Naringenin modulates the metastasis of human prostate cancer cells by down regulating the matrix metalloproteinases -2/-9 via ROS/ERK1/2 pathways

Metastasis is a multifactorial condition that complicates cancer treatment options and widens the target of treatment. Matrix mettalopriteinases (MMPs) of the extracellular matrix (ECM) are involved in metastasis, thus they present as potential targets in halting cancer metastasis. The study was undertaken to investigate the influence of naringenin, a naturally occurring flavonoid on the metastasis of human prostate cancer cells (PC-3 and DU145). Naringenin was observed to be effective in reducing the viability and migratory percentage of PC-3 and DU145 cells. Naringenin significantly reduced the expression and activities of the chief MMPs (MMP-2 and MMP-9) as assessed by western blotting, real-time PCR and gelatin zymography analysis. The influence of naringenin on extracellular signal-regulated kinase (ERK) -ERK1/2 was analysed by western blotting. The results indicated that naringenin was able to effectively inhibit ERK1/2. Naringenin exposure also significantly suppressed the levels of reactive oxygen species (ROS). Naringenin thus stands as an effective chemotherapeutic agent for prostate cancer treatment that could be further explored.

In recent years much attention has been focussed on the natural products derived from plants as a source for the discovery of novel cancer chemopreventive agents (Surh, 2003;Weng and Yen, 2012). Flavonoids are polyphenolic compounds that are found in fruits and vegetables (Nijveldt et al., 2001). Flavonoids are found to possess cardioprotective effects (Mink et al., 2007). Previous reports have demonstrated that dietary flavonoids inhibit the development of various human cancers, such as breast cancer (Bosetti et al., 2005) and colorectal cancer (Rossi et al., 2006). Naringenin, a plant bioflavonoid is present in fruits like oranges, grape fruits and tomatoes. It has been reported to possess anti-estrogenic activity (Ruh et al., 1995) antioxidant (Cheng and Breen, 2000;Cavia-Saiz et al., 2010) hepatoprotective (Hermenean et al., 2014), anticancer (Borradaile et al., 1999;Manthey et al., 2001), anti-inflammatory (Manthey et al., 2001) and hypolipidemic properties (Mulvihill et al., 2010). Naringenin with its health benefits presents a potent therapeutic that could be further explored. The present study was undertaken to investigate the effect of naringenin on metastasis of prostate cancer cells by evaluating the expression of MMPs-2/-9 and ERK1/2.

Materials and Methods
Reagents and antibodies: Naringenin (Sigma Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. The concentrations used in this study were 10, 20, 30, 40 and 50 μM. The controls were treated with the same amount of DMSO as used in the corresponding experiments. Antibodies of MMP-2, MMP-9, GAPDH, and p-ERK 1/2 were obtained from Epitomics Inc. (Burlingame, CA, USA). All other reagents used in the study were purchased from Sigma-Aldrich (St. Loius, MO, USA).
Cell viability assay: The prostate cancer cells (PC-3 and DU145) were seeded in a 96-well titer plate (5x10 3 cells/ well). After 24 hpurs incubation at 37°C in the presence of 5%CO2, various concentrations of naringenin (10 -50 μM) were added and the cells were incubated for 72 h at 37°C. Cell growth was assessed by MTT colorimetric assay (Limtrakul et al., 2004). The cytotoxicity assessment was performed in triplicates.

Measurement of ROS generation:
The levels of ROS generated on exposure to naringenin were assessed by flow cytometric analysis. After incubation with naringenin at different concentrations (10 or 50 μM), the cells (PC-3 and DU145) were harvested, washed with PBS and suspended in RPMI 1640 medium containing 10 μM 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 20 min. After incubation, the cells were washed again with PBS and ROS generation was measured using flow cytometer (Becton-Dickinson, San Jose, CA, USA).
Analysis of MMP-2 and MMP-9 activities by gelatin zymography: The activities of MMP-2/-9 were measured after exposure to naringenin (10 or 50 μM). PC-3 and DU145 cells were (5 x 10 5 cells/well) plated in 12-well tissue culture plates and incubated in serum-free RPMI 1640 medium with or without naringenin for 48 h. The conditioned medium was then collected and used for gelatin zymography. The samples were subjected to electrophoresis in 10%SDS-polyacrylamide gel containing 0.1%gelatin at 100 V for 3 hours at 4°C. After electrophoresis, the gels were rinsed with 2.5% Triton X -100 to remove SDS at 4°C. The gels were incubated overnight in activation buffer (40 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 0.02%NaN3) at 37°C followed by staining with 0.5% Coomassie blue R-250 for 60 min and destained with destaining solution (20% methanol, 10% acetic acid, 70% H2O) until the clear bands were visualized and the band density was quantified by using NIH ImageJ software (NIH, MD, USA).
Western blot analysis: Western blot analysis for MMP-2/-9 expression was performed. PC-3 and DU145 cells incubated with naringenin (10 or 50 μM) for 48 hours were collected. The harvested cells were suspended in 50 mM potassium phosphate buffer (pH 7.4) containing EDTA (2 mM) and 0.1%Triton X-100. The cells were sonicated and the homogenates were centrifuged for 10 min at 13000 x g. The total protein in the supernatant were extracted and separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, San Francisco, CA, USA). The membranes were blocked with blocking solution (1%BSA in Tris-buffered saline Tween 20 (TBST)) and then incubated overnight with primary antibodies for MMP-2, MMP-9, GAPDH, p-ERK1/2. After hybridisation with the primary antibody, the membrane was washed thrice with TBST. The membrane was then incubated with secondary antibody for 1 hour and washed with TBST. The final detection was done using the chemiluminescence western blotting reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Real-time PCR -MMP-2/-9: DU145 and PC-3 cells (1 x 10 6 cells/well) were incubated without and with naringenin (10 or 50 μM) for 24 hours. After treatment, the cells were harvested and total RNA was extracted from each treatment by using the Qiagen RNeasy mini kit (Qiagen Inc., Valencia, CA, USA) as previously described (Lu et al., 2010;Chiang et al., 2011). RNA samples were reverse-transcribed using primers for MMP-2 and MMP-9. Primer sequences (5' to 3') for MMP-2 were CTTCCAAGTCTGGAGCGATGT (forward) and TACCGTCAAAGGGGTATCCAT (reverse). GGGACGCAGACATCGTCATC (forward) and TCGTCATCGTCGAAATGGGC (reverse) were the primers for MMP-9. Reverse transcription was performed using Omniscript reverse transcriptase using Taq PCR master mix kit (Qiagen Inc., Valencia, CA, USA). The PCR reaction was initiated by using 20 pmol of primers. Initial denaturation at 94°C for 3 min was followed by 30 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min and the final extension was at 72°C for 10 min. The amplified PCR products were separated on 0.8%agarose gel. The gel was stained using ethidium bromide and the bands were visualised on a UV transilluminator and the band intensities were measured using Image J software (NIH, MD, USA) In vitro migration assay: PC-3 and DU145 cells were plated at a density of 3.0 × 10 5 cells in 6-well plates and grown for 24 hours, where the cells reach about 90% confluence. The cell monolayer was carefully disturbed by scrapping out a small portion with a sterile 10 μL plastic pipette tip so as to create a scratch. Debris was removed from the culture. The scraped portion was noted and cell migration into the denuded areas was assessed using a phase contrast microscope after 24 hours treatment with naringenin at (10 or 50 μM). The cell migration rate was determined using the formula: migration rate = (average wound distance -average no migration distance) / average wound distance × 100%.
Statistical analysis: Data were expressed as mean ± SD from at least three independent experiments. One-way analysis of variance (ANOVA) and Student's t test were used for statistical analysis. A value of p<0.05 was considered statistically significant. The statistical analyses were performed using SPSS 10.0 software.

Results
The anti-proliferative effect of naringenin on prostate cancer cells (PC-3 and DU145) was assessed by MTT assay. With increasing concentration of naringenin, from 10 μM to 50 μM (Figure 1), the cell survival percentage was found to decrease.
At 10 μM the cell survival percentage was observed to be 88.35 % in PC-3 cells and 81.64 % in DU145 cells. On exposure to 50 μM naringenin the survival percentage decreased almost 3 fold in PC-3 and DU145 cells. The highest concentration of naringenin was most effective in inhibiting survival of prostate cancer cells.
MMPs are crucial in cancer metastasis. They are involved in ECM degradation, which is required for cell invasion. The effect of naringenin on the expression and activation of MMPs namely, MMP-2 and MMP-9 were investigated in this study. PC-3 and DU145 prostate cancer cells were exposed to various concentrations of naringenin (10 or 50 μM). As shown in Figure 2 the results of western blotting revealed that naringenin treatment significantly decreased MMP-2 and MMP-9 expression in a dose-dependent manner. GAPDH expression served as a loading control. Treatment with 10 and 50 μM of naringenin for 48 h showed that the relative MMP-2 and MMP-9 protein expression levels in PC-3 cells decreased. We also observed the similar expression pattern of MMP-2 and MMP-9 in DU145 cells on naringenin treatment.
The enzyme activities of MMP-9 and MMP-2 were observed to be markedly (p<0.05) reduced as analyzed by gelatin zymography (Figure 3). Reduction in the activities of MMPs was found to be dose dependent. Exposure to 50 μM naringenin evidenced multifold decrease in the activities of MMPs as compared to the control cells, which were not exposed to naringenin.
Expression of MMP-2 and MMP-9 mRNAs was assessed by real-time PCR with GADPH as standard (Figure 4). The expression of MMP-2 mRNA dropped to 74.5% at 10 μM in PC-3 cells and 76.3% in DU145 cells as compared to untreated cells. Naringenin at 50 μM was more effective in inhibiting mRNA expression of MMPs as evidenced in the study. The expression of MMP-2 mRNA dropped 36.1% in PC-3 cells and 31.1% in DU145 cells as against control.
These findings suggest that down-regulation of MMP-2 and MMP-9 enzyme activities and expressions might be involved in the inhibition of invasion and migration of the prostate cancer cells. The mitogen-activated protein kinase (MAPK) family are considered to be critical regulators of the various pathways that are involved in cell proliferation and migration (Roberts and Der, 2007). The effect of naringenin on ERK1/2 activation was measured by western blotting (Figure 2). The results observed suggest that naringenin exposure inhibited ERK1/2, in a dosedependent manner in both PC-3 and DU145 cells, thus indicating that the decrease in the expression of MMP-2 and MMP-9 could be via ERK1/2. The level of ROS generation was measured by flow cytometry. Cell-permeable dye DCFH-DA, which is specifically cleaved to emit a fluorescence wave length in the presence of ROS was employed. Treatment with naringenin suppressed the generation of ROS considerably in a dose-dependent manner, corresponding well to the phosphorylated ERK expression (Figure 5).
The effect of naringenin on migration of PC-3 and DU145 cells was studied by means of a wound closure assay. The cells were incubated with different  Figure 6 suggests that naringenin suppressed migration of PC-3 and DU145 cells to the denuded zone in a dosedependent manner. The ratio of migration was reduced from 65.1% to 28.5% in PC-3 cells and from 64.4 to 25.2% in DU145 cells, respectively, when cells were incubated with naringenin at 10 and 50 μM for a 24 hours treatment. These results revealed that naringenin was effectively inhibited the motility of PC-3 and DU145 cells in a dose-dependently.

Discussion
Metastasis and invasion are central characteristics of malignant cells. Proteolysis of ECM is important for cell protrusion. The proteolytic degradation of ECM, a major event in metastasis is mediated by extracellular proteases such as MMPs. MMPs play an important role in various pathologic processes such as angiogenesis and tumor invasion (Kahari and Saarialho-Kere, 1999;Chen et al., 2011). The chief MMPs that are involved in metastasis, MMP-2 and MMP-9 play a crucial role in prostate cancer progression. The intensity of expression of MMP-2 and MMP-9 is associated with the degree of prostate cancer progression (Stearns and Stearns, 1995;Nemeth, 2002). Thus blocking tumor cell expression/ inhibition of MMP-2/-9 could possibly suppress tumour invasion and metastasis of prostate cancer (Chien et al., 2010;Kong et al., 2007). Various plants and plant extracts have been explored widely in the treatment of cancer. Our study aimed to evaluate the efficiency of naringenin, a flavonoid on the expression of MMP-2/-9 in the metastasis of human prostate cancer cells, PC-3 and DU145 and the possible mechanism underlying the effect by evaluating the activation of ERK1/2.
The results of the cell viability analysis suggest that, naringenin at various concentrations on PC-3 and DU145 cells was able to effectively reduce the viability in a dose dependent manner. The results suggest the anti-proliferative activity of naringenin. The migration of cells over the wound area was measured as indicative of the migratory capacity of the cancer cells. The marked decrease in the migration of the cells to the wounded area on exposure to naringenin suggests the efficacy of the compound in suppressing invasion of tumor.
The process of invasion and metastasis has been linked with the expression of MMPs. The expression and the activities of MMP-2 and 9 upon exposure to naringenin at 10 and 50 μM for 48 hours suggests that naringenin significantly lowers the enzyme activities as evidenced by gelatin zymography. It is known that the activities of MMP-2 and MMP-9 are involved in degradation of extracellular matrix and play important roles in cancer cell migration and invasion (Zhang et al., 2004;Bjornland et al., 2005). Our results demonstrated that anti-metastastic effects of naringenin could possibly be associated with the inhibition of enzymatically degradative processes of metastasis in PC-3 and DU-145 cells. The results obtained were similar to Liu et al. (2011).
The levels of MMP-2 and MMP-9 mRNA and the relative expression were analysed by RT-PCR and by western blotting. In line with the results of the gelatin zymography, the expression patterns of MMP -2/-9 and the mRNA levels were significantly reduced in the PC-3 and DU145 cells on naringenin exposure. Previous Data are presented as mean ± SD of three independent experiments studies with phytochemicals, gallic acid (Liu et al., 2011) and magnolol (Hwang and Park, 2010) were found to effectively inhibit MMP-2 and MMP-9 expression.
MAPKs have been implicated in regulating the proteolytic enzymes that degrade the ECM. Numerous studies have reported that the expression of MMPs-MMP-2 andMMP-9 to be critically mediated by MAPK members and the AKT/ mTOR pathway (Kim et al., 2001;Shukla et al., 2007;Fang et al., 2013). MAPK pathways also play a vital role in tumor development and progression (Steelman et al., 2011). In metastatic cells the activity of ERK was observed to be higher than non-metastatic cancer cells. In tumors, the continued activation of MAPK could lead to enhanced induction of the proteolytic enzymes in the ECM, leading to the degradation which underlines a key event in metastasis and is a histological marker of invasive carcinoma. The role of ERK1/2 in cell invasiveness has been elucidated in many recent evidences. Our study investigated the influence of naringenin on ERK1/2 activation and also the possible role of ERK1/2 in modulating MMP-2 and MMP-9 expression.
Naringenin at 50 µM was found to inhibit ERK1/2 more effectively as compared to the lower concentration in PC-3 and DU145 cells. The observed results thus suggest that there might be a direct correlation between the activity of ERK1/2 and MMP -2 and MMP-9 activities.
ROS are important signalling molecules that mediate a wide range of cellular responses, including apoptosis and metastasis (Lu et al., 2005). ROS have been shown to enhance ERK activity (Zhou et al., 2008). In the study, naringenin was observed to inhibit ROS levels thus suggesting the capacity of naringenin in counteracting ROS and thereby exhibiting the antioxidant efficacy. Previous reports have demonstrated the antioxidant capacities of various dietary flavonoids. Kaempferol has been shown to have diverse biological and pharmacological effects such as antioxidant activity and anticancer-related properties (Huang et al., 2014;Kang et al., 2010;Chen et al., 2013).
Kaempferol has been reported to reduce the expression of MMP-2 and MMP-9 and inhibit ERK1/2 in cancer cells . Gallic acid was also reported to inhibit the levels of ERK1/2, the key molecules of the ERK signaling pathway that has been shown to promote tumor invasion and metastasis (Liu et al., 2011). Diosgenin, a naturally occurring steroidal saponin present in a variety of plants including fenugreek (Trigonella foenum graecum) and roots of wild yam (Dioscorea villosa) was observed to inhibit metastasis by inhibiting MMPs and ERK1/2 (Chen et al., 2011).
Thus, the results of the study suggest that naringenin markedly suppresses the migratory ability of prostate cancer cells by suppressing the activities of MMP-2/-9 and their expression. The possible mechanism of this suppression could be via reducing ROS levels and ERK1/2 activities. These observations imply that naringenin has a potent stand as a promising therapeutic drug in the treatment of prostate cancer.