RESUMEN
Loss of the tumor suppressor protein menin is a critical event underlying the formation of neuroendocrine tumors (NET) in hormone-expressing tissues including gastrinomas. While aberrant expression of menin impairs its tumor suppression, few studies explore the structure-function relationship of clinical multiple endocrine neoplasia, type 1 (MEN1) mutations in the absence of a complete LOH at both loci. Here, we determined whether clinical MEN1 mutations render nuclear menin unstable and lead to its functional inactivation. We studied the structural and functional implications of two clinical MEN1 mutations (R516fs, E235K) and a third variant (A541T) recently identified in 10 patients with gastroenteropancreatic (GEP)-NETs. We evaluated the subcellular localization and half-lives of the mutants and variant in Men1-null mouse embryo fibroblast cells and in hormone-expressing human gastric adenocarcinoma and NET cell lines. Loss of menin function was assessed by cell proliferation and gastrin gene expression assays. Finally, we evaluated the effect of the small-molecule compound MI-503 on stabilizing nuclear menin expression and function in vitro and in a previously reported mouse model of gastric NET development. Both the R516fs and E235K mutants exhibited severe defects in total and subcellular expression of menin, and this was consistent with reduced half-lives of these mutants. Mutated menin proteins exhibited loss of function in suppressing tumor cell proliferation and gastrin expression. Treatment with MI-503 rescued nuclear menin expression and attenuated hypergastrinemia and gastric hyperplasia in NET-bearing mice. Clinically defined MEN1 mutations and a germline variant confer pathogenicity by destabilizing nuclear menin expression. Significance: We examined the function of somatic and germline mutations and a variant of MEN1 sequenced from gastroenteropancreatic NETs. We report that these mutations and variant promote tumor cell growth and gastrin expression by rendering menin protein unstable and prone to increased degradation. We demonstrate that the menin-MLL (mixed lineage leukemia) inhibitor MI-503 restores menin protein expression and function in vitro and in vivo, suggesting a potential novel therapeutic approach to target MEN1 GEP-NETs.
Asunto(s)
Neoplasia Endocrina Múltiple Tipo 1 , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Gastrinas/genética , Hormonas , Neoplasia Endocrina Múltiple Tipo 1/genética , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genéticaRESUMEN
The Multiple Endocrine Neoplasia I (MEN1) locus encodes the protein MENIN, which functions as a tumor suppressor protein in neuroendocrine tissues. Gastrinomas are neuroendocrine neoplasms that overproduce the hormone gastrin and can arise sporadically or as part of the MEN1 syndrome, in which mutations in the MEN1 gene lead to loss or inactivation of MENIN protein. Gastrin is a peptide hormone that is primarily synthesized in the gastric antrum and stimulates the secretion of histamine from enterochromaffin-like (ECL) cells and subsequently acid from parietal cells in the gastric corpus. In addition, gastrin exerts a mitogenic function primarily on ECL cells and progenitor cells in the gastric isthmus. Current studies seek to understand how MEN1 mutations generate a mutant MENIN protein that abrogates its tumor suppressor function. Mutations in the MEN1 gene are broadly distributed throughout its nine protein-coding exons, making it difficult to correlate protein structure with its function. Although disruption of the Men1 locus in mice causes functional neuroendocrine tumors in the pituitary and pancreas, gastrinomas do not develop in these transgenic animal models. Prior studies of human gastrinomas suggest that tissue-specific microenvironmental cues in the submucosal foregut may contribute to tumorigenesis by reprogramming of epithelial cells toward the neuroendocrine phenotype. Accordingly, recent studies suggest that neural crest-derived cells are also sensitive to reprogramming when MEN1 is deleted or mutated. Thus, the goal of this report is to review our current understanding of how MENIN modulates gastrin gene expression while highlighting its role in the prevention/suppression of neuroendocrine cell transformation.
Asunto(s)
Gastrinoma , Neoplasia Endocrina Múltiple Tipo 1 , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Gastrinoma/genética , Gastrinoma/patología , Gastrinas/genética , Gastrinas/metabolismo , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasia Endocrina Múltiple Tipo 1/metabolismo , Factores de Transcripción/genética , Neoplasias Pancreáticas/patología , Expresión Génica , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the 'intestine-critical region (ICR)', were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.
Asunto(s)
Codón sin Sentido , Gastrinas , Adolescente , Adulto , Niño , Humanos , Masculino , Adulto Joven , Diarrea/genética , Gastrinas/genética , Mutación , FenotipoRESUMEN
BACKGROUND: A hitherto undescribed form of diabetes mellitus type 2 is reported in a Flemish family. In these patients, markedly elevated gastrin levels were observed, which could not be linked to gastrointestinal symptoms. MATERIALS AND METHODS: Gel permeation chromatography was performed for gastrin, insulin, and proinsulin. Proprotein convertase subtilisin/kexin type (PCSK1 and PCSK2)] were sequenced. Whole-exome sequencing was performed on the genomic DNA extracted from leukocytes of the proband of the family. RESULTS: Gel permeation chromatography revealed that the apparent hypergastrinemia was caused by the accumulation of biologically inactive progastrin. Besides, high serum concentrations of proinsulin and intact fibroblast growth factor 23 (FGF23) were also detected. Sequencing of PCSK1 and PCSK2 genes did not reveal any mutations in these genes. Whole exome sequencing revealed a c.1150C > T (p.Pro384Ser) mutation in G protein-coupled receptor kinase 6 (GRK6), which cosegregated with the disease. Expression of the mutant enzyme in mammalian cells revealed that it was mislocalized compared to the wild-type GRK6. CONCLUSIONS: In the affected patients, prohormone processing is impaired likely due to the altered function of mutant GRK6. Delayed pro-insulin processing causes hypoglycaemia episodes a couple of hours following meals. In addition, increased plasma concentrations of progastrin and intact FGF23 in the affected individuals can be explained by incomplete processing of the precursor hormones.
Asunto(s)
Diabetes Mellitus Tipo 2 , Proinsulina , Animales , Secuencia de Bases , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Gastrinas/genética , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Mutación , Proinsulina/genética , Proinsulina/metabolismoRESUMEN
Abetted by widespread usage of acid-suppressing proton pump inhibitors (PPIs), the mitogenic actions of the peptide hormone gastrin are being revisited as a recurring theme in various gastrointestinal (GI) malignancies. While pathological gastrin levels are intricately linked to hyperplasia of enterochromaffin-like cells leading to carcinoid development, the signaling effects exerted by gastrin on distinct cell types of the gastric mucosa are more nuanced. Indeed, mounting evidence suggests dichotomous roles for gastrin in both promoting and suppressing tumorigenesis. Here, we review the major upstream mediators of gastrin gene regulation, including inflammation secondary to Helicobacter pylori infection and the use of PPIs. We further explore the molecular biology of gastrin in GI malignancies, with particular emphasis on the regulation of gastrin in neuroendocrine neoplasms. Finally, we highlight tissue-specific transcriptional targets as an avenue for targetable therapeutics.
Asunto(s)
Neoplasias Gastrointestinales , Infecciones por Helicobacter , Helicobacter pylori , Humanos , Gastrinas/genética , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/metabolismo , Recurrencia Local de Neoplasia/complicaciones , Inhibidores de la Bomba de Protones/farmacología , Neoplasias Gastrointestinales/tratamiento farmacológicoRESUMEN
Progressive destruction of pancreatic islet ß-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional ß-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase ß-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing ß-cells and ameliorating the disease progression in T1D.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Gastrinas , Islotes Pancreáticos , Trasplante de Células Madre Mesenquimatosas , Animales , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Gastrinas/genética , Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos NODRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Essential oil (EO) is the main extract of patchouli and tangerine peel with antiinflammatory, antiulcer, and other functions. However, the efficacy and mechanism of the combination of EO from patchouli and tangerine peel against gastric ulcer (GU) are unclear. AIM OF THE STUDY: This study aims to reveal the protective effect of the combination of EO from patchouli and tangerine peel against GU in rats, as well as explore the optimal ratio and possible mechanism of EO in GU treatment. MATERIALS AND METHODS: The GU model is executed via water immersion and restraint stress. The repair effect of EO in different proportions on gastric mucosa injury and the effects on serum gastrin (GAS), pepsinogen C (PGC), prostaglandin E2 (PGE2), and 5-hydroxytryptamine in GU rats were observed. The optimal ratio obtained was used in the second part to set different dose groups for further experiment. The effects of the different EO doses on gastric mucosal ulcer formation and gastric acid secretion were evaluated. The morphology of chief and parietal cells were observed via transmission electron microscopy. The contents of GAS, PGC, substance P (SP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cholecystokinin (CCK), PGE2, and motilin (MTL) in serum in different groups were detected via enzyme-linked immunosorbent assay. Expressions of epidermal growth factor (EGF) and trefoil factor 2 (TFF2) protein in gastric tissues were detected via immunohistochemistry, and expressions of c-Jun N-terminal kinase (JNK), P53, Bcl-2-associated X protein (Bax), and Caspase-3 protein in gastric tissues were detected via western blotting. RESULTS: The EO from patchouli and tangerine peel at 1:2 ratio of compatibility significantly improved gastric mucosal injury, decreased serum GAS and PGC contents, and increased the PGE2 level in serum (p < 0.05). The mixture of EO from patchouli and tangerine peel (Mix-EO) can reduce the formation of gastric mucosal ulcers, reduce gastric mucosal injury, improve the expansion of the endoplasmic reticulum of the chief cells, repair mitochondrial damage, and inhibit the secretion of gastric acid by parietal cells. Mix-EO at 300 mg/kg can reduce the expression of serum GAS, PGC, SP, CCK, and cAMP/cGMP (p < 0.05 or 0.01); increase the expression of EGF and TFF2 protein in gastric tissues (p < 0.01); and inhibit the expression of JNK, p53, Bax, and Caspase-3 proteins (p < 0.01). CONCLUSION: The combination of EO from patchouli and tangerine peel can repair the gastric mucosal damage in GU rats and prevent the occurrence of ulcers by inhibiting the secretion of gastric acid, enhancing the defensive ability of gastric mucosa, and suppressing the apoptosis of gastric epithelial cells. Moreover, the optimal compatible ratio of patchouli and tangerine peel is 1:2.
Asunto(s)
Citrus/química , Aceites de Plantas/farmacología , Pogostemon/química , Úlcera Gástrica/tratamiento farmacológico , Animales , Dinoprostona/sangre , Dinoprostona/genética , Dinoprostona/metabolismo , Gastrinas/sangre , Gastrinas/genética , Gastrinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Pepsinógeno C/sangre , Pepsinógeno C/genética , Pepsinógeno C/metabolismo , Aceites de Plantas/química , Sustancias Protectoras/farmacología , Ratas , Ratas Sprague-Dawley , Restricción Física/efectos adversos , Serotonina/sangre , Serotonina/genética , Serotonina/metabolismo , Úlcera Gástrica/etiologíaRESUMEN
The antral hormone gastrin potently regulates gastric acid secretion and fundic mucosal growth. Consequently, appropriate gastrin secretion and plasma concentrations are important for the early phases of digestion. This review describes as the first premise the normal biogenesis of gastrin in the antral mucosa, but also mentions the extraantral expression. Subsequently, the molecular nature and concentration levels of gastrin in serum or plasma are overviewed. Third, assays for accurate measurements of plasma or serum concentrations are commented. Finally, the problem of moderate hypergastrinemia due to Helicobacter pylori infections and/or treatment with proton-pump inhibitors (PPI) is discussed. The review concludes that accurate measurement of the true concentrations of bioactive gastrins in plasma is important. Moreover, it suggests that moderate hypergastrinemias are also essential health issues that require serious attention.
Asunto(s)
Susceptibilidad a Enfermedades/sangre , Susceptibilidad a Enfermedades/etiología , Gastrinas/metabolismo , Animales , Biomarcadores , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Células Secretoras de Gastrina/metabolismo , Gastrinas/sangre , Gastrinas/química , Gastrinas/genética , Regulación de la Expresión Génica , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Helicobacter pylori/fisiología , Humanos , Técnicas de Diagnóstico Molecular , Especificidad de Órganos/genética , Inhibidores de la Bomba de Protones/efectos adversos , Inhibidores de la Bomba de Protones/uso terapéutico , Juego de Reactivos para DiagnósticoRESUMEN
Overexpressed gastrin is reported to promote oncogenesis and development of gastric cancer by inhibiting apoptosis of cancer cells; however, the underlying mechanism remains unclear. Our study is aimed at revealing the mechanism underlying the effect of gastrin on apoptosis of gastric cancer cells. Gastrin-interfering cell line was constructed by stably transfecting gastrin-specific pshRNA plasmid to gastric cancer cell line BGC-823. Then, differentially expressed proteins between untreated BGC-823 and gastrin-interfering BGC-823 cell lines were detected by the iTRAQ technique. GO and KEGG analysis was used to analyze the differentially expressed genes that code these differentially expressed proteins. The Annexin V-FITC staining assay was used to detect gastric cancer cell apoptosis. The DCFH-DA fluorescent probe staining assay was used to measure intracellular ROS. Mitochondrial membrane potential was detected by flow cytometry. Western blot was used to analyze the mitochondria respiratory chain proteins and apoptosis-related proteins. A total of 107 differentially expressed proteins were identified by iTRAQ. GO and KEGG analysis showed that proteins coded by the corresponding differentially expressed genes were mainly enriched in the mitochondrial oxidative respiratory chain, and the expression of three proteins (COX17, COX5B, ATP5J) was upregulated. The three proteins with higher scores were verified by Western blot. The apoptosis rate of the gastrin knockdown cancer cell was significantly increased; meanwhile, gastrin knockdown leads to increase of membrane potential and decrease of intracellular ROS production. Additionally, Bax was significantly increased, whereas NF-κB-p65 and Bcl-2 were downregulated after knockdown of gastrin. Concomitantly, pretreatment with NAC reversed the effect of gastrin on the Bax and Bcl-2 expression. Gastrin promotes the production of ROS from mitochondria, activates NF-κB, and inhibits apoptosis via modulating the expression level of Bcl-2 and Bax.
Asunto(s)
Apoptosis/genética , Gastrinas , Especies Reactivas de Oxígeno , Neoplasias Gástricas , Línea Celular Tumoral , Gastrinas/genética , Gastrinas/metabolismo , Humanos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
Gastrin, secreted by G-cells, and glucagon-like peptide-1 (GLP-1), secreted by L-cells, may participate in the regulation of sodium balance. We studied the effect of sodium in mice in vivo and mouse ileum and human L-cells, on GLP-1 secretion, and the role of NFAT5 and gastrin-releasing peptide receptor (GRPR) in this process. A high-sodium diet increases serum GLP-1 levels in mice. Increasing sodium concentration stimulates GLP-1 secretion from mouse ileum and L-cells. GRP enhances the high sodium-induced increase in GLP-1 secretion. High sodium increases cellular GLP-1 expression, while low and high sodium concentrations increase NFAT5 and GRPR expression. Silencing NFAT5 in L-cells abrogates the stimulatory effect of GRP on the high sodium-induced GLP-1 secretion and protein expression, and the sodium-induced increase in GRPR expression. GLP-1 and gastrin decrease the expression of Na+-K+/ATPase and increase the phosphorylation of sodium/hydrogen exchanger type 3 (NHE3) in human renal proximal tubule cells (hRPTCs). This study gives a new perspective on the mechanisms of GLP-1 secretion, especially that engendered by ingested sodium, and the ability of GLP-1, with gastrin, to decrease Na+-K+/ATPase expression and NHE3 function in hRPTCs. These results may contribute to the better utilization of current and future GLP-1-based drugs in the treatment of hypertension.
Asunto(s)
Gastrinas/genética , Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Hipertensión/genética , Factores de Transcripción/genética , Animales , Células Secretoras de Gastrina/metabolismo , Regulación de la Expresión Génica/genética , Silenciador del Gen , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/patología , Túbulos Renales Proximales/metabolismo , Ratones , Fosforilación/efectos de los fármacos , Sodio/metabolismo , Sodio/farmacología , Intercambiador 3 de Sodio-Hidrógeno/genética , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
BACKGROUND & AIMS: Helicobacter pylori infection in humans typically begins with colonization of the gastric antrum. The initial Th1 response occasionally coincides with an increase in gastrin secretion. Subsequently, the gastritis segues to chronic atrophic gastritis, metaplasia, dysplasia and distal gastric cancer. Despite these well characterized clinical events, the link between inflammatory cytokines and non-cardia gastric cancer remains difficult to study in mouse models. Prior studies have demonstrated that overexpression of the Hedgehog (HH) effector GLI2 induces loss of gastrin (atrophy) and antral hyperplasia. To determine the link between specific cytokines, HH signaling and pre-neoplastic changes in the gastric antrum. METHODS: Mouse lines were created to conditionally direct IL1ß or IFN-γ to the antrum using the Gastrin-CreERT2 and Tet activator. Primary cilia, which transduces HH signaling, on G cells were disrupted by deleting the ciliary motor protein KIF3a. Phenotypic changes were assessed by histology and western blots. A subclone of GLUTag enteroendocrine cells selected for gastrin expression and the presence of primary cilia was treated with recombinant SHH, IL1ß or IFN-γ with or without kif3a siRNA. RESULTS: IFN-γ increased gastrin and induced antral hyperplasia. However, antral expression of IL1ß suppressed tissue and serum gastrin, while also inducing antral hyperplasia. IFN-γ treatment of GLUTAg cells suppressed GLI2 and induced gastrin, without affecting cilia length. By contrast, IL1ß treatment doubled primary cilia length, induced GLI2 and suppressed gastrin gene expression. Knocking down kif3a in GLUTAg cells mitigated SHH or IL1ß suppression of gastrin. CONCLUSIONS: Overexpression of IL1ß in the antrum was sufficient to induce antral hyperplasia coincident with suppression of gastrin via primary cilia. ORCID: #0000-0002-6559-8184.
Asunto(s)
Cilios/patología , Gastrinas/metabolismo , Infecciones por Helicobacter/complicaciones , Hiperplasia/patología , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Antro Pilórico/patología , Animales , Antivirales/farmacología , Cilios/metabolismo , Gastrinas/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Hiperplasia/etiología , Hiperplasia/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antro Pilórico/efectos de los fármacos , Antro Pilórico/metabolismo , Antro Pilórico/microbiologíaRESUMEN
BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.
Asunto(s)
Infecciones por Helicobacter/inmunología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias Experimentales/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias Gástricas/inmunología , Administración Oral , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/inmunología , Mucosa Gástrica/inmunología , Mucosa Gástrica/microbiología , Mucosa Gástrica/patología , Gastrinas/genética , Infecciones por Helicobacter/inducido químicamente , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter felis/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Metilnitrosourea/administración & dosificación , Ratones , Ratones Noqueados , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/microbiología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/microbiología , Microambiente Tumoral/inmunologíaRESUMEN
Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers. Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation. Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of ß-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment. Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.
Asunto(s)
Quimioradioterapia/métodos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Neoplasias/terapia , Fragmentos de Péptidos/farmacología , Radiofármacos/farmacología , Animales , Línea Celular Tumoral , Everolimus/farmacología , Everolimus/uso terapéutico , Femenino , Gastrinas/genética , Gastrinas/farmacología , Gastrinas/uso terapéutico , Humanos , Lutecio , Ratones , Neoplasias/diagnóstico por imagen , Neoplasias/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/uso terapéutico , Radioisótopos , Radiofármacos/uso terapéutico , Ratas , Receptor de Colecistoquinina B/metabolismo , Tomografía Computarizada por Tomografía Computarizada de Emisión de Fotón Único , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIM: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients. PATIENTS AND METHODS: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C). RESULTS: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000). CONCLUSION: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings.
Asunto(s)
Anticuerpos Antibacterianos/aislamiento & purificación , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Adolescente , Adulto , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Biopsia , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Gastrinas/genética , Gastrinas/aislamiento & purificación , Gastritis Atrófica/diagnóstico , Gastritis Atrófica/genética , Gastritis Atrófica/microbiología , Gastritis Atrófica/patología , Gastroscopía/métodos , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Pepsinógeno A/genética , Pepsinógeno A/aislamiento & purificación , Pepsinógeno C/genética , Pepsinógeno C/aislamiento & purificación , Derivación y Consulta , Estómago/microbiología , Estómago/patología , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Adulto JovenRESUMEN
OBJECTIVE: The KRAS gene is the most frequently mutated gene in pancreatic cancer, and no successful anti-Ras therapy has been developed. Gastrin has been shown to stimulate pancreatic cancer in an autocrine fashion. We hypothesized that reactivation of the peptide gastrin collaborates with KRAS during pancreatic carcinogenesis. METHODS: LSL-Kras; P48-Cre (KC) mutant KRAS transgenic mice were crossed with gastrin-KO (GKO) mice to develop GKO/KC mice. Pancreata were examined for 8 months for stage of pancreatic intraepithelial neoplasia lesions, inflammation, fibrosis, gastrin peptide, and microRNA expression. Pancreatic intraepithelial neoplasias from mice were collected by laser capture microdissection and subjected to reverse-phase protein microarray, for gastrin and protein kinases associated with signal transduction. Gastrin mRNA was measured by RNAseq in human pancreatic cancer tissues and compared to that in normal pancreas. RESULTS: In the absence of gastrin, PanIN progression, inflammation, and fibrosis were significantly decreased and signal transduction was reversed to the canonical pathway with decreased KRAS. Gastrin re-expression in the PanINs was mediated by miR-27a. Gastrin mRNA expression was significantly increased in human pancreatic cancer samples compared to normal human pancreas controls. CONCLUSIONS: This study supports the mitogenic role of gastrin in activation of KRAS during pancreatic carcinogenesis.
Asunto(s)
Carcinogénesis/genética , Carcinoma in Situ/genética , Gastrinas/genética , Mutación , Páncreas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Carcinogénesis/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Línea Celular Tumoral , Proliferación Celular/genética , Gastrinas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Páncreas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismoAsunto(s)
Gastrinas/genética , Gastropatías/inducido químicamente , Tamoxifeno/efectos adversos , Administración Oral , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Técnicas de Inactivación de Genes , Humanos , Inyecciones Intraperitoneales , Ratones , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/patología , Gastropatías/genética , Gastropatías/metabolismo , Gastropatías/patología , Tamoxifeno/administración & dosificaciónRESUMEN
We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.
Asunto(s)
Colecistoquinina/metabolismo , Gastrinas/metabolismo , Receptor de Colecistoquinina B/metabolismo , Receptores de Colecistoquinina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Trofoblastos/metabolismo , Animales , Señalización del Calcio , Línea Celular , Colecistoquinina/genética , Colecistoquinina/farmacología , Femenino , Gastrinas/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Colecistoquinina B/genética , Receptores de Colecistoquinina/agonistas , Receptores de Colecistoquinina/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacosRESUMEN
BACKGROUND: Sleeve gastrectomy with ileal transposition has been shown to be superior to sleeve gastrectomy alone for promoting weight loss in rat and porcine models. The absence of a mouse model for this procedure has impeded efforts to understand the molecular physiology underlying its efficacy. This study demonstrates the long-term survivability of sleeve gastrectomy with ileal transposition in mice. MATERIALS AND METHODS: In this study of technical feasibility, a sleeve gastrectomy with ileal transposition (SGIT), sleeve gastrectomy (SG), or sham surgery (SH) was performed on 7- to 8-week-old C57Bl/6J mice (n = 8 for each). To evaluate long-term survivability, mice were placed on an obesogenic diet and weighed weekly for 10 weeks. The intestinal identity of the transposed segment was assessed with gene expression analysis of duodenal-, jejunal-, and ileal-specific hormones using quantitative polymerase chain reaction. RESULTS: Overall, SGIT better prevented weight gain than the SG or sham procedures (10-week post-operative weight: SH 45.3 ± 1.0 g, SG 41.25 ± 1.6 g, SGIT 35.4 ± 0.8 g). Gene expression pattern analysis of three markers of intestinal identity (gastrin, cholecystokinin, and peptide YY) suggests that the ileal identity of the transposed segment is maintained 10 weeks after transposition. CONCLUSIONS: We demonstrate for the first time a reproducible mouse model of sleeve gastrectomy with ileal transposition. Future studies utilizing this model will expand our understanding of the molecular pathways through which the hindgut regulates satiety.
Asunto(s)
Gastrectomía/métodos , Íleon/cirugía , Animales , Biomarcadores , Glucemia/análisis , Colecistoquinina/genética , Colecistoquinina/metabolismo , Modelos Animales de Enfermedad , Estudios de Factibilidad , Gastrinas/genética , Gastrinas/metabolismo , Expresión Génica , Ratones Endogámicos C57BL , Péptido YY/genética , Péptido YY/metabolismo , ARN/metabolismo , Distribución Aleatoria , Pérdida de PesoRESUMEN
Gastric cancer has reduced prevalence, but poor prognoses. To improve treatment, better knowledge of carcinogenesis and cells of origin should be sought. Stomach cancers are typically localized to one of the three mucosae; cardial, oxyntic and antral. Moreover, not only the stem cell, but the ECL cell may proliferate and give rise to tumours. According to Laurén, the classification of gastric carcinomas seems to reflect biological important differences and possible different cell of origin since the two subtypes, intestinal and diffuse, do not transform into the other and show different epidemiology. The stem cell probably gives rise to the intestinal type, whereas the ECL cell may be important in the diffuse type. Elevation of gastrin may be the carcinogenic factor for Helicobacter pylori as well as the recently described increased risk of gastric cancer due to proton pump inhibitor treatment. Therefore, it is essential to determine the role of the gastrin target cell, the ECL cell, in gastric carcinogenesis. Clinical trials with gastrin antagonists could improve prognoses in those with gastrin receptor positive tumours. However, further studies on gastric carcinomas applying relative available methods and with the highest sensitivity are warranted to improve our knowledge of gastric carcinogenesis.
Asunto(s)
Carcinogénesis/genética , Carcinoma/fisiopatología , Neoplasias Gástricas/fisiopatología , Carcinoma/clasificación , Carcinoma/etiología , Carcinoma/microbiología , Proliferación Celular/genética , Mucosa Gástrica/microbiología , Mucosa Gástrica/fisiopatología , Gastrinas/genética , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Humanos , Neoplasias Gástricas/clasificación , Neoplasias Gástricas/etiología , Neoplasias Gástricas/microbiologíaRESUMEN
OBJECTIVES: Modulation of cholecystokinin (CCK) receptors has been shown to influence pancreatic endocrine function. METHODS: We assessed the impact of the CCKA and CCKB receptor modulators, (pGlu-Gln)-CCK-8 and gastrin-17, respectively, on ß-cell secretory function, proliferation and apoptosis and glucose tolerance, and investigating alterations of CCK and gastrin islet expression in diabetes. RESULTS: Initially, the presence of CCK and gastrin, and expression of their receptors were evidenced in ß-cell lines and mouse islets. (pGlu-Gln)-CCK-8 and gastrin-17 stimulated insulin secretion from BRIN-BD11 and 1.1B4 ß-cells, associated with no effect on membrane potential or [Ca]i. Only (pGlu-Gln)-CCK-8 possessed insulin secretory actions in isolated islets. In agreement, (pGlu-Gln)-CCK-8 improved glucose disposal and glucose-induced insulin release in mice. In addition, (pGlu-Gln)-CCK-8 evoked clear satiety effects. Interestingly, islet colocalization of CCK with glucagon was elevated in streptozotocin- and hydrocortisone-induced diabetic mice, whereas gastrin coexpression in α cells was reduced. In contrast, gastrin colocalization within ß-cells was higher in diabetic mice, while CCK coexpression with insulin was decreased in insulin-deficient mice. (pGlu-Gln)-CCK-8 and gastrin-17 also augmented human and rodent ß-cell proliferation and offered protection against streptozotocin-induced ß-cell cytotoxicity. CONCLUSIONS: We highlight the direct involvement of CCKA and CCKB receptors in pancreatic ß-cell function and survival.