Estrogen receptor β suppresses inflammation and the progression of prostate cancer

Abstract

Previous studies demonstrated that estrogen receptor β (ERβ) signaling alleviates systemic inflammation in animal models, and suggested that ERβ‑selective agonists may deactivate microglia and suppress T cell activity via downregulation of nuclear factor κ‑light‑chain‑enhancer of activated B cells (NF‑κB). In the present study, the role of ERβ in lipopolysaccharide (LPS)‑induced inflammation and association with NF‑κB activity were investigated in PC‑3 and DU145 prostate cancer cell lines. Cells were treated with LPS to induce inflammation, and ELISA was performed to determine the expression levels of inflammatory cytokines, including tumor necrosis factor‑α (TNF‑α), monocyte chemoattractant protein 1 (MCP‑1), interleukin (IL)‑1β and IL‑6. MTT and Transwell assays, and Annexin V/propidium iodide staining were conducted to measure cell viability, apoptosis and migration, respectively. Protein expression was determined via western blot analysis. LPS‑induced inflammation resulted in elevated expression levels of TNF‑α, IL‑1β, MCP‑1 and IL‑6 compared with controls. ERβ overexpression significantly inhibited the LPS‑induced production of TNF‑α, IL‑1β, MCP‑1 and IL‑6. In addition, the results indicated that ERβ suppressed viability and migration, and induced apoptosis in prostate cancer cells, which was further demonstrated by altered expression of proliferating cell nuclear antigen, B‑cell lymphoma 2‑associated X protein, caspase‑3, E‑cadherin and matrix metalloproteinase‑2. These effects were reversed by treatment with the ERβ antagonist PHTPP or ERβ‑specific short interfering RNA. ERβ overexpression reduced the expression levels of p65 and phosphorylated NF‑κB inhibitor α (IκBα), but not total IκBα expression in LPS‑treated cells. In conclusion, ERβ suppressed the viability and migration of the PC‑3 and DU145 prostate cancer cell lines and induced apoptosis. Furthermore, it reduced inflammation and suppressed the activation of the NF‑κB pathway, suggesting that ERβ may serve roles as an anti‑inflammatory and anticancer agent in prostate cancer.

Figure 1.

Elevated expression of proinflammatory genes and activation of the NF-κB pathway in prostate cancer cells following LPS treatment. (A) Expression of proinflammatory cytokines was determined using ELISA in LPS-treated and control PC-3 and DU145 prostate cancer cells. (B) Expression of NF-κB pathway-associated proteins was determined via western blot analysis in LPS- and control-treated PC-3 and DU145 cells. Data are presented as the mean ± standard deviation; *P<0.05, **P<0.01 and ***P<0.001 vs. the control group. Control, treatment with dimethyl sulfoxide; ERβ, estrogen receptor β; IL, interleukin; NF-κB, nuclear factor-κ-light-chain-enhancer of activated B cells; IκBα, NF-κB inhibitor α; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; p, phosphorylated; TNF-α, tumor necrosis factor α.

Figure 2.

ERβ-induced reduction in proinflammatory cytokine release via suppression of the NF-κB pathway. (A) Expression of NF-κB pathway-associated proteins in NC empty vector-transfected, ERβ expression vector-transfected and PHTPP-treated PC-3 and DU145 cells was determined via western blot analysis. (B) Production of inflammatory cytokines was determined using ELISA in NC, ERβ-transfected and PHTPP-treated PC-3 and DU145 cells. Data are presented as the mean ± standard deviation; *P<0.05, **P<0.01, ***P<0.001 vs. NC; ^#P<0.05, ^##P<0.01, vs. transfection group. ERβ, estrogen receptor β; IL, interleukin; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells; IκBα, NF-κB inhibitor α; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1p, phosphorylated; TNF-α, tumor necrosis factor-α.

Figure 3.

ERβ overexpression reduces the viability and promotes the apoptosis of prostate cancer cells. (A) Cell viability was investigated using an MTT assay in NC, ERβ-transfected and PHTPP-treated PC-3 and DU145 cells. (B) Apoptosis was determined via an Annexin V/PI assay in NC, ERβ-transfected and PHTPP-treated PC-3 and DU145 cells. Data are presented as the mean ± standard deviation; **P<0.01, ***P<0.001 vs. NC; ^###P<0.001, vs. transfection group. ERβ, estrogen receptor β; NC, negative control; OD, optical density; PI, propidium iodide.

Figure 4.

ERβ activation inhibits the migration of prostate cancer cells and alters the expression of proliferation-, apoptosis- and migration-associated proteins. (A) Cell migration was determined using a Transwell assay in NC, ERβ-transfected and PHTPP-treated transfected PC-3 and DU145 cells. (B) Expression of PCNA, Bax, caspase-3, MMP-2 and E-cadherin was evaluated via western blot analysis in NC, ERβ-transfected and PHTPP-treated transfected PC-3 and DU145 cells. Data are presented as the mean ± standard deviation; **P<0.01, ***P<0.001 vs. NC; ^##P<0.01, vs. transfection group. Bax, B-cell lymphoma 2-associated X protein; ERβ, estrogen receptor β; MMP-2, matrix metalloproteinase-2; NC, negative control; PCNA, proliferating cell nuclear antigen.

Figure 5.

Effects of ERβ-specific siRNA on inflammation, proliferation, apoptosis and migration of DU145 prostate cancer cells. (A) Expression levels of ERβ mRNA following treatment with siRNAs were determined via reverse transcription-quantitative polymerase chain reaction. (B) Expression of proteins associated with the nuclear factor κ-light-chain-enhancer of activated B cells pathway was determined via western blot analysis. (C) Cell viability of DU145 was measured with a MTT assay, with absorbance at 595 nm used as an index. (D) Cell apoptosis was determined via flow cytometry. (E) Secretion of proinflammatory cytokines was measured using ELISA. (F) Migration of DU145 cells was evaluated using a Transwell migration assay. (G) Expression levels of proliferation-, apoptosis- and migration-associated proteins in DU145 cells following transfection with siRNA-ERβ#1 or Scramble were determined via western blot analysis. Data are presented as the mean ± standard deviation; *P<0.05, **P<0.01 vs. Scramble group. Bax, B-cell lymphoma 2-associated X protein; cle, cleaved; ERβ, estrogen receptor β; IL, interleukin; IκBα, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor α; MCP-1, monocyte chemoattractant protein 1; MMP-2, matrix metalloproteinase-2; NC, negative control; NS, not significant; OD, optical density; p, phosphorylated; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; siRNA, short interfering RNA; TNF-α, tumor necrosis factor-α.