Autocrine effect of DHT on FGF signaling and cell proliferation in LNCaP cells: role of heparin/heparan-degrading enzymes.

BACKGROUND: LNCaP cells are androgen-sensitive human prostate cancer cells. They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture. At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT. Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1. The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells. METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum. In serum-free cultures, the characteristic DHT-induced proliferation was not observed. The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation. These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2. However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message. As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media. FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM). In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished. These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity. The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation. Moreover, 0.1 nM DHT caused a significant increase in heparinase activity. CONCLUSIONS: These results provide a possible mechanism for DHT action in LNCaP cells. In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact with LNCaP cells. However, in the presence of 0.1 nM DHT, heparinase activity in the culture was elevated and, as a result, it liberated the trapped FGF-2 which, in turn, stimulated proliferation in LNCaP cells.

Effect of beta-sitosterol on transforming growth factor-beta-1 expression and translocation protein kinase C alpha in human prostate stromal cells in vitro.

BACKGROUND: Today, plant extracts are widely used in the treatment of benign prostatic hyperplasia (BPH). However, the complete mode of action of the active substance, beta-sitosterol, is under investigation. The purpose of this study was to investigate the effect of beta-sitosterol on the expression of transforming growth factor beta 1 (TGF-beta1) and the activity of protein kinase C alpha (PKC-alpha) in primary prostate stromal cell cultures in vitro. METHODS: Tissue samples for primary cell cultures were obtained from patients undergoing transurethral resection of the prostate (TURP). TGF-beta1 levels in stromal cell conditioned media following a culture with beta-sitosterol were detected in a TGF-beta1 specific ELISA assay. Following different incubation periods with beta-sitosterol, cells were lysed and fractionated into a Triton-soluble membrane fraction and a cytosol fraction. PKC-alpha protein was detected using immunoblot analysis. RESULTS: Beta-sitosterol was able to induce the expression and secretion of TGF-beta1 significantly between 1.26- and 1.86-fold compared to a cholesterol and the nonsupplemented control in 6 of 8 individual cultures. The total amount of secreted TGF-beta1 varied in cells from different patients. Based on its presence in both membrane fraction and cytosol, PKC-alpha appeared to be constitutively expressed in stromal cells. In the absence of beta-sitosterol PKC-alpha was predominantly found in its membrane-associated active form. Following a culture with beta-sitosterol, a translocation of PKC-alpha from the membrane to the cytosol was observed. This effect was specific for beta-sitosterol as compared to cholesterol. CONCLUSION: This study describes the effect of beta-sitosterol on the expression of a multifunctional growth factor (TGF-beta1) and the activity of PKC-alpha membrane in stromal cells of the human prostate in vitro.

[Molecular genetic and cell biology principles for the development of malignant tumors]. / Molekulargenetische und zellbiologische Grundlagen der Entstehung von malignen Tumoren.

The development of cancer is one of the most intensively studied areas of medical research resulting in an immense quantity of data. Therefore, the purpose of this article is to give an overview of the basic principles of cancer development. Key words such as multi-step carcinogenesis, cell cycle, protooncogene, tumor suppressor gene, DNA repair gene, apoptosis and telomeres are explained and described in examples. This paper aims to connect recent information of molecular and cellular biology in an overview of cancer origin and development.

Treatment of symptomatic benign prostatic hyperplasia with beta-sitosterol: an 18-month follow-up.

OBJECTIVES: To determine the long-term effects of phytotherapy with beta-sitosterol (the trade name for beta-sitosterol used in this study is Harzol(R)) for symptomatic benign prostatic hyperplasia (BPH). Patient and methods At 18 months after enrolment in a 6-month multicentre double-blind placebo-controlled clinical trial with beta-sitosterol (reported previously), patients were re-evaluated using the modified Boyarsky score, the International Prostate Symptom Score and quality-of-life index, the maximum urinary flow rate (Qmax) and postvoid residual urine volume (PVR). In this open extension of the original trial (after 6 months of treatment or placebo), patients were free to chose their further treatment for BPH. RESULTS: In all, 117 patients (59%) were eligible for analysis during the follow-up. Of the formerbeta-sitosterol group, 38 patients who continued beta-sitosterol treatment had stable values for all outcome variables between the end of the double-blind study and after 18 months of follow-up. The 41 patients choosing no further therapy had slightly worse symptom scores and PVR, but no changes in Qmax. Of the former placebo group, 27 patients who started beta-sitosterol after the double-blind trial improved to the same extent as the treated group for all outcome variables. The 18 patients choosing no further therapy showed no signs of improvement. CONCLUSION: The beneficial effects of beta-sitosterol treatment recorded in the 6-month double-blind trial were maintained for 18 months. Further clinical trials should be conducted to confirm these results before concluding that phytotherapy with beta-sitosterol is effective.

Stromal cells of the human prostate: initial isolation and characterization.

The present study was conducted to isolate and to characterize stromal cells from the human prostate and to study the effects of androgen and different growth factors in this model system. Benign prostatic hyperplasia (BPH) tissue samples were obtained from transurethral resection of the prostate (TURP). Tissue specimens were mechanically and enzymatically dissociated by treatment with DNAse and collagenase. Epithelial cells were separated from stromal cells by discontinuous Percoll gradient centrifugation. The stromal cells obtained were cultured in phenol red-free RPMI-1640 supplemented with 10% fetal bovine serum. Immunocytochemical analysis revealed that the stromal cell cultures were composed of both smooth muscle cells and fibroblasts. The short and broad, smooth muscle cells wee identified by using an antibody directed against alpha-smooth muscle actin. The thin and elongated fibroblasts stained positively for prolyl 4-hydroxylase. Smooth muscle cells were the predominant cell type in the present investigation. Typical cultures contained up to 99% of cells staining positively for alpha-smooth muscle actin. The prostate smooth muscle cultures were treated with dihydrotestosterone (DHT), bovine pituitary extract (BPE), basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). When cells were cultured in serum free RPMI-1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) mitogenic effect in medium supplemented with ITS+. In the presence of 10% charcoal-stripped fetal bovine serum (cFBS) DHT, at a concentration of 0.1 nM, was able to cause a slight but significant (P < 0.05) mitogenic effect on BPH smooth muscle cells growth. Basic FGF was able to stimulate BPH smooth muscle cells in a concentration-dependent fashion. The combination of DHT and 0.1 ng/ml bFGF was able to increase the proliferation of prostate smooth muscle cells above either agents alone. Addition of BPE to serum free RPMI-1640 caused a significant (P < 0.05) stimulation of cell proliferation in a concentration-dependent fashion. Addition to TGF-beta to serum or BPE containing RPMI-1640 caused a significant (P < 0.05) inhibition to cell proliferation in a concentration-dependent fashion. TGF-beta was cytostatic to the benign prostatic smooth muscle cells only in the presence of media containing growth stimulating factors found in charcoal-stripped serum or in bovine pituitary extract. These results demonstrated that stromal fraction isolated from BPH specimens was composed of both fibroblasts and smooth muscle cells. These cells could be cultured and were able to respond to various growth stimulatory and inhibitory agents.