The antibodies used in this study included polyclonal rabbit anti-human MAOA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal rabbit anti-human anti-mTOR (Cell Signaling Technology, Boston, MA, USA), polyclonal rabbit anti-human HIF-1α (Bioworld, St. Louis Park, MN, USA), monoclonal mouse anti-human E-cadherin (Cell Signaling Technology), monoclonal mouse anti-human vimentin (Cell Signaling Technology), monoclonal mouse anti-human α-SMA (Sigma-Aldrich, St. Louis, MO, USA), monoclonal mouse anti-human cytokeratin (Sigma-Aldrich), and monoclonal mouse anti-human β-actin (Santa Cruz Biotechnology). 2′,7′-Dihydrochlorofluorescein diacetate (H₂DCF-DA) was purchased from Molecular Probes (Carlsbad, CA, USA). Curcumin was purchased from Dolcas Biotech LLC (Landing, NJ, USA).

PC3 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human prostate CAFs were isolated from surgically explanted cancer regions of patients with PCa (Gleason 4+5) (30). Four different surgical explants were used for CAF isolation. The study protocol was approved by the relevant ethics committee of the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, China, and the patients provided written informed consent. PC3 cells and fibroblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10 U/ml penicillin, and 10 mg/ml streptomycin. CAFs were used for 15 passages without significant modification of their ability to elicit EMT. Fresh serum-free medium was added for an additional 24 h before collection of conditioned medium (CM) in order to obtain CM free from CAFs. PC3 cells were then incubated with the obtained CM for 72 h and used for the analyses.

PC3 cells (1×10⁶) grown under our experimental conditions were lysed for 20 min on ice in 300 μl radioimmunoprecipitation assay (RIPA) lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenyl-methanesulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin]. Total proteins (100 μg) were loaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels, separated, and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Penzberg, Germany). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 [TBST; 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween-20] and were subsequently incubated with primary antibodies overnight at 4°C. After five washes of 10 min each in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000, Cell Signaling Technology) for 2 h and subsequently washed again. The peroxidase reaction was performed using an enhanced chemiluminescence detection system to visualize the immunoreactive bands.

A chamber-based invasion assay (Millipore, Billerica, MA, USA) was performed to evaluate breast cancer cell invasion. Briefly, the upper surface of the membrane was coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). PC3 cells (1×10⁵) were resuspended in the upper chamber in serum-free medium and allowed to migrate toward a serum gradient (10%) in the lower chamber for 24 h. The media was aspirated from inside the insert, and the non-invasive cells on the upper side were removed by a cotton swab. The membrane was fixed with 4% paraformaldehyde and stained with crystal violet. The number of migrated cells was counted on each membrane in 10 random fields and photographed at x100 magnification. The values reported here are the averages of triplicate experiments.

Total RNAs were extracted from PC3 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed using the PrimeScript RT Reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. Real-time experiments were carried out using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and a SYBR Green PCR kit (Takara). The following PCR program was used: denaturation at 95°C for 30 sec, followed by 40 cycles consisting of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. A melting curve analysis was applied to assess the specificity of the amplified PCR products. The PCR primer sequences for HIF-1α was 5′-AAGTCTAGGGATGCAGCA-3′ (forward) and 5′-CAAGATCACCAGCATCATG-3′ (reverse). The PCR primer sequences for MAOA was 5′-CTGATCGACTTGCTAAGCTAC-3′ (forward) and 5′-ATGCACTGGATGTAAAGCTTC-3′ (reverse). The PCR primer sequences for mTOR was 5′-CTGGGACTCAAATGTGTGCAGTTC-3′ (forward) and 5′-GAACAATAGGGTGAATGATCCGGG-3′ (reverse). The PCR primer sequences for CXCR4 was 5′-TCTGTGACCGCTTCTACC-3′ (forward) and 5′-AGGATGAGGATGACTGTGG-3′ (reverse). The PCR primer sequences for IL-6 receptor was 5′-CATTGCCATTGTTCTGAGGTTC-3′ (forward) and 5′-AGTAGTCTGTATTGCTGATGTC-3′ (reverse). The PCR primer sequences for β-actin was 5′-TTGTTACAGGAAGTCCCTTGCC-3′ (forward) and 5′-ATGCTATCACCTCCCCTGTGTG-3′ (reverse). The amount of each target gene was quantified by the comparative C_T method using β-actin as the normalization control (31).

Intracellular production of H₂O₂ was assayed as previously described (32). Five minutes before the end of the incubation, the cells were treated with 5 μg/ml 2′-7′-dichlorofluorescein diacetate (DCF-DA). After washing with phosphate-buffered saline (PBS), the cells were lysed in 1 ml RIPA buffer and analyzed immediately by fluorimetric analysis at 510 nm. The data were normalized to total protein content.

After the designated treatment, CAFs were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.5% Triton X-100 for 10 min, and blocked in 1% bovine serum albumin (BSA) for 1 h. Fixed cells were then incubated with mouse anti-human-α-SMA antibodies (1:100) or mouse anti-human-cytokeratin antibodies (1:100) at 4°C overnight. Cells were washed and incubated with goat anti-mouse dylight 594 (red) IgG antibody (Qenshare Biological Inc., Xi'an, China) at 1:200 dilution for 60 min. Nuclei were stained with DAPI for 5 min. The cells were visualized using a fluorescent microscope (Observer A1, Carl Zeiss Microscopy GmbH, Jena, Germany) using appropriate excitation and emission filters at x400 magnification.

shRNA against MAOA (Santa Cruz Biotechnology, sc-35847-SH), shRNA against mTOR (Santa Cruz Biotechnology, sc-35409-SH), shRNA against HIF-1α (Santa Cruz Biotechnology, sc-35561-SH), and a negative control shRNA (Santa Cruz Biotechnology, sc-108060) were obtained from GenePharm (Shanghai, China). PC3 cells (2×10⁵ per well) were seeded in 6-well plates and transfected with shRNA. Silencing with shRNA was performed with Lipofectamine (Invitrogen) following the manufacturer's instructions. Silenced cells were selected by puromycin treatment, and a pool of stably transfected cells was generated in order to avoid clonal selection.

The data are presented as the mean ± standard deviations (SD) values obtained from at least three independent experiments. Statistical analysis of the data was performed by Student's t-tests using SPSS software (version 13.0; SPSS, Chicago, IL, USA). P-values <0.05 were considered statistically significant.

To assess the role of MAOA/mTOR/HIF-1α signaling elicited by CAFs in the EMT of cells, we used PC3 cells, a line isolated from a bone metastasis of human PCa and CAFs. CAFs were obtained from the surgically explanted prostate of patients with PCa (Gleason 4+5). Expression of E-cadherin and cytokeratin was assessed to exclude epithelial contamination of CAFs (Fig. 1A).

In order to study the molecular pathways driving the CAF-induced EMT response in PC3 cells, we analyzed the MAOA/mTOR/HIF-1α pathway in PC3 cells treated with CM from CAFs. PC3 cells exhibited CAF-induced EMT with activation of the MAOA/mTOR pathway. In addition, CAF exposure led to activation of HIF-1α (Fig. 1B), a known transcription factor normally induced by hypoxia but already reported to be activated in normoxia by inflammation in an mTOR-dependent manner (23). Silencing of MAOA, mTOR, or HIF-1α in PC3 cells by shRNA (Fig. 1C) after exposure to CAFs indicated that this signaling pathway proceeds from MAOA to mTOR and then transduction to HIF-1α (Fig. 2A). MAOA, mTOR, or HIF-1α knockdown in PC3 cells after exposure to CAFs revealed that this MAOA/mTOR/HIF-1α axis plays a mandatory role in orchestrating CAF-induced EMT in PC3 cells. Indeed, silencing of MAOA, mTOR, or HIF-1α individually reduced the invasiveness and expression of EMT markers (E-cadherin and vimentin) among PC3 cells (Fig. 2B–D).

Inflammation and oxidative stress have already been linked to cancer progression and claimed to be responsible for enhanced malignancy (33,34). Oxidative stress, due to deregulated intracellular ROS production, has been correlated to PCa carcinogenesis, androgen resistance, anchorage-independence, and resistance to anoikis (35,36). We observed that exposure to CAFs led to an increase in the ROS content of PC3 cells (Fig. 3A). ROS production appears to be mainly due to MAOA/mTOR/HIF-1α signaling, as indicated by the ability of MAOA, mTOR, or HIF-1α knockdown to block CAF-induced ROS generation and the invasive phenotype (Figs. 2C–D and 3A).

Moreover, culture in CM from CAFs resulted in a significant increase in CXCR4 and IL-6 receptor expression in PC3 cells (Fig. 3B and C). However, silencing of MAOA, mTOR, or HIF-1α by shRNA in PC3 cells abrogated the elevated CXCR4 and IL-6 receptor expression induced by CAF-derived CM (Fig. 3B and C), which indicates an important role for MAOA/mTOR/HIF-1α signaling in PC3 cells with regard to the regulation of chemotactic and inflammation responses to CAF-derived CM.

Curcumin has been found to possess anticancer activities based on its effect on a variety of biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigenesis, and metastasis; that is, the modulation of several important molecular targets including transcription factors (NF-κB, AP-1, β-catenin), enzymes (e.g., COX-2, MMPs), proinflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6), and cell surface adhesion molecules (e.g., cadherins, integrins) (37–39). Here, we wanted to investigate whether curcumin has a protective effect in prostate cancer via modulation of MAOA/mTOR/HIF-1α signaling.

We observed that 25 μM curcumin [a concentration selected according to a previous study (40)] was able to abolish the EMT response to CAF-derived CM in PC3 cells as revealed by increased E-caderin levels and decreased vimentin levels (Fig. 4A). However, it could not inhibit the EMT response to CAF-derived CM in PC3 cells when MAOA, mTOR, or HIF-1α was silenced by shRNA in PC3 cells (Fig. 4B). Similar results were obtained for the invasiveness of PC3 cells. Curcumin abrogated the invasiveness of PC3 cells induced by CAF-derived CM (Fig. 4C). However, it was not able to suppress the invasiveness of PC3 cells induced by CAF-derived CM when MAOA, mTOR, or HIF-1α was silenced by shRNA in PC3 cells (Fig. 4D).

Curcumin has been shown to have antioxidant activity and anti-inflammatory activity. Here, we aimed to evaluate the effect of curcumin on CAF-induced ROS production and CXCR4 and IL-6 receptor expression in PC3 cells. We found that curcumin abolished the CAF-derived CM-induced ROS production and CXCR4 and IL-6 receptor expression in PC3 cells (Fig. 5). However, it could not inhibit the CAFs in CM-induced ROS production and CXCR4 and IL-6 receptor expression in PC3 cells when MAOA, mTOR, or HIF-1α was silenced by shRNA in PC3 cells (Fig. 5), which indicates that curcumin inhibits CAF-induced ROS production and CXCR4 and IL-6 receptor expression in PC3 cells by impeding MAOA/mTOR/HIF-1α signaling.