Inhibition of mitogenic signaling and induction of apoptosis in human bladder smooth muscle cells treated with doxazosin
ABSTRACT The alpha1 antagonist doxazosin is used to treat lower urinary tract symptoms and is believed to function primarily as a smooth muscle relaxant. However, doxazosin has been shown to inhibit proliferation and induce apoptosis in nonbladder smooth muscle. Consequently, we examined the effects of doxazosin on human bladder smooth muscle cell (SMC) proliferation and apoptosis.
Primary human bladder SMCs were cultured in M199 with 10% fetal bovine serum (FBS) until they reached 65% confluency and then they were made quiescent by serum starvation in M199 with 0.4% FBS for 24 hours. The quiescent bladder SMCs were pretreated for 30 minutes with doxazosin or vehicle (dimethyl sulfoxide) and then stimulated with 10% FBS for 24 hours. Measurement of 5'-bromo-2'-deoxyuridine (BrdU) uptake by flow cytometry was used to determine the effect of doxazosin on cell cycle progression. Western immunoblot was used to examine cell cycle protein expression and phosphorylation of the retinoblastoma protein (Rb) and cyclin A, both of which regulate cell cycle progression.
Cellular proliferation was inhibited in a dose dependent manner by doxazosin. There was nearly a 50% decrease in BrdU uptake at 10 microM doxazosin and an approximately 90% decrease in BrdU at 25 microM doxazosin. Notably, doxazosin inhibited phosphorylation of Rb and expression of cyclin A, both of which are necessary for cell cycle progression. At concentrations of 25 microM doxazosin or greater apoptosis was induced in the bladder SMCs, as indicated by an increase in subG1 DNA content.
Our study demonstrates that doxazosin inhibits mitogen induced proliferation of human bladder SMC by blocking cell cycle progression at the of G1/S border. Doxazosin induced cell cycle inhibition appears to be at least in part due to an inhibition of mitogen induced Rb phosphorylation and cyclin A expression. At higher concentrations doxazosin induces apoptosis in human bladder SMCs.
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ABSTRACT: Finasteride (Fin) and Doxazosin (Dox), alone or in combination, have been widely used in treatment of benign prostatic hyperplasia (BPH) symptoms and recently have been suggested as potential drugs for prostate cancer (PCa)prevention and treatment. However, little is known about the effects of the combination therapy on prostate tissue morphology, physiology and matrix metalloproteinases (MMPs) activity, a special set of enzymes closely related to PCa progression and metastasis. In this study, adult Wistar rats were treated with Fin + Dox (25 mg/kg per day) and the ventral prostate (VP) was excised at days 3 and 30 of treatment to evaluate morphology, cell proliferation, death, transforming growth factor-beta1 (TGF-beta1) protein expression, MMP-2, MMP-9 activities and MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA expression. Fin + Dox treatment induced a transient increase in testosterone (T) plasma concentration and a permanent reduction in dihydrotestosterone (DHT). The VP and epithelial cell proliferation were reduced and the stromal collagen fibre volume fraction and apoptosis of the epithelial cell were increased. Fin + Dox treatment also increased the TGF-beta1 immunoreaction in the epithelium and in the stroma. The mRNAs for MMP-2, TIMPs-1 and -2 expressions after 30 days of treatment were decreased. The mRNA for MMP-9 was not detected in any of the groups analysed. Fin + Dox treatment for 30 days promoted a decrease in gelatinolytic activity of MMP-2 and an increase in MMP-9. In conclusion, combined treatment with Fin and Dox interferes in the epithelial cell behaviour and in the MMPs activity, potentially via TGF-beta1-mediated and androgen pathways. Our results contribute to a better understanding of the clinical data and also of the molecular mechanisms behind isolated or combined Fin and Dox long-term treatment.International Journal of Andrology 04/2009; 33(3):489-99. DOI:10.1111/j.1365-2605.2009.00963.x · 3.21 Impact Factor
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ABSTRACT: Doxazosin and related, quinazoline-based alpha(1)-adrenoceptor antagonists can induce apoptosis in prostate and various other normal, benign, smooth muscle, endothelial and malignant cells. Such apoptosis-inducing effects occur independently of alpha(1)-adrenoceptor antagonism and typically require much high concentrations than those required for receptor occupancy. Several studies have invested efforts towards the elucidation of the molecular mechanisms underlying doxazosin-induced apoptosis. These include various tumor cells, cardiomyocytes, endothelial cells and bladder smooth muscle cells. While the high concentrations of doxazosin required to induce apoptosis challenge the use of this and related drugs for clinical optimization of apoptosis induction, such quinazoline structure may represent chemical starting points to develop more potent apoptosis-inducing agents free of alpha(1)-adrenoceptor antagonistic action and suitable for cancer treatment with minimal and well-tolerated side effects.Archiv für Experimentelle Pathologie und Pharmakologie 11/2009; 380(6):473-7. DOI:10.1007/s00210-009-0462-4 · 2.36 Impact Factor
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ABSTRACT: Alpha1-adrenoceptors antagonists (doxazosin, terazosin, prazosin) are commonly prescribed for benign prostate hyperplasia and hypertension. Doxazosin and terazosin exhibit anti-angiogenic effects and apoptotic activities against multiple cell types and are potential preventive agents for prostate cancer. Prazosin induces apoptosis in three prostate cancer cell lines. We hypothesized that prazosin, a more potent alpha1-adrenoceptor antagonist with a distinct mechanism, exhibits anti-angiogenic activity. We examined the effect of prazosin on growth and tube formation of human umbilical vascular endothelial cells (HUVECs). We used flow cytometry to assess the effect of prazosin on cell cycle progression and Western blotting to assess its effect on the expression of various apoptotic proteins. Prazosin inhibited the growth of HUVEC with an IC(50) of 6.53 µM and suppressed tube formation in a dose-dependent manner. Unlike prostate cancer cells, prazosin did not arrest cell cycle progression at the G2/M checkpoint. We used rhodamine 123 staining to show that prazosin (20 µM) treatment induced a loss of mitochondrial membrane potential by 12 hr. Prazosin treatment of HUVECs resulted in reduced MCL-1 expression, increased Bad, and Bcl-xL expression, cytochrome c release, and induction of apoptosis via the intrinsic apoptosis pathway. Prazosin induced apoptosis in prostate cancer cells and normal HUVEC cells via different mechanisms. These data suggest that prazosin exhibits anti-angiogenic activity and differentially modulates apoptotic pathways depending on the cell type.The Prostate 06/2011; 71(9):976-84. DOI:10.1002/pros.21313 · 3.57 Impact Factor