Dihydrotestosterone synthesis pathways from inactive androgen 5α-androstane-3β,17β-diol in prostate cancer cells: Inhibition of intratumoural 3β-hydroxysteroid dehydrogenase activities by abiraterone

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3β,17β-diol (3β-diol), which is inactive androgen metabolized from DHT. 3β-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3β-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3β-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3β-diol, by the inhibition of the intratumoural 3β-hydroxysteroid dehydrogenase (3β-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3β-diol to DHT was catalysed by 3β-HSD and abiraterone could inhibit this activity of 3β-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3β-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3β-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

In this study, we performed experiments designed to demonstrate intratumoural DHT synthesis pathways form 3β -diol in PC cells and to show the effect of CYP17 inhibitors on intratumoural androgen metabolism by 3β -HSD.

Results
Androgenic activities and the existence of back-conversion of 3β-diol or EpiAND to upstream androgens in the pathway of DHT synthesis in PC cells. In order to assess androgenic activities of 3β -diol, DHEA and EpiAND, we measured PSA secretions, absorbance of MTS assay and changes of DHT levels secreted into the medium by LNCaP cells by using liquid chromatography-mass spectrometry (LC-MS) (Fig. 2). In the presence of each androgen, PSA secretions and absorbance were increased in a concentration-dependent manner. At a concentration of 10 nM, in particular, 3β -diol stimulated LNCaP cells stronger than DHEA or EpiAND. PSA secretions in the presence of 10 nM of DHEA, EpiAND and 3β -diol were 7.4 ± 0.9, 12.1 ± 2.7 and 33.6 ± 7.9 ng/ml (control, 6.7 ± 1.0 ng/ml), respectively, and EpiAND and 3β -diol stimulated PSA secretions significantly (both P < 0.001) (Fig. 2a). The ratios of absorbance in the presence of 10 nM DHEA, EpiAND and 3β -diol to that of the control (1.00 ± 0.11) were 1.04 ± 0.15, 1.13 ± 0.11 and 1.59 ± 0.20, respectively, and EpiAND and 3β -diol stimulated cell proliferations significantly (P < 0.01 and P < 0.01, respectively) (Fig. 2b). DHT levels in the presence of 10 nM of DHEA, EpiAND and 3β -diol were 0.02 ± 0.02, 0.52 ± 0.22 and 1.83 ± 0.57 pg/ml (control, 0.04 ± 0.03 pg/ml), respectively, and DHT levels were increased significantly by the addition of EpiAND and 3β -diol (both P < 0.05) (Fig. 2c). There were strong associations among PSA secretions, DHT and 3β -diol levels. The coefficient of correlation between PSA secretions and DHT levels, PSA secretions and 3β -diol levels, and DHT levels and 3β -diol levels were 0.83, 0.90 and 0.99, respectively (see Supplementary Fig. S1).
In order to assess back-conversion in detail, we measured levels of DHEA, A-dione, Δ 5-Adiol and T in the medium in LNCaP cells treated with 10 nM of 3β -diol or EpiAND for 3 days using LC-MS (Fig. 3). We showed the scheme of back-conversion in Fig. 3a. By the addition of 3β -diol, DHEA, A-dione and Δ 5-Adiol levels were not changed significantly (P = 0.64, P = 0.35 and P = 0.07, respectively), but T level was increased significantly (P = 0.03) (Fig. 3b-e). By the addition of EpiAND, Δ 5-Adiol and T levels were not changed significantly (P = 0.125 and P = 0.09, respectively), but DHEA and A-dione levels were increased significantly (P < 0.001 and P = 0.02, respectively) ( Fig. 3b-e). By the addition of EpiAND, DHEA and A-dione levels were increased higher than by the addition of 3β -diol (Fig. 3b,c). By the addition of 3β -diol, Δ 5-Adiol and T levels were increased higher than in the presence of EpiAND (Fig. 3d,e). These results suggested that 3β -diol and EpiAND tended to be converted back to upstream androgens, Δ 5-Adiol and DHEA, respectively.
Inhibition of intraprostatic 3β-HSD mediated conversion by abiraterone. We checked the cytotoxicity of the CYP17 inhibitors abiraterone, ketoconazole and orteronel. These three agents did not show severe cell toxicity at a concentration of 10 μ M (see Supplementary Fig. S2). PSA secretions into the medium by LNCaP cells in the presence of 10 nM of DHEA, EpiAND and 3β -diol were suppressed by the addition of each CYP17 inhibitor (Fig. 4a-c). Abiraterone suppressed PSA secretions in the presence of EpiAND and 3β -diol (P = 0.03 Optical density (OD). (c) DHT levels secreted into the medium in LNCaP cells (1 × 10 5 cells/ml) treated with 10 nM of DHEA, EpiAND or 3β -diol for 3 days were measured using LC-MS. PSA secretions and absorbances increased in the presence of EpiAND and 3β -diol in a concentration-dependent manner. At concentrations of 1 and 10 nM, 3β -diol exhibited higher androgenic activities than EpiAND in LNCaP cells. DHT levels were also higher in the presence of 3β -diol than in the presence of EpiAND. DHEA at 10 nM slightly stimulated LNCaP cell proliferation, but the stimulation was not significant and increase of DHT level was not measured. Data displayed are the mean ± s.d and are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, vs. CTRL, by using t-test. and P < 0.0001, respectively). Ketoconazole suppressed PSA secretions only in the presence of 3β -diol (P < 0.001) and orteronel did not suppress PSA secretions. These results suggested that abiraterone had the strongest influence on intratumoural androgen metabolism in CTP17 inhibitors.
In order to assess whether abiraterone could inhibit the 3β -HSD activity or not, we measured androgen secretions into the medium by LNCaP cells treated with or without abiraterone 10 μ M in the presence of 3β -diol, DHEA or EpiAND for 3 days using LC-MS (Fig. 5). We showed the schema of the metabolic pathway catalysed 3β -HSD including back-conversion and the inhibition of abiraterone in Fig. 5a. DHEA and Δ 5-Adiol levels were further increased by the addition of abiraterone in the presence of DHEA (P < 0.01 and P < 0.05, respectively) and and T (e) levels secreted into the media by LNCaP cells (1 × 10 5 cells/ml) treated with 10 nM of 3β -diol or EpiAND for 3 days were measured using LC-MS. By the addition of 3β -diol, DHEA, A-dione and Δ 5-Adiol levels were not changed significantly (P = 0.64, P = 0.35 and P = 0.07, respectively), but T level was increased significantly (P = 0.03). By the addition of EpiAND, Δ 5-Adiol and T levels were not changed significantly (P = 0.125 and P = 0.09, respectively), but DHEA and A-dione levels were increased significantly (P < 0.001 and P = 0.02, respectively). Data displayed are the mean ± s.d and are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, vs. CTRL, by using t-test. suppressed PSA secretions only in the presence of 3β -diol (P < 0.001) and orteronel (orte) did not suppress PSA secretions. Data displayed are the mean ± s.d and are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, -androgen (CTRL) vs. + androgen, + androgen vs. + androgen + CYP17 inhibitor, by using t-test.
there were similar tendencies in the presence of 3β -diol (DHEA level; P = 0.125, and Δ 5-Adiol level; P = 0.100) (Fig. 5b,d). The increases of A-dione and T levels were suppressed by the addition of abiraterone in the presence of DHEA (P < 0.001 and P = 0.080, respectively) and there were similar tendencies in the presence of 3β -diol (A-dione level; P = 0.643 and T level; P = 0.242, respectively) (Fig. 5c,e). 3β -diol and EpiAND levels were further increased by the addition of abiraterone in the presence of 3β -diol or EpiAND (3β -diol levels; P < 0.01 or P < 0.01, EpiAND levels; P < 0.05 or P < 0.01, respectively) ( Fig. 5f,g). The increase of 5α -A-dione level was suppressed by the addition of abiraterone in the presence of 3β -diol or EpiAND (P < 0.01 or P = 0.093, respectively) (Fig. 5h). These results suggested that abiraterone could not inhibit back-conversion from 3β -diol, but abiraterone could inhibit the 3β -HSD activities in all three points demonstrated. (a) Androgen metabolic pathway catalysed by 3β -HSD and back-conversion from 3β -diol. Abiraterone (abi). DHEA (b), A-dione (c), Δ 5-Adiol (d), and T (e) levels secreted into the medium in the presence of 10 nM of DHEA or 3β -diol by LNCaP cells (1 × 10 5 cells/ml) treated with or without 10 μ M abi for 3 days were measured using LC-MS. In the presence of 10 nM of EpiAND or 3β -diol, 3β -diol (f), EpiAND (g) and 5α -A-dione (h) levels secreted into the medium by LNCaP cells (1 × 10 5 cells/ml) treated with or without 10 μ M abi for 3 days were measured using LC-MS. DHEA and Δ 5-Adiol levels were further increased by the addition of abi in the presence of DHEA (P < 0.01 and P < 0.05, respectively) and there were similar tendencies in the presence of 3β -diol (DHEA level; P = 0.125, and Δ 5-Adiol level; P = 0.100) (b,d). The increases of A-dione and T levels were suppressed by the addition of abi in the presence of DHEA (P < 0.001 and P = 0.080, respectively) and there were similar tendencies in the presence of 3β -diol (A-dione level; P = 0.643 and T level; P = 0.242, respectively) (c,e). 3β -diol and EpiAND levels were further increased by the addition of abi in the presence of 3β -diol or EpiAND (3β -diol levels; P < 0.01 or P < 0.01, EpiAND levels; P < 0.05 or P < 0.01, respectively) (f,g). The increase of 5α -A-dione level was suppressed by the addition of abi in the presence of 3β -diol or EpiAND (P < 0.01 or P = 0.093, respectively) (h). Data displayed are the mean ± s.d and are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, CTRL vs. + androgen, + androgen vs. +androgen + abi, by using t-test.
Scientific RepoRts | 6:32198 | DOI: 10.1038/srep32198 DHT synthesis pathways from 3β-diol. In order to assess main metabolic pathways of 3β -diol to DHT, we also measured DHT secretion into the medium by LNCaP cells treated with or without abiraterone 10 μ M in the presence of 3β -diol, DHEA or EpiAND for 3 days using LC-MS (Fig. 6). We assumed three metabolic pathways from 3β -diol to DHT; via back-conversion, via EpiAND and 5α -A-dione, and direct-conversion catalysed 3β -HSD (Fig. 6a). In the presence of 3β -diol, DHT level was increased higher than in the presence of DHEA, however the levels of androgens as DHT resource such as DHEA, A-dione, Δ 5-Adiol and T in the presence of 3β -diol were much lower than in the presence of DHEA (Figs 5b-e and 6b). Additionally, the increase of DHT in the presence of 3β -diol was significantly suppressed by the addition of abiraterone. These results suggested that main metabolic pathway of 3β -diol to DHT would not be via back-conversion and the main metabolic pathway should to be inhibited by abiraterone. On the other hand, in the presence of 3β -diol, DHT level was increased higher than in the presence of EpiAND, however 5α -A-dione level as DHT resource was much lower than in the presence of EpiAND (Figs 5h and 6b). These results suggested that the pathway from 3β -diol to DHT via 5α -A-dione was not the main metabolic pathway. Thus, the direct-conversion catalysed by 3β -HSD was the main metabolic pathway of 3β -diol to DHT and abiraterone could inhibit the intratumoural multiple DHT synthesis pathways from 3β -diol by blocking 3β -HSD activities.

Discussion
In this study, we presented three important findings: the identification of intratumoural DHT synthesis pathways from 3β -diol including direct-conversion of 3β -diol to DHT; back conversion of 3β -diol to upstream androgens and inhibition of the intratumoural 3β -HSD-mediated conversion by abiraterone. The androgen 3β -diol is categorised as an inactive androgen because it cannot bind to the AR, and it may have an anti-prostate cancer potential by binding to ERβ [15][16][17][18][19] . In particular, DHT is converted to 3β -diol by 3β -HSD; however, in this study, 3β -diol was converted back to DHT and stimulated LNCaP cell growth. We confirmed three different metabolic pathways by which 3β -diol can be converted to DHT as shown in Fig. 6a. They are the known metabolic pathways via EpiAND and 5α -A-dione; back-conversion to DHEA and direct-conversion to DHT by 3β -HSD [26][27][28][29][30][31][32] . During ADT, DHEA produced in the adrenal gland is the main precursor of androgens for PC cells [9][10][11] . DHEA or Δ 5-Adiol is metabolised to A-dione or T by 3β -HSD and A-dione or T is metabolised to 5α -A-dione or DHT by 5α -reductase. Because these activities of 3β -HSD and 5α -reductase proceed in only one direction, intratumoural androgen metabolism has been thought as a unidirectional pathway. However, as we showed in this study, 3β -diol is converted back to upstream androgens such as DHEA or Δ 5-Adiol which was metabolised by 3β -HSD to A-dione or T, respectively. This back-conversion suggested the existence of a counter-flow in the intratumoural androgen cascade. We could not identify an enzyme working in this metabolic reversal, but the reversed flow was not inhibited by abiraterone, which suggests that the unknown enzyme may not be 3β -HSD.
We compared androgen profiles in the presence of DHEA, EpiAND, and 3β -diol at the same concentration. At concentrations of DHEA lower than physiological concentrations, DHEA was hardly metabolised to DHT via T. EpiAND was also hardly metabolised to DHT via 5α -A-dione. In the presence of 3β -diol, the level of DHT significantly increased, but levels of T or 5α -A-dione were lower than these levels in the presence of DHEA or EpiAND. Several reports showed the capacity of 3β -HSD to directly convert 3β -diol to DHT in the presence of NAD + in human cells, adrenal grand and placenta [29][30][31][32] . This direct-conversion had been seen in mouse prostate cells, but there were no reports about the existence of the direct-conversion in human prostate cells. In this study, we affirmed the direct-conversion of 3β -diol to DHT using LC-MS, and this is the first report concerning direct-conversion in prostate cancer cells. This increase of 3β -diol was suppressed by the addition of abi (P < 0.05). Data displayed are the means ± s.d. and are each representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, CTRL vs. +androgen, + androgen vs. + androgen + abi, by using t-test.
Scientific RepoRts | 6:32198 | DOI: 10.1038/srep32198 It had been suggested the existence of multiple mechanism of castration-resistance for PC. As one mechanism of CRPC progression, Nishiyama et al. had shown residual DHT in PC cells after receiving ADT was enough to stimulate PC cell growth 9 . The metabolism of 3β -diol including back-conversion and direct-conversion would be one reason of residual DHT. This phenomenon could be hardly applied to the mechanism of androgen-independent CRPC, which did not require androgens including DHT for its survival and growth, because our LC-MS results suggested that 3β -diol could not stimulate AR directly. In our real-time polymerase chain reaction (RT-PCR) result, AR related genes such as KLK3 and TMPRSS2 were increased by the addition of 3β -diol in the medium and abiraterone strongly suppressed these increases of mRNA (see Supplementary  Fig. S3). These RT-PCR results supported our findings that 3β -diol could not stimulate AR directly and 3β -diol promoted LNCaP cells due to the conversion to DHT by 3β -HSD. LNCaP cells could survive and proliferate in the CS-FBC medium with 3β -diol, whether LNCaP cells could not grow in the medium without 3β -diol (see Supplementary Fig. S4). We also affirmed that 3β -diol could promote cell growth of VCaP, which expressed wild type AR, not only LNCaP cells, which expressed mutant AR and these cell lines required androgens for its survival (see Supplementary Fig. S5) 46,47 . Thus, our findings would be one approach for the resolution of the mechanism with the object of intratumoral androgen synthesis in CRPC, which had remaining androgen dependence.
Ketoconazole has traditionally been used to treat CRPC in America and Europe; abiraterone and orteronel are among the new therapeutic agents for CRPC patients. Some reports have suggested that ketoconazole prevents intratumoural androgen metabolism, but the effect had not been clear [39][40][41] . Rue et al. showed that abiraterone could inhibit the activity of the 3β -HSD-catalysed conversion of DHEA to A-dione 43 . In this study, we could not demonstrate the significant prevention of intratumoural androgen metabolism by ketoconazole or orteronel (data did not shown). However, abiraterone significantly inhibited the androgenic activity of DHEA, EpiAND and 3β -diol, thereby showing its potential to prevent the effects of intratumoural androgen metabolism.
By adding abiraterone in the presence of those each androgens, androgen profiles were significantly changed. These changes were found at same points of the androgen metabolism pathway catalysed by 3β -HSD 27,32 . 3β -HSD-1 is expressed in prostate tissue, including PC cells. In vitro studies have strongly associated the activity of 3β -HSD-1 with the intratumoural conversion of DHEA to A-dione in the castration environment 27,28,33,34 . We performed knockdown of 3β -HSD-1 by using small interfering RNA (siRNA) technique for the affirmation of importance of 3β -HSD-1. PSA secretions by LNCaP cells treated siRNA of 3β -HSD-1 were significantly decreased (see Supplementary Fig. S6). Additionally, we confirmed the expressions of several androgen metabolic enzyme mRNA using RT-PCR (see Supplementary Fig. S7). The gene expressions of 17β -hydroxysteroid dehydrogenase 6 (HSD17B6), 10 (HSD17B10), retinol dehydrogenase 16 (all-trans) (RDH16) and retinol dehydrogenase 5 (RDH5) were detected in higher level than the expression of HSD3B1, however those enzymes were known as catalysts as 3α -HSD 48 . Therefore, we concluded that 3β -HSD-1 was most important enzyme which convert 3β -diol to DHT especially via direct-conversion. This is the first report to demonstrate that abiraterone could inhibit 3β -HSD-1 activity in all four points in the intratumoural androgen metabolic pathway.
One group has conducted a trial of intratumoural inhibition by abiraterone in CRPC patients in which the abiraterone was taken with food because taking abiraterone with food increases its blood concentration several fold, compared with taking abiraterone without food 44,45,49 . However, this trial did not show significant improvement. There may be several reasons why it is hard to directly apply this intratumoural inhibition to CRPC patients. We thought the strongest factor was the concentration of abiraterone. Looking at our study, it was clear that abiraterone could inhibit intratumoral 3β -HSD-1, but to exert this inhibition in PC cells, we needed a concentration of abiraterone higher than that usually used to treat CRPC patients 44,45 . In our study, intratumoural inhibition of 3β -HSD-1 by abiraterone had occurred in a concentration-dependent manner in the presence of 1-10 μ M abiraterone. However, at abiraterone concentrations lower than 1 μ M, we could not find significant inhibition (data did not shown). When we treat CRPC patients by inhibiting the activity of CYP17A1 in the adrenal gland and suppressing DHEA production, abiraterone is used at blood levels near 1 μ M. Therefore, to successfully apply this intratumoral inhibition strategy using abiraterone to CRPC patients, we need to find a way to increase and maintain the blood abiraterone level. We would also need to monitor the blood abiraterone level for safety purposes. The experience and knowledge described here suggests the importance of thinking about the intratumoural androgen environment and the possibility of inhibiting the intratumoural activity of 3β -HSD-1 as a new anti-prostate cancer treatment. Selective 3β -HSD-1 inhibitors would have stronger efficacy, selectivity for prostate cancer and less toxicity to the adrenal gland than abiraterone.
Recently, Zhenfei et al. reported a new association between abiraterone and 3β -HSD-1 50 . They reported that abiraterone had been converted to Δ 4-abirateorne (Δ 4-abi) by 3β -HSD-1. They also reported that Δ 4-abi had several-fold stronger potential to inhibit the activities of 3β -HSD-1, CYP17A1, 5α -reductase and AR in PC cells than abiraterone. Interestingly, the overexpression of 3β -HSD-1 stimulated PC cell growth; however, in the presence of abiraterone, 3β -HSD-1 would help to suppress PC cell growth by converting abiraterone to Δ 4-abi. These findings may represent another avenue for the discovery of new anti-prostate cancer agents. As described above, clarifying intratumoural androgen metabolism, including the function of 3β -HSD-1, would provide deeper insight into the mechanism of castration resistance in PC and identify possible avenues for the development of new CRPC treatments.
In conclusion, we affirmed that three metabolic pathways of 3β -diol to DHT, back-conversion to DHEA, via 5α -A-dione and direct-conversion by 3β -HSD, and these metabolism formed intratumoural DHT synthesis from inactive androgens. Abiraterone had the potential to inhibit intratumoral androgen metabolisms by the inhibition intratumoural 3β -HSD activities, not only to inhibit adrenal CYP17A1 activities. Intratumoural 3β -HSD activities, especially 3β -HSD-1 would be a new approach to treat CRPC. Cell culture and drug treatment. Cells were cultured in RPMI 1640 (Gibco Invitrogen, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% MEM nonessential amino acids, 1% MEM sodium pyruvate solution 100 mM, 0.14% NaHCO 3 , and 80 mg/L of kanamycin, at 37 °C in a humidified 5% CO 2 atmosphere. Cells were grown to sub-confluence and switched to steroid hormone-depleted medium without phenol-red, containing 10% charcoal-dextran stripped FBS (Biowest, Paris, France), with various concentrations of pharmacological agents for 3 days. In all experiments, the concentration of LNCaP cells and VCaP cells were 1 × 10 5 cells/ml and 2 × 10 5 cells/ml, respectively. For Tandem-R-PSA tests (Beckman Coulter Inc., San Diego, CA, USA), cells were cultured in 25 cm 3 cell culture flasks with 5 ml medium in triplicates. Experiments were performed with each androgen, in the presence or absence of each CYP17 inhibitor. Entire experiment was performed thrice. To determine the levels of androgens, including DHEA, Δ 5A-diol, A-dione, T, DHT, 5α -A-dione, EpiAND and 3β -diol in the culture medium, cells were cultured in 75 cm 3 cell culture flasks with 10 ml medium in the presence of 10 nM of each androgen, with or without 10 μ M of CYP17 inhibitors for 3 days. After the measurement of PSA levels of culture medium, levels of androgens in the culture medium were determined using LC-MS, as described below. Promega, Madison, WI, USA) was added to each well. According to manufacturer's instructions, the absorbance, which indicated relative cell proliferation and was shown as Optical density (OD) in figures, was determined using 490 nm filter and an iMark microplate reader (Bio-Rad Laboratories, Inc.). Results under each set of conditions were determined in triplicate or quadruplicate wells. All experiments were performed thrice.
LC-MS analysis. Levels of androgens, including DHEA, Δ 5-Adiol, A-dione, T, DHT, 5α -A-dione, EpiAND and 3β -diol, in the cell culture medium were determined using LC-MS using the procedure described by Takizawa. et al. 51 . In brief, the culture medium was extracted with ethyl acetate, and then the extracts were purified using a solid-phase extraction cartridge. After derivatization to picolinate ester forms, the concentrations of steroids were determined using LC-MS. Androgen levels were indicated as 'pg/ml' . The limits of quantification of DHEA, A-diol, A-dione, T, DHT, 5α -A-dione, EpiAND and 3β -diol were 2, 0.5, 0.5, 0.5, 0.5, 1, 0.5 and 1 pg/assay, respectively. All experiment were performed thrice.

RNA extraction and RT-PCR.
LNCaP cells were treated with 10 nM 3β -diol with or without 10 μ M abi for 3 days. After incubation, total RNA was isolated from the cells using the RNAqueous ® -4PCR Kit (Ambion, Austin, TX, USA), and cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit ® (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. Incubation conditions for the generation of cDNA were as follows: 10 min at 25 °C and 2 h at 37 °C. RT-PCR experiments were carried out using a standard TaqMan PCR protocol according to the manufacturer's recommendations (Applied Biosystems). Transcript of the housekeeping gene, β -actin (ACTB), was measured as the internal control. Assays were carried out using the ABI 7500 Real Time PCR system, Taqman Gene Expression assay mix ® (Applied Biosystems) and Taqman Gene Expression assay (gene name and assay ID were shown in Supplemental T1). PCRs were carried out after incubation at 50 °C for 2 min and denaturing at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. Quantification of target gene expression in samples was accomplished by measuring the fractional cycle number at which the amount of expression reached a fixed threshold (CT). Relative quantification was given by CT values, determined by triplicate reactions. Triplicate CT values were averaged and ACTB CT was subtracted to obtain Δ CT. Δ Δ CT was then calculated by subtracting Δ CT of the control (cells incubated in CS-FBS medium without any drugs for 3 days) from Δ CT of the sample. Relative expression levels were determined as 2 −ΔΔCT . All experiments were performed thrice.
Cell proliferation. To assess sufficiency of 3β -diol for LNCaP cells survival, cumulative differences in proliferation were measured. LNCaP cells were plated 1 × 10 6 cells per 75 cm 3 flasks in 10 ml phenol-red free medium with 10% CS-FBS with or without 10 nM 3β -diol. The cell culture medium were changed every 3 days and cell numbers were counted every 3 days. For cell counts, trypan blue was used to measure cell viability. All data were reported as numbers of viable cells counted using a cell counter (Countess ® II FL Automated Cell Counter, Invitrogen).
Transfection by using electroporation. LNCaP cells in steroid hormone-depleted medium without phenol-red, containing 10% CS-FBS were prepared for transfection. One × 10 5 LNCaP cells were re-suspended in 100 μ l resuspension buffer R (Neon ® Transfection System; Invitrogen, Carlsbad, CA, USA) with 2 μ M siRNA for HSD3B1 (s6926; Silencer ® select, Ambion) or control non-silencing siRNA (#1 siRNA; Ambion) and transfected in 100 μ l Neon tip with Neon transfection system (Invitrogen) using two pulses (1250 V input pulse voltage/20 ms input pulse width). Five × 10 4 transfected cells in 500 μ l phenol-red free medium containing 10% CS-FBS with 10 nM 3β -diol were plated each well of 24 well plate in triplicate and cultured for 3 days before Tandem-R PSA tests.