CODE: 6.3 - The influence of doxazosin on the expression of membranous receptors inducing apop-tosis in primary prostate epithelial cultures established from BPH patients
Article published in Urologia Polska 2006/59/Suplement 1.
Zbigniew Wolski 1, Tomasz Drewa 1, Bartosz Misterek 1, Robert Dębski 2, Zdzisław Skok 3
- 1 Katedra i Klinika Urologii Ogólnej, Onkologicznej i Dziecięcej Collegium Medicum w Bydgoszczy
2 Klinika Pediatrii, Onkologii i Hematologii Dziecięcej Collegium Medicum w Bydgoszczy
3 Katedra i Zakład Patomorfologii Collegium Medicum w Bydgoszczy
- Introduction. Tumor Necrosis Factor Receptors 1 and 2 (TNFR 1 & 2), FAS and CD40 are membranous receptors present on prostate epithelium. Activation of either TNFR1 or FAS induces apoptosis in human prostate cell lines, from the other side activation of CD 40 and TNFRII inhibits this process.
- The aim of the study was to established the influence of Doxazosin on TNFR1, TNFR2, FAS and CD40 expression in primary cell culture from BPH patients.
- Materials and methods. Prostate epithelial cell culture: 14 BPH patients were involved in the study. The age was 65.8 +6,6 on average. Samples were taken from the transitional zone of an enucleated prostate. Tissue was placed in the enzymatic bath for 8h. Cells were seeded on 25 cm2 culture bottles (Greiner). Dulbecco's Modified Medium (Sigma) was supplemented with foetal bovine serum (Sigma) and microelements. Cultures were confirmed with anti-pancytokeratin antibody (Dako). Cells from each patient were divided into 4 groups. 20, 50 and 80 uM/L of Doxazosin (Pfizer) was added to 3 groups. 20 uM of Doxazosin was equiva-lent to 8mg dose. PBS was added to the control group. Cells were incubated 24h with Doxa-zosin. After 48h cells were detached and 4 x 105 cells were suspended in 25 ul PBS with 1 ug of human IgG. Then cells were incubated for 40 min in 40C with 10 ul of one of the FITC conjugated antibodies; anti-FAS, anti-TNFR 1, anti-TNFR 2 and anti-CD40 (Coulter). 400 ul of PBS was added to cell suspensions before analysis in an Epics XL flow cytometer (System 2 Software Version 1.0). One result was composed of 3 measurements. The average values and standard deviations were calculated from 21 to 45 separate results. The average values were compared using Student t-tests. Correlation between drug concentration and percent of positive cells was additionally calculated.
- Results. Cultures from 10 patients were established. There were 61.3 +28.4 and 77 +5.4% TNFR 1 positive cells in the control and 80uM Doxasosin (equivalent to 32 mg dose), respec-tively (p=0.01). Strong positive correlation between Doxazosin dose and percent of TNFR 1 positive cells was noticed (Correlation Coefficient was 0.994). Doxazosin did not influence TNFR 2 and FAS expression. Significant decrease of CD40 cell population was observed after doxazosin treatment at dose of 20 uM (equivalent 8 mg dose),- 40.4 +6.7% vs. 53.4 +19.9% in the control (p=0.001).
- Conclusions. 1. There is a 80 uM/l dependent increase of TNFR 1 expression in epithelial cells from BPH patients. 2. We hypothesized that TNFR1 activation can be implicated in apoptosis of prostate epithelium after doxazosin treatment. 3. The expression pattern of anti-apoptotic CD40 should be elucidated.