**4. 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 [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 G1 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 p<sup>15</sup>INK4b, p<sup>21</sup>CIP1, and p27KIP1. In this study, the p21CIP1 protein level was increased in PFD-treated LNCaP cells. Increased expression of p<sup>21</sup>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 sugges<sup>t</sup> 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 G1 cell cycle arrest. In addition, our data sugges<sup>t</sup> 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.
