasis . The expression of matrix metalloproteinase-9 is increased in invasive breast cancer, and the cause of this phenomenon is related to the mutual regulation of various intracellular signal transduction factors under different stimulation . MMP-9 transcription is influenced by two major transcriptional factors, NF-κB and AP-1. The most common signals upstream of these two molecules are PI3K/Akt and MAPK [34, 35], which manage survival, metastasis and drug resistance [36, 37]. Inhibitors of the PI3K/Akt signaling pathway have been clinically applied for cancer treatment . The present study showed that MPPa-PDT reduced MMP-9 and MMP-2 transcription and that MPPa-PDT also suppressed Akt phosphorylation and NF-κB activation. We used pharmacologic inhibitors of ROS (NAC), PI3K (LY294002), and Akt (triciribine) and an activator of Akt (SC79) to identify the intrinsic mechanism. Inhibition of PI3K and Akt further reduced Akt and NF-κB protein phosphorylation and regulated downstream signaling molecules, which in turn resulted in a reduction in MMP2 and MMP9 gene expression. At the same time, the capacities of migration and invasion of MCF-7 were subdued. However, suppression of ROS and activation of Akt specifically diminished MPPa-PDT’s effect on the expression of Akt and NF-κB proteins and the MMP-9 and MMP-2 genes, as well as cell metastatic ability. These results suggest that MPPa-PDT inhibited MCF-7 migration and invasion through the Akt/NF-κB/MMP-9/MMP-2 signaling axis by upregulating ROS production.
The F-actin cytoskeleton governs numerous biological functions, including cell contraction, cell motility and vesicle trafficking, with a well-configured network . We investigated the changes in F-actin because it is known to regulate tumor cell invasion and migration [40, 41]. The results showed that MPPa-PDT destroyed the cytoskeleton, in contrast with the three control groups. Additionally, apparent disorganization of the cytoskeleton was discovered after administration of the PI3K inhibitor LY294002 and the Akt inhibitor triciribine. The cells had well-defined actin filaments pretreated with NAC or SC79. These results demonstrated that MPPa-PDT inhibited MCF-7 migration and invasion through the Akt/NF-κB signaling pathway via ROS.
To further examine the effects of MPPa-PDT on tumor growth and metastasis in vivo, MCF-7 cells were xenografted to nude mice. During the treatment period, tumors in the three control groups grew rapidly, while those in the MPPa-PDT group were obviously inhibited, indicating that MPPa-PDT can inhibit tumor growth. The major lung tissue was collected and subjected to H&E staining. There were obvious lung micrometastatic foci in the three control groups but no significant micrometastatic foci in the MPPa-PDT group, indicating that MPPa-PDT can inhibit tumor metastasis.
Cytoskeletal F-actin and collagen were used to evaluate the destruction of the ECM in the present study. The results showed that collagen deposition in the three control groups decreased obviously, while the collagen in the MPPa-PDT group exhibited no significant change. ECM destruction is involved in metastasis, and the primary component of ECM is collagen; thus, the integrity of collagen can frustrate the metastatic capacity of tumors. According to the F-actin staining, the network structure of F-actin was depolymerized significantly after MPPa-PDT treatment; conversely, the network structure of F-actin was notably visible in the other groups. These results showed that the tumor metastasis capacity in the MPPa-PDT group was weaker than that in the other groups. These results also demonstrated that MPPa-PDT can inhibit tumor metastasis.
The present study suggests that MPPa-PDT inhibited the migration and invasion of breast cancer cells through the Akt/NF-κB-dependent MMP-9 signaling pathway by upregulating ROS production. Downregulation of MMP-2 and MMP-9 expression induced by MPPa-PDT may enhance the therapeutic efficacy for breast cancer by restricting metastasis by upregulating cell apoptosis and necrosis. These findings suggest that MPPa-PDT is a promising therapeutic method for breast cancer.
The Chongqing Key Laboratory of Translational Medicine in Major Metabolic Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China.
DB and HL helped design experiments. QC helped write the manuscript. LH generated the data and completed the images. DB and LY revised the manuscript. All authors have read and approved the manuscript.
All animal studies were conducted under a protocol approved by the Animal Ethics Committee of Chongqing Medical University.
Consent for publication
The authors declare that they have no competing interests.
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