Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells

Abstract

Background: Elevated NF-κB activity has been previously demonstrated in prostate cancer cell lines as hormone-independent or metastatic characteristics develop. We look at the effects of piperlongumine (PL), a biologically active alkaloid/amide present in piper longum plant, on the NF-κB pathway in androgen-independent prostate cancer cells.

Methods: NF-κB activity was evaluated using Luciferase reporter assays and Western blot analysis of p50 and p65 nuclear translocation. IL-6, IL-8, and MMP-9 levels were assessed using ELISA. Cellular adhesion and invasiveness properties of prostate cancer cells treated with PL were also assessed.

Results: NF-κB DNA-binding activity was directly down-regulated with increasing concentrations of PL, along with decreased nuclear translocation of p50 and p65 subunits. Expression of IL-6, IL-8, MMP-9, and ICAM-1 was attenuated, and a decrease of cell-to-matrix adhesion and invasiveness properties of prostate cancer cells were observed.

Conclusions: PL-mediated inhibition of NF-κB activity decreases aggressive growth characteristics of prostate cancer cells in vitro.

Keywords: IL-6; IL-8; MMP-9; NF-κB; ROS; adhesion; apoptosis; cytokines; piperlongumine; prostate cancer.

Fig. 1

The effect of Piperlongumine (PL) on proliferation of human prostate cancer cell lines (PC-3 and DU-145). Cells were treated with indicated concentrations of PL for 48 hr. Cellular proliferation was assessed using the Cell Titer Blue assay.

Fig. 2

Levels of NF-κB activity were determined by Luciferase Reporter Assay as described in Materials and Methods section. PC-3, DU-145, and LNCaP cells were pre-incubated with indicated concentrations of PL for 6 hr prior to TNF-α (40 ng/ml) stimulation. Statistical analysis was performed by one-way ANOVA. Statistically significant (P < 0.05) compared with cells cultured in medium alone (*) or TNF-α-induced cells (**).

Fig. 3

PL attenuates degradation of IκBα and blocks nuclear translocation of p50 and p65 in prostate cancer cells primarily via Akt-independent pathway. PL blocks NF-κB translocation in PC-3 (A), and DU-145 cells (B). PL effects on NF-κB translocation in dose-dependent fashion in PC-3 (C), and DU-145 (D) prostate cancer cell lines. E: PL (10 μM, overnight) effectively blocks both Akt and NF-κB pathways. Levels of the nuclear and cytoplasmic proteins were determined by Western blot analysis with specific antibodies. Expression of Topo I (nuclear extract) and β-actin (cytoplasmic extract) were used to control equal protein loading. Representative data from one of three experiments.

Fig. 4

PL dramatically reduces expression of cytokines (A) IL-6, (B) IL-8, and (C) MMP-9 in PC-3 cells after TNF-α-mediated activation of NF-κB pathway. Statistical analysis was performed by one-way ANOVA. Statistically significant (P < 0.05) compared with cells cultured in medium alone (*) or TNF-α-induced cells (**).

Fig. 5

PL reduces invasiveness of PC-3 cells. A: Migration of PC-3 cells through the filters was quantified as described in the Materials and Methods section. Statistical analysis was performed by one-way ANOVA. Statistically significant (P < 0.05) compared with cells cultured in medium alone (*). Representative data from one of three experiments. B: Representative images of PC-3 cells treated under indicated conditions that invaded through the FluoroBlok membrane.

Fig. 6

Effect of PL on the adhesion of PC-3 cells. Adhesion of PC-3 cells was determined as described in the Materials and Methods section. A: Adhesion capacity is depicted as tumor cell binding and related to100% binding of non-treated control. Statistical analysis was performed by one-way ANOVA. Statistically significant (P <0.05) compared with cells cultured in medium alone (*). Representative data from one of three experiments. B: Representative images of attached PC-3 cells.

Fig. 7

PL significantly inhibits TNF-α mediated ICAM-1 expression in PC-3 cell line. Normal mouse IgG-FITC was utilized as a negative control (dotted line). X-axis represents fluorescence intensity; Y-axis represents cell number. Representative data from one of three experiments.