Prognostic significance of α- and ß2-adrenoceptor gene expression in breast cancer patients.

AIMS: Breast cancer is the most frequently diagnosed and leading cause of cancer death among women worldwide. It was classified within molecular intrinsic subtypes: luminal A, luminal B, human epidermal growth factor receptor 2-enriched and basal-like. Epinephrine and norepinephrine, released during stress, bind to adrenoceptors. α2 -adrenoceptors are encoded by the ADRA2A, ADRA2B and ADRA2C genes and ß2 by ADRB2. METHODS: We compiled several publicly available Affymetrix gene expression datasets, obtaining a large cohort of 1924 patients with distant metastasis-free survival (DMFS) data and evaluated the association between adrenoceptor expression, clinicopathological markers and outcome. RESULTS: ADRA2A high expressing tumours also expressed hormone receptors and presented diminished tumour size, grade and not compromised lymph nodes. ADRB2 high expression was found in smaller, low grade, oestrogen receptor-positive tumours. Both were significantly associated with the absence of metastasis. High expression of ADRA2C was positively associated with increased tumour size and metastatic relapse. We observed a significant increase in DMFS of patients with high ADRA2A (hazard ratio 0.54, 95% CI 0.45-0.65, P < .001) and ADRB2 (0.77, 0.64-0.93, P = .006) expression and a decrease with ADRA2C high expression (1.45, 1.16-1.81, P = .001). For patients with luminal tumours, ADRA2A was the only factor that retained its significance as an independent predictor of DMFS while ADRA2C expression was an independent predictor for worse prognosis in basal-like tumours. CONCLUSIONS: We herein provide new insight for a potential role of ADRA2A and ADRA2C in breast cancer. In low- and medium-income countries, their incorporation to routine immunohistochemistry analysis of biopsies or tumour samples, could provide additional low-cost prognostic factors.

[One hundred years after the "Spanish" flu]. / A cien años de la gripe "española".

The "Spanish" flu pandemic, which occurred a century ago, is considered the most devastating in human history. An estimated one third of world population fell ill with flu and more than 2.5% of them died. The course of the epidemic had two main waves (1918 and 1919) and showed an unusual W-shaped morbidity/mortality distribution. Death was not a direct outcome of flu itself but rather a consequence of secondary bacterial bronchopneumonia, for which antibiotics had not yet been discovered. Pre-existing pulmonary tuberculosis was also accountable for increased flu death rates during the pandemic. As it happened in Europe, in Argentina the epidemic had two main waves, with ample variation in mortality by region. Available treatment at the time included diet, throat antiseptic rinses, low doses of quinine valerianate, salicylates, codeine as a cough suppressant, and camphor oil. Primitive anti-pneumococcal vaccines and immune sera were also applied. Upon the disclosure of the whole RNA sequence of the 1918 influenza virus genome, by means of reverse genetics it was possible to assemble viral particles resembling those of the deadly pandemic. The reconstituted virus proved to be extraordinarily virulent for mice. Current seasonal flu vaccines help to reduce, but not to abolish, the risk of another pandemic. The ongoing development of "universal" vaccines against influenza conferring reliable and long-lasting immunity may prevent its global spread in the future.

A Novel Effect of ß-Adrenergic Receptor on Mammary Branching Morphogenesis and its Possible Implications in Breast Cancer.

Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. ß-adrenergic receptors (ß-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in ß2-AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of ß2-AR. To study regulation of the breast cell phenotype by ß2-AR, we over-expressed ß2-AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down ß2-AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the ß2-AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that ß2-AR expression level is a key player in cell behavior. ß-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of ß-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through ß-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context.

Dosage-dependent regulation of cell proliferation and adhesion through dual ß2-adrenergic receptor/cAMP signals.

The role of ß-adrenergic receptors (ß-ARs) remains controversial in normal and tumor breast. Herein we explore the cAMP signaling involved in ß-AR-dependent control of proliferation and adhesion of nontumor human breast cell line MCF-10A. Low concentrations of a ß-agonist, isoproterenol (ISO), promote cell adhesion (87.5% cells remaining adherent to the plastic dishes following specific detachment vs. 35.0% in control, P<0.001), while increasing concentrations further engages an additional 36% inhibition of Erk1/2 phosphorylation (p-Erk1/2)-dependent cell proliferation (P<0.01). Isoproterenol dose response on cell adhesion was fitted to a 2-site curve (EC50(1): 16.5±11.5 fM, EC50(2): 4.08±3.09 nM), while ISO significantly inhibited p-Erk1/2 according to a 1-site model (EC50: 0.25±0.13 nM). Using ß-AR-selective agonist/antagonists and cAMP analogs/inhibitors, we identified a dosage-dependent signaling in which low ISO concentrations target a ß2-AR population localized in raft microdomains and stimulate a Gs/cAMP/Epac/adhesion-signaling module, while higher concentrations engage a concomitant activation of another ß2-AR population outside rafts and inhibit the proliferation by a Gs/cAMP/PKA-dependent signaling module. Our data provide a new molecular basis for the dose-dependent switch of ß-AR signaling. This study also sheds light on a new cAMP pathway core mechanism with a single receptor triggering dual cAMP signaling controlled by PKA or Epac but with different cellular outputs.

Steroid profile in patients with breast cancer and in mice treated with mifepristone.

Progesterone receptors (PRs) are biomarkers used as prognostic and predictive factors in breast cancer, but they are still not used as therapeutic targets. We have proposed that the ratio between PR isoforms A and B (PRA and PRB) predicts antiprogestin responsiveness. The MIPRA trial confirmed the benefit of 200 mg mifepristone, administered to patients with tumors with a high PRA/PRB ratio, but dose-ranging has not been conducted. The aim of this study was to establish the plasma mifepristone levels of patients from the MIPRA trial, along with the resultant steroid profiles, and compare these with those observed in mifepristone-treated mice using therapeutic schemes able to induce the regression of experimental mammary carcinomas with high PRA/PRB ratios: 6 mg pellets implanted subcutaneously, or daily doses of 12 mg/kg body weight. The plasma levels of mifepristone and other 19 plasma steroids were measured by liquid chromatography-tandem mass spectometry. In mifepristone-treated mice, plasma levels were lower than those registered in mifepristone-treated patients (i.e. day 7 after treatment initiation, pellet-treated mice: 8.4 ± 3.9 ng/mL; mifepristone-treated patients: 300.3 ± 31.7 ng/mL (mean ± s.d.; P < 0.001)). The increase in corticoid related steroids observed in patients was not observed in mifepristone-treated mice. The increase in progesterone levels was the most significant side effect detected in mifepristone-treated mice after 14 or 21 days of treatment, probably due to an ovarian compensatory effect not observed in postmenopausal patients. We conclude that in future clinical trials using mifepristone, the possibility of lowering the standard daily dose of 200 mg should be considered.

The Beta2-adrenergic agonist salbutamol synergizes with paclitaxel on cell proliferation and tumor growth in triple negative breast cancer models.

PURPOSE: Globally breast cancer accounts for 24.5% in incidence and 15.5% in cancer deaths in women. The triple-negative subtype lacks any specific therapy and is treated with chemotherapy, resulting in significant side-effects. We aimed to investigate if the dose of chemotherapeutic drugs could be diminished by co-administering it with the ß2-agonist salbutamol. METHODS: Cell proliferation was measured by thymidine incorporation; gene expression, by real-time PCR and protein phosphorylation by WB. Apoptosis was assessed by acridine orange / ethidium bromide and TUNEL tests. Public patient databases were consulted. Cells were inoculated to nude mice and their growth assessed. RESULTS: The ß2-agonist salbutamol synergizes in MDA-MB-231 cells in vitro with paclitaxel and doxorubicin on cell proliferation through ADRB2 receptors, while the ß-blocker propranolol does not. The expression of this receptor was assessed in patient databases and other cell lines. Triple negative samples had the lowest expression. Salbutamol and paclitaxel decreased MDA-MB-231 cell proliferation while their combination further inhibited it. The pathways involved were analyzed. When these cells were inoculated to nude mice, paclitaxel and salbutamol inhibited tumor growth. The combined effect was significantly greater. Paclitaxel increased the expression of MDR1 while salbutamol partially reversed this increase. CONCLUSION: While the effect of salbutamol was mainly on cell proliferation, suboptimal concentrations of paclitaxel provoked a very important enhancement of apoptosis. The latter enhanced transporter proteins as MDR1, whose expression were diminished by salbutamol. The expression of ADRB2 should be assessed in the biopsy or tumor to eventually select patients that could benefit from salbutamol repurposing.

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer.

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.

Adrenergic receptors in breast cancer.

Breast cancer is the most diagnosed malignancy in women worldwide and in the majority of the countries. Breast cancers are classified on the expression of estrogen and progesterone receptor expression and overexpression of human epidermal growth factor receptor 2 (HER2) as luminal, HER2+ and triple negative breast cancer. The intrinsic molecular subtypes match this classification. Cancer diagnosis and treatment cause distress. In both acute and chronic stress, the secreted catecholamines adrenaline and noradrenaline trigger the "fight-or-flight" response. This chapter focuses on the actions of the ß2 and α2 adrenergic receptors in several models of breast cancer. The actions of these receptors depend on the model used to investigate them. The ß2-adrenergic receptors seem to exert a dual action. They can directly act on the epithelial cells inhibiting cell proliferation and migration/invasion and indirectly upon the immune microenvironment. The proportion of ß2 receptors in each compartment could, therefore, lean the scale to an inhibition or to an exacerbation of tumor growth, invasion and metastasis. All the work points to a beneficial or neutral action of ß-blockers on breast cancer. With respect to α2-adrenergic receptors, the investigation performed by our group suggest that the α2B and the α2C receptors are linked to enhanced cell proliferation and tumor growth acting through both the epithelial and the stromal (fibroblastic) compartments while α2A could be beneficial for patients. Some adrenergic compounds could be repurposed for breast cancer treatment due to their very low side effects and very well-known pharmacology.

Classical membrane progesterone receptors in murine mammary carcinomas: agonistic effects of progestins and RU-486 mediating rapid non-genomic effects.

In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10(-11) and 1 × 10(-8) M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10(-8) M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10(-11) M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 µg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.

SARS-CoV-2 variants and the so-called resistance to vaccines.

RNA viruses (except retroviruses) replicate by the action of an RNA-dependent RNA polymerase, which lacks a proofreading exonuclease and, consequently, errors may occur in each replication giving place to viral mutants. Depending on their fitness, these mutants either become extinct or thrive, spawning variants that escape the immune system. The most important SARS-CoV-2 mutations are those that alter the amino acid sequence in the viral S protein because this protein holds the key for the virus to enter the human cell. The more viruses replicate, the more they mutate, and the more likely it is that dominant resistant variants will appear. In such cases, more stringent measures for community protection will be required. Vaccines and polyclonal antibodies, which induce a response directed towards several sites along the S protein, would maintain effective protection against SARS-CoV-2 variants. Furthermore, vaccines appear to induce an increased helper and cytotoxic T-cell response, which may also be a biomarker of protection. In densely populated areas with insufficient protection measures, the virus spreads freely, thus increasing the likelihood of generating escape mutants. India and Manaus exemplify this situation. Natural evolution selects the mutants that multiply most efficiently without eliminating the host, thus facilitating their spread. Contrastingly, the circulation of viruses of high virulence and lethality (Ebola, hantavirus) that eliminate the host remain limited to certain geographic areas, without further dissemination. Therefore, it would be expected that SARS-CoV-2 will evolve into more infectious and less virulent variants.

Los virus ARN, excepto los retrovirus, se replican por acción de una ARN polimerasa ARN-dependiente que carece de exonucleasa correctora y, en consecuencia, en cada replicación puede cometer errores. Así se originan mutantes que, según su menor o mayor fitness, se extinguen o bien prosperan y originan variantes que escapan al sistema inmune. Las mutaciones de SARS-CoV-2 más importantes son las que alteran la proteína viral S, porque ella tiene la llave de ingreso del virus a la célula humana. Cuanto más se replican los virus, más mutan, y se hace más probable que aparezcan variantes resistentes dominantes. En esos casos, se requerirá una aplicación más estricta de las medidas de protección de la comunidad. Las vacunas y los anticuerpos policlonales, que inducen una respuesta dirigida hacia toda la proteína S, mantendrían protección efectiva contra las variantes del SARSCoV-2. Además, las vacunas inducirían una mayor respuesta de células T helper y citotóxicas, lo que puede ser un biomarcador de protección. En áreas densamente pobladas con escasas medidas de protección, el virus se difunde libremente y aumenta la probabilidad de mutaciones de escape. India y Manaos ejemplifican esa situación. La evolución natural selecciona las mutantes que se reproducen con mayor eficiencia sin eliminar al huésped, lo que facilita la propagación. En cambio, la circulación de virus de alta virulencia y letalidad (Ebola, hantavirus), que eliminan al huésped, se circunscribe a determinadas áreas geográficas, sin mayor difusión. Por lo tanto, sería esperable que SARS-CoV-2 evolucione a variantes más infecciosas y menos virulentas.

Expression analysis of epithelial cadherin and related proteins in IBH-6 and IBH-4 human breast cancer cell lines.

Epithelial cadherin (E-cadherin) is a 120 kDa cell-cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as beta-catenin. Loss of E-cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down-regulation of E-cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH-6 and IBH-4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E-cadherin and related proteins in IBH-6 and IBH-4 cell lines was evaluated. In IBH-6 and IBH-4 cell extracts, only an 89 kDa E-cadherin form (Ecad89) was detected, which is truncated at the C-terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E-cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH-6 and IBH-4 cells showed an intracellular scattered E-cadherin localization. beta-catenin accompanied E-cadherin localization, and actin stress fibers were identified in both cell types. E-cadherin mRNA levels were remarkably low in IBH-6 and IBH-4 cells. The E-cadherin mRNA and genomic sequence encoding exons 14-16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up-regulation of Twist mRNA was found in both cell lines. In conclusion, IBH-6 and IBH-4 breast cancer cells show down-regulation of E-cadherin expression with aberrant protein localization, and up-regulation of Twist; these features can be related to their invasive/metastatic characteristics.

[Measles]. / Sarampión.

In April 2019, UNICEF denounced that more than 20 million children worldwide had not been vaccinated and alerted on possible outbreaks of measles which, due to the high transmissibility of this virus, is the first disease preventable by vaccination to emerge. If the decline in vaccinations continues, pertussis, tetanus and other diseases, which require less coverage to achieve population protection, may also reappear. In Argentina, the current outbreak began in late August 2019. Measles virus is transmitted by air, infects multiple organs, and is associated with immunosuppression. Its genome consists of single stranded RNA. Genotyping is carried out by sequencing a 450-nucleotide fragment of the N protein, which contains the highest density of nucleotide variation. In South America, D8 is the circulating genotype and in North America, B3 accounts for 8% of the cases. Each person with measles infects, on average, another 12-18 people in a susceptible population. Vaccination confers direct and indirect protection, and induces both antibodies and cellular immunity. Newborns are protected by maternal antibodies transmitted via the placenta, up to 6 months. In Argentina, the Vaccination Calendar includes two doses of triple viral vaccine, at 12 months and 5 years, and a zero dose (6-11 months of age) in districts with disease cases. The protection conferred by the vaccine is 93% at 12 months with a dose, and with 2 doses 97% for life.

En abril de 2019, UNICEF denunció que más de 20 millones de niños en todo el mundo no habían sido vacunados y alertó sobre posibles brotes de sarampión que, por su alta contagiosidad, es la primera enfermedad en emerger entre las prevenibles mediante vacunación. De continuar el descenso en las vacunaciones, podrían reaparecer también pertussis, tétanos y otras enfermedades con menor requerimiento de cobertura para alcanzar protección poblacional. A fin de agosto de 2019 se inició en la Argentina el actual brote de sarampión. Este virus se transmite por vía respiratoria, infecta múltiples órganos e induce inmunosupresión. Su genoma consiste en ARN de cadena simple. La genotipificación se efectúa por secuenciación de un fragmento de 450 nucleótidos de la proteína N que contiene la mayor densidad de variación de nucleótidos del genoma. En Sudamérica circula el genotipo D8, y en Norteamérica hay, además, un 8% de genotipo B3. Cada persona con sarampión infecta, en promedio, otras 12-18 en una población susceptible. La vacunación confiere protección directa e indirecta, e induce tanto anticuerpos como inmunidad celular. Los recién nacidos tienen protección hasta los 6 meses por anticuerpos maternos transmitidos vía placentaria. En la Argentina, el Calendario de Vacunación incluye dos dosis de triple viral, a los 12 meses y a los 5 años, y una dosis cero (6-11 meses de edad) en distritos con casos de enfermedad. Una dosis protege al 93% de los vacunados a los 12 meses y dos dosis al 97%, de por vida.

Agonist Effects of Propranolol on Non-Tumor Human Breast Cells.

The ß-blocker propranolol (PROP) has been proposed as a repurposed treatment for breast cancer. The similarity of action between ß-agonists and antagonists found on breast cells encouraged us to compare PROP and isoproterenol (ISO, agonist) signaling pathways on a human breast cell line. Cell proliferation was measured by cell counting and DNA-synthesis. Cell adhesion was measured counting the cells that remained adhered to the plastic after different treatments. Changes in actin cytoskeleton were observed by fluorescence staining and Western Blot. ISO and PROP caused a diminution of cell proliferation and an increase of cell adhesion, reverted by the pure ß-antagonist ICI-118551. ISO and PROP induced a reorganization of actin cytoskeleton increasing F-actin, p-COFILIN and p-LIMK. While ISO elicited a marked enhancement of cAMP concentrations and an increase of vasodilator-stimulated phosphoprotein (VASP) and cAMP response element-binding protein (CREB) phosphorylation, PROP did not. Clathrin-mediated endocytosis inhibition or ß-arrestin1 dominant-negative mutant abrogated PROP-induced cell adhesion and COFILIN phosphorylation. The fact that PROP has been proposed as an adjuvant drug for breast cancer makes it necessary to determine the specific action of PROP in breast models. These results provide an explanation for the discrepancies observed between experimental results and clinical evidence.

Novel human breast cancer cell lines IBH-4, IBH-6, and IBH-7 growing in nude mice.

Breast cancer is the most frequent cancer in women. However, in vivo hormone receptor positive and metastatic models are scarce. The aim of the present manuscript was to assess if the novel steroid receptor positive human cell lines IBH-4, IBH-6, and IBH-7 developed in our laboratory from primary infiltrant ductal carcinomas are good models to study in vivo human breast cancer. Cell lines or tumors were inoculated to nude mice in the presence or absence of hormone supplementation. Growth was analyzed by ANOVA followed by Tukey-Kramer's test. Steroid hormone expression was assessed by immunohistochemistry and Western blotting. The histology of the tumors was analyzed. IBH-4 and IBH-6 cells were inoculated to nude mice and 100% of the injected mice developed tumors in the presence or absence of hormone treatment, although tamoxifen inhibited growth. IBH-4 and IBH-6 cell lines in vivo gave rise to poorly differentiated carcinomas with areas of solid growth and sarcomatoid areas showing no morphological signs of epithelial differentiation. Distinct features of malignancy were observed. IBH-7 tumors in animals receiving estradiol were semi-differentiated adenocarcinomas. IBH-7 cells grew only in the presence of estradiol, but even with hormone addition, the tumor take was 20%. These tumors metastasized to the uterus and lung and vascular tumor emboli were evident. IBH-7 tumors were invasive and able to break through the peritoneum. As a conclusion, IBH-4 and IBH-6 are good models for studying tumor progression, whereas IBH-7 is a good model for tumor take, being metastatic and strictly estrogen-dependent.

Contribution of alpha2-adrenoceptors to the mitogenic effect of catecholestrogen in human breast cancer MCF-7 cells.

Catecholestrogens are estrogen metabolites formed by hydroxylation of 17beta-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via alpha(2)-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E(2)), 2-hydroxy-estrone (2-OH-E(1)) and 4-hydroxy-estrone (4-OH-E(1)) competed for [(3)H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E(2)) did not. The contribution of alpha(2)-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific alpha(2)-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E(2). The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the alpha(2)-adrenoceptors and ERs for 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1) was mixed, whereas for 4-OH-E(2), the only receptor implied was an ER. In MDA-MB-231 cells (ER-alpha negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that alpha(2)-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1).