Hispolon inhibits TPA-induced invasion by reducing MMP-9 expression through the NF-κB signaling pathway in MDA-MB-231 human breast cancer cells

Abstract

Hispolon has been demonstrated to possess analgesic, anti-inflammatory and anticancer activities. However, whether hispolon prevents the invasion of breast carcinoma cells and the underlying mechanisms of its action remain unknown. In the present study, various assays, including a matrigel-based Transwell invasion assay and electrophoretic mobility shift assay, were used to investigate the anti-invasion effect of hispolon and explore its mechanism of action. The results revealed that hispolon inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced migration and invasion of MDA-MB-231 human breast cancer cells at non-toxic concentrations. Hispolon also prevented the TPA-induced secretion of matrix metalloproteinase-9 (MMP-9) and reduced its expression at the transcriptional and translational levels. Furthermore, the phosphorylation of IκBα was reduced by hispolon, which resulted in the suppression of nuclear factor-κB (NF-κB), and p65 phosphorylation and nuclear translocation. An electrophoretic mobility shift assay demonstrated that NF-κB DNA-binding activity was induced by TPA and inhibited by hispolon. In addition, Bay 11-7082, which is a specific inhibitor of NF-κB, functioned in a similar manner as hispolon and blocked the secretion and expression of MMP-9. In conclusion, the results of the present study indicated that hispolon inhibited TPA-induced migration and invasion of MDA-MB-231 cells by reducing the secretion and expression of MMP-9 through the NF-κB signaling pathway.

Keywords: MDA-MB-231 cancer cells; hispolon; invasion; matrix metalloproteinase-9; nuclear factor-κB.

Figure 1.

(A) Chemical structure of hispolon. (B) Effects of hispolon on cell viability. MDA-MB-231 cells were treated with 0–60 µM hispolon and/or TPA (160 nM) for 24 h, and the cell viability was measured by an MTT assay. Dox was used as a positive control. All the data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. DMSO control; ^#P<0.05 vs. TPA alone-treated group. TPA, 12-O-tetradecanoylphorbol-13-acetate; Dox, doxorubicin.

Figure 2.

Hispolon inhibited TPA-induced migration and invasion of MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with hispolon for 2 h followed by TPA (160 nM) treatment for 24 h. The anti-invasive properties of hispolon were detected by Matrigel-based Transwell invasion assay. (B) Cells were treated as described previously and the antimigration ability was detected by wound-healing assay. (C) Relative quantification of the cell invasion and migration. All the data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and ^#P<0.05, vs. TPA alone-treated group; **P<0.05 and ^##P<0.05 vs. DMSO control. TPA, 12-O-tetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide; TPA+H-10, 160 nM TPA combined with 10 µM hispolon; TPA+H-20, 160 nM TPA combined with 20 µM hispolon; TPA+H-40, 160 nM TPA combined with 40 µM hispolon.

Figure 3.

Hispolon inhibited TPA-induced MMP-9 activation, as well as inhibited the secretion and expression in a dose-dependent manner. (A) Cells in serum-free medium were pretreated with hispolon at the indicated concentrations for 2 h prior to TPA incubation for another 24 h. Conditioned media were analyzed by gelatin zymography (upper panel) and western blot analysis (middle panel). Cell lysates were collected and subjected to western blot analysis (lower panel). β-actin was used as an internal control. (B) Hispolon inhibited TPA-induced MMP-9 gene expression. MMP-9 expression was detected by RT-PCR and GAPDH was used as an internal control. (C) Relative quantification of MMP-9 gene expression. All the data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. TPA alone-treated group; ^#P<0.05 vs. DMSO control. TPA, 12-O-tetradecanoylphorbol-13-acetate; MMP-9, matrix metalloproteinase-9; RT-PCR, reverse transcription-polymerase chain reaction; DMSO, dimethyl sulfoxide.

Figure 4.

Hispolon inhibited the TPA-induced NF-κB signaling pathway. (A) Hispolon inhibited the phosphorylation of IκBα and p65. Cells were pretreated with hispolon for 2 h prior to TPA incubation and then for another 2 h. Cell lysates were collected and subjected to western blot analysis for total IκBα, total p65, phospho-IκBα and phospho-p65 proteins. β-actin was used as an internal control. (B) Relative quantity of phosphorylated IκBα and p65 expression levels. *P<0.05 and ^#P<0.05 vs. TPA alone-treated group, **P<0.05 and ^##P<0.05 vs. DMSO control. All the data are presented as the mean ± standard deviation of three independent experiments. (C) Hispolon prevented NF-κB p65 nuclear translocation. Cells were preincubated with hispolon for 2 h prior to TPA treatment for another 2 h. Nuclear fractions were collected and histone H3 was used to verify equal loading. (D) Hispolon suppressed TPA-induced NF-κB DNA-binding activity. MDA-MB-231 cells were treated as described in part (A). Nuclear extracts (10 µg) were incubated with biotin-labeled oligonucleotides containing the NF-κB motif of the MMP-9 promoter. Bound mixtures were separated by 6% native-polyacrylamide electrophoresis. (E) Inhibitory effect of Bay 11–7082 on TPA-induced phosphorylations of IκBα and p65. Cells were pretreated with 5 µM Bay 11–7082 for 2 h and then stimulated with TPA for 2 h. (F) Inhibitory effect of Bay 11–7082 on TPA-induced MMP-9 expression. Cells in serum-free medium were pretreated with 2.5 or 5 µM Bay 11–7082 for 2 h and then stimulated with TPA for 24 h. Cell lysates were analyzed by western blot analysis. β-actin was used as an internal control. (G) Inhibitory effect of Bay 11–7082 on TPA-induced MMP-9 secretion. Cells in serum-free medium were preincubated with 5 µM Bay 11–7082 for 2 h, followed by treatment with TPA for 24 h. (H) Schematic diagram of the molecular mechanisms underlying the antimetastasis activity of hispolon in TPA-induced MDA-MB-231 cells. TPA, 12-O-tetradecanoylphorbol-13-acetate; DMSO, dimethyl sulfoxide; NF-κB, nuclear factor-κB; MMP-9, matrix metalloproteinase-9; Comp., competitor.