Estramustine-binding Protein Research Articles

Relationship of thyroid to estramustine binding protein (EMBP) concentration in rat prostate

Alternations of estramustine binding protein (EMBP) concentration in rat prostate in relation to androgen status under hypo- and hyperthyroid conditions was estimated by means of radioimmunoassay (RIA). In hyperthyroid rats, EMBP concentration in dorsal as well as in lateral prostate was not significantly changed, but significantly reduced in ventral prostate (P less than 0.01). Ventral tissue concentration of testosterone(T) + dihydrotestosterone (DHT), DHT and T, and the ventral tissue ratio of DHT to T + DHT were not significantly changed in hypo- and hyper-thyroid conditions compared with that of the control. In hyperthyroid rats, serum T level and EMBP concentration in ventral prostate were significantly reduced in comparison to ones of the control (P less than 0.05 and P less than 0.01, respectively), however, the ratio of ventral tissue (T + DHT) to serum T was significantly increased (P less than 0.05). Moreover, in contrast to the control, EMBP concentration expressed as microgram/microgram ventral DNA was significantly reduced in hyperthyroid rats (P less than 0.05). On the other hand, in hypothyroid rats, change of EMBP concentration in ventral prostate in relation to androgen status was not significant in comparison to the control. The results here obtained revealed that EMBP concentration in ventral prostate under hyperthyroid condition would be affected by (a) a modulation of endogenous androgen-dependency in EMBP synthesis or by (b) a reduction of cell numbers responsible for EMBP synthesis.

Estramustine binding protein in human benign prostatic hypertrophy

Using a highly sensitive radioimmunoassay (RIA) of estramustine binding protein (EMBP) with the antibody raised against rat-EMBP, the concentration of EMBP in human benign prostatic hypertrophy (BPH) tissue was evaluated. Using mechanical separation procedure, EMBP was found significantly higher in the epithelium (10.7 ng/mg protein) than in the stroma (2.9 ng/mg protein). When human BPH was classified into glandular, fibromuscular and mixed type, the concentration of EMBP was the highest in glandular type (15.7 ng/mg protein) and the lowest in fibromuscular type (10.8 ng/mg protein).

Immunoreactive protein to rat estramustine binding protein in human seminal plasma: its relationship to prostatic acid phosphatase and zinc

Immunoreactive estramustine binding protein in human seminal plasma obtained from 34 infertile men was measured by means of radioimmunoassay (RIA) using the antibody raised against EMBP obtained from ventral prostate of rat. The mean concentration of IR-EMBP in human seminal plasma measured by this RIA was 1.092 ng/ml, ranging 258 to 1.859 ng/ml. Both concentrations of prostatic acid phosphatase and zinc in human seminal plasma concurrently measured were statistically significantly negatively correlated with that of IR-EMBP. These results here obtained might reveal that IR-EMBP in human seminal plasma signify androgen status in low possibility.

Evaluation of Lipophilins as Determinants of Tumor Cell Response to Estramustine

Estramustine administered orally as estramustine phosphate (EMP) remains a major tool in hormone refractory prostate cancer chemotherapy. The presence of estramustine binding protein, prostatin, in prostate tissue may be a determinant of response to treatment. Lipophilins are secretory proteins with homology to prostatin. Reverse transcription-polymerase chain reaction was performed to estimate expression patterns of lipophilins A to C in human biopsies and cell lines resistant to estramustine. Although lipophilin A was not expressed in prostate tissue, both lipophilins B and C were expressed in normal and tumor prostate without significant differences. For lipophilin C, a somatic mutation (T to C transition at positions 409 and 412) was found in human tumor samples and absent in normal prostate tissue. No consistent response to EMP was observed in enhanced green fluorescent protein (EGFP)-tagged lipophilin C-transfected PC3 cells compared with parental controls. Among these EGFP-lipophilin C clones, no direct correlation between response to EMP treatment (IC50 values) and EGFP expression was observed (p = 0.73). Lipophilin C mRNA levels did not vary significantly between wild-type and estramustine-resistant cells in prostate (DU145 and PC3) and ovarian (SKOV3) cancer cell lines. Overall, these results suggest that lipophilins are not specific determinants of estramustine efficacy.

Distribution of estramustine-binding protein during postnatal development of rat brain

Estramustine-binding protein (EMBP) is expressed in several types of brain tumors, such as astrocytoma, ependymoma, and meningioma. It binds the cytotoxic drug estramustine with high affinity and is suggested to cause accumulation of the drug in EMBP-expressing tumor cells. In this study, the spatial distribution of EMBP in normal rat brain was studied with immunohistochemistry. Brains from male and female rats of different ages were used. EMBP was found in the cytoplasm of ependymal cells, in the leptomeninges, mainly the arachnoid, and in scattered neurons. Moreover, staining was seen in nuclei of choroid plexus cells, in the granular cell layer in the cerebellum, and in a few scattered endothelial cells. The nuclear staining was more frequent in younger animals. No obvious difference in EMBP expression between male and female rats was observed. The expression of EMBP in rat brain was confirmed with nested RT-PCR. Future studies are justified to elucidate the role of EMBP-like proteins in CNS and in brain tumors.

Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer.

The mammaglobin gene encodes a 10-kDa glycoprotein that is distantly related to a family of proteins that includes rat estramustine binding protein (EMBP)/prostatein and human Clara cell 10-kDa protein (CC10)/uteroglobin. Among normal adult tissues, mammaglobin mRNA expression has been detected only in the mammary gland. As an initial step to determine mammaglobin's clinical utility as a breast tumor marker, we evaluated the frequency and specificity with which mammaglobin expression could be detected in primary breast tumors, metastatic breast tumors, and breast tumor cells present in the peripheral circulation. Approximately 80% of all primary and metastatic breast tumors examined were strongly immunopositive for mammaglobin protein, and staining was independent of tumor grade. Among peripheral stem cell collections from breast cancer patients, mammaglobin mRNA could be detected in 60% of cases. Recent work has identified the secreted mammaglobin protein in the sera of some breast cancer patients using both Western blot and ELISA. This study demonstrates that the detection of mammaglobin protein and mRNA in clinical samples may be a useful marker for primary, metastatic, and occult breast cancer.

Estramustine-binding protein in malignant glioma in rat.

Estramustine is a chemotherapeutic drug, used in the treatment of prostatic carcinoma. In the prostate, it binds specifically to a 46 kDa glycoprotein called estramustine-binding protein (EMBP), which consists of three polypeptide components; C1, C2, and C3, each coded for by a specific gene. Expression of EMBP and binding of estramustine has also been detected in malignant glioma in both rats and humans. Elevated levels of this protein in astrocytoma have proved to correlate with poor prognosis. In the present work, expression of all three polypeptide components of EMBP was confirmed in an orthotopic rat glioma model with nested reverse transcriptase PCR and Western blot (molecular weights of 8, 10, and 12 kDa). Specific binding of estramustine with a Kd of 40 for male and 50 for female rats, and a total number of binding sites of 0.7 and 0.4 pmol/mg proteins for male and female rats respectively, was demonstrated with Scatchard plot analysis. These binding characteristics are similar to those of prostatic EMBP. Further studies to elucidate how EMBP expression affects the effect of estramustine treatment, and its putative prognostic value is of special clinical interest. The confirmation of BMBP expression in BT4C rat glioma demonstrates its suitability as a model system for such studies.

Estramustine-binding protein in meningioma.

The presence of estramustine-binding protein has been suggested to positively correlate with the effect of the cytotoxic drug estramustine, a combination of estradiol and nornitrogen mustard used in the treatment of prostatic carcinoma. This study demonstrates expression of estramustine-binding protein in a series of meningioma using different ligand-based and immunological techniques. Scatchard plot analysis showed specific binding sites for [3H]lestramustine in meningioma tissue with a dissociation constant of 22-26 nM. Immunohistochemistry revealed an immunoreactivity in meningioma comparable to that demonstrated in prostatic carcinoma. The mean concentration (n = 6) of estramustine-binding protein in meningioma, as determined by radioimmunoassay was 159 ng/g tissue (range 18-274 ng/g). Moreover, partial characterization using size exclusion chromatography of [3H]estramustine-labeled tumor extracts and Western blot analysis of immunoprecipitated samples indicated that the structure of the estramustine-binding protein in meningioma is similar to that in rat prostate, with three polypeptide components of 10, 14, and 16 kDa, as compared to 8, 10-11, and 12 kDa in rat prostate. In conclusion, the novel observation of estramustine-binding protein and specific binding of estramustine in meningioma justify further evaluation regarding the role of estramustine-binding protein in the growth behavior of meningioma and the potential for estramustine and similar hormone-related drugs in the treatment of relapsing meningioma.

Lipophilin, a novel heterodimeric protein of human tears

We identified a novel heterodimeric protein, lipophilin AC, in human tears. One of its components, lipophilin A (69 residues; mass, 7575.1; p I, 9.47) was homologous to the C1 and C2 components of prostatein (`estramustine-binding protein'), the major secreted protein of rat prostate. Human lipophilin C (77 residues; mass, 8854.1; p I, 4.94) was homologous to the rat prostatein C3 component and to human mammaglobin, a protein overexpressed in some mammary carcinomas. Tear lipophilins A and C expand the roster of human uteroglobin superfamily members and provide models for exploring these typically steroid-regulated and steroid-binding molecules.

Synthesis and In Vitro Evaluation of [ carbonyl- 11C]Estramustine and [ carbonyl- 11C]Estramustine Phosphate

[ carbonyl- 11C]Estramustine and [ carbonyl- 11C]estramustine phosphate were synthesized from [ 11C]phosgene using a one pot procedure. [ carbonyl- 11C]Estramustine was obtained in 31–43% decay corrected yield based on radioactivity trapped in the reaction vessel. The product was obtained 25 min after the end of radionuclide production with a specific radioactivity of 0.38–1.11 Ci/μmol. A method was developed yielding [ carbonyl- 11C]estramustine phosphate in 29–45% decay corrected yield based on trapped radioactivity, without purification of the [ carbonyl- 11C]estramustine intermediate. The product was obtained within 40 min of the end of radionuclide production with a specific radioactivity of 0.59–0.86 Ci/μmol. Results from in vitro experiments suggest that because of their high nonspecific binding, the compounds are unsuitable for positron emission tomography as imaging agents for the estramustine binding protein in cancer.

Immunohistochemical analysis of estramustine binding protein with particular reference to proliferative activity in human prostatic carcinoma.

The estramustine binding protein (EMBP) specifically binds to estramustine and was first discovered in the rat ventral prostate. However, the physiological property of EMBP in the human prostate still remains to be elucidated. To elucidate whether EMBP is interrelated with cellular proliferation in human prostatic carcinoma (PC), the change in EMBP immunostaining during luteinizing hormone-releasing hormone (LH-RH) analog administration or during Cis-platinum-based chemotherapy, and the difference in EMBP immunostaining between hormone refractory (hr-PC) and untreated PC were analyzed. Forty-six patients with histologically proven untreated PCs (34 were treated with LH-RH analog and 12 were treated with chemotherapy as an initial therapy) and 14 with hr-PC were used in this study. PC tissues were obtained before and 3 months after the initial therapy. The changes in immunostainings for EMBP, proliferating cell nuclear antigen (PCNA), and nm23 protein were compared with the change in serum prostate-specific antigen (PSA) level and the histological response during the treatment. The increased EMBP expression was observed in tumors with high histological grade and high clinical stage as well as in hr-PC. In untreated PC, EMBP expression weakly correlated with PCNA or nm23 protein immunoreactivity. In PC receiving LH-RH analog, EMBP expression was significantly reduced after treatment, however, no significant changes were observed in PCNA or nm23 protein immunoreactivity. In addition, EMBP expression before the treatment significantly correlated with the serum PSA change, while PCNA expression and nm23 protein immunoreactivity did not. On the other hand, no significant relationship was observed between histological changes induced by the LH-RH analog and immunostainings for EMBP, PCNA, and nm23 protein before treatment. In PC patients receiving chemotherapy, immunostainings for EMBP, PCNA, and nm23 protein were not significantly changed during the treatment. EMBP immunoreactivity was significantly higher in hr-PC than in untreated PC with paralleled change of PCNA expression and nm23 protein immunoreactivity. These observations indicate that EMBP is androgen regulated in some PCs. However, EMBP expression is demonstrated even in hr-PC and is interrelated with cellular proliferation especially in hr-PC.

Radioiodinated estramustine phosphate and estramustine binding protein antibody accumulate in the prostate of a mouse.

The purpose of this study was to determine the distribution of radioiodinated estramustine (RI-EMP) and a radioiodinated antibody against estramustine binding protein (RI-EMBP-AB) in mice. RI-EMP and RI-EMBP-AB were injected in male mice intravenously, and the activities of tissue samples were measured 1-31 hr from the injection. Pure iodine-125 (RI) was used as a control. RI-EMP accumulated in the prostate, which contained 2.6% of injected activity (ID) per gram tissue at 7 hr. The liver had an activity of 21.4% ID/g at 1 hr, which decreased as RI-EMP was secreted in bile. The lung contained 2.3% ID/g at 7 hr, and it retained the activity longer than the prostate. RI-EMBP-AB accumulated in the prostate: The activity was 2.9% ID/g at 7 hr. The gallbladder contained 6.5% ID/g at 7 hr. Due to its cytotoxic and radiosensitizing properties, RI-EMP can possibly be used for treating prostate cancer and other tumors.

Radioiodinated estramustine phosphate and estramustine binding protein Antibody accumulate in the prostate of a mouse

Radioiodinated estramustine phosphate and estramustine binding protein Antibody accumulate in the prostate of a mouse

Targeting microtubule-associated proteins in glioblastoma: a new strategy for selective therapy.

This report presents a summary of preclinical data concerning the use of estramustine, an antimicrotubule agent against human glioblastoma cells. The strategy for the investigation of estramustine is predicated on the unique affinity of this agent for microtubule-associated proteins (MAPs). A series of laboratory investigations were used to demonstrate antiproliferative effects (MTT assay, colony forming assay, thymidine incorporation), cell cycle synchronization (flow cytometry), intracellular localization of binding sites (immunocytochemistry, electron microscopy), and activity in subcutaneous xenografts of human glioblastoma. Estramustine has potent in vitro activity against human glioblastoma cells and can enhance the cytotoxic effects of ionizing radiation. Estramustine-binding protein was abundantly expressed in glioblastoma cells and may contribute to the selective effects of estramustine on neoplastic cells. This agent has activity against subcutaneous xenografts of human glioblastoma. Synthesized novel estrogen carbamates also can inhibit proliferation of glioblastoma cells. Cytoskeletal elements (MAPs) of glioblastoma cells may provide a useful target for therapy with agents like estramustine because of the potent antimitotic effects of this agent and its affinity to a protein that is expressed in glioma cells. These observations have stimulated a search for other estrone carbamates with antimitotic activity that exceeds more conventional antimicrotubule agents.

Estramustine in malignant glioma

Estramustine, a carbamate ester combining 17 beta-estradiol and nornitrogen mustard, has primarily been employed in the treatment of advanced prostatic carcinoma. However, a significant amount of preclinical investigation has been directed toward estramustine's activity against human malignant glioma. These studies have demonstrated that estramustine has potent antiproliferative effects against malignant glioma both in vitro and in vivo. Similar antimitotic effects also have been demonstrated for other carbamate esters. Estramustine does not impair proliferation of nonneoplastic astrocytes at concentrations that inhibit glioma cells. Although the reasons for this selective activity remain to be determined, it has been shown that malignant gliomas expresses an estramustine-specific binding site, estramustine-binding protein, more than brain tissue. In the clinical situation, an uptake and accumulation of estramustine in human glioma tissue have been demonstrated. Estramustine has been shown to enhance the cytotoxic effects of irradiation in relatively radioresistant glioma cells both in cell culture and in a rat glioma model. Estramustine has been regarded as mainly an anti-mitotic drug but recently other effects such as inhibition of DNA synthesis, induction of apoptosis, and membrane alterations have been shown. This report summarizes the preclinical observations concerning the effects of estramustine and related compounds on human malignant gliomas. These findings form the basis for proposing further laboratory and clinical investigation regarding estramustine and human malignant gliomas.

Clinical study on estramustine binding protein (EMBP) in human prostate.

To elucidate the characteristics of estramustine binding protein (EMBP) in human prostate, tissue EMBP concentration was examined in 42 benign prostatic hypertrophy (BPH), 34 untreated prostatic carcinoma (PC), 8 hormone refractory PC (hr-PC), as well as 13 control prostate human tissue samples by RIA using rat-EMBP antibody, and the concentration thus obtained was compared with dihydrotestosterone (DHT), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), and zinc, indices exhibiting androgen dependency in the prostate. EMBP concentration correlated significantly with DHT and PSA levels in the control prostate and BPH, but not in untreated PC. In BPH, EMBP concentration increased significantly after administration of fluoxymesterone (4 mg/day for 2 weeks), whereas it decreased significantly after estramustine phosphate (280 mg/day for 2 weeks). The EMBP/DHT ratio in moderately and poorly differentiated, and the hr-PC was significantly higher than in controls, BPH, and well-differentiated PC. In addition, untreated PC with an EMBP/DHT ratio of more than 40 showed significantly lower progression-free probability as compared with PC with an EMBP/DHT of less than 40. These results suggest that (1) EMBP in BPH and well-differentiated PC preserves androgen dependency, but not in moderately and poorly differentiated, nor in hr-PCs, indicating that EMBP is a protein different from PAP and PSA, and (2) that the tissue EMBP/DHT ratio might be useful as a marker for predicting disease progression.

Clinical study on estramustine binding protein (EMBP) in human prostate

Clinical study on estramustine binding protein (EMBP) in human prostate

Effect of aromatase inhibitor, TZA-2209, on the prostate of androstenedione-treated castrated dogs: Changes in prostate volume and histopathological findings

To determine whether the inhibition of estrogen-related effect in the prostate would be of value in the management of benign prostate hyperplasia (BPH), we examined the effect of TZA-2209, a new steroidal aromatase inhibitor, on the prostate in three of six castrated beagles that received 75 mg/week androstenedione. The three other animals served as controls. Sequential measurements of prostate volume by transrectal ultrasonography showed that the volume in TZA-treated dogs was significantly decreased compared with that in the controls. Prostatic aromatase activity was suppressed by TZA administration. Histopathologically, the stromal component was increased and glands were atrophied by androstenedione treatment. TZA administration increased the volume of the glands. Immunohistochemical detection of estramustine-binding protein showed more positive staining of the protein in the glands that were increased in volume by TZA administration. We concluded that the aromatase inhibitor effectively antagonized the estrogen-related stromal changes, however, this action was accompanied by stimulation of the glandular component due to the accumulation of androgens, the substrate of the aromatase. In the light of these findings, we suggest the simultaneous treatment for the androgen-glandular component route in the prostate is necessary for the effective management of BPH.

Uptake and Distribution of the Estramustine-Phosphate Metabolite Estramustine after Single-Dose Injection in Patients with Prostatic Cancer

The concentration of estramustine (EaM) in serum and tumor tissue of 19 patients with prostatic carcinoma was determined after single i. v. injection of estramustine phosphate (300 or 600 mg) administered 12 to 15.5 hours prior to radical prostatectomy. The concentration (mean ± SD) was 21.9 ± 10.3 ng/ml in serum and 234 ± 168 ng/g in the tumor tissue. In patients treated with 300 mg the tumor tissue: serum ratio was decreased (12.0: 1 for 600 mg and 5.4: 1 for 300 mg, respectively). EMBP was detected in 18/19 (95 %) of the specimens, levels were positively correlated both with the tumor: serum ratio (r = 0.48) and the absolute tumor concentration (r = 0.62). Specific binding of EaM was obtained in 18/19 (95 %) of the specimens. The study is the first to show that the cytotoxically active metabolite estramustine is concentrated in prostatic tissue after single dose i.v. injection and that there might be a dose-dependent effect on the concentration. The concept of an estramustine binding protein that causes the selective binding and uptake of EaM in prostatic tissue is further supported by our results.

Estramustine-binding protein to dihydrotestosterone ratio in human prostatic carcinoma: a new marker for predicting disease progression.

To elucidate the clinical significance of estramustine-binding protein (EMBP) in human prostatic carcinoma (PC) as an indicator for predicting disease progression. EMBP concentrations in prostate tissue samples taken from 35 patients with benign prostatic hyperplasia (BPH), 33 patients with prostatic carcinoma (PC) taken before treatment, and from nine patients with hormone-refractory PC (hr-PC) were measured by radioimmunoassay using an antibody raised against rat EMBP. The dihydrotestosterone (DHT), prostatic acid phosphatase (PAP), prostate-specific antigen (PSA) and zinc levels in the tissue were also measured. The EMBP concentration in well differentiated PC (W-PC) samples was no higher than in samples of BPH tissue, whereas concentrations in moderately differentiated PC (M-PC) and poorly differentiated PC (P-PC) were significantly higher (P < 0.01 and P < 0.005, respectively); the highest levels were those in tissue from hr-PC (P < 0.001). Levels of PAP, PSA and zinc were significantly lower in tissue from PC than from BPH, while the differences in levels between W-PC, M-PC and P-PC were not significant. The EMBP to DHT ratio in the tissue increased significantly from W-PC through M-PC to P-PC and was greatest in hr-PC tissue. Conversely, the PAP:DHT, PSA:DHT and zinc:DHT ratios did not correlate with the progress of histological grade. In addition, the pre-treatment EMBP:DHT and zinc:DHT ratios in 14 patients who developed hr-PC within 3 years after the administration of estramustine phosphate were significantly higher when compared with the remaining 19 patients (P < 0.001 and P = 0.0184, respectively), whereas the PAP:DHT and PSA:DHT ratios showed no significant difference between the groups. Moreover, patients with a high EMBP:DHT ratio (> or = 82) had a low progression-free probability compared to those with a lower ratio. The androgen-dependent property of EMBP tends to decline with the transformation of prostatic tissue into biologically more malignant disease. The tissue EMBP:DHT ratio before treatment may be a good indicator of the individual malignant potential of PCs.