RESUMO
This article evaluates and compares the performance and perception of prosthetic devices based on different design principles, a traditional rigid gripper and an adaptable poly-articulated hand, in a pre- and post-training protocol with an individual with bilateral amputation. As a representative of the first class, we use commercial hands (Ottobock's MyoHand VariPlus Speed), which is a widely adopted model by prosthesis users worldwide. We compare these with two SoftHand Pro hands, which are experimental prototypes exhibiting 19 articulations actuated by one single motor, and are inspired by human hand motor control models. Results show that the individual with bilateral amputation, who was a non-expert myoelectric user, achieved better performance with adaptive poly-articulated hands. Furthermore, the acceptation, satisfaction and perceived functionality of the user were considerably higher for the SoftHand Pro. An observational analysis of the patient's behaviour by experienced therapists suggests that adaptable poly-articulated hands reduced compensatory movements and cognitive load. Using soft technologies may be especially advantageous for individuals with bilateral amputation, who present a very limited residual mobility and can largely benefit from the active use of their artificial arms in everyday life.
Assuntos
Membros Artificiais , Biônica , Amputação Cirúrgica , Mãos , Humanos , Percepção , Desenho de PróteseRESUMO
Progress in basic research led to the design of new generations of anticancer drugs with some notable achievements. Over the years, more and more powerful drugs have been developed with the purpose of increasing the rate of response to therapy. As molecular power of chemotherapeutic agents increased, unfortunately also toxicity and undesired side-effects increased. The search for new therapeutic strategies to be used in the management of cancer is one of the more promising strategies to reduce chemotherapy toxicity. Extracorporeal Shock Waves (ESW), widely used for the treatment of urolithiasis, have been reported to cause modifications of cell growth both in vitro and in vivo. They exert an agonist cytotoxic effect with several chemotherapeutic agents, such as cisplatin, doxorubicin, bleomycin, paclitaxel. Moreover, as it has been reported that their main mechanism of action is an increase in cell membrane permeability, ESW are also used to deliver oligonucleotides and other small particles to cells. Recently, it was found that certain dye compounds, in particular porphyrins, can achieve a cytopathogenic effect when the disease site is subjected to ultrasound irradiation. This technique is referred to as sonodynamic therapy. Based on the new knowledge regarding the interaction between ultrasound with bulk liquid, several studies have shown a synergic effect of ESW and porphyrins in vitro, thus opening a new perspective in sonodynamic therapy, able to overcome some drawbacks encountered during conventional anticancer drug treatment. Finally, current advances in bioengineering encouraged the application of nano-scale technologies to medicine. Nanobubbles, composed of an external shell and a gas core, can deliver chemotropic drugs and porfirins, to target tumour tissues in response to physical triggers, and ESW features make them an ideal alternative to ultrasound in combination with drug-loaded nanobubbles in delivery strategies.
Assuntos
Ondas de Choque de Alta Energia/uso terapêutico , Neoplasias/terapia , Terapia por Ultrassom , Ácido Aminolevulínico/farmacocinética , Ácido Aminolevulínico/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Terapia Combinada , Sistemas de Liberação de Medicamentos , Terapia Genética/métodos , Humanos , Microbolhas , Nanocápsulas , Neoplasias/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/terapia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacocinética , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/farmacocinética , Porfirinas/uso terapêutico , Transfecção/métodos , Terapia por Ultrassom/métodosRESUMO
OBJECTIVE: Aim of the study was to evaluate tumour necrosis factor α (TNF-α) axis and oxidative status in patients with anorexia nervosa (AN) seeking a possible correlation with both nutritional status and evolution of the disease. SUBJECTS AND METHODS: Thirty-nine consecutive women with AN and an age-matched healthy control group were studied. Patients were 26±9 yr, with a body mass index (BMI) of 13.9±2 kg/m(2). TNF-α, its receptors TNF-R55 and TNF-R75, and oxidative status markers (selenium, ascorbic/ dehydroascorbic acid, retinol, α-tocopherol, selenium-dependent gluthatione peroxidase, reduced/oxidated gluthatione) were measured. A correlation with both nutritional indexes (body weight, BMI, albumin, prealbumin, transferrin, lymphocyte count) and disease duration was investigated. Pearson's correlation and unpaired Student's t-test were used to compare patients and controls. RESULTS: TNF-α and oxidative status markers were significantly higher in patients than controls and TNF-α was directly related to dehydroascorbic acid (p<0.05). Both TNF-R55 and TNF-R75 were higher in patients with duration of disease longer than one year as compared to controls and patients with shorter duration. Receptors inversely correlated with BMI (p<0.05 and p<0.01) and directly with disease duration (p<0.05). Inverse correlation between disease duration and BMI was present (p<0.01). CONCLUSIONS: The study showed activation of TNF-α axis and oxidative stress in AN patients, as well as correlation between the two systems. Due to the correlation between TNF receptors and both BMI and disease duration, a possible role of pro-inflammatory cytokines in the evolution of the eating disorder is suggested.
Assuntos
Anorexia Nervosa/metabolismo , Estresse Oxidativo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Índice de Massa Corporal , Peso Corporal , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Estado NutricionalRESUMO
New drugs with anti-tumor activity, also able to modify the expression of selected molecules, are under evaluation in breast cancer which is becoming resistant to conventional treatment, or in metastatic disease. The sodium-iodide symporter (NIS), which mediates iodide uptake into thyroid cells, and is the molecular basis of radioiodine imaging and therapy in thyroid cancer, is also expressed in a large portion of breast tumors. Since NIS expression in breast cancer is not sufficient for a significant iodide uptake, drugs able to induce its expression and correct function are under evaluation. In the present study, we report for the first time that the pan-deacetylase (DAC) inhibitor LBH589 (panobinostat) significantly induced NIS, both as mRNA and as protein, through the increase of NIS promoter activity, with the final consequence of obtaining a significant up-take of iodide in MCF7, T47D, and MDA-MB231 breast cancer cells. Moreover, we observed that LBH589 causes a significant reduction in cell viability of estrogen-sensitive and -insensitive breast cancer cells within nanomolar range. The anti-tumor effect of LBH589 is sustained by apoptosis induction and cell cycle arrest in G(2)/M. In conclusion, our data suggest that LBH589 might be a powerful tool in the management of breast cancer due to its multiple effects and support a potential application of LBH589 in the diagnosis and treatment of this disease.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Simportadores/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis , Concentração Inibidora 50 , Radioisótopos do Iodo/metabolismo , Panobinostat , RNA Mensageiro/metabolismo , Simportadores/genética , Transfecção , Regulação para CimaRESUMO
The human serum Sex Hormone-Binding Globulin (SHBG) plays an important role in breast cancer pathophysiology and risk definition, since it regulates the bioavailable fraction of circulating estradiol. We here summarize data reported over the years concerning the involvement of SHBG and SHBG polymorphisms in the definition of breast cancer risk. We also report what is known about the direct action of SHBG in breast cancer cells, illustrating its interaction with these cells and the subsequent initiation of a specific intracellular pathway leading to cross-talk with the estradiol-activated pathway and, finally, to the inhibition of several effects of estradiol in breast cancer cells. In conclusion, as a result of its unique property of regulating the estrogen free fraction and cross-talking with the estradiol pathways, by inhibiting estradiol-induced breast cancer cell growth and proliferation, SHBG is associated with a reduced risk of developing the neoplasm after estrogen exposure.
Assuntos
Neoplasias da Mama/fisiopatologia , Estradiol/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Polimorfismo Genético , Fatores de Risco , Globulina de Ligação a Hormônio Sexual/genética , Transdução de Sinais/fisiologiaRESUMO
Histone deacetylase inhibitors (HDIs) are valuable drugs in breast cancer where estrogen receptor alpha (ER alpha) can be silenced by epigenetic modifications. We report the effect of the clinically available HDI, valproic acid (VPA), on ER alpha expression and function in ER-negative breast cancer cells, MDA-MB-231. VPA induced ER alpha mRNA and protein, while did not modify ER beta. In VPA-treated cells, we also observed: (1) a correct transcriptional response to estradiol after transfection with the luciferase gene under the control of an estrogen-responsive minimal promoter (ERE-TKluc); (2) increased expression of the ER-related transcription factor FoxA1; (3) estradiol-induced up-regulation of several estrogen-regulated genes (e.g. pS2, progesterone receptor); (4) inhibitory effect of tamoxifen on cell growth. In conclusion, the HDI VPA, inducing ER alpha and FoxA1, confers to MDA-MB 231 cells an estrogen-sensitive "phenotype", restoring their sensitivity to antiestrogen therapy.
Assuntos
Neoplasias da Mama , Linhagem Celular Tumoral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Moduladores de Receptor Estrogênico , Receptor alfa de Estrogênio/metabolismo , Ácido Valproico/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/metabolismo , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Tamoxifeno/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.
Assuntos
Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Globulina de Ligação a Hormônio Sexual/farmacologia , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/biossíntese , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Prolactina/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Multimodal treatments do not meaningfully improve survival of anaplastic thyroid cancer. Consequently, new effective therapeutic modalities are needed. The use of paclitaxel is under clinical investigation; it shows about a 50% response rate, but it is not able to alter the fatal outcome for patients with anaplastic carcinoma. High energy shock waves (HESW) have been shown to cause a transient increase in the permeability of cell membranes thus allowing higher intracellular drug concentrations. 5-Aminolevulinic acid (ALA) is used in the photodynamic therapy (PDT) of cancer, and HESW are under evaluation for their use as an activator in ALA-PDT. DESIGN: We investigated the effect of HESW produced by a piezoelectric generator on the sensitivity to paclitaxel and ALA treatments of two different anaplastic thyroid cancer cell lines (ARO and CAL-62). Cells, treated sequentially with ALA and paclitaxel were exposed to HESW; thereafter, cell viability and apoptosis induction were evaluated. MAIN OUTCOME: Combined exposure to ALA, paclitaxel, and shock waves resulted in a significant enhancement of cytotoxicity and induction of apoptosis in thyroid cancer cells with respect to cells treated with paclitaxel alone. CONCLUSIONS: These preliminary data suggest the possibility of using HESW and ALA in combination with paclitaxel as a promising new therapy in the treatment of anaplastic thyroid cancer.
Assuntos
Ácido Aminolevulínico/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Carcinoma/terapia , Permeabilidade da Membrana Celular , Ondas de Choque de Alta Energia , Paclitaxel/farmacologia , Fotoquimioterapia , Neoplasias da Glândula Tireoide/terapia , Ácido Aminolevulínico/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Humanos , Paclitaxel/farmacocinética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that regulates the action of steroid hormones at several levels. SHBG regulates the availability of free androgens and estradiol to hormone-responsive tissues. Moreover, SHBG is also part of a novel steroid signaling system. We report here on the mechanism of action and the biological effects of SHBG in breast cancer cells, especially distinguishing cross-talk between membrane-initiated SHBG and estradiol pathways. After interacting with a specific binding site on breast cancer cell membranes, SHBG activates a specific pathway, and by cAMP induction, inhibits estradiol-mediated activation of ERK. Both estradiol and SHBG membrane-initiated pathways involve cross-talk at MAP kinase level with the ultimate result of inhibiting estradiol-mediated cell growth and antiapoptosis. On the basis of reported evidence, we suggest that SHBG is one of the regulators of growth and apoptosis of estrogen-dependent breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Receptor Cross-Talk/fisiologia , Globulina de Ligação a Hormônio Sexual/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transdução de SinaisRESUMO
Sex hormone-binding globulin, the plasma carrier for sex steroids, inhibits the estradiol-induced proliferation of breast cancer cells. Estradiol induces cell proliferation triggering multiple mechanisms. Besides regulating growth factors, it activates Erk-1/-2, thus inhibiting apoptosis. In the present study, we investigated the effect of SHBG on estradiol-mediated anti-apoptotic effect in MCF-7 breast cancer cells. As expected, estradiol reduced the number of cells undergoing apoptosis. Although no modification of estradiol action was observed in cells treated contemporarily with estradiol and SHBG, pre-incubation with SHBG before estradiol treatment contrasted the anti-apoptotic effect completely. A mutant form of SHBG, lacking the O-linked oligosaccharide in Thr(7), displayed no such effect. Moreover, SHBG prevented the estradiol-induced phosphorylation of Erk-1/-2, whereas it had no effect on estradiol-induced transcription. Taken together, our observations suggest that the interaction of SHBG with MCF-7 cell membranes causes inhibition of the anti-apoptotic effect of estradiol which might account for SHBG's inhibitory effect on breast cancer cell growth.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Estradiol/fisiologia , Antagonistas de Estrogênios/farmacologia , Globulina de Ligação a Hormônio Sexual/farmacologia , Linhagem Celular Tumoral , Estradiol/farmacologia , Feminino , Glicosilação , Humanos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação/genética , Fosforilação/efeitos dos fármacos , Globulina de Ligação a Hormônio Sexual/genética , Globulina de Ligação a Hormônio Sexual/metabolismo , Transcrição GênicaRESUMO
Solid lipid nanoparticles (SLN) carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had previously been developed, and the antiproliferative effect of SLN formulations versus conventional drug formulations was here evaluated on HT-29 cells. The 50% inhibitory concentration (IC(50) values were interpolated from growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN carrying chol-but (IC(50 72 h) 0.3 +/- 0.03 mM vs >0.6 mM) and doxorubicin (IC(50 72 h) 81.87 +/- 4.11 vs 126.57 +/- 0.72 nM) was higher than that of conventional drug formulations. Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the conventional drug formulation, at the highest concentration evaluated by flow cytometry. In vitro cytotoxicities of paclitaxel-loaded SLN and conventional drug formulation (IC(50 72 h) 37.36 +/- 6.41 vs 33.43 +/-1.17 nM) were similar. Moreover, the combination of low concentrations of chol-but SLN (0.1-0.2 mM) and doxorubicin (1.72 nM) or paclitaxel (1.17 nM) exerted a greater-than-additive antiproliferative effect at 24 h exposure, while the combination of Na-but and doxorubicin or paclitaxel did not. These preliminary in vitro results suggest that SLN could be proposed as alternative drug delivery system.
Assuntos
Antineoplásicos/toxicidade , Nanoestruturas/toxicidade , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Ácido Butírico/administração & dosagem , Ácido Butírico/farmacocinética , Ácido Butírico/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ésteres do Colesterol/administração & dosagem , Ésteres do Colesterol/farmacocinética , Ésteres do Colesterol/toxicidade , Neoplasias Colorretais/tratamento farmacológico , Relação Dose-Resposta a Droga , Células HT29 , HumanosRESUMO
Human sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein, and each SHBG monomer may have an O-linked oligosaccharide at Thr(7) and up to two N-linked oligosaccharides at Asn(351) and Asn(367). In addition, a common genetic variant of SHBG exists with an extra site for N-glycosylation at residue 327. In the present study, we isolated MCF-7 derived cell lines expressing human SHBG cDNAs encoding the wild type protein or various glycosylation mutants. Estradiol (1 nM) treatment of parental (untransfected) MCF-7 cells or MCF-7 cells transfected with control expression vectors resulted in an increase in proliferation which was fully abrogated by co-incubation with an equimolar amount of human SHBG. In contrast, the same amount of purified SHBG added to MCF-7 cells expressing wild type SHBG partially inhibited the estradiol-induced cell proliferation. A high affinity binding site for SHBG was detectable on untransfected and control cells, but not on MCF-7 cells expressing wild type SHBG. Moreover, the treatment of MCF-7 cells with the conditioned medium containing wild type SHBG caused the disappearance of the SHBG plasma membrane-binding site. Media containing SHBG N-glycosylation mutants exerted the same effect, but mutants lacking the O-linked oligosaccharide at Thr(7) failed to do so. Estradiol-induced proliferation of parental MCF-7 cells was also inhibited by treatment with conditioned medium containing wild type SHBG or SHBG mutants lacking N-linked oligosaccharides, or containing an additional N-linked oligosaccharide at residue 327. However, MCF-7 conditioned medium containing SHBG mutants lacking an O-linked oligosaccharide at Thr(7) failed to exert this effect. These data suggest that O-glycosylation of SHBG is essential for SHBG binding to a membrane receptor that is responsible for inhibiting the estradiol-induced proliferation of MCF-7 breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Divisão Celular/fisiologia , Estradiol/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Animais , Meios de Cultivo Condicionados , Feminino , Glicosilação , Humanos , Camundongos , Mutação , Ligação Proteica , Globulina de Ligação a Hormônio Sexual/química , Globulina de Ligação a Hormônio Sexual/genética , Globulina de Ligação a Hormônio Sexual/farmacologia , Células Tumorais CultivadasRESUMO
Estradiol controls the gene transcription and expression of many proteins in breast cancer cells, like the progesterone receptor, PR, that is up-regulated by the hormone. Moreover, estradiol is one of the crucial factors inducing the proliferation of breast cancer cells. Sex Hormone-Binding Globulin (SHBG), the plasma carrier for both estradiol and androgens, inhibits the estradiol-induced growth of MCF-7 cells (estrogen-dependent breast cancer cells), through its membrane receptor (SHBG-R), cAMP and PKA. The anti-estrogenic effect of SHBG, which has been described only as far as cell proliferation is concerned, could also play a meaningful role in the estradiol control of other factors in breast cancer cells. In the present study, the effect of SHBG on the estradiol control of PR expression (both mRNA and protein) and function (receptor binding capacity) in MCF-7 cells was examined. SHBG inhibited the estradiol-induced up-regulation of PR mRNA as well as protein level and function. Moreover, the effect of SHBG on estradiol control of PR expression and function was showed to be specific and mediated by PKA. The intermediacy of PKA in the PR expression control, together with the observation that it is effective in the condition in which the SHBG receptor is activated, supports the hypothesis that the anti-estrogenic effect of SHBG could be receptor-mediated. The ability of SHBG to inhibit estradiol action in a specific way in estrogen-dependent breast cancer cells has, therefore, to be taken into account for the development of future therapeutic strategies.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Progesterona/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/patologia , Estradiol/farmacologia , Feminino , Humanos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Globulina de Ligação a Hormônio Sexual/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The human androgen receptor gene contains a polymorphic CAG repeat region ranging from 8 to about 35 repeats in the normal human population. The repeat length is inversely related to the transactivation potential of the receptor. We have analyzed the repeat length in 50 sporadic colon cancer samples in comparison to surrounding healthy mucosa and have found somatic reductions of up to 10 repeats in 5 cases (10%), 3 of which were complex, probably involving both alleles. Alterations occurred in tumors with and without microsatellite instability indicating that they follow an independent mutation pathway. The similar repeat of the huntingtin gene did not show any somatic alterations in the same cases. No correlation to sex, tumor stage, location, or histology was evident. In the tumors that showed somatic reductions, the reduced allele was present in at least half of the cells and thus in most, if not all, of the tumor component of the sample. Somatic reductions of the androgen receptor CAG repeat thus occur frequently, through a pathway distinct from microsatellite instability and early during colon carcinogenesis. The receptor is expressed in most normal and neoplastic tissue samples analyzed. Apparent growth selection of cells bearing shortened AR alleles suggests that androgens contribute to colon carcinogenesis in a yet unknown way.
Assuntos
Neoplasias do Colo/genética , Repetições de Microssatélites/genética , Receptores Androgênicos/genética , Repetições de Trinucleotídeos/genética , Alelos , Neoplasias do Colo/patologia , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Frequência do Gene , Humanos , Masculino , MutaçãoRESUMO
Many groups have examined of androgen the effects on normal and neoplastic colon tissues, but no clear picture has hitherto emerged. In particular, the presence and the function of the androgen receptor (AR) has only partially been investigated in the past. The present study reports analysis of expression of the AR gene as messenger RNA and as protein in surgical samples of neoplastic colon mucosa and of corresponding healthy surrounding tissue. Specific binding for DHT, demonstrating the presence of AR, was observed in almost all the samples (2 samples out of 12 were negative). No significant difference was observed between healthy and neoplastic mucosa, or between male and female patients. A further characterization of AR was performed with Western blot, using 2 different primary antibodies. Both AR isoforms, AR-B and AR-A, were detected in healthy mucosa, while only AR-A, resolving at 87 kDa, was observed in neoplastic mucosa. RT-PCR analysis revealed the transcript for AR in both healthy and neoplastic mucosa in 10 samples; no message was detectable in 2 samples (negative also for binding); 2 additional samples presented AR mRNA only in healthy colon mucosa, 2 others only in neoplastic mucosa. In addition, a variant AR messenger RNA, probabily derived from alternative splicing, was observed. We found that AR is expressed both in healthy and in neoplastic colon mucosa, either as mRNA or as protein. Neoplastic colon tissue shows a characteristic loss of expression of the AR-B isoform, while AR-A expression is maintained. These findings underscore the possible role of androgen and its receptor in colon carcinogenesis.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Receptores Androgênicos/genética , Adulto , Idoso , Androgênios/metabolismo , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Androgênicos/biossínteseRESUMO
Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.
Assuntos
Neoplasias da Mama/metabolismo , AMP Cíclico/metabolismo , Estradiol/farmacologia , Sangue Fetal/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Animais , Bovinos , Meios de Cultura , Feminino , Humanos , Células Tumorais CultivadasRESUMO
The role of human Sex Hormone-Binding Globulin (SHBG), the plasma carrier of sex steroids, and its membrane receptor, SHBG-R, in estrogen-dependent breast cancer has been investigated in our laboratory in the past few years. SHBG-R is expressed in MCF-10 A cells (not neoplastic mammary cells), MCF-7 cells (breast cancer, ER positive) and in tissue samples from patients affected with ER positive breast cancer, but not in estrogen-insensitive MDA-MB 231 cells. The SHBG/SHBG-R interaction, followed by the binding of estradiol to the complex protein/receptor, causes a significant increase of the intracellular levels of cAMP, but does not modify the amount of estradiol entering MCF-7 cells. The estradiol-induced proliferation of MCF-7 cells is inhibited by SHBG, through SHBG-R, cAMP and PKA. Similarly, the proliferation rate of tissue samples positive for SHBG-R was significantly lower than the proliferation rate of negative samples. SHBG and SHBG-R could thus trigger a 'biologic' anti-estrogenic pathway. In order to get a more detailed knowledge of this system, we first examined the frequence of the reported mutated form of SHBG in 255 breast cancer patients. The mutated SHBG is characterized by a point mutation (Asp 327 --> Asn) causing an additional N-glycosylation site, which does not affect the binding of steroids to SHBG. The frequence of the mutation was significantly higher (24.5%) in estrogen-dependent breast cancers than in healthy control subjects (11.6%). This observation confirms the close relationship between SHBG and estrogen-dependent breast cancer and suggests that the mutation could modify SHBG activity at cell site. Lastly, the possibility of using SHBG to modulate the estradiol action in breast cancer was further studied by transfecting MCF-7 cells with an expression vector carrying the SHBG cDNA (study in collaboration with G.L. Hammond). Transfected cells are able to produce significant amount of SHBG in their medium, but their SHBG-R is reduced to undetectable levels. The SHBG produced by transfected MCF-7 cells is, however, able to inhibit estradiol-induced proliferation of MCF-7 cells expressing a functional receptor. Thus, the local production of SHBG obtained with transfection could be a useful tool to control cell growth in estrogen-dependent breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Receptores de Superfície Celular/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Neoplasias da Mama/patologia , Divisão Celular , DNA Complementar , Glicosilação , Humanos , Globulina de Ligação a Hormônio Sexual/genética , TransfecçãoRESUMO
In the last years, an increasing amount of studies described a membrane receptor for the Sex Steroid Binding Protein (SBP) on several androgen-estrogen dependent tissues. One of the suggested biological roles of the interaction between SBP and its receptor seems to be a negative control of the E2 induced proliferation of human breast cancer cells through the cAMP pathway. In the present work, SBP membrane receptor was evaluated on human breast cancer specimens with a radio-binding assay. Each tissue sample was also evaluated for ER and PGR status. Cytosol Thymidine Kinase levels were measured in tissue samples in order to evaluate cell proliferation rate. SBP binding to membranes of ER +/PGR + samples was time and temperature dependent, specific and at high affinity. In addition, SBP recognized on breast cancer membranes two sites at different affinity, as previously described for other human tissues and cultured cells. Membrane SBP-R was detected in a significantly higher number of samples positive for both ER and PGR than in negative samples. SBP-R positive samples showed a significantly lower proliferation rate than SBP-R negative samples as demonstrated by TK activity. The present study contains evidences for the existence of a specific membrane receptor for SBP in breast cancer sample membranes and the presence of SBP-R seems to be strictly related to a lower proliferation rate of the sample.