br In vivo experiments br Male
2.6. In vivo experiments
Male severe combined immune deficiency (SCID) mice (Lab Animal Shared Resource at RPCCC), or Hsd:Athymic Nude-Fox1nu nude mice (Envigo, Indianapolis, IN), were purchased at 5 weeks of age, and ex-perimental procedures were initiated when mice were 6 weeks old. Mice were surgically castrated, and silastic tubing (catalog number 508-008 from VWR, Radnor, PA) packed with T was implanted sub-cutaneously onto the mice during surgery. The procedure produced mice that had minimum inter-individual variations in circulating T; and the established circulating T levels were similar to the levels in adult male humans. The mice were inoculated subcutaneously with 2 × 106 VCaP Methylpiperidino pyrazole suspended in 0.1 mL Matrigel (Cat#356237, Corning Life Sciences, Corning, NY) 3 days after T-silastic tubing implantation. Treatment of each individual mouse was started when the size of the tumor on the mouse reached 300 mm3. T-silastic tubing remained on the mice in the control group. T-silastic tubing was removed and an empty silatic tubing was implanted on each mouse in the castration group. T-silatic tubing was removed and a silastic tubing packed with DHEA was implanted on each mouse in the castration plus DHEA group. Tumor size was monitored twice a week with a caliper. Tumor size was calculated using the equation: π/6 x L x W2, where L and W were length and width of a tumor, respectively (Tomayko and Reynolds, 1989). Mice were treated up to 8 weeks, or when tumor size reached 2000 mm3. The eﬀect on tumor growth between treatments was eval-uated using a growth rate-based approach (Hather et al., 2014). Briefly, the size of each tumor over the time course was transformed to log scale, growth rate of each tumor was calculated using the transformed tumor sizes and the corresponding time intervals. Ratios of growth rates of tumors on mice treated with various conditions were calculated and statistically analyzed. All experimental procedures were approved by the RPCCC Institutional Animal Care and Use Committee (IACUC) and were performed by staﬀ at the RPCCC Mouse Tumor Model Resources (MTMR) core facility.
2.7. Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)
Cell lines were treated without T (as controls) or 1 nM T for 24 h in respective phenol red-free medium supplemented with 10% CS-FBS as described in “Cell culture conditions”. RNA preparation and cDNA preparation were performed as described previously (Wu et al., 2013). Briefly, RNA was prepared using the QiaShredder Kit and the RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was generated using the Su-perScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA). Real-time PCR primers for STS and β-actin were purchased from Thermo Fisher Scientific. Real-time PCR reactions were performed using the Taqman Universal PCR Master Mix (Thermo Fisher Scientific) and an Applied Biosystems 7900HT Fast Real-Time PCR System (Foster City, CA). Expression levels of STS relative to the expression levels of β-actin were calculated using the equation: STS/β-actin = 2-STS(Ct−actinCt), where CtSTS and Ctactin were real-time PCR cycle numbers of STS and β-actin, respectively.
2.8. Immunohistochemistry (IHC)
TMA sections were de-paraﬃnized, rehydrated through an alcohol gradient, and antigen retrieved using a Reveal Decloaker (Biocare Medical, Concord, CA) for 30 min at 110 °C and 5.5–6.0 psi. Sections
Fig. 1. Expression of STS in human prostate cancer tissue and human prostate cancer cell lines, and the conversion of DHEAS to DHEA by prostate cancer cell lines
(A) STS expression in benign human prostate tissue and prostate cancer tissue at mRNA levels (n = 20); (B) STS expression at protein levels in matched benign and malignant prostate tissue specimens; (C) STS expression at mRNA levels in human prostate cancer cell lines; (D) DHEA production by prostate cancer cell lines treated with DHEAS for 1 day or 3 days. DHEA in the culture medium was normalized against cell numbers, which was indicated by units of OD570 measured using the MTT assay.
were blocked for endogenous peroxidase activity using 3% H2O2 in dd-H2O for 15 min at room temperature, washed in 10 mM Tris-HCl (pH 7.8), and blocked with Background Punisher (Biocare Medical) for 10 min at room temperature to reduce non-specific staining. Non-im-mune control rabbit IgG (LSBio, Seattle, WA), or rabbit anti-human STS antibody (Atlas Antibodies, Bromma, Sweden), was diluted in Renoir Red Diluent (Biocare Medical) and used at 0.0025 mg IgG/mL, or 0.002 mg IgG/mL, respectively. Sections were incubated overnight with the diluted antibodies at 4 °C. Following incubation with the primary antibody, slides were incubated with the Biocare MACH4 mouse probe and MACH 4 HRP polymer (Biocare Medical) at room temperature for 15 min, and immunostaining was developed using diaminobenzidine (Sigma-Aldrich). The sections were counterstained with hematoxylin (Vector Laboratories, Burlingame, CA), dehydrated and mounted using