estramustine and 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.

Topics: Animals; Antineoplastic Agents, Hormonal; Brain Neoplasms; Carrier Proteins; Estramustine; Glioma; Humans; Molecular Structure; Prostatic Secretory Proteins; Radiation-Sensitizing Agents

The synthetic estramustine (EM) and natural podophyllotoxin (PODO) anti-mitotic agents that inhibit tubulin polymerization are known anticancer agents. As low bioavailability limits their anticancer properties, we investigated whether conjugation with PAMAM dendrimer (D) could enhance the activity of D-EM and D-PODO by altering their release pattern. Release kinetics indicated synthesized conjugates to be stable against hydrolytic cleavage and showed sustained release characteristics. However, release of D-EM was slow compared to D-PODO conjugate. Antitumor effect of these conjugates on glioma cells revealed (i) increased cell death and cell cycle arrest (ii) decreased migration and (iii) increased tubulin depolymerization as compared to free drug. Importantly, the effects of natural PODO conjugate on glioma cell survival and migration is more pronounced than D-EM.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Dendrimers; Estramustine; Glioma; Humans; Microscopy, Electron, Transmission; Molecular Structure; Podophyllotoxin; Tubulin Modulators

Highly 5-HT(3)-receptor-specific antiemetic agents may have different effects on ion transport of tumour cells during treatment with cytotoxic drugs. Cell volume regulation, which is dependent on potassium ion (K(+)) flux, is involved the control of cell growth, proliferation, and apoptosis. K(+)-flux response mechanisms to the antiemetics ondansetron and granisetron were therefore correlated to malignant glioma cell (Mg251) volume response to estramustine phosphate (EMP) in vitro.. We quantified the influx and efflux of potassium ions (using the K(+) analogue (86)Rb(+)) as well as cell volume changes (with image analysis) of glioma cells incubated with the 5-HT(3)-receptor antagonists ondansetron and granisetron (0.1 micro mol/l) combined with 40 mg/l EMP.. The EMP-induced cell volume increase was fully inhibited by ondansetron but not affected by granisetron. Ondansetron retained high cellular K(+)-efflux and reduced Na(+), K(+), 2Cl(-)-cotransport activity, whereas granisetron (0.1 micro mol/l) reduced K(+)-efflux and retained an augmented Na(+), K(+), 2Cl(-)-cotransport activity in the presence of 40 mg/l EMP.. Ondansetron affects K(+) transport with ensuing effects on cell volume of tumour cells treated with EMP, whereas granisetron does not. Since ondansetron and other 5-HT(3)-receptor antagonists are used routinely to prevent nausea during anticancer treatment, an increased awareness of possible interactions with the antitumour efficacy of anticancer drugs seems warranted.

Topics: Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Size; Estramustine; Glioma; Granisetron; Humans; Ion Transport; Ondansetron; Potassium; Rubidium Radioisotopes; Serotonin; Serotonin Antagonists; Sodium; Sodium-Potassium-Chloride Symporters; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Irradiation is one of the cornerstones used in the treatment of malignant glioma. However, the effect is modest and glioma cells generally display a pronounced radio-resistance. In this study, the effect of irradiation, alone and in combination with the antimicrotubule drug estramustine (EaM), was investigated in vitro using the BT4C rat glioma cell line, and in vivo the BT4C rat intracerebral glioma model was used. Apoptosis was detected by analysing DNA laddering, in situ end labelling (ISEL) and Annexin V reactivity. In addition, phosphorylation status of MAPK, JNK, p38, and AKT, proteins involved in pro- and anti-apoptotic signalling pathways was analysed by Western blotting. Irradiation did not induce apoptosis, neither in vitro nor in vivo. EaM, however, induced apoptosis in vivo and in vitro, regardless of whether EaM was given alone, before or after irradiation. When BT4C cells were treated with the caspase-3 inhibitor Ac-DEVD-CHO prior to EaM, the number of apoptotic cells was decreased, indicating an involvement of caspase-3. The signalling pathways regulating apoptosis are complex and involve kinases such as MAPK, JNK, p38 and AKT. Irradiation did not induce any changes in the expression levels or phosphorylation status of these proteins. On the other hand, the phosphorylation level of AKT was reduced after EaM treatment, which might, in part, propose how EaM induces apoptosis in glioma cells.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Estramustine; Glioma; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Tumor Cells, Cultured

Estramustine phosphate (EMP) is an anti-microtubule agent that depolymerizes microtubules and also causes apoptosis of glioma cells. Both of these pharmacological actions have been previously studied within the same cytotoxic range of EMP concentrations. The purpose of this study was to investigate which of these two phenomena occurred before the other. A preliminary MTT assay was done to distinguish non-cytotoxic (0.005-0.1 microM) and cytotoxic (0.5-10 microM) of EMP for BT4C cells. To investigate apoptotic changes, transmission electron microscopy (TEM), DNA laddering, and in situ endo-labeling (TUNEL) method were employed. A chemotaxis assay was used to assess cell motility. Scanning electron microscopy and TEM immunocytochemistry with an anti-beta tubulin antibody were applied to detect morphological changes of the microtubules. Suppression of cell motility by cytotoxic doses of EMP (0.5-10 microM) group was attributed by the cyto-reductive effect, relating to apoptosis. At 0.01-0.1 microM (non-cytotoxic doses), EMP did not indue apoptosis. At these concentrations, TEM and immunohistochemistry revealed the formation of blebs on the tip of the pseudopodia that contained abnormally depolymerized microtubules, a finding that was not observed at a low temperature or during cell migration. Cell chemotaxis was significantly inhibited by cytostatic EMP doses (0.05 and 0.1 microM). Bleb formation of the pseudopodia might be evidence of the abnormal disassembly of microtubules by cytostatic EMP concentrations, prior to the induction of apoptosis. In glioma cells EMP probably initiates apoptosis by causing the depolymerization of microtubules. Inhibition of cell motility by cytostatic doses of EMP could be beneficial to support other therapies.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Chemotaxis; Estramustine; Extracellular Space; Glioma; Microscopy, Electron; Microscopy, Electron, Scanning; Microtubules; Polymers; Pseudopodia; Rats; Tumor Cells, Cultured

Estramustine phosphate (EMP) is an anti-microtubule agent that induces apoptosis of glioma cells. We investigated whether EMP caused apoptosis through the alkylating effect of its nitrogen mustard component or by phosphorylation of bcl-2 like other anti-microtubule agents in normal human astrocyte and human malignant glioma cell lines. Apoptosis was seen in glioma cells treated either with nitrogen mustard or EMP and expression of bcl-2 mRNA was not changed by exposure to the drug. An immunoprecipitation study only found phosphorylation bcl-2 in glioma cells exposed to EMP and not in cells exposed to nitrogen mustard. These results indicate that induction of apoptosis in glioma cells by EMP is mediated by phosphorylation of bcl-2.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Hormonal; Astrocytes; Brain Neoplasms; Electrophoresis, Agar Gel; Estramustine; Glioma; Humans; Mechlorethamine; Microscopy, Electron; Microtubules; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

The drug effect of estramustine phosphate (EMP), an anti-microtubule agent on human glioma cells has been studied with the focus being mainly its cytotoxity or its targeting of organelles. However, the pharmacological knowledge of estramustine with respect to its cytotoxity and mechanism is limited. To acquire such knowledge, the present study investigates the ability of EMP to induce apoptosis in a human malignant glioma cell line. Transmission electron microscope (TEM) images were examined to monitor periodic changes. Agarose gel electrophoresis was also examined. Cellular DNA fragmentation ELISA was performed to investigate the DNA fragmentation rates and an MTT assay was studied to evaluate the ID50. A TEM study revealed condensing and fragmentation of the chromatin. Laddering of the bands was observed in all EMP exposure groups in agarose gel electrophoresis. DNA fragmentation in all EMP groups began at 0.5 h following an exposure with EMP and increased in a dose- and time-dependent manner as revealed by DNA ELISA fragmentation. ID50 at 24 h was 5.0 microM according to the MTT assay, a value close to 4.8 microM of ID50 was revealed by the DNA fragmentation assay. None of the above mentioned changes was observed in the control group. These results indicated that EMP caused a drug-induced apoptosis in the human malignant glioma cell line, U87MG.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; DNA Fragmentation; DNA, Neoplasm; Dose-Response Relationship, Drug; Electrophoresis, Agar Gel; Enzyme-Linked Immunosorbent Assay; Estramustine; Glioma; Humans; In Vitro Techniques; Microscopy, Electron; Microtubules; Organelles; Survival Analysis; Tumor Cells, Cultured

A technique of microscopy with computerised detection of early morphological changes during continuous perifusion was used to monitor the geometry changes of cultured glioma cells (MG-251) when exposed to 40 mg/L estramustine phosphate (EMP) alone or in combination with granisetron (0.1 mumol/L), ondansetron (0.1 mumol/L), or serotonin (1 mumol/L). When the cells were exposed to EMP, cell volume measured as projected cell area (PCA) rapidly increased. Serotonin and ondansetron, but not granisetron, prevented the acute EMP response (PCA). Serotonin, but none of the 5-HT3 receptor antagonists, protected against the cytotoxicity of EMP to the glioma cells as measured by a fluorometric microculture assay. Our results demonstrate hitherto unknown differences between selective 5-HT3 receptor antagonist on the cellular response to EMP and shows the necessity to study the receptor antagonists from viewpoint of interference with the antitumour drug effects on malignant cells. The perifusion technique could be used to study the effects of serotoninergic agonists and antagonists on cell volume regulation of cells exposed to anticancer drugs.

Topics: Cell Size; Drug Combinations; Drug Interactions; Estramustine; Glioma; Granisetron; Humans; Ondansetron; Serotonin; Serotonin Antagonists; Tumor Cells, Cultured

Tumour angiogenesis is essential for progression of solid tumours and constitutes an interesting target for therapy. However, impaired tumour blood supply may also be an important obstacle for treatment by radiotherapy and chemotherapy. Estramustine has been shown to increase tumour blood flow and potentiate the effect of radiotherapy in experimental glioma. This study investigated the effects of fractionated radiotherapy and estramustine on angiogenesis in malignant glioma. The intracerebral BT4C rat glioma model was used and the animals were given whole brain radiotherapy 4 Gy x 5 days alone or in combination with estramustine 20 mg kg(-1) i.p. daily. Tumour microvascular density (MVD) was assessed by manual and computerized morphometrical analysis. Expression of vascular endothelial growth factor (VEGF) was studied by in situ hybridization. Radiotherapy decreased MVD to 157 vessels per mm2 compared to 217 vessels per mm2 in controls. Estramustine counteracted this anti-angiogenic effect and potentiated the anti-tumoural effect of radiotherapy. In addition, vessel size increased after estramustine treatment. Five days after completion of radiotherapy the expression of VEGF was increased in the centre of the tumours. In conclusion, fractionated radiotherapy decreases microvascular density in experimental malignant glioma. This effect was abolished by estramustine. The anti-vascular effect of irradiation is important to recognize when combining radiotherapy with cytotoxic drugs.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Combined Modality Therapy; Endothelial Growth Factors; Estramustine; Glioma; In Situ Hybridization; Lymphokines; Microcirculation; Neovascularization, Pathologic; Radiation-Protective Agents; Rats; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Estramustine (EaM), a carbamate ester of 17beta-estradiol and nor-nitrogen mustard, is a cytotoxic compound with antitumoral effect in malignant glioma in vitro and in vivo . However, knowledge of the pharmacokinetics of EaM in experimental glioma is limited. The objective of this study was therefore to investigate further the distribution of EaM in the BT4C rat glioma model. Assessment of EaM uptake and distribution was performed by quantitative whole-body autoradiography. In addition, the uptake of EaM and its metabolites estromustine (EoM), estradiol, and estrone were analyzed by gas chromatography. EaM was taken up from the circulation and was found to be the main product in glioma tissue. Whole-body autoradiography after [14C]-EaM administration revealed a strong 14C label simultaneously in tumor and normal brain tissue at 0.5 h after drug administration. In tumor tissue, sustained high levels of 14C label were detected at 12 h after drug administration. In contrast to the tumor, radioactivity in normal brain tissue rapidly leveled off, indicating a retention of radioactivity in the tumor. The tumor/brain radioactivity ratio reached a peak of 4.5 at 12 h after drug administration. High levels of 14C label were also found in pulmonary tissue. By gas chromatography, EoM was found to be the main metabolite in plasma. However, EaM reached higher levels in tumor tissue, with the mean tumor/plasma ratio being 11.7 as compared with 2.0 for EoM. Only low plasma levels of the estrogen metabolites were detected. In conclusion, EaM is taken up in the BT4C rat glioma tissue and is retained in the tumor as compared with normal brain tissue and plasma. EaM showed a greater selectivity for tumor tissue, exhibiting a high tumor/plasma ratio as compared with EoM. The distribution pattern after administration of EaM, as evaluated by both whole-body autoradiography and gas chromatography, supports the earlier suggestion that the uptake is related to a protein with EaM-binding characteristics.

Topics: Animals; Antineoplastic Agents, Alkylating; Autoradiography; Brain; Brain Neoplasms; Chromatography, Gas; Disease Models, Animal; Estramustine; Glioma; Neoplasm Transplantation; Rats; Rats, Inbred Strains; Tissue Distribution

Estramustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, is metabolized and exerts specific cytotoxic effects in malignant glioma in vitro and in vivo. In the present study, we have evaluated the cytotoxic effect of EMP in the clinical situation with regard to appearance of DNA damage and its correlation to the uptake of estramustine (EaM) in human malignant astrocytoma tissue. Ten patients were given 280 mg of EMP p.o. 12 h before surgery. Specimens from brain tumor tissue were collected during surgery and used for detection of fragmented DNA, a hallmark of apoptosis, with in situ end labeling (ISEL) and agarose gel techniques. The main metabolite of EMP in glioma tissue, EaM, was analyzed with gas chromatography. It was demonstrated that EMP induced clusters of ISEL-positive tumor cells and fragmentation of DNA on agarose gels in patients treated with EMP. In the same patients, a significant uptake of EaM in tumor tissue was demonstrated. In control patients, who were not treated with EMP, and in two EMP-treated patients with no uptake of EaM, no signs of fragmented DNA and only a few scattered ISEL-positive cells were seen in the tumor tissue. Signs of apoptosis were also seen in two different experimental models, i.e., in vitro cell cultures of rat glioma cells and an in vivo rat glioma model. It is suggested that EaM can induce apoptosis by a direct effect on a subpopulation of glioma cells in human brain tumors in the clinical situation.

Topics: Adult; Aged; Animals; Antineoplastic Agents, Alkylating; Apoptosis; DNA Fragmentation; Estramustine; Female; Glioma; Humans; Male; Middle Aged; Rats

Estramustine (EaM) is a conjugate of nor-nitrogen mustard (NNM) and 17 beta-estradiol (E2) that has cytotoxic and radiosensitizing effects on experimental malignant glioma. Its mechanism of action is only partly understood. To further investigate the mechanism in vivo, the effects on tumor blood flow (TBF) and tumor growth were analyzed.. TBF was measured by radioactive microspheres, and tumor growth was measured by weight. Apoptosis was evaluated by in situ end labeling and gel electrophoresis. The effects of the constituents NNM and E2 were also evaluated.. EaM increased TBF to 153.8 ml/100 g/min after 3 days and to 153.9 ml/100 g/min after 10 days of treatment, compared with 94.0 ml/100 g/min in untreated controls. Cerebral blood flow did not change after EaM treatment. NNM increased TBF but also showed a tendency to increase cerebral blood flow. E2 increased TBF, whereas cerebral blood flow was unchanged. EaM resulted in a rapid reduction in tumor weight from 230 mg in untreated animals to 146 mg after 3 days of treatment. EaM induced an early transient fragmentation of deoxyribonucleic acid in glioma but not in the normal brain. Neither NNM nor E2 affected tumor weight.. EaM increases TBF in the BT4C rat glioma model with a concomitant rapid antitumoral effect. The increase in TBF could partially be induced by an estrogen-like action of EaM, but the rapid cytotoxic effect of the drug is obviously attributed to the intact EaM compound. This cytotoxic effect might be attributable to the induction of programmed cell death.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Blood Flow Velocity; Brain Neoplasms; Cell Line; Cell Survival; Estramustine; Female; Glioma; Male; Neoplasm Transplantation; Neovascularization, Pathologic; Rats; Rats, Inbred Strains

A perifusion technique for microscopy with computerized detection of early changes in cell morphology during continuous perifusion was used to show that the geometry of cultured glioma cells (MG-251) changes rapidly when they are exposed to estramustine phosphate (EMP). When the cells were exposed to 20 or 40 mg l(-1) EMP, cell volume projected cell area (PCA) rapidly increased. When the Na+,K+-ATPase blocker ouabain (100 micromol l(-1)) was added to the EMP (40 mg l(-1)) perifusion, the acute EMP response was eradicated. When the PCA curve for ouabain alone was subtracted from the curve of combined ouabain and EMP perifusion, the resulting curve showed that ouabain completely blocked the EMP-induced increase in PCA. When the Na+, K+, Cl- co-transport inhibitors bumetanide (10 micromol l(-1)), or furosemide (100 micromol l(-1)), were added to EMP (40 mg l(-1)), the acute increase in PCA seen for EMP alone was also completely blocked. This study shows that inhibitors of ion transmembrane transport can modify EMP-induced cell volume increases. This may be of particular importance since the blockers have been found to interfere also with the cytotoxic function of EMP during cell culture. Thus, it is possible that cell volume changes could serve as a rapid technique for predicting the cytotoxic activity of antineoplastic drugs.

Topics: Bumetanide; Cell Size; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estramustine; Furosemide; Glioma; Humans; Ion Channels; Ouabain; Perfusion; Tumor Cells, Cultured

Estramustine-phosphate (EMP), a combination of nornitrogen mustard and 17 beta-estradiol, has been demonstrated to exert specific antiproliferative effects on human glioma cells in vitro. The cytotoxic effect is, at least partially, mediated by inhibiting microtubule function. In this study the combined effect of EMP and radiation was evaluated in the human glioma cell-lines, 251-MG and 105-MG, in vitro, and in the rat glioma BT4C in vitro and in vivo. In all cell-lines an additive effect of EMP and radiation was obtained in vitro. Assuming equal effect of EMP is obtained in subsequent radiation fractions, the cell kill will be increased from 2-3 to 5-10 logs if delivering 30 fractions of 2 Gy combined with EMP. In the BT4C rat model the combined effect was found to be synergistic. Flow cytometry demonstrated an arrest in G2/M phase in all cell-lines after EMP treatment. This block in G2/M phase in addition to the previously demonstrated induction of free oxygen radicals, and the increase of blood flow with an assumed subsequent increase of oxygenation, might provide an explanation for the observed radiosensitizing effect of estramustine.

Topics: Animals; Brain Neoplasms; Cell Cycle; Cell Line; DNA, Neoplasm; Estramustine; Female; Flow Cytometry; Glioma; Humans; Radiation-Sensitizing Agents; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Estramustine, a combination of 17 beta-oestradiol and nor-nitrogen mustard, has been shown to be metabolised and to induce specific antiproliferative effects in malignant glioma, including arrest of glioma cells in the G2/M phase of the cell cycle, damage to cell membranes and DNA and induction of free oxygen radicals. To evaluate further the effects of estramustine, an in vivo rat glioma model using inbred BD-IX rats and the BT4C cell line was set up. In order to detect cells with fragmented DNA, tumour and brain specimens were, following fixation for histological examination, processed for in situ end labelling (ISEL) with biotin-labelled nucleotides. Fresh tissue fragments were also used for DNA integrity analysis on agarose gels. It was demonstrated that estramustine induced clusters of ISEL-positive cells and a pronounced typical fragmentation of DNA 0.5-8 h after treatment. In tumours examined 24 or 94 h after estramustine treatment, and in untreated tumours, only occasional single ISEL-positive cells were scattered in the tumour. DNA from normal brain tissue did not display any visible sign of fragmentation. These changes are indicative of programmed cell death induced by estramustine in glioma cells but not in normal brain tissue. Further studies are, however, needed to establish in detail the mechanism of cell death following treatment with the antimitotic drug estramustine.

Topics: Animals; Apoptosis; Brain; Brain Neoplasms; DNA; DNA Damage; DNA, Neoplasm; Electrophoresis, Agar Gel; Estramustine; Glioma; Rats; Rats, Inbred Strains

Clinically-used drugs such as furosemide, bumetanide and cardiac glycosides, are modulators of transmembrane fluxes of cations. Recently, it has been suggested that the regulation of intracellular cation concentrations could be a primary target for anti-neoplastic drugs, and that the cytotoxic activity may be altered by inhibitors of cation fluxes at the level of the plasma membrane. Therefore, we investigated the mechanisms by which cations are translocated across the plasma membrane of malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines. The interactions between cation flux inhibitors and the cytotoxicity of estramustine were also evaluated. Ouabain, the classical inhibitor of Na+, K+ATPase, markedly reduced 86Rb (K+) influx in all three lines, indicating that this ion transport system is present in the cells. Furosemide and especially bumetanide inhibited the 86Rb influx, indicating the presence of the Na+, K+, Cl- co-transport system. The potassium channel blocker, tetraethylammonium, but not apamin reduced the influx of 86Rb showing that high conductance K+ channels are present, but that channels of low conductance probably do not exist in these cell lines. The Na+, K+, Cl- co-transport inhibitors furosemide and bumetanide significantly reduced cytotoxicity of estramustine in P31 cells, whereas no interaction between other K+ flux inhibitors and the anti-neoplastic drugs were detected in any of the cell lines investigated. Thus, the data show that Na+, K+, ATPase and NA+, K+, Cl- co-transport systems and K+ channels of high conductance are present in malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines, and that inhibition of the Na+, K+, Cl- co-transport system in P31 is associated with reduced cytotoxicity of estramustine. The results justify further studies evaluating the role of these cation flux pathways in terms of targets for anti-neoplastic therapy.

Topics: Bumetanide; Cell Division; Estramustine; Furosemide; Glioma; Humans; Ion Transport; Lung Neoplasms; Male; Ouabain; Potassium; Potassium Channels; Prostatic Neoplasms; Rubidium Radioisotopes; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Estramustine (EM), an antimicrotubule agent, binds microtubule-associated proteins, causes spindle disassembly, and arrests cells at the late G2/M phase of the cell cycle. Since cells in the G2/M phase are the most radiosensitive and some human cancer cells contain high level of EM-binding protein, experiments were carried out to determine whether radiation sensitization could be obtained in human carcinoma cells.. Cells containing a high level of EM-binding protein such as prostate carcinoma (DU-145), breast carcinoma (MCF-7), and malignant glioma (U-251) were used to demonstrate radiosensitization. Cervical carcinoma (HeLa-S3) and colon carcinoma (HT-29) cells which are not known to contain EM-binding protein were also employed. Cell survival was assayed by the colony forming ability of single plated cells in culture to obtain dose-survival curves.. Pretreatment of DU-145, MCF-7, and U-251 cells to a nontoxic concentration (5 microM) of EM for more than one cell cycle time, substantially enhanced the radiation-induced cytotoxicity. The sensitizer enhancement ratio of these cells ranged from 1.35-1.52. The magnitude of the enhancement was dependent on the drug concentration and exposure time. The rate of cell accumulation in G2/M phase, as determined by flow cytometry, increased with longer treatment time in the cell lines which showed radiosensitization. Other antimicrotubule agents such as taxol and vinblastine caused minimal or no radiosensitization at nontoxic concentrations.. The data provide a radiobiological basis for using EM as a novel radiation enhancer, with the property of tissue selectivity.

Topics: Breast Neoplasms; Cell Cycle; Cell Survival; Colonic Neoplasms; Combined Modality Therapy; Estramustine; Female; Flow Cytometry; Glioma; HeLa Cells; Humans; Male; Microtubules; Neoplasms; Paclitaxel; Prostatic Neoplasms; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Vinblastine

Estraumustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, has been shown to exert cytotoxic effects on glioma cells in vitro. The drug uptake is assumed to depend on a specific estramustine binding protein (EMBP). One of the main difficulties in achieving cytotoxic effect in malignant brain tumours is believed to be due to the poor penetration of cytotoxic drugs into tumour tissue. In patients with malignant supratentorial brain tumours we have analysed the uptake of EMP metabolites in tumour tissue after oral administration and demonstrated EMBP in the same tissue specimens. Sixteen patients were given 280 mg EMP orally 14 h prior to surgery. Specimens from brain tumour tissue, cystic fluid, and serum were collected during surgery. Using gas chromatography the metabolites of EMP, estramustine (EaM) and estromustine (EoM), were quantified, EMBP was demonstrated by immunohistochemistry. The mean concentrations of EaM and EoM, expressed in ng g-1, were 60.3 and 38.4 in tumour tissue and 3.5 and 56.3 in serum, respectively. An accumulation of EaM in tumour tissue was found with a mean concentration gradient of 16.1 versus serum, while the gradient for EoM was 0.76. EMBP was demonstrated with a high degree of staining in all but one tumour. The high concentrations of EaM and EoM found in malignant brain tumour tissue correspond to potentially cytotoxic levels. The present results as well as the earlier in vitro demonstrated cytotoxic effects on glioma cells strengthen the possibility of a therapeutic effect of EMP in the treatment of malignant brain tumours.

Topics: Adult; Aged; Astrocytoma; Brain Neoplasms; Carrier Proteins; Ependymoma; Estramustine; Estrone; Female; Glioma; Humans; Immunohistochemistry; Male; Middle Aged; Nitrogen Mustard Compounds; Prostatic Secretory Proteins

Estramustine, an estradiol-17 beta and nornitrogen mustard complex, is used in the treatment of advanced prostatic carcinoma. A specific estramustine binding protein (EMBP) is important for its cytotoxic action, and the presence of EMBP has previously been demonstrated in rat and human prostatic cancer tissue. Significant levels of EMBP were detected by radioimmunoassay in human brain-tumor tissue. The EMBP concentrations (expressed as ng/mg protein) in 16 astrocytomas (mean 2.6 ng/mg, range 0.5 to 6.2 ng/mg) and seven meningiomas (mean 5.1 ng/mg, range 0.3 to 9.3 ng/mg) were significantly higher than that found in four samples of epileptic brain (mean 0.7 ng/mg, range 0.5 to 1 ng/mg) and 18 samples of normal brain (mean 0.5 ng/mg, range 0.2 to 1.0 ng/mg). The uptake, metabolism, and antiproliferative effects of the prostatic anticancer agent estramustine have been previously demonstrated in cultured glioma cells. The presence of EMBP may suggest a selective binding and effectiveness in human brain-tumor tissue.

Topics: Astrocytoma; Brain; Brain Neoplasms; Carrier Proteins; Epilepsy; Estramustine; Female; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma; Prostatic Hyperplasia; Prostatic Secretory Proteins; Radioimmunoassay; Reference Values

The present study describes a new microscopic perifusion technique for detecting momentary alterations in cell volume and shape. The method has been applied for evaluating early signs of cytotoxicity following chemotherapeutic treatments. The effects of estramustine phosphate (EMP) have been evaluated. EMP is a complex between oestradiol-17 beta and the alkylating agent nor-nitrogen mustard and has recently demonstrated a marked cytotoxicity against malignant glioma cells. The results showed a concentration-dependent increase in cell size and a concomitant decrease in shape factor following EMP-treatment of glioma cells. These changes correlated with cytotoxicity evaluated as cell proliferation and cell membrane alterations shown by 86Rb fluxes and ultrastructural visible membrane damage. The colon cancer line HT-29 displayed no reactions at all following EMP treatment. It is suggested that acute alterations in cell morphology and shape display a strong correlation to the cytotoxicity of EMP encountered by traditional cell culture systems. The findings are discussed with respect to cell membrane disturbances caused by EMP and its potential role as an early test of cytotoxicity.

Topics: Cell Line; Cell Membrane; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Estramustine; Glioma; Histological Techniques; Humans; Microscopy, Electron, Scanning; Rubidium Radioisotopes; Time Factors; Tumor Cells, Cultured

Estramustine phosphate (EMP), a complex between estradiol-17 beta and nor-nitrogen mustard, commonly used in treatment of prostatic cancer, also exerts marked antiproliferative effects on cultured human malignant glioma cells. The mechanism of action is unknown but has previously been considered to be mediated through non-DNA targets, specifically via the mitotic spindle, and related to the intact estramustine complex. EMP cytotoxicity was studied on the malignant glioma cell line U-251 MG. A dose-dependent increase in DNA strand breaks was demonstrated at EMP-concentrations ranging 10-40 mg/l. The uptake of 86Rb, used as a tracer for potassium to study ion transport and membrane permeability, was reduced after incubation with EMP. The mean decline in 86Rb accumulation by U-251 MG cells was 12, 20 and 32% at EMP concentrations 10, 20 and 40 mg/l respectively. Scanning electron microscopy gave further evidence for cell membrane damage. In conclusion, EMP seems to affect malignant glioma cells on several vital functions and the results indicate the the cytotoxic potential may at least partially be related to effects on DNA and cell membrane.

Topics: Cell Membrane; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Estramustine; Glioma; Humans; Microscopy, Electron, Scanning; Rubidium Radioisotopes; Tumor Cells, Cultured

Four human cell lines derived from malignant gliomas were immunohistochemically examined for their content of estramustine-binding protein (EMBP). EMBP was detected in a large amount in all glioma cells during the entire cell cycle. EMBP has previously been demonstrated to be the major receptor protein in prostatic cancers for the cytostatic drug estramustine-phosphate (EMP). EMP caused a dose-dependent inhibition of exponentially growing cells by increasing the number of cells in G2/M stage of the cell cycle as monitored by flow cytofluorometry. The effect may be coupled to arrest of the glioma cells at metaphase. The presence of EMBP may suggest a selective binding and effect of EMP in glioma cells.

Topics: Carrier Proteins; Cell Division; Cell Line; Estramustine; Glioma; Humans; Immunoenzyme Techniques; Neoplasm Proteins; Nitrogen Mustard Compounds; Prostatic Secretory Proteins; Tumor Cells, Cultured