Anti-oncogenic effects of dutasteride, a dual 5-alpha reductase inhibitor and a drug for benign prostate hyperplasia, in bladder cancer

Cell lines and treatments

Two human urothelial carcinoma cell lines, T24 and J82, were obtained from the Korea Cell Line Bank (Seoul, Korea) and maintained in RPMI 1640 medium (Sigma-Aldrich, MO, USA), containing 10% fetal bovine serum (FBS) (Gibco, NY, USA) and 1% penicillin/streptomycin (Gibco). The human normal urothelial cell line SV-HUC1 was obtained from the American Type Culture Collection (ATCC, Manassas, USA) and cultured in Nutrient Mixture F-12 K medium (Gibco, NY, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were cultured in a humidified atmosphere of 5% CO2 at 37 °C.

For treatment with 5α-reductase inhibitor and testosterone, T24 and J82 cell lines were cultured with phenol-red-free RPMI 1640 medium (Gibco) containing 10% charcoal striped-FBS (CS-FBS) for 48 h. Dutasteride was selected among a group of 5α-reductase inhibitors because of its dual 5α-reductase inhibition effect. The optimal concentration of 5α-reductase inhibitor was determined by evaluating harmless cytotoxic conditions in normal bladder and BCa cell lines using concentrations ranging from 0 to 5 μM.

Reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted using Labozol (CMRZ001, Labopass, Seoul, Korea) according to the manufacturer's instructions. RNA concentration was measured using a NanoPhotometer (IMPLEN, Müchen, Germany), and 2 µg of total RNA was used to synthesized cDNA using the oligo dT primer with M-MuLV reverse transcription kit (CMRT010, Labopass). The cDNA sequences of interest were amplified by PCR using rTaq Plus 5× PCR Master Mix (EBT-1319, Elpisbiotech, Daejeon, Korea) and the primers listed in Additional file 1: Table S1, and the PCR products were analyzed with 1–2% agarose gel electrophoresis. Band densities were measured using Image J (version 1.52p, National Institutes of Health, MD, USA) and normalized against the housekeeping gene GAPDH to calculate relative RNA expression.

PCR products were quantified by quantitative real-time PCR (qPCR) using HiPi Real-Time PCR 2× Master Mix (SYBR green, ROX) (EBT-1802, Elpisbiotech). The primer sequences for the genes analyzed in this study are listed in Additional file 1: Table S2.

Lentivirus production

The lentiviral plasmid for AR overexpression (pAR) and lentiviral control plasmid (pLenti) were purchased from Addgene (Watertown, MA, USA). Lentiviral packaging and envelope plasmids (Addgene) were used to produce lentiviruses from the packaging cells using Invitrogen Lipofectamine 3000 reagent (Carlsbad, CA, USA). HEK293T cells were cultured in DMEM high medium (Sigma-Aldrich, MO, USA) supplemented with 10% fetal bovine serum and transfected with the viral packaging plasmids and specific lentiviral vector in 6well plate according to the manufacturer’s instructions. After 12–16 h of transfection, culture media were replaced with fresh media and cells were cultured for 48–72 h. Culture media were collected and passed through a syringe filter with a 0.45 µm pore size to obtain the lentiviral soup utilized for infection of the T24 cell line.

To evaluate the SRD5A1-silencing effects in T24 and J82 cell lines, an additional lentiviral plasmid (shSRD5A1) was purchased from Vectorbuilder Inc. (Chicago, USA) and lentiviral samples were prepared as explained above using the same packaging and envelop plasmids.

Western blot

Total cell lysates were isolated using RIPA lysis buffer (Biosesang, R2002), incubated on ice for 15 min, vortexed every 3 min, and centrifuged at 13,000 rpm for 15 min. Protein concentration in the supernatant was estimated using a BCA protein assay kit (Thermofisher Scientific). Protein samples (10 µg) were separated by electrophoresis using Bolt™ 4–12% Bis–Tris Plus 15-well gels (Invitrogen, NW04125BOX, CA, USA) and transferred to nitrocellulose membranes using iBolt2 Transfer Stacks (Invitrogen, IB23001) and iBolt 2 Dry Blotting System (Invitrogen). Next, the membranes were incubated with primary antibodies against anti-β-actin (1:1000, Santa Cruz, sc-47778), Bcl-2 (1:500, Santa Cruz, sc-509, TX, USA), MMP2 (1:500, Santa Cruz, sc-13595), MMP9 (1:500, Santa Cruz, sc-13520), IκBα (1:500, Cell Signaling Technology, 4814S, Danvers, MA,), phosphorylated-IκBα (1:500, Cell Signaling Technology, 2859S), p65 (1:500, Cell Signaling Technology, 8242S), and phosphorylated-p65 (1:500, Cell Signaling Technology, 3033S) at 4 °C overnight. The membranes were washed three times for 10 min using Tris-buffered saline and Tween 20 buffer (TBS-T; 1:1000) and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) including anti-mouse (1:3000, Santa Cruz, sc-2005), rabbit (1:3000, Santa Cruz, sc-2004), or goat (1:3000, Santa Cruz, sc-2020) at RT for 2 h. Membranes were then washed with TBS-T for 30 min and the immunoreactive proteins were visualized using an ECL detection kit (Advanstar, K-12045-D50) and measured using iBright Analysis Software (Invitrogen).

Cell growth and viability assay

The 5α-reductase inhibitor dutasteride (Santa Cruz, sc-207600) was selected for the experiments because it inhibits both the Type-1 and Type-2 isoforms. SV-HUC1, T24, and J82 cell lines were seeded in 12-well culture plates to analyze cell proliferation. The culture medium was replaced with fresh medium containing different dutasteride concentrations (0, 0.01, 0.1, 0.1, 2.5, and 5 µM) to determine the optimal concentration for the experiments. After 24 and 48 h, the number of cells at each concentration was counted using a hemocytometer via trypan blue exclusion. To evaluate the effects on cell growth of 5α-reductase inhibitor treatment, T24 and J82 were cultured in phenol-red free RPMI1640 (Gibco) containing 10% charcoal striped-FBS (CS-FBS) with or without testosterone (10 nM, Abmole, M6105, USA) and 5α-reductase inhibitor for 4 days, and the number of cells was counted every 24 h.

To measure cell viability, T24 pLenti and pAR cells (2 \(\times \) 103 cells/well) were cultured into 96-well culture plates, and 10% (v/v) EZ-cytox (DoGeneBio, Seoul, Korea) was added to the medium after 24 and 48 h. After incubation for 1 h, the absorbance in each well was measured at 450 nm using a Bio-RAD x-MarkTM microplate spectrophotometer (Bio-Rad Laboratories, CA, USA), as previously described [17,18,19,20].

Colony forming unit (CFU) assay

T24 and J82 cells (200 cells/well) were cultured with phenol-red free RPMI1640 containing 10% CS-FBS in 6-well plates and changed every 3 days into fresh media with or without testosterone and dutasteride treatment in a humidified atmosphere of 5% CO2 at 37 °C for 2 weeks. Then each well was fixed using 4% paraformaldehyde (PFA) (Biosesang, HP2031, Seoul, Korea) and stained with 0.5% crystal violet (Sigma-Aldrich, V5265). Photographs of the colonies were obtained using a light microscope (ZEISS, Axiovert 40 C). and colony numbers were determined using Image J software, as previously described [20].

Wound closure assay

Cell lines were precultured in phenol-red free RPMI 1640 containing 10% CS-FBS for 48 h with or without testosterone and dutasteride treatment. Then cells were seeded into 6-well culture plates at approximately 90% confluence and treated with 10 µg/mL of mitomycin for 2 h. The cell monolayer was scratched using the end of a 1000-µL pipette tip, and debris caused by the scratch was completely removed. The wound closure areas in the wells were photographed every 12 h for up to 2 days. The closure area was estimated using TScratch (version 1.0, Swiss Federal Institute of Technology, Zurich, Switzerland) and the closure percentage (%) was calculated, as previously described [17,18,19].

Migration assay

To analyze the migratory ability, 5 \(\times \) 104 cells in 100 µL serum-free media were placed into the upper chamber with 0.8 µm pore size of a transwell plate (Corning, 3413, NY, USA), and culture media containing serum were filled into the bottom chamber. After incubation for 24 h, transwell plates were fixed using 4% PFA and stained with 0.5% crystal violet. Photographs were obtained using a a light microscope (ZEISS, Axiovert 40C). and analyze using ImageJ to estimate the relative migrated area, as previously described [21].

Clinical expression and survival dataset

The mRNA microarray data of steroid 5 alpha reductase 1, 2 (SRD5A1 and SRD5A2) in BCa and normal tissues were acquired from the Oncomine database version 4.5 (Thermo Fisher Scientific Inc., MI, USA) [22, 23] and analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA, Beijing, China) [24]. Differences in mRNA expression of paired bladder tumor and corresponding normal bladder tissues were considered statistically significant at p-value < 0.05 and fold-change cutoff > 2.

The R2: Genomics Analysis and Visualization Platform (Academic Medical Center, Amsterdam, The Netherlands) was used to analyze the survival rate of BCa patients based on SRD5A1 and SRD5A2 mRNA expression. A threshold with a Cox p-value < 0.05 was considered statistically significant.

Gene correlation analysis

Correlated genes of the SRD5A1 were investigated from TCGA BLCA and Hoglund datasets, using the R2 database. The analysis was performed with the adjustment of the Bonferroni test using a threshold p-value < 0.01. Subsequently, Venn diagrams were used to classify positively correlated genes between TCGA BLCA and Hoglund datasets in BCa, using Venny 2.1.0 (Spanish National Biotechnology Centre (CNB)-CSIC, Madrid, Spain).

Enrichr gene ontology and signaling pathway analysis

To analyze the ontology and signaling pathway of SRD5A1 and co-expressed genes, the Enrichr database was used [25]. GO and pathway analyses were visualized as a bar graph. Kyoto Encyclopedia of Genes and Genomes information (KEGG) pathway, Reactome pathway, GO biological process, molecular function, and cellular component were also included in the analysis.

Statistical analysis

All statistical analyses were performed with GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA). Statistical significance was analyzed by multiple t-test, one-way ANOVA, and two-way ANOVA. p < 0.05 was considered to be statistically significance.

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