Pirfenidone, an Anti-Fibrotic Drug, Suppresses the Growth of Human Prostate Cancer Cells by Inducing G1 Cell Cycle Arrest

Pirfenidone (PFD) is an anti-fibrotic drug used to treat idiopathic pulmonary fibrosis by inducing G1 cell cycle arrest in fibroblasts. We hypothesize that PFD can induce G1 cell cycle arrest in different types of cells, including cancer cells. To investigate the effects of PFD treatment on the growth of human prostate cancer (PCa) cells, we used an androgen-sensitive human PCa cell line (LNCaP) and its sublines (androgen-low-sensitive E9 and F10 cells and androgen-insensitive AIDL cells), as well as an androgen-insensitive human PCa cell line (PC-3). PFD treatment suppressed the growth of all PCa cells. Transforming growth factor β1 secretion was significantly increased in PFD-treated PCa cells. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G0/G1 phase, which was accompanied by a decrease in the S/G2 cell population. CDK2 protein expression was clearly decreased in PFD-treated LNCaP and PC-3 cells, whereas p21 protein expression was increased in only PFD-treated LNCaP cells. In conclusion, PFD may serve as a novel therapeutic drug that induces G1 cell cycle arrest in human PCa cells independently of androgen sensitivity. Thus, in the tumor microenvironment, PFD might target not only fibroblasts, but also heterogeneous PCa cells of varying androgen-sensitivity levels.


Introduction
The number of males diagnosed with prostate cancer (PCa) is increasing worldwide [1]. Most patients with early-stage PCa can be treated with therapies such as radical prostatectomy or irradiation, whereas androgen deprivation therapy (ADT) is the standard systemic therapy given to patients with advanced PCa. Even though ADT induces temporary remission, the majority of patients (approximately 60%) eventually progress to castration-resistant PCa (CRPC), which is associated with a high mortality rate [2,3].
PCa is characterized by multifocal and heterogeneous progression of the primary tumor. In PCa progression, a decrease or loss of androgen sensitivity in PCa cells is a significant clinical concern. CRPC, a heterogeneous disease, exhibits varying degrees of androgen sensitivity. Once PCa cells lose sensitivity to ADT, effective therapies are limited [4]. In the past few years, however, several new

Cell Culture
The androgen-sensitive, AR-positive human PCa cell line LNCaP, and the androgen-insensitive, AR-negative human PCa cell line PC-3 were obtained from the American Type Culture Collection (Manassas, VA, USA). LNCaP and PC-3 cells were authenticated by the short tandem repeat method and cultured in RPMI 1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co.). Androgen-low-sensitive E9 and F10 cells were established from androgen-sensitive LNCaP cells using a limiting dilution method under regular culture conditions [24,25]. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted conditions [26]. The androgen sensitivity of the parental LNCaP cells and the E9, F10, and AIDL cells were confirmed by the change in KLK3 (PSA) mRNA expression after treatment with the synthetic androgen R1881 [20].

Cell Viability Assay
To assess cell viability after the PFD treatments, LNCaP, E9, F10, AIDL, and PC-3 cells were plated in 12-well plates at 5 × 10 4 to 1 × 10 5 cells/well. PFD (0.1 and 0.3 mg/mL) or vehicle-only (0.1% dimethyl sulfoxide [DMSO]) was added on day two, and the cells were cultured for an additional three days. The cells were detached by trypsinization and counted using the Countess II Automated Cell Counter (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell viability was assessed by trypan blue exclusion assay.

Cell Cycle Analysis
LNCaP or PC-3 cells (1.5 × 10 5 cells) were seeded into 100-mm culture dishes (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). Twenty-four hours after seeding, the cells were treated with 0.1 or 0.3 mg/mL PFD or vehicle (0.1% DMSO) for 24 h. After treatment, the cells were isolated, and the nuclei were stained using the BD Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA). To determine the cell cycle distribution, the DNA content of the stained cells was analyzed using the BD FACS Canto II flow cytometer (BD Biosciences), as described previously [28].

Apoptosis Assay
LNCaP cells (6 × 10 5 cells) and PC-3 cells (4 × 10 5 cells) were seeded in 100 mm culture dishes (Sumitomo Bakelite Co., Ltd.). 24 h after seeding, the cells were treated with 0.1 or 0.3 mg/mL PFD, or vehicle (0.1% DMSO), for 48 h (LNCaP cells) or 72 h (PC-3 cells). After treatment, the cells were trypsinized, collected, and stained with annexin V-fluorescein isothiocyanate and propidium iodide simultaneously using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences). The cell suspensions were analyzed using the BD FACS Canto II flow cytometer (BD Biosciences) to determine the percentage of apoptotic (annexin V-fluorescein isothiocyanate staining) and necrotic (propidium iodide staining) cells, as described previously [28]. A minimum of 20,000 cells were collected for all samples.

ELISA
For quantitative determination of TGFβ1 and PSA proteins, aliquots of conditioned medium from PCa cells were collected and subjected to ELISA using the Quantikine ® human TGF-β1 immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) and PSA Enzyme Immunoassay Test Kit (Hope Laboratories, Belmont, CA, USA), respectively.

Statistical Analysis
Results are expressed as means ± standard deviation. Differences between two groups were determined using Student's t-test. Values of p < 0.05 were considered statistically significant.

Effects of Pirfenidone Treatment on the Growth of Prostate Cancer Cells (LNCaP, LNCaP Sublines, and PC-3)
First, we confirmed that PFD treatment suppresses the growth of fibroblasts. PFD treatment (0.3 mg/mL) for 72 h suppressed the growth of commercially available prostate stromal cells (data not shown). Using these experimental conditions, we treated the PCa cells (LNCaP, E9, F10, AIDL, and PC-3) with PFD and found that PFD treatment suppressed the growth of all cell lines ( Figure 1A). Among the LNCaP cells and sublines, growth suppression was more pronounced in LNCaP and E9 cells than in F10 and AIDL cells. We also assessed TGFβ1 secretion from PCa cells because of its relationship to cell cycle and apoptosis. TGFβ1 levels were measured in the culture medium of the PCa cells using ELISA. TGFβ1 secretion was significantly increased by PFD treatment in all PCa cells evaluated ( Figure 1B). Among the LNCaP cells and sublines, the increase in TGFβ1 secretion was greater in LNCaP and E9 cells than in F10 and AIDL cells. collected. The protein concentration was measured using the NanoDrop 2000 instrument (Thermo Fisher Scientific Inc.).

Statistical Analysis
Results are expressed as means ± standard deviation. Differences between two groups were determined using Student's t-test. Values of p < 0.05 were considered statistically significant.

Effects of Pirfenidone Treatment on the Growth of Prostate Cancer Cells (LNCaP, LNCaP Sublines, and PC-3)
First, we confirmed that PFD treatment suppresses the growth of fibroblasts. PFD treatment (0.3 mg/mL) for 72 h suppressed the growth of commercially available prostate stromal cells (data not shown). Using these experimental conditions, we treated the PCa cells (LNCaP, E9, F10, AIDL, and PC-3) with PFD and found that PFD treatment suppressed the growth of all cell lines ( Figure 1A). Among the LNCaP cells and sublines, growth suppression was more pronounced in LNCaP and E9 cells than in F10 and AIDL cells. We also assessed TGFβ1 secretion from PCa cells because of its relationship to cell cycle and apoptosis. TGFβ1 levels were measured in the culture medium of the PCa cells using ELISA. TGFβ1 secretion was significantly increased by PFD treatment in all PCa cells evaluated ( Figure 1B). Among the LNCaP cells and sublines, the increase in TGFβ1 secretion was greater in LNCaP and E9 cells than in F10 and AIDL cells.

Pirfenidone Antiproliferative Mechanisms in LNCaP and PC-3 Cells
To investigate whether PFD treatment affects the cell cycle, we performed flow cytometric and Western blot analyses of cell-cycle regulatory proteins. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G0/G1 phase, which was accompanied by a decrease in S/G2 phase cells (Figure 2, Tables 1 and 2). p21 protein expression was increased by PFD treatment in LNCaP cells, but was not detected in PC-3 cells (Figure 3). Of note, PFD-increased p21 protein expression was the highest in E9 cells ( Figure S1). In contrast, CDK2 protein expression was clearly decreased in both PFD-treated LNCaP and PC-3 cells. Of note, PFD treatment did not induce early apoptosis in either LNCaP or PC-3 cells [29]. Table 1. Effects of pirfenidone (PFD) treatment on cell cycle progression in LNCaP cells.

Pirfenidone Antiproliferative Mechanisms in LNCaP and PC-3 Cells
To investigate whether PFD treatment affects the cell cycle, we performed flow cytometric and Western blot analyses of cell-cycle regulatory proteins. In both LNCaP and PC-3 cells, PFD treatment increased the population of cells in the G 0 /G 1 phase, which was accompanied by a decrease in S/G 2 phase cells (Figure 2, Tables 1 and 2). p21 protein expression was increased by PFD treatment in LNCaP cells, but was not detected in PC-3 cells (Figure 3). Of note, PFD-increased p21 protein expression was the highest in E9 cells ( Figure S1). In contrast, CDK2 protein expression was clearly decreased in both PFD-treated LNCaP and PC-3 cells. Of note, PFD treatment did not induce early apoptosis in either LNCaP or PC-3 cells.    with PFD for two days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDSpolyacrylamide gel. After separation, the proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. p21 and CDK2 protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring βactin protein levels.

Effects of Pirfenidone Treatment on Androgen Receptor Signaling-Related Protein Levels in LNCaP and PC-3 Cells
To confirm specific inhibition of PFD treatment on the AR signaling pathway, we evaluated the protein levels of AR and PSA in LNCaP cells by Western blot analysis. AR protein expression was not changed, but PSA protein expression was decreased by PFD treatment (Figure 4). In both LNCaP and PC-3 cells, PFD treatment slightly decreased the level of phospho-Akt (Ser473), suggesting slight  with PFD for two days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDSpolyacrylamide gel. After separation, the proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. p21 and CDK2 protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring βactin protein levels.

Effects of Pirfenidone Treatment on Androgen Receptor Signaling-Related Protein Levels in LNCaP and PC-3 Cells
To confirm specific inhibition of PFD treatment on the AR signaling pathway, we evaluated the protein levels of AR and PSA in LNCaP cells by Western blot analysis. AR protein expression was not changed, but PSA protein expression was decreased by PFD treatment (Figure 4). In both LNCaP and PC-3 cells, PFD treatment slightly decreased the level of phospho-Akt (Ser473), suggesting slight Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDS-polyacrylamide gel. After separation, the proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. p21 and CDK2 protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring β-actin protein levels.

Effects of Pirfenidone Treatment on Androgen Receptor Signaling-Related Protein Levels in LNCaP and PC-3 Cells
To confirm specific inhibition of PFD treatment on the AR signaling pathway, we evaluated the protein levels of AR and PSA in LNCaP cells by Western blot analysis. AR protein expression was not changed, but PSA protein expression was decreased by PFD treatment (Figure 4). In both LNCaP and PC-3 cells, PFD treatment slightly decreased the level of phospho-Akt (Ser473), suggesting slight inhibition of Akt phosphorylation. Of note, AR and PSA protein expression was not detected in PC-3 cells as reported previously [20].
inhibition of Akt phosphorylation. Of note, AR and PSA protein expression was not detected in PC-3 cells as reported previously [20]. with PFD for an additional two days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDS-polyacrylamide gel. After separation, proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. Androgen receptor, prostate-specific antigen, phospho-Akt (Ser473), and total Akt protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring β-actin levels.
We further evaluated the effects of PFD treatment on PSA secretion by measuring PSA protein levels in conditioned medium from PFD-treated LNCaP cell cultures using ELISA. The PSA protein level was significantly reduced in LNCaP cell culture medium, suggesting that PSA secretion was inhibited by PFD treatment (Figure 5).

Discussion
Drug repositioning of the anti-fibrotic compound PFD to cancer treatment is not a novel idea, but investigation of the effects of PFD on PCa progression, considering the extracellular-matrix-rich microenvironment of PCa, would provide meaningful information for this potential application of PFD. In this study, we demonstrated that PFD treatment suppressed the growth and induced G1 cell cycle arrest in various PCa cell lines that differed in androgen sensitivity, suggesting that PFD may target not only fibroblasts but also heterogeneous PCa cells within the tumor microenvironment.
In animal models of fibrosis, PFD induced anti-fibrotic effects mainly via inhibition of TGFβ signaling in fibroblasts. TGFβ is a multifunctional cytokine that regulates cell proliferation, extracellular matrix production and degradation, cell differentiation, and apoptosis [30]. In animal Figure 4. Effects of pirfenidone (PFD) treatment on androgen receptor signaling-related protein levels in human prostate cancer cells. Both LNCaP and PC-3 cells were plated in 100 mm dishes and treated with PFD for an additional two days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDS-polyacrylamide gel. After separation, proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. Androgen receptor, prostate-specific antigen, phospho-Akt (Ser473), and total Akt protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring β-actin levels.
We further evaluated the effects of PFD treatment on PSA secretion by measuring PSA protein levels in conditioned medium from PFD-treated LNCaP cell cultures using ELISA. The PSA protein level was significantly reduced in LNCaP cell culture medium, suggesting that PSA secretion was inhibited by PFD treatment (Figure 5).
inhibition of Akt phosphorylation. Of note, AR and PSA protein expression was not detected in PC-3 cells as reported previously [20]. with PFD for an additional two days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDS-polyacrylamide gel. After separation, proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. Androgen receptor, prostate-specific antigen, phospho-Akt (Ser473), and total Akt protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring β-actin levels.
We further evaluated the effects of PFD treatment on PSA secretion by measuring PSA protein levels in conditioned medium from PFD-treated LNCaP cell cultures using ELISA. The PSA protein level was significantly reduced in LNCaP cell culture medium, suggesting that PSA secretion was inhibited by PFD treatment (Figure 5).

Discussion
Drug repositioning of the anti-fibrotic compound PFD to cancer treatment is not a novel idea, but investigation of the effects of PFD on PCa progression, considering the extracellular-matrix-rich microenvironment of PCa, would provide meaningful information for this potential application of PFD. In this study, we demonstrated that PFD treatment suppressed the growth and induced G1 cell cycle arrest in various PCa cell lines that differed in androgen sensitivity, suggesting that PFD may target not only fibroblasts but also heterogeneous PCa cells within the tumor microenvironment.
In animal models of fibrosis, PFD induced anti-fibrotic effects mainly via inhibition of TGFβ signaling in fibroblasts. TGFβ is a multifunctional cytokine that regulates cell proliferation, extracellular matrix production and degradation, cell differentiation, and apoptosis [30]. In animal

Discussion
Drug repositioning of the anti-fibrotic compound PFD to cancer treatment is not a novel idea, but investigation of the effects of PFD on PCa progression, considering the extracellular-matrix-rich microenvironment of PCa, would provide meaningful information for this potential application of PFD. In this study, we demonstrated that PFD treatment suppressed the growth and induced G 1 cell cycle arrest in various PCa cell lines that differed in androgen sensitivity, suggesting that PFD may target not only fibroblasts but also heterogeneous PCa cells within the tumor microenvironment.
In animal models of fibrosis, PFD induced anti-fibrotic effects mainly via inhibition of TGFβ signaling in fibroblasts. TGFβ is a multifunctional cytokine that regulates cell proliferation, extracellular matrix production and degradation, cell differentiation, and apoptosis [29]. In animal models of fibrosis, PFD treatment inhibited fibrosis, which was associated with down-regulation of TGFβ, platelet-derived growth factor, and collagen synthesis in various types of cells, including human lung fibroblasts [30], rat hepatic stellate cells [14], human pancreatic stellate cells [31], rat renal fibroblasts [32], human Tenon fibroblasts [15], and rat cardiac fibroblasts [33].
In contrast, our results showed that PFD treatment significantly increased TGFβ secretion from all PCa cells evaluated, regardless of androgen sensitivity. Our previous study reported that TGFβ1 secretion from PCa cells was quite low compared with that from fibroblasts, especially carcinoma-associated fibroblasts [27]. TGFβ participates in cell proliferation and differentiation not only in normal processes such as embryonic development and wound healing, but also abnormal processes such as cancer progression and angiogenesis [34]. Although a number of studies have investigated the role of TGFβ, the results are still controversial. Importantly, the TGFβ signaling pathway is involved in both tumor-suppressive and tumor-promoting roles. The presence of TGFβ in the tumor microenvironment may promote tumor growth by enhancing stromal support and angiogenesis and by impairing immune surveillance [35]. In contrast, TGFβ plays a tumor-suppressive role by inducing G 1 cell cycle arrest in various cell types, such as epithelial, endothelial, and hematopoietic cells and fibroblasts [36]. Cell-cycle inhibition by TGFβ is mediated in part by the up-regulation of antiproliferative proteins such as p15 INK4b , p21 CIP1 , and p27 KIP1 . In this study, the p21 CIP1 protein level was increased in PFD-treated LNCaP cells. Increased expression of p21 CIP1 , a cell-cycle-inhibitory protein, is not only associated with cell cycle inhibition, but also cell differentiation and senescence [37].
Lin et al. reported that the Akt pathway is associated with AR activation in LNCaP cells [38]. Iguchi et al. demonstrated that inhibition of Akt phosphorylation by the PI3K inhibitor LY294002 reduced PSA expression in LNCaP cells [39]. Previous studies reported that PFD treatment inhibits phosphorylation of Akt in rat hepatocytes [40], human lung fibroblasts [30], and human Tenon fibroblasts [16]. Similarly, in this study, PFD treatment slightly inhibited the phosphorylation of Akt in both LNCaP and PC-3 cells. In addition, PFD treatment reduced PSA protein expression and secretion in LNCaP cells.
Serum PSA levels are influenced by a number of drugs, such as non-steroidal anti-inflammatory drugs and statins [41,42]; for example, the serum PSA level was found to be lower in aspirin users than non-users [41]. In contrast, Iguchi et al. reported that betamethasone, an agonist of the glucocorticoid receptor, increased PSA mRNA expression in LNCaP cells [43]. Similar to PFD, the antidiabetic drug troglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma, reduced PSA expression in LNCaP cells [7]. Previous studies and our results suggest that PFD treatment reduces PSA expression, which is associated with inhibition of Akt phosphorylation in LNCaP cells.
The tumor microenvironment of the prostate is highly complex and heterogeneous, and is composed of carcinoma-associated fibroblasts as well as epithelial cancer cells that infiltrate into the surrounding tumor stroma, referred to as reactive stroma [44]. This heterogenous stromal component of the prostate contains multiple populations of fibroblasts that are associated with tumorigenesis [45,46]. In this study, we demonstrated that the anti-fibrotic drug PFD suppressed the growth of human PCa cells by inducing G 1 cell cycle arrest. In addition, our data suggest that PFD-induced growth suppression occurs independently of androgen sensitivity. Therefore, PFD may provide a novel therapeutic option for targeting not only fibroblasts surrounding cancer cells, but also heterogeneous PCa cells of varying androgen sensitivities within patients with CRPC.

Conclusions
In our studies of drug repositioning, we demonstrate that PFD may serve as a novel therapeutic drug that induces G 1 cell cycle arrest in human PCa cells independently of androgen sensitivity.
Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0383/8/1/44/s1, Figure S1: Effects of pirfenidone (PFD) treatment on the expression of cell cycle-related proteins in human prostate cancer cells. E9, F10, and AIDL cells were plated in 100-mm dishes and treated with PFD for 2 days. Cell lysates (50 µg) were separated by electrophoresis using a 12.5% SDS-polyacrylamide gel. After separation, the proteins in the gel were transferred to a polyvinylidene difluoride membrane by electroblotting. p21 and CDK2 protein levels were determined by Western blot analysis using specific antibodies. Equal loading of the samples was confirmed by measuring β-actin protein levels.