Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis

Recent evidence from our laboratory has demonstrated that alpha1-adrenoceptor antagonists doxazosin and terazosin induced apoptosis in prostate epithelial and smooth muscle cells in patients with benign prostatic hypertrophy (BPH; J. Urol., 159: 1810-1815, 1998; J. Urol., 161: 2002-2007, 1999). In this study, we investigated the biological action of three alpha1-adrenoceptor antagonists, doxazosin, terazosin, and tamsulosin, against prostate cancer cell growth. The antigrowth effect of the three alpha1-adrenoceptor antagonists was examined in two human prostate cancer cell lines, PC-3 and DU-145, and a prostate smooth muscle cell primary culture, SMC-1, on the basis of: (a) cell viability assay; (b) rate of DNA synthesis; and (c) induction of apoptosis. Our results indicate that treatment of prostate cancer cells with doxazosin or terazosin results in a significant loss of cell viability, via induction of apoptosis in a dose-dependent manner, whereas tamsulosin had no effect on prostate cell growth. Neither doxazosin nor terazosin exerted a significant effect on the rate of cell proliferation in prostate cancer cells. Exposure to phenoxybenzamine, an irreversible inhibitor of alpha1-adrenoceptors, does not abrogate the apoptotic effect of doxazosin or terazosin against human prostate cancer or smooth muscle cells. This suggests that the apoptotic activity of doxazosin and terazosin against prostate cells is independent of their capacity to antagonize alpha1-adrenoceptors. Furthermore, an in vivo efficacy trial demonstrated that doxazosin administration (at tolerated pharmacologically relevant doses) in SCID mice bearing PC-3 prostate cancer xenografts resulted in a significant inhibition of tumor growth. These findings demonstrate the ability of doxazosin and terazosin (but not tamsulosin) to suppress prostate cancer cell growth in vitro and in vivo by inducing apoptosis without affecting cell proliferation. This evidence provides the rationale for targeting both drugs, already in clinical use and with established adverse-effect profiles, against prostatic tumors for the treatment of advanced prostate cancer.     

Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptor-mediated pathway

Quinazoline-based alpha1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an alpha1-adrenoceptor-independent pathway, involving activation of transforming growth factor-beta1 (TGF-beta1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 micromol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins alpha2, alphaV, beta1, and beta8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor-mediated mechanism with a potential integrin contribution towards cell survival outcomes.     

Polymorphisms in DNA repair genes and associations with cancer risk.

Common polymorphisms in DNA repair genes may alter protein function and an individual's capacity to repair damaged DNA; deficits in repair capacity may lead to genetic instability and carcinogenesis. To establish our overall understanding of possible in vivo relationships between DNA repair polymorphisms and the development of cancer, we performed a literature review of epidemiological studies that assessed associations between such polymorphisms and risk of cancer. Thirty studies of polymorphisms in OGG1, XRCC1, ERCC1, XPC, XPD, XPF, BRCA2, and XRCC3 were identified in the April 30, 2002 MEDLINE database (National Center for Biotechnology Information. PubMed Database: http://www.ncbi.nlm.nih.gov/entrez). These studies focused on adult glioma, bladder cancer, breast cancer, esophageal cancer, lung cancer, prostate cancer, skin cancer (melanoma and nonmelanoma), squamous cell carcinoma of the head and neck, and stomach cancer. We found that a small proportion of the published studies were large and population-based. Nonetheless, published data were consistent with associations between: (a) the OGG1 S326C variant and increased risk of various types of cancer; (b) the XRCC1 R194W variant and reduced risk of various types of cancer; and (c) the BRCA2 N372H variant and increased risk of breast cancer. Suggestive results were seen for polymorphisms in other genes; however, small sample sizes may have contributed to false-positive or false-negative findings. We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of cancer in the presence of reduced DNA repair capacity.     

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.     

Defective DNA strand break repair after DNA damage in prostate cancer cells: implications for genetic instability and prostate cancer progression

Together with cell cycle checkpoint control, DNA repair plays a pivotal role in protecting the genome from endogenous and exogenous DNA damage. Although increased genetic instability has been associated with prostate cancer progression, the relative role of DNA double-strand break repair in malignant versus normal prostate epithelial cells is not known. In this study, we determined the RNA and protein expression of a series of DNA double-strand break repair genes in both normal (PrEC-epithelial and PrSC-stromal) and malignant (LNCaP, DU-145, and PC-3) prostate cultures. Expression of genes downstream of ATM after ionizing radiation-induced DNA damage reflected the p53 status of the cell lines. In the malignant prostate cell lines, mRNA and protein levels of the Rad51, Xrcc3, Rad52, and Rad54 genes involved in homologous recombination were elevated approximately 2- to 5-fold in comparison to normal PrEC cells. The XRCC1, DNA polymerase-beta and -delta proteins were also elevated. There were no consistent differences in gene expression relating to the nonhomologous end-joining pathway. Despite increased expression of DNA repair genes, malignant prostate cancer cells had defective repair of DNA breaks, alkali-labile sites, and oxidative base damage. Furthermore, after ionizing radiation and mitomycin C treatment, chromosomal aberration assays confirmed that malignant prostate cells had defective DNA repair. This discordance between expression and function of DNA repair genes in malignant prostate cancer cells supports the hypothesis that prostate tumor progression may reflect aberrant DNA repair. Our findings support the development of novel treatment strategies designed to reinstate normal DNA repair in prostate cancer cells.     

Variant XRCC3 implicated in cancer is functional in homology-directed repair of double-strand breaks

Polymorphisms in DNA repair genes, including double-strand break (DSB) repair genes, are postulated to confer increased cancer risk. A variant of the XRCC3 gene, which is involved in DSB repair, has been associated with increased risk of malignant skin melanoma and bladder cancer. We tested the hypothesis that this variant, Thr241Met, may affect cancer risk by disrupting a critical function of XRCC3, i.e., promoting homology-directed repair (HDR) of chromosomal DSBs. Using a quantitative fluorescence assay, we find that the variant XRCC3 protein is functionally active for HDR, complementing the HDR defects of an XRCC3 mutant cell line as well as the wild-type protein. We also examined cells expressing this variant for sensitivity to the interstrand cross-linking agent, mitomycin C (MMC), as HDR mutant cell lines, including the XRCC3 mutant, have been found to be hypersensitive to this DNA damaging agent. Cells expressing the variant protein were found to be no more sensitive than cells expressing the wild-type protein. These results suggest that the increased cancer risk associated with this variant may not be due to an intrinsic HDR defect.     

Quinazoline-derived alpha1-adrenoceptor antagonists induce prostate cancer cell apoptosis via an alpha1-adrenoceptor-independent action.

Recent evidence suggests that the quinazoline-based alpha1-adrenoceptor antagonists, doxazosin and terazosin, exhibit a potent apoptotic effect against prostate tumor epithelial cells, whereas tamsulosin, a sulfonamide-based alpha1-adrenoceptor antagonist, was ineffective in inducing a similar apoptotic effect against prostate cells (Cancer Res., 60: 4550-4555, 2000). In this study, to identify the precise molecular mechanism underlying this apoptosis induction, we examined whether doxazosin and terazosin (both piperazinyl quinazolines) affect prostate growth via an alpha1-adrenoceptor-independent action. Transfection-mediated overexpression of alpha1-adrenoceptor in human prostate cancer cells, DU-145 (that lack alpha1-adrenoceptor), did not alter the ability of prostate cancer cells to undergo apoptosis in response to quinazolines. Significantly enough, there was no modification of the apoptotic threshold of the androgen-sensitive prostate cancer cells, LNCaP, to either quinazoline-based alpha1-agonist by androgens. Furthermore, human normal prostate epithelial cells exhibited a very low sensitivity to the apoptotic effects of doxazosin compared with that observed for the malignant prostate cells. These findings provide the first evidence that the apoptotic activity of the quinazoline-based alpha1-adrenoceptor antagonists (doxazosin and terazosin) against prostate cancer cells is independent of: (a) their capacity to antagonize alpha1-adrenoceptors; and (b) the hormone sensitivity status of the cells. This may have potential therapeutic significance in the use of quinazoline-based alpha1-adrenoceptor antagonists (already in clinical use for the treatment of hypertension and benign prostate hyperplasia) for the treatment of androgen-independent human prostate cancer.     

DNA repair gene XRCC1 and XPD polymorphisms and risk of prostate cancer.

The X-ray repair cross-complementing group 1 (XRCC1) and xeroderma pigmentosum group D (XPD) genes are involved in base excision repair and nucleotide excision repair of DNA repair pathways, respectively. A growing body of evidence suggests that XRCC1 and XPD are important in environmentally induced cancers, and polymorphisms in both genes have been identified. To determine whether the XRCC1 (codon Arg399Gln) and XPD (codon Asp312Asn and codon Lys751Gln) polymorphisms are associated with prostate cancer susceptibility, we genotyped these polymorphisms in a primarily Caucasian sample of 506 sibships (n = 1,117) ascertained through a brother with prostate cancer. Sibships were analyzed with a Cox proportional hazards model with age at prostate cancer diagnosis as the outcome. Of the three polymorphisms investigated, only the XPD codon 312 Asn/Asn genotype had an odds ratio (OR) significantly different from one (OR, 1.61; 95% CI, 1.03-2.53). Analyses stratified by the clinical characteristics of affected brothers in the sibship did not reveal any significant heterogeneity in risk. In exploring two-way gene interactions, we found a markedly elevated risk for the combination of the XPD codon 312 Asn/Asn and XRCC1 codon 399 Gln/Gln genotypes (OR, 4.81; 95% CI, 1.66-13.97). In summary, our results suggest that the XPD codon 312 Asn allele may exert a modest positive effect on prostate cancer risk when two copies of the allele are present, and this effect is enhanced by the XRCC codon 399 Gln allele in its recessive state.     

Analysis of gene expression during staurosporine-induced neuronal differentiation of human prostate cancer cells

Staurosporine induces neuronal differentiation and suppresses malignancy of human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying neuronal differentiation and suppression of malignancy, we used cDNA microarrays to examine gene expression profiles in TSU-Pr1 cells treated with staurosporine. mRNAs isolated from untreated and staurosporine-treated TSU-Pr1 cells were hybridised to microarrays consisting of approximately 9100 genes. Changes in gene expression were verified by Northern blot analysis. Staurosporine-responsive genes were involved in a variety of cellular functions including growth regulation, differentiation, replication, DNA repair, G2/M transition and inhibition of apoptosis. Interestingly, expression of genes associated with cell proliferation and malignancy, such as Cyr61 and CTGF, was reduced. Expression of CD73/NT5E, which is involved in neuronal differentiation, was increased. In the present study, we identified various staurosporine-responsive genes in TSU-Pr1 cells. Further studies of the roles of these genes may clarify the mechanisms underlying neuronal differentiation and inhibition of malignancy by staurosporine and identify better approaches for the prevention and treatment of prostate cancer.     

Polymorphisms of DNA repair genes are risk factors for prostate cancer

DNA repair gene alterations have been shown to cause a reduction in DNA repair capacity. We hypothesised that DNA repair gene polymorphisms may be risk factors for prostate cancer (PC). To test this hypothesis, DNA samples from 165 cases of prostate cancer and healthy controls were analyzed by PCR-RFLP to determine the genotypic frequency of three DNA repair genes (XRCC1, XPC and XRCC7). We found that the frequency of 939Gln variant at XPC Lys939Gln was significantly lower in PC cases (OR=0.39, P=0.016). Haplotype analysis of XRCC1 Arg194Trp (C/T) and Arg399Gln (G/A) revealed that the frequency of the T-A haplotype was significantly higher in PC patients. This is the first report on the studies of XPC and XRCC1 Arg194Trp polymorphisms in PC, and our present data suggest that XPC Lys939Gln and the T-A haplotype of XRCC1 Arg194Trp and Arg399Gln may be risk factors for PC in Japanese.     

Novel quinazoline-based compounds impair prostate tumorigenesis by targeting tumor vascularity

Previous evidence showed the ability of the quinazoline-based alpha(1)-adrenoreceptor antagonist doxazosin to suppress prostate tumor growth via apoptosis. In this study, we carried out structural optimization of the chemical nucleus of doxazosin and a subsequent structure-function analysis toward the development of a novel class of apoptosis-inducing and angiogenesis-targeting agents. Our lead compound, DZ-50, was effective at reducing endothelial cell viability via a nonapoptotic mechanism. Treatment with DZ-50 effectively prevented in vitro tube formation and in vivo chorioallantoic membrane vessel development. Confocal microscopy revealed a significantly reduced ability of tumor cells to attach to extracellular matrix and migrate through endothelial cells in the presence of DZ-50. In vivo tumorigenicty studies using two androgen-independent human prostate cancer xenografts, PC-3 and DU-145, showed that DZ-50 treatment leads to significant suppression of tumorigenic growth. Exposure to the drug at the time of tumor cell inoculation led to prevention of prostate cancer initiation. Furthermore, DZ-50 resulted in a reduced formation of prostate-tumor derived metastatic lesions to the lungs in an in vivo spontaneous metastasis assay. Thus, our drug discovery approach led to the development of a class of lead (quinazoline-based) compounds with higher potency than doxazosin in suppressing prostate growth by targeting tissue vascularity. This new class of quinazoline-based compounds provides considerable promise as antitumor drugs for the treatment of advanced prostate cancer.     

Alpha1-adrenoceptor antagonists radiosensitize prostate cancer cells via apoptosis induction

BACKGROUND: Androgen-independent prostate cancer cells can undergo apoptosis in response to non-androgen ablative means such as ionizing radiation. Recent evidence documented the ability of alpha-adrenoceptor antagonists, a widely used medical therapy for the treatment of benign prostatic hypertrophy (BPH), to induce apoptosis in benign and malignant prostate cells. In this study, we evaluated the potential additive/synergistic apoptotic effect of alpha1-adrenoceptor antagonists with ionizing radiation against human prostate cancer cells in vitro. MATERIALS AND METHODS: Androgen-independent human prostate cancer cells (PC-3) were treated with two alpha1-adrenoceptor antagonists, doxazosin and terazosin, for various periods of time prior to and after exposure to ionizing radiation. Apoptosis induction, cell viability and clonogenic assays were then performed to determine loss of clonogenic survival Hoechst staining was performed to detect the apoptotic morphology in prostate cancer cells and the temporal protein expression of the apoptosis regulators bax and caspase-3, was determined using Western blot analysis. RESULTS: No significant difference in cell death of PC-3 cells was detected when either doxazosin or terazosin was combined with ionizing radiation. Terazosin treatment however, 24 hours prior to, or 24 hours post-irradiation resulted in a significant enhancement of radiation-induced loss of clonogenic survival compared to radiation alone (p<0.05). Furthermore, there was a further significant increase in apoptosis induction when cells were pre-treated with terazosin (15%), compared to treatment with radiation alone (6%). Western blot analysis revealed a significant increase in bax protein expression (but not caspase-3) in response to radiation, with no additional effect with the combination treatment (terazosin and ionizing irradiation) compared to radiation alone. CONCLUSION: This is the first study to document the ability of alpha1-adrenoceptor antagonists to enhance the apoptotic effect of ionizing radiation against human prostate cancer cells. As this alpha1-adrenoceptor-mediated elevation of the apoptotic threshold involves neither bax deregulation nor caspase-3 activation, a differential mechanism might be underlying this radiosensitizing effect. The present findings may have important clinical relevance in identifying a more effective therapeutic approach for androgen-independent prostate cancer based on the combined apoptotic effects of quinazoline-based alpha1-adrenoceptor-antagonsists and radiotherapy.