Ability of Group IVB metallocene polyethers containing dienestrol to arrest the growth of selected cancer cell lines.

BACKGROUND: Monomeric Group IVB (Ti, Zr and Hf) metallocenes represent a new class of antitumor compounds. There is literature on the general biological activities of some organotin compounds. Unfortunately, there is little information with respect to the molecular level activity of these organotin compounds. We recently started focusing on the anti-cancer activity of organotin polymers that we had made for other purposes and as part of our platinum anti-cancer effort. METHODS: For this study, we synthesized a new series of metallocene-containing compounds coupling the metallocene unit with dienestrol, a synthetic, nonsteroidal estrogen. This is part of our effort to couple known moieties that offer antitumor activity with biologically active units hoping to increase the biological activity of the combination. The materials were confirmed to be polymeric using light scattering photometry and the structural repeat unit was verified employing matrix assisted laser desorption ionization mass spectrometry and infrared spectroscopy results. RESULTS: The polymers demonstrated the ability to suppress the growth of a series of tumor cell lines originating from breast, colon, prostrate, and lung cancers at concentrations generally lower than those required for inhibition of cell growth by the commonly used antitumor drug cisplatin. CONCLUSION: These drugs show great promise in vitro against a number of cancer cell lines and due to their polymeric nature will most likely be less toxic than currently used metal-containing drugs such as cisplatin. These drugs also offer several addition positive aspects. First, the reactants are commercially available so that additional synthetic steps are not needed. Second, synthesis of the polymer is rapid, occurring within about 15 seconds. Third, the interfacial synthetic system is already industrially employed in the synthesis of aromatic nylons and polycarbonates. Thus, the ability to synthesize large amounts of the drugs is straight forward.

Effects of some diethylstilbestrol metabolites and analogs on cytotoxicity and aneuploidy induction in Chinese hamster V79 cells.

We have previously reported the inhibitory effects of diethylstilbestrol (1) and optically active indenestrol derivatives on microtubule polymerization in vitro and their disruptive effect on cytoplasmic microtubules and cytotoxicity in cultured Chinese hamster V79 cells. In the present study, the cytotoxicities of (+-)-diethylstilbestrol oxide (2), (+)-, (-)- and (+-)-monomethyl ethers (4) of 2, (+-)-dimethyl ether (5) of 2, diethylstilbestrol pinacolone (3), E,E-dienestrol (6), Z,Z-dienestrol (7), meso-hexestrol (8), a mixture of (1R,1'S)4-hydroxyhexestrol and (1R,1'S)4'-hydroxyhexestrol (9), and the 4-hydroxy derivative (10) of diethylstilbestrol dimethyl ether were investigated in Chinese hamster V79 cells. The results indicated that the cytotoxic activity of 10 was the strongest of the compounds tested, although its activity was the almost same as that of 1. Moreover, as the activity of (-)-4 was greater than those of 2 and 1 monomethyl ethers, the effect of 4 on cytotoxic activities was elucidated. In conclusion, the present results indicate that the cytotoxic activities of hydroxylated metabolites are greater than those of each mother compound, although epoxidation of 1 leads to a product which can be broken down more readily than the parent compound.

Promotive effects of diethylstilbestrol, its metabolite (Z,Z-dienestrol) and a stereoisomer of the metabolite (E,E-dienestrol) in tumorigenesis of rat mammary glands pregnancy-dependently initiated with radiation.

Wistar-MS rats received whole body irradiation with 260 cGy gamma-rays at day 20 of pregnancy and then were treated with diethylstilbestrol (DES), E,E-dienestrol (E,E-DIES) or Z,Z-dienestrol (Z,Z-DIES) for 1 year. DES administration caused the highest incidence of mammary tumors with a concomitant reduction of gain in body weight. When E,E-DIES or Z,Z-DIES in pellet form was implanted, the incidence of tumors was significantly lower than that observed in rats treated with DES. To clarify the increased susceptibility to mammary tumorigenesis after DES administration we measured hormone levels in the serum of rats implanted with pellets containing derivatives of the synthetic estrogens. The serum prolactin concentration was significantly increased by DES administration. When E,E-DIES or Z,Z-DIES pellets were implanted the prolactin level was markedly reduced to 4.5% and 0.7% of that observed in DES-treated rats, respectively. In addition, the serum concentrations of estradiol-17 beta and progesterone in rats with Z,Z-DIES pellets were higher than those of rats with DES or E,E-DIES pellets. A large number of DES-induced mammary tumors were positive for both estrogen and progesterone receptors, but no tumors negative for both receptors were obtained. The findings suggest that DES acts directly on radiation-initiated mammary cells via binding with estrogen receptors and/or stimulates the secretion of prolactin from the pituitary glands.

[The characteristics of the estrogen reaction of the resident macrophages of the dermis and of the macrophages in an inflammatory focus in rats]. / Osobennosti reaktsii na éstrogen rezidentnykh makrofagov dermy i makrofagov ochaga vospaleniia u krys.

The method of morphometry was used to study specific features of the ultrastructure of derma resident macrophages in 2-weeks-old and mature rats and macrophages of an experimental inflammation focus caused by a subcutaneous introduction of a celloidin ball. Synthetic nonsteroid estrogen was injected subcutaneously to experimental animals: synestrol in dose 25 mg/kg of body mass 3 times a week. Animals not treated with the drug were taken as controls. Resident macrophages of the inflammation focus reacted to synestrol differently. The reaction of resident macrophages to synestrol depends on the age. Synestrol causes an increase of the summary section area of mature animals and slightly decreases the size of the lysosomal apparatus and intensity of the formation of microgrowings of the macrophage cytoplasm in young rats. Unlike the resident macrophages in which the apparatus of synthesis and secretion is not changed, the size of Golgi complex and the summary area of profiles of the granular endoplasmic reticulum in the cytoplasm of macrophages of the inflammation focus are increased under the influence of synestrol. All this seems to be a manifestation of the estrogen stimulation of secretory activity of the inflammation focus macrophages.

Diethylstilbestrol stimulates persistent phosphatidylinositol lipid turnover by an estrogen receptor-mediated mechanism in immature mouse uterus.

The effect of estrogen on phosphoinositide (PI) metabolism was evaluated in the immature mouse uterus, a tissue which undergoes estrogen-induced proliferation. Uteri isolated from untreated mice or from mice injected ip with diethylstilbestrol (DES) were incubated with [3H]myo-inositol and assessed for incorporation of label into PI lipids or inositol phosphate generation. DES administration elicited a rapid increase in [3H]myo-inositol incorporation, which persisted until at least 18 h post treatment. This effect could not be duplicated by incubation of uteri with DES in vitro, although [3H]myo-inositol incorporation in uteri removed from DES-treated mice remained elevated for 3 h of in vitro incubation. Stimulation of PI lipid metabolism by DES was blocked by ICI 164,384, a specific estrogen receptor antagonist. The effect of DES on PI metabolism consisted of a time-dependent increase in the specific activity of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and a significant increase of inositol (1,4,5)-trisphosphate mass by 12 h post treatment. These changes occur before the onset of estrogen-induced DNA synthesis. The results indicate that estrogens rapidly modulate PI lipid turnover through an estrogen receptor-mediated mechanism. Since the metabolic products of PI lipids are important for signal transduction and cellular proliferation, altered metabolism of these lipids may play an integral role in estrogen-induced mitogenesis.

Qualitative study of the interaction mechanism of estrogenic drugs with tubulin.

Synthetic estrogenic drugs (E-diethylstilbestrol, erythro-hexestrol and E,E-dienestrol) inhibit tubulin assembly and erythro-hexestrol and E,E-dienestrol lead to the formation of twisted ribbon structures. For the inhibitory effect on tubulin assembly, estrogenic drugs seem to interact directly with tubulin 6S on site(s) analogous to the colchicine-site, but independent of the GTP- and vinblastine-sites. This binding does not involve tubulin tryptophanyl residues or sulfhydryl groups. The influence of temperature, calcium and magnesium on the formation of twisted ribbon structures induced by the binding of estrogenic drugs to microtubular protein and tubulin has also been studied. This formation is strongly magnesium-dependent whereas preformed twisted ribbon structures are calcium- and chilling-insensitive.

[Functional morphology of capillary bed of the uterus after administration of synestrol]. / Funktsional'naia morfologiia kapilliarnogo rusla matki posle vvedeniia sinestrola.

The capillary vessels of rats uterus were examined. The animals who received 0.2% solution were examined 1, 3, 5, 10, 20, 30, 60 days after the last injection. As a marker for capillary vessels the histochemical method for finding the Mg++ adenosine triphosphatase was used. The studies have found prominent qualitative and quantitative changes (activity of enzyme, length and diameter of capillaries) not less than for two months after the last injection of the solution. Particularly prominent changes were found in endometrium between 10-20 days of the restorative period.

[The functional morphology of the uterine capillary bed after synestrol administration]. / Funktsional'naia morfologiia kapilliarnogo rusla matki posle vvedeniia sinestrola.

The capillary vessels of rats uterus was examined which were receiving for 7 days 0.2% solution of synoestrol i/m (2.0 mg per 1 kg of body weight). The animals were examined 1, 3, 5, 10, 20, 30, 60 days after the last injection of the solution. As the marker for the capillary vessels the histochemical method for finding the Mg2(+)-adenosintriphosphatase was used. The investigations have found prominent qualitative and quantitative changes (activity of enzyme, length and diameter of capillaries) not less than for two months after the last injection of the solution. Particularly prominent changes were found in endometrium between 10-20 days of the restorative period.

Performance of Leghorn type hens fed two levels of energy and a synthetic estrogen during the growing period.

Feeding of an estrogen, dienestrol diacetate, at 352 mg/kg of diet to December-hatched White Leghorn type pullets, from 16 to 20 wk of age, caused onset of production to be delayed approximately 3 wk. The dietary dienestrol diacetate also resulted in increased body weights at 30 and 46 wk in one experiment. Hens receiving the estrogen laid significantly (P less than .05) heavier but fewer eggs during most of the production year than did those fed diets without the estrogenic compound. The addition of 2.2% fat to diets of pullets from 0 to 20 wk of age failed to influence their performance in the layer house.

Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media.

It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. The cells of tissue explant outgrowths exhibited ultrastructural features typical of proximal tubules including junctional complexes, numerous mitochondria, peroxisomes, and microvilli. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17 beta-estradiol or diethylstilbestrol. Maximal proliferative response was observed at hormone concentrations of 0.6-1 nM. A similar 3-fold increase in cell number was also seen at 1 nM 17 beta-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Neither progesterone, 5 alpha-dihydrotestosterone, nor the inactive diethylstilbestrol metabolite beta-dienestrol elicited this mitogenic effect. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17 beta-estradiol. Tubular cells obtained from hamster medulla did not exhibit this proliferative response when exposed similarly to 17 beta-estradiol or diethylstilbestrol. The proliferative effect of estrogens on proximal tubular cell growth appears to be species specific since 17 beta-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17 beta-estradiol, [3H]thymidine labeling of hamster tubular cells was enhanced 3-fold. A similar increase in mitoses was also observed in cultures containing these potent estrogens during the same time interval of estrogen exposure. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney.

Inhibition of the H+-ATPase in bovine adrenal chromaffin granule ghosts by diethylstilbestrol. Evidence for a tight coupling between ATP hydrolysis and proton translocation.

Diethylstilbestrol (DES) was found to inhibit reversibly the hydrolysis of MgATP (80% at 100 microM) and proton pump activity (I50 approximately equal to 15 microM, complete at 100 microM) in chromaffin granule ghosts. The parallel inhibition suggests a tight kinetic coupling between the two activities. The Mg2+-ATPase activity, but not proton pumping, was partially restored by N,N'-dicyclohexylcarbodiimide, indicating that the two inhibitors in combination cause a partial uncoupling. The non-competitive type of inhibition shows that the action of DES is distal to the site of ATP binding and hydrolysis. Although unspecific, the interaction of DES with the chromaffin granule membrane seems primarily to affect the H+-ATPase.

Interaction of some estrogenic drugs with tubulin. Formation of twisted ribbon structures.

We studied the action of E-diethylstilbestrol (E-DES), erythro-hexestrol (erythro-HES), and E,E-dienestrol (E,E-DIES) on microtubule formation. The three drugs inhibit this formation from microtubular protein; the percentages of inhibition were, respectively, 15% and 45% for 1.25 X 10-s M E-DES and E,E-DIES. With purified tubulin 6S, 7.5 X 10(-6) M E,E-DIES and erythro-HES induced a 20% inhibition. In the case of E-DES, our results are in good agreement with previous ones. These drugs partially disrupt preformed microtubules. Moreover, when E,E-DIES (5 X 10(-5) M) is added to tubulin, loosely organized aggregates composed of twisted ribbon structure are formed. In the case of erythro-HES, similar structures were observed but at higher concentrations. With E-DES, no organized structures are present.

Estrogen stimulation of phosphatidylinositol metabolism in mouse uterine tissue.

The effect of estrogens on incorporation of labeled precursor into inositol-containing phospholipids (PI) from ovariectomized mouse uterus was investigated. The results indicate that diethylstilbestrol (DES) a potent mitogen, stimulated incorporation of myo-[3H]inositol into uterine PI in 1-3 h, with maximal incorporation occurring at 6 h. This activity followed a dose-dependent increase, with a maximal effect at 5 micrograms/kg. Incorporation of radiolabeled phosphorous into the polyphosphoinositides was also increased in estrogen-stimulated uterine tissue. Studies using a weak uterotropic DES derivative, Z,Z-dienestrol, produced an early stimulation of the PI response comparable to DES. This activity was increased by Z,Z-dienestrol, with minimal estrogen receptor occupancy, and did not result in stimulation of DNA synthesis. These findings would suggest that uterine PI stimulation may not occur via an estrogen receptor-mediated mechanism related to tissue proliferation induced by estrogens.

Reduction of irreversible binding of diethylstilbestrol in hamster renal cortex by inhibitors of cytochrome P-450.

Hamster renal cortical slices metabolized [3H]diethylstilbestrol (DES) to reactive intermediates that irreversibly bound to macromolecules. Protein isolated from cortical slices following incubation with 50 nM [3H]DES for 90 min at 37 degrees C had 0.160 pmol [3H]DES eq/mg protein irreversibly bound. Samples of protein analyzed by gel electrophoresis revealed several radioactive peaks, indicating that specific adduct formation had occurred. No radioactivity was associated with DNA isolated from the same tissue slices. Incubation of the slices with [3H]DES under an atmosphere enriched in carbon monoxide decreased the nonextractable binding of [3H]DES metabolites to protein. The cytochrome P-450 inhibitors diethylaminoethyl 2,2-diphenylpentanoate HCl (SKF 525-A), metyrapone, butylated hydroxytoluene (BHT), and dicumarol decreased the irreversible binding of [3H]DES by 38 to 72%. Analysis of metabolites isolated from the incubation medium by high-pressure liquid chromatography indicated that carbon monoxide, BHT, and dicumarol inhibited the hydroxylation of [3H]DES. Arachidonic acid and indomethacin did not alter the irreversible binding of [3H]DES, indicating a lack of involvement of prostaglandin H synthetase in the metabolism of DES to reactive intermediates. These findings suggest that cytochrome P-450 isozymes in the hamster renal cortex metabolize DES to reactive species that covalently bind to macromolecules.

Toxoplasma gondii: decreased resistance to infection in mice due to estrogen.

Exposure to pharmacological concentrations of potent estrogenic compounds, including 17 beta-estradiol, diethylstilbestrol, and alpha-dienestrol, increased the susceptibility of mice to Toxoplasma gondii as measured by brain cyst formation. Compounds with weak estrogenic activity or other hormonal activity, including 5 alpha-dihydrotestosterone, progesterone, and zearalanol, did not alter host resistance to infection. The ability of estrogens to alter susceptibility was inhibited by the estrogen antagonist, tamoxifen. The restoration of ovariectomized mice with normal physiological concentrations of estrogen had no effect on subsequent infection with T. gondii. These results indicate that pharmacological, but not physiological, levels of estrogen selectively alter host resistance to T. gondii, possibly through hormonal events.

Reduction in hepatic lipid and plasma estradiol in estrogenized chicks injected with ascorbic acid.

Injecting White Leghorn chicks every other day with 20 mg ascorbic acid significantly reduced the increase in liver weight and lipids caused by feeding a diet with 0.1% dienestrol diacetate. In chicks fed two different basal diets containing 0.1% dienestrol diacetate, injecting chicks every other day with 20 mg alpha-tocopherol did not significantly reduce liver weight or lipids while the ascorbic acid injections did. Injecting meat-type chicks implanted with estradiol with 10 mg ascorbic acid daily significantly reduced liver weight, liver lipids, and plasma estradiol, but injecting with 8 mg alpha-tocopherol daily had no significant effect.

Estrogen immunosuppression is regulated through estrogenic responses in the thymus.

Previous studies have clearly established that physiologic and pharmacologic levels of estrogens modulate immunologic responses that result in simultaneous activation of the reticuloendothelial system and depression of cell-mediated immunity. The mechanisms of estrogen immunoregulation were examined in adult female mice administered pharmacologic levels of exogenous estrogens. Evaluation of steroidal and nonsteroidal compounds with varying degrees of estrogenicity (i.e., uterotrophic activity) provided evidence that their immunotoxicity, for the most part, correlates with estrogenicity. The mechanisms responsible for these effects appear to be complex, mediated through a direct chemical interaction with lymphoid target cells, as well as with nonlymphoid tissue, resulting in the release of soluble immunoregulatory factors. The latter phenomenon was examined in detail and it appears to constitute a regulatory factor(s) produced by thymic epithelium in response to an estrogen stimulus. This response is not only estrogen specific but may involve specific binding to estrogen receptors or receptor-like structures present in cytosol preparations from thymic epithelial cells.

[Carcinogenesis in rats under the combined transplacental action of sex hormones and nitrosoethylurea]. / Kantserogenez u krys pri kombinirovannom transplatsentarnom vozdeistvii polovyk gormonov i nitrosoétilmocheviny.

Transplacental treatment with synestrol exerted a slight modifying effect on nitrosoethylurea-induced carcinogenesis in random-bred albino rats: prenatal treatment with synestrol reduced the rates of Leydig-cell and uterine tumor incidence to those of spontaneous tumors.

The influence of environmental temperature, thyroid status and a synthetic oestrogen on the induction of fatty livers in chicks.

1. Hepatic lipid content, lipogenic enzyme activity and plasma lipid concentration were measured in chicks reared at 21 degrees or 34 degrees C and after thyroxine (T4), thiouracil (TU), propylthiouracil (PTU), dienestrol diacetate (DD) or PTU with DD had been given for 14 d. 2. At 34 degrees C there was a significant increase in the total liver lipid and triglyceride content. 3. Injections of T4 decreased liver lipid content whereas it was increased by feeding PTU or DD. The effects of PTU were more pronounced at 21 degrees C while those of DD were more pronounced at 34 degrees C. 4. There were significant interactions between temperature, thyroid status and synthetic oestrogen treatments on total lipid and triglyceride content of the liver. Fatty liver with marked steatosis could be produced through synergic actions of PTU and DD in chicks maintained at 21 degrees C.

Hormonal regulation of a new female-specific serum protein (FP) of the laboratory rat.

This paper reports evidence for a hormonal regulation of a new rat serum protein (female specific protein, FP) which is only demonstrable in female rats. Male and female rats were treated with testosterone and estrogen. The FP was assayed with immunological methods. The following results were obtained: 1. In testosterone-treated females the serum level of FP is reduced significantly 2. In estrogenized males the FP was distinctly demonstrable but not in the control males.