CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9–3 μM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.     

Tamoxifen decreases the estradiol induced progesterone receptors by interfering with nuclear estrogen receptor accumulation

The retention time of the estrogen receptor in the nucleus of target cells after antiestrogen treatment has been shown to be longer than after estradiol. This paper describes the accumulation of nuclear estrogen receptors and the obtention of estrogenic responses (i.e. synthesis of cytosolic progesterone receptors and DNA) in the rat uterus after tamoxifen treatment in the presence or absence of estradiol. One-week ovariectomized adult rats were implanted with a silicone elastomer capsule containing corn oil or 25 micrograms estradiol/capsule (0 h). 48 h after implantation rats were injected with corn oil or 2 mg tamoxifen/kg and decapitated at 72, 96 or 120 h after implantation. In parallel experiments the implants were removed just before the injections of tamoxifen or oil. Tamoxifen injected into rats implanted with oil increased both the occupied nuclear receptors and the progesterone receptors at 96 h. In rats implanted with estradiol, tamoxifen did not increase the occupied nuclear receptors and decreased the levels of progesterone receptor and DNA at 96 h. In rats whose estradiol implants were removed at 48 h tamoxifen did not change the level of occupied nuclear receptors at 72 h but it increased them abruptly at 96 and 120 h. In these rats progesterone receptors decreased at 72 h but they increased at 96 and 120 h, and DNA decreased at 120 h to a lower level than before implantation. The results suggest that when estradiol is acting, tamoxifen is not able to increase the level of occupied estrogen receptor and it acts as an antiestrogen by decreasing the high level of progesterone receptors previously induced by estradiol. When estradiol is not acting tamoxifen behaves as a partial estrogen agonist by inducing progesterone receptors. However, the antiestrogenic action of tamoxifen on the rat uterus DNA does not seem to be affected by estradiol.     

Ion channels and receptors: molecular targets for behavioral evolution

Ion channels and receptors play critical roles in shaping neuronal activity, and thus are appropriate targets for evolutionary change to generate new behaviors. In this review, the evolution and differentiation of the many voltage-gated ion channels and transmitter-activated receptors is summarized; these channels and receptors evolved very early, and with some exceptions all species with nervous systems use similar sets of channels and receptors. Several examples are given of mechanisms for species-specific behavioral evolution that arise from mutations involving the structure, alternative splicing, level of expression, targeting and modulation of these important neural proteins.     

Insect ryanodine receptors: molecular targets for novel pest control chemicals

Ryanodine receptors (RyRs) are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. They are located on the sarcoplasmic reticulum of muscle and the endoplasmic reticulum of neurons and many other cell types. Ryanodine, a plant alkaloid and an important ligand used to characterize and purify the receptor, has served as a natural botanical insecticide, but attempts to generate synthetic commercial analogues of ryanodine have proved unsuccessful. Recently two classes of synthetic chemicals have emerged resulting in commercial insecticides that target insect RyRs. The phthalic acid diamide class has yielded flubendiamide, the first synthetic ryanodine receptor insecticide to be commercialized. Shortly after the discovery of the phthalic diamides, the anthranilic diamides were discovered. This class has produced the insecticides Rynaxypyr(R) and Cyazypyrtrade mark. Here we review the structure and functions of insect RyRs and address the modes of action of phthalic acid diamides and anthranilic diamides on insect ryanodine receptors. Particularly intersting is the inherent selectivity both chemical classes exhibit for insect RyRs over their mammalian counterparts. The future prospects for RyRs as a commercially-validated target site for insect control chemicals are also considered.     

Expression of estrogen and progesterone receptors and estrogen metabolizing enzymes in different breast cancer cell lines

Prolonged exposure to estrogens is a significant risk factor for the development of breast cancer. Estrogens exert carcinogenic effects by stimulating cell proliferation or through oxidative metabolism that forms DNA-damaging species. In the present study, we aimed to provide a better understanding of estrogen metabolism and actions in breast cancer, and to characterize model breast cancer cell lines. We determined the expression profiles of the genes for the estrogen and progesterone receptors, and for 18 estrogen-metabolizing enzymes in eight cell lines: MCF-7, MCF-10A, T47D, SKBR3, MDA-MB-231, MDA-MB-361, Hs-578T and Hs-578Bst cells. Similar gene expression profiles of these receptors and enzymes for the formation of estradiol via the aromatase and sulfatase pathways were observed in the MCF-7 and T47D metastatic cell lines. The MDA-MB-361 cells expressed ESR1, ESR2 and PGR as well, but differed in expression of the estrogen-metabolizing enzymes. In the MDA-MB-231 and SKBR3 cells, all of these estrogen-forming enzymes were expressed, although the lack of ESR1 and the low levels of ESR2 expression suggested that the estrogens can only act via non-ER mediated pathways. In the non-tumorigenic MCF-10A cell line, the key enzymes of the aromatase pathway were not expressed, and the sulfatase pathway also had a marginal role. The comparison between gene expression profiles of the non-tumorigenic Hs-578Bst cells and the cancerous Hs-578T cells revealed that they can both form estrogens via the sulfatase pathway, while the aromatase pathway is less important in the Hs-578Bst cells. The Hs-578T cells showed low levels of ESR1, ESR2 and PGR expression, while only ESR1 and ESR2 expression was detected in the Hs-578Bst cells. Our data show that the cell lines examined provide the full range of model systems and should further be compared with the expression profiles of breast cancer specimens.     

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.     

Tectoridin from Maackia amurensis modulates both estrogen and thyroid receptors

AimThe stem bark of Maackia amurensis has been used as folk medicine for the treatment of cancer, cholecystitis, arthritis, and hyperthyroidism in females. In this study we examined the effects of the ethyl acetate fraction obtained from the 70% ethanol extract of M. amurensis and tectoridin, an active constituent isolated from the ethyl acetate fraction on thyroid and estrogen hormone activity.MethodsThe effect of the ethanolic extract of M. amurensis stem bark on thyroid hormone activity was evaluated using thyroid hormone responsive-luciferase assay. We isolated tectoridin from the ethyl acetate fraction using a recrystallization method. T-screen assays were used to confirm thyroid hormone activity. The estrogenic activity of the ethyl acetate fraction of M. amurensis and tectoridin was evaluated by estrogen responsive-luciferase assay and estrogen receptor alpha regulation as compared to 17β-estradiol.ResultsBoth the ethyl acetate fraction and tectoridin activated thyroid-responsive reporters and increased thyroid hormone-dependent proliferation of rat pituitary GH3 cells, indicating modulation of thyroid hormone receptors. In parallel, the estrogenic activity of the fraction and tectoridin were characterized in a transient transfection system using estrogen-responsive luciferase plasmids in MCF-7 cells. The ethyl acetate fraction and tectoridin activated reporter gene expression and decreased the estrogen receptor protein level.ConclusionsThese data indicate that tectoridin acts as a weak phytoestrogen as well as a thyroid hormone-like agent by activating both estrogen and thyroid hormone receptors.     

Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes

Estrogen receptor (ER) analysis of breast cancer tissue has been shown to be very useful in predicting which patients will respond to hormone therapy and have a better prognosis. The ER assay is, however, tedious and time consuming. Measurement of ER by flow cytometry would be rapid and based on either an average fluorescence-E2 probe intensity per cell or the percentage of the ER+ cells per cell suspension. Analysis of E2 modified structures for relative binding affinity to the ER determined by competition studies and for fluorescence uptake into cell suspensions determined by flow cytometry was performed. Lack of high affinity to the ER and purity of the compound were major problems for the fluorescein-labeled estrogen probes. Base hydrolysis of the ester linkage in fluorescein-E2 compounds demonstrated by HPLC very little estradiol derivative in the parent compounds compared to total components present. A second type of fluoresceinated estrogen which has a peptide bond between the steroid and the chromophore was also tested. It was less contaminated but was unable to get into the cell and showed no binding activity to the ER. A pure plant fluorescent estrogen, coumestrol, has Ka of 6 X 10(8) M-1 for the ER and is a single component as determined by HPLC. Specific fluorescent uptake of coumestrol was performed on ER+ and ER- viable cell suspensions. When these coumestrol-cell suspensions were excited at 350-360 nm and the blue emission was measured using flow cytometry, the result was a fluorescence uptake that was not highly displaceable by excess nonfluorescence E2 probes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The histochemistry of estrogen receptors

Summary Estrogen receptors in frozen section of the rat uterus were demonstrated by a radiolabeled ligand binding technique. The bound hormone was extracted with ethanol and measured by liquid scintillation. The binding of ³H-estradiol-17 at various molar concentrations was inhibited by a 100-fold excess of DES, and the bound ³H-estradiol resisted exhaustive washings at 4°C for 16 h. These binding sites were not present in the sections of the spleen, and perhaps at a very low concentration in the myocardium. Thus their binding behavior and distribution pattern are consistent with those of specific estrogen receptors. The hydrophilic fluorescent estradiol conjugate, 17-estradiol-6-CMO-BSA-FITC was found to be a highly effective competitor against binding of ³H-estradiol to its receptors in tissue sections, and is considered a useful histochemical reagent for localizing target cells with high concentrations of estrogen receptors. Estrogen receptor sites in frozen sections of human breast cancer were also measured by this radiolabeled ligand binding technique, and expressed in femtomoles of hormone bound per 1,000 cancer cells. The values were parallel to the histochemical findings in terms of percentage of the estrogen receptor-positive in the cancer cell population.     

Immunochemical studies of estrogen receptors

Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.