Regulation of HER expression and transactivation in human prostate cancer cells by a targeted cytotoxic bombesin analog (AN‐215) and a bombesin antagonist (RC‐3095)

Bombesin (BN) and gastrin‐releasing peptide (GRP) have been shown to stimulate the growth of human prostate cancer in vivo and in vitro by mechanisms initiated by binding of the peptide to BN/GRP receptor (GRPR). GRPR is overexpressed in a variety of human cancers, including human prostatic carcinoma. This led us to evaluate the effectiveness of blocking GRPR and of chemotherapy targeted to GRPR in androgen‐dependent (LNCaP) and androgen‐independent (PC‐3) prostate cancer cells, which exhibit different features of disease progression. Thus, we used a cytotoxic BN/GRP analog, AN‐215, consisting of 2‐pyrrolinodoxorubicin (AN‐201) linked to BN‐like carrier peptide, and a BN/GRP receptor antagonist, RC‐3095. Semiquantitative RT‐PCR and Western blotting revealed that mRNA and protein levels for GRPR increased in prostate cancer cells as compared with nonneoplastic RWPE‐1 cells. Immunofluorocytochemistry and Western blot assays revealed that AN‐215 was the most effective analog decreasing both the expression of epidermal growth factor receptor family members and the activation of epidermal growth factor receptor and HER‐2, which are associated to a poor prognosis. Furthermore, analogs targeted to BN/GRP receptors, AN‐215 and RC‐3095, blocked the effect of BN on cell growth in RWPE‐1, LNCaP and PC‐3 cells. These findings shed light on the mechanisms of action of these analogs and support the view that the use of AN‐215 and RC‐3095 for blocking BN/GRP receptors for targeted therapy may be of benefit for treatment of advanced prostate cancer.     

Targeted cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), AEZS-108 (AN-152), inhibits the growth of DU-145 human castration-resistant prostate cancer in vivo and in vitro through elevating p21 and ROS levels

Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone (LHRH) analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108, consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog, AEZS-108, on LHRHR positive castration-resistant prostate cancer cells.     

Effective treatment of experimental U-87MG human glioblastoma in nude mice with a targeted cytotoxic bombesin analogue, AN-215

Some brain tumours, such as glioblastomas express high levels of receptors for bombesin/gastrin releasing peptide. We investigated whether bombesin/gastrin releasing peptide receptors found in glioblastoma cell lines can be utilised for targeting of a cytotoxic bombesin analogue, AN-215 consisting of a potent derivative of doxorubicin, 2-pyrrolino-doxorubicin (AN-201) linked to a bombesin-like peptide carrier. This study reports the effect of AN-215 on the growth of U-87MG human glioblastomas xenografted into nude mice. High affinity binding of AN-215 to U-87MG tumours was characterised by an IC(50) value of 4.0+/-0.1 nM, as determined by radioreceptor assays. mRNA analyses revealed the presence of mRNA for BN receptor subtypes 1 and 2. Treatment with AN-215 significantly (P<0.05) extended tumour doubling time from 4.54+/-0.2 days to 8.18+/-1.8 days and inhibited tumour growth as demonstrated by a 69.6% reduction in final tumour volume (P<0.001) and a 64.6% decrease in tumour weight as compared to controls. Cytotoxic radical AN-201 at the same dose was ineffective. The antitumour effect of AN-215 could be blocked by pretreatment with an excess of a bombesin antagonist, indicating that the action of this cytotoxic analogue is receptor-mediated. Our results suggest that patients with inoperable brain tumours such as malignant gliomas may benefit from targeted chemotherapy based on cytotoxic bombesin analogue AN-215.     

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.     

Inhibition of PC-3 human prostate cancers by analogs of growth hormone-releasing hormone (GH-RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity

Vasoactive intestinal peptide (VIP) stimulates the proliferation and invasiveness of malignant prostatic cells. Receptors for VIP and the closely related growth hormone-releasing hormone (GH-RH) show considerable homology and are found in prostatic and other carcinomas. Among various analogs of GH-RH synthesized, JV-1-52 is a non-selective VIP/GH-RH antagonist, whereas JV-1-53 is a VIP antagonist devoid of GH-RH antagonistic effect. In our study, nude mice bearing PC-3 human androgen-independent prostate carcinomas were treated with JV-1-52 or JV-1-53 (20 microg/day, s.c.) for 28 days. Both antagonists produced a similar reduction in tumor volume (62-67%, p < 0.01) and tumor weight (59-62%; p < 0.05) vs. controls and extended tumor doubling-time from 9.1 to about 16 days (p < 0.05). To investigate the mechanisms involved, in another study we compared the effects of JV-1-53 with those of somatostatin analog RC-160. VIP antagonist JV-1-53 reduced tumor weight by 67% (p < 0.01) and suppressed the expression of mRNA for c-fos and c-jun oncogenes by about 34% (p < 0.05), without affecting serum levels of insulin-like growth factor-I (IGF-I). In contrast, RC-160 (50 microg/day) reduced serum IGF-I by 19% (p < 0.05), but did not significantly decrease tumor weight. mRNA for VIP and high affinity receptors for VIP were detected on PC-3 tumors. Our results suggest that VIP/GH-RH antagonists can inhibit the growth of androgen-independent prostate cancer by abrogating the autocrine/paracrine mitogenic stimuli of VIP. The ability of GH-RH antagonists to block tumoral VIP receptors, in addition to GH-RH receptors, could be potentially beneficial for prostate cancer therapy.     

Administration of a targeted cytotoxic analog of luteinizing hormone-releasing hormone inhibits growth of estrogen-independent MDA-MB-231 human breast cancers in nude mice

Receptor targeted chemotherapy is less toxic and more effective than conventional chemotherapy. Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in about 50% of human breast cancers. Highly potent cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) was linked to the agonistic analog [D-Lys⁶]LH-RH to form cytotoxic LH-RH analog AN-207. We evaluated whether AN-207 could be targeted to the hormone-independent MDA-MB-231 human breast cancers. Nude mice bearing MDA-MB-231 tumors were injected i.v. with 250 nmol/kg doses of cytotoxic radical AN-201, cytotoxic LH-RH analog AN-207, the unconjugated mixture of AN-201 and carrier [D-Lys⁶]LH-RH, [D-Lys⁶]LH-RH alone and vehicle (control). The growth of MDA-MB-231 tumors in animals given a single dose of AN-207 was inhibited significantly (p=0.01) for 3 weeks after injection, whereas tumors in all the other groups grew steadily. All cytotoxic compounds produced leukopenia, but the strongest lymphocyte suppression was caused by cytotoxic radical AN-201. Three weeks after treatment, the presence of mRNA for LH-RH receptors was demonstrated by RT-PCR in all the groups and radioreceptor assays demonstrated high-affinity binding sites for LH-RH on tumor cell membranes of control animals and those treated with AN-201, the carrier peptide alone or in combination with AN-201. At this time point binding assays did not reveal the expression of membrane proteins in tumors treated with AN-207, but 60 days after administration of AN-207, high affinity LH-RH binding sites were found again in MDA-MB-231 tumors. These results indicate that cytotoxic LH-RH analog AN-207 could be utilized for receptor targeted chemotherapy of breast cancers expressing receptors for LH-RH.     

Targeted cytotoxic somatostatin analog AN-162 inhibits growth of human colon carcinomas and increases sensitivity of doxorubicin resistant murine leukemia cells

The effect of the targeted cytotoxic somatostatin (SST) analog AN-162, consisting of doxorubicin (DOX) conjugated to SST carrier RC-121, was investigated on the growth of human colorectal cancer (CRC) cell lines HT-29, HCT-15, and HCT-116 and a DOX-resistant mouse leukemia cell line P388/R84. mRNA for SST-receptors and high affinity binding sites for SST were detected in all CRC cell lines and in P388/R84 cells. In contrast to DOX alone, AN-162 blocked HCT-116 cells and P388/R84 cells in S/G2 phase and increased the number of apoptotic cells. In vivo, AN-162 reduced the volume of CRC xenografts more effectively than its unconjugated components. Our results suggest that AN-162 inhibits growth of experimental CRC more effectively than DOX and increases sensitivity of DOX resistant human leukemia cells.     

Additive antitumor effects of the epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), and the nonsteroidal antiandrogen, bicalutamide (Casodex), in prostate cancer cells in vitro

Progression from an androgen-dependent to an androgen-independent state often occurs in patients with prostate cancer (PCa) who undergo hormonal therapy. We have investigated whether inhibition of the epidermal growth factor receptor (EGFR) signaling pathway affects the antitumor effect of a nonsteroidal antiandrogen. Gefitinib (Iressa), an EGFR tyrosine kinase inhibitor, and bicalutamide (Casodex), a nonsteroidal antiandrogen [androgen receptor (AR) antagonist], were administered alone and in combination to AR-positive human PCa cell lines. FACS analysis showed lower EGFR expression levels on AR-positive cells (LNCaP, CWR22, CWR22R 2152 and AR-transfected DU145 cell lines) compared with AR-negative cells (DU145, PC3 and TSU-Pr1). Moreover, in AR-transfected DU145 cells, chronic treatment with bicalutamide increased EGFR expression to levels similar to androgen-independent DU145 cells. All AR-positive PCa cell lines were sensitive to gefitinib (IC50 = 0.1-0.6 microM), whereas higher concentrations of bicalutamide were needed to reduce AR-positive PCa cell line proliferation (IC50 = 0.8-2.0 microM). Low doses of gefitinib increased the antitumor effects of bicalutamide by strongly reducing the IC50 of bicalutamide (approximately 10-fold). Similarly, bicalutamide increased the antiproliferative effects of gefitinib by reducing the IC50 of gefitinib (approximately 5-fold). Taken together, our data suggest that in androgen-dependent cell lines, addition of gefitinib in combination with bicalutamide results in concurrent dual inhibition of AR and EGFR/HER2 pathways. This causes a significant delay in the onset of EGFR-driven androgen independence.     

Molecular mechanisms for apigenin-induced cell-cycle arrest and apoptosis of hormone refractory human prostate carcinoma DU145 cells

Development of effective agents for treatment of hormone-refractory prostate cancer has become a national medical priority. We have reported recently that apigenin (4',5,7-trihydroxyflavone), found in many common fruits and vegetables, has shown remarkable effects in inhibiting cell growth and inducing apoptosis in many human prostate carcinoma cells. Here we demonstrate the molecular mechanism of inhibitory action of apigenin on androgen-refractory human prostate carcinoma DU145 cells that have mutations in the tumor suppressor gene p53 and pRb. Treatment of cells with apigenin resulted in a dose- and time-dependent inhibition of growth, colony formation, and G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin D1, D2, and E and their activating partner, cyclin-dependent kinase (cdk)2, 4, and 6, with concomitant upregulation of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18. The induction of WAF1/p21 and its growth inhibitory effects by apigenin appears to be independent of p53 and pRb status of these cells. Apigenin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1 (Apaf-1). This effect was found to result in a significant increase in cleaved fragments of caspase-9, -3, and poly(ADP-ribose) polymerase (PARP). Further, apigenin treatment resulted in downmodulation of the constitutive expression of nuclear factor-kappaB (NF-kappaB)/p65 and NF-kappaB/p50 in the nuclear fraction that correlated with an increase in the expression of IkappaB-alpha (IkappaBalpha) in the cytosol. Taken together, we concluded that molecular mechanisms during apigenin-mediated growth inhibition and induction of apoptosis in DU145 cells was due to (1) modulation in cell-cycle machinery, (2) disruption of mitochondrial function, and (3) NF-kappaB inhibition.     

Targeted therapy with a cytotoxic somatostatin analog, AN-238, inhibits growth of human experimental endometrial carcinomas expressing multidrug resistance protein MDR-1

BACKGROUND: Chemoresistance mediated by membrane transporters such as multidrug resistance (MDR-1) glycoprotein remains a challenge in the chemotherapy treatment of advanced or recurrent endometrial carcinoma. Targeted chemotherapy might overcome this resistance. The cytotoxic somatostatin (SST) analog, AN-238, consists of a superactive derivative of doxorubicin (DOX), 2-pyrrolino-DOX (AN-201), linked to the SST analog carrier, RC-121. This conjugate binds strongly to SST receptor subtypes (sst) 2a (sst2(a)) and 5 (sst(5)) and can be targeted to tumors that express these receptors. METHODS: The presence of sst2(a) and sst(5) was determined in 3 human endometrial carcinoma cell lines (HEC-1A, RL-95-2, and AN3CA). Nude mice bearing xenografts of these cancers were treated with AN-238 and its radical, AN-201. The antitumor effects and toxicity were compared. The authors studied the effects of AN-238 and AN-201 on the expression levels of MDR-1, multidrug resistance related protein (MRP-1), and breast carcinoma resistance protein (BCRP) by real-time polymerase chain reaction. RESULTS: The authors demonstrated the presence of mRNA and receptor protein for sst(2a) and sst(5) on HEC-1A, RL-95-2, and AN3CA tumors. AN-238 significantly (P < 0.05) inhibited the growth of these tumors, whereas AN-201 had no effect. Blockade of SST receptors nullified the effects of AN-238. In all 3 endometrial carcinoma lines, AN-238 caused a weaker induction of MDR-1 than AN-201. No major induction of MRP-1 and BCRP occurred after treatment with AN-238 or AN-201. CONCLUSIONS: Targeted chemotherapy with the cytotoxic SST analog, AN-238, inhibited powerfully the growth of endometrial carcinoma, which express SST receptors, regardless of their expression level of MDR-1.     

Induction of apoptosis in BCL-2-expressing rat prostate cancer cells using the Semliki Forest virus vector.

The Semliki Forest virus (SFV) vector is a transient RNA expression vector that has an inherent p53-independent apoptosis-inducing property. It is administered as recombinant SFV particles (rSFV) that undergo 1 round of replication only and express a gene cloned into the multicloning site. In our study we have investigated the ability of the SFV vector to induce apoptosis and inhibit tumour growth in rat prostate cancer (AT3-Neo) cells expressing the Bcl-2 oncogene (AT3-Bcl-2 cells), which normally inhibits apoptosis. rSFV expressing the enhanced green fluorescent protein (EGFP) gene (rSFV-EGFP), or recombinant RNA transfected into cells by electroporation, induced delayed apoptosis in AT3-Bcl-2 cells. SFV-mediated expression of a cloned pro-apoptotic Bax gene by the vector, however, enhanced apoptosis induction both in AT3-Bcl-2 cells and standard BHK-21 cells. Such Bax-expressing particles could be produced only at low titers compared to EGFP-expressing particles under standard conditions for particle production, but lowering the incubation temperature for particle production to 33 degrees C partially alleviated this effect. Bax-expressing particles were shown to inhibit the growth of AT3-Neo and AT3-Bcl-2 tumours in nude mice, as did high titre EGFP-expressing particles. It is concluded that SFV recombinant particles have potential as anti-tumour agents to treat apoptosis-resistant tumours.     

Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth

G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity.     

Simultaneous targeting of androgen receptor (AR) and MAPK-interacting kinases (MNKs) by novel retinamides inhibits growth of human prostate cancer cell lines

Androgen receptor (AR) and MNK activated eIF4E signaling promotes the development and progression of prostate cancer (PCa). In this study, we report that our Novel Retinamides (NRs) target both AR signaling and eIF4E translation in androgen sensitive and castration resistant PCa cells via enhancing AR and MNK degradation through ubiquitin-proteasome pathway. Dual blockade of AR and MNK initiated eIF4E activation by NRs in turn induced cell cycle arrest, apoptosis, and inhibited cell proliferation. NRs also inhibited cell migration and invasion in metastatic cells. Importantly, the inhibitory effects of NRs on AR signaling, eIF4E translation initiation and subsequent oncogenic program were more potent than that observed with clinically relevant retinoids, established MNK inhibitors, and the FDA approved PCa drugs. Our findings provide the first preclinical evidence that simultaneous inhibition of AR and eIF4E activation is a novel and efficacious therapeutic approach for PCa, and that NRs hold significant promise for treatment of advanced prostate cancer.     

Equol inhibits prostate cancer growth through degradation of androgen receptor by S‐phase kinase‐associated protein 2

Chemopreventive and potential therapeutic effects of soy isoflavones have been shown to be effective in numerous preclinical studies as well as clinical studies in prostate cancer. Although the inhibition of androgen receptor signaling has been supposed as one mechanism underlying their effects, the precise mechanism of androgen receptor inhibition remains unclear. Thus, this study aimed to clarify their mechanism. Among soy isoflavones, equol suppressed androgen receptor as well as prostate‐specific antigen expression most potently in androgen‐dependent LNCaP cells. However, the inhibitory effect on androgen receptor expression and activity was less prominent in castration‐resistant CxR and 22Rv1 cells. Consistently, cell proliferation was suppressed and cellular apoptosis was induced by equol in LNCaP cells, but less so in CxR and 22Rv1 cells. We revealed that the proteasome pathway through S‐phase kinase‐associated protein 2 (Skp2) was responsible for androgen receptor suppression. Taken together, soy isoflavones, especially equol, appear to be promising as chemopreventive and therapeutic agents for prostate cancer based on the fact that equol augments Skp2‐mediated androgen receptor degradation. Moreover, because Skp2 expression was indicated to be crucial for the effect of soy isoflavones, soy isoflavones may be applicable for precancerous and cancerous prostates.     

Inhibition of the Akt, cyclooxygenase-2, and matrix metalloproteinase-9 pathways in combination with androgen deprivation therapy: potential therapeutic approaches for prostate cancer

Prostate cancer cells are generally dependent on androgen stimulation mediated by the androgen receptor (AR) for growth and survival, and, therefore, hormonal manipulation, such as castration and/or the use of AR antagonists, results in a regression of the cancer. However, this treatment very rarely leads to the "cure" of advanced disease, and cancers eventually become androgen-independent. A number of genes/pathways have been reported to be activated in prostate cancer, most of which are possibly associated with disease progression. In this article, among them, we focus on Akt (also known as protein kinase B), cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9, whose activities or expressions have been found to be regulated by androgens/AR. Previous studies by us and others, with androgen-sensitive prostate cancer cell lines, have demonstrated that androgen deprivation results in activation/overexpression of Akt, COX-2, and MMP-9 in cells. This suggests that androgen deprivation in clinical settings activates the Akt, COX-2, and MMP-9 pathways in prostate cancer, which may increase cell growth and in turn promote the transition to the androgen-independent state. We hypothesize that androgen deprivation, in combination with inhibition of the Akt, COX-2, and MMP-9 pathways, delays the androgen-independent transition and has more beneficial effects than hormonal therapy alone.     

Three independently deleted regions at chromosome arm 16q in human prostate cancer: allelic loss at 16q24.1-q24.2 is associated with aggressive behaviour of the disease, recurrent growth, poor differentiation of the tumour and poor prognosis for the patient.

Loss of heterozygosity at chromosome arm 16q is a frequent event in human prostate cancer. In this study, loss of heterozygosity at 16q was studied in 44 prostate cancer patients exhibiting various clinical features. Fifteen polymorphic polymerase chain reaction (PCR) markers were used to identify the separately deleted areas and the findings were compared with clinicopathological variables and 5-year survival of the patients. The results indicated that there are at least three independently deleted regions at 16q. Allelic losses at the central and distal areas were associated significantly with aggressive behaviour of the disease (16q24.1-q24.2, P < 0.01, and 16q24.3-qter, P < 0.05), and the central area of deletion was further significantly associated with poorly differentiated tumour cells (P < 0.05) and with recurrent (P < 0.01) growth of the tumour. During the follow-up period, 28% of the patients initially with M0 disease developed distant metastases. Of the patients showing allelic loss at 16q24.1-q24.2, distant metastasis were found in 45% during the 5-year follow-up period, and 31% of the patients showing loss at 16q21.1 also developed distant metastases. After the 5-year follow-up period, 14 (32%) of the patients remained alive, whereas 19 (43%) had died because of their prostate cancer. The overall survival rate of the patients showing allelic loss at 16q21.1 or 16q24.1-q24.2 was significantly lower than that of the patients with retained heterozygosity.