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.
Chronic inflammation is the leading cause of epithelial malignancies, such as prostate cancer (
The adaptive immune system regulates antitumor effects via immunosurveillance (
Estrogen receptor β (ERβ) was reported to be expressed in prostate carcinoma cells; ERβ-regulated estrogen signaling served to inhibit tumor progression in patients with prostate cancer (
PC-3 and DU145, human prostate cancer cell lines, were obtained from the Chinese Academy of Sciences (Shanghai, China). PC-3 and DU145 prostate cancer cells were cultured in RPMI-1640 culture medium (BD Biosciences, San Jose, CA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 100 U/ml penicillin (Sigma-Aldrich; Merck KGaA). The cell lines were maintained in humidified incubators with 5% CO2 at 37°C. PC-3 and DU145 cells were transfected with an ERβ expression plasmid and empty vector was used as negative control using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Construction of receptor expression vectors was made as previously described (
The sequences of the siRNAs targeting ERβ and scramble RNA were as follows: siRNA-ERβ#1, 5′-GCCCUGCUGUGAUGAAUUAdTdT-3′; siRNA-ERβ#2, 5′-CCACCUUCCUUUCUAUUAUdTdT-3′; siRNA-ERβ#3, 5′-CGGGCUUCAUAAGCUAGAUdTdT-3′; and scramble, 5′-GAACUGAUGACAGGGAGGCTT-3′. Cells (5×104/well) were seeded in 96-well plates. Since the expression level of ERβ was most pronounced with siRNA-ERβ#1, this cell line was selected for the following experiments. DU145 cells were transfected with siRNA (150 pmol/ml) using Lipofectamine 2000® at 37°C overnight. Prior to transfection, the culture medium was replaced with Opti-MEM® Reduced Serum Medium (Gibco; Thermo Fisher Scientific, Inc.). The resulting cell populations were selected for further experiments. The levels of ERβ mRNA and protein expression were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively.
Total RNA was extracted from cells using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). RNA purity and concentration were determined using a NanoDrop™ 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Total RNA was reverse transcribed into cDNA using a PrimeScript Master Mix kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocols. The cDNA was amplified with SYBR Green Master Mix (Takara Bio, Inc.), which was performed using a LightCycler® 96 system (Roche Applied Science, Penzberg, Germany). The primers (5′→3′) used in qPCR were:
Cells cultured in RPMI-1640 medium were seeded into 96-well plates at a density of 5×103 cells/well and incubated at 37°C for 24 h. PC-3 and DU145 parental cells (negative vector transfected cells used as the control), and ERβ plasmid-transfected cells were treated with LPS for 24, 48, 72 and 96 h. MTT assay solution (20 µl) was applied to the medium, which was subsequently incubated for 4 h at 37°C. Then, 150 µl DMSO was added to cells for 10 min to solubilize the formazan crystals. Vehicle (0.1% DMSO) was used as the control. The optical density values in each well were measured at 595 nm with an absorption spectrophotometer (Olympus Corporation, Tokyo, Japan). All treatments were performed in triplicate.
Cells (5×104 cells/well) were cultured in serum-free RPMI-1640 medium (500 µl) and plated in the upper chambers of Transwell plates. RPMI-1640 with 15% FBS (600 ml; Beijing Transgen Biotech Co., Ltd., Beijing, China) was plated in the lower chambers. Plates were incubated in humidified conditions with 5% CO2 for 24 h at 37°C. Then, the inserts were washed with PBS three times, and cells on the upper surface were removed with a cotton swab. The cells remaining on the bottom surface of the inserts were treated with methanol (500 µl, 50 µg/ml) at 4°C for 10 min prior to staining with hematoxylin at room temperature for 10 min. A total of five predetermined fields (magnification, ×200) of each well were selected for cell counting. Images were captured by a photomicroscope (Axiovert 200 M; Carl Zeiss AG, Oberkochen, Germany).
Following rinsing in cold PBS, total protein was extracted from the cells using lysis buffer (0.1% SDS, 50 mmol Tris-base, 1% Triton X-100, 150 mmol sodium chloride, 1 mmol sodium orthovanadate, 0.5% sodium deoxycholate, 1% protease inhibitor cocktail and 10 mmol sodium fluoride) and quantified using the Bradford protein assay. Proteins (20 µg) were separated via 10% SDS-PAGE at 100 V and electrotransferred onto 0.45 µm polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in TBS-Tween 20 (10%) at room temperature for 2 h, and then incubated with primary antibodies against ERβ (1:500; cat. no. ab3576), p65 (1:100; cat. no. ab16502), NF-κB inhibitor α (IκBα, 1:200; cat. no. ab32518), phosphorylated (p)-IκBα (1:200; cat. no. ab133462), proliferating cell nuclear antigen (PCNA; 1:50; cat. no. ab92552), E-cadherin (1:100; cat. no. ab40772), B-cell lymphoma 2-associated X protein (Bax; 1:200; cat. no. 32503), caspase-3 (1:200; cat. no. ab32351), matrix metalloproteinase-2 (MMP-2; 1:200; cat. no. ab37150) and β-actin (1:50; cat. no. ab8226; Abcam, Cambridge, UK) overnight at 4°C. Anti-rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific, Inc.) was then added at room temperature for 1 h, and membranes were incubated for 20 min. Protein bands were visualized using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). Protein expression was quantified using Total Lab Nonlinear Dynamic Image (version 2009) analysis software (MathWorks, Natick, MA, USA).
High binding microtiter plates were coated overnight at 4°C with 100 µl per well of III-BSA coating antigen (0.05 µg/ml in coating buffer). Cells were then rinsed with PBS and blocked with 3% bovine serum albumin (BSA; Beijing Transgen Biotech Co., Ltd.) in PBS for 1 h at room temperature. Anti-tumor necrosis factor-α (TNF-α; cat. no. ab91235), anti-interleukin (IL)-1β (cat. no. ab242234), anti-monocyte chemoattractant protein-1 (MCP-1; cat. no. ab9669) and anti-IL-6 (cat. no. ab178013) monoclonal antibodies were purchased from Abcam, and were diluted in PBS + 3% BSA, added to cells and incubated at room temperature for 2 h. Following washing with PBS, cells were incubated with goat anti-mouse secondary antibodies (1:100; cat. no. BMS2007INST; Thermo Fisher Scientific, Inc.), and incubated for 60 min at room temperature. Cells expressing TNF-α, IL-1β, MCP-1 and IL-6 were detected using an ELISA Kit according to the manufacturer's protocols; protein concentrations were determined using a Spectramax M5 plate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).
Apoptotic cell death was determined using the Annexin V (conjugated to Alexa Fluor 594; excitation/emission: 590/617 nm) Apoptosis Detection Kit (BioVision, Inc., Milpitas, CA, USA) and PI. Following transfection, cells were incubated for 48 h, and then collected and stained with Annexin V/PI according to the manufacturer's protocols. The percentage of early apoptotic, late apoptotic and necrotic cells were determined using a flow cytometer (BD Bioscience) and analyzed with guavaSoft 3.1.1 software (EMD Millipore, Billerica, MA, USA). The occurrence of apoptosis in each group was investigated in ≥3 independent experiments.
SPSS 19.0 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Data are presented as the mean ± standard deviation. Comparisons across three or more groups were performed using one-way analyses of variance followed by Tukey's test, while a Student's t-test was used to determine significant differences between two groups. P<0.05 was considered to indicate a statistically significant difference. All tests were investigated in ≥3 independent experiments.
Following incubation with LPS for 24 h, the expression levels of proinflammatory cytokines in the supernatants of the prostate cancer cell lines PC-3 and DU145 were determined using ELISA. Stimulation with LPS led to significantly elevated expression levels of TNF-α, MCP-1, IL-1β and IL-6 in PC-3 and DU145 cells compared with controls treated with DMSO (P<0.05;
To investigate whether p65 is regulated by ERβ signaling, the expression levels of proteins involved in the NF-κB pathway were determined. As presented in
To investigate the role of the NF-κB pathway in the regulation of ERβ-induced inflammation, western blot analysis was performed to determine the expression of NF-κB pathway-associated proteins in PC-3 and DU145 cells transfected with an ERβ expression vector or treated with the ERβ antagonist PHTPP. It was revealed that ERβ overexpression suppressed the expression of p65 and p-IκBα, but not total IκBα expression in LPS-stimulated PC-3 and DU145 cells compared with control cells. This expression profile was reversed following treatment with PHTPP (
Providing ERβ is a suppressor of prostate inflammation, ERβ activation may suppress the expression of certain proinflammatory genes. To investigate the involvement of ERβ signaling in LPS-induced inflammation, the expression of the proinflammatory cytokines TNF-α, IL-1β, MCP-1 and IL-6, was determined in cells overexpressing ERβ and transfected cells treated with PHTPP via ELISA. The results demonstrated that the LPS-induced production of TNF-α, MCP-1, IL-1β and IL-6 was significantly reduced in prostate cancer cells following overexpression of ERβ compared with negative control vector cells (P<0.05;
To determine the effects of ERβ overexpression on prostate cancer cell viability, apoptosis and migration, MTT, Annexin V/PI and Transwell assays were performed using control, ERβ-overexpressing, and ERβ-inhibited cells. As presented in
As ERβ signaling appeared to be involved in the regulation of cell viability, apoptosis and migration, and the expression of proteins associated with these properties was determined. PCNA, Bax and caspase-3, and MMP-2 and E-cadherin were selected as markers of proliferation (
To further investigate the effects of ERβ on the progression of prostate cancer, ERβ-targeting siRNAs were employed to downregulate ERβ expression in DU145 prostate cancer cells. The silencing effects of three siRNAs compared with a negative control scramble siRNA were reported via RT-qPCR analysis (
In the present study, it was revealed that ERβ overexpression reduced the inflammation induced by LPS treatment via regulating the levels of proinflammatory cytokines, including TNF-α, MCP-1, IL-1β and IL-6. Furthermore, it was demonstrated that the ERβ antagonist PHTPP increased the expression of proinflammatory cytokines. Additionally, it was observed that ERβ overexpression suppressed the viability and migration of PC-3 and DU145 prostate cancer cells, and promoted apoptosis. These findings may improve understanding of the possible mechanisms by which ERβ, a tumor suppressor, may contribute to inhibition of the NF-κB pathway.
LPS is a common inducer of inflammation; exposure leads to the activation of a number of components involved in chronic inflammation processes, such as altered cytokine levels (
ERβ has been associated with the differentiation of prostatic epithelial cells, and was reported to exhibit antiproliferative effects on prostate cancer cells (
It was reported that, under physiological conditions, estrogen signaling via ERβ inhibited prostate gland growth (
NF-κB is activated by various signals via phosphorylation and degradation of the inhibitory IκBα protein (
In conclusion, the present study reported that activation of ERβ suppressed the NF-κB signaling pathway, and suggested that ERβ may be a potential anti-inflammatory and anticancer agent in the treatment of prostate cancer.
Not applicable.
The present study was funded by the Internal Research Institutions Fund of Health and Technology Planning Commission of Yunnan Province (grant no. 2016NS209).
All data generated or analyzed during this study are included in this manuscript.
LX and YL made substantial contributions to data acquisition and analysis, and were major contributors in drafting the manuscript. RT was responsible for interpretation of data; NZ conceived and designed the study, and revised the manuscript critically for important intellectual content. All authors have read and agreed with the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
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.
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.
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.
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.
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.