RPMI-1640 and Ham’s F12 media for cell culture were supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Life Technologies (Carlsbad, CA, USA), respectively. Fetal bovine serum (FBS) was obtained from Nichirei Biosciences, Inc. (Tokyo, Japan). Monoclonal antibody against human β-actin and polyclonal rabbit antibodies against human ObR, and phosphorylated forkhead box O1 (FOXO1) (Ser256) were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan) and Abcam (Tokyo, Japan), respectively. LY294002, used as an PI3K inhibitor, and polyclonal rabbit antibodies against phosphorylated Akt, Akt, cyclin D1, p21Cip1 and lamin A/C were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Polyclonal rabbit antibody against FOXO1 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA USA).

The AS human PCa cell line LNCaP and the AI human PCa cell lines DU145 and PC-3 used in this study were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). LNCaP and DU145 cells were maintained in RPMI-1640 medium and PC-3 cells were maintained in Ham’s F-12 medium. Media were supplemented with 10% heat-inactivated FBS in the presence or absence of human recombinant leptin (Sigma Aldrich) at the indicated concentrations, in a humidified incubator at 37°C and 5% CO₂. The medium was changed every 4 days. Cells were passaged when they were 50–70% confluent. Cells were transferred into serum-free medium and cultured for 24 h under the same conditions except for the absence of serum.

Cells were lysed with ice-cold lysis buffer and protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). Samples were centrifuged at 12,000 × g for 10 min at 4°C, and supernatants were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the membranes were probed with primary antibodies overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK) for 1 h at room temperature. The immune complexes were visualized with the Enhanced Chemiluminescence Plus detection system (GE Healthcare) according to the manufacturer’s instructions.

For small interfering RNA (siRNA) experiments, siRNA cocktails were obtained from, and functionally annotated by Cosmo Bio Co., Ltd. (Tokyo, Japan) and Bioneer, Inc. (Alameda, CA, USA). The siRNA cocktails are indicated in Table I. For the control, non-targeting siRNA (cat. no. 4390844) was obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cells were transiently transfected with each siRNA or control siRNA cocktail using the Neon Transfection System (Life Technologies). After 48-h incubation, cells were utilized for each assay.

The relative effect of leptin on the proliferation of each cell line was assessed using WST-8 assays and a Cell Counting kit-8 (Dojindo, Kumamoto, Japan). Cells that had been cultured in the presence or absence of graded concentrations of leptin for each day were re-seeded in 96-well plates at a density of 3×10³ cells/well and were cultured in the same graded concentrations of leptin in at least three replicate wells at 37°C for 48 h. The WST-8 reagent was added to each well, and the cells were further incubated for another 3 h. Absorbance was determined using a spectrophotometer (Thermo Scientific Multiskan FC; Thermo Fisher Scientific, Inc.) at 450 nm. Absorbance values were expressed as percentages relative to untreated controls.

The cell invasion assay was performed using a Matrigel Invasion Chamber from Becton-Dickinson (BD Biosciences, Tokyo, Japan) according to the manufacturer’s instructions. Approximately 3×10⁴ cells that had been cultured in the presence or absence of 100 ng/ml of leptin for 28 days were seeded onto the top of chamber and were incubated at 37°C in a 5% CO₂ incubator. After 48-h incubation, the media were aspirated, and the cells remaining on the upper surface of the membrane and the Matrigel Matrix were removed with a sterile cotton swab. Invasive cells on the bottom surface of the membrane that had passed through the Matrigel were fixed in methanol and stained with Diff-Quik stain (Sysmex Corp., Hy go, Japan). The number of invaded cells on four representative sections of each membrane was counted in high-power fields under a light microscope (BX63; Olympus Corp., Tokyo, Japan). Percent invasion, which was the percent of the seeded cells that had passed through the Matrigel, was calculated. The assay was repeated at least three times.

Cells that had been cultured in the presence or absence of 100 ng/ml of leptin for 28 days were seeded into 6-well plates and cultured in at least three replicate wells at 37°C. When cells were grown to 90% or above confluence, a crossing linear scratch was made with a 200-μl pipette tip, and floating cells were removed by washing with phosphate-buffered saline (PBS). These plates were photographed at the identical location both before and 18 h after incubation. The average area of the open scratch area was calculated using ImageJ ver 1.46r (NIH, Bethesda, MD, USA).

Approximately 1×10⁴ cells were seeded in a Lab-Tek II Chamber Slide (Thermo Fisher Scientific, Inc.). Cells were then further cultured in serum-free media for 48 h with the same leptin concentration. For PI3K inhibition, cells were pre-treated with LY294002 for 2 h. The cells were then fixed with 4% paraformaldehyde and permeabilized with PBS containing 0.2% Triton X-100 followed by blocking with 1% bovine serum albumin. Thereafter, the cells were incubated with polyclonal anti-FOXO1 antibody followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Labs, West Grove, PA, USA). Trihydrochloride (Hoechst 33342; Dojindo) staining was used for nuclear detection. Fluorescence was photographed using an Olympus IX71 microscope (Olympus Corp.).

Subcellular fractionation was carried out using Nuclear/Cytosol Fractionation kits (BioVision, Inc., Milpitas, CA, USA) as described by the manufacturer. Equal volumes of each fraction were loaded onto an SDS-polyacrylamide gel, and western blotting was performed as previously described.

All values are expressed as the means ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using Student’s unpaired t-test. P<0.05 was considered statistically significant.

To clarify the effect of leptin on PCa cell proliferation, LNCaP, DU145 and PC-3 were treated with various concentrations of leptin for 48 h. Fig. 1A–C shows only results derived from LNCaP cells, because the similar tendency was found in their experiments using three PCa cells examined. The incubation for 48 h did not affect the growth (Fig. 1A). Because short-term leptin exposure may not reflect the in vivo situation, we tried to examine the effects of long-term leptin exposure. Interestingly, time-course experiments showed that cell growth reached maximum after 28-day incubation with 100 ng/ml leptin (Fig. 1B). The most effective concentration of leptin was at 100 ng/ml (Fig. 1C), thus, 100 ng/ml was selected as the leptin concetration for further experiments. Maximum physiological concentration of serum leptin in human is ~100 ng/ml (10). These results indicated that treatment with 100 ng/ml leptin for 28 days might be the relevant condition for induction of proliferation of all PCa cells. We tested the effect of long-term leptin treatment (1 month) on the ability of PCa cells to invade through Matrigel. As shown in Fig. 1D, PCa cells that were treated long-term with leptin showed significantly higher transit to the bottom surface of a Matrigel-coated membrane than non-leptin-treated cells. In addition, the effect of long-term leptin treatment on the migration ability of PCa cells was estimated using a scratch wound-healing migration assay. As shown in Fig. 1E, PCa cells treated long term with leptin showed a significantly larger healed area after scratching compared to non-treated cells. Thus, long-term exposure to leptin enhanced the malignant potential of PCa cell lines.

Next, the effect of long-term leptin treatment on leptin-induced intracellular signaling pathways in PCa cells was investigated. We first determined if the ObR and intracellular Akt phosphorylation played a role in the enhanced proliferation observed in long-term leptin-treated cells. Akt phosphorylation was assayed because several groups have previously demonstrated that leptin enhances cell proliferation via the PI3K/Akt signaling pathway. Expression of the ObR, as assessed by western blotting, was significantly increased by long-term leptin treatment of all PCa cell lines, compared to non-treated cells, as shown in Fig. 2A. Furthermore, we confirmed that the level of Akt phosphorylation increased in all PCa cell lines that were treated with leptin for 1 month compared to non-treated cells.

To confirm that ObR signaling played a role in leptin-induced proliferation of PCa cells, ObR specific siRNA was transfected into PCa cells that had been exposed to leptin for 1 month. After 48-h transfection, ObR protein expression was markedly decreased in comparison to cells transfected with scrambled non-specific siRNA (Fig. 2B). The silencing of ObR significantly decreased the proliferation of all PCa cell lines compared to control siRNA cells. This result suggests that the ObR receptor mediates leptin effects on proliferation.

To further analyze the role of Akt activation in leptin- enhanced PCa cell proliferation, we used LY294002 to inhibit Akt activation. Following long-term leptin culture, LY294002 or control dimethyl sulfoxide (DMSO) was added for 2 h. And cells were incubated in serum-free media for 48 h. As shown in Fig. 2C, inhibition of Akt phosphorylation with LY294002 decreased cell proliferation in the absence of leptin and further suppressed leptin-induced proliferation in LNCaP cells. We observed similar results in DU145 and PC-3 (data not shown). These results suggest that the PI3K/Akt signaling pathway is important for leptin-induced cell proliferation.

The PI3K/Akt signaling pathway has been reported to phosphorylate FOXO1, which mediates some of its downstream signaling. To determine if FOXO1 was involved in leptin-mediated cellular effects, we first determined whether long-term exposure to leptin induces phosphorylation of FOXO1 using western blotting. As shown in Fig. 3A, long-term exposure to leptin induced a higher level of FOXO1 phosphorylation than that of non-treated cells. We further assayed if this phosphorylation is mediated by PI3K/Akt by assaying the effect of adding the specific PI3K inhibitor LY294002 (25 μM) for the last 2 h of long-term leptin culture of PCa cells on FOXO1 phosphorylation. Following LY294002 treatment FOXO1 phosphorylation induced by leptin treatment was greatly reduced compared to control cells (Fig. 3A). These results suggested that long-term exposure to leptin induced phosphorylation of FOXO1 via the PI3K/Akt signaling pathway.

To clarify the role of FOXO1 in the proliferation of these PCa cell lines, we transiently transfected leptin-untreated cells with FOXO1 siRNA. After 48-h transfection, FOXO1 protein expression was decreased in comparison to cells transfected with scrambled non-specific siRNA (Fig. 3B). FOXO1 gene silencing significantly increased the proliferation of these cells compared to control cells (Fig. 3B). These results indicated that FOXO1 functions to suppress PCa cell proliferation.

One possible mechanism by which Akt-mediated phosphorylation of FOXO1 might mediate FOXO1 effects on leptin-induced proliferation is by regulation of FOXO1 cellular localization. Thus, if FOXO1 is phosphorylated on specific residues by Akt, FOXO1 can be excluded from the nucleus, thereby inactivating its transcriptional activity. To confirm the effect of long-term exposure to leptin on the intracellular localization of FOXO1, immunocytochemistry was performed. As shown in Fig. 4A, FOXO1 was expressed in both the nucleus and the cytoplasm in DU145 cells. In contrast, FOXO1 expression in the nucleus was strongly decreased by long-term leptin treatment. The addition of LY294002 to the medium with leptin restored the intra-nuclear expression of FOXO1 protein. We observed similar results in LNCaP and PC-3 cells (data not shown). To further confirm the intracellular distribution of the FOXO1 protein, we compared FOXO1 expression in isolated nuclear fractions of leptin-treated and non-treated cells by western blotting. Fig. 4B shows that FOXO1 expression in the nuclei of leptin-treated cells was significantly decreased to ~50% of that in non-treated cells.

As described above, long-term exposure of leptin-induced PCa cell proliferation resulted from FOXO1 exclusion out of a nuclear region. We therefore next determined if the nuclear exclusion of FOXO1 that resulted from long-term exposure to leptin might affect the expression of cell cycle-related proteins. Increased cyclin D1 expression and decreased p21 expression was shown by western blotting of cells following long-term leptin treatment compared to non-treated cells (Fig. 5), suggesting that genes that regulate the G1/S transition were modulated towards cell cycle progression by leptin treatment.