Search Results (13)

The stomach has been a highly conserved organ throughout vertebrate evolution; however, there are now over 20 lineages composed of monotremes, lungfish and teleost fish displaying a secondary loss of stomach function and morphology. This “agastric phenotype” has evolved convergently and is typified by a loss of gastric glands and gastric acid secretion and a near-to-complete loss of storage capacity of the stomach. All agastric species have lost the genes for gastric enzymes (Pga and Pgc) and proton pump subunits (Atp4a and Atp4b), and gastrin (Gast) has been lost in monotremes. As a key gastric hormone, the conservation of gastrin has not yet been investigated in the lungfish or agastric teleosts, and it is unclear how the loss of gastrin affects the evolution and selection of the native receptor (Cckbr), gastrin-releasing peptide (Grp) and gastrin-releasing peptide receptor (Grpr) in vertebrates. Furthermore, there are still many genes implicated in gastric development and function which have yet to be associated with the agastric phenotype. We analysed the evolution, selection and conservation of the gastrin pathway and a novel gastric gene repertoire (Gkn1, Gkn2, Tff1, Tff2, Vsig1 and Anxa10) to determine the correlation with the agastric phenotype. We found that the loss of gastrin or its associated genes does not correlate with the agastric phenotype, and their conservation is due to multiple pleiotropic roles throughout vertebrate evolution. We found a loss of the gastric gene repertoire in the agastric phenotype, except in the echidna, which retained several genes (Gkn1, Tff2 and Vsig1). Our findings suggest that the gastrin physiological pathway evolved differently in pleiotropic roles throughout vertebrate evolution and support the convergent evolution of the agastric phenotype through shared independent gene-loss events. Full article

Knowledge of how novel antigens or dietary stimuli affect stomach development and function in pigs remains limited. This study aimed to investigate stomach characteristics, parietal cell numbers, and the expression of genes essential to the functioning of the fundic and pyloric gland regions at weaning compared to seven days post-weaning and to examine whether maternal probiotic supplementation or piglet dietary tryptophan (Trp) levels influence these stomach parameters. This study has a 2 × 3 factorial design, with 48 sows assigned to one of two diets: basal or basal supplemented with Bacillus subtilis and Bacillus amyloliquefaciens. Their litters received creep diets containing 0.22, 0.27, or 0.33% standardized ileal digestible (SID) Trp. In total, 96 pigs were sacrificed for gastric sampling, 48 on the day of weaning and 48 on day 7 post-weaning. At 7 days post-weaning, pigs had an increased number of parietal cells and expression of parietal cell activity and digestive enzyme (PGA5 and CHIA) genes in the fundic gland region (p < 0.05), although the expression of signaling molecules involved in the regulation of acid secretion was unchanged in the fundic gland region (p > 0.05) and reduced in the pyloric gland region (p < 0.05), compared to the day of weaning. Overall, maternal probiotic supplementation had a significant impact on gene expression in the fundic gland region of the offspring, elevating several genes related to parietal cell activity (CLIC6, HRH2, KCNE1, KCNQ1, CHRM3, CCKBR, and SSTR2) (p < 0.05). Additionally, there were time × maternal interactions, where certain acid secretion pathway (ATP4A and HDC), chitinase enzyme (CHIA), and ghrelin (GHRL) genes were increased in offspring from probiotic sows compared to control sows at weaning (p < 0.05), but not at 7 days post-weaning (p > 0.05). Maternal probiotic supplementation did not influence growth performance pre-weaning or during the 7-day post-weaning period. There was a limited effect of creep Trp level or maternal × creep interactions on performance, gene expression, or parietal cell counts. Low pre-weaning creep intake may have confounded this analysis. In conclusion, maternal probiotic supplementation accelerated the maturation of the offspring’s stomach, particularly in terms of the expression of genes linked to acid secretion from parietal cells. Full article

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III–IV than stage I–II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion. Full article

Despite playing a key role in digestion, there is only a broad characterization of the spatiotemporal development of the three glandular regions of the stomach (cardiac, fundic and pyloric) in the weaned pig. Hence, the objective of this experiment was to explore the differential expression (DE) of a panel of key genes within the three glandular regions of the stomach. Eight pigs were sacrificed at d 8 post-weaning, and three mucosal samples were collected from each stomach’s glandular regions. The expression of a panel of genes were measured using QPCR. The true cardiac gland region was characterized by increased expression of PIGR, OLFM4, CXCL8 and MUC2 relative to the two other regions (p < 0.05). The fundic gland region was characterized by increased expression of ATP4A, CLIC6, KCNQ1, HRH2, AQP4, HDC, CCKBR, CHIA, PGA5, GHRL and MBOAT4 compared to the two other regions (p < 0.05). The pyloric gland region was characterized by exclusive expression of GAST (p < 0.05). A transition region between the cardiac and fundic region (cardiac-to-oxyntic transition) was observed with a gene expression signature that resembles a cross of the signatures found in the two regions. In conclusion, unique gene expression signatures were identifiable in each of the glandular regions, with a cardiac-to-oxyntic transition region clearly identifiable in the post-weaned pigs’ stomachs. Full article

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 K_D = 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. Full article

In order to optimize elevated kidney retention of previously reported minigastrin derivatives, we substituted (R)-DOTAGA by DOTA in (R)-DOTAGA-rhCCK-16/-18. CCK-2R-mediated internalization and affinity of the new compounds were determined using AR42J cells. Biodistribution and µSPECT/CT imaging studies at 1 and 24 h p.i. were carried out in AR42J tumor-bearing CB17-SCID mice. Both DOTA-containing minigastrin analogs exhibited 3- to 5-fold better IC₅₀ values than their (R)-DOTAGA-counterparts. ^natLu-labeled peptides revealed higher CCK-2R affinity than their ^natGa-labeled analogs. In vivo, tumor uptake at 24 h p.i. of the most affine compound, [¹⁹F]F-[¹⁷⁷Lu]Lu-DOTA-rhCCK-18, was 1.5- and 13-fold higher compared to its (R)-DOTAGA derivative and the reference compound, [¹⁷⁷Lu]Lu-DOTA-PP-F11N, respectively. However, activity levels in the kidneys were elevated as well. At 1 h p.i., tumor and kidney accumulation of [¹⁹F]F-[¹⁷⁷Lu]Lu-DOTA-rhCCK-18 and [¹⁸F]F-[^natLu]Lu-DOTA-rhCCK-18 was high. We could demonstrate that the choice of chelators and radiometals has a significant impact on CCK-2R affinity and thus tumor uptake of minigastrin analogs. While elevated kidney retention of [¹⁹F]F-[¹⁷⁷Lu]Lu-DOTA-rhCCK-18 has to be further addressed with regard to radioligand therapy, its radiohybrid analog, [¹⁸F]F-[^natLu]Lu-DOTA-rhCCK-18, might be ideal for positron emission tomography (PET) imaging due to its high tumor accumulation at 1 h p.i. and the attractive physical properties of fluorine-18. Full article

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15–20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC. Full article

Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRAS^G12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-Kras^G12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues. Full article

In order to enable ¹⁸F- and ¹⁷⁷Lu-labelling within the same molecule, we introduced a silicon-based fluoride acceptor (SiFA) into the hexa-D-glutamate chain of DOTA-PP-F11N. In addition, minigastrin analogues with a prolonged as well as γ-linked D-glutamate chain were synthesised and evaluated. CCK-2R affinity (IC₅₀, AR42J cells) and lipophilicity (logD_7.4) were determined. Biodistribution studies at 24 h post-injection (p.i.) and µSPECT/CT imaging at 1, 4 and 24 h p.i. were carried out in AR42J tumour-bearing CB17-SCID mice. CCK-2R affinity of (R)-DOTAGA-rhCCK-1 to 18 was enhanced with increasing distance between the SiFA building block and the binding motif. Lipophilicity of [¹⁷⁷Lu]Lu-(R)-DOTAGA-rhCCK-1 to 18 was higher compared to that of [¹⁷⁷Lu]Lu-DOTA-PP-F11N and [¹⁷⁷Lu]Lu-CP04. The respective α- and γ-linked rhCCK derivatives revealing the highest CCK-2R affinity were further evaluated in vivo. In comparison with [¹⁷⁷Lu]Lu-DOTA-PP-F11N, [¹⁷⁷Lu-]Lu-(R)-DOTAGA-rhCCK-9 and -16 exhibited three- to eight-fold increased activity levels in the tumour at 24 h p.i. However, activity levels in the kidneys were elevated as well. We could show that the introduction of a lipophilic SiFA moiety into the hydrophilic backbone of [¹⁷⁷Lu]Lu-DOTA-PP-F11N led to a decelerated blood clearance and thus improved tumour retention. However, elevated kidney retention has to be addressed in future studies. Full article

This study aimed to provide insights into molecular regulation and mitochondrial functionality under hypoxia by exploring the mechanism of adaptation to hypoxia, blood indexes, tissue morphology, mRNA/miRNA regulation, mitochondrial dynamics, and functional changes in Tibetan sheep raised at different altitudes. With regard to blood indexes and myocardial morphology, the HGB, HCT, CK, CK-MB, LDH, LDH1, SOD, GPX, LDL level, and myocardial capillary density were significantly increased in the sheep at higher altitudes (p < 0.05). The RNA-seq results suggested the DEmRNAs and DEmiRNAs are mainly associated with the PI3K-Akt, Wnt, and PPAR signaling pathways and with an upregulation of oncogenes (CCKBR, GSTT1, ARID5B) and tumor suppressor factors (TPT1, EXTL1, ITPRIP) to enhance the cellular metabolism and increased ATP production. Analyzing mRNA–miRNA coregulation indicated the mitochondrial dynamics and functions to be significantly enriched. By analyzing mitochondrial dynamics, mitochondrial fusion was shown to be significantly increased and fission significantly decreased in the heart with increasing altitude (p < 0.05). There was a significant increase in the density of the mitochondria, and a significant decrease in the average area, aspect ratio, number, and width of single mitochondrial cristae with increasing altitudes (p < 0.05). There was a significant increase in the NADH, NAD+ and ATP content, NADH/NAD+ ratio, and CO activity, while there was a significant decrease in SDH and CA activity in various tissues with increasing altitudes (p < 0.05). Accordingly, changes in the blood indexes and myocardial morphology of the Tibetan sheep were found to improve the efficiency of hemoglobin-carrying oxygen and reduce oxidative stress. The high expression of oncogenes and tumor suppressor factors might facilitate cell division and energy exchange, as was evident from enhanced mitochondrial fission and OXPHOS expression; however, it reduced the fusion and TCA cycle for the further rapid production of ATP in adaptation to hypoxia stress. This systematic study has for the first time delineated the mechanism of hypoxia adaptation in the heart of Tibetan sheep, which is significant for improving the ability of the mammals to adapt to hypoxia and for studying the dynamic regulation of mitochondria during hypoxia conditions. Full article

The inhibition of the mammalian target of rapamycin complex 1 (mTORC1) by everolimus (RAD001) was recently shown to enhance the tumor uptake of radiolabeled minigastrin. In this paper, we investigate if this finding can improve the in vivo therapeutic response to [¹⁷⁷Lu]Lu-PP-F11N treatment. The N-terminal DOTA-conjugated gastrin analogue PP-F11N (DOTA-(DGlu)₆-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) was used to evaluate treatment efficacy in the human A431/CCKBR xenograft nude mouse model in combination with RAD001. Both RAD001 and [¹⁷⁷Lu]Lu-PP-F11N single treatments as well as their combination inhibited tumor growth and increased survival. In concomitantly treated mice, the average tumor size and median survival time were significantly reduced and extended, respectively, as compared to the monotherapies. The histological analysis of kidney and stomach dissected after treatment with RAD001 and [¹⁷⁷Lu]Lu-PP-F11N did not indicate significant adverse effects. In conclusion, our study data demonstrate the potential of mTORC1 inhibition to substantially improve the therapeutic efficacy of radiolabeled minigastrin analogues in CCKBR-positive cancers. Full article

Survival from pancreatic cancer remains extremely poor, in part because this malignancy is not diagnosed in the early stages, and precancerous pancreatic intraepithelial neoplasia (PanIN) lesions are not seen on routine radiographic imaging. Since the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in PanIN lesions, it may serve as a target for early detection. We developed a biodegradable fluorescent polyplex nanoparticle (NP) that selectively targets the CCK-BR. The NP was complexed to a fluorescent oligonucleotide with Alexa Fluor 647 for far-red imaging and to an oligonucleotide conjugated to Alexa Fluor 488 for localization by immunohistochemistry. Fluorescence was detected over the pancreas of five- to ten-month-old LSL-Kras^G12D/+; P48-Cre (KC) mice only after the injection of the receptor target-specific NP and not after injection of untargeted NP. Ex vivo tissue imaging and selective immunohistochemistry confirmed particle localization only to PanIN lesions in the pancreas and not in other organs, supporting the tissue specificity. A human pancreas tissue microarray demonstrated immunoreactivity for the CCK-BR only in the PanIN lesions and not in normal pancreas tissue. The long-term goal would be to develop this imaging tool for screening human subjects at high risk for pancreatic cancer to enable early cancer detection. Full article

The overexpression of cholecystokinin B receptor (CCKBR) in human cancers led to the development of radiolabeled minigastrin analogues for targeted radionuclide therapy, which aims to deliver cytotoxic radiation specifically to cancer cells. Alpha emitters (e.g., actinium-225) possess high potency in cancer cell-killing and hold promise for the treatment of malignant tumors. In these preclinical studies, we developed and evaluated CCKBR-targeted alpha particle therapy. The cellular uptake and cytotoxic effect of actinium-225 labeled and HPLC-purified minigastrin analogue [²²⁵Ac]Ac-PP-F11N were characterized in the human squamous cancer A431 cells transfected with CCKBR. Nude mice bearing A431/CCKBR tumors were used for biodistribution and therapy studies followed by histological analysis and SPECT/CT imaging. In vitro, [²²⁵Ac]Ac-PP-F11N showed CCKBR-specific and efficient internalization rate and potent cytotoxicity. The biodistribution studies of [²²⁵Ac]Ac-PP-F11N revealed CCKBR-specific uptake in tumors, whereas the therapeutic studies demonstrated dose-dependent inhibition of tumor growth and extended mean survival time, without apparent toxicity. The histological analysis of kidney and stomach indicated no severe adverse effects after [²²⁵Ac]Ac-PP-F11N administration. The post-therapy SPECT-CT images with [¹¹¹In]In-PP-F11N confirmed no CCKBR-positive tumor left in the mice with complete remission. In conclusion, our study demonstrates therapeutic efficacy of [²²⁵Ac]Ac-PP-F11N without acute radiotoxicity in CCKBR-positive cancer model. Full article