Human prostate carcinoma cell lines PC-3 and LNCaP were a kind gift from Dr Dan Mercola (The SKCC; Sidney Kimmel Cancer Center, La Jolla, CA, USA). The cells were cultured in RPMI-1640 medium supplemented with 100 ml/l fetal bovine serum (FBS), 8×10⁵ U/l penicillin and 0.1 g/l streptomycin in humidified incubator containing 50 ml/l CO₂ at 37°C.

Tris-borate-EDTA and acrylamide:bisacrylamide (29:1) were obtained from Bio-Rad Laboratories (Richmond, CA, USA). Egr-1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Lipofectamine was purchased from Life Technologies, Inc. (Carlsbad, CA, USA) and TNF-α from Sigma Chemical Co. (St. Louis, MO, USA). Complete Mini-EDTA-Free Protease Inhibitor Cocktail Tablets and Annexin-V-Fluos were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Other reagents were purchased from Sigma Chemical Co. (Taufkirchen, Germany). Phorbol 12-myristate 13-acetate (TPA), TNF-α and TGF-β1 were purchased from Stratagene Inc. (La Jolla, CA, USA). Anti-Egr-1, anti-p14^ARF, anti-TGF-β1, and β-actin antibodies were purchased from Santa Cruz Biotechnology.

The sequences designed used to specifically target human p14^ARF and Egr-1 RNAs were: for p14^ARF, 5′-GAACAUGGU GCGCAGGUUCTT-3′; and for Egr-1, 5′-AACGCAAGAGGC AUACCAAGA-3′. The scrambled small interfering RNA (siRNA) oligonucleotides used as controls for all RNA interference experiments were: 5′-AAAGGUGACGCUGACGAA GTT-3′ and 5′-CAAGAAAGGCCAGUCCAAGTT-3′. The cells were transfected with siRNA oligonucleotide duplexes and cultured in medium without antibiotics 24 h prior to transfection resulting in a confluence of the cell monolayer by 60–80%. Egr-1-siRNA or non-silencing siRNA (70 nmol) were mixed with Lipofectamine™ 2000 (Invitrogen-Life Technologies) according to the manufacturer’s recommendation and added to the cells. After 6 h at 37°C, the medium was replaced and the cells were cultivated in RPMI-1640 supplemented with 10% heat-inactivated FBS (9,16,17).

PC-3 and LNCaP cells (5×10⁵) were placed in 96-well plates in RPMI-1640 containing 10% FBS at a final volume of 0.1 ml. The following day, the cells were treated with the pTER siRNA. MTT was added (20 ml/well of 5 g/l solution in PBS) after culture for 24, 48 and 72 h. Following incubation at 37°C for 4 h, the reaction was stopped by the addition of 100 ml DMSO. The reaction product was quantified by measuring the absorbance at 490 nm using an ELISA reader (WALLAC 1420 VICTOR 2; Wallac, Turku, Finland) and the Software HT-Soft (Perkin-Elmer). The samples were assayed repeatedly in 6-wells.

PC-3 and LNCaP prostate carcinoma cells lines (5×10⁵) were seeded in 6-well plates. Forty-eight hours after transfection, the cells were collected and washed twice by cold PBS, and each well was treated with 50 ml lysis buffer (2 mmol/l Tris-HCl pH 7.4, 50 mmol/l NaCl, 25 mmol/l EDTA, 50 mmol/l NaF, 1.5 mmol/l Na₃VO₄, 1% Triton X-100, 0.1% SDS, supplemented with protease inhibitors 1 mmol/l phenylmethylsulfonylfluoride, 10 mg/l pepstatin, 10 mg/l aprotinin and 5 mg/l leupeptin) (all from Sigma). Protein concentrations were determined using the Bradford protein assay. Equal amounts of protein (40 mg) were separated on a 15% SDS polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond C; Amersham, Freiburg, Germany). The membranes were blocked in 5% non-fat dry milk in TBS for 1 h at room temperature and probed with rabbit anti-Egr-1 antibodies (1:500 dilution; Santa Cruz Biotechnology) overnight at 4°C. After being washed 3 times with TBS containing 0.1% Tween-20, the membranes were incubated with anti-rabbit IgG-horseradish-peroxidase (1:5,000; Santa Cruz Biotechnology), and developed by luminol-mediated chemiluminescence (Appylgen Technologies Inc., Beijing, China). To confirm equal protein loading, membranes were reprobed with a 1:1,000 dilution of an anti-actin antibody (Santa Cruz Biotechnology). Densitometric analyses were performed using Scion Image software. Intestinal alkaline phosphatase (IAP) activity was detected using an IAP kit from Sigma-Aldrich.

The cells were plated in 96-well plates 24 h prior to treatment. Following treatment, DNA fragmentation was evaluated by examination of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) using a Cell Death Detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions.

PC-3 and LNCaP prostate carcinoma (5×10⁵) cell lines were seeded in triplicate in 6-well plates, and cultured in RPMI-1640 supplemented with 100 ml/l FBS. After transfection for 48 h, the cells were collected and washed with ice-cold PBS, and fixed in 70% ethanol overnight at 4°C. The fixed cells were pelleted, washed in PBS, resuspended in PBS containing 0.1 mg/ml of propidium iodide and analysed by flow cytometry.

EGR-1 is an early response nuclear factor that is important in the regulation of several genes (Fig. 1). To determine the effect of overexpressing Egr-1 in the expression of EGR-1, p14^ARF and TGF-β1 in PC-3 (Fig. 1A) and LNCaP (Fig. 1B), prostate carcinoma cell lines the cells were treated with FCS 0.5%, FCS 10%, TPA (30 nM) and TNF-α (20 ng/ml). The protein expression of EGR-1, p14^ARF and TGF-β1 was assessed by western blot analysis (Fig. 1).

PC-3 (Fig. 2A) and LNCaP (Fig. 2B) prostate carcinoma cells were transfected with a wild-type Egr-1-cDNA and the expression of EGR-1, TGF-β1 and p14^ARF proteins was assessed by western blot analysis (Fig. 2A and B). We observed a strong induction of Egr-1 and p14^ARF after the treatment of cells with TPA and TNF-α treatment (Fig. 2A and B, lanes 1 and 2). By contrast, TGF-β1 was significantly induced only after TNF-α treatment in PC-3 cells (Fig. 2A, lane 3). The expression of TGF-β1 in LNCaP cells was observed after treatment with TPA and TNF-α treatment (Fig. 2B, lanes 2, 3 and 5). A decrease in the cell protein expression in the PC-3 (Fig. 2A) and LNCaP (Fig. 2B) prostate carcinoma cells was observed following Egr-1 siRNA treatment (Fig. 2A and B, lane 4). Nevertheless, Egr-1-siRNA was unable to reverse the effect of TPA in the induction of TGF-β1 in LNCaP cells (Fig. 2B, lane 5).

To determine whether blocking the expression of p14arf by a siRNA against p14arf affected the expression of EGR-1, p14arf or TGF-β1, PC-3 (Fig. 2C) and LNCaP (Fig. 2D) cells were transfected with a vector carrying a p14arf cDNA. At 72 h after transfection, the cells were treated with TPA (30 mM), TNF-α (20 ng/ml) and siRNA against p14arf and cultured for an additional 12 h. At the indicated time point, the cells were harvested and analyzed for expression of EGR-1, p14^ARF and TGF-β1. The protein expression was assessed by western blot analysis. As shown in Fig. 2C and D, p14arf-siRNA strongly decreased EGR-1 and p14^ARF expression but was unable to reverse the effect of TPA.

We determined whether blocking Egr-1 or p14arf expression exerted an apoptotic effect on PC-3 or LNCaP cells treated with siRNA against the abovementioned proteins. To this effect, LNCaP cells were transfected with siRNA against Egr-1 (Fig. 3A) or against p14arf (Fig. 3B) and with a non-specific siRNA as the control. After culturing at the indicated times, the cells were harvested and analysed for induction of apoptosis. Results of the flow cytometric analysis showed that knocking down Egr-1 using siRNA against Egr-1, significantly induced apoptosis in PC-3 and LNCaP cells (Fig. 3A and B). However, when PC-3 and LNCaP cells were treated with p14arf-siRNA and analysed for induction of apoptosis, the effect was only moderate (Fig. 3C and D) and decreased after a 48-h transfection, suggesting a moderate role for p14arf inhibition in controlling apoptosis. However, unlike Egr-1-siRNA-treated PC-3 cells, the apoptotic activity decreased after 48 h as compared to that observed in Fig. 3A and B. The results suggested that the differential expression of EGR-1 and p53 was responsible for the differences in apoptotic response demonstrated in PC-3 and LNCaP cells (Fig. 3).

Since EGR-1 is a potential tumor inducer for prostate cancer (1) and inhibition of Egr-1 by siRNA-Egr-1 decreases TGF-β1-mediated proliferation (6), we analysed the role of Egr-1 in TGF-β1-induced cell survival in PC-3 and LNCaP cells. Treatment with TGF-β1 (10 ng/ml) increased IAP activity (Fig. 4A and B), an inhibitor of apoptosis that plays a key role in preventing cell death by apoptosis. However, this increase was attenuated by the transfection of siRNA against Egr-1 (Egr-1-siRNA), suggesting a role for Egr-1 in TGF-β1-mediated cell viability.

The aforementioned results showed that inhibition of Egr-1 by siRNA enhanced TGF-β1-mediated cell viability in human prostate cancer cells (Fig. 4A and B). The effect of Egr-1 knockdown on TGF-β1-induced PC-3 and LNCaP cell survival was assessed. TGF-β1 induced obvious cell survival as shown by the decreased DNA fragmentation in the PC-3 and LNCaP cells (Fig. 4C and D) and this decrease was partially attenuated by the knockdown of Egr-1 using Egr-1-siRNA transfection. Inhibition of Egr-1 expression induced obvious cell death as shown by the increased DNA fragmentation (Fig. 4C and D), suggesting a survival role of Egr-1 in prostate cancer progression. Collectively, the results suggested that Egr-1 is involved in TGF-β-mediated prostate cell death, survival and differentiation.

The role of p14^ARF-induced cell death in PC-3 and LNCaP cells was assessed. Treatment with TGF-β1 (10 ng/ml) increased IAP activity (Fig. 5A and B), an inhibitor of apoptosis. However, unlike Egr-1 (Fig. 4A and B), this increase was not attenuated by the transfection of siRNA against p14^ARF (p14arf-siRNA) suggesting a key role of TGF-β1-mediated proliferation in allowing cell viability in human prostate cancer cells (Fig. 5A and B).

The effect of p14arf knockdown on TGF-β1-induced PC-3 and LNCaP cell survival was examined. TGF-β1 induced obvious cell survival as shown by the decreased DNA fragmentation in the PC-3 and LNCaP cells (Fig. 5C and D). This decrease was not attenuated by the knockdown of p14arf using p14arf-siRNA transfection. Inhibition of p14arf expression induced obvious cell survival as shown by the decreased DNA fragmentation (Fig. 5C and D), suggesting a cell death role for p14arf in prostate cancer. Collectively, our results suggested that Egr-1 plays a role in TGF-β1 and p14arf-mediated prostate cell death, survival and differentiation.