RESUMEN
Vasoactive intestinal peptide (VIP) and its receptors (VPACs) are involved in proliferation, survival, and differentiation in human breast cancer cells. Its mechanism of action is traditionally thought to be through specific plasma membrane receptors. There is compelling evidence for a novel intracrine mode of genomic regulation by G-protein-coupled receptors (GPCRs) that implies both endocytosis and nuclear translocation of peripheral GPCR and/or the activation of nuclear-located GPCRs by endogenously-produced, non-secreted ligands. Regarding to VPAC receptors, which are GPCRs, there is only a report suggesting them as a dynamic system for signaling from plasma membrane and nuclear membrane complex. In this study, we show that VPAC(1) receptor is localized in cell nuclear fraction whereas VPAC(2) receptor presents an extranuclear localization and its protein expression is lower than that of VPAC(1) receptor in human breast tissue samples. Both receptors as well as VIP are overexpressed in breast cancer as compared to non-tumor tissue. Moreover, we report the markedly nuclear localization of VPAC(1) receptors in estrogen-dependent (T47D) and independent (MDA-MB-468) human breast cancer cell lines. VPAC(1) receptors are functional in plasma membrane and nucleus as shown by VIP stimulation of cAMP production in both cell lines. In addition, VIP increases its own intracellular and extracellular levels, and could be involved in the regulation of VPAC(1)-receptor traffic from the plasma membrane to the nucleus. These results support new concepts on function and regulation of nuclear GPCRs which could have an impact on development of new therapeutic drugs.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/metabolismo , Núcleo Celular/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Adulto , Anciano , Línea Celular Tumoral , Membrana Celular/metabolismo , AMP Cíclico/biosíntesis , Femenino , Humanos , Persona de Mediana Edad , Receptores Acoplados a Proteínas G/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) bind similarly to VPAC1 and VPAC2 receptors, whereas PACAP binds with higher affinity than VIP to PAC1 receptors. Here we demonstrate by different approaches the expression of the three subclasses of PACAP/VIP receptors in human normal and malignant breast tissue. At the mRNA level, reverse transcription-polymerase chain reaction experiments showed VPAC1 and VPAC2 receptors as well as various isoforms (null, hip/hop) of PAC1 receptors due to alternative splicing. At the protein level, Western blot experiments revealed the three subclasses of receptor although no conclusive differences could be established when comparing control, peritumoral and tumoral tissue samples. Immunohistochemistry showed the distribution of these receptors: they were located at epithelial cells in normal and cancer conditions but also in leukocytes at the stromal level in carcinomatous tissue. A weaker immunostaining of PAC1 receptors in normal tissue and a strong density of the three PACAP/VIP receptor subclasses in cancer tissue may be related to differential expression patterns during breast tumor progression but more samples need to be studied to validate this hypothesis. PAC1, VPAC1 and VPAC2 receptors were functional, as shown by their coupling to adenylate cyclase stimulation: VIP, PACAP-27 and PACAP-38 behaved similarly at this level, whereas both VPAC receptors acted alike as shown by means of specific peptide agonists and antagonists. The present results together with the known presence of PACAP and VIP in the mammary gland support a paracrine/autocrine involvement of both peptides at this level in physiological and pathological conditions, i.e. during malignant transformation.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glándulas Mamarias Humanas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Transformación Celular Neoplásica , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vasoactive intestinal peptide (VIP) functions as a mitogenic agent in the human prostate gland acting by autocrine/paracrine mechanisms. Here we extend knowledge on the VIP system (expression of VIP and VIP receptors, functionality of VIP receptors) at this level by analyzing the differences between human normal prostate and prostate carcinoma specimens. RT-PCR showed the expression of mRNA for VIP in normal and malignant tissues, whereas VIP levels, as measured by enzyme immuno-analysis, were about two times higher in adenocarcinoma samples. Real-time RT-PCR indicated a minor expression of VPAC2 receptors in the prostate gland, as well as the overexpression of VPAC1 and PAC1 receptors in malignant tissue specimens. Radio-labeled binding experiments with [125I]VIP showed an increased number of VIP binding sites (2.5 times for the high- and 1.7 times for the low-affinity sites) during malignant transformation, whereas the affinity values were unaffected. The receptors were functional in control and cancer tissues as shown by the ability of increasing VIP doses to stimulate adenylate cyclase activity. Interestingly, JV-1-53 (a GHRH-related peptide analog) (at 0.1 microM) behaved as a potent VIP antagonist since it inhibited by 60% the maximal VIP effect upon the enzyme activity. The results further explain the mechanisms of the autocrine/paracrine actions of VIP in human prostate and prostatic carcinoma through the observation of differences between healthy tissue and malignant transformation. Moreover, present data support the potential usefulness of VIP and/or synthetic peptide analogs for diagnostic or radiotherapeutic purposes as well as for long-term peptide therapy in this malignancy.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/análogos & derivados , Hormona Liberadora de Hormona del Crecimiento/farmacología , Neoplasias de la Próstata/química , Receptores de Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Péptido Intestinal Vasoactivo/análisis , Anciano , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/terapia , ARN Mensajero/análisis , Receptores de Superficie Celular/análisis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pituitary adenylate cyclase-activating peptide (PACAP) is involved in various biological processes including cell growth, proliferation and differentiation. Here, we demonstrate for the first time the presence of both PACAP mRNA and PACAP immunoreactivity in human normal breast and breast carcinoma. Control, peritumoral and tumoral tissue pieces expressed preproPACAP mRNA since reverse transcription-polymerase chain reaction analysis gave an amplification product of the expected size (226 bp), which was further identified by analysis of the sequence. A main 19.9-kDa product (preproPACAP protein) was identified by Western blot in the three classes of breast tissue together with a small amount of a 14.6-kDa product (conceivably a result of partial processing by proprotein convertases). However, the mature peptide PACAP-38 was absent. The levels of both PACAP mRNA and PACAP immunoreactivity increased from normal to peritumoral and tumoral breast tissues but more patients must be considered to reinforce this feature. Immunohistochemistry showed the localization of PACAP immunoreactivity in alveolar epithelial cells in normal and carcinomatous tissues but also, at high density, in duct-like structures and in leucocytes in the connective tissue from breast cancer pieces. The results suggest that PACAP may play a role by autocrine/paracrine mechanisms in both normal human breast physiology and breast tumorigenesis.
Asunto(s)
Neoplasias de la Mama/química , Mama/química , Neuropéptidos/análisis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Neuropéptidos/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.
Asunto(s)
Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Anciano , Empalme Alternativo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neuropéptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The presence, expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) in human prostate cancer and healthy tissue were investigated by means of biochemical and morphological procedures. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the presence of its precursor encoding mRNA in both normal and pathological conditions (amplification products with 577 or 226 bp were identified). Immunochemistry using an appropriate antibody served to detect in both classes of tissues a 19.9-kDa product corresponding to the PACAP preproprotein and another protein of 14.6 kDa that may represent a product partially processed by convertases. However, a 5-kDa band characteristic of PACAP-38 peptide was not observed. Immunohistochemistry on tissue sections indicated the location of PACAP in the epithelial layer of prostate glands (and in some scarce leucocytes) but not in the stroma, either in normal or carcinomatous tissues. No clear differences could be established when comparing samples from patients with different tumor Gleason grades. These results are the first demonstration of the localization of PACAP or its precursors and its mRNA in the human prostate gland and their presence during the progression of prostate carcinoma.
Asunto(s)
Adenocarcinoma/metabolismo , Neuropéptidos/biosíntesis , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Anciano , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neuropéptidos/genética , Neuropéptidos/inmunología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Próstata/citología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Precursores de Proteínas/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
BACKGROUND: Adult granulosa cell tumors (AGCT), unilocular or multilocular, with thin-walled cysts, are extremely rare. They can be erroneously diagnosed as follicular cysts by sonography and fine needle aspiration and therefore may be a source of false negative results. CASE: Laparoscopy was performed on a 29-year-old woman in whom a cystic ovarian mass, probably benign, was diagnosed by transvaginal sonography. The smears obtained from the cyst revealed a moderate amount of regular, small cells with scant cytoplasm and round to oval nuclei with finely granular chromatin without longitudinal grooves. These granulosa cells were arranged singly and in small groups. Very scarce cellular aggregates, microfollicular or rosettelike, some containing amorphous material and resembling Call-Exner bodies, were identified. Histologic study of the cystic wall confirmed the diagnosis of multilocular cystic AGCT with a microfollicular pattern. CONCLUSION: The finding of a moderate to abundant amount of regular granulosa cells, with or without nuclear longitudinal grooves, during cytologic examination of ovarian cysts considered benign by ultrasonography requires a careful search for microfollicular differentiation and Call-Exner bodies due to the rare but possible occurrence of cystic AGCT.