Evidence for Steroidogenic Potential in Human Prostate Cell Lines and Tissues
2012; Elsevier BV; Volume: 181; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.06.009
ISSN1525-2191
AutoresNigel C. Bennett, John D. Hooper, Duncan Lambie, Cheok S. Lee, Tao Yang, David A. Vesey, Hemamali Samaratunga, David W. Johnson, Glenda C. Gobé,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoMalignant prostate cancer (PCa) is usually treated with androgen deprivation therapies (ADTs). Recurrent PCa is resistant to ADT. This research investigated whether PCa can potentially produce androgens de novo, making them androgen self-sufficient. Steroidogenic enzymes required for androgen synthesis from cholesterol (CYP11A1, CYP17A1, HSD3β, HSD17β3) were investigated in human primary PCa (n = 90), lymph node metastases (LNMs; n = 8), and benign prostatic hyperplasia (BPH; n = 6) with the use of IHC. Six prostate cell lines were investigated for mRNA and protein for steroidogenic enzymes and for endogenous synthesis of testosterone and 5α-dihydrotestosterone. All enzymes were identified in PCa, LNMs, BPH, and cell lines. CYP11A1 (rate-limiting enzyme) was expressed in cancerous and noncancerous prostate glands. CYP11A1, CYP17A1, HSD3β, and HSD17β3 were identified, respectively, in 78%, 52%, 16%, and 82% of human BPH and PCa samples. Approximately 10% of primary PCa, LNMs, and BPH expressed all four enzymes simultaneously. CYP11A1 expression was stable, CYP17A1 increased, and HSD3β and HSD17β3 decreased with disease progression. CYP17A1 expression was significantly correlated with CYP11A1 (P = 0.0009), HSD3β (P = 0.0297), and HSD17β3 (P = 0.0090) in vivo, suggesting CYP17A1 has a key role in prostatic steroidogenesis similar to testis and adrenal roles. In vitro, all cell lines expressed mRNA for all enzymes. Protein was not always detectable; however, all cell lines synthesized androgen from cholesterol. The results indicate that monitoring steroidogenic metabolites in patients with PCa may provide useful information for therapy intervention. Malignant prostate cancer (PCa) is usually treated with androgen deprivation therapies (ADTs). Recurrent PCa is resistant to ADT. This research investigated whether PCa can potentially produce androgens de novo, making them androgen self-sufficient. Steroidogenic enzymes required for androgen synthesis from cholesterol (CYP11A1, CYP17A1, HSD3β, HSD17β3) were investigated in human primary PCa (n = 90), lymph node metastases (LNMs; n = 8), and benign prostatic hyperplasia (BPH; n = 6) with the use of IHC. Six prostate cell lines were investigated for mRNA and protein for steroidogenic enzymes and for endogenous synthesis of testosterone and 5α-dihydrotestosterone. All enzymes were identified in PCa, LNMs, BPH, and cell lines. CYP11A1 (rate-limiting enzyme) was expressed in cancerous and noncancerous prostate glands. CYP11A1, CYP17A1, HSD3β, and HSD17β3 were identified, respectively, in 78%, 52%, 16%, and 82% of human BPH and PCa samples. Approximately 10% of primary PCa, LNMs, and BPH expressed all four enzymes simultaneously. CYP11A1 expression was stable, CYP17A1 increased, and HSD3β and HSD17β3 decreased with disease progression. CYP17A1 expression was significantly correlated with CYP11A1 (P = 0.0009), HSD3β (P = 0.0297), and HSD17β3 (P = 0.0090) in vivo, suggesting CYP17A1 has a key role in prostatic steroidogenesis similar to testis and adrenal roles. In vitro, all cell lines expressed mRNA for all enzymes. Protein was not always detectable; however, all cell lines synthesized androgen from cholesterol. The results indicate that monitoring steroidogenic metabolites in patients with PCa may provide useful information for therapy intervention. The term castrate-resistant prostate cancer (CRPC) implies that this type of prostate cancer (PCa) can proliferate and survive without the testes, the principal androgen-producing tissue. Androgens function via androgen receptor (AR), a ligand-activated nuclear transcription factor. Most CRPCs continue to express AR and androgen-regulated proteins such as prostate-specific antigen.1Yuan X. Li T. Wang H. Zhang T. Barua M. Borgesi R.A. Bubley G.J. Lu M.L. Balk S.P. Androgen receptor remains critical for cell-cycle progression in androgen-independent CWR22 prostate cancer cells.Am J Pathol. 2006; 169: 682-696Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar PCa may survive independently of androgens because of AR gene amplification, somatic missense mutations of the AR, AR coregulator mutations, altered growth factor signaling and kinase activation of AR, and AR activation by adrenal androgens.2Feldman B.J. Feldman D. The development of androgen-independent prostate cancer.Nat Rev Cancer. 2001; 1: 34-45Crossref PubMed Scopus (1953) Google Scholar, 3Attard G. Sarker D. Reid A. Molife R. Parker C. de Bono J.S. Improving the outcome of patients with castration-resistant prostate cancer through rational drug development.Br J Cancer. 2006; 95: 767-774Crossref PubMed Scopus (69) Google Scholar In CRPC, the androgens testosterone and dihydrotestosterone (DHT) are recorded at levels sufficiently high for activation of AR in patients who have undergone chemical or physical castration.4Mohler J.L. Gregory C.W. Ford III, O.H. Kim D. Weaver C.M. Petrusz P. Wilson E.M. French F.S. The androgen axis in recurrent prostate cancer.Clin Cancer Res. 2004; 10: 440-448Crossref PubMed Scopus (583) Google Scholar, 5Titus M.A. Schell M.J. Lih F.B. Tomer K.B. Mohler J.L. Testosterone and dihydrotestosterone tissue levels in recurrent prostate cancer.Clin Cancer Res. 2005; 11: 4653-4657Crossref PubMed Scopus (435) Google Scholar Recent results that show de novo androgen synthesis with the use of a PCa cell line (LNCaP) xenograft mouse model suggest PCa cells possess steroidogenic properties that enable survival in androgen-depleted environments.6Locke J.A. Guns E.S. Lubik A.A. Adomat H.H. Hendy S.C. Wood C.A. Ettinger S.L. Gleave M.E. Nelson C.C. Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer.Cancer Res. 2008; 68: 6407-6415Crossref PubMed Scopus (616) Google Scholar Therefore, CRPCs that express AR are likely to be androgen dependent, with the original source of androgen being replaced by adrenal or intratumoral androgen synthesis. These PCa cases are considered to be androgen self-sufficient.7Bennett N.C. Gardiner R.A. Hooper J.D. Johnson D.W. Gobe G.C. Molecular cell biology of androgen receptor signalling.Int J Biochem Cell Biol. 2010; 42: 813-827Crossref PubMed Scopus (196) Google Scholar Cholesterol is central to steroid hormone synthesis for normal reproductive function and bodily homeostasis. Steroid hormones, including androgens, are produced typically within the testes, adrenal glands, ovaries, placenta, and the brain.8Stocco D.M. Wang X. Jo Y. Manna P.R. Multiple signaling pathways regulating steroidogenesis and steroidogenic acute regulatory protein expression: more complicated than we thought.Mol Endocrinol. 2005; 19: 2647-2659Crossref PubMed Scopus (407) Google Scholar The rate-limiting enzyme in the steroidogenic pathway is CYP11A1, a member of the cytochrome P450 family localized to the inner membrane of mitochondria and predominantly expressed in the gonads and adrenal cortex.9Guo I.C. Hu M.C. Chung B.C. Transcriptional regulation of CYP11A1.J Biomed Sci. 2003; 10: 593-598PubMed Google Scholar CYP11A1 is responsible for side chain cleavage of cholesterol, converting it to pregnenolone. The following enzymes are required to convert pregnenolone to testosterone: CYP17A1 which contains both 17 α-hydroxylase (hydroxyl addition to pregnenolone and progesterone) and 17,20-lyase (side-chain cleavage from 17-hydroxyprogesterone and 17-hydroxypregnenolone) activity and is expressed in the testes and adrenal glands,10Chung B.C. Picado-Leonard J. Haniu M. Bienkowski M. Hall P.F. Shively J.E. Miller W.L. Cytochrome P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues.Proc Natl Acad Sci U S A. 1987; 84: 407-411Crossref PubMed Scopus (409) Google Scholar ovaries,11Wickenheisser J.K. Quinn P.G. Nelson V.L. Legro R.S. Strauss III, J.F. McAllister J.M. 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A novel communication role for CYP17A1 in the progression of castration-resistant prostate cancer.Prostate. 2009; 69: 928-937Crossref PubMed Scopus (31) Google Scholar; HSD3β which converts dehydroepiandrosterone to androstenedione and is expressed predominantly in adrenal gland, testis, and ovary15Simard J. Ricketts M.L. Gingras S. Soucy P. Feltus F.A. Melner M.H. Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.Endocr Rev. 2005; 26: 525-582Crossref PubMed Scopus (409) Google Scholar; and HSD17β3 which catalyzes the conversion of androstenedione and dehydroepiandrosterone to testosterone and androstenediol, respectively, is expressed in the cytoplasm and nucleus of cells within the seminiferous tubules of the testes.16Labrie F. Luu-The V. Lin S.X. Labrie C. Simard J. Breton R. Belanger A. The key role of 17 beta-hydroxysteroid dehydrogenases in sex steroid biology.Steroids. 1997; 62: 148-158Crossref PubMed Scopus (418) Google Scholar CYP11A1, CYP17A1, HSD3β, and HSD17β3 have received limited investigation in prostate tissues. The CYP17A1 protein was confirmed in prostate tissue by immunohistochemistry (IHC).14Locke J.A. Fazli L. Adomat H. Smyl J. Weins K. Lubik A.A. Hales D.B. Nelson C.C. Gleave M.E. Tomlinson Guns E.S. A novel communication role for CYP17A1 in the progression of castration-resistant prostate cancer.Prostate. 2009; 69: 928-937Crossref PubMed Scopus (31) Google Scholar HSD3β transcripts and protein have been reported in normal prostate and benign prostatic hyperplasia (BPH), but no isotype was described.17El-Alfy M. Luu-The V. Huang X.F. Berger L. Labrie F. Pelletier G. Localization of type 5 17beta-hydroxysteroid dehydrogenase, 3beta-hydroxysteroid dehydrogenase, and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry.Endocrinology. 1999; 140: 1481-1491Crossref PubMed Google Scholar HSD3β2 RNA, but not protein, has been reported in PCa.18Stanbrough M. Bubley G.J. Ross K. Golub T.R. Rubin M.A. Penning T.M. Febbo P.G. Balk S.P. Increased expression of genes converting adrenal androgens to testosterone in androgen-independent prostate cancer.Cancer Res. 2006; 66: 2815-2825Crossref PubMed Scopus (879) Google Scholar HSD17β3 was, until recently, found only in testis but has now been detected in some CRPCs.19Pfeiffer M.J. Smit F.P. Sedelaar J.P. Schalken J.A. Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer.Mol Med. 2011; 17: 657-664Crossref PubMed Scopus (93) Google Scholar Protein and mRNA expression for HSD17β3 has been confirmed in mouse LNCaP xenografts and mRNA in human PCa.6Locke J.A. Guns E.S. Lubik A.A. Adomat H.H. Hendy S.C. Wood C.A. Ettinger S.L. Gleave M.E. Nelson C.C. Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer.Cancer Res. 2008; 68: 6407-6415Crossref PubMed Scopus (616) Google Scholar, 20Castagnetta L.A. Carruba G. Traina A. Granata O.M. Markus M. Pavone-Macaluso M. Blomquist C.H. Adamski J. Expression of different 17beta-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells.Endocrinology. 1997; 138: 4876-4882PubMed Google Scholar RNA transcripts for CYP11A1 were found in human PCa tissues, but the protein was not analyzed.21Hofland J. van Weerden W.M. Dits N.F. Steenbergen J. van Leenders G.J. Jenster G. Schroder F.H. de Jong F.H. Evidence of limited contributions for intratumoral steroidogenesis in prostate cancer.Cancer Res. 2010; 70: 1256-1264Crossref PubMed Scopus (147) Google Scholar Recent reports show that mRNA for these enzymes and other steroidogenic enzymes are expressed weakly and variably in PCa cell lines and mouse xenograft models, but no IHC analysis has been performed for these enzymes, as a group, in localized PCa.6Locke J.A. Guns E.S. Lubik A.A. Adomat H.H. Hendy S.C. Wood C.A. Ettinger S.L. Gleave M.E. Nelson C.C. Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer.Cancer Res. 2008; 68: 6407-6415Crossref PubMed Scopus (616) Google Scholar, 21Hofland J. van Weerden W.M. Dits N.F. Steenbergen J. van Leenders G.J. Jenster G. Schroder F.H. de Jong F.H. Evidence of limited contributions for intratumoral steroidogenesis in prostate cancer.Cancer Res. 2010; 70: 1256-1264Crossref PubMed Scopus (147) Google Scholar The present investigation tested the hypothesis that prostate cell lines express mRNA and proteins for CYP11A1, CYP17A1, HSD3β2, and HSD17β3 and are able to produce androgens from cholesterol. In addition, human primary PCa, lymph node metastases (LNMs), and BPH were analyzed with IHC for expression and localization of these proteins. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Approval for use of human tissues was obtained from the Human Research Ethics Committee of the Royal Prince Alfred Hospital and The University of Queensland. Prostate cell lines LNCaP, 22Rv1, PC3, DU145, RWPE1 (American Type Culture Collection, Manassas, VA), and ALVA41 (provided by K.A. Landers, Queensland Institute of Medical Research, Herston, Australia) were used. LNCaP, 22Rv1, PC3, DU145, and ALVA41 are PCa cell lines, whereas RWPE1 was immortalized from normal prostate cells. The cells were routinely maintained in RPMI 1640 media with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen, Life Technologies, Mt. Waverley, Australia). RNA was collected twice from each cell line, and each collection was separated by at least two passages. RNA was extracted from cells with the use of an RNeasy Mini Kit (Qiagen, Clifton Hill, Australia) and reverse transcribed with a Superscript First Strand Synthesis System (Invitrogen, Carlsbad CA). Quantitative RT-PCR primers for the candidate genes were as previously published.22Montgomery R.B. Mostaghel E.A. Vessella R. Hess D.L. Kalhorn T.F. Higano C.S. True L.D. Nelson P.S. Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth.Cancer Res. 2008; 68: 4447-4454Crossref PubMed Scopus (1114) Google Scholar The following primers were synthesized: CYP11A1 forward, 5′-CTGCATCTTCAGTCGTCTGTCC-3′; CYP11A1 reverse, 5′-GGTGACCACTGAGAACCCATTC-3′; CYP17A1 forward, 5′-TCCCCAAGGTGGTCTTTCTGAT-3′; CYP17A1 reverse, 5′-GTGGACAGGGGCTGTGAGTTAC-3′; HSD3B2 forward, 5′-CTGCTGCCTCTCTTTCACACAA-3′; HSD3B2 reverse, 5′-AGAAAGTTCTGGTTGGGCCAGT-3′; HSD17B3 forward, 5′-CTGAAGCTCAACACCAAGGTCA-3′; HSD17B3 reverse, 5′-CTGCTCCTCTGGTCCTCTTCAG-3′; GAPDH forward, 5′-ACGACCACTTTGTCAAGCTC-3′; and GAPDH reverse, 5′-TCACAGTTGCCATGTAGACC-3′. Real-time PCR was performed in 15-μL volumes [cDNA, 2× QuantiTect SYBR Green PCR Master Mix (Qiagen Pty Ltd, Chadstone Centre, Australia), and 5 pmol of each primer] with the use of a 72-well Rotor-Gene 6000 Real-Time Rotary Analyzer (Corbett Life Science, Mortlake, Australia). Cycles consisted of a hot start (95°C for 15 minutes), denaturing step (20 seconds at 95°C), annealing step (20 seconds at 58°C), and an extension step (20 seconds at 72°C) for 35 to 40 cycles. The PCR finished with a melt curve between 65°C and 99°C. Standard curves for each gene were generated from control cDNA, then expression for each gene in each individual cell line was normalized to the expression of the housekeeping gene GAPDH in the same cell line. Data were analyzed by the Pfaffl method and are presented as mean ± SEM.23Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25091) Google Scholar For Western blot analysis, cell lysates were collected in RIPA buffer [150 mmol/L NaCl, 25 mmol/L NaF, 0.5 mmol/L EDTA, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% IGEPAL CA-630, in 50 mmol/L Tris-Cl buffer (pH 7.5)], containing protease and phosphatase inhibitors (2 μg/mL phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mmol/L sodium orthovanadate), and then sonicated briefly to disrupt cell membranes. Protein concentration was determined with a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Equal amounts of protein (40 μg) were separated with 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and probed with the selected antibodies. Polyclonal primary anti-human antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were as follows: CYP11A1 (sc-18043), CYP17A1 (sc-46084), HSD3β (sc-30820), and HSD17β3 (sc-66415). Duplicate Western blot analyses with blocking peptides (Santa Cruz Biotechnology) to inhibit specific antibody binding were done as antibody controls (data not shown). Horseradish peroxidase-conjugated secondary antibodies (Invitrogen, Thornton, Australia) were detected with chemiluminescence substrate (Pierce, Rockford, IL; Catalog numbers 1859674 and 1859675) and exposed to X-ray film. For treatments with 14C-labeled cholesterol, phenol red-free RPMI 1640 growth medium (no serum, no antibiotics) was used. Cells were grown to ≥80% confluence in 6-well tissue culture plates. Before treatment, cells were washed twice in PBS to remove residual serum components, then incubated for 24 hours in phenol red-free RPMI 1640 (no serum, no antibiotics). Then 14C-labeled cholesterol (1 μCi/well) in fresh phenol red-free RPMI 1640 (no serum, no antibiotics) was delivered to the cells and incubated for a further 7 days. Cells were lysed with 1 mol/L NaOH; lysates were collected, vortexed for 1 minute, and then incubated at room temperature for ≥90 minutes to allow base hydrolysis of steroid esters. Control lysates were spiked with 14C-labeled cholesterol immediately before diethyl ether extraction. Radiolabeled experiments were done in triplicate and repeated. Lysates from LNCaP, 22Rv1, PC3, DU145, ALVA41, and RWPE1 cells were extracted twice with diethyl ether. The organic fraction was evaporated under nitrogen gas. Precipitates were resuspended in 50% methanol/25% ethanol/25% water spiked with 10 μg/mL testosterone, 250 μg/mL DHT, and 50 μg/mL cholesterol. Waters high-performance liquid chromatography (HPLC) system, including an Alltima C18, 5 μm, 150 mm × 4.6 mm (Alltech, Baulkham Hills, Australia) column and Millenium version 2.10 software, was used for chromatographic separation. Each chromatography run was at 1 mL/min and as follows: isocratic flow 40:60 acetonitrile (ACN)/H2O (0 to 20 minutes), linear gradient to 50:50 can/EtOH (20 to 30 minutes), isocratic flow 50:50 ACN/EtOH (30 to 41 minutes), linear gradient to 40:60 ACN/H2O (41 to 42 minutes), and isocratic flow 40:60 ACN/H2O (42 to 50 minutes). Fractions for testosterone, DHT, and cholesterol were collected as spiked molecules eluted from samples treated with 14C-cholesterol and untreated. Fractions were vacuum evaporated, and precipitates were reconstituted in liquid scintillant (PerkinElmer, Waltham, MA) before radio-counting (Wallac MicroBeta 1450 LSC Counter; PerkinElmer). Samples were obtained from the Royal Prince Alfred Hospital and Aquesta Pathology (Toowong, Australia) and consisted of 6 BPH samples obtained by transurethral resection of the prostate, 90 samples of clinically localized PCa (79 from radical prostatectomy, 11 from transurethral resection of the prostate) of Gleason scores 5 to 10, and 8 samples of LNMs. Sections (4 μm) on Superfrost Plus histology slides were dewaxed and rehydrated to buffer. Primary antibodies were those used for Western blot analysis except for HSD3β, whereby sc-100466 was used. Antibody concentration was 4 μg/mL in Renaissance Antibody Diluent (Biocare Medical, Concord, CA). Secondary antibodies were from Dako (Dako Australia Pty Ltd, Botany, Australia). Positive control sections were from either human adrenal or testicular tissue. Negative controls were performed routinely without primary antibodies. Staining intensity and localization for CYP11A1, CYP17A1, HSD3β, and HSD17β3 were analyzed by N.C.B. and D.L. from de-identified slides. A positive result was recorded only if a sample had staining in PCa cells or glandular epithelium of BPH. When appropriate, data are presented as means ± SEMs. Linear regression was used to examine changes in steroidogenic enzyme expression correlating with increasing Gleason score and disease progression. Pearson's χ2 test with Fisher's exact test was used to ascertain relations in paired enzyme expression. P < 0.05 was considered significant. A one-tailed t-test was used for comparisons of samples treated with 14C-cholesterol, based on observations whereby changes in treated groups were only positive relative to the control. To determine whether the prostate cell lines were capable of synthesizing androgens de novo, we quantified transcripts encoding the enzymes responsible for synthesis of testosterone from cholesterol (CYP11A1, CYP17A1, HSD3β2, and HSD17β3). All four enzymes analyzed were expressed in all six prostate cell lines (Figure 1). In the cell lines LNCaP, 22Rv1, DU145, and RWPE1, expression for each enzyme was generally low relative to GAPDH. However, the cell lines PC3 and ALVA41 showed strong expression of all four enzymes compared with GAPDH. Western blot analysis was performed to examine protein expression of the selected steroidogenic enzymes in the six prostate cell lines. All four enzymes were expressed (Figure 2). The enzymes CYP17A1, HSD3β, and HSD17β3 were expressed in all six cell lines. CYP11A1 expression was found in PC3, DU145, ALVA41, and RWPE1 but not in LNCaP and 22Rv1. Human adrenal tissue was used as a positive control for CYP11A1, CYP17A1, and HSD3β. HSD17β3 is not expressed in human adrenal tissue. Fresh human testis tissue for a positive control of HSD17β3 protein was not available to us. However, positive staining for HSD17β3 in paraffin-embedded testis tissue was seen (Figure 3I).Figure 3IHC. For each antibody, positive controls for CYP11A1, CYP17A1, and HSD3β antibodies were prepared with human adrenal tissue (A, E, and I, respectively) and with human testis for HSD17β3 (M). Scale bars: 100 μm (A–P). CYP11A1: Positive staining can be seen in the adrenal cortex, and no staining was present in the medulla (A). CYP11A1 staining is shown in human BPH (B) and PCa (C and D) tissues. Staining in glands with BPH was predominantly in the cytoplasm of luminal epithelial cells and occasionally basal cells. The PCa staining was variable, with moderate-to-low level staining mainly in the cytoplasm of luminal epithelial cells. There was little nuclear staining. CYP17A1: Staining is evident in the cytoplasm of cells in the adrenal cortex, and no staining was present in the medulla (E). CYP17A1 staining is shown in glands with BPH (F) and PCa (G and H). Staining in BPH was predominantly cytoplasmic of luminal epithelial cells. The PCa staining was variable, with moderate-to-low level staining and mainly cytoplasmic of luminal epithelial cells. There was little nuclear staining. HSD17β3: Nuclear and cytoplasmic staining were seen in spermatogonia (along the basement membrane) and primary and secondary spermatocytes within the seminiferous tubules (I). The nucleus and cytoplasm of spermatids and spermatozoa cannot be differentiated, but these cells are positively stained (I). HSD17β3 staining is also shown in human BPH (J) and PCa (K and L). Staining in BPH was high and was predominantly cytoplasmic of luminal epithelial cells. There was occasional stromal cell staining. The cancer tissue staining was low to high and mainly cytoplasmic of luminal epithelial cells, but weak nuclear staining was also evident. Cytoplasmic staining in stromal and endothelial cells was generally low, but uniform across the sections. HSD3β: HSD3β was cytoplasmic in cells of the adrenal cortex glands, and no staining was present in the medulla (M). IHC staining is shown for HSD3β in human BPH (N) and PCa (O and P) tissues. Positive staining in BPH was moderate to high and cytoplasmic in the luminal epithelium. Staining in the cancer tissue was variable (minimal or absent through to moderate) and was mainly cytoplasmic in the epithelial cells. There was little nuclear staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IHC was used to investigate intensity and locality of protein expression of CYP11A1, CYP17A1, HSD3β, and HSD17β3 in BPH, primary PCa of varying Gleason scores, and LNMs (Table 1). IHC showed that all enzymes were expressed in well-differentiated and poorly differentiated PCa glands in samples of Gleason scores from 5 to 10. Only CYP11A1, CYP17A1, and HSD17β3 were present in LNMs, and only CYP11A1, HSD3β, and HSD17β3 were expressed in BPH. Each series of micrographs (Figure 3) has expression of each enzyme in positive tissue (adrenal gland or testis), BPH, and PCa. Negative controls for all antibodies were routinely clear of any stain (data not shown).Table 1Summary of Patient Samples Positive by Immunohistochemical Analysis for Expression of CYP11A1, CYP17A1, HSD3β and HSD17β3 in BPH, Primary PCa, and LMNNo. of positive samples (%)No. of samplesCYP11A1CYP17A1HSD3βHSD17β3BPH66 (100)02 (33%)6 (100%)Gleason score 522 (100)1 (50)02 (100) 62415 (62)15 (62)8 (33)20 (83) 73119 (61)7 (23)3 (10)26 (84) 81515 (100)12 (80)2 (13)12 (80) 91616 (100)12 (75)1 (6)13 (81) 1022 (100)1 (50)1 (50)2 (100)LNM86 (75)6 (75)04 (50)Total10481 (78)54 (52)17 (16)85 (82) Open table in a new tab In Figure 3, A–D, the positive control (Figure 3A; human adrenal tissue) had moderate-to-high expression in the glandular cortex. Low-to-high staining in BPH (Figure 3B) was predominantly cytoplasmic in the glandular luminal epithelium. Occasional epithelial basal cell cytoplasmic staining was also identified. There was little nuclear staining in these cells. In PCa, the staining intensity was variable (Figure 3, C and D), with moderate-to-low staining, mainly in the luminal epithelium. Staining in noncancerous regions was relatively consistent and of moderate intensity, particularly in the stroma, endothelial cytoplasm, smooth muscle, and nerve cells. CYP11A1 was expressed in 78% (100% BPH, 77% primary PCa, 75% LNMs) of the human prostate and PCa tissue samples investigated (Table 1). The number of positively stained cells within each positive tissue section ranged from 5% to 90% within glandular structures and cancerous foci. All Gleason score groups, BPH, and LNMs contained samples that were positive for CYP11A1. In Figure 3, E–H, expression was low in the cytoplasm of human adrenal tissue (Figure 3E). BPH (Figure 3F) staining was predominantly cytoplasmic in luminal epithelial cells and was variable, with low-to-high staining. PCa staining (Figure 3, G and H) was variable, with low-to-moderate staining mainly in the cytoplasm of the luminal epithelium. CYP17A1 was expressed in 52% (0% BPH, 53% primary PCa, 75% LNMs) of the human prostate samples investigated (Table 1). The number of positively stained cells within each positive tissue section ranged from 10% to 90% within glandular structures and cancer foci. PCa with a Gleason score of 6 frequently showed weak-to-intermediate cytoplasmic reactivity in most cells. Frequently in these cases, the noncancerous glands were also positive and showed a similar intensity of staining, in contrast to the lack of any staining in the BPH samples. In Figure 3, I–L, human testis was used as positive tissue (Figure 3I) and was localized to the cytoplasm and nucleus of cells within the seminiferous tubules as reported previously.24Abalain J.H. Quemener E. Carre J.L. Simon B. Amet Y. Mangin P. Floch H.H. Metabolism of androgens in human hyperplastic prostate: evidence for a differential localization of the enzymes involved in the metabolism.J Steroid Biochem. 1989; 34: 467-471Crossref PubMed Scopus (27) Google Scholar Spermatogonia along the basement membrane, primary and secondary spermatocytes, spermatids, and spermatozoa all stained positively for HSD17β3. Cells positive for HSD17β3 were found in human BPH (Figure 3J) and PCa (Figure 3, K and L). Staining in BPH was variable and predominantly cytoplasmic in luminal epithelial cells. PCa staining was variable, with low-to-high staining mainly in the cytoplasm of luminal epithelial cells. Cytoplasmic stromal staining was generally low but uniform across the sections. There was little nuclear staining. HSD17β3 was expressed in 82% (100% BPH, 83% primary PCa, 50% LNMs) of the human prostate tissue samples (Table 1). The number of positively stained cells within each positive tissue section ranged from 5% to 95% within glandular structures and cancer foci. All Gleason score groups, the BPH group, and the LNM group contained samples positive for HSD17β3 expression. The cancerous glands displayed more uniform cytoplasmic staining compared with the noncancerous glands that had a paler, slightly granular, cytoplasmic appearance and occasionally luminal membrane accentuation. In Figure 3, M–P, the human adrenal gland was used for positive control tissue (Figure 3M), where it
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