Peroxiredoxin-3 is overexpressed in prostate cancer and promotes cancer cell survival by protecting cells from oxidative stress

Abstract

Objective: We have previously identified peroxiredoxin-3 (PRDX-3) as a cell-surface protein that is androgen regulated in the LNCaP prostate cancer (PCa) cell line. PRDX-3 is a member of the peroxiredoxin family that are responsible for neutralising reactive oxygen species.

Experimental design: PRDX-3 expression was examined in tissue from 32 patients using immunohistochemistry. Subcellular distribution was determined using confocal microscopy. PRDX-3 expression was determined in antiandrogen-resistant cell lines by western blotting and quantitative RT-PCR. The pathways of PRDX-3 overexpression and knockdown on apoptosis and response to oxidative stress were investigated using protein arrays.

Results: PRDX-3 is upregulated in a number of endocrine-regulated tumours; in particular in PCa and prostatic intraepithelial neoplasia. Although the majority of PRDX-3 is localised to the mitochondria, we have confirmed that PRDX-3 at the cell membrane is androgen regulated. In antiandrogen-resistant LNCaP cell lines, PRDX-3 is upregulated at the protein but not RNA level. Resistant cells also possess an upregulation of the tricarboxylic acid (TCA) pathway and resistance to H₂O₂-induced apoptosis through a failure to activate pro-apoptotic pathways. Knockdown of PRDX-3 restored H₂O₂ sensitivity.

Conclusion: Our results suggest that PRDX-3 has an essential role in regulating oxidation-induced apoptosis in antiandrogen-resistant cells. PRDX-3 may have potential as a therapeutic target in castrate-independent PCa.

Figure 1

PRDX-3 expression in tissue. A TMA containing matched normal/tumour cores from a variety of different organs was probed with anti-PRDX-3 antibody and visualised using Alexafluor 488 (green). Nuclei were counterstained with DAPI (blue). Examples of different paired normal and tumour tissue are shown (A). A prostate tissue TMA was also stained in an identical manner. Examples from a single patient are shown (B). PRDX-3-stained prostate TMAs were scanned and quantified. Total pixel count for each core was calculated using the following equation: total pixel intensity × total pixel area/1000. Data were grouped according to pathology, for example, benign, prostatic intraepithelial neoplasia (PIN) or prostate cancer (PCa) (C) or subdivided by Gleason grade (D). N numbers indicate the number of cores analysed. P-values were calculated using a Kruskal–Wallis test.

Figure 2

PRDX-3 is localised to the cell membrane and can be detected by ELISA. Mitochondrial PRDX-3 was confirmed by colocalisation of COX IV (green) with PRDX-3 (red) (A). For androgen regulation, LNCaP cells were grown in RPMI containing 10% charcoal stripped serum and treated for another 24 h with vehicle or R1881. Cells were fixed with methanol and probed for E-cadherin (green) or PRDX-3 (red). Nuclei are shown in blue. Scale bars represent 10 μM (B). Gene expression data were generated as previously described in LNCaP cells (Massie, 2011) by treating cells with the androgen, R1881 and harvesting over a timecourse of 24 h. Points represent log-expression relative to the 0 time point. The autocorrelation is 0.61. The probability of seeing that whether there were not a trend is 0.00005 (from 1 000 000 permutations). Hence, this is the P-value for the hypothesis that there is no change after androgen treatment (C).

Figure 3

PRDX-3 is predominantly mitochondrial and upregulated at the protein level by antiandrogen treatment. Twenty micrograms protein lysates from parental LNCaP cells treated with R1881 or vehicle for 24 h, fibroblasts from primary benign prostatic hyperplasia (BPH) COS, PC3 or the antiandrogen-resistant cell lines grown with1 μM hydroxyflutamide alone (LNCaP-OHF), 1 μM hydroxyflutamide and 10 pM R1881 (LNCaP-OHF/R1881), 1 μM bicalutamide alone (LNCaP-BIC), 1 μM bicalutamide and 10pM R1881 (LNCaP-BIC/R1881) were separated by SDS–PAGE and probed for PRDX-3 and actin (A). qRT–PCR was performed on samples from the same cell lines. Relative expression levels were calculated based on the difference in Ct values between the test samples and the control and results normalised with the expression of GAPDH. All results were expressed relative to vehicle-treated cells. Mean results and s.d. are shown relative to vehicle-treated parental cell lines (*). P-values were calculated using a two-tailed Students t-test (B). Twenty micrograms of identical cell lysates were also analysed by western blotting (WB) for COX IV and actin as before (C). wtLNCaP cells were grown in the presence of 0 μM, 1 μM or 10 μM bicalutamide for 3 days and 20 μg cell lysate separated by SDS–PAGE, transferred to nitrocellulose and probed for oxidized PRDX-3 (SO₂/SO₃), total PRDX-3 and actin (D, top panel). Images were taken showing the more etiolated appearance of the bicalutamide treated cells (D, lower panels).

Figure 4

PRDX-3 protects cells from apoptosis. For the citrate synthase assay, wtLNCaP cells and antiandrogen-resistant cells were lysed on ice and 10 μg total protein assayed immediately for citrate synthase activity. P-values were determined using a two-way ANOVA test (A). wtLNCaP cells (solid line, squares) and LNCaP-BIC cells (dashed line, circles) were serum starved for 24 h and treated with varying concentrations of H₂O₂ (0.00016–0.001%) for 24 h before measuring cell viability (B). wtLNCaP cells and LNCaP-BIC (BIC) were serum starved and treated as before with (+) or without (−) 0.00033% H₂O₂ and 200 μg of total protein used on an apoptosis protein array. Selected changes are highlighted in the lower panel (C). PRDX-3 knockdown was performed on wtLNCaP and LNCaP-BIC cells using 0, 50 or 100 μM siRNA. Seventy-two hours after transfection cells were harvested, lysed and probed for PRDX-3 or actin (D, right panel). Cells were treated with 0.00033% H₂O₂ for 1 h or 24 h before harvesting and assaying for cell viability as before (D, left panel). All results were calculated as relative to the no treatment controls. P-values were determined using a two-tailed Student's t-test.