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PubMed1991

The effect of estramustine derivatives on microtubule assembly in vitro depends on the charge of the substituent.

Estramustine, and derivatives of estramustine with a charged substituent at position 17 on the estrogen moiety, have been investigated for their effects on bovine brain microtubules in vitro. The negatively charged estramustine phosphate has been found previously to be a microtubule-associated protein (MAP)-dependent microtubule inhibitor [Wallin M, Deinum J and Fridén B, FEBS Lett 179: 289-293, 1985]. In the present study the binding of estramustine phosphate to MAP2 and tau was investigated. Both these MAPs were found to have two to three binding sites for estramustine phosphate which is compatible with the reported number of basic amino acid repeats of these MAPs, considered to be the ultimate tubulin binding domains. The Kd for the binding of estramustine phosphate to MAP2 was estimated to be 20 microM at 4 degrees, and for the binding of tau, 200 microM. The rate of dissociation was very low (T1/2 greater than 2 hr), which indicates that the binding of estramustine phosphate may stabilize the protein-drug complex by changing the protein conformation. Two new negatively charged estramustine derivatives, estramustine sulphate and estramustine glucuronide, were found to be similar MAP-dependent microtubule inhibitors. The concentration for 50% inhibition of assembly was 100 microM for the sulphate derivative, the same as found previously for estramustine phosphate, and 250 microM for the more bulky estramustine glucuronide. A positively charged derivative, estramustine sarcosinate, did not inhibit microtubule assembly or alter the composition of the coassembled MAPs. The morphology of the microtubules was, however, affected. The uncharged estramustine bound to both tubulin and MAPs, but no effects were seen on microtubule assembly, the composition of coassembled MAPs or the microtubule morphology. Our results suggest that only negatively charged estramustine derivatives have a MAP-dependent microtubule inhibitory effect. The two new negatively charged derivatives could therefore be valuable tools in the study of tubulin-MAP interactions. The results also confirm that these interactions between tubulin and MAPs are mainly electrostatic.

Animals↗
PubMed1998

Androgen antagonistic effect of estramustine phosphate (EMP) metabolites on wild-type and mutated androgen receptor.

Estramustine phosphate is used frequently, alone or in combination with other antitumor agents, for the treatment of hormone-refractory prostate cancer. Estramustine phosphate is metabolically activated in vivo, and its metabolites, estramustine, estromustine, estrone, and beta-estradiol inhibit the assembly of microtubules [for review see: Kreis W, In: Concepts, Mechanisms, and New Targets for Chemotherapy (Ed. Muggia FM), pp. 163-184. Kluwer Academic Publishers, Boston, 1995]. We investigated, by displacement of [3H]methyltrienolone in the presence of 2.5 mM of triamcinolone acetonide, the binding of estramustine phosphate and its metabolites, estramustine, estromustine, estrone, and beta-estradiol, as well as other antiandrogen agents including alpha-estradiol, bicalutamide, and hydroxyflutamide, to the mutated androgen receptor (m-AR) in LNCaP cells and to the wild-type androgen receptor in wild-type AR cDNA expression plasmid (w-pAR0) cDNA-transfected HeLa cells. Analogous to the antiandrogens, bicalutamide and hydroxyflutamide, binding of estramustine phosphate metabolites to the androgen receptor was observed. The EC50 values (in microM) were: estramustine phosphate, > 10; estramustine, 3.129 +/- 0.312; estromustine; 2.612 +/- 0.584; estrone, 0.800 +/- 0.090; alpha-estradiol, 1.051 +/- 0.096; beta-estradiol, 0.523 +/- 0.028; bicalutamide, 4.920 +/- 0.361; and hydroxyflutamide, 0.254 +/- 0.012. The transactivation assay demonstrated that, analogous to bicalutamide, estramustine could not induce luciferase activity in either w-pAR0 or m-pARL transfected HeLa cells. In contrast, a strong induction of the reporter activity by dihydrotestosterone was observed. Down-regulation of prostate-specific antigen (PSA) expression, an AR-target gene, by estramustine and bicalutamide was accompanied by the blockade of the mutated androgen receptor. Exposure of LNCaP cells to estramustine for 24 hr caused transcriptional inhibition of PSA in a concentration-dependent manner. The levels of PSA mRNA decreased 56 and 90% when LNCaP cells were treated with 5 and 10 microM of estramustine, respectively (IC50 = 10.97 +/- 1.68 microM). Binding of hydroxyflutamide to m-AR in LNCaP cells resulted in a concentration-dependent stimulation of PSA expression, suggesting that hydroxyflutamide acted as an agonist of the m-AR. Our data indicate that estramustine phosphate metabolites perform as androgen antagonists of AR, an additional mechanism involved in the therapeutic effect of estramustine phosphate in patients with prostate cancer.

Androgen Antagonists↗
PubMed1997

Interaction of estramustine with tubulin isotypes.

The interaction of the antimitotic agent estramustine with bovine microtubule proteins and purified tubulin was investigated. Direct photoaffinity labeling of microtubule protein with [14C]estramustine resulted in the labeling of both alpha- and beta-tubulin, and this was inhibited with unlabeled estramustine in a dose-dependent manner. [14C]Estramustine was incorporated into both the soluble and polymerized forms of tubulin. The affinity constant for estramustine binding to tubulin was determined by equilibrium dialysis to be 23 +/- 5 mM. Estramustine did not affect [3H]vinblastine binding, and vinblastine had no effect on direct labeling with [14C]estramustine. Both rhizoxin and paclitaxel decreased the covalent labeling of tubulin with [14C]estramustine in a dose-dependent fashion and were noncompetitive inhibitors of the binding of estramustine to tubulin. The binding of colchicine to tubulin was not inhibited by estramustine as detected by fluorescence and DEAE filter assays. The estramustine binding site on tubulin is therefore distinct from that of colchicine and vinblastine and may at least partially overlap with the binding site for paclitaxel. In both bovine brain microtubules and cytoskeletal proteins from human prostatic carcinoma cells, the incorporation of [14C]estramustine into the beta III isotype of tubulin was found to occur with a reduced efficiency compared to that of the other beta-tubulin isotypes and alpha-tubulin. Since this isotype is overexpressed in estramustine resistant human prostate carcinoma cells, these results indicate that beta III-tubulin may play a role in the response to the effects of estramustine.

Animals↗
PubMed1996

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.

Animals↗
PubMed1999

Synergistic effect of estramustine and [3'-keto-Bmtl]-[Val2]-cyclosporine (PSC 833) on the inhibition of androgen receptor phosphorylation in LNCaP cells.

Estramustine phosphate has been used frequently alone or in combination with other drugs for the treatment of hormone-refractory prostate cancer. Estramustine is one of the major active metabolites of estramustine phosphate in vivo. We recently demonstrated that estramustine acts as an androgen antagonist, and the combination of estramustine with [3'-keto-Bmtl]-[Val2]-cyclosporine (PSC 833) results in synergistic cytotoxicity. Unlike other regulators of microtubules, such as paclitaxel, the present study demonstrated that estramustine alone or in combination with PSC 833 did not induce bcl-2 phosphorylation in LNCaP cells. No synergism between estramustine and PSC 833 in the induction of bcl-2 phosphorylation was obtained in MCF-7 cells exposed for 16 hr to estramustine (5-15 microM) and PSC 833 (5 microM). A significant synergistic antiandrogenic effect as measured by the inhibition of dihydrotestosterone-induced reporter gene luciferase expression in both wild-type and mutated androgen receptor (AR) cDNA-transfected HeLa cells was observed when the cells were exposed to estramustine and PSC 833. Treatment of LNCaP cells with estramustine alone (5-15 microM) resulted in a decrease of AR expression and phosphorylation. This effect was enhanced markedly by PSC 833. A strong correlation between AR phosphorylation and expression of the AR target gene PSA was obtained in dihydrotestosterone-stimulated LNCaP cells. The up-regulated PSA expression is a function of the level of the phosphorylated AR (r = 0.9814), but not the dephosphorylated form of the receptor protein (r = 0.4808). Thus, our studies suggest that the synergism between estramustine and PSC 833 in LNCaP cells is a consequence of inhibition of AR expression and phosphorylation, thus leading to interruption of AR-mediated gene expression.

Antineoplastic Agents, Hormonal↗
PubMed1995

Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins.

BACKGROUND: Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. METHODS: The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. RESULTS: Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. CONCLUSION: Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Uptake of estramustine phosphate (estracyt) metabolites in prostatic cancer.

Plasma and tumour concentrations of estramustine, estromustine, estradiol and estrone, the major metabolites of estramustine phosphate (estracyt), were determined in patients with prostatic carcinoma treated between one and nine years with repeated oral doses of estracyt (560 to 840 mg./day). The last dose was given 12 to 16 hours before sampling. The binding of radioactive estramustine and estromustine was determined in the tumour tissue to examine the possible role of estramustine-binding protein for the accumulation of these metabolites into the tumour. Comparison was made with benign prostate hyperplastic tissue from untreated patients. Estromustine was the main metabolite in plasma as well as in the tumour (range 235 to 450 and 205 to 485 ng./gm., respectively), whereas estramustine (20 to 45; 95 to 370), estrone (62 to 140; 63 to 160) and estradiol (8 to 15; 7 to 36) were found in lower concentrations. Interestingly the concentration of estramustine was as an average six times higher in the tumour than in plasma contrasting with the other metabolites which were present in equal amounts of the two localities. Binding of 3H-estramustine and 3H-estromustine was two to three times higher in the tumour than in benign hyperplastic tissue and negligible in plasma samples. The present study is the first where substantial amounts of cytotoxically active estramustine and estromustine are demonstrated in tumour tissue from estracyt treated patients. Our findings suggest a mechanism for selective uptake of these metabolites in prostatic cancer (estramustine-binding protein). The uptake and binding of estramustine and estromustine in the tumour may account for the clinical effects of estracyt in prostatic carcinoma.

Aged↗
PubMed1993

Estramustine depolymerizes microtubules by binding to tubulin.

To investigate the mechanism of action of the antineoplastic drug estramustine, we compared its effects on human prostate cancer cells with those of vinblastine. At their respective concentrations that result in 50% inhibition of clonogenic growth, both drugs caused an accumulation of cells blocked at mitosis and similar dose- and time-dependent depolymerization of interphase microtubules. Also, colcemid-resistant and colcemid-hypersensitive Chinese hamster ovary cells with tubulin mutations were collaterally cross-resistant or -sensitive to estramustine. Thus, the cytotoxicity of estramustine is due to its microtubule depolymerization properties. This could be caused by interaction with tubulin and/or with microtubule-associated proteins (MAPs). Previous investigations have shown that high concentrations of estramustine phosphate can inhibit microtubule polymerization in vitro by binding to MAPs. However, estramustine phosphate is the clinical prodrug to estramustine, the intracellular active compound. In this study, we investigated the effects of estramustine on the binding of MAPs to taxol-stabilized microtubules in vivo. In contrast to previous reports, no effect of estramustine on the binding of MAPs to microtubules was found. Furthermore, we found that polymerization of purified tubulin could be inhibited by estramustine in vitro. Taken together, these results demonstrate that estramustine causes depolymerization of microtubules by direct interaction with tubulin.

Adenocarcinoma↗
PubMed1999

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.

Blotting, Western↗
PubMed1995

Estramustine phosphate sodium. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in prostate cancer.

Estramustine phosphate sodium (estramustine phosphate), a unique antitumour agent, is selectively taken up by prostate cells and exerts antineoplastic effects by interfering with microtubule of dynamics and by reducing plasma levels of testosterone. In noncomparative studies of estramustine phosphate in patients with hormone-refractory disease, objective response rates ranging from 19 to 69% have been reported. Preliminary clinical investigations indicate that combining estramustine phosphate with vinblastine, etoposide or paclitaxel improves objective response rates over single-agent treatment, although no survival benefit over single-agent treatment has been demonstrated to date. In comparative studies, estramustine phosphate produces similar objective response rates to conventional antineoplastic agents in patients with hormone-refractory prostate cancer. In previously untreated patients with advanced metastatic hormone-responsive prostate cancer, objective responses are achieved in approximately 80% of patients. Estramustine phosphate appears to be at least as effective as estrogen or flutamide therapy in these patients. Nausea and vomiting are the most frequently observed adverse effects of treatment with estramustine phosphate. While these symptoms are usually mild to moderate in nature, they may occasionally be more troublesome to the patient and necessitate withdrawal of treatment. Cardiovascular complications are a more serious, though less frequently encountered, adverse effect of the drug. However, these complications may be avoided by careful patient selection and prophylactic treatment measures. Unlike some other antineoplastic agents, estramustine phosphate is rarely associated with myelosuppression. In addition to producing similar objective response rates to other established agents, estramustine phosphate improves the subjective status of many patients and has been shown to reduce the intensity of pain and improve the performance status of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1981

Intracellular localization of estramustine in rat ventral prostate in vitro.

With the aim of studying the mechanism behind the effect of estramustine in the treatment of prostatic carcinoma, the intracellular fate of the drug has been investigated in rat ventral prostate in vitro. Minced tissue was incubated with [3H] estramustine under different conditions, homogenized, and submitted to isopycnic centrifugation on a sucrose gradient using the recently introduced vertical tube rotor. The subcellular localization of the drug was determined by comparison between the distribution of radioactivity in the gradient fractions and the activities of a number of marker enzymes. No metabolism of estramustine occurred as judged by thin-layer chromatography. After incubation of the minced prostate tissue for 1 hour at 30 degrees C with 0.15 microM of [3H] estramustine, most of the drug was recovered in the cytosol fractions which also contained the highest concentrations of the estramustine-binding protein. However, after incubation with 220 microM of estramustine, most drug equilibrated in heavier fractions with high concentrations of N-acetyl-beta-glucosaminidase and cathepsin B, marker enzymes for the lysosomes as well as NADPH:cytochrome c reductase, marker enzyme for the endoplasmic reticulum. Extending the incubation time and increasing the temperature reduced the amount of estramustine equilibrating in the heavy fractions and concomitantly increased the portion localized in the cytosol. During all incubation conditions, very little drug seemed to accumulate in the nuclei since the drug distribution was completely different from that of DNA. This suggests that effects other than interaction with nuclear DNA might be of importance for the cytotoxic effect of estramustine.

Acetylglucosaminidase↗
PubMed1994

Estramustine potentiates the radiation effect in human prostate tumor transplant in nude mice.

In this study, we have investigated the combined effect of estramustine treatment and external beam radiation on human prostatic cancer tumor cells (DU 145) transplanted in nude mice. The treatment was given according to two different schedules. In the first treatment regimen, estramustine was administered intraperitoneally (i.p.) intermittently for 20 days. The radiation therapy, which was started on day 9, was given with 6 Gy fractions during an 11-day-long period to a total dose of 36 Gy. The combination treatment (estramustine + radiation) resulted in a significant tumor growth retardation as compared to the control group. This pronounced effect was seen neither with radiation alone nor with estramustine alone. In order to further extend the radiation treatment time, a second therapy regimen was employed. In this part of the study, estramustine was administered i.p. intermittently for 26 days. The radiation therapy, which was started on day 6, was given with 4 Gy fractions during a 21-day-long period to a total dose of 40 Gy. Under these conditions, a significant tumor growth retardation was disclosed, when comparing the combination treatment (estramustine + radiation) with radiation alone. The tumors were analyzed for content of necrosis and proliferative activity. The largest proportion of necrosis was seen in the combination (estramustine + radiation) treatment group. Also, the tumors from this group expressed a decreased proliferative activity. The data indicate that estramustine acts as a radiosensitizing agent in human prostatic cancer cells in vivo. The radiosensitizing properties of the drug encourage further studies with respect to clinical application.

Animals↗
PubMed1996

Effect of concomitant administration of clodronate and estramustine phosphate on their bioavailability in patients with metastasized prostate cancer.

Estramustine phosphate is generally used as a second-line treatment in patients with advanced prostate cancer. The bone metastases due to the cancer are often treated simultaneously with clodronate in order to relieve the bone pain. Therefore, the interaction of clodronate (800 mg orally four times daily) and estramustine phosphate (280 mg orally twice daily) on their bioavailability was studied in twelve patients with prostate carcinoma and bone metastases. The drugs were first given separately, each to six patients, for five days, and then concomitantly for the same period. The bioavailabilities of the drugs were calculated on the last day of each treatment period. When clodronate was given alone, its concentrations in serum and AUC for one dose interval (6 hr) did not differ from those obtained with the drug given concomitantly with estramustine phosphate, nor did the combination of estramustine phosphate change the excretion of clodronate in urine. The serum concentrations of estramustine phosphate were elevated by about 80% when the drug was given together with clodronate. The AUC for one dose interval (12 hr) was also significantly higher for estramustine phosphate with clodronate than without clodronate. The urinary excretion of estrone, a major metabolite of estramustine phosphate, was also significantly higher after the admission with clodronate. The results suggest that clodronate increases the oral bioavailability of estramustine phosphate.

Administration, Oral↗
PubMed1993

Estramustine and estrone analogs rapidly and reversibly inhibit deoxyribonucleic acid synthesis and alter morphology in cultured human glioblastoma cells.

Estramustine is an estradiol-based agent that has been shown to accumulate in human glioma cells, resulting in a concentration-dependent alteration in cell size and shape within minutes and an inhibition of proliferation over 3 to 6 days. We evaluated human glioblastoma cultures with [3H]thymidine incorporation assays to determine estramustine's early effects on deoxyribonucleic acid synthesis in these tumors. Because estramustine shares a common structural motif with other antimicrotubule drugs, we synthesized four A-ring conjugates of estrone that contained a carbamate moiety but lacked nitrogen mustard. These analogs were examined by [3H]thymidine incorporation and compared with vinblastine. Greater than 70% inhibition of [3H]thymidine incorporation occurred within 1 hour of treatment with estramustine at 10(-5) mol/L, which increased to 80% inhibition at 4 hours. Ethyl carbamate JE208 was nearly as effective as estramustine in inhibiting deoxyribonucleic acid synthesis, and both were more effective than vinblastine. The inhibitory effects of estramustine and estrone analogs were reversible; vinblastine was not reversible. Although estramustine and JE208 induced similar antiproliferative and morphological changes in glioblastoma cells that persisted for at least 4 days, there was a modest recovery of morphology and thymidine incorporation with JE208 after prolonged treatment. The common findings with estramustine and JE208 suggest that these agents may have a similar mechanism of action and form the basis for the investigation of new agents that may rapidly and reversibly inhibit glioblastoma.

Brain Neoplasms↗
PubMed1994

Selective antimitotic effects of estramustine correlate with its antimicrotubule properties on glioblastoma and astrocytes.

Estramustine is an estradiol-based agent that accumulates in cells containing estramustine binding protein. Previous studies have shown that this binding site is expressed in human glioblastoma cells and that estramustine accumulates in glioma cells, resulting in a concentration-dependent inhibition of proliferation. We have shown that estramustine treatment results in a rapid inhibition of deoxyribonucleic acid synthesis (within 4 h) in human glioblastoma cells associated with an alteration of cell size and shape, consistent with its known antimicrotubule activity. To extend these findings, we performed an immunohistochemical analysis of microtubules with a monoclonal antibody to beta-tubulin, using a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to measure the antimitotic effects of estramustine on both human glioblastoma and astrocyte cultures. Within 4 hours, estramustine (10 mumol/L) caused a dramatic alteration in the tubulin staining in glioma cells, characterized by a disorganization in microtubules. Cell shape and microtubule staining in astrocytes were relatively preserved. Estramustine had a concentration-dependent cytotoxic effect in tumor cultures, whereas it had no effect on astrocyte viability at any concentration. Differences in the antimitotic effects do not appear to be related to variations in proliferation rates among these different types of cells. These data suggest that although estramustine is a potent inhibitor of proliferation in glioblastoma cells, it has modest antiproliferative effects on astrocytes and its selective activity is closely correlated with its antimicrotubule properties.

Astrocytes↗
PubMed1988

Expression of an estramustine-binding associated protein in human lung cancer cell lines.

Estramustine-binding protein has previously been demonstrated in normal rat prostatic tissue, in normal human prostate epithelium, and in prostatic carcinomas. It binds specifically estramustine and estromustine, the cytotoxic metabolites of estramustine phosphate (Estracyt), a drug which is used in the treatment of prostatic carcinoma. In this study we have examined the presence of an estramustine-binding associated protein in a panel of human cell lines, representing the major histopathological types of lung cancer. A mouse (murine) monoclonal antiserum developed against rat estramustine-binding protein was used for immunohistochemical detection. Fast protein liquid chromatography was used for biochemical characterization. As judged from the immunohistochemical investigation, estramustine-binding protein was present in large amounts in five of six non-small cell carcinoma cell lines, while seven of eight small cell carcinoma cell lines were essentially negative. Fast protein liquid chromatography analyses of lysated cells from the lung cancer cell lines, incubated with [3H]estromustine, concurred with the results from the immunohistochemical stainings. These data strongly indicate a convincing connection between the immunoreactivity and ligand-binding properties of estramustine-binding protein in the cell lines examined. The presence of an estramustine-binding associated protein in human lung cancer cell lines has implications for further investigations into the biological relevance and the potential for eventual therapeutic applications.

Antibodies, Monoclonal↗
PubMed1986

Relationship of glutathione depletion and inhibition of glutathione-S-transferase activity to the antimitotic properties of estramustine.

A depletion of intracellular glutathione (GSH) is accompanied by a subsequent inhibition of GSH-S-transferase activity in a DU145 human prostatic carcinoma cell line treated with cytotoxic concentrations of estramustine. When GSH depletion reached approximately 50% of normal (approximately 2 hours after 10 microM estramustine), the mitotic index of logarithmically dividing cultures began to increase. A linear increase from 3.14% to 22.5% occurred during the period of 2-24 hours following 10 microM estramustine. Morphological studies showed that mitotic figures accumulated in metaphase and had abnormalities consistent with spindle malformation. Nocodazole and cytochalasin B also possess anticytoskeletal properties, but had little effect upon the intracellular levels of GSH or its associated transferase enzymes. The constituent moieties of estramustine, estradiol, and nor-mechlorethamine had effects on thiol metabolism which were dissimilar from estramustine, confirming previous findings that the unmetabolized parent drug is responsible for pharmacological activity. Estramustine had no effect upon GSH reductase activity, suggesting that drug toxicity was not a general thiol phenomenon. Buthionine sulfoximine decreased intracellular GSH in DU145 cells and enhanced the cytotoxic potential of estramustine in this cell line. The anticytoskeletal and antimitotic properties of estramustine may be enhanced by the drug-induced GSH imbalance and subsequent effects on GSH-S-transferase enzymes.

Buthionine Sulfoximine↗
PubMed1984

[Estracyt (estradiol-nitrogen mustard complex)--estramustine binding protein and its specificity].

Estracyt (estramustine phosphate) is a nitrogen mustard derivative of estradiol-17 beta which is rapidly dephosphorylated to yield estramustine. Estramustine is metabolized to estromustine mainly in the liver. Both estramustine and estromustine are retained in rat prostate with a high degree of specificity. This retention is due to the binding of estramustine and/or estromustine to a protein called EMBP (estramustine binding protein). When estimated by HPLC, the molecular weights of these estramustine binding components in rat prostate are 45,000-50,000 and 25,000-30,000, respectively. HPLC and glycerol density gradient analysis clearly demonstrated the occurrence of EMBP in a cytosol preparation from human prostatic cancer tissue. When estimated by HPLC, the molecular weights of estramustine binding components are 45,000 and 25,000 daltons, respectively. In addition, results of effectiveness of Estracyt studied under a cooperative research group in Japan, are reported in this paper. Effectiveness was evaluated at 3 months of the treatment on 121 patients with untreated prostatic cancer (Study I) and 95 patients with reactivated cancer (Study II), at 12 months of the treatment on 68 patients in Study I and 85 patients in Study II, and at 24 months on 37 patients in Study I and 23 patients in Study II. At 3 months of the treatment, Estracyt was effective in 89% of untreated prostatic cancers, and 38% of reactivated prostatic cancers. At 24 months of the treatment, this drug was effective in 65% of untreated prostatic cancers and 30% of reactivated ones. Estracyt is especially recommended as a first-choice drug for both the untreated patients with poorly differentiated adenocarcinoma and reactivated cancer.

Animals↗