**3. Results**

### *3.1. 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.

**Figure 1.** *Cont*.

**Figure 1.** Effects of pirfenidone (PFD) treatment on the growth and secretion of transforming growth factor β1 (TGFβ1) of human prostate cancer (PCa) cells. Human PCa cells (parental LNCaP cell line and the LNCaP sublines E9, F10, and AIDL; and the PC-3 cell line) were plated in 12-well plates and treated with PFD for three days. Effects of PFD treatment on the (**A**) growth and (**B**) TGFβ1 secretion of human PCa cells. Data are representative of three independent experiments, and the values represent the means ± standard deviation. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 versus the vehicle-treated control.

### *3.2. 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.


**Table 1.** Effects of pirfenidone (PFD) treatment on cell cycle progression in LNCaP cells.

\*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 versus vehicle-treated control.

**Table 2.** Effects of pirfenidone (PFD) treatment on cell cycle progression in PC-3 cells.


\*\*, *p* < 0.01 versus vehicle-treated control.

**Figure 2.** Cell cycle analysis by flow cytometry of human prostate cancer cells treated with pirfenidone (PFD). The cell cycle was determined by propidium iodide (PI) staining, as detailed in the "Material and Methods" section. The proportions of cells in the G0/G1, S, and G2/M phase were calculated from one representative experiment (*n* = 3).

**Figure 3.** Effects of pirfenidone (PFD) treatment on the expression of cell cycle-related proteins in human prostate cancer cells. Both LNCaP and PC-3 cells were plated in 100 mm dishes and treated with PFD for two 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.

### *3.3. 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].

**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).

**Figure 5.** Effects of pirfenidone (PFD) treatment on prostate-specific antigen (PSA) secretion from human prostate cancer cells. The level of PSA secreted from LNCaP cells was determined by measuring the PSA level in LNCaP conditioned medium by ELISA. Values represent the means ± standard deviation. \*, *p* < 0.05 versus the vehicle-treated control.
