Inhibition of Specificity Protein 1 Is Involved in Phloretin-Induced Suppression of Prostate Cancer

Phloretin is a flavonoid existed in various plants and has been reported to possess anticarcinogenic activity. However, the anticancer mechanism of phloretin in prostate cancer (PCa) remains unclear. Here, our in vitro and in vivo experimental data demonstrate that phloretin inhibits the phosphorylation and the activation of EGFR and then inhibits its downstream PI3K/AKT and MEK/ERK1/2 pathways in PCa cells. Inhibition of these two pathways further decreases expression of Sp1 by inhibiting Sp1 gene transcription, induces degradation of Sp1 protein by inhibiting GSK3β phosphorylation, suppresses nucleolin-enhanced translation of Sp1 mRNA by inhibiting nucleolin phosphorylation, and directly inactivates transcription activity of Sp1. Inhibition of Sp1 subsequently decreases the expression of Sp3/4, VEGF, and Survivin and then upregulates apoptosis-related proteins and downregulates cell cycle-related proteins in PCa cells. Finally, phloretin treatment in PCa cells induces cell growth inhibition and apoptosis, suggesting that phloretin may be an effective therapy compound in the treatment of prostate cancer.


Introduction
Prostate cancer is a commonly diagnosed cancer and the fifth leading cause of cancer deaths in men in the world [1]. Chemoprevention is a promising approach in prostate cancer research, in which natural or synthetic compounds are often used to prevent this malignant disease [2]. Phloretin, a natural flavonoid found mostly in plants [3,4], has been reported to possess anticancer activity by inducing apoptosis in human glioblastoma cells, Hep G2 cells, and lung carcinoma cells [5][6][7], while its anticancer molecular mechanism on prostate cancer is still not well known.
Nucleolin, a multifunctional protein localized not only in the nucleus but also in the cytoplasm and cell membrane, plays an important role in many cellular processes, such as chromatin remodeling, translation of mRNA, transcription of ribosomal RNA (rRNA), rRNA maturation, and ribosome assembly [28]. Usually, nucleolin binds to the G-rich sequence were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

MTT Assay and CCK-8 Assay for Cell Viability and
Proliferation. It mainly referred our previous report [35]. In detail, cells were seeded in a 96-well plate at a density of 1 × 10 4 cells/well overnight and treated with different concentrations of phloretin (0, 20, 50, and 100 μM) for 24 h, and then culture medium was removed and fresh medium (100 μl) was added with 10 μl of MTT (5 mg/ml) or 5 μl of CCK-8 solution. The plate was incubated at 37°C for 4 h in the dark. For CCK-8 assay, the absorbance of the incubations was measured using a microplate reader (Thermo Scientific, Fremont, CA, USA) at 450 nm. For MTT assay, the medium was removed again, and 100 μl of DMSO was added to each well and the absorbance at 570 nm was measured by a microplate reader (Thermo Scientific, Fremont, CA, USA).
All the measured OD values were converted into cell viability and compared with the value of the control well.

Cell Apoptosis Analysis by Flow Cytometry and DAPI
Staining Assay. Cells cultured in 6-well plates were treated with phloretin (0, 20, 50, and 100 μM) for 24 h and then harvested for flow cytometry analysis (cell apoptosis assay) by using an Annexin V-FITC Apoptosis Detection Kit (Keygentec, Nanjing, China) according to the instruction of manufacturer. Flow cytometry analysis for cell apoptosis was performed using the EPICS Elite ESP high-performance cell sorter (Coulter Electronics, Ltd., England, UK) and analyzed by ModFit LT (version 2.0; Verity Software), and a minimum of 30,000 events were collected for each sample.
For DAPI staining assay, cells cultured in 12-well plates were incubated with phloretin (0, 20, 50, and 100 μM) for 24 h. Cells were briefly washed with 1× PBS and fixed in 4% formaldehyde for 15 min, and then washed three times with 1× PBS and permeabilized in 0.2% Triton X-100 for 15 min. Finally, cells were stained with DAPI (1 μg/ml) at 37°C for 30 min in the dark and then observed and photographed by fluorescence microscopy (Nikon, IX-71, Japan).  Figure 1(a). After 24 h, cells were washed with 1× PBS and harvested for extractions of cytosolic and nuclear proteins by using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology). The cytosolic and nuclear extracts were then analyzed by western blot.

Cell
2.9. RNA Immunoprecipitation Assay (RIP Assay) and Western Blotting Analysis. Cells were treated with phloretin as indicated in Figure 1(c) and then harvested and lysed with lysis buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl 2 , 5% glycerol, 0.5% NP-40, and 1 U/μl RNaseOUT) for 30 min on ice. The cell lysates were divided into two parts: one part was for extracting the total RNA and then doing RT-PCR for Sp1 5′-UTR (5′-UTR of Sp1 mRNA, as input) and β-actin (as internal control); another part was for RNA immunoprecipitation (RIP) by incubating with IgG (Beyotime Biotechnology) and anti-nucleolin antibody, and then protein A/G agarose beads (Santa Cruz Biotechnology) at 4°C overnight. Immunoprecipitated complexes were washed with lysis buffer and RNA was extracted for RT-PCR of Sp1  For western blotting analysis, cells were harvested and lysed in radio immunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology) by adding Roche complete protease inhibitor cocktail. After centrifugation and protein quantification, the supernatants (containing total protein 15-30 μg) were submitted to western blotting assay with related primary antibodies.
2.10. Antitumor Assay of Phloretin In Vivo. PC-3 cells were calculated by using trypan blue, and finally 2 × 10 6 cells in 100 μl 1× PBS were subcutaneously injected into male nude mice (4-5 weeks, supplied by the animal center in the College of Medicine, Nanjing University, Nanjing, China). After tumors grew to 24-30 mm 3 , mice were randomly divided into four groups (5 mice in each group) and treated every two days by intragastrical administration with 1× PBS (NC group), 5-FU (PC group, 20 mg/kg), and phloretin (including the LD group, low dose of 10 mg/kg; the HD group, highdose of 50 mg/kg) for six weeks. After 42 days, mice were sacrificed and the subcutaneous tumors were isolated and weighted, and then the tumors were equally dissected into two parts. One part of the tumor tissues was stored at -80°C for western blot assay and another part was formalin fixed and paraffin embedded for immunohistochemistry.
2.11. Immunohistochemistry Analysis. The formalin-fixed tissues were sectioned in 4-5 μm thick. Each tissue section was deparaffinized and rehydrated with upgraded ethanol, and then tissue sections were boiled in EDTA for 15 min, quenched with 0.3% hydrogen peroxide solution for 10 min at room temperature, and blocked with BSA in PBS for 30 min. The sections were subsequently incubated with special primary antibodies as indicated in figures overnight at 4°C and then counterstained with hematoxylin. Antibody binding was detected with an Envision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (Gene Tech, Shanghai, China). The expression levels of specific proteins were observed and photographed under a microscope at a magnification of 400x (CTR 6000; Leica, Wetzlar, Germany).

Statistical Analysis.
All data were expressed as the means ± SD and analyzed using Student's t-test. Comparison between groups was made by the Dunnett test of SPSS in figures. A P value of <0.05 was statistically significant. All experiments were replicated three times. Western blot data further identified that phloretin treatment in LNCaP and PC-3 cells decreased the protein levels of Cyclin B1, XIAP, and Bcl-2, while increased the protein levels of c-Caspase 3 (cleaved Caspase 3), c-PARP-1 (cleaved PARP-1), c-Caspase 8 (cleaved Caspase 8), and c-Caspase 9 (cleaved Caspase 9). The change degrees were all in a phloretin dose-dependent manner (Figure 3(d)). Although phloretin increased the level of BAX in a dose-dependent manner only in LNCaP cells (no changes in PC-3 cells), the BAX/Bcl-2 ratios in both cell lines were upregulated by phloretin in a dose-dependent manner. Furthermore, we found that phloretin treatment did not change the protein level of p53 in LNCaP cells (undetectable in PC-3 cells) (Figure 3(d)), suggesting that phloretin-induced PCa cell apoptosis was p53 independent.

Phloretin Inhibited the Activation of EGFR and Its Downstream PI3K/AKT and MEK/ERK Pathways and Then
Decreased the Activities of GSK-3β and Sp1. In exploring the molecular mechanism of phloretin-induced cell growth inhibition, cell cycle arrest, and apoptosis in PCa cells, we found that phloretin treatment substantially downregulated the autophosphorylation levels of EGFR at Y1173, but not the total protein level of EGFR (Figure 4(a)), suggesting the activity of EGFR was inhibited by phloretin (it is the same as isorhapontigenin treatment in PCa cells we reported previously [35]). As the downstream of EGFR, the PI3K/AKT and MEK/ERK1/2 pathways have been reported to play the crucial roles in regulating cell survival, growth, migration, and invasion [20,21]. From the western blot data, we also found that phloretin downregulated the levels of p-PI3K, p-AKT(S473), p-AKT(T308), p-C-RAF, p-MEK, and p-ERK1/2 in a concentration-dependent manner, while there is almost no effect on the total protein levels of PI3K, AKT, RAF, MEK1/2, and ERK1/2 in PCa cells (Figures 4(a) and 4(b)).
GSK3β is a downstream target of AKT and inactivated by AKT-induced phosphorylation of GSK3β proteins at Ser9 [25]. Our results showed that phloretin treatment in PCa cells decreased the phosphorylation level of GSK3β at Ser9 in a concentration-dependent manner, while the total protein level of GSK3β unchanged (Figure 4(c)), suggesting that phloretin increased the activity of GSK3β via inhibiting the PI3K/AKT pathway.
As reported, the activity of Sp1 was regulated by AKT, ERK1/2, and GSK3β, respectively. AKT could upregulate the expression of Sp1 gene [36,37], ERK1/2 could phosphorylate Sp1 at Thr453/Thr739 and then facilitate its binding to promoters of the targeted genes [26,27], and GSK3β could induce the degradation of Sp1 proteins [24,25]. Here, phloretin treatment in LNCaP and PC-3 cells decreased the total protein level of Sp1 and the phosphorylation levels of Sp1 at Thr453/Thr739 in a concentration-dependent manner, and the quantification data of the ratios of p-Sp1/Sp1 (including p-Sp1 at both T453 and T739) further demonstrated that phloretin-induced decrease of p-Sp1(T453/T739) levels was also concentration dependent (Figure 4(c)). Therefore, phloretin-induced inhibition of AKT and ERK1/2 inevitably inhibited Sp1 expression, decreased the phosphorylation of Sp1(T453/T739) and GSK3β(S9), induced Sp1 degradation, and finally decreased Sp1 activity and impaired Sp1 binding to the promoters of its downstream genes.
Together, phloretin inhibited AKT and ERK1/2 and activated GSK3β, and subsequently decreased the transcriptional activity of Sp1 by downregulating the expression of Sp1 gene, inducing the degradation of Sp1 and decreasing Sp1-binding to the promoters of its target genes in PCa cells.

Phloretin Decreased the Level of Sp1 by Increasing the Degradation of Sp1 Protein in PCa Cells.
To identify the degradation of Sp1 protein by phloretin-induced inhibition of PI3K/AKT and activation of GSK3β, LNCaP and PC-3 cells were cultured and treated with CHX (10 μg/ml) and/or phloretin (50 μM) for different time as indicated Figure 6(a), and then cells were harvested for western blot assay. The data showed that treatment with CHX alone caused the decrease of Sp1 protein level in a time-dependent manner; cotreatment with CHX and phloretin resulted in a more decrease of Sp1 protein level. The quantification data also demonstrated that the levels of Sp1 in CHX-treated cells were further decreased by phloretin treatment in a time-dependent manner, suggesting that phloretin could induce the degradation of Sp1 proteins. In addition, the Sp1-targeted genes Sp3/4 were also decreased with the downregulation of Sp1 (Figure 6(a)).
To further identify phloretin-induced degradation of Sp1, LNCaP and PC-3 cells were cultured and treated with phloretin (50 μM) and/or MG132 (10 μM) as indicated in Figure 6(b) and then harvested for western blot assay. Our data showed that phloretin-induced decrease of Sp1 and Sp3/4 could be partially rescued by cotreated MG132 (Figure 6(b)). Moreover, dual-luciferase assay showed that MG132 treatment could also partially reverse the phloretin-  Figure 1(a) and then checked the protein levels of Sp1 and its downstream proteins, including VEGF and Cyclin D1. From the experimental data, we found that overexpression of nucleolin upregulated the protein levels of Sp1 and its downstream proteins in both LNCaP and PC-3 cells, and phloretin treatment in PCa cells obviously suppressed nucleolin-induced upregulation of protein levels of Sp1 and its targeted proteins (Figure 1(a)).
It is reported that nucleolin could be recruited to the 5′-UTR of Sp1 mRNA in the cytoplasm as an IRES (internal ribosomal entry site, nucleolin binding site is +1/250 bp of 5′-UTR of Sp1 mRNA) transacting factor to enhance the translation of Sp1 mRNA during lung cancer formation [31], and PI3K/AKTinduced phosphorylation of nucleolin at Thr76 and Thr84 promoted nucleolin translocation from the nucleus to the cytoplasm in the cells of colorectal carcinoma [30]. To identify the effect of phloretin on the phosphorylation of nucleolin at Thr76 and Thr84 and the distribution of nucleolin in the nucleus and the cytoplasm, LNCaP and PC-3 cells were incubated with 50 μM or different concentrations of phloretin (as indicated in Figures 1(b) and 1(c)) for 24 h. Then, cells were harvested for nucleus/cytoplasm separation and/or western blot assays to detect the nucleus/cytoplasm distribution and phosphorylation (Thr76/Thr84) levels of nucleolin. From the results, we found that the protein levels of nucleolin were decreased in cytoplasm and increased in the nucleus with the treatment of phloretin (Figure 1(b)). Levels of p-Nucleolin(Thr76) and p-Nucleolin(Thr84) were distinctly decreased by the treated phloretin in a concentration-dependent manner, while the total protein levels of nucleolin were almost not changed in LNCaP and PC-3 cells (Figure 1(c)).
In addition, RNA-IP experiment was employed to identify the effect of phloretin on nucleolin binding to 5 ′ -UTR of Sp1 mRNA. LNCaP and PC-3 cells (treated with different concentrations of phloretin as indicated in Figure 1(d)) were harvested for direct RT-PCR of 5 ′ -UTR of Sp1 mRNA and for RNA-IP with IgG/nucleolin antibodies. Then, the RNAs of IP were also extracted for RT-PCR of 5 ′ -UTR of Sp1 mRNA. The direct RT-PCR data showed that the mRNA levels of β-actin (as internal control) were not changed with the treatment of phloretin, while the total levels of 5′-UTR of Sp1 mRNA (as input) were decreased with the treatment of phloretin in a concentration-dependent manner in both PCa cell lines (Figure 1(d)). The RNA-IP experimental data showed that the levels of nucleolin-bound 5 ′ -UTR of Sp1  Figure 6: Phloretin induced the degradation of Sp1 protein in PCa cells. (a) Cells were treated with 10 μg/ml CHX alone or cotreated with 10 μg/ml CHX and 50 μM phloretin (CHX pretreated for 30 min) for western blot assays to detect the protein levels of Sp1 and Sp3/4. The quantification: first, the densities of bands of Sp1 and β-actin were quantified using ImageJ software, and the ratios of Sp1/β-actin (the Sp1 and β-actin with no treatment of phloretin and CHX as control) were calculated; second, the ratios of Sp1/β-actin vs. Con were obtained by using the values of Sp1/β-actin vs. the value of control Sp1/β-actin (control Sp1/β-actin vs. control Sp1/β-actin as 1). (b) Cells were treated with phloretin (50 μM) and/or MG132 (10 μM), and then cells were harvested for western blotting assay to detect the protein levels of Sp1 and Sp3/4. All western blotting assays used β-actin as loading control. (c) PC-3 cells were transfected with the various luciferase constructs and treated with phloretin (50 μM) or MG132 (10 μM) alone, or cotreated with phloretin (50 μM) and MG132 (10 μM), and then dual-luciferase activities (Firefly and Renilla) were measured. The fold inductions of luciferases were calculated by using relative luciferases (the relative luciferase of control as 1). * P < 0:01; * * P < 0:05. Con: control. mRNA were obviously decreased with the treatment of phloretin in a concentration-dependent manner in both PCa cell lines, and the decreasing degree was a little higher than that of input of 5 ′ -UTR (Figure 1(d)). Besides, no RT-PCR products were detected in the negative control samples of RNA-IP with IgG (Figure 1(d)).
These results suggested that phloretin treatment in PCa cells decreased the levels of cytoplasmic nucleolin via downregulating the phosphorylation levels of nucleolin at Thr76 and Thr84 and then resulted in the reduction of nucleolin binding to 5 ′ -UTR of Sp1 mRNA in the cytoplasm. It might inevitably lead to the decrease of nucleolin-induced Sp1 mRNA translation.

Phloretin Suppressed Tumor Growth and Induced
Apoptosis of Prostate Cancer Cells In Vivo. To investigate the efficacy of phloretin on prostate cancer cells in vivo, subcutaneous xenotransplanted tumor models of PC-3 cell in nude mice were established to evaluate the inhibition of tumor growth by phloretin treatment. Compared with the negative control group, our results showed that phloretin treatment in vivo inhibited growth of the transplanted prostate tumors in a time-dependent manner in both the highdose and low-dose phloretin-treated groups, while the body weights of mice had no significant changes in all groups (Figures 7(a) and 7(b)). At the end of experiments, the tumor volumes of mice in the high-dose (HD) group were much smaller than those in both the negative control (NC) and low-dose (LD) groups and almost the same as those in the positive control (PC) group (Figures 7(a) and 7(c)). Immunohistochemistry (IHC) data showed that the levels of Ki-67 (marker of cell proliferation), Sp1, Sp3/4, and Survivin in the cells of PC-3-transplanted tumor tissues were all decreased with the treatment of phloretin, and these protein levels in the tumor cells of the HD group were almost the same as those of the PC group (5-FU treatment) (Figure 7(d)). In addition, the western blot data of PC-3transplanted tumor tissues demonstrated that the protein levels of Sp1, Sp3/4, Survivin, and Cyclin D1 were decreased, while the protein levels of c-Caspase 3 and c-PARP-1 were increased in the HD and PC groups (compared with the NC and LD groups) (Figure 7(e)).
These results demonstrated that phloretin treatment could also inhibit PC-3 cell growth and induce PC-3 cell apoptosis in vivo, and the high-dose phloretin had a greater inhibition efficacy on the growth of PCa cell-transplanted tumors by comparing with low-dose phloretin in vivo.

Discussion
Phloretin has been recently attracted extensive interest and extensively studied in treating many diseases including in anticancer research. Phloretin could induce apoptosis of many types of cancer cells in vitro and inhibit growth of the transplanted tumors of multiple human cancer cell lines in vivo [5,7,42]. However, the molecular mechanism of the anticancer effect of phloretin in prostate cancer remained unclear. In this study, we reported that phloretin could inhibit the activation of EGFR and its downstream signal pathways, including the PI3K/AKT, MEK/ERK1/2, and GSK3β pathways. Inhibition of these pathways further inhibited the activation of Sp1 and subsequently resulted in the downregulation of Sp3/4 proteins, cell growth, and antiapoptosis-related proteins (including Cyclin D1, Cyclin B1, Bcl-2, Survivin, AR, VEGF, and XIAP) and the upregulation of cell apoptosis-related proteins (including c-Caspase 3, c-Caspase 8, c-Caspase 9, and c-PARP-1), and finally resulted in cell growth inhibition and apoptosis in prostate cancer cells both in vitro and in vivo (Figure 7(f)).
Usually, p53 is the key protein in cell growth inhibition and cell apoptosis promotion involved in the inhibition of pathways that related to cell cycle and the activation of pathways that related to cell apoptosis [43,44]. In current study, the protein levels of p53 were almost not changed in LNCaP cells and almost not detected in PC-3 cells (Figure 3(d)). In addition, the phosphorylation levels of p53(Ser15) in LNCaP cells which related to the activity of p53 protein were decreased with the treatment of phloretin (data not shown). It indicated that p53 was not the key factor in phloretininduced cell growth inhibition and apoptosis in prostate cancer cells. Thus, our experimental results indicated that phloretin-induced cell apoptosis was in a p53-independent manner in PCa cells. Based on the capability of Sp1 in modulating the expression of Cyclin B1 gene [45], we could conclude that phloretin-induced downregulation of cyclin B1 and cell cycle arrest at G2/M phase in PCa cells was not via the p53/cyclin B1 pathway, but via the Sp1/cyclin B1 pathway.
Nucleolin, the RNA-binding protein, is involved in mRNA processing including the regulation of mRNA stability and translational efficiency [46]. During lung cancer formation, nucleolin was recruited to the 5 ′ -UTR of Sp1 mRNA to enhance cap-independent translational activity in the cytoplasm. Phosphorylation of nucleolin was also important to increase the distribution of nucleolin protein in the cytoplasm and enhance nucleolin binding to 5 ′ -UTR of Sp1 mRNA [30,31]. Our results here identified that phloretin treatment in PCa cells downregulated the phosphorylation levels of nucleolin and resulted in the decreased distribution of nucleolin protein in the cytoplasm and reduced nucleolin binding to 5′-UTR of Sp1 mRNA, and finally decreased the translational efficiency of Sp1 mRNA. Of course, more details of the molecular mechanism of the relationship between phloretin and nucleolin needed to be further explored.
Sp1, usually overexpressed in many human tumors and cancer cell lines [8,9], plays an important role in tumorigenesis and cancer progress and is a potential target for development of drugs in cancer chemotherapy [47]. Regulation of Sp1 activity should be an effective strategy for cancer drug screening. As reported, Sp1 is positively regulated by AKT and ERK and negatively regulated by GSK3β, and GSK3β is negatively regulated by the PI3K/AKT pathway again [24-27, 36, 37]. Our study demonstrated that phloretin-induced inhibition of Sp1 in PCa cells included at least three pathways: first, phloretin induced inhibition of EGFR/PI3K/AKT and then downregulated expression of Sp1 gene (phloretin-EGFR/-PI3K/AKT-Sp1 pathway); second, phloretin induced inhibition of EGFR/PI3K/AKT and then decreased the phosphorylation levels of GSK3β(S9) and activated GSK3β, and finally induced the degradation of Sp1 proteins (by combined with reported results in [48]); and third, phloretin induced inhibition of EGFR/PI3K/AKT and then downregulated the phosphorylation levels of nucleolin(Thr76/Thr84) and might suppress the translation of Sp1 mRNA (by combined with reported results in [31]). Of course, the detailed molecular mechanisms of phloretin in regulating Sps needed to be further studied in the future studies.
In conclusion, phloretin treatment in PCa cells downregulated the protein levels of Sp1 by inhibiting the expression of Sp1 genes, increasing the degradation of Sp1 proteins and suppressing the translation of Sp1 mRNAs via inhibiting the activity of EGFR and its downstream pathways, including the PI3K/AKT, MEK/ERK, AKT/GSK3β, and AKT/nucleolin pathways, and finally induced cell cycle arrest, cell growth inhibition, and apoptosis in PCa cells by decreasing the levels of cell cycle/growth-related proteins and increasing the levels of cell apoptosis-related proteins. Our study suggested that phloretin had the potential to be a candidate compound to treat the patients with prostate cancer in clinic in the future.

Data Availability
The data used to support this study are available from the corresponding author upon request.