**1. 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 options for the treatment of CRPC have been approved, including CYP17 inhibitors, androgen receptor (AR) antagonists, and taxane [5]. Despite progress in the development of new drugs, there is a strong medical need to optimize the sequence and combination of approved drugs.

Drug repositioning or repurposing is the process of finding new uses for existing drugs [6], provided that additional clinical trials are relatively easy to perform, and the drug safety profiles have been established. In PCa, there have been a number of drug repositioning studies of non-cancer drugs, including the antidiabetic drug troglitazone, which is a ligand for peroxisome proliferator-activated receptor gamma [7]; the antihypertensive drug candesartan, which is an angiotensin II receptor blocker [8]; naftopidil, which is a selective <sup>α</sup>1-adrenoceptor antagonist used to treat benign prostatic hyperplasia [9]; and the antiallergy drug, tranilast [10]. A drug repositioning approach helps identify new pharmaceutical processes to transform existing drugs into useful sources of new anticancer drugs [11].

Pirfenidone (PFD) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis, an interstitial lung disease characterized by accumulation of fibroblasts/ myofibroblasts, excessive extracellular matrix production, and altered transforming growth factor β (TGFβ)/bone morphogenetic protein signaling [12,13]. A number of studies have reported that PFD treatment suppresses the growth of and induces G1 cell cycle arrest in stromal cells, rat hepatic stellate cells [14], and human Tenon fibroblasts [15,16]. Interestingly, PFD treatment has also been reported to suppress the growth of epithelial cells/cancer cells, including human lens epithelial cells [17] and human hepatocellular carcinoma cells [18]. Epidemiologically, Miura et al. reported a reduced incidence of lung cancer in patients with idiopathic pulmonary fibrosis treated with PFD [19]; however, the mechanism of PFD-induced cancer cell suppression is not well characterized.

Many studies on CRPC have used androgen-insensitive PCa cell lines, such as PC-3 and DU145 cells, which do not express AR [20]. These cell lines were derived from highly anaplastic tumors from different metastatic sites in the bone and brain [21,22]. The PC-3 and DU145 cell lines both differ strongly in aggressiveness compared with the androgen-sensitive, AR-positive LNCaP cell line, which was derived from a lymph node metastasis [23]. Comparisons between androgen-sensitive LNCaP cells and androgen-insensitive PC-3 and DU145 cell lines may not be relevant to the acquisition of androgen insensitivity in clinical PCa, because many clinical androgen-insensitive PCa cases express AR. A more accurate model of clinical cancer requires, at the very least, an androgen-insensitive, AR-positive cancer cell line. To compare the biochemical characteristics of androgen-insensitive and sensitive PCa cells, we generated three sublines from androgen-sensitive LNCaP cells: E9 and F10 (androgen-low-sensitive) and AIDL (androgen-insensitive) cells [24–26]. The parental LNCaP cell line and its derivative E9, F10, and AIDL sublines express similar levels of the AR protein, but androgen-dependent secretion of the prostate-specific antigen (PSA) is only detected in LNCaP cells [27]. In this study, we used the LNCaP cell line and its sublines to investigate the effects of PFD treatment on the growth of human PCa cells, focusing on androgen sensitivity.

### **2. Materials and Methods**
