BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.     

Peptides and antitumor activity. Development and investigation of some peptides with antitumor activity

We developed a group of synthetic analogs of GnRH and Somatostatin to inhibit the tumor growth of different kind. The GnRH analogs decreasing the gonadotroph and steroid hormone levels act on the hormone dependent tumors and influence their growth. One of the most effective antitumor analog was patented under the name FOLLIGEN which inhibited the breast cancer caused by DMBA in rats without any side-effects. Other inhibitory analogs of GnRH with long-lasting effect were effective in the treatment of breast, ovary and prostate tumors. Another analog [alpha-Asp(DEA)]6,Gln8-hGnRH showed a very low endocrine but high antitumor effect in both in vitro and in vivo experiments. Its tritium labeled derivative exhibited specific binding sites on human tumor cell lines. We synthesized the analogs of GnRH-III with effective selective antitumor activity which does not alter the ovarian cycle of rats but inhibits the colony-formation of human breast cancer cell lines and has a significant antiproliferative effect. We also synthesized conjugates of potent GnRH analogs with a branched chain polylysine backbone which induce a 33-35% decrease of cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines and 45-50% inhibition of cell proliferation. Another conjugate decreased the tumor growth of MDA-MB-231 xenografts by 80% in a treatment of 9 weeks and even tumor free animals could be found among the ones treated. Using these radiolabeled peptide hormone analogs we found that human tumor cell lines and xenografts specifically bind the GnRH conjugates. We also synthesized a series of Somatostatin analogs which inhibit tyrosine kinases and the growth of several breast, prostate and colon tumor cell lines. One of our best analogs was a heptapeptide, TT-232, which strongly inhibited the tyrosine kinase activity and the cell-proliferation in different colon tumor cells. However, it did not inhibit the growth hormone release either in vitro or in vivo from rat pituitary cells. The TT-232 was found to be effective on 60 human tumor cell lines, it significantly inhibited the tumor growth on different animal tumor models, and induced apoptosis, as a result of which some animals became tumor free. The TT-232 inhibited the tumor growth of PC3 prostate xenografts with 60% and caused a 100% survival of mice 60 days after the transplantation. It is being preclinically tested at present. We have shown that the new GnRH analogs acting without any hormonal effect and the Somatostatin analogs with strong antitumor and tyrosine kinase inhibitory activity but no hormonal effect may represent a breakthrough in the research of the antitumor peptides, having direct effect on tumor cells.     

Parasite regulation by host hormones: an old mechanism of host exploitation?

Recent experimental evidence suggests that parasites can not only evade immune responses actively but also exploit the hormonal microenvironment within the host to favor their establishment, growth and reproduction. The benefit for parasites of hormonal exploitation is so great that they have evolved structures similar to the steroid and protein hormone receptors expressed in upper vertebrates that can bind to the hormonal metabolites synthesized by the host. This strategy is exemplified by two parasites that respond to adrenal steroids and sexual steroids, respectively: Schistosoma mansoni and Taenia crassiceps. Understanding how the host endocrine system can, under certain circumstances, favor the establishment of a parasite, and characterizing the parasite hormone receptors that are involved might aid the design of hormonal analogs and drugs that affect the parasite exclusively.     

Growth hormone-releasing hormone antagonists inhibit growth of human ovarian cancer

Epithelial ovarian carcinoma is the leading cause of cancer-related deaths among women with gynecologic malignancies. Antagonists of the growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of various cancers through endocrine, autocrine, and paracrine mechanisms. In this study, we have investigated the effects of GHRH antagonists (GHRHa) in ES-2 human clear cell ovarian cancer and in UCI-107 human serous ovarian cancer in vitro and in vivo. We evaluated the expression of mRNA for GHRH receptor, the binding to GHRH receptors, in specimens of ES-2 ovarian cancer. We evaluated also the in vitro effects of GHRHa on ES-2 cells and the in vivo effect of 2 different GHRHa on ES-2 and UCI-107 tumors. Nude mice bearing xenografts on ES-2 and UCI-107 ovarian cancer were treated with JMR-132 and MZ-J-7-118, respectively. Tumor growth was compared to control. ES-2 cells expressed mRNA for the functional splice variant SV1 of the GHRH receptor. JMR-132 inhibited cell proliferation in vitro by 42% and 18% at 10 and 1 μM concentration, respectively. Specific high affinity receptors for GHRH were detected in ES-2 cancer samples. In vivo daily subcutaneous injections of GHRHa significantly reduced tumor growth compared to a control group in both animal models. Our results indicate that GHRHa such as JMR-132 and MZ-J-7-118 can inhibit the growth of human ovarian cancer. The efficacy of GHRHa in ovarian cancer should be assessed in clinical trials.     

Novel approaches for the study of vertebrate steroid hormone receptors

Steroid hormones are essential for the normal function of most organ systems in vertebrates. Reproductive activities in females and males, such as the differentiation, growth and maintenance of the reproductive system, require signaling by sex steroid hormones. Although extensively studied in mammals and a few fish and bird species, the evolution and molecular mechanisms associated with the nuclear steroid hormone receptors are still poorly understood in amphibians and reptiles. Given our interest in environmental signaling of sex determination as well as a major interest in environmental contaminants that can mimic steroid hormone signaling, we have established an approach to study the molecular function (ligand binding and trans-activation) of steroid hormone receptors cloned from reptiles. This approach involves molecular cloning and sequencing of steroid hormone receptors, phylogenic analysis and in vitro trans-activation assays using endogenous or exogenous ligands. Comparing the in vitro trans-activation induced by different ligands with receptors cloned from different species would develop additional functional relationships (classification) among steroid hormone receptors. This approach can provide insight into understanding why each species could have different responses to exogenous ligands. Further, we have developed a novel and less invasive approach to obtaining mRNA for molecular cloning and sequencing of steroid hormone receptors in reptiles and other non-mammalian species, using blood cells as a source of genetic material. For example, white blood cells (WBCs) and red blood cells (RBCs) of the American alligator both express steroid hormone receptors and have adequate amounts of mRNA for molecular cloning. This approach would allow us to analyze components of endocrine function of steroid hormones without sacrificing animals. Especially in endangered species, this approach could provide an understanding of endocrine functions, elucidate the phylogenic relationships of various receptors in vitro, such as the steroid hormone receptors, and determine possible effects of environmental contaminants in a minimally invasive manner.     

Insulin like growth factor 1 and regulation of ovarian function in mammals

Various growth factors have been proposed to play endocrine and/or paracrine role in mammalian ovarian follicular development. The insulin like growth factor 1 (IGF-1) is one such factor. More and more reports now support the existence of an intra-ovarian IGF system including receptors and binding proteins. The role of IGF-1 in ovary is to amplify gonadotropin hormone action in terms of increased steroidogenesis by ovarian granulosa cell and granulosa cell proliferation. The synthesis and proteolysis of insulin like growth factor binding proteins, under the control of follicle stimulating hormone, regulate the intra-follicular availability of IGF-1, which further determines the sensitivity of granulosa cells to gonadotropins. Besides gonadotropins, IGF-1 has been implicated in somatotropin hormone action in the ovarian function. Exact mechanism of IGF-1 action in the ovarian follicles needs to be worked out to elucidate whether or not IGF-1 is indispensable in addition to know endocrine factors like gonadotropic and ovarian steroid hormones. This will pave the way for better understanding of control(s) which ensure final development of dominant follicle(s) and atresia of other follicles of the cohort.     

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.     

Heterogeneous Regulation of Steroid Hormone Receptors in the Brain

SYNOPSIS. The ovarian steroid hormones, estradiol and progesterone,act in the guinea pig brain to regulate the expression of sexualbehavior. In studies of the cellular mechanisms of steroid hormoneaction, we have used an immunocytochemical technique to studythe regulation of these receptors in different neuroanatomicalregions. We have observed that progestin receptor-immunoreactivityin cells in certain neuroanatomical regions are more responsiveto particular steroid hormone treatments than are cells in otherregions. Similarly, we have observed selective regulation ofprogestin receptor-immunoreactivity in neurons identified onthe basis of their neuropeptide content. Finally, in the rostralpart of the ventrolateral hypothalamus, a site involved in hormonalregulation of female sexual behavior, estrogen receptor-immunoreactiveneurons that have dopamine-ß-hydroxylase varicositiesclosely-associated have higher levels of immunostaining forestrogen receptors than neurons without this relationship. Takentogether, these studies demonstrate the possibility of studyingthe microregulation of steroid hormone receptors in subsetsof neurons defined by neuroanatomical location, neuropeptide/neurotransmittercontent, afferent input and projection sites. The ability tostudy interactions among different systems at the cellular levelmay help us to understand more clearly the cellular processesinvolved in hormonal regulation of fundamental neuroendocrineprocesses, including the neuroendocrine regulation of sexualbehavior     

Ovariectomy has minimal effects on neuroadaptations associated with ethanol dependence in female rats

We previously found gender selective alterations in gene expression for GABA_A and NMDA receptors associated with the development of ethanol dependence. Males and females have a differing hormonal environment, including steroid hormone derivatives (neuroactive steroids) that exert effects at GABA_A and NMDA receptors. Therefore, we explored whether the removal of ovarian steroids would alter gender differences in response to chronic ethanol exposure. We found that ovariectomy reduced ethanol drinking levels by 15%, comparable to earlier observations between intact female and male rats. However, investigation of the effects of chronic ethanol exposure on intact versus ovariectomized female rats uncovered few differences in chronic ethanol-induced alterations in selected GABA_A or NMDA receptor subunit peptide levels. In general, findings for both groups of females were similar to previous observations. There was no reduction in GABA_A receptor 1 subunit levels in cerebral cortex in either intact or ovariectomized female rats, in contrast to the significant reduction observed in male rats. In addition, both intact and ovariectomized female rats had increased levels of the NMDA NR1 subunit in cerebral cortex and hypothalamus, but not in hippocampus, whereas ethanol dependent male rats displayed significant increases in the NR1 subunit only in hippocampus. Radioligand binding analysis with [³⁵S]TBPS found no differences in modulation of the GABA_A receptor by neuroactive steroids between ethanol dependent male, intact female or ovariectomized female rats. Seizure susceptibility was not different between intact or ovariectomized female rats during ethanol withdrawal. We did observe differential effects on brain allopregnanolone and plasma corticosterone levels between ethanol dependent intact and ovariectomized female rats, suggesting that ovarian steroids influence HPA axis adaptations to prolonged ethanol exposure. Overall, these data suggest that ovarian steroids do not significantly impact the gender selective alterations of GABA_A and NMDA receptors associated with ethanol dependence.     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Cancers du sein hormono-dépendants: quels biomarqueurs tissulaires utiles dans la prise en charge ?

Breast cancer is the second most frequently diagnosed cancer in women, in France. It accounts for almost 37% of new cancer diagnosed in women. Despite a decrease of the mortality during the last 15 years, breast cancer remains the first cause of cancer death in women and a public health problem. Historically, there are two very different types of cancers based on their content of hormone receptors (HR): estrogen receptor (ER) and progesterone receptor (PR). Nearly 70% of breast tumors are HR-positive, called “hormone-dependent” because their growth depends on steroid hormones. Their treatment includes the first targeted therapy: the hormone therapy. Initially, the clinical benefits of hormone therapy were demonstrated on patients whose HR content was evaluated by quantitative ligand-binding (LBA) or immunoassays (EIA). Today, the hormone dependence is determined by testing the presence of the receptors by immunohistochemistry, a semi quantitative evaluation of content, on fixed tissue section. But this evaluation of HR, seems not to be sufficient to predict the sensitivity or resistance to hormonal treatment. These technical evolutions of RH mesurement and the loss of quantitative assessement remain an open question. Also the discovery of many other players in the estrogen signaling complicates the understanding of this tumor subtype. Thus, determination of the hormonal dependence remains a unresolved problem. The discovery of new potential relevant biomarkers and new technologies allows us to reconsider the RH question and to move towards the most personalized medicine.     

Topographic recognition of cyclic hydrocarbons and related compounds by receptors for androgens, estrogens, and glucocorticoids

The structural requirements for the interaction of about 80 cyclic hydrocarbons and related compounds with the androgen receptor of rat ventral prostate, the estrogen receptor of human breast tumor MCF-7 cells, and the glucocorticoid receptor of rat liver were examined by comparing their abilities to compete with radioactive hormones for binding to the respective receptors. The results indicate that the receptor-binding affinity of a compound is dependent on its electronic configuration and geometrical similarity to a portion of a natural steroid hormone which can be recognized by local ligand-binding sites in the receptor. For the estrogen receptor, beta-phenols are more active than the corresponding alpha-phenols, whereas nonphenolic compounds are totally inactive. For androgen and glucocorticoid receptors, alpha-phenols are more active than beta-phenols. The androgen receptor can interact stereospecifically with nonoxygenated and nonalkylated cyclic hydrocarbons, such as 10,11-dihydro-5H-dibenzo[a,d] cycloheptene or 9,10-dihydrophenanthrene, which can, in vivo, inhibit the androgen-dependent growth of the male accessory reproductive organs. The affinities of naphthalene, anthracene, phenanthrene, biphenyl, and adamantane toward glucocorticoid and androgen receptors can be enhanced by acetylation or ethanolization of these ligands. Our results also indicate that, while the hormonal action of a steroid may be dependent on the interaction of a functional group on the hormone with a specific group on the receptor, the presence of such a group may not be required for the antagonistic activity of a compound that can physically block hormone binding to the receptor. Thus, many small molecules that were hitherto considered to be biologically inert may interact with steroid receptors specifically and affect hormonal activities in vivo.     

A cell surface protein controls endocrine ring gland morphogenesis and steroid production

Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. We show in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. Our data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, we propose that this mechanism is evolutionary conserved.     

Regulation of tumor-host interactions in breast cancer

Breast cancer has a striking dependence upon steroid and other endocrine hormones in its onset, regulation, and malignant progression to its most deadly forms. The epithelium of the normal mammary gland is also regulated by the ovarian endocrine steroids estrogen and progesterone, by other endocrine hormones, and by poorly defined influences of the stromal cells and basement membrane. The onset and development of cancer appears to involve tumor misinterpretation of and/or desensitization to host regulatory signals, and finally to releasing its own hormonal signal to reorganize the host for its own benefit. Current studies are beginning to examine mediators of tumor-host interaction and their regulation by steroid hormones. Important tumor-host interactions under investigation include desmoplasia, angiogenesis, metastases and immunosuppression.     

Evidence for a pituitary site of gonadal steroid stimulation of GnRH receptors in female mice

Treatment of GnRH-deficient (hpg) female mice with oestradiol-17 beta (E2) for 7 days increased GnRH receptors from 4.1 +/- 0.4 fmol/pituitary (control) to 7.2 +/- 0.7 fmol/pituitary (GnRH-treated), and consistently increased pituitary FSH content. Treatment of hpg female mice with E2 plus progesterone (P) for 14 days stimulated GnRH receptors more than did E2 alone, although values still remained lower than those of normal intact female mice. In contrast, GnRH treatment of intact hpg female mice alone, or combined with E2 + P, increased GnRH receptors to values similar to those of intact normal female mice. In contrast, the receptor rise after GnRH treatment alone of ovariectomized hpg mice was significantly less than in intact hpg mice similarly treated. However, the combination of GnRH + E2 + P treatment of ovariectomized hpg mice increased GnRH receptors to normal intact female values, indicating the synergistic actions of these hormones on GnRH receptor up-regulation at the pituitary. Oestradiol treatment of ovariectomized normal female mice prevented the receptor fall after ovariectomy, and when combined with exogenous GnRH further increased receptors to values identical to those of intact female mice receiving GnRH alone. Ovariectomy of hpg mice had no effect on GnRH receptor, serum or pituitary LH and FSH values. There was no change in serum LH concentration after GnRH treatment of hpg female mice, but serum FSH increased and this was accentuated by ovariectomy, indicating that in intact mice an ovarian factor(s) normally inhibits GnRH-stimulated FSH release. This factor did not appear to be an ovarian steroid since serum FSH was not suppressed in intact or ovariectomized GnRH-treated hpg mice concurrently receiving E2 + P treatment. These results suggest that: (1) gonadal steroids alone have a major direct stimulatory action on the pituitary to increase GnRH receptors; (2) the oestrogen-induced increase in GnRH receptors is enhanced in the presence of GnRH; (3) steroids exert inhibitory feedback on gonadotrophin secretion that is mediated at some cellular regulatory locus other than the GnRH-receptor complex.     

Hormonal treatment of endometrial carcinoma: An overview and new development in biology

Progestins are routinely used in the treatment of endometrial carcinomas with about 30% response rate. After a 10–12 month mean response time, the tumors begin to regrow. This clinical situation has been reproduced in the experimental model for human endometrial carcinomas, developed by us. The model consists of growth and maintenance of human endometrial carcinomas of different histologic grade and sex steroid receptor content, in defined hormonal milieu, by serial transplantation in athymic nude mice. Biologically and clinically relevant information on the role of steroid receptors in eliciting hormonal responses, the effect of combination treatment with tamoxifen and progestin and the mechanism of resistance to this treatment after an initial response have been obtained. These studies form the basis for designing and testing rational treatment strategies for human endometrial carcinomas.