Androgen-independent effects of Serenoa repens extract (Prostasan®) on prostatic epithelial cell proliferation and inflammation.

Extracts from Serenoa repens are widely used for the treatment of benign prostatic hyperplasia (BPH) and traditionally for prostatitis. In the present study we evaluated the biological effects of Serenoa repens extract (Prostasan®) on prostate cells beyond its known antiandrogenic actions. Prostasan® inhibited epidermal growth factor (EGF) and lipopolysaccharide (LPS) induced proliferation of the prostatic epithelial, androgen independent cell line PC-3. At effective concentrations of 50 µg/mL, Prostasan® partly displaced EGF from EGF receptor (EGFR) but fully blocked EGF-induced cell proliferation of PC-3 cells. Similarly, Prostasan® inhibited LPS-induced proliferation of PC-3 cells without affecting LPS activation of the NFĸB pathway via toll-like receptor-4 (TLR-4). Additionally, Prostasan® reduced the constitutive secretion of monocyte chemotactic protein-1 (MCP-1), the LPS-induced secretion of IL-12 and inhibited MCP-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the presence of LPS on PC-3 cells. Taken together, our results suggest that S. repens extracts, in addition to other reported effects on BPH development and prostatitis, inhibits EGF-dependent growth and proinflammatory responses of the prostate epithelial cells.

Gene expression profiling of androgen deficiency predicts a pathway of prostate apoptosis that involves genes related to oxidative stress.

Androgens are critical for prostate development, growth, and functions. In general, they support proliferation and prevent cell death of prostatic epithelial cells. Here, we studied changes of gene expression after castration and testosterone replacement therapy in the rat ventral prostate using cDNA microarrays analysis. We could identify 230 genes that were regulated in either experimental condition. Using hierarchical clustering analysis, different groups of genes could be detected according to their expression pattern. This enabled us to distinguish the putative androgen-responsive genes from the secondary-responsive ones. Among genes that altered during castration and testosterone replacement, a set of oxidative stress-related genes, including thioredoxin, peroxiredoxin 5, superoxide dismutase 2, glutathione peroxidase 1, selenoprotein 15 kDa, microsomal glutathione-S-transferase, glutathione reductase, and epoxide hydrolase, were changed by castration. We hypothesize that modulation of redox status can be a factor of relevance in androgen withdrawal-induced prostate apoptosis. In selective cases, quantitative RT-PCR was used to confirm changes in gene expression. Immunohistochemistry was performed to detect thioredoxin and ezrin. Both of these were detected in the prostate and seem to be regulated in a similar manner as shown by gene expression analysis. In conclusion, gene expression profiling provides a unique opportunity for understanding the molecular mechanisms of androgen actions in prostate gland.

Regulation of matrix metalloproteinase 13 expression by androgen in prostate cancer.

The matrix metalloproteinases (MMPs) are members of a family of endopeptidases that are able to degrade extra-cellular matrix. MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), play a key role in the migration of normal and malignant cell. Interaction of MMPs and TIMPs has been involved in the process of tumor invasion and metastasis. Using cDNA microarray as a screening tool to find androgen regulated gene in prostate cancer, we have found that expression of MMP-13 is regulated by androgen in prostate cancer derived cell line LNCaP. This regulation was further confirmed and quantified by real-time RT-PCR. In addition, the upregulation of MMP-13 mRNA by androgen could be abolished by the androgen antagonist Casodex but not the protein inhibitor cycloheximide. Western blot and immunohistochemistry of MMP-13 confirmed the androgen regulation at the protein level. We have furthermore shown that MMP-13 expression is presented in human prostate cancer obtained from aspiration biopsy. In summary, MMP-13 is androgen regulated and detectable in prostate cancer. Further study of MMP-13 in prostate cancer may help us to understand the progression of the cancer and can lead to new therapeutic options.

Left-sided grade 3 varicocele may affect the biological function of the epididymis.

OBJECTIVE: Varicocele is present in approximately 30-40% of men evaluated for infertility and in 10-20% of the general male population, but the association between varicocele, compromised semen quality and male infertility remains unclear. This indicates that varicocele may impair spermatogenesis or in some other way mitigate sperm quality. Better understanding of criteria for the selection of men who will benefit from varicocele repair would improve the clinical management of men with impaired semen quality and varicocele. MATERIAL AND METHODS: To further understand the effect of varicocele repair by embolization in men with grade 3 varicocele, 50 men referred for infertility with varicocele were evaluated before and after embolization. RESULTS: There was a significant improvement in total sperm count (25.5 ± 4.7 million to 78 ± 11 million, p < 0.001) and sperm motility (slowly and rapidly progressive) (31.5 ± 2.9% to 45 ± 2.5%, p < 0.001) after spermatic vein embolization, comparing baseline to 3 month post-treatment follow-up data. Furthermore, alpha-glucosidase (specific epididymis-derived protein) increased from 61.7 ± 5.7 U to 84.7 ± 7.0 U (p < 0.05) and sperm droplets decreased from 14.2 ± 1.5% to 11.0 ± 1.0% (p < 0.05). CONCLUSION: The results indicate improved epididymal function and suggest that left-sided grade 3 varicocele may affect the epididymis in addition to effects on the testis.

Androgen induction of prostate cancer cell invasion is mediated by ezrin.

Ezrin is a key signaling molecule that regulates cell survival, adhesion migration, and invasion. We have previously shown that ezrin is regulated by androgen in rat prostate and that its expression is increased in prostate cancer and in prostate intraepithelial neoplasia. We have used the androgen-sensitive cell line LNCaP-FGC to investigate the role of ezrin in androgen-induced cell invasion. We found that androgen treatment of LNCaP-FGC cells induces ezrin expression, an effect that is inhibited by the androgen receptor antagonist, bicalutamide. In addition, androgen treatment induces the phosphorylation of ezrin in Thr-567 and Tyr-353 in a sequential manner. This is mediated through protein kinase C alpha and Src tyrosine kinase, respectively. Androgen treatment induces the translocation of both protein kinase C alpha and ezrin to the cell membrane and their association. Inhibition of ezrin function using short interference RNA or the overexpression of T567A and Y353F-ezrin mutants significantly reduces androgen-induced Matrigel invasion but does not affect cell proliferation or cell adhesion. Matrigel invasion of the androgen-insensitive prostate cancer cell lines PC-3 and LNCaP-R is also dependent on ezrin. In summary, we have shown that androgens regulate ezrin at transcriptional and posttranscriptional levels. Hormonal regulation of ezrin phosphorylation is required for androgen-induced cell invasion.

Cytogenetic and expression profiles associated with transformation to androgen-resistant prostate cancer.

BACKGROUND: The mechanisms underlying the progression of prostate cancer to androgen-resistant cancer are still not fully understood. Here, we studied the genetic events associated with this transformation. METHODS: The androgen sensitive prostate cancer cells line LNCaP-FGC and its androgen resistant subline LNCaP-r were investigated using SKY, CGH, and cDNA microarray. RESULTS: Karyotypically, several additional chromosomal aberrations were seen in LNCaP-r as compared to the parental line. CGH also revealed unique net chromosomal alterations in LNCaP-r compared to LNCaP-FGC, including gain of 2p13-23, 2q21-32, and 13q and loss of 6p22-pter. cDNA microarray analysis identified several genes involved in DNA methylation, such as DNMT2, DNMT3a, and methyl-CpG binding domain protein 2 and 4 that were higher expressed in LNCaP-r. Interestingly, androgen responsiveness of LNCaP-r was restored after treated with DNA methyltransferase inhibitor. CONCLUSIONS: Our findings may serve as a basis for molecular dissection of the mechanisms involved in development of androgen resistant prostate cancer.

Proteomic comparison of prostate cancer cell lines LNCaP-FGC and LNCaP-r reveals heatshock protein 60 as a marker for prostate malignancy.

BACKGROUND: Androgen-sensitive prostate cancer cell-line LNCaP-FGC and androgen-resistant line LNCaP-r constitute a model for development of androgen resistance in prostate cancer. METHODS: Proteins differently expressed in the two cell-lines were identified by two-dimensional (2-D) electrophoresis and mass spectrometry. HSP60, more abundant in LNCaP-r, was studied by RT-PCR and immunohistochemistry in specimens of human prostate cancer. RESULTS: HSP60 was upregulated in LNCaP-r, nm23 in LNCaP-FGC, and titin (two isoforms) in either LNCaP-r or LNCaP-FGC. In non-malignant prostate, HSP60-staining was in the glandular compartment, particularly basal epithelial cells. In prostate cancer, most epithelial cells showed moderate-strong staining without apparent correlation between staining intensity and Gleason grade. CONCLUSIONS: The LNCaP-FGC/LNCaP-r model, characterized by 2-D electrophoresis, reveals distinct proteomic alterations. With HSP60, results from cell-lines correlated with clinical results, indicating that this model can be used for dissection of mechanisms involved in transformation to androgen resistance and assignment of protein markers in prostate cancer.

Changes in tissue prostatic acidic phosphatase during endocrine treatment of patients with prostatic carcinoma.

OBJECTIVE: We have previously developed methods for the quantification of different macromolecules in aspiration biopsy material and described the changes in prostate-specific antigen (T-PSA) during cancer treatment. We have now studied the changes in tissue prostatic acidic phosphatase (T-PAP) in 58 endocrine-treated patients with prostatic carcinoma and compared these data with cancer development data and tissue PSA (T-PSA) levels. MATERIAL AND METHODS: PAP and PSA were quantified in aspiration biopsies taken before treatment and after 6 and 12 months of treatment. Patients were followed until death or for >98 months. RESULTS: Pretreatment T-PSA was more strongly associated with survival than T-PAP. Both T-PSA and T-PAP decreased in responders during treatment. In non-responders, T-PSA and T-PAP increased after 12 months in 17/18 and 7/13 patients, respectively. Estrogen-treated responders had significantly higher T-PSA, but not T-PAP, treatment values than those treated with orchidectomy or gonadotropin-releasing hormone. CONCLUSIONS: The inferiority of serum PAP compared to PSA for monitoring cancer treatment may reflect its less pronounced changes at the tissue level, indicating different in vivo regulation of the two markers. Estrogen stimulation of PSA synthesis in vivo may underlie the higher PSA levels observed during estrogen treatment.

Estrogens affect endothelin-1 mRNA expression in LNCaP human prostate carcinoma cells.

OBJECTIVE: To study effects of estrogens on endothelin-1 (ET-1) mRNA expression in the androgen-sensitive LNCaP-FGC cell line and its androgen-resistant derivative LNCaP-r. Further, if effects of estrone sulfate (E1S) are mediated via conversion to estradiol-17beta (E2). Estrogens have been shown to down-regulate ET-1, a mediator of the osteoblastic response of bone to metastatic prostate cancer. METHODS: Cells were grown in steroid-depleted medium and incubated for 2-4 and 48 hours with 0, 1, 10, and 100 nM of either E1S or E2. mRNA levels were measured with an RT-PCR technique. Estrogen metabolism by LNCaP-FGC cells was studied by incubation with estrone (E1) and E1S at the same conditions, followed by determination of E1 and E2. RESULTS: ET-1 mRNA expression in LNCaP-FGC cells was significantly suppressed by E2 and E1S following incubation for 2-4h but after 48 h only by E2 at 1 and 10nM and in LNCaP-r cells only by E2 at 100 nM following 2-4h of incubation. ET-1 mRNA expression was significantly higher in untreated LNCaP-r than in untreated LNCaP-FGC cells. E1 was efficiently transformed into E2 by LNCaP-FGC cells but very little to E1 and no E2 was formed from E1S. CONCLUSION: ET-1 mRNA expression in LNCaP-FGC can be inhibited by E2, but also by its prehormone E1S. The lack of formation of E2 from E1S suggests a mode of action not related to classical steroid receptors. The higher level of ET-1 mRNA expression found in LNCaP-r cells may reflect the capability of a hormone refractory tumor to maintain activity on its own, independently of known regulatory mechanisms such as sex steroids.

Expression of ezrin in prostatic intraepithelial neoplasia.

OBJECTIVES: To study the protein expression and gene copy number of ezrin in a set of high-grade prostatic intraepithelial neoplasia (HGPIN) samples with concomitant prostate cancer. Ezrin is a cytoskeleton linker protein that is actively involved in regulating the growth and metastatic capacity of cancer cells. METHODS: Nineteen HGPIN samples obtained from radical prostatectomy specimens were used for the study. Among them, 13 samples also contained invasive prostate cancer. The expression of ezrin was studied by immunohistochemistry. The same samples were also used for fluorescence in situ hybridization to study the gene copy number of ezrin. RESULTS: Immunoreactivity for ezrin was absent or weak in benign prostatic epithelial cells. Weak or moderate immunostaining was detected in 11 of 13 prostate cancer specimens. However, the immunostaining was moderate or strong in all HGPIN samples. In addition, whenever HGPIN and prostate cancer were found in the same sample, the staining was always more intense in the HGPIN cells than in the cancer cells. No alteration was found in the gene copy number detected by fluorescence in situ hybridization. CONCLUSIONS: We have shown that ezrin is overexpressed in HGPIN and prostate cancer compared with adjacent benign prostatic epithelium. In addition, HGPIN has a greater expression level of ezrin compared with that of prostate cancer. Our results indicate that aberrant expression of ezrin might be involved in the pathogenesis of prostate cancer, and ezrin expression may be useful for the diagnosis of HGPIN.

Parenteral estrogen versus combined androgen deprivation in the treatment of metastatic prostatic cancer -- Scandinavian Prostatic Cancer Group (SPCG) Study No. 5.

OBJECTIVE: In the mid-1980s, interest in parenteral estrogen therapy for prostate cancer was renewed when it was found that it influenced liver metabolism only marginally and had very few cardiovascular side-effects. In this study high-dose polyestradiol phosphate (PEP; Estradurin) was compared to combined androgen deprivation (CAD) for the treatment of patients with metastatic prostate cancer. The aim of the study was to compare anticancer efficacy and adverse events, especially cardiovascular side-effects. MATERIAL AND METHODS: A total of 917 patients with T0-4, NX, M1, G1-3 prostate cancer and an Eastern Cooperative Oncology Group performance status of 0-2 were randomized to treatment with either PEP 240 mg i.m. twice a month for 2 months and thereafter once a month or flutamide (Eulexin) 250 mg t.i.d. per os in combination with either triptorelin (Decapeptyl) 3.75 mg per month i.m. or, on an optional basis, bilateral orchidectomy. A total of 556 patients had died at the time of this analysis. RESULTS: There was no difference between the treatment arms in terms of time to biochemical or clinical progression and overall or disease-specific survival. There was no increase in cardiovascular mortality in the PEP arm. The PEP group had a higher prevalence of cardiovascular disease prior to the study and a significantly higher incidence of non-fatal ischemic heart events and heart decompensation during the study. CONCLUSIONS: PEP has an equal anticancer efficacy to CAD and does not increase cardiovascular mortality. Final evaluation of cardiovascular morbidity is awaiting further analysis and follow-up. PEP is considerably cheaper than CAD.