Benign Prostatic Hypertrophy Tissue Research Articles

PLC\u03b5 regulates prostate cancer mitochondrial oxidative metabolism and migration via upregulation of Twist1

BackgroundMetabolic rewiring is a common feature of many cancer types, including prostate cancer (PCa). Alterations in master genes lead to mitochondrial metabolic rewiring and provide an appealing target to inhibit cancer progression and improve survival. Phospholipase C (PLC)ε is a regulator of tumor generation and progression. However, its role in mitochondrial metabolism remains unclear.MethodsThe GEO, The Cancer Genome Atlas, and the GTEx databases were used to determine Twist1 mRNA levels in tumors and their non-tumor counterparts. Fifty-five PCa and 48 benign prostatic hypertrophy tissue samples were tested for the presence of PLCε and Twist1 immunohistochemically. An association between PLCε and Twist1 was determined by Pearson’s correlation analysis. PLCε was knocked down with a lentiviral short hairpin RNA. Mitochondrial activity was assessed by measuring the oxygen consumption rate. Western blotting analyses were used to measure levels of PPARβ, Twist1, phosphorylated (p)-Twist1, p-MEK, p-ERK, p-P38, and p-c-Jun N-terminal kinase (JNK). Cells were treated with inhibitors of MEK, JNK, and P38 MAPK, and an agonist and inhibitor of peroxisome proliferator activated receptor (PPAR) β, to evaluate which signaling pathways were involved in PLCε-mediated Twist1 expression. The stability of Twist1 was determined after blocking protein synthesis with cycloheximide. Reporter assays utilized E-cadherin or N-cadherin luciferase reporters under depletion of PLCε or Twist1. Transwell assays assessed cell migration. Finally, a nude mouse tumor xenograft assay was conducted to verify the role of PLCε in tumor formation.ResultsOur findings revealed that the expression of PLCε was positively associated with Twist1 in clinical PCa samples. PLCε knockdown promoted mitochondrial oxidative metabolism in PCa cells. Mechanistically, PLCε increased phosphorylation of Twist1 and stabilized the Twist1 protein through MAPK signaling. The transcriptional activity of Twist1, and the Twist1-mediated epithelial-to-mesenchymal transition, cell migration, and transcription regulation, were suppressed by PLCε knockdown and by blocking PPARβ nuclear translocation. The tumor xenograft assay demonstrated that PLCε depletion diminished PCa cell tumorigenesis.ConclusionsThese findings reveal an undiscovered physiological role for PLCε in the suppression of mitochondrial oxidative metabolism that has significant implications for understanding PCa occurrence and migration.

Neoadjuvant immunotherapy for prostate cancer with GM-CSF and tumor infiltration by antigen presenting cells

3063 Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for antigen presenting cells that may enhance anti-tumor immune responses. Although this cytokine is being evaluated in immunotherapy trials for different malignancies, the impact of GM-CSF dose and schedule has not been directly addressed. Because systemic GM-CSF could potentiate anti-tumor immunity by enhancing presentation of endogenous tumor antigens, we examined whether increasing duration of GM-CSF treatment prior to radical prostatectomy could alter the composition of antigen presenting cells within the prostate tissue. Methods: A phase I trial in patients with localized prostate cancer was performed in subjects who were planned to undergo radical prostatectomy (RP). Two cohorts of 6 patients were sequentially accrued and treated with either 2 weeks or 3 weeks of daily GM-CSF (sargramostim, Bayer) 250 mg/m2/d SC. Patients were monitored for toxicity as well as for changes in circulating immune cells. RP tissues were assessed for immune cells by flow cytometry or immunohistochemistry (IHC) and digital slide scanning. In addition, IHC was also performed on RPs from untreated control Gleason grade- matched patients. Results: 12 patients were treated with neoadjuvant GM-CSF for either 2 weeks or 3 weeks. While significant leukocytosis was observed with treatment, no dose-limiting toxicities were encountered. As expected, circulating monocytes and dendritic cells (DC) were expanded following treatment. Compared to RPs from 11 untreated control patients, there was an increase in prostate-resident CD1a+ interstitial DC (Kruskal-Wallis test: p = 0.04). The increase was most pronounced in the 3-week treatment cohort. Moreover, tumor-resident DC expressed higher levels of the activation markers CD83 and 86 compared to DC residing in adjacent normal prostate and benign prostatic hypertrophy tissue. Conclusions: Systemically administered neoadjuvant GM-CSF can enhance the frequency of DC in prostate cancer. These effects appear to be dose dependent as they were most pronounced with 3 weeks of treatment. The composition of resident DC and monocytes also differs between malignant, benign, and hypertrophic prostate tissue. No significant financial relationships to disclose.

Estradiol/Androgen Receptors During Aging: Microsomal Distribution in Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor lev...

721Alterations in calcium-activated potassium channel expression in human prostate cancer

Duk Yoon Kim, Eun Kyoung Yang, Jung Wook Kim Department of Urology, Catholic University of Daegu School of Medicine, Department of Physiology, Kyungpook National University School of Medicine, Daegu, Korea Purpose: Recent studies have shown that potassium (K) and sodium channels are involved in prostate cell growth. However, a great many of the studies have been done in prostate cancer cell lines and there are only scant studies on prostate cancer and benign prostatic hypertrophy (BPH) tissue. The present study was aimed to evaluate the alterations of the calcium-activated K channel (KCa) expression in prostate cancer, and to compare them with the expression profiles in human BPH tissue to understand their potential role in the progression of prostate cancer. Materials and Methods: The prostate tissues obtained from radical prostatectomy (n=10) and transurethral resection of the prostate (n=18) were quickly frozen in liquid nitrogen for the RNA measurements. The protein and mRNA levels of the KCa subtypes and connexins were measured by performing immunoblot analysis and reverse-transcription polymerase chain reaction, respectively. Results: The mRNA levels of type 2 (SK2) and type 3 (SK3) small-conductance and large-conductance (BK) KCas in the prostate cancer tissues were decreased more than 50% compared with those in the BPH samples. In addition, the BK and SK2 protein levels in prostate cancer were also significantly lower than those in the BPH. As reported previously, the connexin 26 and 43 transcript signals in the prostate cancer were significantly reduced compared with those in the BPH samples. Conclusions: These results suggest that the impaired expression of KCas may have a role in tumor progression via aberrant and uncontrolled prostate cell growth. (Korean J Urol 2006;47:548-552) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

Estradiol/Androgen Receptors During Aging: Microsomal Distribution In Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor levels (mean percentage +/- 50%). These findings suggest that the biological effects due to the subcellular distribution of AR or ER and that their microsomal distribution would constitute a reserve mechanism and activation of receptors for the continued growth of benign prostatic hypertrophy.

Expression of vascular endothelial growth factor receptors in human prostate cancer

Objectives. We recently reported the expression and cytokine regulation of vascular endothelial growth factor (VEGF) in human prostate cancer (PCa). VEGF exerts its angiogenic and pro-tumorigenic properties by way of two high affinity receptors, fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1). We hypothesized that these receptors are expressed and control VEGF functions in the PCa microenvironment. Herein, we evaluate the expression of these receptors in ex vivo PCa tissue, benign prostatic hypertrophy (BPH) tissue, and cultured PCa cell lines. Methods. Ex vivo PCa specimens were obtained from patients undergoing radical retropubic prostatectomy. Specimens were selected to contain both PCa and BPH tissue (n = 15). Immunohistochemical analysis using antihuman FLT-1 and FLK-1 was performed and specimens were analyzed to characterize the expression and distribution of both receptors. Immunocytochemical analysis for FLT-1 and FLK-1 was also performed on cultured PCa cell lines (DU-145 and LNCaP). Results. PCa cells expressed the VEGF receptor FLT-1 in 100% of specimens evaluated. Expression of FLK-1 was variable and related to tumor grade; high-grade tumors displayed little or no FLK-1 expression. Vascular endothelial cells (VECs) within areas of PCa consistently expressed both FLT-1 and FLK-1 receptors. FLT-1 and FLK-1 were both expressed in BPH tissue. FLT-1 was expressed in the glandular epithelial cells in BPH, but in most cases FLK-1 was localized specifically to the basal cell layer of hypertrophic glands. FLT-1, but not FLK-1, was expressed by the DU-145 and LNCaP cell lines. Conclusions. Although they are differentially expressed, both FLT-1 and FLK-1 are present in PCa and BPH. Expression of receptors on VECs of tumor vessels supports the well-established role of VEGF in paracrine stimulation of VECs in the tumor microenvironment. The expression of FLT-1 and FLK-1 on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), in addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development.

N-Acetyltransferase expression and DNA binding of N-hydroxyheterocyclic amines in human prostate epithelium.

Intact prostate epithelial cells prepared from benign prostatic hypertrophy tissues from two patients were incubated for 2 h with N-hydroxy derivatives of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (N-OH-PhIP) or 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (N-OH-MeIQx). (32)P-post-labeling analysis detected PhIP and MeIQx adducts in the DNA of these cells but not in the untreated control. Adduct levels were approximately 100 times greater in N-OH-PhIP- than in N-OH-MeIQx-treated cells. Repair synthesis of DNA was observed in cells, prepared from two additional patients, treated for 24 h with these carcinogens and was greater for N-OH-PhIP than for N-OH-MeIQx. PhIP, MeIQx and their nitro derivatives did not produce repair synthesis of DNA in this system. The difference in the activity of N-OH-PhIP and N-OH-MeIQx may be due to their stability, since N-OH-MeIQx decomposed rapidly in neutral solution. Transcripts of NAT1 and NAT2 were detected by an in situ hybridization method in prostate epithelial cells, but were absent from stromal tissues. These results suggest that PhIP may be a potential carcinogen for human prostate, since cooked meats, which contain this heterocyclic amine, have been associated with human prostate cancer.

Genistein inhibits the growth of human-patient BPH and prostate cancer in histoculture.

There is strong epidemiological evidence that prostate disease is significantly less prevalent in the Orient, where the intake of soy products is very high, than in the United States. We therefore undertook a study of the effects of genistein, a major component of soy, on growth of human-patient benign prostatic hypertrophy (BPH) and prostate cancer tissue in three-dimensional collagen gel-supported histoculture. Surgical specimens of human BPH and cancer were histocultured for 5 days to study the effects of genistein on growth, as measured by inhibition of 3H-thymidine incorporation per microgram protein on day 5. Genistein in doses of 1.25-10 micrograms/ml decreased the growth of BPH tissue in histoculture in a dose-dependent manner, with little additional effect at higher doses. Prostate cancer tissue in histoculture was similarly inhibited by these doses of genistein. Genistein decreases the growth of both BPH and prostate cancer tissue in histoculture. The data suggest that genistein has potential as a therapeutic agent for BPH and prostate cancer.

Frequent abnormalities of TSG101 transcripts in human prostate cancer.

TSG101 has been identified as a candidate tumor suppressor gene and abnormal transcripts have been identified in a substantial fraction of breast cancers. To determine whether TSG101 expression is commonly altered in other tumors, a series of 15 primary and metastatic prostate cancers were analysed by reverse transcriptase-PCR amplification. Abnormal transcripts with extensive deletions in the coding region were found in nine of these tumors, while only the normal transcript was found in control and benign prostatic hypertrophy tissues. More than one abnormal transcript was found in four of these nine cases and distinct abnormal TSG101 transcripts were found in separate biopsies taken from one tumor. Importantly, the normal TSG101 transcript was undetectable in two metastatic prostate cancers, indicating the absence of TSG101 protein. Sequence analysis demonstrated that there were at least six distinct deletions, with four of these deletions found in more than one tumor sample. The most commonly identified deletion, from bp 153 to 1055, was identical to a deletion reported previously in breast cancer. These results demonstrate that TSG101 transcripts are frequently abnormal in prostate cancer and suggest that loss of TSG101 protein contributes to disease development or progression.

Alpha‐1 adrenoceptor subtypes (high, low) in human benign prostatic hypertrophy tissue according to the affinities for prazosin

BACKGROUND A novel classification of α-1 adrenoceptor subtypes (High, Low) was applied to human benign prostatic hypertrophy (BPH)tissue. METHODS Human BPH specimens were examined by a radioligand binding assay method using 3H-prazosin, and those data were compared with preoperative therapies. RESULTS (1) Scatchard analysis showed a high-affinity site (Kd:27.18 ± 6.41 pM; Bmax:9.29 ± 0.98 fM/mg protein; mean ± SE) as α1H, and a low-affinity site (Kd:4088.0 ± 744.34 pM; Bmax:140.81 ± 19.98 fM/mg protein; mean ± SE) as α1L subtype, for prazosin. (2) The Kd and Bmax were not different in the nontreated group (n = 5), α1 blocker group (n = 5), and antiandrogen group (n = 5), in either α1-high affinity or α1-low affinity subtype. (3) Phenoxbenzamine had different pKi value for the above two adrenoceptor subtypes. Scatchard analysis showed that α1-high affinity binding site disappeared in the presence of 1 μM of phenoxbenzamine, and the Kd and Bmax values in the prescence of 1 μM of phenoxybenzamine were almost identical to the α1-low affinity site of the two subtypes. CONCLUSIONS Human BPH tissue possesses both α1H-and α1L-adrenoceptor subtypes according to the affinities for prazosin, and only the α1H subtype can be completely inhibited by some concentration of phenoxybenzamine. Treatent by α1 blocker may not change the conditons of α1-adrenoceptors in prostatic tissue.Prostate 31:216–222, 1997. © 1997 Wiley-Liss, Inc.

Alpha-1 adrenoceptor subtypes (high, low) in human benign prostatic hypertrophy tissue according to the affinities for prazosin.

A novel classification of alpha-1 adrenoceptor subtypes (High, Low) was applied to human benign prostatic hypertrophy (BPH) tissue. Human BPH specimens were examined by a radioligand binding assay method using 3H-prazosin, and those data were compared with preoperative therapies. (1) Scatchard analysis showed a high-affinity site (Kd:27.18 +/- 6.41 pM; Bmax:9.29 +/- 0.98 fM/mg protein; mean +/- SE) as alpha 1H, and a low-affinity site (Kd: 4088.0 +/- 744.34 pM, Bmax: 140.81 +/- 19.98 fM/mg protein) as alpha 1L subtype, for prazosin. (2) The Kd and Bmax were not different in the nontreated group (n = 5), alpha 1 blocker group (n = 5), and antiandrogen group (n = 5), in either alpha 1-high affinity or alpha 1-low affinity subtype. (3) Phenoxybenzamine had different pKi values for the above two adrenoceptor subtypes. Scatchard analysis showed that alpha 1-high affinity binding site disappeared in the presence of 1 microM of phenoxybenzamine, and the Kd and Bmax values in the presence of 1 microM of phenoxybenzamine were almost identical to the alpha 1-low affinity site of the two subtypes. Human BPH tissue possesses both alpha 1H- and alpha 1L-adrenoceptor subtypes according to the affinities for prazosin, and only the alpha 1H subtype can be completely inhibited by some concentration of phenoxybenzamine. Treatment by alpha 1 blocker may not change the conditions of alpha 1-adrenoceptors in prostatic tissue.

Role of m1 receptor-G protein coupling in cell proliferation in the prostate

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

Procoagulant properties of benign and malignant prostatic tissue.

To determine the factor-X activating procoagulant activity (FXAA) of prostatic tissue removed at transurethral resection in a specific chromogenic assay. FXAA was extracted from transurethrally resected prostatic tissue using a cryofragmentation technique. Tissue from 50 patients with prostate cancer was analysed and compared with that from 36 control patients with benign prostatic-hypertrophy. Enzyme activities were assayed in a two stage chromogenic assay and correlated with conventional markers of tumour aggressiveness. FXAA was found to be significantly lower (P < 0.001) in tissue from malignant prostates compared with benign prostatic hypertrophy tissue. Loss of FXAA was also related to Gleason grade (P < 0.03), percentage of chips involved by tumour (P < 0.04) and bone scan status (P < 0.02). Using antibody inhibition tests this procoagulant was characterized as being a factor VII/tissue factor complex. Malignant change in the prostate is associated with a reduction in FXAA and this may be an important factor in prostatic tumour growth and dissemination.

Estradiol causes the rapid accumulation of cAMP in human prostate.

Androgens are widely acknowledged to be central to the pathogenesis of benign prostatic hypertrophy (BPH). However, BPH increases in prevalence as men age, at precisely the stage of life when plasma androgens are decreasing. The decrease in total plasma androgens is amplified by an age-related increase in plasma sex hormone-binding globulin (SHBG) that results in a relatively greater decrease in free androgens than in total androgens. In addition, estrogens have long been suspected to be important in BPH, but a direct effect on the human prostate has never been demonstrated. We present data that are consistent with a role for estradiol, and for a decrease in androgens and an increase in SHBG, in the pathogenesis of BPH. We show that estradiol, but not dihydrotestosterone, acts in concert with SHBG to produce an 8-fold increase in intracellular cAMP in human BPH tissue. This increase is not blocked by an antiestrogen and is not provoked by an estrogen (diethylstilbestrol) that does not bind to SHBG, thus excluding the classic estrogen receptor as being operative in these events. Conversely, dihydrotestosterone, which blocks the binding of estradiol to SHBG, completely negates the effect of estradiol. Finally, we demonstrate that the SHBG-steroid-responsive second-messenger system is primarily localized to the prostatic stromal cells and not to the prostatic epithelial cells. Thus, we have shown a cell-specific, powerful, nontranscriptional effect of estradiol on the human prostate.

Analysis of hypoxia-associated gene expression in prostate cancer: lysyl oxidase and glucose transporter-1 expression correlate with Gleason score

Prostate cancer cells exist under hypoxic conditions. Hypoxia has a detrimental effect on the efficacy of treatment and final outcome in patients with prostate cancer. There have been a large number of endogenous markers of hypoxia described previously across a range of cancer types, both in vitro and in vivo. The aim of this study was to evaluate the expression of a range of hypoxia-associated genes within benign prostatic hypertrophy (BPH) and prostate cancer tissue. Messenger RNA was extracted from primary prostate tissue obtained from 67 men with benign prostatic hypertrophy or prostate cancer (Gleason score 5 to 10). Real-time polymerase chain reaction was performed to quantify the expression levels of 12 hypoxia-associated genes in these tissues. Expression of lysyl oxidase (LOX) and glucose transporter-1 (GLUT-1) genes were significantly higher in prostate cancer compared with BPH tissue (P<0.05) and correlated with Gleason score (LOX: R=0.297, P=0.015; GLUT-1: R=0.274, P=0.026). HIF-2alpha had a negative correlation with Gleason score (R= -0.309, P=0.012). The remaining hypoxia-associated genes did not show any specific pattern of expression in prostate tissue. Numerous molecules have been proposed as endogenous markers of hypoxia. The findings of this study illustrate that not all hypoxia-associated molecules are relevant to prostate cancer in vivo. However, LOX and GLUT-1 are candidate markers of hypoxia in prostate cancer and may prove useful in identifying patients with hypoxic prostate cancer. Not all hypoxia-associated molecules are relevant in prostate cancer in vivo.

Quantitative Measurements of Prostatic Blood Flow and Blood Volume by Positron Emission Tomography

Prostatic blood flow was measured with 15oxygen-water by positron emission tomography using a 1-compartment model. A dynamic study method was applied to 9 normal subjects, 6 with benign prostatic hypertrophy (BPH) and 11 with advanced stages C to D2 prostatic adenocarcinoma. Prostatic blood flow was 15.7 ± 7.5ml. per minute per 100gm. in normal controls, 17.7 ± 5.2ml. per minute per 100gm. in BPH patients and 29.4 ± 7.8ml. per minute per 100gm. in prostatic cancer patients. Prostatic blood flow negatively correlated well with age in the normal subjects. Prostatic blood volume was also estimated by the steady state method using 15oxygen-carbon monoxide. Prostatic blood volume was 8.1 ± 2.6% in normal controls, 8.9 ± 1.1% in BPH patients and 6.1 ± 2.1% in prostatic cancer patients. Blood flow in the prostatic cancer tissue was higher than that in the normal (p <0.001) or BPH (p <0.01) tissue. A significant difference in prostatic blood volume was also observed between BPH and cancer tissues (p <0.02).

Aromatase and other inhibitors in breast and prostatic cancer

Estrogens have an important role in the growth of breast and other hormone-sensitive cancers. We have shown that 4-hydroxyandrostenedione (4-OHA) selectively blocks estrogen synthesis by inhibiting aromatase activity in ovarian and peripheral tissues and reduces plasma estrogen levels in rat and non-human primate species. In postmenopausal men and women, estrogens are mainly of peripheral origin. When postmenopausal breast cancer patients were administered either by daily oral or parenteral weekly treatment with 4-OHA, plasma estrogen concentrations were significantly reduced. Complete or partial response to treatment occurred in 34% of 100 patients with advanced breast cancer, while the disease was stabilized in 12%. We recently studied the effects of 4-OHA and other aromatase inhibitors, 10-propargylestr-4-ene-3,17-dione (PED) and imidazo[1,5-α]3,4,5,6-tetrahydropyrin-6-yl-(4-benzonitrile) (CGS 16949A) as well as 5α-reductase inhibitors, N,N-diethyl-4-methyl-3-oxo-4-aza-5α-androstane-17β-carboxyamide (4-MA) and 17β-hydroxy-4-aza-4-methyl-19norandrost-5-en-3-one (L651190) in prostatic tissue from 11 patients with prostatic cancer and six patients with benign prostatic hypertrophy (BPH), and from normal men at autopsy. We attempted to measure aromatase activity in tissue incubation by quantitating 3H 2O released during aromatization of androstenedione or testosterone labeled at the C-1 position. The amount of 3H 2O released from all samples was at least twice that of the heat inactivated tissue samples. The 3H 2O release was significantly inhibited by 4-OHA and 4-MA, but not by the other aromatase inhibitors. However, when HPLC and TLC were used to isolate steroid products, no estrone or estradiol was detected in the incubates. Furthermore, no aromatase mRNA was detected following amplification by PCR. The 4-OHA was found to inhibit 5α-reductase in both BPH and cancer tissue, although to a lesser extent than 4-MA. The other aromatase inhibitors were without effect. Although a mechanism involving intraprostatic aromatase is not likely, inhibitors may act to reduce peripherally-formed estrogens. In postmenopausal breast cancer, the results indicate that 4-OHA is of significant benefit.

前立腺癌患者における粗核内DHT濃度測定の有用性に関する研究

The nuclear and crude nuclear 5 alpha-dihydrotestosterone (DHT) levels were measured in patients with benign prostatic hypertrophy (BPH) and prostatic cancer (PC). In the fundamental study using BPH tissues, nuclear DHT levels were not influenced by the treatment for nuclear purification such as DTT, Triton X-100 or DNase I. The correlation between DHT levels and androgen receptor contents was found in only the crude nuclear salt extractable fractions (r = 0.748, p less than 0.01). In the relation of DHT levels to prostatic cancer patients, crude nuclear salt extractable and salt resistant DHT in both the low grade and in early stage group were significantly higher than those in the high grade and in advanced stage. Moreover, DHT levels in the both crude nuclear salt extractable and salt resistant fractions were below 5 pg/mg protein in all of clinical non-responders to endocrine therapy, and the total crude nuclear DHT, the sum of the crude extractable and salt resistant DHT, was below 10 pg/mg protein in all of the non-responders to endocrine therapy. When the relation between the total crude nuclear DHT level and the clinical course of 34 prostatic cancer patients followed for over 12 months was studied, the elevated DHT group (over 20 pg/ml protein) responded to endocrine therapy and experienced long-term remissions, but the low DHT group (below 10 pg/ml protein) did not respond to endocrine therapy. Therefore, it is suggested that the total crude nuclear DHT levels were appropriate biochemical indicators for androgen dependency in prostatic cancer patients.

Analysis of the Androgen Binding in Human Benign Prostatic Hypertrophy

In order to delineate optimal conditions for the determination of androgen specific binding in human benign prostatic hypertrophy tissues (BPH), extracts of these tissues were prepared using buffers of different compositions. The binding of 5 alpha-dihydrotestosterone (DHT) and of methyltrienolone (R 1881) was analyzed using agar gel electrophoresis. This method allowed the detection of 3 different high affinity tissular binding peaks with similar specificity. Moreover, the inhibition by each of the competitors was also the same for both ligands. It could be demonstrated that none of the observed peaks resulted from the binding of 1 of the ligands to sex hormone binding globulin (SHBG) or to a progesterone receptor. Hypotheses about the possible origin of these peaks are discussed.

Determination of Androgen Receptors in Human Benign Prostatic Hypertrophy with Two Synthetic Radiolabeled Ligands

Nuclear and cytosolic androgen receptor concentrations in tissues of human benign prostatic hypertrophy (BPH) were determined by use of methyltrienolone (R-1881) and 7 alpha,17 alpha-dimethyl-19-nortestosterone (DMNT) as radiolabeled ligands. Cytosolic R-1881-binding sites were 46.1 +/- 43 fmol/mg protein and nuclear R-1881-binding sites were 51.8 +/- 42 fmol/mg protein. DMNT-binding sites in cytosol were 44.3 +/- 38 fmol/mg protein and in nuclear extract 73.4 +/- 64 fmol/mg protein. No significant correlation was found between the number of R-1881- and DMNT-binding sites in either cytosol or nuclear extracts. Cytosolic or nuclear androgen receptor content was not significantly correlated with the percentage of epithelial or stromal cells as determined from the corresponding histological sections. In BPH tissue with marked cystic degeneration, very low androgen receptor levels were found.