Somatostatin receptors in the gastrointestinal tract in health and disease.

The multiple actions of somatostatin are mediated by specific membrane-bound receptors present in all somatostatin target tissues, such as brain, pituitary, pancreas, and gastrointestinal tract. Three different types of tissues in the human gastrointestinal tract express somatostatin receptors: (1) the gastrointestinal mucosa, (2) the peripheral nervous system, and (3) the gut-associated lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogs. Somatostatin receptors are also expressed in pathological states, particularly in neuroendocrine tumors of the gastrointestinal tract. Ninety percent of the carcinoids and a majority of islet-cell carcinomas, including their metastases, usually have a high density of somatostatin receptors. Only 10 percent of the colorectal carcinomas and none of the exocrine pancreatic carcinomas, however, contain somatostatin receptors. The somatostatin receptors in tumors are identified with in vitro binding methods or with in vivo imaging techniques; the latter allow the precise localization of the tumors and their metastases in the patients. Since somatostatin receptors in gastroenteropancreatic tumors are functional, their identification can be used to assess the therapeutic efficacy of octreotide to inhibit excessive hormone release in the patients.     

Somatostatin receptors in malignant tissues

High affinity somatostatin receptors (SS-R) have been identified in membrane homogenates or tissue sections from several hundred human tumors. SS-R were found in most tumors originating from SS target tissues, i.e. GH- and TSH-producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors (including metastases) and brain tumors, including gliomas and neuroblastomas. SS-R were also expressed in several tumors originating from various other tissues, i.e. breast and small cell lung carcinomas, some colorectal cancers, and medullary tyroid carcinomas. In general, most of the SS-R+ tumors are well-differentiated and/or have neuroendocrine features. They often have low or absent epidermal growth factor receptor (EGF-R) expression. In some tumors (i.e. breast tumors) SS-R are not homogeneously distributed, making SS-R autoradiography a particularly useful tool for assessing SS-R status. SS-R are functional in pituitary and GEP tumors where they mediate hormone secretion inhibition. In these and in the other SS-R+ tumors, SS-R may also mediate antiproliferative effects of SS, as evidenced, in animals where growth of SS-R+ tumor xenografts is inhibited by SS analogs. For diagnosis, SS-R+ tumors and metastases can be localized in vivo by scanning techniques after ¹²³I-labelled SS analog injection.     

Perspectives of new potential therapeutic applications of somatostatin analogs

At the present time only two long-acting somatostatin (SS) analogs, octreotide and lanreotide, are commonly used in the routine therapy. Both analogs have a high affinity mainly to a somatostatin receptor subtype 2 (SSTR2). The established indications for SS analogs treatment include acromegaly, neuroendocrine tumors of the pancreas and gastrointestinal tract, and some gastro-enterologic diseases (pancreatitis, gastrointestinal bleedings, refractory diarrheas, pancreatic and intestinal fistulas). The recent investigations allow to predict the enlargement of therapeutic applications of SS analogs. It concerns pituitary tumors other than somatotropinoma, tumors of other endocrine glands like thyroid and adrenal gland, as well as some non-endocrine tumors. The progress depends on the introduction of new SS analogs with high affinity for SS receptor subtypes other than SSTR2, because some tumors present the high expression of SSTR1 (e.g. prostatic cancers) or SSTR5 (e.g. colonic cancers). Great hopes are connected with the coupling of SS analogs with the radioactive isotopes or non-radioactive cytotoxic agents to destruct the neoplastic cells highly expressing the specific subtypes of SS receptors. The pre- or postoperative in vivo imaging of SS receptors by means of the receptor scintigraphy, as well as the post-operative identification of SS receptor subtypes in the excised tumor tissues using immunohistochemistry, should play an important role in the prediction of the effects of SS analog treatment. Beside oncology, new therapeutic applications of SS analogs could be presumed among others in ophthalmology; it concerns the treatment of progressive Graves-Basedow ophtalmopathy, diabetic retinopathy, glaucoma and corneal diseases connected with corneal vascularization.     

Genetics of pituitary tumors: Focus on G-protein mutations

In recent years the demonstration that human pituitary adenomas are monoclonal in origin has provided further evidence that pituitary neoplasia arise from the replication of a single mutated cell in which growth advantage results from either activation of proto-oncogenes or inactivation of tumor suppressor genes. While common oncogenes, such as Ras, are only exceptionally involved, the only mutations identified in a significant proportion of pituitary tumors, and particular in GH-secreting adenomas, occur in the Gsalpha gene (GNAS1) and cause constitutive activation of the cAMP pathway (gsp oncogene). Moreover, pituitary tumors overexpress hypothalamic releasing hormones, growth factors, and their receptors as well as cyclins involved in cell cycle progression. As far as the role of tumor suppressor genes in pituitary tumorigenesis is concerned, reduced expression of these genes seems to frequently occur in pituitary tumors as a consequence of abnormal methylation processes. Although the only mutational change so far identified in pituitary tumors is the gsp oncogene, this oncogene is not associated with a clear phenotype in patients bearing positive tumors. Mechanisms able to counteract the cAMP pathway, such as high sensitivity to somatostatin, and induction of genes with opposite actions, such as phosphodiesterases, CREB end ICER, or instability of mutant Gsalpha, have been proposed to account for the lack of genotype/phenotype relationships.     

Somatostatin receptors in human cancer: incidence, characteristics, functional correlates and clinical implications.

Somatostatin receptors (SS-R) have been identified in membrane homogenates or tissue sections from several hundred tumors. SS-R were found in most neuroendocrine tumors, i.e. GH and TSH producing pituitary tumors, endocrine gastroenteropancreatic (GEP) tumors, paragangliomas, pheochromocytomas, medullary thyroid carcinomas (MTC) and small cell lung carcinomas. SS-R were also expressed in a majority of malignant lymphomas, in several brain tumors (all meningiomas, most astrocytomas) and in breast tumors. The majority of tumors expressing SS-R are rather differentiated (i.e. astrocytomas vs glioblastomas), but exceptions exist (high grade malignant lymphomas). An inverse relationship exists between SS-R and receptors for epidermal growth factor (EGF-R) incidence in lung tumors, glial tumors and most breast tumors, whereas meningiomas express simultaneously both receptors. A minority of tumors (ovarian tumors, MTC, insulinomas) express a subtype of SS-R, characterized by low affinity for the octapeptide SS analog octreotide. The function mediated by SS-R in human tumors may differ according to the tumor type. SS-R in pituitary and GEP tumor mediate hormone secretion inhibition with, in addition, possibly some antiproliferative effects. In meningiomas, however, activation of SS-R inhibits forskolin-stimulated adenylate cyclase activity, and weakly stimulates proliferation. Whereas SS-R seem to mediate antiproliferative effects in animal models and cell lines of lymphomas, breast and lung tumors, such an effect has not yet been convincingly documented in human primary tumors. The clinical implications of the presence of SS-R in tumors are manyfold: (1) as a predictive marker for efficient therapy with octreotide in pituitary and GEP tumors; (2) as a diagnostic marker: for pathobiochemical classification of tumors, using in vitro detection methods; for clinical evaluation using in vivo scanning techniques; (3) as a prognostic marker; and (4) as a potential radiotherapeutic target.     

In vivo application of [111In-DTPA-D-Phe1]-octreotide for detection of somatostatin receptor-positive tumors in rats.

Radioiodinated somatostatin analogues are useful ligands for the in vitro and in vivo detection of somatostatin receptors. [111In-DTPA-D-Phe1]-octreotide, a somatostatin analogue labeled with a different radionuclide, also binds specifically to somatostatin receptors in vitro. In this study we investigated its in vivo application in the visualization of somatostatin receptor-positive tumors in rats. The distribution of the radiopharmaceutical was investigated after intravenous injection in normal rats and in rats bearing the somatostatin receptor-positive rat pancreatic carcinoma CA 20948. After injection the radiopharmaceutical was rapidly cleared (50% decrease in maximal blood radioactivity in 4 min), predominantly by the kidneys. Excreted radioactivity was mainly in the form of the intact radiopharmaceutical. Ex vivo autoradiographic studies showed that specific accumulation of radioactivity occurred in somatostatin receptor-containing tissue (anterior pituitary gland). However, in contrast to the adrenals and pituitary, the tracer accumulation in the kidneys was not mediated by somatostatin receptors. Increasing radioactivity over the somatostatin receptor-positive tumors was measured rapidly after injection and the tumors were clearly visualized by gamma camera scintigraphy. In rats pretreated with 1 mg octreotide accumulation of [111In-DTPA-D-Phe1]-octreotide in the tumors was prevented. Because of its relatively long effective half-life, [111In-DTPA-D-Phe1]-octreotide is a radionuclide-coupled somatostatin analogue which can be used to visualize somatostatin receptor-bearing tumors efficiently after 24 hr, when interfering background radioactivity is minimized by renal clearance. This is an advantage over the previously used [123I-Tyr3]-octreotide which has a shorter effective half-life and shows high abdominal interference due to its hepato-biliary clearance. Therefore, [111In-DTPA-D-Phe1]-octreotide seems a better alternative for scintigraphic imaging of somatostatin receptor-bearing tumors.     

Imaging of pediatric brain tumors using somatostatin analogue 111Ih-DTPA-D-Phe1-octreotide

Malignant solid tumors and leukemias are the second most common causes of death in childhood. The most frequent pediatric solid tumors are brain tumors. Brain tumors, especially medulloblastoma should be treated by surgery, irradiation and chemotherapy. However, chemotherapy has only moderate effect. Pediatric brain tumors, especially medulloblastomas, express somatostatin receptors. The aim of this study was the investigation of the expression of somatostatin receptors in pediatric brain tumors for diagnostic and therapeutic purpose. Fifty-six scintigraphic imagings (111In-DTPA-D-Phe1-octreotide) made in 45 children treated with brain tumor at the Unit of Oncology of the 2nd Department of Pediatrics, Semmelweis University. The diagnosis was medulloblastoma in 21 cases (46.7%). MRI scans have been performed parallel with the Octreoscan images. Octreoscan images were positive in 27 of 56 (48.2%) cases. The 27 positive Octreoscan images consisted of 16 medulloblastomas, 4 ependymomas, 4 astrocytomas and 3 glioblastomas. In 37 (66.1%) cases the results of Octreoscans were the same as those of the MRI scans. However, in 19 scans (33.9%) the outcome was different. Octreoscan imaging is not suitable for differential diagnosis in pediatric brain tumors, including medulloblastomas. Isotopes specifically binding to the somatostatin receptors (111In-DTPA-D-Phe1-octreotide) can be applied in medulloblastomas for diagnosis and follow-up treatment. In Octreoscan-positive tumors the Octreoscan images establish the opportunity to somatostatin analogue and/or specifically targeted radiation therapies.     

111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptor-positive tumors: synthesis, radiolabeling and in vitro validation.

Somatostatin receptor-positive human tumors can be detected using radioiodinated analogues of somatostatin, both in vitro and in vivo. [123I-Tyr3]-octreotide has been successfully used in the visualization of somatostatin receptor-positive tumors by gamma camera scintigraphy, but this radiopharmaceutical has some major drawbacks, which can be overcome with other radionuclides such as 111In. As starting material for a potentially convenient radiopharmaceutical, a diethylenetriaminopentaacetic acid (DTPA) conjugated derivative of octreotide (SMS 201-995) was prepared. This peptide, [DTPA-D-Phe1]-octreotide (SDZ 215-811) binds more than 95% of added 111In in an easy, single-step labeling procedure without necessity of further purification. The specific somatostatin-like biologic effect of these analogues was proven by the inhibition of growth hormone secretion by cultured rat pituitary cells in a dose-dependent fashion by octreotide, [DTPA-D-Phe1]-octreotide and non-radioactive [115In-DTPA-D-Phe1]-octreotide. The binding of [111In-DTPA-D-Phe1]-octreotide to rat brain cortex membranes proved to be displaced similarly by natural somatostatin as well as by octreotide, suggesting specific binding of [111In-DTPA-D-Phe1]-octreotide to somatostatin receptors. The binding of the indium-labeled compound showed a somewhat lower affinity when compared with the iodinated [Tyr3]-octreotide, but indium-labeled [DTPA-D-Phe1]-octreotide still binds with nanomolar affinity. In conjunction with in vivo studies, these results suggest that [111In-DTPA-D-Phe1]-octreotide is a promising radiopharmaceutical for scintigraphic imaging of somatostatin receptor-positive tumors.     

The incidence of somatostatin receptors in human neoplasms in the light of ex vivo-in vitro studies

Human neoplastic cells express and often even over-express the somatostatin receptors (sstr). It concerns not only pituitary tumors and the so-called neuroendocrine tumors of the gut, but many other neoplasms, including the non-endocrine cancers. The incidence of sstr is important because it determines the possibility of treatment with SST (somatostatin) analogs. The examination of sstr is possible under the in vivo conditions, by means of the receptor scintigraphy using the radiolabeled SST analogs. The receptors can be also examined under the ex vivo-in vitro conditions, using the post-surgical or biopsy specimens. Among the in vitro (ex vivo) methods, the immunohistochemical investigation with specific anti-receptor antibodies seems to be particularly useful for routine clinical diagnostics. This review presents the data, obtained by means of different in vitro techniques, on the incidence of five sstr subtypes in the different human tumors, deriving from endocrine glands, diffuse neuroendocrine cells as well as in neoplasms considered as non-endocrine.     

Phenotypical and Pharmacological Characterization of Stem-Like Cells in Human Pituitary Adenomas

The presence and functional role of tumor stem cells in benign tumors, and in human pituitary adenomas in particular, is a debated issue that still lacks a definitive formal demonstration. Fifty-six surgical specimens of human pituitary adenomas were processed to establish tumor stem-like cultures by selection and expansion in stem cell-permissive medium or isolating CD133-expressing cells. Phenotypic and functional characterization of these cells was performed (1) ex vivo, by immunohistochemistry analysis on paraffin-embedded tissues; (2) in vitro, attesting marker expression, proliferation, self-renewal, differentiation, and drug sensitivity; and (3) in vivo, using a zebrafish model. Within pituitary adenomas, we identified rare cell populations expressing stem cell markers but not pituitary hormones; we isolated and expanded in vitro these cells, obtaining fibroblast-free, stem-like cultures from 38 pituitary adenoma samples. These cells grow as spheroids, express stem cell markers (Oct4, Sox2, CD133, and nestin), show sustained in vitro proliferation as compared to primary cultures of differentiated pituitary adenoma cells, and are able to differentiate in hormone-expressing pituitary cells. Besides, pituisphere cells, apparently not tumorigenic in mice, engrafted in zebrafish embryos, inducing pro-angiogenic and invasive responses. Finally, pituitary adenoma stem-like cells express regulatory pituitary receptors (D2R, SSTR2, and SSTR5), whose activation by a dopamine/somatostatin chimeric agonist exerts antiproliferative effects. In conclusion, we provide evidence that human pituitary adenomas contain a subpopulation fulfilling biological and phenotypical signatures of tumor stem cells that may represent novel therapeutic targets for therapy-resistant tumors.     

Localization of somatostatin receptors at the light and electron microscopial level by using antibodies raised against fusion proteins

Somatostatin mediates its multiple biological effects via specific plasma membrane receptors belonging to the family of G-protein coupled receptors with seven putative membrane-spanning domains. Five somatostatin receptor subtypes (sst1-sst5) have been cloned in human, mouse, and rat. We have raised specific antibodies against the five human somatostatin receptors by using the fusion protein technique. DNA sequences encoding C-terminal parts of the somatostatin receptors were inserted into a pGEX-2T plasmid vector. E. coli bacteria were transformed with the recombinant plasmid and fusion proteins were expressed and purified using the glutathione S-transferase Gene Fusion System. The fusion proteins were emulsified with Freund's complete adjuvant and polyclonal antibodies were raised in rabbits. The antisera were tested for specificity in Western blot analysis of membrane preparations from cell lines expressing the receptors and in membrane preparations of brain tissues. The receptors were visualized at the light microscopical level in paraformaldehyde fixed tissue sections by use of biotin labelled secondary antibodies as well as by amplification with biotinylated tyramide. The final step in the immunohistochemical visualization of the receptors was done by both peroxidase labelled streptavidin/biotin and different fluorophores. At the electron microscopical level, some of the receptors could be visualized in tissues fixed with a combination of paraformaldehyde and low concentrations of glutaraldehyde. In the hamster brain, sst2 receptors labelling was observed in both neuronal processes and perikarya. The staining was present in neo-, and allocortical areas of the forebrain, the hypothalamus, brain stem, and spinal cord. In the rat and human, sst1 receptor was shown to be an auto receptor on somatostatinergic neurons located in the hypothalamus. In the retina both sst1 and sst2 receptors were present. sst1 receptors were confined to amacrine cells, few ganglionic cells, and Müller cell-end feet. sst2 receptors were more widespread than the sst1 receptors. sst2-immunoreactivity was present in dopaminergic amacrine cells, the Müller cell-end feet, and in the inner segments of the cone photoreceptors. Thus, the availability of subtype specific antibodies against the five somatostatin receptors makes it possible to identify the receptors involved in the multiple somatostatinergic system in the body.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Somatostatin receptors in brain and pituitary

Somatostatin (SRIF) actions in the brain and pituitary are mediated by specific receptors. Using radioiodinated ligands it has been possible to characterize the kinetics of specific binding sites in the brain and pituitary, and to determine their cellular localization by autoradiography. At the pituitary level, the inhibition of growth hormone, prolactin and thyrotropin secretions induced by SRIF is mediated through a single binding site which is coupled to the inhibition of adenylate cyclase. In the brain, SRIF receptors are localized on neurons and glial cells and are also coupled to adenylate cyclase inhibition. Two sites are differentiated in the brain with an analogue of somatostatin, SMS 201995. In humans, SRIF-binding sites have been related to a number of pathologies. At the pituitary level, it has been shown that the number of binding sites was negatively correlated to growth hormone levels in acromegaly. Furthermore, SRIF-binding sites were undetectable in a patient which did not respond to SMS 201995 therapy. In the brain, meningiomas and gliomas are rich in SRIF binding sites. This suggests a possible role for SRIF on glia. In neurodegenerative diseases, cortical SRIF concentrations are decreased in Alzheimer's and Parkinson's disease associated with dementia while SRIF-binding sites are only affected in Alzheimer's disease. In conclusion, the physiological role of SRIF in the brain and pituitary can be evaluated by studying the receptors of the peptide. Such studies allow to question the implication of SRIF in endocrine and neuropathologies.     

Somatostatin: a historical perspective

Following the discovery and biochemical characterization of natural somatostatin its action profile has been thoroughly investigated. Although the name somatostatin was coined in virtue of its growth hormone release-inhibiting properties, a number of central and peripheral endocrine and paracrine actions have been ascribed to this peptide. Its inhibitory effect on a series of pituitary and gastrointestinal hormones has characterized somatostatin as a classical brain-gut hormone. Circulating and tissue levels of somatostatin and its possible physiological role are analyzed and clinical implications are drawn.     

Human relaxin increases cyclic AMP levels in cultured anterior pituitary cells

Although relaxin acts at several abdominal sites and mammary tissue associated with pregnancy and parturition, the scope of target tissues and the signals conveying the relaxin message into the cell are poorly defined. We found that human relaxin rapidly elevates the cyclic AMP content of cultured rat anterior pituitary cells. This is a graded response (EC50 0.3 nM relaxin) that can be blocked by anti-relaxin antibodies or the hormones somatostatin and dopamine. Furthermore, other hormones with some sequence homology to relaxin, such as insulin and insulin-like growth factor-I, have no such action. We conclude that the anterior pituitary may be a target tissue for relaxin and that cyclic AMP may act as an intracellular messenger for relaxin in these cells.     

生长抑素受体介导的肿瘤靶向治疗

许多肿瘤,如神经内分泌肿瘤、甲状腺癌、乳腺癌等能高表达生长抑素受体,从而为生长抑素及其类似物的治疗提供了靶点。生长抑素受体介导的肿瘤靶向治疗主要包括放射性核素治疗、放射导向手术治疗、细胞毒素治疗和溶瘤病毒治疗。目前,生长抑素受体介导的放射性核素治疗已应用于神经内分泌肿瘤,尤其在胃肠胰神经内分泌肿瘤的诊断和治疗中占据重要地位。另外,生长抑素受体介导的放射导向手术治疗、细胞毒素治疗和溶瘤病毒治疗的潜在价值也越来越引起研究者们的重视。本文通过查阅近五年来关于生长抑素受体介导的肿瘤靶向治疗的国内外文献,综述了生长抑素受体介导的肿瘤靶向治疗的最新研究进展。     

Receptors and neurohormones in human pituitary adenomas

In order to go further into the pathogenesis of human pituitary adenomas, we studied receptors for neurohormones (thyroliberin, TRH; dopamine, DA; somatostatin, SRIH), for estradiol and epidermal growth factor (EGF) thought to influence hormone secretion and/or cell growth. The following results were obtained: (1) the receptors listed above, with the exception of EGF receptors in the adenomas, are present in normal pituitary tissue and in prolactin (PRL)- and growth hormone (GH)-secreting adenomas; (2) they are functional and their affinities are not different in normal or tumoral tissues; (3) their density is variable and depends on the type of secreting adenoma (GH or PRL), the size of the tumor and the plasma level of the hormone which is secreted, and (4) in nonsecreting adenomas, only TRH receptors are found with characteristics identical to those observed in secreting adenomas. We also showed that TRH is contained in normal and tumoral pituitary tissues. TRH and SRIH are released in vitro from adenomatous cells in large amounts, suggesting their possible synthesis by the pituitary. In both cases a local regulation is observed. TRH release is stimulated in the presence of DA while SRIH is inhibited in the presence of TRH. This neuropeptide release may be implicated in the pituitary hormone regulation through a paracrine or an autocrine mechanism. Thus, the neurohormone receptors found in pituitary adenomas should be dependent on a more complex regulation than it has been envisaged till now.