RESUMO
The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.
Assuntos
Receptor de Colecistocinina B , Humanos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Linhagem Celular Tumoral , Trissacarídeos/química , Lactose/análogos & derivados , Lactose/química , Glucose/metabolismoRESUMO
Cholecystokinin (CCK) increases core body temperature via CCK2 receptors when administered intracerebroventricularly (icv). The mechanisms of CCK-induced hyperthermia are unknown, and it is also unknown whether CCK contributes to the fever response to systemic inflammation. We studied the interaction between central CCK signaling and the cyclooxygenase (COX) pathway. Body temperature was measured in adult male Wistar rats pretreated with intraperitoneal infusion of the nonselective COX enzyme inhibitor metamizol (120 mg/kg) or a selective COX-2 inhibitor, meloxicam, or etoricoxib (10 mg/kg for both) and, 30 min later, treated with intracerebroventricular CCK (1.7 µg/kg). In separate experiments, CCK-induced neuronal activation (with and without COX inhibition) was studied in thermoregulation- and feeding-related nuclei with c-Fos immunohistochemistry. CCK increased body temperature by â¼0.4°C from 10 min postinfusion, which was attenuated by metamizol. CCK reduced the number of c-Fos-positive cells in the median preoptic area (by â¼70%) but increased it in the dorsal hypothalamic area and in the rostral raphe pallidus (by â¼50% in both); all these changes were completely blocked with metamizol. In contrast, CCK-induced satiety and neuronal activation in the ventromedial hypothalamus were not influenced by metamizol. CCK-induced hyperthermia was also completely blocked with both selective COX-2 inhibitors studied. Finally, the CCK2 receptor antagonist YM022 (10 µg/kg icv) attenuated the late phases of fever induced by bacterial lipopolysaccharide (10 µg/kg; intravenously). We conclude that centrally administered CCK causes hyperthermia through changes in the activity of "classical" thermoeffector pathways and that the activation of COX-2 is required for the development of this response.NEW & NOTEWORTHY An association between central cholecystokinin signaling and the cyclooxygenase-prostaglandin E pathway has been proposed but remained poorly understood. We show that the hyperthermic response to the central administration of cholecystokinin alters the neuronal activity within efferent thermoeffector pathways and that these effects are fully blocked by the inhibition of cyclooxygenase. We also show that the activation of cyclooxygenase-2 is required for the hyperthermic effect of cholecystokinin and that cholecystokinin is a modulator of endotoxin-induced fever.
Assuntos
Temperatura Corporal/efeitos dos fármacos , Colecistocinina/administração & dosagem , Ciclo-Oxigenase 2/metabolismo , Hipertermia/induzido quimicamente , Hipertermia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anorexia/induzido quimicamente , Benzodiazepinas/administração & dosagem , Regulação da Temperatura Corporal/efeitos dos fármacos , Colecistocinina/efeitos adversos , Inibidores de Ciclo-Oxigenase 2/administração & dosagem , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Febre/induzido quimicamente , Febre/tratamento farmacológico , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Injeções Intraventriculares , Lipopolissacarídeos/efeitos adversos , Masculino , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Receptor de Colecistocinina B/antagonistas & inibidores , Resultado do TratamentoRESUMO
Cholecystokinin-2 receptor (CCK2R) has been proven to be a specific biomarker for colorectal malignancies. Immunotoxins are a valuable class of immunotherapy agents consisting of a targeting element and a bacterial or plant toxin. Previous work demonstrated that targeting CCK2R is a good therapeutic strategy for the treatment of colorectal cancer (CRC). In the present study, we developed a new version of CCK2R-targeting immunotoxin GD9P using a targeted peptide, GD9, as the binding motif and a truncated Pseudomonas exotoxin A (PE38) as the cytokiller. BALB/c nude mice were treated with different doses of GD9P, and pharmacodynamics, pharmacokinetic, and toxicological data were obtained throughout this study. Compared to the parental immunotoxin rCCK8PE38, GD9P exhibited about 1.5-fold yield, higher fluorescence intensity, and increased antitumor activity against human CRC in vitro and in vivo. The IC50 values of GD9P in vitro ranged from 1.61 to 4.55 nM. Pharmacokinetic studies were conducted in mice with a T1/2 of 69.315 min. When tumor-bearing nude mice were treated with GD9P at doses ≥2 mg/kg for five doses, a rapid shrinkage in tumor volume and, in some cases, complete remission was observed. A preliminary safety evaluation demonstrated a good safety profile of GD9P as a Pseudomonas exotoxin A-based immunotherapy. The therapy in combination with oxaliplatin can increase the antitumor efficacy and reduce the toxic side effects caused by chemotherapy. In conclusion, the data support the use of GD9P as a promising immunotherapy targeting CCK2R-expressing colorectal malignancies.
Assuntos
ADP Ribose Transferases/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Exotoxinas/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Virulência/farmacologia , ADP Ribose Transferases/genética , ADP Ribose Transferases/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/genética , Toxinas Bacterianas/uso terapêutico , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Exotoxinas/genética , Exotoxinas/uso terapêutico , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Distribuição Tecidual , Testes de Toxicidade Aguda , Fatores de Virulência/genética , Fatores de Virulência/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Exotoxina A de Pseudomonas aeruginosaRESUMO
We previously reported that exposure of human colon adenocarcinoma (Caco-2) cells to the bitter substance phenylthiocarbamide (PTC) rapidly enhanced the transport function of P-glycoprotein (P-gp). In this study, we investigated the short-term effect of etoposide, another bitter-tasting P-gp substrate, on P-gp transport function in the same cell line. We found that etoposide exposure significantly increased both the P-gp protein level in the plasma membrane fraction and the efflux rate of rhodamine123 (Rho123) in Caco-2 cells within 10 min. The efflux ratio (ratio of the apparent permeability coefficient in the basal-to-apical direction to that in the apical-to-basal direction) of Rho123 in etoposide-treated cells was also significantly increased compared with the control. These results indicated that etoposide rapidly enhances P-gp function in Caco-2 cells. In contrast, P-gp expression in whole cells at both the mRNA and protein level was unchanged by etoposide exposure, compared with the levels in non-treated cells. Furthermore, etoposide increased the level of phosphorylated ezrin, radixin and moesin (P-ERM) proteins in the plasma membrane fraction of Caco-2 cells within 10 min. P-gp functional changes were blocked by YM022, an inhibitor of cholecystokinin (CCK) receptor. These results suggest that etoposide induces release of CCK, causing activation of the CCK receptor followed by phosphorylation of ERM proteins, which recruit intracellular P-gp for trafficking to the gastrointestinal membrane, thereby increasing the functional activity of P-gp.
Assuntos
Etoposídeo/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Benzodiazepinas/farmacologia , Células CACO-2 , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colecistocinina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosforilação/efeitos dos fármacos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismoRESUMO
Hypergastrinemia has been associated with high-grade dysplasia and adenocarcinoma in patients with Barrett's esophagus, and experimental studies suggest proinflammatory and proneoplastic effects of gastrin on Barrett's esophagus. This is of potential concern, as patients with Barrett's esophagus are treated with medications that suppress gastric acid production, resulting in increased physiologic levels of gastrin. We aimed to determine whether treatment with the novel gastrin/CCK2 receptor antagonist netazepide reduces expression of markers associated with inflammation and neoplasia in Barrett's esophagus. This was a randomized, double-blind, placebo-controlled trial of netazepide in patients with Barrett's esophagus without dysplasia. Subjects were treated for 12 weeks, with endoscopic assessment at baseline and at end of treatment. The primary outcome was within-individual change in cellular proliferation as assessed by Ki67. Secondary analyses included changes in gene expression, assessed by RNA-sequencing, and safety and tolerability. A total of 20 subjects completed the study and were included in the analyses. There was no difference between arms in mean change in cellular proliferation (netazepide: +35.6 Ki67+ cells/mm2, SD 620.7; placebo: +307.8 Ki67+ cells/mm2, SD 640.3; P = 0.35). Netazepide treatment resulted in increased expression of genes related to gastric phenotype (TFF2, MUC5B) and certain cancer-associated markers (REG3A, PAX9, MUC1), and decreased expression of intestinal markers MUC2, FABP1, FABP2, and CDX1 No serious adverse events related to study drug occurred. The gastrin/CCK2 receptor antagonist netazepide did not reduce cellular proliferation in patients with nondysplastic Barrett's esophagus. Further research should focus on the biological effects of gastrin in Barrett's esophagus.Prevention Relevance: Treatment of patients with Barrett's esophagus with a gastrin/CCK2 receptor antagonist did not have obvious chemopreventive effects.
Assuntos
Adenocarcinoma/prevenção & controle , Esôfago de Barrett/tratamento farmacológico , Benzodiazepinonas/administração & dosagem , Neoplasias Esofágicas/prevenção & controle , Compostos de Fenilureia/administração & dosagem , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Idoso , Esôfago de Barrett/patologia , Benzodiazepinonas/efeitos adversos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Método Duplo-Cego , Mucosa Esofágica/diagnóstico por imagem , Mucosa Esofágica/efeitos dos fármacos , Mucosa Esofágica/patologia , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Esofagoscopia , Feminino , Gastrinas/antagonistas & inibidores , Humanos , Masculino , Pessoa de Meia-Idade , Compostos de Fenilureia/efeitos adversos , Receptor de Colecistocinina B/antagonistas & inibidoresRESUMO
The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [111In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.
Assuntos
Benzodiazepinas/química , Radioisótopos de Índio/farmacocinética , Neoplasias Pulmonares/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Radioisótopos de Índio/administração & dosagem , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Compostos Radiofarmacêuticos/administração & dosagem , Receptor de Colecistocinina B/metabolismo , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) is the fastest growing cancer worldwide in part due to the obesity epidemic and fatty liver disease, particularly nonalcoholic steatohepatitis (NASH). Chronic inflammation with the release of cytokines and chemokines with activation of hepatic stellate cells results in changes of the liver extracellular matrix (ECM) that predisposes to the development of HCC. Blood levels of the gastrointestinal peptide cholecystokinin (CCK) are increased in humans and mice consuming a high-fat diet. We found that the CCK-B receptor (CCK-BR) expression increased in the livers of mice with NASH. Treatment of mice with a CCK-BR antagonist, proglumide, prevented NASH, lowered hepatic inflammatory cytokines and chemokines, reduced oxidative stress, decreased F4/80+ hepatic macrophages, and prevented HCC. CCK-AR and CCK-BR expression was increased in both murine and human HCC cell lines compared with that of normal liver, and CCK stimulated the growth of wild-type and CCK-A receptor knockout HCC cells in vitro, but not CCK-BR knockout cells suggesting that the CCK-BR mediates proliferation. Proglumide therapy significantly reduced growth by 70% and 73% in mice bearing Dt81Hepa1-6 or in RIL-75 HCC tumors, respectively. IHC of a human liver tissue array with a selective CCK-BR antibody revealed staining of human HCC and no staining in normal liver. PREVENTION RELEVANCE: This investigation demonstrates the role of the gastrointestinal peptide cholecystokinin (CCK) in hepatocellular carcinoma (HCC) and how CCK-BR blockade reverses the premalignant state of the hepatic extracellular matrix hence, rendering it less susceptible to the development of HCC. Thereby, CCK-BR blockade is a novel approach for the prevention/treatment of HCC.
Assuntos
Carcinoma Hepatocelular/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Proglumida/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colecistocinina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/patologia , Proglumida/uso terapêutico , Receptor de Colecistocinina B/genética , Receptor de Colecistocinina B/metabolismoRESUMO
The cholecystokinin-2/gastrin receptor (CCK2 R) is considered a suitable target for the development of radiolabelled antagonists, due to its overexpression in various tumours, but no such compounds are available in clinical use. Therefore, we designed novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated ligands based on CCK2 R antagonist Z360/nastorazepide. As a proof of concept that CCK2 R antagonistic activity can be retained by extending the Z360/nastorazepide structure using suitable linker, we present herein three compounds containing various PEG linkers synthesised on solid phase and in solution. The antagonistic properties were measured in a functional assay in the A431-CCK2 R cell line (in the presence of agonist G17), with IC50 values of 3.31, 4.11 and 10.4â nM for compounds containing PEG4 , PEG6 and PEG12 , respectively. All compounds were successfully radiolabelled with indium-111, lutetium-177 and gallium-68 (incorporation of radiometal >95 %). The gallium-68-labelled compounds were stable for up to 2â h (PBS, 37 °C). log D7.4 values were determined for indium-111- and gallium-68-labelled compounds, showing improved hydrophilicity compared to the reference compound.
Assuntos
Desenho de Fármacos , Polietilenoglicóis/química , Compostos Radiofarmacêuticos/síntese química , Receptor de Colecistocinina B/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Radioisótopos de Gálio/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Radioisótopos de Índio/química , Lutécio/química , Simulação de Acoplamento Molecular , Radioisótopos/química , Compostos Radiofarmacêuticos/metabolismo , Receptor de Colecistocinina B/metabolismo , Técnicas de Síntese em Fase SólidaRESUMO
The widespread involvement of the cholecystokinin-2/gastrin receptor (CCK2R) in multiple (patho)physiological processes has propelled extensive searches for nonpeptide small-molecule CCK2R antagonists. For the past three decades, considerable research has yielded numerous chemically heterogeneous compounds. None of these entered into the clinic, mainly because of inadequate biological effects. However, it appears that the ultimate goal of a clinically useful CCK2R antagonist is now just around the corner, with the most promising compounds, netazepide and nastorazepide, now in Phase II clinical trials. Here, we illustrate the structure-activity relationships (SARs) of stablished CCK2R antagonists of various structural classes, and the most recent proof-of-concept studies where new applicabilities of CCK2R antagonists as visualizing agents are presented.
Assuntos
Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Benzodiazepinonas/química , Benzodiazepinonas/uso terapêutico , Ensaios Clínicos como Assunto , Furanos/química , Furanos/uso terapêutico , Humanos , Lactamas/química , Lactamas/uso terapêutico , Receptor de Colecistocinina B/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/uso terapêutico , Tetragastrina/química , Tetragastrina/uso terapêuticoRESUMO
BACKGROUND & AIMS: In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling. METHODS: We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGSGR gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples. RESULTS: Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGSGR cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGSGR cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5. CONCLUSIONS: In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide.
Assuntos
Benzodiazepinonas/farmacologia , Tumores Neuroendócrinos/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Proteína Plasmática A Associada à Gravidez/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Animais , Benzodiazepinas/farmacologia , Benzodiazepinonas/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Mucosa Gástrica/citologia , Mucosa Gástrica/patologia , Gastrinas/antagonistas & inibidores , Gastrinas/sangue , Gastrinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Transgênicos , Tumores Neuroendócrinos/sangue , Tumores Neuroendócrinos/patologia , Organoides , Compostos de Fenilureia/uso terapêutico , Proteína Plasmática A Associada à Gravidez/análise , Proteína Plasmática A Associada à Gravidez/antagonistas & inibidores , Proteína Plasmática A Associada à Gravidez/genética , Cultura Primária de Células , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Neoplasias Gástricas/sangue , Neoplasias Gástricas/patologia , Resultado do TratamentoRESUMO
BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.
Assuntos
Carcinoma Hepatocelular/prevenção & controle , Antagonistas de Hormônios/farmacologia , Neoplasias Hepáticas/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Proglumida/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Deficiência de Colina/complicações , Modelos Animais de Doenças , Epigênese Genética , Etionina , Feminino , Regulação Neoplásica da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Colecistocinina B/genética , Receptor de Colecistocinina B/metabolismo , Transdução de SinaisRESUMO
Cholecystokinin (CCK), through the CCK-2 receptor, exerts complex effects on anxiety. While CCK agonists are panicogenic, CCK-2 antagonists fail to alleviate human anxiety. Preclinical studies with CCK-2 antagonists are also inconsistent because their anxiolytic effects largely depend on the behavioral paradigm and antecedent stress. The controversy might be accounted by the neuromodulatory role for CCK in anxiety which is ill-defined. If this is its actual role, blocking CCK-2 will have carry-over effects on the anxiety baseline over time. To test this hypothesis, the consequences of acute administration of the CCK-2 antagonist Ly225.910 (0.1â¯mgâ¯Kg-1) was evaluated in the temporal expression of aversion toward exploration-conflicting tasks. Ly225.910 effects were evaluated in rats exposed to the elevated plus-maze (EPM) twice, an approach-avoidance anxiety-like test. While LY225.910-treated rats had less anxiety than vehicle-treated rats, the difference was reversed during the EPM retest 24â¯h later without drug. Moreover, Ly225.910 effects in stress-induced cognitive impairment was measured giving the novel-object discrimination (NOD) test to rats not habituated to the exploration apparatus to elicit neophobia. After a first encounter with objects ("old"), Ly225.910-treated rats did not recognize the "novel" object introduced 6â¯h later. Ly225.910-exposed rats did not discriminate the new location of the "novel object" when it was repositioned in the arena 24â¯h later. Ly225.910-treated rats also failed to explore objects. In line with its neuromodulatory role, aversive carry-over effects of Ly225.910 suggest that CCK-2 activation by endogenous CCK, rather than triggering anxiety, may return the anxiety state to its normal level.
Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Quinazolinonas/uso terapêutico , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Ansiolíticos/farmacologia , Ansiedade/metabolismo , Ansiedade/psicologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Quinazolinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologiaRESUMO
Gastrin signaling mediated through cholecystokinin-2 receptor (CCK2R) and its downstream molecules is altered in pancreatic cancer. CCK2R antagonists, YF476 (netazepide) and JNJ-26070109, were tested systematically for their effect on pancreatic intraepithelial neoplasia (PanIN) progression to pancreatic ductal adenocarcinoma (PDAC) in KrasG12D mice. After dose selection using wild-type mice, six-week-old p48Cre/+ -LSL-KrasG12D (22-24 per group) genetically engineered mice (GEM) were fed AIN-76A diets containing 0, 250, or 500 ppm JNJ-26070109 or YF-476 for 38 weeks. At termination, pancreata were collected, weighed, and evaluated for PanINs and PDAC. Results demonstrated that control-diet-fed mice showed 69% (males) and 33% (females) incidence of PDAC. Administration of low and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% and 71% (P < .004) in male mice and by 100% and 24% (P > .05) in female mice, respectively. Low and high dose YF476 inhibited the incidence of PDAC by 74% (P < .02) and 69% (P < .02) in male mice and by 45% and 33% (P > .05) in female mice, respectively. Further, transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18, and Gpr85 genes in JNJ-26070109-treated mice compared with untreated mice. YF476-treated mouse pancreas showed downregulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn gene expressions compared with untreated mouse pancreas. Overall, JNJ-26070109 showed better chemopreventive efficacy than YF476. However, caution is recommended when selecting doses, as the agents appeared to exhibit gender-specific effects.
Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Receptor de Colecistocinina B/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Benzodiazepinonas/farmacologia , Carcinoma in Situ , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Quimioprevenção/métodos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinoxalinas/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Urocortin-3 (UCN3) as a brain-gut peptide inhibits food intake of animal, but the underlying mechanism is not clear. To explore the appetite mechanism about the action of UCN3 in fish, intraperitoneal injection of UCN3 with CCK8, Lorglumide (CCK1R antagonist) or LY225910 (CCK2R antagonist) were conducted. Siberian sturgeon administrated with UCN3 and CCK8 showed a drastic reduction in food intake. The anorectic effect of UCN3 was significantly blocked by LY225910, but not affected by Lorglumide. Furthermore, LY225910 could effectively reverse appetite factor mRNA expressions, including cck, pyy, cart, npy, ucn3, apelin and nucb2 in the whole brain, stomach and intestinum valvula, but Lorglumide could only partially reverse these effects, suggesting the anorectic effect of UCN3 may be primarily mediated CCK2R in Siberian sturgeon. This study indicates for the first time in fish that UCN3 may inhibit food intake in coordination with CCK and CCK2R.
Assuntos
Ingestão de Alimentos/genética , Peixes/fisiologia , Receptor de Colecistocinina B/genética , Urocortinas/genética , Animais , Peixes/genética , Proglumida/análogos & derivados , Proglumida/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Urocortinas/antagonistas & inibidoresRESUMO
AIMS: RPR 102681, a cholecystokinin-B antagonist, increased dopamine (DA) release and reduced cocaine self-administration in animals. This pilot study sought to assess the safety and pharmacokinetics (PK) of co-administration of RPR 102681 and cocaine, and to confirm the DA release mechanism of RPR 102681. METHODS: Sixteen cocaine-dependent participants were randomized to either placebo or RPR102681 at 3 ascending doses; cocaine was co-administered at steady state of RPR 102681. [11 C]raclopride positron emission tomography scans were conducted at baseline and at each RPR102681 dose. RESULTS: RPR 102681 was well tolerated, and safe to co-administer with cocaine. RPR 102681 did not alter the PK of either cocaine or its metabolite benzoylecgonine and showed no intrinsic abuse liability. There was a trend toward reduction of cocaine craving scores. In contrast to animal studies, RPR 102681 significantly increased the binding potential of [11 C]raclopride in the ventral striatum (t test, P < .001) and caudate nucleus (t test, P < .0001) in a small subset of patients, suggesting that it may reduce intrasynaptic striatal DA. CONCLUSION: Overall, this pilot study suggests that RPR 102681 would be unlikely candidate, as an agonist medication for the treatment for cocaine addiction but worth investigating further for possible role in reducing craving.
Assuntos
Acetamidas/farmacologia , Fármacos do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Compostos de Fenilureia/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Acetamidas/efeitos adversos , Acetamidas/farmacocinética , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Fármacos do Sistema Nervoso Central/efeitos adversos , Fármacos do Sistema Nervoso Central/farmacocinética , Cocaína/farmacocinética , Transtornos Relacionados ao Uso de Cocaína/diagnóstico por imagem , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Fissura/efeitos dos fármacos , Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacocinética , Relação Dose-Resposta a Droga , Método Duplo-Cego , Interações Medicamentosas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Compostos de Fenilureia/efeitos adversos , Compostos de Fenilureia/farmacocinética , Projetos Piloto , Tomografia por Emissão de Pósitrons , Racloprida , Compostos RadiofarmacêuticosRESUMO
Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.
Assuntos
Tonsila do Cerebelo/metabolismo , Colecistocinina/metabolismo , Dor/patologia , Receptor de Colecistocinina B/metabolismo , Transdução de Sinais/fisiologia , Tonsila do Cerebelo/patologia , Animais , Adaptação à Escuridão/efeitos dos fármacos , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Adjuvante de Freund/toxicidade , Gastrinas/uso terapêutico , Glutamato Descarboxilase/metabolismo , Inflamação/induzido quimicamente , Inflamação/complicações , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nociceptividade/efeitos dos fármacos , Dor/etiologia , Limiar da Dor/efeitos dos fármacos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B/agonistas , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/genética , Transdução de Sinais/efeitos dos fármacos , Sincalida/uso terapêutico , Tetragastrina/análogos & derivados , Tetragastrina/uso terapêuticoRESUMO
BACKGROUND: Ischemia/reperfusion injury (IRI) is one of the most predominant complications of ischemic heart disease. Gastrin has emerged as a regulator of cardiovascular function, playing a key protective role in hypoxia. Serum gastrin levels are increased in patients with myocardial infarction, but the pathophysiogical significance of this finding is unknown. The purpose of this study was to determine whether and how gastrin protects cardiac myocytes from IRI. METHODS AND RESULTS: Adult male Sprague-Dawley rats were used in the experiments. The hearts in living rats or isolated Langendorff-perfused rat hearts were subjected to ischemia followed by reperfusion to induce myocardial IRI. Gastrin, alone or with an antagonist, was administered before the induction of myocardial IRI. We found that gastrin improved myocardial function and reduced the expression of myocardial injury markers, infarct size, and cardiomyocyte apoptosis induced by IRI. Gastrin increased the phosphorylation levels of ERK1/2 (extracellular signal-regulated kinase 1/2), AKT (protein kinase B), and STAT3 (signal transducer and activator of transcription 3), indicating its ability to activate the RISK (reperfusion injury salvage kinase) and SAFE (survivor activating factor enhancement) pathways. The presence of inhibitors of ERK1/2, AKT, or STAT3 abrogated the gastrin-mediated protection. The protective effect of gastrin was via CCK2R (cholecystokinin 2 receptor) because the CCK2R blocker CI988 prevented the gastrin-mediated protection of the heart with IRI. Moreover, we found a negative correlation between serum levels of cardiac troponin I and gastrin in patients with unstable angina pectoris undergoing percutaneous coronary intervention, suggesting a protective effect of gastrin in human cardiomyocytes. CONCLUSIONS: These results indicate that gastrin can reduce myocardial IRI by activation of the RISK and SAFE pathways.
Assuntos
Gastrinas/farmacologia , Coração/efeitos dos fármacos , Hormônios/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Idoso , Angina Instável/sangue , Angina Instável/cirurgia , Animais , Apoptose/efeitos dos fármacos , Feminino , Gastrinas/sangue , Humanos , Preparação de Coração Isolado , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Intervenção Coronária Percutânea , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Troponina I/sangueRESUMO
AIM: Cholecystokinin (CCK) participates in the storage of dietary triglycerides in white adipose tissue (WAT). Our goal was to characterize, both in subcutaneous (Sc-WAT) and visceral WAT (Vis-WAT), the functional expression of the two known CCK receptors, CCK-1 (CCK-1R) and CCK-2 (CCK-2R), as well as of CCK. MAIN METHODS: Gene and protein expression was assessed in different cell types of rat and human WAT by means of RT-PCR and western-blot, respectively. The functionality of CCK-Rs was tested by quantifying protein kinase B (Akt) phosphorylation after treatment of pre-adipocytes with the bioactive fragment of CCK, CCK-8. The CCK receptor subtype involved in Akt phosphorylation was investigated by using selective CCK-1R (SR-27,897) and CCK-2R antagonists (L-365,260). KEY FINDINGS: In rats, CCK-1R (Cckar) and CCK-2R (Cckbr) gene expression was detected in the two types of WAT analyzed as well as in isolated adipocytes, mesenchymal stem cells and pre-adipocytes. CCK-1R and CCK-2R proteins were identified in adipocytes and, to a minor extent, in pre-adipocytes. In addition, CCK-2R were detected in subcutaneous mesenchymal stem cells. Gene expression of the CCK precursor preproCCK as well as CCK immunoreactivity were also found in Sc-WAT and Vis-WAT. In human WAT, CCK gene expression as well as CCK-2Rs and CCK were also identified. CCK-8 evoked Akt phosphorylation in rat pre-adipocytes, and this effect was antagonized by SR-27,897 and L-365,260. SIGNIFICANCE: Our data show that both human and rat WAT express a complete CCK system, and suggest that CCK may have an autocrine/paracrine role in regulating adipose tissue biology.
Assuntos
Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/fisiologia , Colecistocinina/metabolismo , Colecistocinina/fisiologia , Adipócitos/metabolismo , Animais , Benzodiazepinonas/farmacologia , Regulação da Expressão Gênica/genética , Inativação Gênica , Humanos , Ácidos Indolacéticos/farmacologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Compostos de Fenilureia/farmacologia , Fosforilação , Ratos , Ratos Wistar , Receptor de Colecistocinina A/antagonistas & inibidores , Receptor de Colecistocinina A/biossíntese , Receptor de Colecistocinina A/genética , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/biossíntese , Receptor de Colecistocinina B/genética , Tiazóis/farmacologiaRESUMO
The cytoprotective protein clusterin is often dysregulated during tumorigenesis, and in the stomach, upregulation of clusterin marks emergence of the oxyntic atrophy (loss of acid-producing parietal cells)-associated spasmolytic polypeptide-expressing metaplasia (SPEM). The hormone gastrin is important for normal function and maturation of the gastric oxyntic mucosa and hypergastrinemia might be involved in gastric carcinogenesis. Gastrin induces expression of clusterin in adenocarcinoma cells. In the present study, we examined the expression patterns and gastrin-mediated regulation of clusterin in gastric tissue from: humans; rats treated with proton pump (H+/K+-ATPase) inhibitors and/or a gastrin receptor (CCK2R) antagonist; H+/K+-ATPase ß-subunit knockout (H/K-ß KO) mice; and Mongolian gerbils infected with Helicobacter pylori and given a CCK2R antagonist. Biological function of secretory clusterin was studied in human gastric cancer cells. Clusterin was highly expressed in neuroendocrine cells in normal oxyntic mucosa of humans and rodents. In response to hypergastrinemia, expression of clusterin increased significantly and its localization shifted to basal groups of proliferative cells in the mucous neck cell-chief cell lineage in all animal models. That shift was partially inhibited by antagonizing the CCK2R in rats and gerbils. The oxyntic mucosa of H/K-ß KO mice contained areas with clusterin-positive mucous cells resembling SPEM. In gastric adenocarcinomas, clusterin mRNA expression was higher in diffuse tumors containing signet ring cells compared with diffuse tumors without signet ring cells, and clusterin seemed to be secreted by tumor cells. In gastric cancer cell lines, gastrin increased secretion of clusterin, and both gastrin and secretory clusterin promoted survival after starvation- and chemotherapy-induced stress. Overall, our results indicate that clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and stimulates gastric cancer cell survival.
Assuntos
Clusterina/fisiologia , Regulação Neoplásica da Expressão Gênica , Células Parietais Gástricas/patologia , Neoplasias Gástricas/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Clusterina/genética , Clusterina/metabolismo , Feminino , Gastrinas/metabolismo , Gastrinas/fisiologia , Perfilação da Expressão Gênica , Gerbillinae , Humanos , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , Células Parietais Gástricas/metabolismo , Inibidores da Bomba de Prótons/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B/antagonistas & inibidores , Neoplasias Gástricas/metabolismoRESUMO
BACKGROUND/AIM: The aim of the study was to evaluate the anti-tumor mechanism of Z-360, a gastrin/cholecystokinin-2 receptor (CCK2R) antagonist, in MIA PaCa-2 cells and in a subcutaneous xenograft mice model. MATERIALS AND METHODS: The anti-tumor effects of Z-360 and/or gemcitabine were monitored using a MIA PaCa-2 xenograft model. The effect of Z-360 on apoptosis in the model was examined by TUNEL staining and real-time PCR analysis and the effect in MIA PaCa-2 cells stably expressing human CCK2R was also evaluated by caspase-3/7 activity. RESULTS: In this xenograft model, Z-360 significantly reduced the tumor weight, increased TUNEL-positive cells and suppressed the expression of anti-apoptosis factors such as survivin, XIAP and Mcl-1, and these effects of Z-360 combined with gemcitabine were more effective. Furthermore, gastrin-17 and gastrin-34 inhibited apoptosis in vitro and Z-360 dose-dependently abrogated this effect. CONCLUSION: These results suggest that Z-360 exerts an anti-tumor effect through a reduction in anti-apoptosis factors by blocking CCK2R.