RESUMO
BACKGROUND AND PURPOSE: ß-Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours. EXPERIMENTAL APPROACH: ß-Adrenoceptor expression was analysed by immunofluorescence and RT-PCR. Cell proliferation was assessed by [(3) H]-thymidine incorporation, tumour growth by measuring with a calliper and ERK 1/2 phosphorylation by Western blotting. KEY RESULTS: ß(2) -Adrenoceptor expression was confirmed in the mouse and human cells tested. Cell proliferation was increased by adrenaline (by α(2) -adrenoceptor action) and decreased in every tested cell line by the ß-adrenoceptor agonist isoprenaline and the ß(2) -adrenoceptor agonist salbutamol. Isoprenaline and salbutamol reduced tumour growth in every tumour tested (mouse C4-HD and CC4-3-HI and human IBH-4, IBH-6 and MDA-MB-231 cell lines growing as xenografts in nude mice). These effects were reversed by the ß-adrenoceptor antagonist propranolol. The α(2) -adrenoceptor antagonist rauwolscine and the ß(2) -adrenoceptor agonist salbutamol were equally effective in diminishing tumour growth. ERK 1/2 activation analysed in IBH-4 tumours correlated with tumour growth, with the ß-adrenoceptor agonists decreasing its activation. Inhibition of ERK 1/2 phosphorylation in vitro was mainly mediated by the PKA pathway. CONCLUSIONS AND IMPLICATIONS: In our experimental models, the ß-adrenoceptor agonists inhibited breast cancer cell proliferation and tumour growth, probably mediated by inhibition of ERK 1/2 phosphorylation. The ß-adrenoceptor agonists were as effective as the α(2) -adrenoceptor antagonist rauwolscine, providing possible novel adjuvant treatments for breast cancer.
Assuntos
Agonistas Adrenérgicos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/uso terapêutico , Albuterol/farmacologia , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Propranolol/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Ioimbina/farmacologiaRESUMO
Epinephrine and Norepinephrine, typically released during stress bind to nine different adrenoceptors (AR) which classically control the cardiovascular and respiratory systems. New targets were described for the many agonists and antagonists developed for these AR, as the central nervous system. During the last three decades, AR expression and action on the mammary gland/breast were extensively investigated. In the cow mammary gland, good milkability was associated with low density of beta(2)-AR and high density of alpha(2)-AR. In the rat normal mammary gland, beta-AR are expressed in the epithelial cells, alveoli, ducts, and adipocytes showing an exquisite regulation by steroid hormones and prolactin. In rat dimethylbenz(a)anthracene (DMBA) tumors, a close correlation was observed between tumor growth and beta-AR concentration. beta(2)-AR were described in numerous human cell lines and breast tumors. The action of beta-adrenergic compounds on cell proliferation is contradictory. While some authors found that beta-agonists significantly inhibit cancer cell proliferation and tumor growth in mice, others described a significant reduction in DNA synthesis by beta-blockers. Also, positive effects of beta-AR on human carcinoma cell migration have been described. alpha(2)-AR are expressed in human breast cancer and non-cancer cell lines, their stimulation being associated with increased cell proliferation. In vivo clonidine increased tumor growth and alpha (2)-adrenergic antagonists completely reversed this effect. When administered alone, rauwolscine inhibited tumor growth behaving as an inverse agonist. Therefore, the numerous adrenergic beta- and alpha-AR agonists or antagonists could prove to be unexpected therapeutic options for mammary gland/ breast and mainly breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptores Adrenérgicos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Transdução de SinaisRESUMO
BACKGROUND AND PURPOSE: Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described alpha(2)-adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on alpha(2)-adrenoceptors in a reliable model of breast cancer. EXPERIMENTAL APPROACH: The expression of alpha(2)-adrenoceptors was confirmed by immunocytochemistry, immunofluorescence and reverse transcription-PCR in the mouse mammary tumour cell line MC4-L5. Proliferation was assessed by [(3)H]thymidine incorporation and tumours were measured daily. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling. KEY RESULTS: Incubation for 2 days with alpha(2)-adrenoceptor agonists (clonidine and dexmedetomidine) significantly enhanced proliferation of the mouse mammary tumour cell line MC4-L5. These agonists also significantly stimulated tumour growth of the progestin-dependent tumour C4-HD even in the presence of medroxyprogesterone acetate (MPA). In every tumour tested (C4-HD, CC4-2-HD and CC4-3-HI), regardless of MPA sensitivity, clonidine significantly enhanced tumour growth in the absence of MPA. The alpha(2)-adrenoceptor antagonists, yohimbine and rauwolscine, completely reversed the effects of clonidine. However, the group receiving yohimbine alone showed a nonsignificant but constant increase in tumour growth, whereas rauwolscine alone diminished tumour growth significantly, behaving as a reverse agonist. In CC4-3-HI tumours, rauwolscine treatment enhanced apoptosis and diminished the mitotic index, whereas clonidine had the inverse effect. CONCLUSIONS AND IMPLICATIONS: Alpha(2)-adrenoceptor agonists enhanced tumour growth and rauwolscine behaved in vivo as a reverse agonist, suggesting that it may be tested for adjuvant treatment.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Linhagem Celular Tumoral , Clonidina/farmacologia , Dexmedetomidina/farmacologia , Agonismo Inverso de Drogas , Feminino , Neoplasias Mamárias Experimentais/fisiopatologia , Acetato de Medroxiprogesterona/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores Adrenérgicos alfa 2/metabolismo , Ioimbina/farmacologiaRESUMO
The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.
Assuntos
Hormônios/fisiologia , Melanoma/metabolismo , Melanoma/secundário , Receptores Androgênicos/metabolismo , Adulto , Ligação Competitiva , Biópsia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Interpretação Estatística de Dados , Di-Hidrotestosterona/farmacologia , Feminino , Hormônios/farmacologia , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patologia , Melanoma Amelanótico/secundário , Receptores de Esteroides/metabolismo , Células Tumorais CultivadasRESUMO
(-)Epinephrine (Epi) and (-)Norepinephrine (NEpi) significantly stimulated tritiated Thymidine incorporation in MCF-7 cells at concentrations 10-30pM to 10nM, with an EC50 of 10pM for Epi and 14.2pM for NEpi. To characterize this action, cells were incubated in the presence of NEpi or Epi and different antagonists. The beta-adrenergic antagonist Propanolol showed no effect on the agonist's stimulation, whereas the alpha-adrenergic antagonist Phentolamine, reverted it completely at high concentrations (100 microM). The alpha1-adrenergic antagonist Prazosin (Pra) acted only at high concentrations, while the alpha2-adrenergic antagonist Yohimbine (Yo) reverted the stimulation at an EC50 of 0.11 microM. Likewise, when the cells were incubated in the presence of the specific alpha2-adrenergic agonist Clonidine (Clo), Thymidine incorporation was significantly stimulated at an EC50 of 0.298 pM. Again, the incubation of the cells in the presence of the alpha1-adrenergic antagonist Pra exerted its action at high concentrations, whereas the alpha2-adrenergic antagonist Yo showed a clear reversal of the agonist's enhancement at an EC50 of 0.136 microM. Moreover, Clo caused a clear and significant inhibition of stimulated cAMP levels both in the intracellular and the extracellular fractions. Yo showed a complete reversion of cAMP levels to control values in the presence of Clo, while Pra had the opposite effect. These data suggest that the stimulation provoked in Thymidine incorporation by the agonists Epi, NEpi, and Clo is, at least in part, due to an alpha2-adrenergic mechanism directly on tumoral cells, and that the effect is coupled with inhibition of cAMP levels, as described for this kind of receptors.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Neoplasias da Mama/patologia , Antagonistas Adrenérgicos beta/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Clonidina/farmacologia , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Epinefrina/farmacologia , Epinefrina/fisiologia , Feminino , Humanos , Norepinefrina/farmacologia , Norepinefrina/fisiologia , Fentolamina/farmacologia , Prazosina/farmacologia , Propranolol/farmacologia , Células Tumorais Cultivadas , Ioimbina/farmacologiaRESUMO
We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Receptores de Progesterona/metabolismo , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/tratamento farmacológico , Androgênios/metabolismo , Animais , Sítios de Ligação , Estradiol/farmacologia , Feminino , Flutamida/farmacologia , Glucocorticoides/metabolismo , Gonanos/farmacologia , Antagonistas de Hormônios/farmacologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/tratamento farmacológico , Acetato de Medroxiprogesterona/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Mifepristona/farmacologia , Neoplasias Hormônio-Dependentes/induzido quimicamente , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ovariectomia , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/genética , Timidina/metabolismo , Células Tumorais CultivadasRESUMO
Scatchard analysis of prolactin binding sites (PRL-BS) from ejaculated spermatozoa showed a single population of binding sites (apparent association constant: 2.51+/-0.186 nmol/l[-1]) with 0.317+/-0.0743 fmol/10(6) sperm binding sites. Different pools of spermatozoa were incubated with increasing concentrations of several hormones. There was a decrease in [125I]-oPRL binding with purified ovine prolactin (oPRL) and human growth hormone (hGH) which was not observed in the presence of synthetic ACTH and recombinant FSH, suggesting that binding was hormone specific. When the patient's samples were analyzed using the single point assay at saturation concentration, asthenospermic patients showed a significantly higher concentration of binding sites compared to normospermic ones. Both groups of patients displayed similar PRL levels in seminal plasma measured by DELFIA. Moreover, individual values of PRL levels in seminal plasma did not correlate with PRL-BS concentrations. We thus conclude that [125I]-oPRL binding to ejaculated spermatozoa was hormone specific and with similar parameters as seen in other target tissues. PRL-BS concentration in asthenospermic patients was significantly higher than in normospermic but this was not due to different levels of PRL in seminal plasma.
Assuntos
Infertilidade Masculina/metabolismo , Prolactina/metabolismo , Espermatozoides/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Sítios de Ligação , Ligação Competitiva , Estudos de Coortes , Hormônio Foliculoestimulante/farmacologia , Hormônio do Crescimento Humano/farmacologia , Humanos , Infertilidade Masculina/patologia , Radioisótopos do Iodo , Masculino , Prolactina/análise , Sêmen/química , OvinosRESUMO
Repeated isolation stress and prazosin effect were evaluated in 7,12-dimetylbenz[A]anthracene (DMBA) mammary tumors. Tumor volume was significantly lower in stressed than in control animals from 10 to 52 days considering day 1 the moment when tumors became palpable and treatment began. Control Prazosin (0.5 mg/kg) rats showed diminished tumor volume after 40 days. Stress Prazosin curve was similar to stress alone. The proportion of progressing tumors in control was significantly higher than in stressed groups, regardless of Prazosin administration. Body weight gain was similar in every group throughout the experiment. Behavioral studies were performed when stress effect was no longer evident. Grooming and the number of fecal boli were similar in all groups, as well as prolactin serum levels, suggesting that habituation took place. No significant differences were observed between groups for estrogen receptors. However, a greater concentration of progesterone receptors was found in Stressed rats, compared to all other groups. We conclude that the decrease of tumor volume provoked by stress could not be reversed by the alpha 1-adrenergic antagonist prazosin. Then, it appears that the main effect of stress is not mediated by the alpha 1-adrenergic receptors. Higher progesterone receptors in stressed rats could explain the differences observed.
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Prazosina/farmacologia , Estresse Fisiológico/fisiopatologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Feminino , Prolactina/análise , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/fisiologia , Receptores de Progesterona/fisiologiaRESUMO
The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.
Assuntos
Adenocarcinoma/patologia , Neoplasias Mamárias Experimentais/patologia , Acetato de Medroxiprogesterona/farmacologia , Adenocarcinoma/sangue , Adenocarcinoma/induzido quimicamente , Antagonistas de Androgênios/farmacologia , Animais , Fenômenos Fisiológicos Sanguíneos , Divisão Celular/efeitos dos fármacos , Dexametasona/farmacologia , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Feminino , Flutamida/análogos & derivados , Flutamida/farmacologia , Neoplasias Mamárias Experimentais/sangue , Camundongos , Camundongos Endogâmicos BALB C , Mifepristona/farmacologia , Ovariectomia , Progesterona/farmacologia , Células Tumorais CultivadasRESUMO
To evaluate the presence of androgen receptors in the human melanoma cell line IIB-MEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (KD) at 37 degrees C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [3H]-R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [3H]-R1881 binding, but also estradiol, progesterone, and cortisol at 500-fold excess concentration. Binding of [3H]-estradiol and [3H]-R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained. Different doses of flutamide (FLU) and OH-FLU tested on cultured IIB-MEL-J cells in the presence of serum inhibited significantly cell proliferation in a dose-dependent manner. When cells were incubated with 10 nM DHT and 1% charcoal-adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 microM OH-FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.
Assuntos
Melanoma/metabolismo , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/farmacologia , Animais , Anticorpos Monoclonais , Antineoplásicos Hormonais/farmacologia , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Di-Hidrotestosterona/metabolismo , Estradiol/metabolismo , Flutamida/análogos & derivados , Flutamida/metabolismo , Flutamida/farmacologia , Humanos , Hidrocortisona/metabolismo , Imuno-Histoquímica , Masculino , Metribolona/metabolismo , Camundongos , Camundongos Nus , Progesterona/metabolismo , Testosterona/metabolismo , Células Tumorais CultivadasRESUMO
In prepubertal male rats, the injection of bromocriptine (Br) for 10 days caused an increase in adrenal weight (Br 0.75 mg/kg BW (Br I): 2.83%; Br 1.5 mg/kg BW (Br II): 12.1% and Br 3 mg/kg BW (Br III): 24.7%), and this effect was only significant at the highest dose. Sulpiride (S, 30 mg/kg BW/day) for 10 days produced a significant decrease in adrenal weight (18.6%), whereas ovine prolactin (oPRL) administered at doses of 0.5 or 5 mg/kg BW/day for 10 had no effect on this parameter. The action of these drugs on corticosterone serum levels was for Br III a 50.6% increase and for S a 29.2% decrease. Bromocriptine caused a significant increment of cytosolic available estrogen receptors C: 7.65 +/- 0.36 (SE); Br I: 10.2 +/- 0.36; Br II: 11.0 +/- 0.23 and Br III: 13.3 +/- 0.35) and total lactogenic receptors in the adrenal gland (C: 125.2 +/- 2.84; Br I: 203.8 +/- 4.43; Br II: 213.1 +/- 7.58 and Br III: 251.3 +/- 10.4), and this effect was dose-related. oPRL diminished adrenal estrogen receptors only at the highest dose used (C: 11.1 +/- 1.73; PRL 5: 8.2 +/- 0.75) as did S (C: 11.2 +/- 1.84 and S: 5.2 +/- 1.07); while the former originated a marked decrease in lactogenic adrenal binding sites at both doses (C: 198.7 +/- 12.2; PRL 0.5: 52.9 +/- 5.00 and PRL 5: 38.8 +/- 4.76), S also had a highly significant diminution over these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)