CCK-B Receptor Research Articles

Synthesis and biological evaluation of novel isobenzofuran-1(3H)-one derivatives as antidepressant agents

A series of novel isobenzofuran-1(3H)-one derivatives were designed and synthesized as antidepressants. Firstly, the serotonin reuptake inhibition of these compounds was tested in vitro, and most of them exhibited activity. Particularly, compounds 9d, 10a, and 10c demonstrated superior inhibitory effects and possibly avoided addiction via the μ-opioid receptor and CCK-B receptor. Secondly, the antidepressant effect of compound 10a was evaluated using chronic restraint stress (CRS)-induced mice. The results showed that compound 10a significantly improved CRS-induced depression-like behavior by increasing the neurotransmitters 5-HT in the cortex and THP2 expression in the hippocampus. Thirdly, compound 10a was further investigated and found to enhance CRS-induced hippocampal neuron damage recovery and elevate the expression of synaptic-associated proteins such as BDNF, TrkB, PSD95, and Spinophilin in CRS-induced mice. These findings suggested that novel isobenzofuran-1(3H)-one derivative showed efficacy in treating depression, with compound 10a emerging as a potential lead compound warranting further investigation.

Role of Spinal Cholecystokinin Octapeptide, Nociceptin/Orphanin FQ, and Hemokinin-1 in Diabetic Allodynia.

A complication of diabetes is neuropathic pain, which is difficult to control with medication. We have confirmed that neuropathic pain due to mechanical allodynia in diabetic mice is mediated by a characteristic neuropeptide in the spinal cord. We evaluated the strength of mechanical allodynia in mice using von Frey filaments. When mice were intravenously injected with streptozotocin, mechanical allodynia appeared 3 days later. Antibodies of representative neuropeptides were intrathecally (i.t.) administered to allodynia-induced mice 7 days after the intravenous administration of streptozotocin, and allodynia was reduced by anti-cholecystokinin octapeptide antibodies, anti-nociceptin/orphanin FQ antibodies, and anti-hemokinin-1 antibodies. In contrast, i.t.-administered anti-substance P antibodies, anti-somatostatin antibodies, and anti-angiotensin II antibodies did not affect streptozotocin-induced diabetic allodynia mice. Mechanical allodynia was attenuated by the i.t. administration of CCK-B receptor antagonists and ORL-1 receptor antagonists. The mRNA level of CCK-B receptors in streptozotocin-induced diabetic allodynia mice increased in the spinal cord, but not in the dorsal root ganglion. These results indicate that diabetic allodynia is caused by cholecystokinin octapeptide, nociceptin/orphanin FQ, and hemokinin-1 released from primary afferent neurons in the spinal cord that transmit pain to the brain via the spinal dorsal horn.

Cholecystokinin B receptor agonists alleviates anterograde amnesia in cholecystokinin-deficient and aged Alzheimer's disease mice

BackgroundAs one major symptom of Alzheimer’s disease (AD), anterograde amnesia describes patients with an inability in new memory formation. The crucial role of the entorhinal cortex in forming new memories has been well established, and the neuropeptide cholecystokinin (CCK) is reported to be released from the entorhinal cortex to enable neocortical associated memory and long-term potentiation. Though several studies reveal that the entorhinal cortex and CCK are related to AD, it is less well studied. It is unclear whether CCK is a good biomarker or further a great drug candidate for AD.MethodsmRNA expressions of CCK and CCK-B receptor (CCKBR) were examined in two mouse models, 3xTg AD and CCK knock-out (CCK−/−) mice. Animals’ cognition was investigated with Morris water maze, novel object recognition test and neuroplasticity with in-vitro electrophysiological recording. Drugs were given intraperitoneally to animals to investigate the rescue effects on cognitive deficits, or applied to brain slices directly to explore the influence in inducement of long-term potentiation.ResultsAged 3xTg AD mice exhibited reduced CCK mRNA expression in the entorhinal cortex but reduced CCKBR expression in the neocortex and hippocampus, and impaired cognition and neuroplasticity comparable with CCK−/− mice. Importantly, the animals displayed improved performance and enhanced long-term potentiation after the treatment of CCKBR agonists.ConclusionsHere we provide more evidence to support the role of CCK in learning and memory and its potential to treat AD. We elaborated on the rescue effect of a promising novel drug, HT-267, on aged 3xTg AD mice. Although the physiological etiology of CCK in AD still needs to be further investigated, this study sheds light on a potential pharmaceutical candidate for AD and dementia.

Cholecystokinin facilitates motor skill learning by modulating neuroplasticity in the motor cortex.

Cholecystokinin (CCK) is an essential modulator for neuroplasticity in sensory and emotional domains. Here, we investigated the role of CCK in motor learning using a single pellet reaching task in mice. Mice with a knockout of Cck gene (Cck-/-) or blockade of CCK-B receptor (CCKBR) showed defective motor learning ability; the success rate of retrieving reward remained at the baseline level compared to the wildtype mice with significantly increased success rate. We observed no long-term potentiation upon high-frequency stimulation in the motor cortex of Cck-/- mice, indicating a possible association between motor learning deficiency and neuroplasticity in the motor cortex. In vivo calcium imaging demonstrated that the deficiency of CCK signaling disrupted the refinement of population neuronal activity in the motor cortex during motor skill training. Anatomical tracing revealed direct projections from CCK-expressing neurons in the rhinal cortex to the motor cortex. Inactivation of the CCK neurons in the rhinal cortex that project to the motor cortex bilaterally using chemogenetic methods significantly suppressed motor learning, and intraperitoneal application of CCK4, a tetrapeptide CCK agonist, rescued the motor learning deficits of Cck-/- mice. In summary, our results suggest that CCK, which could be provided from the rhinal cortex, may surpport motor skill learning by modulating neuroplasticity in the motor cortex.

Rapid Generation and Molecular Docking Analysis of Single-Chain Fragment Variable (scFv) Antibody Selected by Ribosome Display Targeting Cholecystokinin B Receptor (CCK-BR) for Reduction of Chronic Neuropathic Pain.

A robust cell-free platform technology, ribosome display in combination with cloning, expression, and purification was utilized to develop single chain Fragment variable (scFv) antibody variants as pain therapy directed at the mouse cholecystokinin B (CCK-B) receptor. Three effective CCK-B peptide-specific scFvs were generated through ribosomal display technology. Soluble expression and ELISA analysis showed that one antibody, scFv77-2 had the highest binding and could be purified from bacterial cells in large quantities. Octet measurements further revealed that the CCK-B scFv77-2 antibody had binding kinetics of KD = 1.794 × 10-8 M. Molecular modeling and docking analyses suggested that the scFv77-2 antibody shaped a proper cavity to embed the whole CCK-B peptide molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force, and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic intermolecular interactions and functional in vivo studies of CCK-BR. The high affinity scFv77-2 antibody showed good efficacy with binding to CCK-BR tested in a chronic pain model. In vivo studies validated the efficacy of the CCK-B receptor (CCK-BR) scFv77-2 antibody as a potential therapy for chronic trigeminal nerve injury-induced pain. Mice were given a single dose of the CCK-B receptor (CCK-BR) scFv antibody 3 weeks after induction of a chronic trigeminal neuropathic pain model, during the transition from acute to chronic pain. The long-term effectiveness for the reduction of mechanical hypersensitivity was evident, persisting for months. The anxiety- and depression-related behaviors typically accompanying persisting hypersensitivity subsequently never developed in the mice given CCK-BR scFv. The effectiveness of the antibody is the basis for further development of the lead CCK-BR scFv as a promising non-opioid therapeutic for chronic pain and the long-term reduction of chronic pain- and anxiety-related behaviors.

Cholecystokinin B receptor antagonists for the treatment of depression via blocking long-term potentiation in the basolateral amygdala.

Depression is a common and severe mental disorder. Evidence suggested a substantial causal relationship between stressful life events and the onset of episodes of major depression. However, the stress-induced pathogenesis of depression and the related neural circuitry is poorly understood. Here, we investigated how cholecystokinin (CCK) and CCKBR in the basolateral amygdala (BLA) are implicated in stress-mediated depressive-like behavior. The BLA mediates emotional memories, and long-term potentiation (LTP) is widely considered a trace of memory. We identified that the cholecystokinin knockout (CCK-KO) mice impaired LTP in the BLA, while the application of CCK4 induced LTP after low-frequency stimulation (LFS). The entorhinal cortex (EC) CCK neurons project to the BLA and optogenetic activation of EC CCK afferents to BLA-promoted stress susceptibility through the release of CCK. We demonstrated that EC CCK neurons innervate CCKBR cells in the BLA and CCK-B receptor knockout (CCKBR-KO) mice impaired LTP in the BLA. Moreover, the CCKBR antagonists also blocked high-frequency stimulation (HFS) induced LTP formation in the BLA. Notably, CCKBR antagonists infusion into the BLA displayed an antidepressant-like effect in the chronic social defeat stress model. Together, these results indicate that CCKBR could be a potential target to treat depression.

Cholecystokinin Signaling can Rescue Cognition and Synaptic Plasticity in the APP/PS1 Mouse Model of Alzheimer's Disease.

Synaptic impairment and loss are an important pathological feature of Alzheimer's disease (AD). Memory is stored in neural networks through changes in synaptic activity, and synaptic dysfunction can cause cognitive dysfunction and memory loss. Cholecystokinin (CCK) is one of the major neuropeptides in the brain, and plays a role as a neurotransmitter and growth factor. The level of CCK in the cerebrospinal fluid is decreased in AD patients. In this study, a novel CCK analogue was synthesized on the basis of preserving the minimum bioactive fragment of endogenous CCK to investigate whether the novel CCK analogue could improve synaptic plasticity in the hippocampus of the APP/PS1 transgenic mouse model of AD and its possible molecular biological mechanism. Our study found that the CCK analogue could effectively improve spatial learning and memory, enhance synaptic plasticity in the hippocampus, normalize synapse numbers and morphology and the levels of key synaptic proteins, up-regulate the PI3K/Akt signaling pathway and normalize PKA, CREB, BDNF and TrkB receptor levels in APP/PS1 mice. The amyloid plaque load in the brain was reduced by CCK, too. The use of a CCKB receptor antagonist and targeted knockdown of the CCKB receptor (CCKBR) attenuated the neuroprotective effect of the CCK analogue. These results demonstrate that the neuroprotective effect of CCK analogue is achieved by activating the PI3K/Akt as well as the PKA/CREB-BDNF/TrkB signaling pathway that leads to protection of synapses and cognition.

Cholecystokinin Receptor Antagonist Induces Pancreatic Stellate Cell Plasticity Rendering the Tumor Microenvironment Less Oncogenic.

CCK receptors are expressed on pancreatic cancer epithelial cells, and blockade with receptor antagonists decreases tumor growth. Activated pancreatic stellate cells or myofibroblasts have also been described to express CCK receptors, but the contribution of this novel pathway in fibrosis of the pancreatic cancer microenvironment has not been studied. We examined the effects of the nonselective CCK receptor antagonist proglumide on the activation, proliferation, collagen deposition, differential expression of genes, and migration in both murine and human PSCs. CCK receptor expression was examined using western blot analysis. Collagen production using activated PSCs was analyzed by mass spectroscopy and western blot. Migration of activated PSCs was prevented in vitro by proglumide and the CCK-B receptor antagonist, L365,260, but not by the CCK-A receptor antagonist L365,718. Proglumide effectively decreased the expression of extracellular matrix-associated genes and collagen-associated proteins in both mouse and human PSCs. Components of fibrosis, including hydroxyproline and proline levels, were significantly reduced in PSC treated with proglumide compared to controls. CCK peptide stimulated mouse and human PSC proliferation, and this effect was blocked by proglumide. These investigations demonstrate that targeting the CCK-B receptor signaling pathway with proglumide may alter the plasticity of PSC, rendering them more quiescent and leading to a decrease in fibrosis in the pancreatic cancer microenvironment.

High resolution retrograde tracing, morphological analysis, RNA sequencing and differential expression analysis of identified vagal afferent neurons innervating the muscle or mucosa in 3 different stomach regions in the rat

The functions of the stomach are powerfully influenced by the vagus nerve and vago‐vagal reflexes. The vagal afferent neurons that innervate the stomach and monitor its functional state have clearly demonstrated roles in appetite regulation, gastric accommodation and gastric emptying. In mammals with single compartment stomachs, including humans, the stomach can be divided into functional regions and the muscle and mucosal layers show region‐specific specializations. Electrophysiological and morphological studies suggest that the vagal sensory axons innervating the different regions and layers of the stomach wall have distinct properties and participate in different types of reflex controls. We therefore hypothesized that these functional and structural differences would be reflected in distinct gene expression profiles of afferent neurons, depending on the stomach region and tissue innervated. We developed methods to selectively retrogradely label afferent neurons innervating the muscle or mucosa in the corpus and antrum and the muscle in the fundus in the rat, using local microinjections of fluorescent nanospheres that diffuse minimally. Up to 30 microinjections of 30‐100nL of tracer beads were made into either the muscle or the mucosa in one stomach region in each rat under anesthesia. The animal was allowed to recover and kept for 1‐2 weeks before being humanely killed and tissues harvested. Labelled neurons were studied in situ in the nodose ganglion using confocal microscopy, immunohistochemistry and fluorescence in situ hybridization, and individual neurons collected after dissociation for RNAseq analysis. RNA sequencing was performed using pooled samples of 5‐12 neurons labelled from each region and deep sequencing (30‐50 million reads per sample), and then analyzed using minimal filtering to allow detection of genes expressed at low copy number.Nodose neurons labelled from the mucosa were significantly larger than those labelled from the muscle in both the corpus and antrum, whether quantified in situ or after dissociation, confirming that microinjections into the different stomach wall layers labelled different populations of neurons. Pairwise differential expression analyses of RNAseq data showed distinct mRNA expression profiles depending on the region innervated, and between muscle and mucosal innervating neurons in each region, based on Benjamini/Hochberg adjusted P values (Limma analysis tool). Some genes displayed region specific expression patterns while others showed tissue specific patterns (eg muscle vs mucosa) and some showed both. Among differentially expressed genes were some previously identified to specifically label gastric mucosal and muscle afferent neurons, such as somatostatin, CCKB receptor and GRP65. In addition, many other genes were differentially expressed and mRNA for genes known to be functional in vagal afferent neurons, such as ghrelin, leptin and galanin receptors, were detected. Differential expression patterns for various genes were confirmed in labelled neurons in situ using immunohistochemistry and/or in situhybridization. The data provide a rich resource for further studies of the visceral sensation in the different stomach regions and how region‐specific signaling might regulate appetite and gastric function in both normal and disease states.

Proglumide Reverses Nonalcoholic Steatohepatitis by Interaction with the Farnesoid X Receptor and Altering the Microbiome.

Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide’s interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.

The CCK-B receptor: A novel target for therapy of hepatocellular carcinoma.

466 Background: Today, the fastest growing cause of cancer-related death is hepatocellular carcinoma (HCC). Treatment with immune checkpoint antibodies has shown promise in HCC, but the response is still low at about 15%-20% as monotherapy. Our research laboratory discovered that the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in the injured/ inflamed liver and is up-regulated in HCC. The primary aim of this study is to evaluate the effects of a CCK-BR antagonist, proglumide, alone and in combination with a PD-1 antibody (PD-1 Ab) on survival in HCC tumor-bearing mice and the tumor infiltrating T-cells. The secondary aim was to evaluate the frequency of CCK-BR expression in human HCC. Methods: RNA was extracted from murine RIL-175 HCC cells and subjected to qRT-PCR for gene expression of CCK-BRs, gastrin, and PD-L1. C57BL/6 mice (N = 40) were injected with RIL-175 HCC cells (100,000) subcutaneously. After one week when the mice had palpable tumors, they were divided into 4 groups (N = 10ea) of equal tumor size. Treatment included: controls-untreated drinking water; proglumide (0.1mg/ml) in drinking water; PD-1Ab (50µg, ip, days 7, 14, 21 with untreated water); and combination therapy with PD-1 Ab (as above) and proglumide water. Mice were euthanized when tumors reached a diameter of 20mm according to IACUC guidelines. Tumors were stained with a CD8 antibody to evaluate CD8+ tumor infiltrating T-cells. CCK-BR protein expression in human liver tissues and human HCC was evaluated by immunohistochemistry using a tissue microarray (US Biomax). Results: RIL-175 murine HCC cells exhibited increased expression of CCK-BR, gastrin, and PD-L1 by qRT-PCR. Survival of mice treated with the combination of proglumide and the PD-1 Ab was double that of the control mice and greater than proglumide or PD-1 Ab monotherapy. Compared to tumors of control mice, tumors of mice receiving proglumide monotherapy exhibited a 5-fold increase in CD8+ cells (P = 2.5x10-8), and there was a 12-fold increase in CD8+ T-cells in tumors of mice treated with the combination therapy (P = 4.7x10-17). In the human liver tissue microarray, immunoreactivity for the CCK-BR was negative in the normal human liver tissues and increased with the grade of HCC tumor (P = 0.0045) with 84.6% of grade-3 human HCC tissues staining positive for CCK-BR. Conclusions: The CCK-BR may provide a novel target for therapy in HCC. Treatment with the CCK-BR antagonist, proglumide, in combination with a PD-1 Ab appears to be synergistic improving the survival of mice by increasing the number of intratumoral CD8+ T-cells.

Cholecystokinin B receptor gene polymorphism (rs2941026) is associated with anxious personality and suicidal thoughts in a longitudinal study.

Cholecystokinin is a neuropeptide with a role in the neurobiology of adaptive behaviour that is implicated in anxiety disorders, while the underlying mechanisms currently remain insufficiently explained. The rs2941026 variation in the cholecystokinin B receptor gene has previously been associated with trait anxiety. Our aim was to investigate associations between the CCKB receptor gene polymorphism rs2941026 with anxiety, personality, depressiveness and suicidality in a longitudinal study of late adolescence and early adulthood. We used reports on trait and state anxiety, depressiveness and suicidal thoughts, as well as Affective Neuroscience Personality Scales, from the two birth cohorts of the Estonian Children Personality, Behaviour and Health Study. We measured associations between the CCKBR gene rs2941026 and anxiety-related phenotypes both longitudinally and cross-sectionally at ages 15, 18, 25 and 33. Homozygosity for both alleles of the CCKBR rs2941026 was associated with higher trait and state anxiety in the longitudinal analysis. Cross-sectional comparisons were statistically significant at ages 18 and 25 for trait anxiety and at ages 25 and 33 for state anxiety. Higher depressiveness and suicidal thoughts were associated with the A/A genotype at age 18. Additionally, homozygosity for the A-allele was related to higher FEAR and SADNESS in the Affective Neuroscience Personality Scales. The genotype effects were more apparent in females, who displayed higher levels of negative affect overall. CCKBR genotype is persistently associated with negative affect in adolescence and young adulthood. The association of the CCKBR rs2941026 genotype with anxiety-related phenotypes is more pronounced in females.

Inhibition of Geranylgeranylacetone on cholecystokinin-B receptor, BDNF and dopamine D1 receptor induced by morphine

Morphine is the pain releasing and abusing drug. Morphine leads to addiction by activating dopaminergic rewarding system consisted of the ventral tegmental area (VTA) and nucleus accumbens (NAc). Cholecystokinin (CCK) is a gut–brain neuropeptide and involved in morphine dependence. Brain-derived neurotrophic factor (BDNF) is a neurotrophin and plays roles in regulating addiction. Geranylgeranylacetone (GGA) is a medicine of protecting gastric mucosal injury and protecting neurons. Our previous study showed that GGA blocked morphine-induced withdrawal and relapse through inducing thioredoxin 1(Trx1). In this study, we investigated that whether cholecystokinin-B receptor (CCKB receptor) and BDNF were related to GGA inhibition on morphine addiction. At first, we made conditioned place preference (CPP) model and confirmed again that GGA blocked the expression of morphine-CPP in present study. Then, our results showed that morphine increased the expressions of dopamine D1 receptor, tyrosine hydroxylase (TH), CCKB receptor and BDNF in the VTA and NAc in mice, which was inhibited by GGA. These results suggest that CCK and BDNF in dopaminergic systems are associated with the role of GGA blocking morphine-CPP.

Abstract 1931: Evaluation of cholecystokinin receptor expression and function in non-small cell lung cancer

Abstract Introduction: Lung cancer is the leading cause of cancer-related death in the United States, with nearly 85% of cases histologically classified as non-small cell lung cancer (NSCLC). A recent epidemiological study provides evidence of an increased risk ratio for NSCLC in association with adherence to a Western dietary pattern, which has a higher dietary fat content, compared to vegetarian or Tex-Mex dietary patterns. Our laboratory and others have measured increased tumor burden in murine models of NSCLC when feeding diets are high in fat compared to a standard rodent cereal diet. In our laboratory, increased tumor burden is associated with increased tumor multiplicity, but no change in proliferative markers (Ki-67 and pS6) were observed. Dietary fat increases both tumorigenesis in mouse models of pancreatic cancer and circulating levels of the gastrointestinal hormone cholecystokinin (CCK). Inhibition of CCK receptors antagonizes progression of precursor lesions in murine models of pancreatic cancer. Although CCK receptor expression has not been detected in normal human pulmonary tissue, the CCK-B receptor has been identified immunohistologically in human lung tumors. Further, the CCK-A receptor is expressed in normal murine pulmonary epithelium. Hypothesis: At concentrations approximating circulating levels in mice fed a high fat diet, CCK reduces apoptosis of NSCLC cells in vitro. Materials and Methods: The murine NSCLC cell line IO33 was grown in serum-replete RPMI 1640 or serum-deprived RPM 1640 with 0 to 1000 pM exogenous CCK-8 peptide added. Expression of CCK-A and CCK-B receptors were measured in IO33 and human NSCLC cell lines by qPCR. Apoptosis was assessed using a Caspase-3/7 peptide cleavage assay. Results: Expression of CCK-A and CCK-B receptors are reported in IO33 cells and 11 human lung cancer cell lines. Apoptosis of IO33 cells was significantly decreased by greater than 30% at CCK concentrations greater than 100pM (P < 0.03), which is the circulating concentration of CCK in mice fed a high fat diet. Conclusions: CCK receptors are expressed in human and murine NSCLC cells and circulating CCK may reduce NSCLC cell apoptosis. Citation Format: Alexander C. Miller, Shelby J. Subry, Manijeh M. Assar, Krista Pearman, Jeffrey William Norris. Evaluation of cholecystokinin receptor expression and function in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1931.

Aptamer-Targeted Calcium Phosphosilicate Nanoparticles for Effective Imaging of Pancreatic and Prostate Cancer

PurposeAccurate tumor identification and staging can be difficult. Aptamer-targeted indocyanine green (ICG)-nanoparticles can enhance near-infrared fluorescent imaging of pancreatic and prostate tumors and could improve early cancer detection. This project explored whether calcium-phosphosilicate nanoparticles, also known as NanoJackets (NJs), that were bioconjugated with a tumor-specific targeting DNA aptamer could improve the non-invasive detection of pancreatic and prostate tumors.MethodsUsing in vivo near-infrared optical imaging and ex vivo fluorescence analysis, DNA aptamer-targeted ICG-loaded NJs were compared to untargeted NJs for detection of tumors.ResultsNanoparticles were bioconjugated with the DNA aptamer AP1153, which binds to the CCK-B receptor (CCKBR). Aptamer bioconjugated NJs were not significantly increased in size compared with unconjugated nanoparticles. AP1153-ICG-NJ accumulation in orthotopic pancreatic tumors peaked at 18 h post-injection and the ICG signal was cleared by 36 h with no evidence on uptake by non-tumor tissues. Ex vivo tumor imaging confirmed the aptamer-targeted NJs accumulated to higher levels than untargeted NJs, were not taken up by normal pancreas, exited from the tumor vasculature, and were well-dispersed throughout pancreatic and prostate tumors despite extensive fibrosis. Specificity for AP1153-NJ binding to the CCK-B receptor on pancreatic tumor cells was confirmed by pre-treating tumor-bearing mice with the CCK receptor antagonist proglumide. Proglumide pre-treatment reduced the in vivo tumoral accumulation of AP1153-NJs to levels comparable to that of untargeted NJs.ConclusionThrough specific interactions with CCK-B receptors, tumor-targeted nanoparticles containing either ICG or rhodamine WT were well distributed throughout the matrix of both pancreatic and prostate tumors. Tumor-targeted NJs carrying various imaging agents can enhance tumor detection.

Targeting the Cholecystokinin Receptor: A Novel Approach for Treatment and Prevention of Hepatocellular Cancer.

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.

Distinct CCK-positive SFO neurons are involved in persistent or transient suppression of water intake

The control of water-intake behavior is critical for life because an excessive water intake induces pathological conditions, such as hyponatremia or water intoxication. However, the brain mechanisms controlling water intake currently remain unclear. We previously reported that thirst-driving neurons (water neurons) in the subfornical organ (SFO) are cholecystokinin (CCK)-dependently suppressed by GABAergic interneurons under Na-depleted conditions. We herein show that CCK-producing excitatory neurons in the SFO stimulate the activity of GABAergic interneurons via CCK-B receptors. Fluorescence-microscopic Ca2+ imaging demonstrates two distinct subpopulations in CCK-positive neurons in the SFO, which are persistently activated under hyponatremic conditions or transiently activated in response to water drinking, respectively. Optical and chemogenetic silencings of the respective types of CCK-positive neurons both significantly increase water intake under water-repleted conditions. The present study thus reveals CCK-mediated neural mechanisms in the central nervous system for the control of water-intake behaviors.

Contribution of Histamine to Nociceptive Behaviors Induced by Intrathecally Administered Cholecystokinin-8.

The involvement of spinal release of histamine in the nociceptive behaviors induced by cholecystokinin-8 (CCK-8) was investigated in mice. Intrathecal (i.t.) injection of CCK-8 elicited the nociceptive behaviors consisting of biting and licking. The nociceptive behaviors induced by i.t. treatment with CCK-8 showed two bell-shaped patterns. The histamine H3 receptor antagonist significantly promoted the nociceptive behaviors induced by CCK-8 at doses of 1–100 fmol and 100 pmol. The nociceptive behaviors elicited by CCK-8 was inhibited by i.t. administration of the CCK-B receptor antagonist in a dose-dependent manner, but not by the CCK-A receptor antagonist. The nociceptive behaviors induced by CCK-8 were markedly suppressed by i.t. pretreatment with antiserum against histamine and were abolished in histidine decarboxylase-deleted gene mice. In histamine H1 receptor-deleted gene mice, the nociceptive behaviors induced at both 10 amol and 10 pmol of CCK-8 were not affected. The tachykinin neurokinin-1 (NK1) receptor antagonists inhibited CCK-8 (10 pmol)-induced nociceptive behaviors in a dose-dependent manner. CCK-8 (10 amol)-induced nociceptive behaviors was not antagonized by co-administration with the tachykinin NK1 receptor antagonists. The nociceptive behaviors elicited by CCK-8 were inhibited by i.t. administration of the antagonist for the N-methyl-D-aspartate (NMDA) receptor in a dose-dependent manner. Our results suggest that the nociceptive behaviors induced by i.t. administration of CCK-8 (10 pmol) are mediated through the spinal release of histamine and are elicited via activation of the tachykinin NK1 and NMDA receptors, whereas the nociceptive behaviors induced by i.t. administration of CCK-8 (10 amol) are mediated through the spinal release of histamine and elicited via NMDA receptor activation.

Abstract 5193: Cholecystokinin-B receptor mediates growth of hepatocellular carcinoma

Abstract Background: The fastest-growing cause of cancer-related death is hepatocellular carcinoma (HCC), which is in part attributable to the obesity epidemic and nonalcoholic steatohepatitis (NASH). The gastrointestinal (GI) peptides, gastrin and cholecystokinin (CCK), regulate digestive functions and growth of the GI tract through the G-protein coupled receptors CCK- receptors (CCK-Rs). Both peptides activate the CCK-B receptor (CCK-BR) whereas only CCK peptide activates the CCK-A receptor (CCK-AR). CCK-Rs and gastrin are low or absent in normal mouse hepatocytes. We have previously shown that CCK blood levels increase and CCK-Rs become overexpressed in the livers of mice eating a high saturated fat diet that induced NASH and HCC. Hypothesis: CCK and gastrin mediate the growth of HCC through the CCK-R and that interruption of this signaling pathway could decrease growth of HCC. Methods: RNA was extracted from murine Hepa1-6, RIL-175, and human HepG2 cells and was evaluated by qRT-PCR for expression of CCK-AR, CCK-BR, and gastrin. CCK-R protein expression was analyzed by flow cytometry. In order to determine which CCK-R mediated the growth effects, growth was studied in vitro using selective receptor antagonists or CRISPR Cas9 technology to knock-out each receptor. HCC cells were treated in vitro with CCK peptide (1-10nM), the CCK-AR antagonist (L364,718), or the CCK-BR antagonist (L365,260). Proliferation of selective CCK-R KO cells was compared to that of wild-type cells. To determine the effect of a CCK-R antagonist on tumor growth in vivo, two cohorts of mice bearing subcutaneous Hepa1-6 or RIL-175 HCC tumors were treated with an oral bioavailable CCK-R antagonist, proglumide (1 mg/ml) or untreated water (controls) for 3-4 weeks. The mice bearing Hepa1-6 tumors were placed on a high-fat diet to raise blood CCK levels. Mice bearing RIL-175 tumors were fed standard chow to determine if proglumide could block autocrine growth by gastrin. Results: The mRNA expression of CCK-AR, CCK-BR and gastrin were increased 80-90-fold in all HCC cell lines compared to that of normal liver. CCK-BRs were detected on >85% of the cells by flow cytometry. CCK peptide (1nM) stimulated HCC growth in vitro in both wild-type cells and in CCK-AR KO cells but not in CCK-BR KO cells. CCK-BR antagonist blocked CCK-stimulated growth in vitro but the CCK-AR antagonist did not, suggesting that the CCK-BR was responsible for mediating proliferation. In vivo tumor growth was significantly reduced with proglumide treatment by 70% (p<0.05) in Hepa1-6 and by 73% (p<0.001) in RIL-75 tumors, respectively. Conclusion: CCK-Rs are overexpressed in HCC and proliferation appears to be mediated through the CCK-BR. Downregulation with CRISPR Cas9 or blockade of the CCK-BR with an antagonist decreases growth in vitro and proglumide therapy decreases tumor growth in vivo. Strategies that block signaling at the CCK-BR may provide a novel therapeutic option for HCC treatment. Citation Format: Martha D. Gay, Anita Safronenka, Hong Cao, Robin Tucker, Narayan Shivapurkar, Jill P. Smith. Cholecystokinin-B receptor mediates growth of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5193.

4119 Cholecystokinin (CCK) Receptor Antagonist Reverses Nonalcoholic Steatohepatitis (NASH) by Reducing Hepatic Macrophages and Inflammatory Cytokines

OBJECTIVES/GOALS: NASH increases the risk of cirrhosis and liver cancer. High-fat diets increase CCK levels and CCK receptors have been identified on fibroblasts and immune cells. We hypothesized that CCK receptor blockade could prevent NASH by altering the hepatic microenvironment and macrophage activation. METHODS/STUDY POPULATION: Female mice were fed a Choline Deficient Ethionine supplemented (CDE) saturated fat diet or control high-fat diet for 18 weeks. Mice in each group were treated with a CCK receptor antagonist, proglumide (0.1 mg/ml) in the drinking water or regular water. Resected livers were stained for H&E for features of NASH and F4/80 for macrophages analysis. Liver RNA was evaluated for the expression of cytokines and chemokines using an 84-gene Profiler array (Qiagen). Oxidative stress was analyzed by qRT-PCR for heat shock proteins (HSPs) 27, 60, 70 and 90 and for glutathione by a fluorometric assay. Differences in CDE fed and CDE/proglumide-treated mouse livers were evaluated. RESULTS/ANTICIPATED RESULTS: Livers from mice on the CDE diet displayed histologic features of NASH that were prevented by proglumide. Cytokines and chemokines expression, especially CCL20 and CCL2, were increased in the CDE fed mice and these levels were reduced greater than 20-fold with proglumide. Infiltration of F4/80+ macrophages was markedly increased in the CDE livers and these were reduced by > 50% (p < 0.0001) with proglumide. RNA expression of HSP70 (p = 0.006) and HSP27 (p = 0.011) were reduced with proglumide. Hepatic glutathione concentration more than doubled in the CDE/proglumide treated mice compared to CDE mice. CCK-B receptor expression increased in the CDE-fed mouse livers compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CCK receptor blockade decreases NASH by reducing hepatic macrophages, oxidative stress, and blocking inflammatory cytokines and chemokines. This data supports our novel hypothesis that CCK receptors play a role in NASH and proglumide may provide an innovative treatment for this condition.