Inflammation induced by lipopolysaccharide advanced androgen receptor expression and epithelial-mesenchymal transition progress in prostatitis and prostate cancer

Abstract

Background: To explore the mechanism of prostatic inflammation on prostate cancer (PCa) by comparing the changes of prostate epithelial cells and PCa cells in an inflammatory environment.

Methods: First, immunohistochemistry (IHC) was used to compare the level of expression of TNF-α, IL-1β, IL-6, and TGF-β between benign prostatic hyperplasia (BPH), prostatitis, and PCa. Then primary prostate epithelial cells were sampled from patients who were suspected of PCa and had histological prostatitis (HP) confirmed by pathological biopsy. Lipopolysaccharide (LPS) or BAY11-7082 were used to investigate the change of androgen receptor (AR) and AR-mediated transcription, epithelial-mesenchymal transition (EMT) in primary prostate epithelial cells, and lymph node carcinoma of the prostate (LNCap) cells.

Results: TNF-α, IL-1β, IL-6, and TGF-β were significantly increased in HP and PCa compared with those in BPH patients. The proliferation of primary prostate epithelial cells and LNCap cells got the inflection point at LPS 10 µg/mL. In an inflammatory environment with 10 µg/mL LPS, both primary prostate epithelial cell and LNCap cell viability increased, and AR, AR-mediated transcription, and EMT processes were significantly increased. Inhibitors of NF-κB with 10 nM BAY11-7082 decreased AR, AR-mediated transcription, and EMT processes.

Conclusions: NF-κB regulates AR expression and EMT in prostatitis and PCa, and NF-κB inhibitors may have potential therapeutic value.

Keywords: Prostatitis; epithelial-mesenchymal transition (EMT); lipopolysaccharide (LPS); prostate cancer (PCa).

Figure 1

IHC results (A) compared the expression level of TNF-α, IL-1β, IL-6, and TGF-β in HP, PCa, and BPH tissues (×200). (B) IOD analysis of TNF-α, IL-1β, IL-6, and TGF-β protein levels in HP, PCa, and BPH tissues. IHC, immunohistochemical; HP, histological prostatitis; PCa, prostate cancer; BPH, benign prostatic hyperplasia; IOD, integrated optical density.

Figure 2

Identification of prostate epithelial cells. Immunofluorescence staining showed most cells expressed pan-cytokeratin (A), the cell nucleus was blue (B), and pan-cytokeratin expression was mainly expressed in the cytoplasm which was in line with the localization of cytoskeletal proteins (C).

Figure 3

The increasing doses of LPS influenced proliferation (A) and cytokine levels (B) of primary prostate epithelial cells and LNCap cells. LPS, lipopolysaccharide; LNCap, lymph node carcinoma of the prostate.

Figure 4

After 48 h, both in primary prostate epithelial cells (A) and LNCap cells (B), cell growth, which is mediated by LPS was blocked and cytokines (TNF-α, IL-1β, IL-6, and TGF-β) were significantly lower after treatment with BAY11-7082. LNCap, lymph node carcinoma of the prostate; LPS, lipopolysaccharide.

Figure 5

WB assesses the protein level of β-catenin, N-cadherin, p65, AR, Capase-3, and MMP-9 in primary prostate epithelial cells (A) and LNCap cells (B). LPS activated the NF-κB signaling pathway (p65) and promoted the EMT process, increased AR and MMP-9 in both primary prostate epithelial cells and LNCap cells, and trends were reversed by blocking the NF-κB signaling pathway with BAY11-7082. WB, western blot; AR, androgen receptor; LNCap, lymph node carcinoma of the prostate; LPS, lipopolysaccharide; EMT, epithelial-mesenchymal transition; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

Both in primary prostate epithelial cells (A) and LNCap cells (B), the mRNA level of AR and PSA was significantly increased, while NKX3.1 was significantly decreased. Also, these trends were reversed by BAY11-7082. LNCap, lymph node carcinoma of the prostate; mRNA, messenger RNA; AR, androgen receptor; PSA, prostate-specific antigen; LPS, lipopolysaccharide.

Figure 7

Fluorescent staining of E-cadherin in primary prostate epithelial cells (A) and vimentin in primary prostate epithelial cells (B), E-cadherin in LNCap cells (C), and vimentin in LNCap cells (D) after exposure to 10 µg/mL LPS or 10 nM BAY11-7082 for 12, 24 and 48 h. LNCap, lymph node carcinoma of the prostate; LPS, lipopolysaccharide; PECs, prostate epithelial cells.