MPPa-PDT suppresses breast tumor migration/invasion by inhibiting Akt-NF-κB-dependent MMP-9 expression via ROS

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Breast tumor; Invasion; Migration; Photodynamic therapy; Reactive oxygen species.

Fig. 1

MPPa-PDT influences the cell viability of MCF-7 cells. a Uptake of MPPa for different times measured by fluorescence microscopy (magnification, × 200). b The effect of MPPa-PDT on the cell viability measured by CCK-8 assay after 24 h. c The effect of MPPa-PDT on ROS production detected by DCFH-DA staining (magnification, × 200). (n = 3; *P < 0.05 versus control group, **P < 0.01 versus control group)

Fig. 2

MPPa-PDT inhibits the migration and invasion of MCF-7 cells. a The effect of MPPa-PDT on migration in MCF-7 cells was detected by wound healing assay after 24 h (magnification, × 100). b The effect of MPPa-PDT on invasion in MCF-7 cells was measured by Matrigel invasion assay after 48 h (magnification, × 200). c The expression of MMP2 was measured by RT-PCR after 24 h. d The expression of MMP9 was measured by RT-PCR after 24 h. (n = 3; *P < 0.05 versus control group, **P < 0.01 versus control group)

Fig. 3

MPPa-PDT reduces the phosphorylation of Akt and NF-κB. a The expression levels of Akt, p-Akt, p65, and p-p65 were detected by western blotting. b Cytoskeleton was significantly reduced after MPPa-PDT, as detected by confocal microscopy (magnification, × 600). (n = 3; *P < 0.05 versus control group)

Fig. 4

MPPa-PDT inhibits metastasis in MCF-7 cells through the Akt/NF-κB-dependent MMP9 signaling pathway. a The effect of NAC and SC79 on migration after MPPa-PDT (magnification, × 100). b The effect of LY294002 and triciribine on migration after MPPa-PDT (magnification, × 100). c The effect of NAC, SC79, LY294002 and triciribine on invasion after MPPa-PDT (magnification, × 200). d The effect of SC79 on MMP2 expression after MPPa-PDT. e The effect of SC79 on MMP9 expression after MPPa-PDT. f The effect of LY294002 on MMP2 expression after MPPa-PDT. g The effect of triciribine on MMP2 expression after MPPa-PDT. h The effect of LY294002 on MMP9 expression after MPPa-PDT. i The effect of triciribine on MMP9 expression after MPPa-PDT. (n = 3; *P < 0.05 versus control group, **P < 0.01 versus control group)

Fig. 5

MPPa-PDT inhibits metastasis in MCF-7 cells through the Akt/NF-κB signaling pathway. a The effect of NAC on the expression levels of Akt, p-Akt, p65, and p-p65 after MPPa-PDT. Quantifications of the proteins are shown. b The effect of SC79 on the expression levels of Akt, p-Akt, p65, and p-p65 after MPPa-PDT. Quantifications of the proteins are shown. c The effect of LY294002 on the expression levels of Akt, p-Akt, p65, and p-p65 after MPPa-PDT. Quantifications of the proteins are shown. d The effect of triciribine on the expression levels of Akt, p-Akt, p65, and p-p65 after MPPa-PDT. Quantifications of the proteins are shown. e The effect of NAC, SC79, LY294002 and triciribine on the F-actin cytoskeleton after MPPa-PDT (magnification, × 600). (n = 3; *P < 0.05 versus control group, **P < 0.01 versus control group)

Fig. 6

MPPa-PDT inhibits tumor metastasis in vivo. a Representative photos of the tumor-bearing mice in the four groups. b Tumor volume changes of the four groups over the course of treatments. c Body weight changes of the four groups over the course of treatments. d Representative H&E staining images of lung tissues from the mice in the four groups. e Collagen and cytoskeleton of tumor tissues were measured by immunofluorescence staining (scale bar, 200 μm)