Comparative characterization of the canine normal prostate in intact and castrated animals

BACKGROUND: Prostate diseases in the dog are generally regarded as representative for their human counterparts. We characterized the normal canine prostate in comparison to the normal human prostate. METHODS: Prostates of dogs were examined histomorphologically and by immunohistochemical detection of the markers CK14, HMWCK, CK5, CK18, CK7, UPIII, PSA, and PSMA. RESULTS: Histomorphologically, the canine prostate lacks the human zonal differentiation, has much more prominent acini, while comprising less stromal tissue. In general, the canine prostate epithelium displayed a highly differentiated character, with no cells expressing CK14, minimal amounts of cells expressing HMWCK/CK5 and the vast majority of cells expressing CK18 and PSA. After castration, the prostate epithelium regressed, and the remaining tubules were largely populated by cells showing a ductal phenotype (HMWCK+/CK5+/CK18+/CK7+). CONCLUSIONS: The human and canine prostate are histologically differently organized. The general scheme of cellular differentiation of the prostate epithelium may however be applicable to both species.     

Treatment with soybean-derived Bowman Birk inhibitor increases serum prostate-specific antigen concentration while suppressing growth of human prostate cancer xenografts in nude mice.

BACKGROUND: Bowman Birk inhibitor (BBI) is an anticarcinogenic serine protease inhibitor that may inhibit the protease activity of prostate-specific antigen (PSA) and the growth of human prostate cancer xenografts in nude mice. METHODS: Human prostate cancer xenografts were established by implanting LNCaP cells into the prostate glands of NCRNU-M athymic nude mice. The animals with established tumors were maintained on a control diet or diets supplemented with 1% BBI or 1%, 2%, or 3% BBI concentrate (BBIC) for 6 weeks. The serum PSA concentrations were determined before and after the BBI or BBIC treatment period. The final tumor loads were determined at autopsy. RESULTS: Treatment with BBI or BBIC decreased the final tumor load and increased the tumor doubling time and PSA density in the nude mice bearing human prostate cancer xenografts. CONCLUSIONS: BBI and/or BBIC could be useful for prostate cancer treatment.     

Comparative study of PSMA expression in the prostate of mouse, dog, monkey, and human

BACKGROUND: Intraprostatic PSMA targeted prodrugs/protoxins are under development in our laboratory. Future toxicologic studies of these therapies require identification of animal models that express PSMA within the prostate. METHOD: PSMA enzymatic activity and protein expression was determined. PSMA expression in the prostates of mouse, dog, and monkey were compared to humans by real-time PCR analysis. RESULTS: No substrate hydrolysis was observed in dog or monkey prostate homogenates. Monkey prostate was negative for PSMA protein expression. No significant PSMA mRNA levels were detected by real time PCR in mouse, dog, or monkey prostate tissue compared to PSMA negative tissues. CONCLUSIONS: PSMA is not expressed in any significant amount in the prostates of mouse, beagle dog, or macaque monkeys in this study but is expressed in high levels by human prostate. These non-human species, therefore, are not suitable toxicologic models to assess prostate damage from PSMA-activated intraprostatic prodrug/protoxin therapies.     

Anti-tumor effects and lack of side effects in mice of an immunotoxin directed against human and mouse prostate-specific membrane antigen

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature in humans, but not on the vasculature of normal tissues. Expression of low levels of PSMA has also been found on non-vascular cells in several normal tissues, most prominently on the brain and kidney in humans. PSMA is an excellent candidate for targeting prostate cancer or targeting tumor vasculature of various solid tumors. The high potential clinical benefit of these agents has prompted the search for an animal model in which to assess the efficacy and safety of anti-PSMA monoclonal antibody (mAb)-based therapies. METHODS: A rat monoclonal antibody, E6 that recognizes both mouse and human PSMA was generated using conventional hybridoma techniques. The antibody was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry. An immunotoxin composed of E6, antibody and deglycosylated ricin A-chain (dgA) was prepared chemically. The anti-tumor effects of the immunotoxin were determined in vitro and in mice bearing subcutaneous LnCaP human prostate tumors, which express PSMA on the tumor cell surface. RESULTS: E6 recognizes the extracellular domain of both human and mouse PSMA in ELISA, immunoblot and by immunohistochemistry. E6 strongly stained the vascular endothelium of tumors from humans but not from mice. E6 stained proximal tubules in mouse and human kidneys, and neurons in the mouse and human hippocampus but, unlike the human, did not detectably stain epithelial cells in mouse prostate or small intestine. An E6-dgA immunoconjugate strongly inhibited the growth of LnCaP tumor xenografts without causing apparent toxicity to the mice. Histological observation indicated that the anti-tumor effects were mediated through direct cytotoxic effects on the tumor cells. CONCLUSIONS: We have generated and characterized a rat mAb (E6) that reacts specifically with both human and mouse PSMA and have demonstrated that an immunotoxin constructed from E6 is safe and effective against human prostatic carcinoma cells growing subcutaneously in nude mice.     

A new generation of monoclonal and recombinant antibodies against cell-adherent prostate specific membrane antigen for diagnostic and therapeutic targeting of prostate cancer

BACKGROUND: Prostate-specific membrane antigen (PSMA) is an excellent candidate for targeting prostate cancer by virtue of its restricted expression on prostatic epithelial cells and its upregulation on prostatic carcinoma cells. PSMA is expressed on the cell surface displaying a specific three-dimensional structure. Therefore, only antibodies with a high cell binding activity will have an important impact on antibody-based imaging and therapy. METHODS: Monoclonal antibodies (mAbs) and single chain antibody fragments (scFvs) were prepared from spleen cells of mice that had been immunized either with purified PSMA or a cell lysate of prostate cancer LNCaP cells containing native PSMA. mAbs and scFvs were screened for reactivity with purified PSMA and binding to PSMA-expressing LNCaP cells. RESULTS: From mice immunized with purified PSMA, we obtained three mAbs (K7, K12, D20) and four scFvs (G0, G1, G2, G4), which were highly reactive with the isolated antigen, but showed weak or no reaction with viable LNCaP cells. From mice immunized with unpurified LNCaP lysate, we obtained three mAbs (3/E7, 3/F11, 3/A12), and one scFv (A5), which were reactive with purified PSMA, also showing a strong and specific binding to viable LNCaP cells and PSMA-transfected cells. CONCLUSIONS: Our results suggest that only the mAbs and scFvs, that were elicited with unpurified LNCaP lysate and not with purified PSMA will be useful agents for diagnostic imaging and therapeutic applications of prostate cancer.     

High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP.

BACKGROUND: An animal model of human prostate cancer LNCaP demonstrating high rates of spontaneous metastasis from the orthotopic site after tumor implantation would be very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice in order to develop high metastatic mouse models of human cancer. METHODS: Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP, was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological examination of primary tumors and relevant organs was done to identify and quantitate micrometastasis. RESULTS: Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis. Mean survival time was 72 days, indicating a high degree of malignancy of the tumor. CONCLUSIONS: The extensive and widespread lung metastasis as well as lymph node metastasis following orthotopic implantation of LNCaP in nude mice and the short survival time provide a high-malignancy nude model of the LNCaP human prostate tumor.     

survivin启动子与PSMA启动子/增强子在前列腺癌细胞系中的转录活性比较

目的:通过研究并比较前列腺特异性膜抗原(PSMA)基因启动子、增强子和survivin基因启动子在不同前列腺癌细胞系(LNCaP和PC-3细胞)中的转录活性,为前列腺癌的靶向性基因治疗提供依据。方法:采用PCR扩增PSMA基因的启动子、增强子和survivin基因启动子,分别克隆入pGL3-Basic,脂质体转染前列腺癌细胞和张氏肝细胞,检测各启动子在细胞中的转录活性。结果:survivin基因启动子在前列腺癌细胞中均具有较强活性,均明显高于PSMA启动子/增强子,其中S2pro启动活性最强,达到CMV启动子活性的1/3,然而,survivin启动子及PSMA启动子/增强子在肝细胞系中几乎不表达。结论:survivin启动子在前列腺癌细胞中具有较强启动活性,可望成为新的前列腺癌靶向性基因治疗工具。     

利用裸鼠建立人前列腺梭形细胞瘤细胞系PC-98106的研究

目的：建立人前列腺肿瘤细胞系PC－98106，为前列腺肿瘤基础研究提供实验模型。方法：由前列腺肿瘤手术标本中取组织块，分别包埋在3只裸鼠右后肢皮下，瘤块增殖后再次包埋于另1只裸鼠皮下，取移植瘤进行体外培养。结果：3个移植瘤均增殖，裸鼠体内传代后增殖速率加快。体外培养建立的前列腺肿瘤细胞系PC－98106其形态结构、分化程度与原发瘤保持一致，染色体形态人类核型，众数维持在64－73之间，占80％，细胞周期分析G1期53．2％，G2期17．0％，S期29．8％，G2／G1＝1．876，为亚三倍体细胞，细胞群体培增时间为16．7h，前列腺特异性抗原（PSA）表达阴性。结论：PC－98106与原发癌保护相同的生物学特性，体外连续传代1年以上，传代120代，细胞形态不变，生长周期恒定，已成为一个稳定细胞系。     

In vitro and preclinical targeted alpha therapy of human prostate cancer with Bi-213 labeled J591 antibody against the prostate specific membrane antigen

Limited options for the treatment of prostate cancer have spurred the search for new therapies. One innovative approach is the use of targeted alpha therapy (TAT) to inhibit cancer growth, using an alpha particle emitting radioisotope such as (213)Bi. Because of its short range and high linear energy transfer (LET), alpha-particles may be particularly effective in the treatment of cancer, especially in inhibiting the development of metastatic tumors from micro-metastases. Prostate-specific membrane antigen (PSMA) is expressed in prostate cancer cells and the neovasculature of a wide variety of malignant neoplasms including lung, colon, breast and others, but not in normal vascular endothelium. The expression is further increased in higher-grade cancers, metastatic disease and hormone-refractory prostate cancer (PCA). J591 is one of several monoclonal antibodies (mabs) to the extracellular domain of PSMA. Chelation of J591 mab with (213)Bi forms the alpha-radioimmunoconjugate (AIC). The objective of this preclinical study was to design an injectable AIC to treat human prostate tumors growing subcutaneously in mice. The anti-proliferative effects of AIC against prostate cancer were tested in vitro using the MTS assay and in vivo with the nude mice model. Apoptosis was documented using terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end-labeling (TUNEL) assay, while proliferative index was assessed using the Ki-67 marker. We show that a very high density of PSMA is expressed in an androgen-dependent human PCA cell line (LNCaP-LN3) and in tumor xenografts from nude mice. We also demonstrate that the AIC extensively inhibits the growth of LN3 cells in vitro in a concentration-dependent fashion, causing the cells to undergo apoptosis. Our in vivo studies showed that a local AIC injection of 50 microCi at 2 days post-cell inoculation gave complete inhibition of tumor growth, whereas results for a non-specific AIC were similar to those for untreated mice. Further, after 1 and 3 weeks post-tumor appearance, a single (100 microCi/100 microl) intra-lesional injection of AIC can inhibit the growth of LN3 tumor xenografts (volume<100 mm(3)) in nude mice. Tumors treated with AIC decreased in volume from a mean 46+/-14 mm(3) in the first week or 71+/-15 mm(3) in the third week to non-palpable, while in control mice treated with a non-specific AIC using the same dose, tumor volume increased from 42 to 590 mm(3). There were no observed side effects of the treatment. Because of its in vitro cytotoxicity and these anti-proliferative properties in vivo, the (213)Bi-J591 conjugate has considerable potential as a new therapeutic agent for the treatment of prostate cancer.     

Canine prostate stimulates osteoblast function using the endothelin receptors

BACKGROUND: Bone metastases are common in humans and dogs with late-stage prostate cancer. A unique feature of prostate cancer metastases is new bone formation at metastatic sites ("osteoblastic metastases"). Many carcinomas that metastasize to bone cause bone destruction, not new bone formation. The mechanisms by which prostate cancer induces bone formation at sites of bone metastasis are not well understood. We hypothesized that stimulation of osteoblasts by prostate tissue at metastatic sites was due to the paracrine actions of growth factors produced by prostate epithelial cells. METHODS: We have previously shown that normal canine prostate tissue induced new bone formation when implanted adjacent to the calvarium of nude mice. To complement this in vivo model, we developed an in vitro system of prostate-stimulated osteoblast function to investigate mechanisms of prostate-induced new bone formation. RESULTS: We found that treatment of cultured rat calvaria for 24 hr with proteins from normal dog prostate stimulated alkaline phosphatase activity in a dose-dependent manner 4-6 fold compared to controls. Stimulation began approximately 8 hr after treatment, and was diminished after 72 hr. Calvaria treated with homogenates of normal dog salivary gland, kidney, bladder, and muscle did not increase ALP activity. Pretreatment of the calvaria for 1 hr with endothelin antagonists, but not anti-parathyroid hormone-related protein (PTHrP) antibody or indomethacin, abrogated the stimulation of ALP. CONCLUSIONS: Our results indicated that osteoblast activation by canine prostate occurs via an endothelin-dependent mechanism, and that PTHrP or prostaglandin synthase-mediated pathways are likely not involved. This is a reliable, reproducible assay for determining the roles of molecules important in the activation of osteoblasts by the prostate.     

Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer

PURPOSE: We investigate the potential use of the phytotherapeutic PC-SPES to treat human prostate cancer, and evaluate its in vivo and in vitro activity, and clinical efficacy. MATERIALS AND METHODS: PC-SPES was evaluated for its ability to induce apoptosis on prostate cancer cell lines LNCaP, PC3 and DU145. The effect of oral PC-SPES on growth of PC3 tumors present in male immunodeficient mice was studied. A total of 30 male nude mice were divided in 5 groups. In groups 1 control and 2 full dose therapy was started the same day of the tumor injection. In groups 3 control, 4 half dose and 5 full dose PC-SPES therapy was initiated 1 week after tumor injection. A total of 69 patients with prostate cancer were treated with 3 capsules of 320 mg. PC-SPES daily. Serum prostate specific antigen (PSA) responses and side effects were evaluated. RESULTS: All of the cultured prostate cancer cell lines had a significant dose dependent induction of apoptosis following exposure to an alcoholic PC-SPES extract. Immunodeficient mice xenografted with the PC3 cell line had reduced tumor volume compared with sham treated controls when they were treated with a PC-SPES extract from the time of tumor cell implantation (931 +/- 89 versus 1,424 +/- 685 mm.3, p not significant) but not when the treatment was begun 1 week after tumor cell implantation. The testis, prostate, bladder and seminal vesicles of the treated mice were significantly reduced in weight compared with the sham treated animals. Of the patients with prostate cancer 82% had decreased serum PSA 2 months, 78% 6 months and 88% 12 months after treatment with PC-SPES. Side effects in the treated patient population included nipple tenderness in 42% and phlebitis requiring heparinization in 2%. CONCLUSIONS: An extract of the phytotherapeutic agent PC-SPES proved to be active in inducing apoptosis of hormone sensitive and insensitive prostate cancer cells in vitro, and in suppressing the growth rate of a hormone insensitive prostate cancer cell line in vivo. The overwhelming majority of patients with prostate cancer treated with the agent experienced a decrease in serum PSA but also demonstrated a side effect profile comparable to estrogen treatment.     

Histopathological and immunohistochemical characterization of canine prostate cancer

BACKGROUND: In this study we try to identify the origin of canine prostate cancer (cPC) by classifying the tumors histological subtypes and relate these subtypes to their combined expressional characteristics of several tissue specific and differentiation markers. METHODS: cPCs were examined histomorphologically and by immunohistochemical detection of the cytokeratin markers CK14, HMWCK, CK5, CK18, and CK7, and of the markers UPIII, PSA and PSMA. RESULTS: Histopathologically, six growth patterns could be differentiated. The most frequent patterns were solid, cribriform and micropapillary growth patterns, while sarcomatoid, small acinar/ductal, and tubulo-papillary growth patterns were less frequent present. Solid growth patterns were significantly (P = 0.027) more often seen in castrated dogs. Immunohistochemically, about half of the cPC cases showed expression of PSA (8/20) and PSMA (10/20); 85% and 60% of the cPC expressed UPIII (17/20) and CK7 (12/20), while 13 and 12 cPC expressed CK5 and CK14, respectively; all cPC expressed CK18. CK14 was significantly more often and UPIII less frequent expressed in the solid growth patterns than in the micropapillary and cribriform patterns, respectively. CONCLUSIONS: Canine prostate cancer appear to be more aggressive and of a less differentiated type than most common human prostate cancers. Comparing the expression patterns of the markers in cPC to those in normal canine prostate tissue, cPC most likely originates from the collecting ducts rather than from the peripheral acini. Given also the fact that canine prostate cancer is unresponsive to androgen withdrawal therapy, canine prostate cancer mostly resembles human, androgen refractory, poorly differentiated prostate cancer.     

Overexpression of the EphA2 tyrosine kinase in prostate cancer.

BACKGROUND: Molecules that are highly expressed by human prostate cancers may serve as therapeutically relevant targets or tumor markers. Tyrosine kinases are frequently overexpressed in metastatic tumor cells and this prompted us to screen for tyrosine kinases that are overexpressed in prostate cancer cells. METHODS: Expression levels of the EphA2 receptor tyrosine kinase were determined by Western blot analysis in canine and human prostate cancer cell lines and in immortalized and transformed variants of 267B1 prostatic epithelial cells. EphA2 levels in benign human prostate and prostate cancers were also determined in formalin-fixed, paraffin-embedded tissues using immunohistochemical staining. RESULTS: Metastatic prostate cancer cells overexpressed EphA2 by 10-100 fold as compared with non-invasive prostatic epithelial cells. EphA2 immunoreactivity in vivo was also significantly greater in human prostate cancers as compared with benign prostate epithelium. CONCLUSIONS: The EphA2 receptor tyrosine kinase is differentially expressed in human and canine prostate cancer cell lines and overexpressed in human prostate cancers as compared with benign prostate tissues. Metastasis-derived canine prostate carcinoma cell lines overexpress EphA2 and may provide pre-clinical models to further evaluate the role of EphA2 in prostate carcinogenesis. Further investigations are needed to determine the utility of EphA2 as a tumor marker and a novel target in human prostate cancer.     

Radioimmunotherapy of prostate cancer in human xenografts using monoclonal antibodies specific to prostate specific membrane antigen (PSMA): studies in nude mice

BACKGROUND: Prostate specific membrane antigen (PSMA), expressed by virtually all prostate cancers is an ideal target for targeted therapy of prostate cancer. Radiolabeled J591 monoclonal antibody (MAb) binds with high affinity to an extracellular epitope of PSMA and localizes specifically in PSMA positive LNCaP tumors in vivo. METHODS: Pre-clinical radioimmunotherapy (RIT) studies using (131)I-huJ591 and (90)Y-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-huJ591 MAbs were studied in nude mice bearing LNCaP xenografts. RESULTS: A 15-90% reduction in mean tumor volume was observed after a single dose of (131)I-huJ591 (3.7-11.1 MBq) or (90)Y-DOTA-huJ591 (3.7-7.4 MBq). The median survival time increased 2-3 times relative to untreated controls. Multiple administrations of fractionated doses of (90)Y-DOTA-huJ591 were even more effective with minimal toxicity. Radiation dose to blood and tumor was higher with (90)Y than with (131)I. The maximum tolerated dose (MTD) is 5.55 MBq for (90)Y-DOTA-huJ591 and more than 11.1 MBq for (131)I-huJ591. For (90)Y-DOTA-huJ591 at MTD, dose to the tumor was 2,753 cGy. CONCLUSIONS: In nude mice bearing PSMA positive tumors, radiation dose to the tumor with (90)Y-DOTA-J591 is greater for large tumors than with (131)I-J591. The theoretical and practical considerations strongly suggest that (90)Y-DOTA-huJ591 may be a suitable radiopharmaceutical for the treatment of prostate cancer.     

Targeting metastatic prostate cancer with radiolabeled monoclonal antibody J591 to the extracellular domain of prostate specific membrane antigen

PURPOSE: We performed an interim analysis of imaging data collected in 2 phase I radioimmunotherapy trials to determine the ability of monoclonal antibody (mAb) J591 directed to the extracellular domain of prostate specific membrane antigen (PSMA) to target sites of known metastatic prostate cancer accurately. MATERIALS AND METHODS: Patients with progressing hormone independent prostate cancer were entered in 2 phase I dose finding trials with radiolabeled mAb J591. J591 is the first mAb targeting the extracellular domain of PSMA as well as the first de-immunized (humanized) mAb to PSMA to be tested in humans. These trials were primarily designed to assess dose limiting toxicity, maximum tolerated dose, pharmacokinetics and organ dosimetry. Planar gamma camera imaging studies obtained on the first 53 patients were reviewed and compared to sites of metastatic prostate cancer visualized on conventional imaging studies including bone scan, computerized tomography and/or magnetic resonance imaging. In 1 trial 29 patients received 111indium-J591 for imaging followed by 90yttrium-J591 for therapy. In the parallel trial 24 patients were treated with 177lutetium-J591, an isotope that can be imaged directly. RESULTS: Of 53 patients reviewed 46 (87%) had evidence of metastatic disease on conventional scans. Overall, of the 43 evaluable patients J591 accurately targeted bone and/or soft tissue lesions in 42 (98%). J591 accurately targeted bone lesions in 32 of 34 (94%) and soft tissue lesions in 13 of 18 (72%) evaluable patients. CONCLUSIONS: Radiolabeled J591 accurately targets bone and soft tissue metastatic prostate cancer sites, and may be useful for targeting therapeutic and/or diagnostic imaging agents.     

1alpha,25-Dihydroxyvitamin D3 down-regulates expression of prostate specific membrane antigen in prostate cancer cells

BACKGROUND: Prostate specific membrane antigen (PSMA) expression correlates with prostate cancer grade and is increased in hormone-refractory prostate cancer. The increased expression of PSMA following androgen deprivation therapy may be a consequence of the down-regulation of PSMA expression by androgen. Moreover, 1alpha,25-dihydroxyvitamin D3 (1,25-VD) has been shown to suppress prostate cancer progression as well as cell motility and invasion. Since PSMA is positively correlated with both of these characteristics, we hypothesized that 1,25-VD would regulate PSMA expression. METHODS: LNCaP prostate cancer cells were treated with 1,25-VD, followed by analysis of cell surface PSMA expression. The PSMA enhancer, located within the third intron of the PSMA gene, was cloned into a reporter vector and regulation by 1,25-VD was investigated. The role of the androgen receptor (AR) in 1,25-VD mediated suppression of PSMA expression was examined using Casodex and AR specific siRNA. RESULTS: Surface expression of PSMA was significantly decreased in a dose-dependent manner by 10 nM 1,25-VD or greater. Regulation by 1,25-VD occurred at the level of the PSMA enhancer. Over-expression of the vitamin D receptor (VDR) also decreased expression of PSMA. Additionally, suppression of AR translation using siRNA technology blocked the suppressive effect of 1,25-VD on PSMA expression, however inhibition of PSMA expression by 1,25-VD occurred in the absence of androgens. CONCLUSIONS: Suppression of PSMA by 1,25-VD occurs at the level of the PSMA enhancer and is elevated by over-expression of the VDR. This regulation involves the AR, but is not dependent on the presence of androgens.