We purchased a human-androgen-dependent prostate cancer cell line (LNCaP) from the American Type Culture Collection (Rockville, MD, USA). These cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% fetal bovine serum (FBS, Sigma-Aldrich), 50 IU/ml penicillin, and 50 mg/ml streptomycin sulfate (Gibco, Grand Island, NY, USA) at 37°C with 5% CO₂ and 95% room air (normoxia), or 5% CO₂, 94% N₂, and 1% O₂ (hypoxia). LNCaP/CH cells were cultured under hypoxia for at least 6 months.

The cells were seeded into 12-well plates at a density of 1×10⁵ cells per well. They were then trypsinized, collected, and counted using a hemocytometer 24, 48, and 72 h after seeding.

The cells were seeded into 96-well plates at density of 5,000 cells per well. After culturing overnight, a fresh standard medium containing various concentrations of ANGPTL4 recombinant proteins (rANGPTL4) (0–1000 pg/ml) was added to each well. After incubation for 48 h, 5 mM WST-1 (Dojindo, Kumamoto, Japan) and 3 nM 1-methoxy-5-methylphenazinium methyl sulfate (Dojindo) were added to each well. After incubation for 1–4 h, absorbance was measured at 450 nm with a microplate reader (Tecan Japan Co., Ltd., Kanagawa, Japan).

Cell migration was assessed using a 24-well plate with Corning^® FluoroBlok Inserts (Corning Inc., Corning, NY, USA). The cells were seeded at 5×10⁴ cells per well in 0.5-ml serum-free medium. The outer chambers were filled with 0.75 ml of media containing 15% FBS. After 24 or 48 h, migrating cells were labeled using Cellstain^®-Calcein-AM solution (Dojindo). After incubation for 1 h under normoxia or hypoxia, the cell fluorescence was detected using a microplate reader.

This method was performed as previously described (6). The primers comprised ANGPTL4 (Hs_ANGPTL4_1_SG QuantiTect Primer Assay, QT00003631) and β-actin (HS_ACTB_1_SG QuantiTect Primer Assay, QT00095431). The quantification of mRNA expression was normalized using β-actin.

The cells were cultured in six-well plates at a density of 5×10⁵ cells per well for 24, 48, and 72 h. The medium was then collected before adding fresh medium to the cultures. The collected medium was used to quantify ANGPTL4 protein concentration using a commercial ELISA according to the manufacturer's instructions (Human ANGPTL4 ELISA; Raybiotech, Inc., Norcross, GA, USA).

To create stably transfected lines, LNCaP cells were transduced with either FLAG-tagged ANGPTL4-expressing (Addgene) or control vectors (Cell Biolabs, Inc., San Diego, CA, USA) using Lipofectamine 3000 (Invitrogen), according to the manufacturer's protocol. The transfected cell lines were selected in 0.2-µg/ml puromycin dihydrochloride (Santa Cruz Biotechnology, Dallas, TX, USA).

This method was performed as previously described (6). The primary antibodies included anti-Bcl-2 (cat. no. SASC509, Santa Cruz Biotechnology), anti-Bad (cat. no. 9292T, Cell Signaling), anti-phospho-Akt (Set473; cat. no. 9271S, Cell Signaling), anti-Akt (cat. no. 9272S, Cell Signaling), anti-β-tubulin (cat. no. #MAB3408, EMD Millipore), and anti-ANGPTL4 (cat. no. GTX114198, GeneTex).

LNCaP cells were transiently transfected with an ANGPTL4 siRNA duplex [siANGPTL4, final concentration: 60 nmol/l (Qiagen Inc.)] or control siRNA [random scrambled sequence: siScr, final concentration: 60 nmol/l (Qiagen Inc.)] using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer's instructions. The siRNA sequences against ANGPTL4 generated by Qiagen were 5′-GGGACAAGAACUGCGCCAATT-3′ and 5′-UUGGCGCAGUUCUUGUCCCTG −3′.

This method was performed as previously described (6). The cell cycles were analyzed using a BD FACSVerse™ flow cytometer and BD FACSuite™ software (Becton Dickinson, San Jose, CA, USA).

The cells were seeded into 12-well plates at a density of 1×10⁵ cells per well. After culturing overnight, a fresh standard medium containing 2 nM docetaxel (Sigma-Aldrich) was added to the plates. The phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, was obtained from Calbiochem (Darmstadt, Germany). Docetaxel is the standard chemotherapy for men with advanced prostate cancer.

As this study was a retrospective chart review, no patient consent was required. From June 2009 to December 2012, 92 patients underwent curative surgery involving a radical prostatectomy for localized prostate cancer at Oita University Hospital (Oita, Japan). Patients who received therapy before and/or immediately after surgery were excluded, and data from the remaining 70 patients were analyzed (Table I). After surgery, the serum prostate specific antigen (PSA) levels were monitored, and PSA recurrence was defined as an elevation in serum PSA levels (≥0.2 ng/ml) in two consecutive measurements.

This method was performed as previously described (15). The primary polyclonal goat anti-human ANGPTL4 (cat. no. 18374-1-AP, Proteintech™) antibody diluted to 100X with PBS containing 1% bovine serum albumin was applied.

To evaluate ANGPTL4 staining, we counted the proportion of positively stained cells out of 1,000 cells from five randomly selected areas. ANGPTL4 expression was divided according to the percentage of positive tumor cells (negative, <20%; weak: 20–50%; and strong, >50%). Two authors independently evaluated in a blinded manner to the clinical findings, the level of immunoreactivity using an ECLIPSE E600 research microscope (Nikon Corp., Tokyo, Japan).

Statistical analyses were performed using a Student's t-test, Fisher's exact test, Mann-Whitney U-test, or Spearman's rank correlation test. A two-tailed test was used for all analyses. Biochemical recurrence-free survival was estimated using the Kaplan-Meier method and compared the groups using the log-rank test. A multivariate analysis was performed using a Cox's proportional hazards model. The P-values of <0.05 were considered to be statistically significant. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (16).

LNCaP/CH cells were grown under hypoxic conditions for over 6 months, and relative ANGPTL4 mRNA expression was 14.4-, 9.0-, and 11.4-fold higher in LNCaP/CH cells than in LNCaP, LNCaP/AH 24 h and LNCaP/AH 48 h cells, respectively (Fig. 1A). Similarly, ANGPTL4 protein expression was 6.6-fold higher in LNCaP/CH cells than in LNCaP cells (Fig. 1B). Fig. 1B showed a doublet band and the molecular weight of the detected bands was 41 and 45 kDa. ANGPTL4 could be cleaved to an active form and the lower band indicates the active form of ANGPTL4.

After 72 h, ANGPTL4 secretion was 7.0-fold higher in LNCaP/CH cells than in LNCaP cells (Fig. 1C); however, no statistically significant differences were found after 48 h (data not shown).

ANGPTL4 siRNA (siANGPTL4) effectively downregulated ANGPTL4 expression in both LNCaP and LNCaP/CH cells (Fig. 2A). After incubation for 72 h with siANGPTL4, cellular proliferation decreased to 82.0 and 74.2% in LNCaP and LNCaP/CH cells, respectively (Fig. 2B and C). Similarly, siANGPTL4 decreased ANGPTL4 secretion to 41.2% after 72 h in LNCaP/CH cells (Fig. 2D). Flow cytometry revealed an increase in hypodiploid DNA (sub-G1 population) cells among the siANGPTL4 transfected cells, concomitant to a significant growth inhibition (Fig. 2E).

To investigate the precise behavior of ANGPTL4 in prostate cancer, we established a stable cell line overexpressing ANGPTL4 (LNCaP/ANGPTL4). LNCaP/ANGPTL4 increased ANGPTL4 expression by 5.1-fold compared to the control (Fig. 3A). Furthermore, cellular migration was 2.0-fold and 3.0-fold higher in LNCaP/ANGPTL4 cells than in LNCaP and LNCaP/neo-transfected cells with a control vector, respectively (Fig. 3B). In contrast, cellular proliferation did not differ significantly among the cell lines (data not shown).

To assess the potential role of ANGPTL4, we added different concentrations of rANGPTL4 to LNCaP cells (Fig. 4A). Treatment with 100 pg/ml of rANGPTL4 for 72 h increased cellular proliferation 1.72-fold (Fig. 4B). The immunoblot assay also revealed that Akt phosphorylation at Ser 473, a site required for Akt activation, was upregulated both time- and dose-dependently (Fig. 4C and D). However, cellular proliferation was suppressed in the cells subsequently treated with LY294002 (a PI3K inhibitor) 6 h later (Fig. 4E). Akt phosphorylation peaked at 5–15 min (Fig. 4C), but the effect on cellular proliferation was observed 48–72 h later (Fig. 4B). It is considered that with the activation of Akt, various signaling molecules are activated resulting in cell survival.

Cellular proliferation was successfully repressed in LNCaP/neo cells following 2 nM docetaxel treatment; however, LNCaP/ANGPTL4 cells maintained their proliferative capabilities even after treatment (Fig. 3C). The immunoblot assay revealed that anti-apoptotic B-cell lymphoma 2 (Bcl-2) expression was upregulated and that of Bcl-2-associated death promoter (Bad) was downregulated in LNCaP/ANGPTL4 cells compared with that in the controls (Fig. 3D). Thus, these results indicate that ANGPTL4 expression is linked to docetaxel resistance by inhibiting apoptosis.

Table I presents the clinicopathological characteristics of the patients included in this study. The median age of the patients was 67.5 (range: 49–76) years. The median PSA at diagnosis was 9.0 (range: 3.7–23.9) ng/ml. The pathological T (pT) stage was ≤pT2 in 61 cases (87.1%) and ≥pT3 in nine cases (12.9%). The Gleason score (GS) was ≤6 in 20 cases (28.6%) and ≥7 in 47 cases (67.1%). The remaining three cases were unremarkable. During a median follow-up of 53.5 (range: 13–82) months, 19 patients (27.1%) had PSA recurrence.

We evaluated ANGPTL4 expression by immunohistochemistry in 70 prostate tissue samples. Normal prostate gland tissue did not express ANGPTL4. In contrast, we observed negative staining in 12 of the 70 prostate cancer samples (17.1%) and positive staining in 58 samples (82.9%). ANGPTL4 expression was particularly strong in 11 samples. Fig. 5A shows the representative images.

Table II lists the correlation between ANGPTL4 expression levels and different clinicopathological factors that were analyzed. ANGPTL4 expression levels were significantly correlated with PSA recurrence (P=0.02). However, PSA at diagnosis, GS, pT stage, and the resection margin were not correlated with ANGPTL4 expression. An extraprostatic extension tended to be higher in cells with positive ANGPTL4 expression than in cells with negative ANGPTL4 expression; however, this difference was not significant (P=0.091).

We performed univariate and multivariate analyses to determine the factors that could be used as indicators of PSA recurrence after surgery (Table III). A univariate analysis revealed that both pT stage (P=0.007) and positive ANGPTL4 expression of the tumor (P=0.04) were indicators of PSA recurrence. The multivariate analysis using the Cox's proportional hazards model revealed that positive ANGPTL4 expression was an independent prognostic indicator of PSA recurrence (P=0.03, hazard ratio = 2.02). The 3-year PSA recurrence-free survival rate for patients with positive ANGPTL4 expression was significantly lower than that for other patients (P=0.01) (Fig. 5B).