Psoralen Promotes Proliferation, Migration, and Invasion of Human Extravillous Trophoblast Derived HTR-8/Svneo Cells in vitro by NF-κB Pathway

Abstract

Recurrent spontaneous abortion (RSA) is a kind of pathological pregnancy, and abnormal function of trophoblast cells may be related to a variety of pregnancy complications including RSA. Psoralen is an effective ingredient extracted from Cullen corylifolium (L.) Medik. with multiple bioactivities mainly including anti-osteoporotic, anti-tumor, anti-inflammatory, and estrogen-like effects. However, the exact role of psoralen on trophoblast invasiveness has not been investigated thus far. In the present study, the effects of psoralen on the proliferation, migration, and invasion abilities of HTR-8/SVneo cells were evaluated by the CCK-8 and Transwell assays. The expression patterns of nuclear factor κB (NF-κB)/p65 and metalloproteinases (MMP)-2 and MMP-9 were characterized by further experiments including real-time quantitative polymerase chain reaction and Western blot. Indirect immunofluorescence was applied to track the NF-κB p65 translocation. Herein, we found that cell viability and invasive ability were promoted by psoralen in a concentration-dependent manner. Psoralen concentration-dependently enhanced both MMP-2 and MMP-9 expression and their activity of HTR-8/SVneo cells. Additionally, we observed accelerated nuclear accumulation and enhanced nuclear translocation of p65 in the presence of psoralen. Furthermore, invasiveness enhancement of psoralen on HTR-8/SVneo cells was partly eliminated by a NF-κB pathway inhibitor. Thus, our findings suggest that psoralen may serve as a potential repurpose drug candidate that can be used to induce migration and invasion of trophoblast cells through strengthening the NF-κB pathway.

Keywords: NF-κB pathway; human extravillous trophoblast; invasion; migration; proliferation; psoralen.

FIGURE 1

Psoralen enhances HTR-8/SVneo cell viability. (A) Chemical structure of psoralen. Cell viability (B) and LDH release (C) of HTR-8/SVneo cells after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 0, 24, 48, and 72 h. Values are mean ± SD. Experiments were performed in triplicate and repeated three times. ^* p < 0.05 vs. 0 μmol/L; ^# p < 0.05 vs. 0.01 μmol/L; ^† p < 0.05 vs. 0.05 μmol/L; ^‡ p < 0.05 vs. 0.1 μmol/L.

FIGURE 2

Psoralen strengthens migration and invasion ability of HTR-8/SVneo cells. (A) Representative images of Transwell assay of HTR-8/SVneo cells after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 24 h. (B) Changes in the number of migrating cells in relation to control cells. (C) Changes in the number of invading cells in relation to control cells. Values are mean ± SD. Experiments were performed in triplicate and repeated three times. ^* p < 0.05 vs. 0 μmol/L; ^# p < 0.05 vs. 0.01 μmol/L; ^† p < 0.05 vs. 0.05 μmol/L; ^‡ p < 0.05 vs. 0.1 μmol/L.

FIGURE 3

Psoralen enhances both MMP-2 and MMP-9 expression and their activity of HTR-8/SVneo cells. (A) Representative images of Western blot. Relative protein levels of MMP-2 (B) and MMP-9 (C) after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 48 h. Relative mRNA levels of MMP-2 (D) and MMP-9 (E) after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 48 h. (F) Activity of MMP-2 and MMP-9 after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 48 h. Values are mean ± SD. Experiments were performed in triplicate and repeated three times. ^* p < 0.05 vs. 0 μmol/L; ^# p < 0.05 vs. 0.01 μmol/L; ^† p < 0.05 vs. 0.05 μmol/L; ^‡ p < 0.05 vs. 0.1 μmol/L.

FIGURE 4

Psoralen promotes NF-κB activity of HTR-8/SVneo cells. (A) Representative images of Western blot. (B) Relative protein level of NF-κB p65 protein in HTR-8/SVneo cells after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 48 h. (C) Representative images of immunofluorescence staining. (D) Fluorescence intensity of p65 in nuclei of HTR-8/SVneo cells after exposure to different concentrations (0, 0.01, 0.05, 0.1, and 0.5 μmol/L) of psoralen for 48 h. Values are mean ± SD. Experiments were performed in triplicate and repeated three times. ^* p < 0.05 vs. 0 μmol/L; ^# p < 0.05 vs. 0.01 μmol/L; ^† p < 0.05 vs. 0.05 μmol/L; ^‡ p < 0.05 vs. 0.1 μmol/L.

FIGURE 5

Involvement of NF-κB in psoralen-induced enhancement of cell invasiveness analyzed by Transwell assay. (A) Representative images of Transwell assay of HTR-8/SVneo cells after exposure to psoralen (0.5 μmol/L), JSH-23 (10 μmol/L) alone or in combination for 24 h. (B) Changes in the number of migrating cells in relation to control cells. (C) Changes in the number of invading cells in relation to control cells. Values are mean ± SD. ^* p < 0.05 vs. control; ^# p < 0.05 vs. psoralen; ^† p < 0.05 vs. JSH-23.