Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes.

PubMed

Sekar, M C; Sambandam, V; McDonald, J M

1993-05-14

Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery

PubMed Central

Liu, Shan; Yang, Hao; Wan, Lin; Cai, Hua-wei; Li, Sheng-fu; Li, You-ping; Cheng, Jing-qiu; Lu, Xiao-feng

2011-01-01

Aim: To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells. Methods: A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models. Results: The IC50 of MG2B for cancer cells (10–15 μmol/L) was at least 10 times lower than the IC50 of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC50 ranging from 10 to 15 μmol/L, which was about 6–10 times lower than the IC50 for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively. Conclusion: The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy. PMID:21131998

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery.

PubMed

Liu, Shan; Yang, Hao; Wan, Lin; Cai, Hua-wei; Li, Sheng-fu; Li, You-ping; Cheng, Jing-qiu; Lu, Xiao-feng

2011-01-01

To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells. A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models. The IC(50) of MG2B for cancer cells (10-15 μmol/L) was at least 10 times lower than the IC(50) of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC(50) ranging from 10 to 15 μmol/L, which was about 6-10 times lower than the IC(50) for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively. The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.

Targeted Type 1 phototherapeutic agents using azido-peptide bioconjugates

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Achilefu, Samuel I.; Jimenez, Hermo N.; Webb, Elizabeth G.; Schmidt, Michelle A.; Bugaj, Joseph E.; Dorshow, Richard B.

2001-07-01

Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.

Expression of matrix metalloproteinase-9 and bombesin/gastrin-releasing peptide in human prostate cancers and their lymph node metastases.

PubMed

Ishimaru, Hisashi; Kageyama, Yukio; Hayashi, Tetsuo; Nemoto, Tetsuo; Eishi, Yoshinobu; Kihara, Kazunori

2002-01-01

Neuroendocrine differentiation and subsequent excretion of neuropeptides have been demonstrated to be associated with progression of human prostate cancer. Among neuropeptides found to exist in the prostate, bombesin/gastrin-releasing peptide has been shown to upregulate matrix metalloproteinase-9 (MMP-9) in human prostate cancer cell lines. Expression levels of bombesin, MMP-9, and neuron-specific enolase were examined by immunohistochemistry in 41 cases of clinically organ-confined prostate cancers including 9 with microscopic lymph node metastases. Twenty-seven (64%) of the 41 radical prostatectomy specimens were positive for both MMP-9 and bombesin. Expression of these molecules was observed in almost the same population of the cancer cells. The remaining 14 cases were negative for both MMP-9 and bombesin. High-grade tumors (Gleason sum > or = 7) were more likely to express MMP-9 and bombesin (21/24:88%) than low-grade tumors (Gleason sum > or = 6) (7/17:41%). In eight of the nine cases with pathological lymph node metastases, expression of MMP-9 and bombesin was also noted in metastatic sites. Neuron-specific enolase was positive in 16 cases (39%) and not always associated with the expression of bombesin. Expression of bombesin and expression of MMP-9 are common in human prostate cancers and may be related to an aggressive phenotype.

Imaging of Prostate Cancer Using Gallium-68-Labeled Bombesin.

PubMed

Sonni, Ida; Baratto, Lucia; Iagaru, Andrei

2017-04-01

Nuclear medicine can play an important role in evaluating prostate cancer combining anatomical and functional information with hybrid techniques. Various PET radiopharmaceuticals have been used for targeting specific biological markers in prostate cancer. Research is ideally oriented towards the development of radiopharmaceuticals targeting antigens overexpressed in prostate cancer, as opposed to normal prostate tissue. In this regard, gastrin-releasing peptide receptors (GRPR) are excellent candidates. Bombesin analogues targeting the GRPR have been investigated. Gallium-68 ( 68 Ga) is an interesting PET radioisotope due to several advantages, such as availability, ease of radiochemistry, half-life, and costs. The focus of this review is on 68 Ga-labeled bombesin analogues in prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Peptide-modified liposomes for selective targeting of bombesin receptors overexpressed by cancer cells: a potential theranostic agent

PubMed Central

Accardo, Antonella; Salsano, Giuseppina; Morisco, Anna; Aurilio, Michela; Parisi, Antonio; Maione, Francesco; Cicala, Carla; Tesauro, Diego; Aloj, Luigi; De Rosa, Giuseppe; Morelli, Giancarlo

2012-01-01

Objectives Drug delivery systems consisting of liposomes displaying a cell surface receptor-targeting peptide are being developed to specifically deliver chemotherapeutic drugs to tumors overexpressing a target receptor. This study addresses novel liposome composition approaches to specifically target tissues overexpressing bombesin (BN) receptors. Methods A new amphiphilic peptide derivative (MonY-BN) containing the BN(7–14) peptide, the DTPA (diethylenetriaminepentaacetate) chelating agent, a hydrophobic moiety with two C18 alkyl chains, and polyethylene glycol spacers, has been synthesized by solid-phase methods. Liposomes have been generated by co-aggregation of MonY-BN with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The structural and biological properties of these new target-selective drug-delivery systems have been characterized. Results Liposomes with a DSPC/MonY-BN (97/3 molar ratio) composition showed a diameter of 145.5 ± 31.5 nm and a polydispersity index of 0.20 ± 0.05. High doxorubicin (Dox) loading was obtained with the remote pH gradient method using citrate as the inner buffer. Specific binding to PC-3 cells of DSPC/MonY-BN liposomes was obtained (2.7% ± 0.3%, at 37°C), compared with peptide-free DSPC liposomes (1.4% ± 0.2% at 37°C). Incubation of cells with DSPC/ MonY-BN/Dox showed significantly lower cell survival compared with DSPC/Dox-treated cells, in the presence of 100 ng/mL and 300 ng/mL drug amounts, in cytotoxicity experiments. Intravenous treatment of PC-3 xenograft-bearing mice with DSPC/MonY-BN/Dox at 10 mg/kg Dox dose produced higher tumour growth inhibition (60%) compared with nonspecific DSPC/ Dox liposomes (36%) relative to control animals. Conclusion The structural and loading properties of DSPC/MonY-BN liposomes along with the observed in-vitro and in-vivo activity are encouraging for further development of this approach for target-specific cancer chemotherapy. PMID:22619538

Regulation of lung branching morphogenesis by bombesin-like peptides and neutral endopeptidase.

PubMed

Aguayo, S M; Schuyler, W E; Murtagh, J J; Roman, J

1994-06-01

The expression of bombesin-like peptides (BLPs) by pulmonary neuroendocrine cells is transiently upregulated during lung development. A functional role for BLPs is supported by their ability to stimulate lung growth and maturation both in vitro and in vivo during the late stages of lung development. In addition, the cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP), which inactivates BLPs and other regulatory peptides, is also expressed by developing lungs and modulates the stimulatory effects of BLPs on lung growth and maturation. We hypothesized that, in addition to expressing BLPs and CD10/NEP, embryonic lungs must express BLP receptors, and that BLPs may also regulate processes that occur during early lung development such as branching morphogenesis. Using reverse transcriptase-polymerase chain reaction and oligonucleotide primers designed for amplifying a BLP receptor originally isolated from Swiss 3T3 mouse fibroblasts, we found that embryonic mouse lungs express a similar BLP receptor mRNA during the pseudoglandular stage of lung development when branching morphogenesis take place. Subsequently, we evaluated the effects of ligands for this BLP receptor using embryonic mouse lungs in an in vitro model of lung branching morphogenesis. We found that, in comparison with control lungs, treatment with bombesin (1 to 100 nM) resulted in a modest increase in clefts or branching points. In contrast, embryonic mouse lungs treated with the BLP analog [Leu13-psi(CH2NH)Leu14]bombesin (1 microM), which also binds to this BLP receptor but has predominantly antagonistic effects, demonstrated fewer branching points.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

PubMed

Schaffer, K; Herrmuth, H; Mueller, J; Coy, D H; Wong, H C; Walsh, J H; Classen, M; Schusdziarra, V; Schepp, W

1997-09-01

In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2

Exploiting cancer’s phenotypic guise against itself: targeting ectopically expressed peptide G-protein coupled receptors for lung cancer therapy

PubMed Central

Khan, Mahjabin; Huang, Tao; Lin, Cheng-Yuan; Wu, Jiang; Fan, Bao-Min; Bian, Zhao-Xiang

2017-01-01

Lung cancer, claiming millions of lives annually, has the highest mortality rate worldwide. This advocates the development of novel cancer therapies that are highly toxic for cancer cells but negligibly toxic for healthy cells. One of the effective treatments is targeting overexpressed surface receptors of cancer cells with receptor-specific drugs. The receptors-in-focus in the current review are the G-protein coupled receptors (GPCRs), which are often overexpressed in various types of tumors. The peptide subfamily of GPCRs is the pivot of the current article owing to the high affinity and specificity to and of their cognate peptide ligands, and the proven efficacy of peptide-based therapeutics. The article summarizes various ectopically expressed peptide GPCRs in lung cancer, namely, Cholecystokinin-B/Gastrin receptor, the Bombesin receptor family, Bradykinin B1 and B2 receptors, Arginine vasopressin receptors 1a, 1b and 2, and the Somatostatin receptor type 2. The autocrine growth and pro-proliferative pathways they mediate, and the distinct tumor-inhibitory effects of somatostatin receptors are then discussed. The next section covers how these pathways may be influenced or ‘corrected’ through therapeutics (involving agonists and antagonists) targeting the overexpressed peptide GPCRs. The review proceeds on to Nano-scaled delivery platforms, which enclose chemotherapeutic agents and are decorated with peptide ligands on their external surface, as an effective means of targeting cancer cells. We conclude that targeting these overexpressed peptide GPCRs is potentially evolving as a highly promising form of lung cancer therapy. PMID:29262666

Insights into Bombesin receptors and ligands: highlighting recent advances

PubMed Central

Ramos-Álvarez, Irene; Moreno, Paola; Mantey, Samuel A.; Nakamura, Taichi; Nuche-Berenguer, Bernardo; Moody, Terry W.; Coy, David H.; Jensen, Robert T.

2015-01-01

This following article is written for Prof. Abba Kastin’s Festschrift, to add to the tribute to his important role in the advancement of the role of peptides in physiological, as well as pathophysiological processes. There have been many advances during the 35 years of his prominent role in the Peptide field, not only as editor of the journal Peptides, but also as a scientific investigator and editor of two volumes of the Handbook of Biological Active Peptides [146,147]. Similar to the advances with many different peptides, during this 35 year period, there have been much progress made in the understanding of the pharmacology, cell biology and the role of (Bombesin) Bn receptors and their ligands in various disease states, since the original isolation of bombesin from skin of the European frog Bombina bombina in 1970 [76]. This paper will briefly review some of these advances over the time period of Prof Kastin 35 years in the peptide field concentrating on the advances since 2007 when many of the results from earlier studies were summarized [128,129]. It is appropriate to do this because there have been 280 articles published in Peptides during this time on Bombesin-related peptides and it accounts for almost 5% of all publications. Furthermore, 22 Bn publications we have been involved in have been published in either Peptides [14,39,55,58,81,92,93,119,152,216,225,226,231,280,302,309,355,361,362] or in the Prof Kastin’s Handbook of Biological Active Peptides [137,138,331]. PMID:25976083

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

PubMed

Chanda, Nripen; Kattumuri, Vijaya; Shukla, Ravi; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R; Kan, Para; Fent, Genevieve M; Casteel, Stan W; Smith, C Jeffrey; Boote, Evan; Robertson, J David; Cutler, Cathy; Lever, John R; Katti, Kattesh V; Kannan, Raghuraman

2010-05-11

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Dual integrin and gastrin-releasing peptide receptor targeted tumor imaging using 18F-labeled PEGylated RGD-bombesin heterodimer 18F-FB-PEG3-Glu-RGD-BBN.

PubMed

Liu, Zhaofei; Yan, Yongjun; Chin, Frederic T; Wang, Fan; Chen, Xiaoyuan

2009-01-22

Radiolabeled RGD and bombesin peptides have been extensively investigated for tumor integrin alpha(v)beta(3) and GRPR imaging, respectively. Due to the fact that many tumors are both integrin and GRPR positive, we designed and synthesized a heterodimeric peptide Glu-RGD-BBN, which is expected to be advantageous over the monomeric peptides for dual-receptor targeting. A PEG(3) spacer was attached to the glutamate alpha-amino group of Glu-RGD-BBN to enhance the (18)F labeling yield and to improve the in vivo kinetics. PEG(3)-Glu-RGD-BBN possesses the comparable GRPR and integrin alpha(v)beta(3) receptor-binding affinities as the corresponding monomers, respectively. The dual-receptor targeting properties of (18)F-FB-PEG(3)-Glu-RGD-BBN were observed in PC-3 tumor model. (18)F-FB-PEG(3)-Glu-RGD-BBN with high tumor contrast and favorable pharmacokinetics is a promising PET tracer for dual integrin and GRPR positive tumor imaging. This heterodimer strategy may also be an applicable method to develop other molecules with improved in vitro and in vivo characterizations for tumor diagnosis and therapy.

Bombesin-induced changes in expression of pancreatic enzymes in young and old rats.

PubMed

Dubick, M A; Cornell, T; Majumdar, A P

1993-01-01

Bombesin is known to induce pancreatic growth. In aged animals, reduced responsiveness of tissues of the gastrointestinal tract to a number of hormones/peptides, including bombesin, has been demonstrated, yet the effects of chronic bombesin administration on the aging pancreas is poorly understood. In the present study, groups of 4- and 20- to 22-month-old male Fischer 344 rats were infused by osmotic minipump with saline (control) or bombesin (300 ng/kg/h) for 14 days. In young rats, bombesin administration increased trypsin activity in the pancreas, which was accompanied by an increase in trypsinogen steady-state mRNA levels. However, this response to bombesin was not observed in aged rats. Bombesin also increased pancreatic glutathione peroxidase and reductase, but not superoxide dismutase activity in young rats, whereas activity of these antioxidant enzymes was not affected by bombesin in old rats. These data further support the observation that responsiveness of the pancreas to hormones is diminished with advancing age.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-11-15

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

PubMed Central

Wang, J L; Kalyanaraman, S; Vivo, M D; Gautam, N

1996-01-01

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals. PMID:8947471

Brain RVD-haemopressin, a haemoglobin-derived peptide, inhibits bombesin-induced central activation of adrenomedullary outflow in the rat.

PubMed

Tanaka, Kenjiro; Shimizu, Takahiro; Yanagita, Toshihiko; Nemoto, Takayuki; Nakamura, Kumiko; Taniuchi, Keisuke; Dimitriadis, Fotios; Yokotani, Kunihiko; Saito, Motoaki

2014-01-01

Haemopressin and RVD-haemopressin, derived from the haemoglobin α-chain, are bioactive peptides found in brain and are ligands for cannabinoid CB1 receptors. Activation of brain CB1 receptors inhibited the secretion of adrenal catecholamines (noradrenaline and adrenaline) induced by i.c.v. bombesin in the rat. Here, we investigated the effects of two haemoglobin-derived peptides on this bombesin-induced response Anaesthetised male Wistar rats were pretreated with either haemoglobin-derived peptide, given i.c.v., 30 min before i.c.v. bombesin and plasma catecholamines were subsequently measured electrochemically after HPLC. Direct effects of bombesin on secretion of adrenal catecholamines were examined using bovine adrenal chromaffin cells. Furthermore, activation of haemoglobin α-positive spinally projecting neurons in the rat hypothalamic paraventricular nucleus (PVN, a regulatory centre of central adrenomedullary outflow) after i.c.v. bombesin was assessed by immunohistochemical techniques. Bombesin given i.c.v. dose-dependently elevated plasma catecholamines whereas incubation with bombesin had no effect on spontaneous and nicotine-induced secretion of catecholamines from chromaffin cells. The bombesin-induced increase in catecholamines was inhibited by pretreatment with i.c.v. RVD-haemopressin (CB1 receptor agonist) but not after pretreatment with haemopressin (CB1 receptor inverse agonist). Bombesin activated haemoglobin α-positive spinally projecting neurons in the PVN. The haemoglobin-derived peptide RVD-haemopressin in the brain plays an inhibitory role in bombesin-induced activation of central adrenomedullary outflow via brain CB1 receptors in the rat. These findings provide basic information for the therapeutic use of haemoglobin-derived peptides in the modulation of central adrenomedullary outflow. © 2013 The British Pharmacological Society.

Effect of bombesin receptor subtype-3 and its synthetic agonist on signaling, glucose transport and metabolism in myocytes from patients with obesity and type 2 diabetes

PubMed Central

GONZÁLEZ, NIEVES; MARTÍN-DUCE, ANTONIO; MARTÍNEZ-ARRIETA, FÉLIX; MORENO-VILLEGAS, ZAIDA; PORTAL-NÚÑEZ, SERGIO; SANZ, RAÚL; EGIDO, JESÚS

2015-01-01

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) member of the bombesin receptor family. Several studies have suggested an association between obesity, alterations in glucose metabolism, diabetes and the BRS-3 receptor. In this study, we focused on patients simultaneously diagnosed with obesity and type 2 diabetes (OB/T2D). The analysis of BRS-3 expression in the skeletal muscle of these patients revealed a marked decrease in the expression of BRS-3 at the mRNA (23.6±1.3-fold downregulation, p<0.0001) and protein level (49±7% decrease, p<0.05) compared to the normal patients (no obesity and diabetes). Moreover, in cultured primary myocytes from patients with OB/T2D, the synthetic BRS-3 agonist, [D-Try6,β-Ala11,Phe13,Nle14]bombesin6–14, significantly increased the phosphorylation levels of mitogen-activated protein kinase (MAPK), p90RSK1, protein kinase B (PKB) and p70s6K. Specifically, the ligand at 10−11 M induced the maximal phosphorylation of MAPKs (p42, 159±15% of the control; p44, 166±11% of the control; p<0.0001) and p90RSK1 (148±2% of the control, p<0.0001). The basal phosphorylation levels of all kinases were reduced (p<0.05) in the patients with OB/T2D compared to the normal patients. Furthermore, the BRS-3 agonist stimulated glucose transport, which was already detected at 10−12 M (133±9% of the control), reached maximal levels at 10−11 M (160±9%, p<0.0001) and was maintained at up to 10−8 M (overall mean, 153±7%; p<0.007). This effect was less promiment than that attained with 10−8 M insulin (202±9%, p=0.009). The effect of the agonist on glycogen synthase a activity achieved the maximum effect at 10−11 M (165±16% of the control; p<0.0001), which did not differ from that observed with higher concentrations of the agonist. These results suggest that muscle cells isolated from patients with OB/T2D have extremely high sensitivity to the synthetic ligand, and the effects are particularly observed on

Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate platform

NASA Astrophysics Data System (ADS)

Bugaj, Joseph E.; Achilefu, Samuel I.; Dorshow, Richard B.; Rajagopalan, Raghavan

2001-04-01

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst2) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide- dye conjugates are effective as contrast agents for optical imaging of tumors.

Bombesin-like peptides and receptors in normal fetal baboon lung: roles in lung growth and maturation.

PubMed

Emanuel, R L; Torday, J S; Mu, Q; Asokananthan, N; Sikorski, K A; Sunday, M E

1999-11-01

Previously, we have shown that bombesin-like peptide (BLP) promotes fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60 (ED60), peaked on approximately ED90, and then declined before term (ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs detected from ED125 until birth. BLP (0.1-10 nM) stimulated type II cell differentiation in organ cultures as assessed by [(3)H]choline incorporation into surfactant phospholipids, electron microscopy, and increased surfactant protein (SP) A- and/or SP-C-immunopositive cells and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60. Cell proliferation was induced by GRP, peaking on ED90. Similarly, blocking BLP degradation stimulated lung growth and maturation, which was completely reversed by a BLP-specific antagonist. The dissociation between GRP and GRP-receptor gene expression during ontogeny suggests that novel BLP receptors and/or peptides might be implicated in these responses.

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

PubMed

Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

Cloning of cDNAs encoding amphibian bombesin: evidence for the relationship between bombesin and gastrin-releasing peptide.

PubMed Central

Spindel, E R; Gibson, B W; Reeve, J R; Kelly, M

1990-01-01

Bombesin is a tetradecapeptide originally isolated from frog skin; its mammalian homologue is the 27-amino acid peptide gastrin-releasing peptide (GRP). cDNAs encoding GRP have been cloned from diverse species, but little is yet known about the amphibian bombesin precursor. Mass spectrometry of HPLC-separated skin exudate from Bombina orientalis was performed to demonstrate the existence of authentic bombesin in the skin of this frog. A cDNA library was prepared from the skin of B. orientalis and mixed oligonucleotide probes were used to isolate cDNAs encoding amphibian bombesin. Sequence analysis revealed that bombesin is encoded in a 119-amino acid prohormone. The carboxyl terminus of bombesin is flanked by two basic amino acids; the amino terminus is not flanked by basic amino acids but is flanked by a chymotryptic-like cleavage site. Northern blot analysis demonstrated similarly sized bombesin mRNAs in frog skin, brain, and stomach. Polymerase chain reaction was used to show that the skin and gut bombesin mRNAs encoded the identical prohormones. Prohormone processing, however, differed between skin and gut. Chromatography showed the presence of only authentic bombesin in skin whereas gut extracts contained two peaks of bombesin immunoreactivity, one consistent in size with bombesin and one closer in size to mammalian GRP. Thus the same bombesin prohormone is processed solely to bombesin in skin but is processed to a peptide similar in size to bombesin and to a peptide similar in size to mammalian GRP in stomach. Images PMID:2263631

Radiolabeled bombesin derivatives for preclinical oncological imaging

PubMed Central

de Aguiar Ferreira, Carolina; Fuscaldi, Leonardo Lima; Townsend, Danyelle M.; Rubello, Domenico; de Barros, André Luís Branco

2017-01-01

Despite efforts, cancer is still one of the leading causes of morbidity and mortality worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths each year, according to the World Health Organization. Among the strategies to reduce cancer progression and improving its management, implementing early detection technologies is crucial. Based on the fact that several types of cancer cells overexpress surface receptors, small molecule ligands, such as peptides, have been developed to allow tumor identification at earlier stages. Allied with imaging techniques such as PET and SPECT, radiolabeled peptides play a pivotal role in nuclear medicine. Bombesin, a peptide of 14 amino acids, is an amphibian homolog to the mammalian gastrin-releasing peptide (GRP), that has been extensively studied as a targeting ligand for diagnosis and therapy of GRP positive tumors, such as breast, pancreas, lungs and prostate cancers. In this context, herein we provide a review of reported bombesin derivatives radiolabeled with a multitude of radioactive isotopes for diagnostic purposes in the preclinical setting. Moreover, since animal models are highly relevant for assessing the potential of clinical translation of this radiopeptides, a brief report of the currently used GRP-positive tumor-bearing animal models is described. PMID:28040598

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

PubMed Central

Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

Receptor stimulated formation of inositol phosphates in cultures of bovine adrenal medullary cells: the effects of bradykinin, bombesin and neurotensin.

PubMed

Bunn, S J; Marley, P D; Livett, B G

1990-04-01

The ability of a number of drugs and neuropeptides to stimulate phosphoinositide metabolism in cultured bovine adrenal medullary cells has been assessed. Low concentrations (10 nM) of angiotensin II, bradykinin, histamine, arginine-vasopressin, and bombesin, and high (10 microM) concentrations of oxytocin, prostaglandins E1, and E2, beta-endorphin, and neurotensin stimulated significant accumulation of [3H]inositol phosphates in adrenal medullary cells preloaded with [3H)]inositol. Bradykinin stimulated a significant response at concentration as low as 10pM, with an EC50 of approximately 0.5 nM. The response was markedly inhibited by the bradykinin B2 antagonist [Thi5,8,D-Phe7] bradykinin but not the B1 antagonist [Des-Arg9,Leu8] bradykinin. Higher concentrations of bombesin and neurotensin were required to elicit a response (10 nM and 10 microM respectively). The bombesin response was sensitive to inhibition by the bombesin antagonist [D-Arg1,D-Pro2,D-Trp7,9Leu11]-substance P. In contrast, the neurotensin response was not reduced by the NT1 antagonist [D-Trp11]-neurotensin. These results indicate there are a number of agents that can stimulate phosphatidylinositide hydrolysis in the adrenal medullary cells by acting on different classes of receptors. Such a range of diverse agonists that stimulate inositol phosphate formation will facilitate further analysis of the phosphatidylinositide breakdown in chromaffin cell function.

In vivo inhibition of PC-3 human androgen-independent prostate cancer by a targeted cytotoxic bombesin analogue, AN-215.

PubMed

Plonowski, A; Nagy, A; Schally, A V; Sun, B; Groot, K; Halmos, G

2000-11-15

The effectiveness of chemotherapy targeted to bombesin (BN) receptors was evaluated in nude mice bearing PC-3 human androgen-independent prostate cancers. Cytotoxic BN analogue AN-215, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to BN-like carrier peptide RC-3094, was injected i.v. at 150 nmol/kg on days 1, 11 and 21. After treatment with AN-215, tumor volume was 69% (p < 0.01) smaller than that in controls and tumor doubling time was extended from 8.5 +/- 0.7 days to 20.3 +/- 3.5 days (p < 0.05). Cytotoxic radical AN-201, carrier RC-3094 and their unconjugated mixture administered at the same dosage were ineffective. The mortality rate was 12.5% in the AN-201 group and 16.7% in the group treated with the mixture, but no deaths occurred in mice receiving AN-215. Because the ester bond linking AN-201 to the carrier molecule is hydrolyzed much faster in mouse serum than in human serum, in the second experiment we investigated the tolerance to AN-215 and its effect in nude mice bearing PC-3 tumors after pharmacological inhibition of serum carboxylesterases. Two applications of AN-201 at 200 nmol/kg were lethal, whereas no mortality was observed after 4 injections of AN-215 at the same dose. Administration of 200 nmol/kg AN-215 on days 1, 7, 17 and 26 again produced 69% tumor inhibition. BN receptors on membranes of PC-3 tumors were detected by (125)I-[Tyr(4)]BN binding, and expression of mRNA for BRS-3 and GRP-R subtypes was also found. AN-215 showed a high affinity to PC-3 tumors, displacing the radioligand at an IC(50) of 12.95 +/- 0.35 nM. Because BN receptors are present on primary and metastatic prostate cancer, targeted chemotherapy with AN-215 might benefit patients with advanced prostatic carcinoma who relapsed androgen ablation. Copyright 2000 Wiley-Liss, Inc.

[Physiological effects of bombesin].

PubMed

Klimov, P K; Mar'ianovich, A T; Poliakov, E L; Kuranova, I L; Churkina, S I

1985-02-01

Central or peripheral administration of bombesin--tetradecapeptide obtained from amphibian skin, acted upon the nervous system, behaviour, thermoregulation and gastro-intestinal tract. New experimental data on synthesis, immunohistochemistry and physiological effects of bombesin and related peptides are presented.

Gastrin-releasing peptide receptor-induced internalization, down-regulation, desensitization, and growth: possible role for cyclic AMP.

PubMed

Benya, R V; Fathi, Z; Kusui, T; Pradhan, T; Battey, J F; Jensen, R T

1994-08-01

Stimulation of the gastrin-releasing peptide receptor (GRP-R) in Swiss 3T3 cells resembles that of a number of other recently described G protein-coupled receptors, insofar as both the phospholipase C and adenylyl cyclase signal transduction pathways are activated. GRP-R activation induces numerous alterations in both the cell and the receptor, but because two signal transduction pathways are activated it is difficult to determine the specific contributions of either pathway. We have found that BALB/3T3 fibroblasts transfected with the coding sequence for the GRP-R are pharmacologically indistinguishable from native receptor-expressing cells and activate phospholipase C in a manner similar to that of the native receptor but fail to increase cAMP in response to bombesin; thus, they may be useful cells to explore the role of activation of each pathway in altering cell and receptor function. Swiss 3T3 cells and GRP-R-transfected BALB/3T3 cells expressed identically glycosylated receptors that bound various agonists and antagonists similarly. G protein activation, as determined by evaluation of agonist-induced activation of phospholipase C and by analysis of the effect of guanosine-5'-(beta,gamma-imido)triphosphate on GRP-R binding affinity, was indistinguishable. Agonist stimulation of GRP-R caused similar receptor changes (internalization and down-regulation) and homologous desensitization in both cell types. Bombesin stimulation of Swiss 3T3 cells that had been preincubated with forskolin increased cAMP levels 9-fold, but no bombesin-specific increase in cAMP levels was detected in transfected cells, even though forskolin and cholera toxin increased cAMP levels in these cells. Quiescent Swiss 3T3 cells treated with bombesin rapidly increased c-fos mRNA levels and [3H]thymidine incorporation, whereas both effects were potentiated by forskolin. The specific protein kinase A inhibitor H-89 blocked increases in c-fos levels and [3H]thymidine incorporation induced by low

Identification and functional characterization of hemorphins VV-H-7 and LVV-H-7 as low-affinity agonists for the orphan bombesin receptor subtype 3

PubMed Central

Lammerich, Hans-Peter; Busmann, Annette; Kutzleb, Christian; Wendland, Martin; Seiler, Petra; Berger, Claudia; Eickelmann, Peter; Meyer, Markus; Forssmann, Wolf-Georg; Maronde, Erik

2003-01-01

The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines. Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed. PMID:12721098

The effects of the phyllolitorin analogue [desTrp3,Leu8]phyllolitorin on scratching induced by bombesin and related peptides in rats

PubMed Central

Johnson, Mark D.; Ko, Mei-Chuan; Choo, Kevin S.; Traynor, John R.; Mosberg, Henry I.; Naughton, Norah N.; Woods, James H.

2010-01-01

Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue wdesTrp3,Leu8]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu8]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 μg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism. PMID:10482814

External pancreatic secretion after bombesin infusion in man.

PubMed

Basso, N; Giri, S; Improta, G; Lezoche, E; Melchiorri, P; Percoco, M; Speranza, V

1975-12-01

The effect of bombesin on external pancreatic secretion was studied in seven healthy volunteers and intwo patients with a two-thirds gastrectomy and a pancreatic fistula. After bombesin infusion (15 ng/kg/min), gastrin levels were significantly raised in all volunteers, but remained at basal levels in the gastrectomized patients. Bombesin was effective in stimulating pancreatic secretion in all patients. The volume of secretion increased tow-fold when compared with basal volume. Amylase and trypsin concentrations and outputs in the duodenal juice were greatly agumented (amylase concentration: basal, 70 dye U/ml; post-bombesin, 620 dye U/ml. Amylase output: basal, 1000 dye U/15 min; post-bombesin, 15,800 dye U/15 min). Secretin, when administered in conjunction with bombesin, partially inhibited its secretory effect. Bicarbonate secretion was slightly stimulated by bombesin, but at a very low level. A similar pattern of results was obtained in the two gastrectomized patients. In man, bombesin exerts an effect on pancreatic secretion that mimics the effect of CCK-PZ, thus confirming the results obtained in the experimental animal. Gastrin does not play a fundamental role in this phenomenon.

External pancreatic secretion after bombesin infusion in man.

PubMed Central

Basso, N; Giri, S; Improta, G; Lezoche, E; Melchiorri, P; Percoco, M; Speranza, V

1975-01-01

The effect of bombesin on external pancreatic secretion was studied in seven healthy volunteers and intwo patients with a two-thirds gastrectomy and a pancreatic fistula. After bombesin infusion (15 ng/kg/min), gastrin levels were significantly raised in all volunteers, but remained at basal levels in the gastrectomized patients. Bombesin was effective in stimulating pancreatic secretion in all patients. The volume of secretion increased tow-fold when compared with basal volume. Amylase and trypsin concentrations and outputs in the duodenal juice were greatly agumented (amylase concentration: basal, 70 dye U/ml; post-bombesin, 620 dye U/ml. Amylase output: basal, 1000 dye U/15 min; post-bombesin, 15,800 dye U/15 min). Secretin, when administered in conjunction with bombesin, partially inhibited its secretory effect. Bicarbonate secretion was slightly stimulated by bombesin, but at a very low level. A similar pattern of results was obtained in the two gastrectomized patients. In man, bombesin exerts an effect on pancreatic secretion that mimics the effect of CCK-PZ, thus confirming the results obtained in the experimental animal. Gastrin does not play a fundamental role in this phenomenon. PMID:1218823

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

PubMed

Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Bombesin receptor-activated protein regulates neutrophil elastase-induced mucin5AC hypersecretion in human bronchial epithelial cells.

PubMed

Xu, Qing; Chen, Ling-Xiu; Ran, Dan-Hua; Xie, Wen-Yue; Li, Qi; Zhou, Xiang-Dong

2017-08-15

Bombesin receptor-activated protein (BRAP) is highly expressed in human bronchial epithelial cells. Recent studies have shown that BRAP reduces oxidative stress, inhibits airway inflammation and suppresses nuclear factor kappaB (NF-κB) activity. Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. Neutrophil elastase (NE) is a potent inducer of mucin5AC (MUC5AC), which is considered the predominant mucin secreted by human airway epithelial cells. Here, we hypothesize that BRAP may regulate NE-induced MUC5AC hypersecretion in a bronchial epithelial cell line (HBE16). We also investigated the underlying mechanism involved in the process. In this study, we found that BRAP was present in HBE16 human bronchial epithelial cells and was significantly increased by NE. Next, we found that the up-regulation of BRAP by pEGFP-N1-BRAP caused a significant decrease in the increased levels of MUC5AC expression, NF-κB activity, and the phosphorylation of extracellular signal-regulated kinases (ERK) and epidermal growth factor receptor (EGFR) induced by NE. Meanwhile, there was a significant decrease in ROS, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels when BRAP was up-regulated by pEGFP-N1-BRAP. Moreover, when cells were transfected with pEGFP-N1-BRAP and pretreated with NF-κB, ERK or EGFR inhibitors before the NE stimulation, there were further decreased in MUC5AC expression, NF-κB activity, and the phosphorylation of ERK and EGFR. These results suggest that BRAP plays an important role in airway inflammation and its overexpression may regulate NE-induced MUC5AC hypersecretion in HBE16 cells via the EGFR/ERK/NF-κB signaling pathway. Copyright © 2017. Published by Elsevier Inc.

Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement.

PubMed

Festuccia, Claudio; Angelucci, Adriano; Gravina, Giovanni; Eleuterio, Enrica; Vicentini, Carlo; Bologna, Mauro

2002-10-15

Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptide, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma tissues and are likely to be related to the progression of this neoplastic disease. Previously, we demonstrated that bombesin increased migration and protease expression in androgen-independent cells. In this work we show that bombesin is able to activate pro-MMP-9 through a mechanism involving the beta1 integrin subunit. In fact, MMP-9 processing was evident only when beta1 integrin was engaged with specific adhesive substrates, such as type I collagen, or when cells were seeded on dishes coated with antibodies against beta1 integrin, resulting in activation of the surface ligand. When exogenous pro-MMP-9 was added to PC3 cells, MMP-9 active forms were produced within 30 min by bombesin-treated cultures while control cultures expressed activated forms only after a longer time and at lower levels. MMP-9 activation required cytoskeleton integrity since this effect was abolished by cytochalasin D. Engagement of beta1 integrin caused an increased membrane-linked uPA activity which was required for MMP-9 activation. The cross talk between bombesin- and beta1-integrin-engaged signals seems to be crucial for the modulation of both membrane-linked uPA activity and MMP-9 activation and triggers complex intracellular signaling pathways requiring activation of tyrosine kinase activity, including that of src and PI3K. The beta1 integrin may be considered an important mechanism by which bombesin induces MMP-9 activation. This finding supports the idea that cellular responses to growth factors may be driven by cell-matrix interactions and stresses the role of neuroendocrine factors in prostate carcinoma progression.

Targeted imaging of gastrin-releasing peptide receptors with 99mTc-EDDA/HYNIC-[Lys3]-bombesin: biokinetics and dosimetry in women.

PubMed

Santos-Cuevas, Clara L; Ferro-Flores, Guillermina; Arteaga de Murphy, Consuelo; Pichardo-Romero, Pablo A

2008-08-01

The gastrin-releasing peptide receptor (GRP-R) is expressed in several normal human tissues and is overexpressed in various human tumors including breast, prostate, small-cell lung cancer and pancreatic cancer. Recently, 99mTc-EDDA/HYNIC-[Lys]-bombesin (99mTc-HYNIC-BN) was reported as a radiopharmaceutical with high stability in human serum, specific cell GRP-R binding and rapid cell internalization. The aim of this study was to determine the biokinetics and dosimetry of 99mTc-HYNIC-BN and the feasibility of using this radiopharmaceutical to image GRP-R in four early breast cancer patients and seven healthy women. Whole-body images were acquired at 20, 90, 180 min, and 24 h after 99mTc-HYNIC-BN administration. The same regions of interest were drawn around source organs on each time frame and regions of interest were converted to activity (conjugate view counting method). The image sequence was used to extrapolate 99mTc-HYNIC-BN time-activity curves in each organ to calculate the total number of disintegrations (N) that occurred in the source regions. N data were the input for the OLINDA/EXM code to calculate internal radiation dose estimates. 99mTc-HYNIC-BN had a rapid blood clearance with mainly renal excretion. No statistically significant differences (P>0.05) in the radiation-absorbed doses among cancer patients and healthy women were observed. The average equivalent doses (n=11) were 24.8+/-8.8 mSv (kidneys), 7.3+/-1.8 mSv (lungs), 6.5+/-4.0 mSv (breast), 2.0+/-0.3 mSv (pancreas), 1.6+/-0.3 mSv (liver), 1.2+/-0.2 mSv (ovaries), and 1.0+/-0.2 mSv (red marrow). The effective dose was 3.3+/-0.6 mSv. The images showed well-differentiated concentration of 99mTc-HYNIC-BN in cancer mammary tissue. All the absorbed doses were comparable with those known for most of the 99mTc studies. 99mTc-HYNIC-BN shows high tumor uptake in breasts with malignant tumors so it is a promising imaging radiopharmaceutical to target site-specific early breast cancer. The results obtained

A coupling of homology modeling with multiple molecular dynamics simulation for identifying representative conformation of GPCR structures: a case study on human bombesin receptor subtype-3.

PubMed

Nowroozi, Amin; Shahlaei, Mohsen

2017-02-01

In this study, a computational pipeline was therefore devised to overcome homology modeling (HM) bottlenecks. The coupling of HM with molecular dynamics (MD) simulation is useful in that it tackles the sampling deficiency of dynamics simulations by providing good-quality initial guesses for the native structure. Indeed, HM also relaxes the severe requirement of force fields to explore the huge conformational space of protein structures. In this study, the interaction between the human bombesin receptor subtype-3 and MK-5046 was investigated integrating HM, molecular docking, and MD simulations. To improve conformational sampling in typical MD simulations of GPCRs, as in other biomolecules, multiple trajectories with different initial conditions can be employed rather than a single long trajectory. Multiple MD simulations of human bombesin receptor subtype-3 with different initial atomic velocities are applied to sample conformations in the vicinity of the structure generated by HM. The backbone atom conformational space distribution of replicates is analyzed employing principal components analysis. As a result, the averages of structural and dynamic properties over the twenty-one trajectories differ significantly from those obtained from individual trajectories.

Comparative in vivo evaluation of two novel 99mTc labelled bombesin derivatives

NASA Astrophysics Data System (ADS)

Gourni, Eleni; Bouziotis, Penelope; Zikos, Christos; Loudos, George; Xanthopoulos, Stavros; Fani, Melpomeni; Archimandritis, Spyridon C.; Varvarigou, Alexandra D.

2006-12-01

Bombesin (BN), a 14 amino acid peptide, is an analogue of human gastrin-releasing-peptide (GRP) that binds to GRP receptors (GRP-R) with high affinity and specificity. In addition to this physiological role, GRP, through its interaction with GRP-R, promotes tumour growth in a number of human cancer cell lines. The GRP receptors are over-expressed on a variety of human cancer cells. Aim of the present work is the study of two novels BN-like peptides, by investigating the radiochemical and radiopharmacological behaviour of their complexes with metals. The derivatives under study are: Gly-Gly-Cys-Aca-BN [2-14] where Aca: 6-amino-hexanoic acid. Pyroglutamic acid in the bombesin molecule has been replaced by the chemical group Gly-Gly-Cys-Aca, which bears an amino-acid combination capable of complexing a variety of radiometals. The other derivative under study is: Gly-Gly-Cys-Aca-BN [7-14]. This moiety of the peptide has been chosen because it has been proven to be a potent GRP agonist. The peptide derivatives were synthesized by SPPS, according to the Fmoc strategy and were identified by reverse phase high performance liquid chromatography (RP-HPLC). Radiolabelling with 99mTc was performed via the precursor 99mTc-gluconate. The stability of the radiolabelled species was examined with time. In vivo studies of the two 99mTc-labelled derivatives were performed, comparatively, in normal mice, attention being focused on GRP receptor-bearing organs, and in experimentally induced prostate cancer models. Experimental tumours were imaged in a small field-of-view animal gamma camera.

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer.

PubMed

Craft, Jeffrey M; De Silva, Ravindra A; Lears, Kimberly A; Andrews, Rebecca; Liang, Kexian; Achilefu, Samuel; Rogers, Buck E

2012-07-01

Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide. p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies. The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues. These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

In vitro action of bombesin and bombesin-like peptides on amylase secretion, calcium efflux, and adenylate cyclase activity in the rat pancreas: a comparison with other secretagogues.

PubMed Central

Deschodt-Lanckman, M; Robberecht, P; De Neef, P; Lammens, M; Christophe, J

1976-01-01

Bombesin (a tetradecapeptide), the C-terminal nonapeptide of bombesin (bombesin-NP), and litorin (a parent nonapeptide), each stimulated amylase secretion from rat pancreatic fragments. These responses were not affected by atropine. The concentrations that produced half-maximal stumulation of secretion were 0.25 nM for bombesin, 0.30 nM for bombesin-NP, and 0.07 nM for litorin, as compared to 0.12 nM for caerulein and 0.80 muM for the cholinergic agent carbamylcholine. When used at maximal concentrations, bombesin, bombesin-NP, and litorin showed no action on cyclic AMP levels in the presence of 5 mM theophylline. By contrast, caerulein and secretin increased cyclic AMP levels by 27 and 208%, respectively. Bombesin, bombesin-NP, and litorin did not activate adenylate cyclase in a purified pancreatic plasma membrane preparation, whereas caerulein and secretin increased this activity 20 and 16-times, respectively... PMID:184111

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

PubMed

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

Bombesin, somatostatin, and related peptides: actions on thermoregulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, M.R.

1981-11-01

Bombesin acts within the anterior hypothalamic preoptic area to interfere with thermoregulation in the rat. The body temperature (T/sub b/) of animals receiving bombesin varies in parallel with ambient temperature (T/sub a/). Bombesin-induced reduction of T/sub b/ in animals at low T/sub a/ is associated with a marked reduction of oxygen consumption (Vo/sub 2/). Some somatostatin-related peptides, e.g., desAA/sup 1,2,4,5,12,13/ (D-Trp/sup 8/)-somatostatin (ODT8-SS), act within the brain to prevent bombesin-induced reduction of Vo/sub 2/ and T/sub b/. ODT8-SS also produces hyperthermia not associated with an increase in Vo/sub 2/.

Rhodium-105 Bombesin Analogs for Prostate Cancer Radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silvia S. Jurisson, PhD

2005-12-31

Over the period of this grant (11/01/2001 to 12/31/2005), the consistent and reproducible production of Rh-105, synthesis and evaluation of three new chelate systems based on hydroxymethyl phosphines, development of a new non-hydroxymethyl phosphine N{sub 2}P{sub 2} chelate system, conjugation of two of the chelates to the bombesin peptide analog BBN[7-14]NH{sub 2}, evaluation of the bombesin conjugates and their Rh-105 complexes for stability, cell binding affinity, and in vivo biodistribution in normal mice has been developed. The BBN analogs bind to GRP receptors that are overexpressed on PC-3 prostate tumor cells. A dedicated glove box is used for the separationmore » and isolation of {sup 105}Rh from the target ({sup 104}Ru). All tubing/connections/valves from the point of the Cl{sub 2} tank are made of Teflon to minimize/eliminate the introduction of any metal into the process (e.g., iron from stainless steel corrosion). The separation of {sup 105}Rh produced from the enriched {sup 104}Ru target involves oxidation of the enriched {sup 104}Ru metal target to ruthenium tetroxide with chlorine gas and sodium hydroxide solution to generate hypochlorite in situ. The RuO4 is removed by distillation and the {sup 105}Rh remaining in the reaction vial is converted into {sup 105}Rh-chloride by acidification with hydrochloric acid and heating. The {sup 105}Rh production process has become reproducible over the past year to consistently make 10-30 mCi of {sup 105}Rh from 1-3 mg of an enriched (99.21%) {sup 104}Ru target. The process itself involves irradiation of the enriched {sup 104}Ru target in the core of the reactor (University of Missouri Research Reactor (MURR)) for one week to yield 16-40 mCi of {sup 105}Rh. The irradiated target is processed to separate the Rh-105 in high specific activity from the {sup 104}Ru target. The irradiated target is dissolved in NaOH (2M, 3 mL) by bubbling Cl{sub 2} gas through the solution (generating NaOCl in situ) to generate Ru

Targeted Delivery of Proteasome Inhibitors to Somatostatin-Receptor-Expressing Cancer Cells by Octreotide Conjugation.

PubMed

Beck, Philipp; Cui, Haissi; Hegemann, Julian D; Marahiel, Mohammed A; Krüger, Achim; Groll, Michael

2015-12-01

Clinical application of proteasome inhibitors (PIs) is so far limited to peripheral blood cancers due to the pronounced cytotoxicity towards all cell types. Targeted delivery of PIs could permit the treatment of other cancers along with decreasing side effects. Herein we describe the first small-molecule proteasome inhibitor conjugate for targeted delivery, created by fusing PIs to a synthetic ligand of somatostatin receptors, which are highly expressed in a variety of tumors. X-ray crystallographic studies and in vitro IC50 measurements demonstrated that addition of the cyclopeptide octreotide as a targeting vehicle does not affect the PI's binding mode. The cytotoxicity of the conjugate against somatostatin-receptor-expressing cells was up to 11-fold higher than that of a non-targeting surrogate. We have therefore established PIs as a new payload for drug conjugates and have shown that targeted delivery thereof could be a promising approach for the broader application of this FDA-approved class of compounds. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inappropriate activation of the androgen receptor by nonsteroids: involvement of the Src kinase pathway and its therapeutic implications.

PubMed

Desai, Sonal J; Ma, Ai-Hong; Tepper, Clifford G; Chen, Hong-Wu; Kung, Hsing-Jien

2006-11-01

The inappropriate activation of androgen receptor (AR) by nonsteroids is considered a potential mechanism in the emergence of hormone-refractory prostate tumors, but little is known about the properties of these "pseudoactivated" AR. Here, we present the first comprehensive analysis closely examining the properties of AR activated by the neuropeptide bombesin that distinguish it from androgen-activated AR. We show that bombesin-activated AR (a) is required for bombesin-induced growth of LNCaP cells, (b) has a transcriptional profile overlapping with, but not identical to, androgen-activated AR, (c) activates prostate-specific antigen by preferentially binding to its proximal promoter, and (d) assembles a distinct coactivator complex. Significantly, we found that Src kinase is critical for bombesin-induced AR-mediated activity and is required for translocation and transactivation of AR. Additionally, we identify c-Myc, a Src target gene, to be activated by bombesin and a potential coactivator of AR-mediated activity specific to bombesin-induced signaling. Because Src kinase is often activated by other nonsteroids, such as other neuropeptides, growth factors, chemokines, and cytokines, our findings have general applicability and provide rationale for investigating the efficacy of the Src kinase pathway as a target for the prevention of relapsed prostate cancers.

CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

PubMed

King, K A; Hua, J; Torday, J S; Drazen, J M; Graham, S A; Shipp, M A; Sunday, M E

1993-05-01

Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs.

The gastrin-releasing peptide analog bombesin preserves exocrine and endocrine pancreas morphology and function during parenteral nutrition

PubMed Central

Pierre, Joseph F.; Neuman, Joshua C.; Brill, Allison L.; Brar, Harpreet K.; Thompson, Mary F.; Cadena, Mark T.; Connors, Kelsey M.; Busch, Rebecca A.; Heneghan, Aaron F.; Cham, Candace M.; Jones, Elaina K.; Kibbe, Carly R.; Davis, Dawn B.; Groblewski, Guy E.; Kudsk, Kenneth A.

2015-01-01

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis. PMID:26185331

Fluorescent, Plasmonic, and Radiotherapeutic Properties of the 177Lu–Dendrimer-AuNP–Folate–Bombesin Nanoprobe Located Inside Cancer Cells

PubMed Central

Mendoza-Nava, Héctor; Ramírez, Flor de María; Ocampo-García, Blanca; Santos-Cuevas, Clara; Azorín-Vega, Erika; Jiménez-Mancilla, Nallely; Luna-Gutiérrez, Myrna; Isaac-Olivé, Keila

2017-01-01

The integration of fluorescence and plasmonic properties into one molecule is of importance in developing multifunctional imaging and therapy nanoprobes. The aim of this research was to evaluate the fluorescent properties and the plasmonic–photothermal, therapeutic, and radiotherapeutic potential of 177Lu–dendrimer conjugated to folate and bombesin with gold nanoparticles in the dendritic cavity (177Lu–DenAuNP–folate–bombesin) when it is internalized in T47D breast cancer cells. The intense near-Infrared (NIR) fluorescence emitted at 825 nm from the conjugate inside cells corroborated the usefulness of DenAuNP–folate–bombesin for optical imaging. After laser irradiation, the presence of the nanosystem in cells caused a significant increase in the temperature of the medium (46.8°C, compared to 39.1°C without DenAuNP–folate–bombesin, P < 0.05), resulting in a significant decrease in cell viability (down to 16.51% ± 1.52%) due to the 177Lu–DenAuNP–folate–bombesin plasmonic properties. After treatment with 177Lu–DenAuNP–folate–bombesin, the T47D cell viability decreased 90% because of the radiation-absorbed dose (63.16 ± 4.20 Gy) delivered inside the cells. The 177Lu–DenAuNP–folate–bombesin nanoprobe internalized in cancer cells exhibited properties suitable for optical imaging, plasmonic–photothermal therapy, and targeted radiotherapy. PMID:28654384

Analysis of argentinated peptide complexes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: Peptide = oxytocin, arg(8) -vasopressin, bradykinin, bombesin, somatostatin, neurotensin.

PubMed

Gupta, Shyam L; Dhiman, Vikas; Jayasekharan, T; Sahoo, N K

2016-06-15

The increased use of silver nanoparticles (AgNPs) for various biological applications, and over-expression of various peptide receptors in different tumors/cancer cells, necessitate the need for dedicated investigations on the intrinsic binding ability of Ag with various biologically important peptides for better understanding of AgNPs-peptide interactions and for the future development of contrasting agents as well as drugs for imaging/biomedical applications. The [M+(Ag)n ](+) complexes are prepared and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Silver complexes of the peptides [M+(Ag)n ](+) , where M = oxytocin, arg(8) -vasopressin, bradykinin, bombesin, somatostatin, and neurotensin, have been investigated for their intrinsic Ag(+) -binding ability. Unusual binding of up to seven Ag(+) with these small peptides is observed. The mass spectra show n = 1-5 for bombesin and somatostatin, n = 1-6 for bradykinin and arg(8) -vasopressin, and n = 1-7 for oxytocin and neurotensin. In addition, oxytocin and arg(8) -vasopressin show the formation of dimers and their complexes [M2 +(Ag)n ](+) with n = 1-8 and n = 1-5, respectively. The possible amino acid residues responsible for Ag(+) binding in each peptide have been identified on the basis of density functional theory (DFT)-calculated binding energy values of Ag(+) towards individual amino acids. Mass spectrometric evidence indicates that the peptides, viz., oxytocin, arg(8) -vasopressin, bradykinin, bombesin, somatostatin, and neurotensin, show greater affinity for Ag(+) . Hence, they may be used as carriers for AgNPs in targeted drug delivery as well as an alternative for iodinated contrasting agents in dual energy X-ray imaging techniques. Radio-labeled Ag with these peptides can also be used in radio-pharmaceuticals for diagnostic and therapeutic applications. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

CD10/neutral endopeptidase 24.11 regulates fetal lung growth and maturation in utero by potentiating endogenous bombesin-like peptides.

PubMed Central

King, K A; Hua, J; Torday, J S; Drazen, J M; Graham, S A; Shipp, M A; Sunday, M E

1993-01-01

Bombesin-like peptides (BLPs) are mitogens for bronchial epithelial cells and small cell lung carcinomas, and increase fetal lung growth and maturation in utero and in organ cultures. BLPs are hydrolyzed by the enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) which is expressed in bronchial epithelium and functions to inhibit BLP-mediated growth of small cell lung carcinomas. To determine whether CD10/NEP regulates peptide-mediated lung development, we administered a specific CD10/NEP inhibitor, SCH32615, to fetal mice in utero from gestational days e15-17. Fetal lung tissues were evaluated on e18 for: (a) growth using [3H]thymidine incorporation into nuclear DNA; and (b) maturation using: [3H]-choline incorporation into surfactant phospholipids, electron microscopy for type II pneumocytes, and Northern blot analyses for surfactant apoproteins A, B, and C. Inhibition of CD10/NEP stimulated [3H]thymidine incorporation into DNA (70% above baseline, P < 0.005), [3H]choline incorporation into surfactant phospholipids (38% above baseline, P < 0.005), increased numbers of type II pneumocytes (36% above baseline, P = 0.07), and fivefold higher surfactant protein A transcripts (P < 0.05). CD10/NEP-mediated effects were completely blocked by the specific bombesin receptor antagonist, [D-Phe12, Leu14]bombesin. These observations suggest that CD10/NEP regulates fetal lung growth and maturation mediated by endogenous BLPs. Images PMID:8486767

Bombesin and G-17 dose responses in duodenal ulcer and controls.

PubMed

Hirschowitz, B I; Tim, L O; Helman, C A; Molina, E

1985-11-01

Gastric acid and pepsin secretion and serum gastrin concentrations were measured in nine patients with uncomplicated duodenal ulcer (DU) and 10 normal controls in the fasting state and in response to graded doses of bombesin, a tetradecapeptide gastrin releaser, and, for reference, synthetic gastrin G-17. Serum gastrin with bombesin stimulation was significantly greater in duodenal ulcer (maximum 467 pg/ml) than in controls (153 pg/ml), while in seven of the DU group tested gastrin levels after a meal were not different from that seen in five of the normal controls. Gastric acid concentrations and outputs were greater in duodenal ulcer with both stimuli. Secretory responses were then related to serum gastrin levels; despite increasing gastrin levels with bombesin stimulation, peak outputs achieved with bombesin were only 50% of G-17 maximum in normals and up to 90% of maximum in duodenal ulcer. Up to the point of peak response to bombesin, acid and pepsin outputs were the same with exogenous and endogenous gastrin, ie, bombesin acted only via G-17. Furthermore, in direct comparison of duodenal ulcer and normals with G-17 infusion, acid and pepsin outputs related to serum gastrin were congruent up to 75% of duodenal ulcer maximum, at which point normals reached their maximum level. These data have shown that duodenal ulcer patients are not more sensitive to either exogenous or endogenous gastrin; we have also shown regulatory defects in duodenal ulcer patients not previously described: an exaggerated release of gastrin with bombesin stimulation, and a defective inhibition of acid and pepsin secretion with higher doses of bombesin.

Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway.

PubMed

Sumitomo, M; Milowsky, M I; Shen, R; Navarro, D; Dai, J; Asano, T; Hayakawa, M; Nanus, D M

2001-04-15

G-protein coupled receptor (GPCR) agonists such as neuropeptides activate the insulin-like growth factor-1 receptor (IGF-IR) or the serine-threonine protein kinase Akt, suggesting that neuropeptides-GPCR signaling can cross-communicate with IGF-IR-Akt signaling pathways. Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that cleaves and inactivates the neuropeptides endothelin-1 (ET-1) and bombesin, which are implicated in progression to androgen-independent prostate cancer (PC). We investigated the mechanisms of NEP regulation of neuropeptide-mediated cell survival in PC cells, including whether neuropeptide substrates of NEP induce phosphorylations of IGF-IR and Akt in PC cells. Western analyses revealed ET-1 and bombesin treatment induced phosphorylation of IGF-IRbeta and Akt independent of IGF-I in TSU-Pr1, DU145, and PC-3 PC cells, which lack NEP expression, but not in NEP-expressing LNCaP cells. Recombinant NEP and induced NEP expression in TSU-Pr1 cells using a tetracycline-repressive expression system inhibited ET-1-mediated phosphorylation of IGF-IRbeta and Akt, and blocked the protective effects of ET-1 against apoptosis induced by serum starvation. Incubation of TSU-Pr1 cells with specific kinase inhibitors together with ET-1 or bombesin showed that IGF-IR activation is required for neuropeptide-induced Akt phosphorylation, and that neuropeptide-induced Akt activation is predominantly mediated by Src and phosphatidylinositol 3-kinase but not by mitogen-activated protein kinase or protein kinase C. These data show that the neuropeptides ET-1 and bombesin stimulate ligand-independent activation of the IGF-IR, which results in Akt activation, and that this cross-communication between GPCR and IGF-IR signaling is inhibited by NEP.

Stimulatory effects of bombesin on plasma trypsin release and exocrine pancreatic secretion in dogs.

PubMed

Kiriyama, S; Hayakawa, T; Kondo, T; Shibata, T; Kitagawa, M; Sakai, Y; Sobajima, H; Ikei, N; Kodaira, T; Hamaoka, T

1990-01-01

We examined the effect of bombesin on plasma trypsin release and exocrine pancreatic secretion in dogs. Bombesin significantly increased plasma immunoreactive trypsin (IRT). Atropine significantly inhibited the response of plasma IRT to bombesin. Pancreatic trypsin secretion was also increased by bombesin, as well as bicarbonate and protein outputs. Atropine failed to inhibit pancreatic trypsin secretion. In conclusion, bombesin has a stimulatory effect on plasma trypsin release mediated by a cholinergic mechanism and different from pancreatic secretion.

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

NASA Astrophysics Data System (ADS)

Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

2013-08-01

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

Effects of bombesin on erythropoietin production in the anaesthetized dog.

PubMed

Melchiorri, P; Sopranzi, N; Roseghini, M

1976-08-01

Bombesin, a tetradecapeptide isolated from the skin of some European discoglossid frogs, has been reported previously to reduce renal blood flow and glomerular filtration rate and to increase plasma renin activity in anaesthetized dogs. In the present study bombesin was infused intravenously in anaesthetized dogs at dose levels of 3, 6 and 12 ng/kg/min for 6 h and renal blood flow, glomerular filtration rate, oxygen consumption, oxygen extraction by the kidney tissue, as well as plasma erythropoietin levels (ESF) and plasma renin activity were measured. Plasma levels of ESF increased during bombesin infusion only when renal blood flow was reduced to a level of 1 ml/g/min or less. In this situation glomerular filtration was blocked, renal oxygen consumption was decreased to 10% of normal and oxygen extraction by the kidney was increased by 2 times. No correlation was found between plasma renin activity and ESF concentrations during bombesin infusion. It is concluded that the stimulant action of bombesin on ESF production is a consequence of the renal hypoxia induced by the reduction in renal blood flow.

The action of bombesin on the kidney of the anaesthetized dog.

PubMed

Erspamer, V; Melchiorri, P; Sopranzi, N

1973-07-01

1. In the anaesthetized dog bombesin had a potent antidiuretic effect, and sometimes arrested urine flow completely. Threshold doses, by i.v. infusion, were of the order of 0.5-1 (ng/kg)/minute. Antidiuresis was the result of a reduction in glomerular filtration rate provoked by a fall in intraglomerular hydrostatic pressure. This, in its turn, was due to afferent vasoconstriction.2. The spasmogenic effect of bombesin on the smooth muscle of the afferent arterioles was directly demonstrated by the radioactive microspheres technique and indirectly by the (85)Kr washout method and by [(3)H]-p-aminohippurate clearance. The vascular compartment most sensitive to bombesin was that of the outer cortical zone, especially in its external half.3. Filtration fraction decreased under the influence of bombesin, indicating that the effect of the polypeptide on postglomerular arterioles was, if present, only of minor importance.4. At high infusion rates (above 6 (ng/kg)/min), bombesin produced a decrease in [(3)H]-p-aminohippurate extraction. The effect of the polypeptide on fractional distal delivery of sodium varied with the dose: at moderate infusion rates it decreased, at high infusion rates it increased. The total glucose appearing in urine following a glucose load was sharply reduced by bombesin. However, the glomerular filtration rate/maximum tubular glucose transport ratio did not show any appreciable change.5. Afferent vasoconstriction produced by bombesin was accompanied by an intense activation of the renin-angiotensin system, as shown by a conspicuous increase in renin secretion, followed by increases in renin activity and angiotensin II concentration in arterial blood. When bombesin was infused into one renal artery only the infused kidney showed afferent vasoconstriction and increased renin secretion. The time-course of renin secretion produced by bombesin depended upon the rate of infusion of the polypeptide. At low rates an increased renin secretion was observed

Phosphoramidon potentiates the bronchoconstriction induced by inhaled bombesin in guinea pigs.

PubMed

Lagente, V; Boichot, E; Mencia-Huerta, J M; Braquet, P

1993-01-01

The effect of the neutral endopeptidase inhibitor, phosphoramidon, on the bronchoconstriction induced by aerosolized bombesin in the guinea pig was investigated. Administered by aerosol for 1 min, bombesin (0.01 or 0.1 mg/ml) induced a dose-dependent increase in pulmonary inflation pressure. Pretreatment of the guinea-pigs with phosphoramidon (0.1 mM), administered by aerosol for 15 min, 15 min prior to challenge, markedly potentiated the increase in pulmonary inflation pressure induced by bombesin (0.01 mg/ml) and substance P (0.1 mg/ml). This result suggests a local hydrolysis of bombesin by airway neutral endopeptidase reducing the activity of this peptide on smooth muscle.

Alterations in receptor expression or agonist concentration change the pathways gastrin-releasing peptide receptor uses to regulate extracellular signal-regulated kinase.

PubMed

Chen, Pei-Wen; Kroog, Glenn S

2004-12-01

G protein-coupled receptors activate extracellular signal-regulated kinases (ERKs) via different pathways in different cell types. In this study, we demonstrate that gastrin-releasing peptide receptor (GRPr) regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration. We examined stably transfected BALB/c 3T3 fibroblasts expressing GRPr constructs at different levels and treated the cells with several concentrations of bombesin (BN, a GRPr agonist) to activate a variable number of GRPr per cell. GRPr induced two waves of ERK activation and one wave of ERK inhibition. One wave of activation required an intact GRPr carboxyl-terminal domain (CTD). It peaked 6 min after addition of high BN concentration ([BN]) in cells with high GRPr expression. Another wave of activation was CTD-independent. It peaked 2 to 4 min after BN addition in cells when [BN] and/or GRPr expression were lower. The early wave of ERK activation was more sensitive than the later one to pretreatment with Bisindolylmaleimide I (GF 109203X) (a protein kinase C inhibitor) or hypertonic sucrose. Because these two waves of activation differ in time course, dose-response curve, requirement for GRPr CTD, and sensitivity to inhibitors, they result from different signaling pathways. A third pathway in these cells inhibited ERK phosphorylation 2 min after addition of high [BN] in cells with high GRPr expression. Furthermore, a GRPr-expressing human duodenal cancer cell line showed differential sensitivity to GF 109203X throughout BN-induced ERK activation, indicating that GRPr may activate ERK via multiple pathways in cells expressing endogenous GRPr.

Targeting tachykinin receptors in neuroblastoma.

PubMed

Henssen, Anton G; Odersky, Andrea; Szymansky, Annabell; Seiler, Marleen; Althoff, Kristina; Beckers, Anneleen; Speleman, Frank; Schäfers, Simon; De Preter, Katleen; Astrahanseff, Kathy; Struck, Joachim; Schramm, Alexander; Eggert, Angelika; Bergmann, Andreas; Schulte, Johannes H

2017-01-03

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.

Bombesin Encapsulated in Long-Circulating pH-Sensitive Liposomes as a Radiotracer for Breast Tumor Identification.

PubMed

De Barros, André Luís Branco; Mota, Luciene Das Graças; Coelho, Marina Melo Antunes; Corrêa, Natássia Caroline Resende; De Góes, Alfredo Miranda; Oliveira, Mônica Cristina; Cardoso, Valbert Nascimento

2015-02-01

Bombesin (BBN) is a tetradecapeptide that binds specifically to gastrin-releasing peptide receptors in humans. These receptors are over-expressed in several forms of cancer; radiolabeled BBN could therefore be used to detect such cancers. However, the degradation of peptides is a critical issue in the development of tumor tracers. Liposomes can be used to overcome this problem and improve the uptake of tracers by tumors. Therefore, the purpose of this study was to prepare and characterize long-circulating and pH-sensitive liposomes (SpHL) containing 99mTc-HYNIC-βAla-Bombesin(7-14) (99mTc-BBN(7-14). In addition, the ability of this system to identify human breast cancer tissue was evaluated using biodistribution studies and scintigraphic images. Long-circulating and pH-sensitive liposomes (SpHL) were prepared and freeze-dried in the presence of cryoprotectants (glucose, mannitol, and trehalose). They were subsequently reconstituted with a solution of 99mTc-HYNIC-βAla-Bombesin(7-14) (99mTc-BBN(7-14)). The liposomes were evaluated for size, encapsulation percentage, radiotracer leakage, and storage stability. In addition, in vivo studies were performed in breast tumor-bearing nude mice. Liposomes in the presence of glucose (SpHLG), exhibited a mean diameter of 164.5 ± 6.5 nm and exhibited a 99mTc-BBN(7-14) encapsulation percentage of 30%. In addition, they remained highly stable for up to 120 days of storage. SpHLG- 99mTc-BBN(7-14) showed longer blood circulation than free 99mTc-BBN(7-14), did. The tumor-to-muscle and tumor-to-blood ratios for SpHLG-99mTc-BBN(7-14 were high at 4 h post-injection (9.31%ID/g and 7.93%ID/g, respectively). Furthermore, scintigraphic images revealed a strong signal in the tumor area, indicating tumor specificity of SpHLG-99mTc-BBN(7-14). In summary, SpHLG-99mTc-BBN(7-14) presented characteristics suitable for a diagnostic agent, and is a potential tool for tumor identification.

Cholecystokinin receptor antagonism by peptidergic and non-peptidergic agents in rat pancreas.

PubMed Central

Dembinski, A; Jaworek, J; Konturek, P K; Konturek, S J; Warzecha, Z

1989-01-01

1. Graded doses of bombesin infused I.V. into conscious rats with chronic pancreatic fistulae induced a dose-dependent stimulation of protein secretion, similar to that obtained with caerulein. This stimulation does not appear to be mediated by cholecystokinin (CCK) receptors because peptidergic (CR-1409) and non-peptidergic (L-364718) CCK antagonists failed to affect protein secretion at a dose range which caused almost complete suppression of caerulein-induced pancreatic secretion. 2. Studies in vitro on isolated rat pancreatic acini revealed that caerulein, pentagastrin and bombesin all showed the same efficacy in their ability to stimulate amylase release. In contrast, CCK antagonists competitively inhibited amylase release induced by caerulein and pentagastrin but not by bombesin or urecholine, indicating that the latter two agents act directly on acinar cells via receptors which are separate from those involved in stimulation induced by caerulein and pentagastrin. 3. DNA synthesis, measured by the incorporation of [3H]thymidine into DNA, was significantly stimulated by caerulein, soybean trypsin inhibitor (FOY 305), pentagastrin and by bombesin in a dose-dependent manner. CCK receptor antagonists prevented stimulation of DNA synthesis induced by caerulein, FOY 305 and pentagastrin but not by bombesin. 4. This study indicates that bombesin strongly stimulates pancreatic enzyme secretion, with an efficacy similar to that of caerulein, and also exerts a potent growth-promoting action on the pancreas, both effects appearing to be mediated by mechanisms independent of the CCK receptors. PMID:2614728

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

PubMed

Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Effect of bombesin and cholecystokinin on plasma immunoreactive trypsin in humans.

PubMed

de Jong, A J; Klamer, M; Jansen, J B; Hopman, W P; Lamers, C B

1987-01-01

Since bombesin is a potent stimulus of the release of cholecystokinin (CCK), it has been suggested that the stimulatory effect of bombesin on pancreatic enzyme secretion is mediated by CCK. The present study was undertaken to determine the role of CCK in the bombesin-induced stimulation of plasma immunoreactive trypsin. Plasma CCK was measured by radioimmunoassay using the antibody T204, which binds to all biologically active sulfated COOH-terminal CCK-peptides. Plasma trypsin was also measured by radioimmunoassay. Infusion of 5 ng/kg/min bombesin in 6 healthy volunteers increased plasma CCK from 1.2 +/- 0.2-8.9 +/- 0.7 pM (p less than 0.0001). The peak increment in plasma CCK during bombesin (9.3 +/- 0.6 pM) was accompanied by a significant rise in plasma trypsin from 206 +/- 21-334 +/- 44 ng/ml (p less than 0.01). However, when similar increases in plasma CCK were achieved by infusion of 0.018 CU/kg/min CCK-33 (9.9 +/- 0.8 pM) or by intraduodenal instillation of 250 ml 20% Intralipid (9.7 +/- 1.9 pM), no significant changes in plasma trypsin were observed. It is therefore concluded that the stimulatory effect of bombesin on plasma immunoreactive trypsin is not mediated by CCK.

Migration of guinea pig airway epithelial cells in response to bombesin analogues.

PubMed

Kim, J S; McKinnis, V S; White, S R

1997-03-01

Bombesin-like peptides within neuroepithelial cells elicit proliferation of normal and malignant airway epithelial cells. It is not clear that these peptides also elicit epithelial cell migration, a necessary component of airway repair after injury. We studied the effects of the bombesin analogues, gastrin releasing peptide (GRP) and neuromedin B (NMB), on guinea pig tracheal epithelial cell (GPTEC) migration. Primary GPTEC were allowed to migrate through 8-microm-pore gelatin-coated filters for 6 h in a chemotaxis chamber, after which the number of migrated cells per 10 high power fields (10 hpf) were counted. Both neuropeptides elicited migration of GPTEC: 24.8 +/- 4.5 cells for 10(-11) M NMB (P < 0.001 versus control, n = 4) and 16.8 +/- 1.2 cells for 10(-12) M GRP (P < 0.001 versus control, n = 8). Migration was attenuated substantially by a bombesin receptor antagonist. To investigate further the relationship of migration through a filter to the repair of a damaged epithelium, we studied the repair of epithelial cells by video microscopy. A 0.3- to 0.5-microm2 wound was created in a confluent monolayer of GPTEC, and wound closure was followed over 24 h. There was no significant acceleration in the rate of repair of GRP- or NMB-stimulated monolayers compared to control. These data demonstrate that GRP and NMB elicit migration of airway epithelial cells but may not play a significant role in the early repair of the airway epithelium in culture.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

PubMed

Zheng, R; Iwase, A; Shen, R; Goodman, O B; Sugimoto, N; Takuwa, Y; Lerner, D J; Nanus, D M

2006-09-28

The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

Effect of bombesin on gastric secretion and motility in the cat.

PubMed

Vagne, M; Gelin, M L; McDonald, T J; Chayvialle, J A; Minaire, Y

1982-01-01

The effect of bombesin on acid and pepsin secretion and antral motility was compared to that of pentagastrin in conscious cats. Bombesin stimulated acid secretion to 65% of the maximal response to pentagastrin but induced a stronger pepsin secretion than any dose of pentagastrin. As to antral motility, bombesin first induced an effect comparable to that of pentagastrin, with an increase of low-amplitude and a decrease of high-amplitude contractions. After about 30 min of continuous infusion, the effect of bombesin changed with a return to basal frequency for the low-amplitude contractions and an increase of high-amplitude contractions. This effect was not observed with pentagastrin nor cholecystokinin and was not explained by the variations of plasma insulin concentration.

Effect of bombesin on pancreatic secretion and gall bladder motility of the chicken.

PubMed

Linari, G; Linari, M B

1975-12-01

Bombesin strongly stimulated the chicken pancreatic secretion. When given by i.v. infusion, the threshold dose was of the order of 7.5-45.0 ng/kg/min and maximum enzyme output was obtained at a rate of 60 ng/kg/min. In addition to total enzyme output, enzyme concentration was also increased. Caerulein displayed a more potent stimulant effect, but composition of juice produced by the two polypeptides was similar. Tachyphylaxis occurred only with bombesin. Neither atropine nor gastric acidification affected the response to bombesin. Bombesin was totally ineffective in promoting gall bladder emptying. It is suggested that in the chicken, bombesin acts on the exocrine pancreas indirectly through release of an endogenous pancreozymin possibly devoid of cholecystokinetic activity.

Effect of a new potent CCK antagonist, lorglumide, on caerulein- and bombesin-induced pancreatic secretion and growth in the rat.

PubMed

Scarpignato, C; Varga, G; Dobronyi, I; Papp, M

1989-03-01

1. The effect of lorglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic secretion and growth induced by caerulein and bombesin was studied in the rat. 2. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (5 days) administration of the peptides and/or lorglumide. 3. Both caerulein and bombesin stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Although the efficacy of both peptides was virtually identical, the potency of caerulein was higher than that of bombesin. Lorglumide displaced the concentration-response curves to caerulein to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope not significantly different from unity. The calculated pA2 for lorglumide was 7.31 +/- 0.45. The antagonist, however, was completely ineffective when tested against bombesin-induced amylase release. 4. In vivo experiments confirmed results from in vitro studies since lorglumide (5 and 10 mg kg-1) significantly reduced pancreatic exocrine secretion induced by caerulein without affecting the response to bombesin. 5. Administration of either peptide increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Lorglumide (10 mg kg-1), administered together with caerulein, reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. On the contrary, when lorglumide was given together with bombesin, all the parameters that were examined were not altered by concomitant administration of the antagonist. 6. These results have demonstrated the ability of lorglumide to antagonize the effects on the pancreas of a CCK-analogue, caerulein, and its inability to affect bombesin

Comparison of [(11)C]Choline ([(11)C]CHO) and [(18)F]Bombesin (BAY 86-4367) as Imaging Probes for Prostate Cancer in a PC-3 Prostate Cancer Xenograft Model.

PubMed

Schwarzenböck, Sarah Marie; Schmeja, Philipp; Kurth, Jens; Souvatzoglou, Michael; Nawroth, Roman; Treiber, Uwe; Kundt, Guenther; Berndt, Sandra; Graham, Keith; Senekowitsch-Schmidtke, Reingard; Schwaiger, Markus; Ziegler, Sibylle I; Dinkelborg, Ludger; Wester, Hans-Jürgen; Krause, Bernd Joachim

2016-06-01

Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [(18)F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [(11)C]Choline ([(11)C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT). We carried out a dual-tracer small animal PET/CT study comparing [(18)F]Bombesin and [(11)C]CHO. The androgen-independent human prostate tumor cell line PC-3 was implanted subcutaneously in the flanks of nu/nu NMRI mice (n = 10) (PET/CT measurements of two [(11)C]Choline mice could not be analyzed due to technical reasons). [(18)F]Bombesin and [(11)C]CHO PET/CT imaging was performed about 3-4 weeks after the implantation of PC-3 cells on two separate days. After the intravenous tail vein injection of 14 MBq [(18)F]Bombesin and 37 MBq [(11)C]CHO, respectively, a dynamic study over 60 min was acquired in list mode using an Inveon animal PET/CT scanner (Siemens Medical Solutions). The sequence of [(18)F]Bombesin and [(11)C]CHO was randomized. Image analysis was performed using summed images as well as dynamic data. To calculate static and dynamic tumor-to-muscle (T/M), tumor-to-blood (T/B), liver-to-blood (L/B), and kidney-to-blood (K/B) ratios, 4 × 4 × 4 mm(3) volumes of interest (VOIs) of tumor, muscle (thigh), liver, kidney, and blood derived from transversal slices were used. The mean T/M ratio of [(18)F]Bombesin and [(11)C]CHO was 6.54 ± 2.49 and 1.35 ± 0.30, respectively. The mean T/B ratio was 1.83 ± 0.79 for [(18)F]Bombesin and 0.55 ± 0.10 for [(11)C

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Powerful inhibition of in-vivo growth of experimental hepatic cancers by bombesin/gastrin-releasing peptide antagonist RC-3940-II.

PubMed

Szepeshazi, Karoly; Schally, Andrew V; Rick, Ferenc G; Block, Norman L; Vidaurre, Irving; Halmos, Gabor; Szalontay, Luca

2012-10-01

Hepatic carcinoma is a major health problem worldwide. Its incidence is increasing in Western countries and there is currently no effective systemic therapy against it. Targeted treatment modalities developed in the past few years have provided very limited success. Development of new treatment strategies is therefore essential. We investigated the effects of bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on experimental human liver cancers in nude mice. SK-Hep-1 and Hep-G2 cancers transplanted subcutaneously into nude mice were treated daily with 10 or 20 µg of RC-3940-II. Tumor growth was monitored for 50-184 days in five experiments. Tumor gene expression was analyzed with PCR array and protein expression by immunoblotting. Characteristics of BN/GRP receptors in the tumors were analyzed by binding assays. Effects of RC-3940-II on cell proliferation were investigated in vitro. RC-3940-II inhibited the growth of SK-Hep-1 cancers in nude mice by 65-98%, with total regression in 9 of 36 tumors in three experiments. The BN/GRP antagonist inhibited the growth of Hep-G2 cancers as well by 73-82% in two experiments, being effective even on originally large tumors. Gene expression analysis showed an increase in several angiogenesis inhibitors and decrease in proangiogenic genes after RC-3940-II treatment. Receptor assays demonstrated high-affinity binding sites for BN/GRP in both tumor lines. BN/GRP antagonist RC-3940-II powerfully inhibits growth of SK-Hep-1 and Hep-G2 cancers in nude mice. Its effect may be linked to changes in expression of those cancer genes important in angiogenesis, invasion, and metastasis. RC-3940-II may be considered for further investigations in treatment of liver cancers.

Diagnostic value of 99mTc-bombesin scintigraphy for differentiation of malignant from benign breast lesions.

PubMed

Shariati, Farzaneh; Aryana, Kamran; Fattahi, Asiehsadat; Forghani, Mohammad N; Azarian, Azita; Zakavi, Seyed R; Sadeghi, Ramin; Ayati, Narjes; Sadri, Keyvan

2014-06-01

In this study, we evaluated the diagnostic accuracy of (99m)Tc-bombesin scintigraphy for differentiation of benign from malignant palpable breast lesions. (99m)Tc-Bombesin is a tracer with high affinity for gastrin-releasing peptide receptor, which is overexpressed on a variety of human tumors including breast carcinoma. We examined 33 consecutive women who were referred to our center with suspicious palpable breast lesions but had no definitive diagnosis in other imaging procedures. A volume of 370-444 MBq of (99m)Tc-bombesin was injected and dynamic 1-min images were taken for 20 min immediately after injection in anterior view. Thereafter, two static images in anterior and prone-lateral views were taken for 5 min. Finally, single-photon emission computed tomography images were taken for each patient. Definitive diagnosis was based on biopsy and histopathological evaluation. The scan findings were positive in 19 patients and negative in 11 on visual assessment of the planar and single-photon emission computed tomography images. Pathologic examination confirmed breast carcinoma in 12 patients with positive scans and benign pathology for 18 patients. The overall sensitivity, specificity, negative and positive predictive values, and accuracy of this radiotracer for diagnosis of breast cancer were 100, 66.1, 100, 63, and 76%, respectively. Semiquantitative analysis improved the specificity of the visual assessment from 66 to 84%. Our study showed that (99m)Tc-bombesin scintigraphy has a high sensitivity and negative predictive value for detecting malignant breast lesions, but the specificity and positive predictive value of this radiotracer for differentiation of malignant breast abnormalities from benign ones are relatively low.

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin.

PubMed

Festuccia, C; Guerra, F; D'Ascenzo, S; Giunciuglio, D; Albini, A; Bologna, M

1998-01-30

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.

G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like receptors and cellular signaling.

PubMed

Abdulkhalek, Samar; Guo, Merry; Amith, Schammim Ray; Jayanth, Preethi; Szewczuk, Myron R

2012-11-01

The mechanism(s) behind GPCR transactivation of TLR receptors independent of TLR ligands is unknown. Here, GPCR agonists bombesin, bradykinin, lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and -2, but not thrombin induce Neu1 activity in live macrophage cell lines and primary bone marrow macrophage cells from wild-type (WT) mice but not from Neu1-deficient mice. Using immunocytochemistry and NFκB-dependent secretory alkaline phosphatase (SEAP) analyses, bombesin induced NFκB activation in BMC-2 and RAW-blue macrophage cells, which was inhibited by MyD88 homodimerization inhibitor, Tamiflu, galardin, piperazine and anti-MMP-9 antibody. Bombesin receptor, neuromedin B (NMBR), forms a complex with TLR4 and MMP9. Silencing MMP9 mRNA using siRNA transfection of RAW-blue macrophage cells markedly reduced Neu1 activity associated with bombesin-, bradykinin- and LPA-treated cells to the untreated controls. These findings uncover a molecular organizational GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of TLR receptors and subsequent cellular signaling. Copyright © 2012 Elsevier Inc. All rights reserved.

Novel DOTA-based prochelator for divalent peptide vectorization: synthesis of dimeric bombesin analogues for multimodality tumor imaging and therapy.

PubMed

Abiraj, Keelara; Jaccard, Hugues; Kretzschmar, Martin; Helm, Lothar; Maecke, Helmut R

2008-07-28

Dimeric peptidic vectors, obtained by the divalent grafting of bombesin analogues on a newly synthesized DOTA-based prochelator, showed improved qualities as tumor targeted imaging probes in comparison to their monomeric analogues.

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-12-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

Regulation of body temperature and brown adipose tissue thermogenesis by bombesin receptor subtype-3

PubMed Central

Lateef, Dalya M.; Abreu-Vieira, Gustavo; Xiao, Cuiying

2014-01-01

Bombesin receptor subtype-3 (BRS-3) regulates energy homeostasis, with Brs3 knockout (Brs3−/y) mice being hypometabolic, hypothermic, and hyperphagic and developing obesity. We now report that the reduced body temperature is more readily detected if body temperature is analyzed as a function of physical activity level and light/dark phase. Physical activity level correlated best with body temperature 4 min later. The Brs3−/y metabolic phenotype is not due to intrinsically impaired brown adipose tissue function or in the communication of sympathetic signals from the brain to brown adipose tissue, since Brs3−/y mice have intact thermogenic responses to stress, acute cold exposure, and β3-adrenergic activation, and Brs3−/y mice prefer a cooler environment. Treatment with the BRS-3 agonist MK-5046 increased brown adipose tissue temperature and body temperature in wild-type but not Brs3−/y mice. Intrahypothalamic infusion of MK-5046 increased body temperature. These data indicate that the BRS-3 regulation of body temperature is via a central mechanism, upstream of sympathetic efferents. The reduced body temperature in Brs3−/y mice is due to altered regulation of energy homeostasis affecting higher center regulation of body temperature, rather than an intrinsic defect in brown adipose tissue. PMID:24452453

In Vitro and In Vivo Evaluation of a 18F-Labeled High Affinity NOTA Conjugated Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging

PubMed Central

Velikyan, Irina; Lindeberg, Gunnar; Sörensen, Jens; Larhed, Mats; Antoni, Gunnar; Sandström, Mattias; Tolmachev, Vladimir; Orlova, Anna

2013-01-01

Expression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2, RM26) conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethylene glycol (PEG2) spacer (NOTA-P2-RM26) labeled with 68Ga and 111In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a 18F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with 18F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC50) of the [natF]AlF-NOTA-P2-RM26 was compared to that of the natGa-loaded peptide using 125I-Tyr4-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with 18F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol). The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC50=4.4±0.8 nM). The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [18F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p.i. The initial biological

Novel receptor-targeted contrast agents for optical imaging of tumors

NASA Astrophysics Data System (ADS)

Becker, Andreas; Hessenius, Carsten; Bhargava, Sarah; Ebert, Bernd; Sukowski, Uwe; Rinneberg, Herbert H.; Wiedenmann, Bertram; Semmler, Wolfhard; Licha, Kai

2000-04-01

Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP- labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor- expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-medicated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in RIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle.

PubMed

Zhao, Hongqiong; Matsuda, Seinosuke; Thanthan, Sint; Yannaing, Swe; Kuwayama, Hideto

2012-10-01

This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 μg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 μg/kg NMC; (3) six steers were IV injected with 2.5μg/kg bovine gastrin-releasing peptide (GRP), 1.0 μg/kg NMC combined with 20.0 μg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 μg/kg NMC combined with 20.0 μg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0 μg/kg N-GRP-EE alone, 1.0 μg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 μg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 μg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion in humans.

PubMed

Lam, W F; Masclee, A A; Muller, E S; Souverijn, J H; Lamers, C B

1998-08-01

This study was undertaken to investigate the effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion. Seven healthy subjects participated in two experiments performed in random order during normoglycemia and hyperglycemic clamping at 15 mM. Duodenal outputs of bilirubin, trypsin, amylase, and bicarbonate were measured by aspiration with a recovery marker under basal conditions for 60 min and during continuous infusion of bombesin (1 ng/kg x min) for 60 min. Plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels were determined at regular intervals. Compared to normoglycemia, during hyperglycemia basal outputs of bilirubin (17 +/- 3 vs. 0.9 +/- 0.4 micromol/60 min), trypsin (24 +/- 4 vs. 4 +/- 1 U/60 min), amylase (12 +/- 1 vs. 3 +/- 1 kU/60 min), and bicarbonate (2.9 +/- 0.5 vs. 1.2 +/- 0.2 mmol/60 min) were significantly p < 0.05) reduced. Bombesin significantly (p < 0.05) increased pancreaticobiliary output during both normo- and hyperglycemia. During hyperglycemia bombesin-stimulated 60-min outputs of bilirubin, trypsin, amylase, and bicarbonate were not significantly different compared to those during normoglycemia. Basal and bombesin-stimulated plasma PP concentrations were significantly (p < 0.05) reduced during hyperglycemia, but plasma CCK levels were not significantly different. It is concluded that acute hyperglycemia reduces basal but does not affect bombesin-induced pancreaticobiliary secretion.

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed Central

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-01-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung. Images PMID:1660144

In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems.

PubMed

Garrison, Jered C; Rold, Tammy L; Sieckman, Gary L; Figueroa, Said Daibes; Volkert, Wynn A; Jurisson, Silvia S; Hoffman, Timothy J

2007-08-01

The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, beta+: 18%, E(beta+ max) = 653 keV; beta-: 37%, E(beta- max) = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N',N'',N'''-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. The CB-TE2A-8-AOC-BBN(7-14)NH2 and DOTA-8-AOC-BBN(7-14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 +/- 2.27 and 4.95 +/- 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 +/- 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 +/- 1.51 %ID

Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

PubMed

Kullmann, A; Weber, P S; Bishop, J B; Roux, T M; Norby, B; Burns, T A; McCutcheon, L J; Belknap, J K; Geor, R J

2016-09-01

Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation. © 2015 EVJ

Time-Resolved Spectroscopy and Near Infrared Imaging for Prostate Cancer Detection: Receptor-targeted and Native Biomarker

NASA Astrophysics Data System (ADS)

Pu, Yang

Optical spectroscopy and imaging using near-infrared (NIR) light provides powerful tools for non-invasive detection of cancer in tissue. Optical techniques are capable of quantitative reconstructions maps of tissue absorption and scattering properties, thus can map in vivo the differences in the content of certain marker chromophores and/or fluorophores in normal and cancerous tissues (for example: water, tryptophan, collagen and NADH contents). Potential clinical applications of optical spectroscopy and imaging include functional tumor detection and photothermal therapeutics. Optical spectroscopy and imaging apply contrasts from intrinsic tissue chromophores such as water, collagen and NADH, and extrinsic optical contrast agents such as Indocyanine Green (ICG) to distinguish disease tissue from the normal one. Fluorescence spectroscopy and imaging also gives high sensitivity and specificity for biomedical diagnosis. Recent developments on specific-targeting fluorophores such as small receptor-targeted dye-peptide conjugate contrast agent offer high contrast between normal and cancerous tissues hence provide promising future for early tumour detection. This thesis focus on a study to distinguish the cancerous prostate tissue from the normal prostate tissues with enhancement of specific receptor-targeted prostate cancer contrast agents using optical spectroscopy and imaging techniques. The scattering and absorption coefficients, and anisotropy factor of cancerous and normal prostate tissues were investigated first as the basis for the biomedical diagnostic and optical imaging. Understanding the receptors over-expressed prostate cancer cells and molecular target mechanism of ligand, two small ICG-derivative dye-peptides, namely Cypate-Bombesin Peptide Analogue Conjugate (Cybesin) and Cypate-Octreotate Peptide Conjugate (Cytate), were applied to study their clinical potential for human prostate cancer detection. In this work, the steady-state and time

Bombesin receptors and transplanted stem cells in rat brain: High-resolution scan with 99mTc BN1.1

NASA Astrophysics Data System (ADS)

Scopinaro, F.; Paschali, E.; Di Santo, G.; Antonellis, T.; Massari, R.; Trotta, C.; Gourni, H.; Bouziotis, P.; David, V.; Soluri, A.; Varvarigou, A. D.

2006-12-01

The aim of this work is to detect the presence of transplanted stem cells (TSC) in rat brain with high-resolution (HR) scintigraphy and labelled bombesin (BN). BN is a morphogen for Central Nervous System (CNS) as well as for other organs: CNS-oriented TSC over-express BN Receptors (BNR). BN is also a neurotransmitter and modulates several functions of CNS. 99mTc labelled BN-like peptide scan of CNS is the ideal method to detect growing TSC once knowing normal distribution of BNRs in CNS. HR Planar and single photon emission computerized tomography (SPECT) images of rat brain were performed with new HR detectors (Li-tech, Italy). Pertechnetate, 99mTc HMPAO and the new 99mTc BN1.1 (patented) were i.v. administered in five rats. HR SPECT of 99mTc BN1.1 detected olfactory tract, fronto-lateral cortex, cerebellum, basal ganglia and amygdale. Results of SPECT were confirmed by bio-distribution study performed after autopsy of three of the five rats. The remaining two rats underwent cerebral lesions followed by transplant of TSC. Three months later, HR scintigraphy was repeated and showed images completely different from previous basal study, with hot spot of 99mTc BN1.1 corresponding to the site of TSC transplant. Immuno-histochemistry confirmed the presence of viable TSC. Not only 99mTc BN1.1 HR scan showed viability of transplanted TSC but also the "background brain" was the still now unknown map of BNR in mammalian brain.

Targeting the TAM Receptors in Leukemia.

PubMed

Huey, Madeline G; Minson, Katherine A; Earp, H Shelton; DeRyckere, Deborah; Graham, Douglas K

2016-11-08

Targeted inhibition of members of the TAM (TYRO-3, AXL, MERTK) family of receptor tyrosine kinases has recently been investigated as a novel strategy for treatment of hematologic malignancies. The physiologic functions of the TAM receptors in innate immune control, natural killer (NK) cell differentiation, efferocytosis, clearance of apoptotic debris, and hemostasis have previously been described and more recent data implicate TAM kinases as important regulators of erythropoiesis and megakaryopoiesis. The TAM receptors are aberrantly or ectopically expressed in many hematologic malignancies including acute myeloid leukemia, B- and T-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma. TAM receptors contribute to leukemic phenotypes through activation of pro-survival signaling pathways and interplay with other oncogenic proteins such as FLT3, LYN, and FGFR3. The TAM receptors also contribute to resistance to both cytotoxic chemotherapeutics and targeted agents, making them attractive therapeutic targets. A number of translational strategies for TAM inhibition are in development, including small molecule inhibitors, ligand traps, and monoclonal antibodies. Emerging areas of research include modulation of TAM receptors to enhance anti-tumor immunity, potential roles for TYRO-3 in leukemogenesis, and the function of the bone marrow microenvironment in mediating resistance to TAM inhibition.

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat.

PubMed

Pollack, P F; Adamson, C; Koldovsky, O

1989-04-15

Because of the presence of bombesin-like immunoreactivity in milk, we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 micrograms/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.

Effect of bombesin on serum immunoreactive trypsin in healthy subjects and in patients with chronic pancreatitis.

PubMed

Labò, G; Vezzadini, P; Gullo, L; Sternini, C; Bonora, G

1983-08-01

We studied the effect of bombesin (9 ng/kg X min for 30 min by intravenous infusion) on serum immunoreactive trypsin in healthy subjects and in chronic pancreatitis patients. Bombesin administration caused a marked and significant increase of serum immunoreactive trypsin concentration in healthy subjects. The increase occurred in the first 15 min after the beginning of bombesin infusion and persisted for the duration of the study (2 h). In patients with chronic pancreatitis, the increase was much less pronounced. In these patients, the integrated immunoreactive trypsin response to bombesin was significantly correlated with bicarbonate, lipase, and chymotrypsin outputs into the duodenum. The response of serum immunoreactive trypsin to bombesin stimulation seems to vary according to the degree of pancreatic exocrine dysfunction and to reflect the functional capacity of acinar cell mass.

Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

PubMed

Tolmachov, Oleg E

2015-01-01

Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

Inhibition of growth of PC-82 human prostate cancer line xenografts in nude mice by bombesin antagonist RC-3095 or combination of agonist [D-Trp6]-luteinizing hormone-releasing hormone and somatostatin analog RC-160.

PubMed

Milovanovic, S R; Radulovic, S; Groot, K; Schally, A V

1992-01-01

The effects of treatment with a bombesin receptor antagonist [D-Tpi6, Leu13 psi (CH2NH) Leu14]BN(6-14)(RC-3095) and the combination of an agonist of luteinizing hormone-releasing hormone [D-Trp6]-LH-RH and somatostatin analog D-Phe-Cys-Tyr-D-Trp-Lys-Val- Cys-Trp-NH2 (RC-160) were studied in nude mice bearing xenografts of the hormone-dependent human prostate tumor PC-82. During the 5 weeks of treatment, tumor growth was decreased in all treated groups compared with controls. Bombesin antagonist RC-3095 and the combination of [D-Trp6]-LH-RH and RC-160 caused a greater inhibition of tumor growth than [D-Trp6]-LH-RH or RC-160 alone as based on measurement of tumor volume and percentage change in tumor volume. The largest decrease in tumor weight was also seen in the groups treated with the bombesin antagonist and with the combination of RC-160 and [D-Trp6]-LH-RH. Serum prostatic-specific antigen levels were greatly decreased, and insulin-like growth factor I (IGF-I) as well as growth hormone levels were reduced in all treated groups. Specific binding sites for [D-Trp6]-LH-RH, epidermal growth factor (EGF), IGF-I, and somatostatin (SS-14) were found in the tumor membranes. Receptors for EGF were significantly down-regulated by treatment with the bombesin antagonist or RC-160. Combination of LH-RH agonists with somatostatin analog RC-160 might be considered for improvement of hormonal therapy for prostate cancer. The finding that bombesin antagonist RC-3095 inhibits the growth of PC-82 prostate cancer suggests the merit of further studies to evaluate the possible usefulness of antagonists of bombesin in the management of prostatic carcinoma.

Effect of bile salt binding or protease inactivation on plasma cholecystokinin and gallbladder responses to bombesin.

PubMed

Thimister, P W; Hopman, W P; Sloots, C E; Rosenbusch, G; Tangerman, A; Willems, H L; Lamers, C B; Jansen, J B

1994-12-01

Bombesin-stimulated plasma cholecystokinin levels decrease after an initial increase despite continuous infusion of bombesin. The aim of this study was to determine if a feedback mechanism, mediated by bile salts or proteolytic enzymes, is responsible for this decline. Bombesin (1.0 ng.kg-1.min-1) was infused into volunteers for 180 minutes on separate occasions. Cholestyramine, colestipol, camostate, or saline were perfused intraduodenally during the second hour of the tests. Cholestyramine was also administered without infusion of bombesin. Colestipol and cholestyramine, dependent on their bile salt-binding capacity, markedly enhanced (P < 0.05) bombesin-stimulated plasma cholecystokinin from 2.1 +/- 0.5 pmol/L to 6.4 +/- 2.2 pmol/L and 12.1 +/- 3.3 pmol/L (P < 0.05 vs. colestipol), respectively, and further decreased gallbladder volume (P < 0.05) from 9.4 +/- 1.6 mL to 2.0 +/- 0.4 mL and 2.2 +/- 0.5 mL, respectively. The protease inhibitor camostate had no effect. Bile salt precipitation also enhanced plasma pancreatic polypeptide responses (P < 0.01) but did not alter gastrin responses. Plasma cholecystokinin responses to cholestyramine without bombesin infusion varied considerably, but increments were highly correlated to decreases in gallbladder volume (r = 0.91; P < 0.005). Bile salt sequestration but not protease inactivation enhances plasma cholecystokinin and gallbladder responses to bombesin infusion in humans.

Differential expression of folate receptor 1 in medulloblastoma and the correlation with clinicopathological characters and target therapeutic potential.

PubMed

Liu, Hailong; Sun, Qianwen; Zhang, Mingshan; Zhang, Zhihua; Fan, Xinyi; Yuan, Hongyu; Li, Cheng; Guo, Yuduo; Ning, Weihai; Sun, Youliang; Song, Yongmei; Yu, Chunjiang

2017-04-04

Medulloblastoma is the most common malignant brain tumor in children. Folate receptor 1 (Folr1) was abundantly expressed in some epithelial malignancies. However the expression profile and the role of clinicopathological significance and therapeutic target potential in medulloblastoma still remain elusive. Currently we detected the expression of Folr1 in medulloblastoma and identified the diagnostic application by evaluating the clinical, pathological and neuroimaging values. Then we developed a target therapeutic compound with Folr1, which exhibited promising efficiency in treatment of medulloblastoma. Folr1 expression was up-regulated in medulloblastoma and positively correlated with percentage of Ki-67 and MMP9 labeling, pathological subtypes, serum Folr1 levels and CSF spreading on MRI. The level of serum Folr1 showed rational sensitivity and specificity in predicting histological subgroups. Strong Folr1 expression was recommended as the independent value regarding the prognosis of patients with medulloblastoma. Folr1 targeted therapy attenuated the tumor growth and metastasis with down-regulation of MMPs proteins and activation of apoptosis. Immunostaining analysis in the xenograft samples showed the decreased Ki-67 and MMP9 index providing the strong evidences that Folr1 targeted application can suppress the proliferation and invasion. Our findings uncovered in Folr1 a predictive candidate and therapeutic target for medulloblastoma.

Effect of atropine and somatostatin on bombesin-stimulated plasma immunoreactive trypsin release in man.

PubMed

de Jong, A J; Klamer, M; Lamers, C B

1987-01-01

This study was undertaken to determine the effect of atropine and somatostatin, two inhibitors of intraduodenal pancreatic enzyme secretion, on bombesin-stimulated release of plasma immunoreactive trypsin in 6 healthy volunteers. Infusion of 5 ng/kg.min bombesin during 30 min induced significant increases in plasma trypsin from 206 +/- 20 to 334 +/- 44 ng/ml (p less than 0.01). Atropine (15 ng/kg as i.v. bolus followed by 5 ng/kg.h) had no influence on the bombesin-stimulated increase in plasma immunoreactive trypsin (207 +/- 20 to 326 +/- 54 ng/ml). Somatostatin (125 micrograms as i.v. bolus followed by 125 micrograms/h) also failed to inhibit the plasma trypsin response to bombesin (207 +/- 18 to 663 +/- 166 ng/ml). These results point to major differences in the regulation of plasma and intraduodenal trypsin secretion.

Evidence for a direct trophic effect of bombesin on the mouse pancreas: in vivo and cell culture studies.

PubMed

Lhoste, E F; Aprahamian, M; Balboni, G; Damgé, C

1989-01-01

The present work studied the effect of chronic bombesin on the mouse pancreas and analyzed whether or not this effect was direct. Bombesin administered s.c. 3 times daily for 4 days at various concentrations (0.1, 1, 10, 20 micrograms/kg b. wt.) induced pancreatic growth in a dose-dependent manner. This growth was characterized by an increase in pancreatic weight, its protein and RNA contents suggesting cellular hypertrophy. Pancreatic enzyme content was also increased, especially for amylase (14-fold) and at a lesser degree for chymotrypsin and lipase (2.5-fold). The DNA content of the gland increased significantly after a 1 microgram/kg bombesin treatment suggesting hyperplasia. [3H]thymidine incorporation into DNA increased slightly from 24 h after the first bombesin injection and more obviously at 72 and 96 h indicating DNA synthesis. To determine the direct effect of bombesin on pancreatic acinar cell growth cells were cultured as monolayers on collagen gels in media lacking added hormones and containing 2.5% FBS with or without bombesin (1 microM-1 nM) or caerulein (10 nM). [3H]thymidine incorporation into DNA was increased by caerulein (10 nM) and bombesin (100 nM and 1 microM). Therefore, it is concluded that bombesin is a pancreaticotrophic peptide in mice. Moreover, it is suggested that this effect occurs directly on pancreatic cells.

A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

PubMed Central

Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

2015-01-01

Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

Androgen Receptor: A Complex Therapeutic Target for Breast Cancer

PubMed Central

Narayanan, Ramesh; Dalton, James T.

2016-01-01

Molecular and histopathological profiling have classified breast cancer into multiple sub-types empowering precision treatment. Although estrogen receptor (ER) and human epidermal growth factor receptor (HER2) are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR) is evolving as a molecular target for cancers that have developed resistance to conventional treatments. The high expression of AR in breast cancer and recent discovery and development of new nonsteroidal drugs targeting the AR provide a strong rationale for exploring it again as a therapeutic target in this disease. Ironically, both nonsteroidal agonists and antagonists for the AR are undergoing clinical trials, making AR a complicated target to understand in breast cancer. This review provides a detailed account of AR’s therapeutic role in breast cancer. PMID:27918430

Peptide conjugated polymeric nanoparticles as a carrier for targeted delivery of docetaxel.

PubMed

Kulhari, Hitesh; Pooja, Deep; Shrivastava, Shweta; V G M, Naidu; Sistla, Ramakrishna

2014-05-01

The aim of this research work was to develop Bombesin peptide (BBN) conjugated, docetaxel loaded nanocarrier for the treatment of breast cancer. Docetaxel loaded nanoparticles (DNP) were prepared by solvent evaporation method using sodium cholate as surfactant. BBN was conjugated to DNP surface through covalent bonding. Both DNP and BBN conjugated DNP (BDNP) were characterized by various techniques such as dynamic light scattering, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and thermogravimetric analysis. The particle diameter and zeta potential of BDNP were 136±3.95 nm and -10.8±2.7 mV, respectively. The change in surface charge and FTIR studies confirmed the formation of amide linkage between BBN and DNP. AFM analysis showed that nanoparticles were spherical in shapes. In nanoparticles, docetaxel was present in its amorphous form as confirmed by DSC and PXRD analysis and was stable during the thermal studies. The formulations showed the sustained release of DTX over the period of 120 h. During cellular toxicity assay in gastrin releasing peptide receptor positive breast cancer cells (MDA-MB-231), BDNP were found to be 12 times more toxic than pure DTX and Taxotere. The IC50 value for DTX, Taxotere, DNP and BDNP was >375, >375, 142.23 and 35.53 ng/ml, respectively. The above studies showed that Bombesin conjugated nanocarrier system could be a promising carrier for active targeting of anticancer drugs in GRP receptor over expressing cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE PAGES

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

2017-04-19

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Divergent effects of bombesin and bethanechol on stimulated gastric secretion in duodenal ulcer and in normal men.

PubMed

Helman, C A; Hirschowitz, B I

1987-06-01

To further investigate differences in the responses of normals and patients with duodenal ulcer with respect to gastrin release and acid and pepsin secretion, we infused bombesin (1 microgram/kg X h) or bethanechol (40 micrograms/kg X h) during the middle hour of a 3-h infusion of pentagastrin and compared the results with a pentagastrin infusion without added drug. Pentagastrin dosage (0.1 microgram/kg X h) was set to give about half-maximal response, to detect either inhibition or further stimulation of gastric secretion, whereas the dose of bombesin was chosen to give maximal gastrin but less than maximal acid secretion. Serum gastrin and somatostatin were also measured. In all subjects tested, bethanechol produced no effects on acid, gastrin, or somatostatin release but increased pepsin output. By contrast, bombesin inhibited pentagastrin-stimulated acid output in all 6 normal men by an average of 55%, whereas it inhibited acid output in only 2 of the 9 men with duodenal ulcer. Serum gastrin increases after bombesin in duodenal ulcer were three to four times greater than in normals. Although bombesin stimulates acid only by releasing gastrin, we postulate that bombesin may also simultaneously limit acid and pepsin secretion and speculate that this effect could be mediated by bombesin-induced somatostatin release. The cause for differences between duodenal ulcer and normal remain speculative.

BI-09EphA3 RECEPTOR IS A MOLECULAR TARGET EXPRESSED IN MULTIPLE COMPARTMENTS OF GBM

PubMed Central

Ferluga, Sara; Gibo, Denise; Debinski, Waldemar

2014-01-01

Eph receptor A3 belongs to the Eph family of receptor tyrosine kinases playing critical roles in cancer. We and others found this receptor to be over-expressed in Glioblastoma (GBM), but not in normal brain. EphA3 is a plasma membrane receptor, which is internalized upon ligand binding making it as an attractive target for specific drug delivery. EphA3 overexpression was found in tumor cells and tumor-initiating cells in GBM. However, we noted that EphA3-positive cells localize around the neovasculature, being consistent with tumor-infiltrating cells. Therefore, we decided to analyze EphA3 in relation to microglia/macrophages, as these cells highly infiltrate GBM favoring tumor progression. It has been demonstrated that glioma-infiltrating microglia acquire the M2 phenotype expressing CD163 and CD204 markers. Co-localization studies using immunofluorescence on tumor-derived primary cells showed that EphA3 co-localizes with CD163 on a sub-population of cells. The two markers also highly co-localize in snap-frozen sections of human GBM specimens, mainly in the perivascular region, as well as on cells within the bulk of the tumor and in the invasive ring, but not on the contralateral side of the diseased brain. EphA3 on snap-frozen specimens co-localized also with CD68, a more general macrophages marker, confirming the presence of EphA3 on these bone marrow-derived cells. Microglia/ macrophages have been shown also around tumor necrotic areas. We cultured GBM cells under normoxia, hypoxia and anoxia conditions and found that the levels of EphA3 receptor increased under anoxia compared to hypoxia, following the same pattern seen with CD163 and CD204. We have already generated a novel and specific cytotoxin capable of activating and internalizing the receptor and potently killing EphA3-overexpressing cells. In this study we demonstrate that by utilizing the EphA3 receptor, we will target not only tumor and tumor-initiating cells, but also infiltrating cells active in

Relation of gastric acid and pepsin secretion to serum gastrin levels in dogs given bombesin and gastrin-17.

PubMed

Hirschowitz, B I; Molina, E

1983-05-01

To quantitate bombesin stimulation of gastric acid and pepsin via release of gastrin, five gastric fistula dogs were given graded doses (60-1,250 pmol X kg-1 X h-1) of bombesin tetradecapeptide and 40-2,000 pmol X kg-1 X h-1 of synthetic gastrin-17 (G-17). Acid and pepsin output and serum gastrin were proportional to the dose of stimulant. The half-maximal dose of bombesin for gastrin release was 200 pmol X kg-1 X h-1. Bombesin-stimulated acid secretion related to serum gastrin concentrations was congruent with the G-17 curve, but with a maximum of only 62% of the G-17 maximum before declining by 27% despite higher serum gastrin levels. This suggested that bombesin stimulates acid secretion only via gastrin release and inhibits at higher doses by releasing another inhibitory peptide, most likely somatostatin, which is also released by bombesin. The same mechanism could apply to supramaximal inhibition of acid and pepsin seen with high doses of G-17. Because the pepsin curve related to serum gastrin was to the left of the G-17 curve, we concluded that another secretagogue released by bombesin acts synergistically with gastrin on pepsin secretion. Therefore, bombesin stimulates gastric secretion through gastrin release, but its effects are modified by peptides coreleased to a) increase pepsin output at low doses and b) limit the output of acid and pepsin to 50-60% of the G-17 maximum.

Somatostatin and CXCR4 chemokine receptor expression in hepatocellular and cholangiocellular carcinomas: tumor capillaries as promising targets.

PubMed

Kaemmerer, Daniel; Schindler, Robin; Mußbach, Franziska; Dahmen, Uta; Altendorf-Hofmann, Annelore; Dirsch, Olaf; Sänger, Jörg; Schulz, Stefan; Lupp, Amelie

2017-12-28

Hepatocellular (HCC) and cholangiocellular carcinomas (CCC) display an exceptionally poor prognosis. Especially for advanced disease no efficient standard therapy is currently available. Recently, somatostatin analogs have been evaluated for the treatment of HCC, however, with contradictory results. Besides, for both malignancies the chemokine receptor CXCR4 has been discussed as a possible new target structure. Expression of somatostatin receptor (SSTR) subtypes 1, 2A, 3, 4, and 5, and of CXCR4 was evaluated in a total of 71 HCCs and 27 CCCs by immunohistochemistry using well-characterized novel monoclonal antibodies. In HCC tumor cells, frequency and intensity of expression of SSTRs and CXCR4 were only low. CXCR4 was present in about 40% of the HCCs, although at a low intensity. SSTR5, SSTR2, and SSTR3 were detected in about 15%, 8%, and 5% of the HCC tumors, respectively. SSTR and CXCR4 expression was much higher in CCC than in HCC. CXCR4 and SSTR1 were present in 60% and 67% of the CCC samples, respectively, followed by SSTR2 and SSTR5, which were detected in 30% and 11% of the tumors, respectively. Most notably, CXCR4 was intensely expressed on the tumor capillaries in about 50% of the HCCs and CCCs. CXCR4 expression on tumor vessels was associated with poor patient outcomes. CCC, but not HCC, may be suitable for SSTR-based treatments. Because of the predominant expression of SSTR1, pan-somatostatin analogs should be preferred. In both HCC and CCC, indirect targeting of tumors via the CXCR4-positive tumor capillaries may represent a promising additional therapeutic strategy.

Theranostic Prospects of Gastrin-Releasing Peptide Receptor-Radioantagonists in Oncology.

PubMed

Maina, Theodosia; Nock, Berthold A; Kulkarni, Harshad; Singh, Aviral; Baum, Richard P

2017-07-01

Gastrin-releasing peptide receptors (GRPRs) represent attractive targets for cancer diagnosis and therapy owing to their overexpression in widespread human tumors. Bombesin (BBN) analogues coupled to suitable chelators for stable radiometal binding have been proposed for diagnostic imaging and radionuclide therapy (theranostics) of GRPR-positive tumors. Recently, interest has shifted from BBN-like receptor agonists to GRPR-radioantagonists, because radioantagonists do not induce adverse effects after injection to patients and display superior pharmacokinetic in vivo profiles. Thus, they seem more advantageous for clinical use compared to agonists. Newer developments highlighting the theranostic potential of GRPR-radioantagonists in cancer patient management are presented herein. Copyright © 2017 Elsevier Inc. All rights reserved.

Sigma receptors as potential therapeutic targets for neuroprotection.

PubMed

Nguyen, Linda; Kaushal, Nidhi; Robson, Matthew J; Matsumoto, Rae R

2014-11-15

Sigma receptors comprise a unique family of proteins that have been implicated in the pathophysiology and treatment of many central nervous system disorders, consistent with their high level of expression in the brain and spinal cord. Mounting evidence indicate that targeting sigma receptors may be particularly beneficial in a number of neurodegenerative conditions including Alzheimer׳s disease, Parkinson׳s disease, stroke, methamphetamine neurotoxicity, Huntington׳s disease, amyotrophic lateral sclerosis, and retinal degeneration. In this perspective, a brief overview is given on sigma receptors, followed by a focus on common mechanisms of neurodegeneration that appear amenable to modulation by sigma receptor ligands to convey neuroprotective effects and/or restorative functions. Within each of the major mechanisms discussed herein, the neuroprotective effects of sigma ligands are summarized, and when known, the specific sigma receptor subtype(s) involved are identified. Together, the literature suggests sigma receptors may provide a novel target for combatting neurodegenerative diseases through both neuronal and glial mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

PubMed

Bitar, K N; Hillemeier, C; Biancani, P

1990-01-01

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.

Somatostatin, prostaglandin E2 and atropine inhibition of the gastric actions of bombesin in the dog.

PubMed

Hirschowitz, B I; Molina, E

1984-01-01

Bombesin, acetylcholine, prostaglandins and somatostatin are all thought to be involved in the regulation of gastrin release and gastric secretion. We have studied the effects of low doses of atropine, 16-16(Me)2-prostaglandin E2 (PGE2) and somatostatin-14 on bombesin-stimulated gastrin release and gastric acid and pepsin secretion in conscious fistula dogs. For reference, synthetic gastrin G-17 was studied with and without somatostatin. Bombesin, in a dose-related manner, increased serum gastrin, which in turn stimulated gastric acid and pepsin secretion in a serum gastrin, concentration-dependent manner. Somatostatin inhibited gastrin release by bombesin as well as the secretory stimulation by G-17; the combination of sequential effects resulted in a marked inhibition of bombesin-stimulated gastric acid and pepsin secretion. PGE2 also strongly inhibited gastrin release and acid and pepsin secretion. Atropine had no significant effect on gastrin release, but greatly inhibited gastric secretion. Thus somatostatin and PGE2 inhibited at two sites, gastrin release and gastrin effects, while atropine affected only the latter.

Canine bombesin-like gastrin releasing peptides stimulate gastrin release and acid secretion in the dog.

PubMed Central

Bunnett, N W; Clark, B; Debas, H T; Del Milton, R C; Kovacs, T O; Orloff, M S; Pappas, T N; Reeve, J R; Rivier, J E; Walsh, J H

1985-01-01

The synthetic mammalian bombesin-like peptides, canine gastrin releasing peptide 27, 23 and 10, and porcine gastrin releasing peptide 27 were compared with amphibian bombesin 14 and 10 during intravenous infusions into six conscious dogs with chronic gastric cannulae. Gastrin and gastrin releasing peptide were measured in peripherally sampled venous blood by radioimmunoassay and gastric acid secretions were collected. All forms of gastrin releasing peptide stimulated gastrin release and gastric acid secretion in a dose-dependent manner. The larger canine and porcine peptides were more potent than the decapeptide. Bombesin 14 was more potent than bombesin 10. A rise in the venous concentration of immunoreactive gastrin releasing peptide of only 20 fmol ml-1 stimulated gastrin release to about 50% of maximal. Gastrin releasing peptide 10 was cleared from the circulation three times faster than the larger forms and this may account for the apparent differences in potency. PMID:3839849

Modulation of inflammation by vasoactive intestinal peptide and bombesin: lack of effects on neutrophil apoptosis.

PubMed

Djanani, Angela M; Kähler, Ch M

2002-01-01

Inhibition of neutrophil apoptosis has been identified as a prominent feature in chronic inflammation, parenchymal damage, and unresolved organ dysfunction. Lung injury animal models suggest that the neuropeptides vasoactive intestinal peptide and bombesin are protective. Therefore, in vitro effects of VIP and bombesin on apoptosis of normal human neutrophils were tested. For measuring effects on cell survival and apoptosis, trypan dye exclusion, colorimetric MTT assay to assess cell survival, and caspase-3 assay and annexin-V binding for analysing apoptosis rates were used. Foetal calf serum, Fas ligand, and tumour necrosis factor-alpha served as modulatory control agents; survival-promoting and apoptosis-inducing activities of the respective agents were confirmed. Vasoactive intestinal peptide and bombesin, however, failed to significantly affect cell death in neutrophils. Data suggest that direct regulation of neutrophil apoptosis is unlikely to be among the mechanisms of lung-protective actions of VIP and bombesin.

Placental expression of D6 decoy receptor in preeclampsia

PubMed Central

Cho, Geum Joon; Lee, Eun Sung; Jin, Hye Mi; Lee, Ji Hye; Kim, Yeun Sun; Seol, Hyun-Joo; Hong, Soon-Cheol; Kim, Hai-Joong

2015-01-01

Objective The purpose of this study was to investigate the expression of the D6 decoy receptor that can bind chemokines and target them for degradation, resulting in inhibition of inflammation in placentas from preeclamptic and normal pregnancies. Methods The current study was carried out in 35 pregnant women (23 patients with preeclampsia and 12 healthy, normotensive pregnant women) during the third trimester of pregnancy. The expressions of D6 decoy receptor in the placenta were determined with real time reverse transcriptase polymerase chain reaction and western blotting. Results The mRNA and protein of D6 decoy receptor were detected in all of placentas from preeclamptic and normal pregnancies. Placental D6 decoy receptor mRNA expression was significantly lower in patients with preeclampsia than in patients with normal pregnancies. Western blot analyses revealed decreased protein expression in cases of preeclampsia. Conclusion The expression of the D6 decoy receptor in preeclamptic placentas was significantly lower than in normal placentas. Further studies are needed to clarify the underlying mechanisms that link decreased expression of placental D6 decoy receptor and preeclampsia. PMID:26430656

Receptor-Mediated Drug Delivery Systems Targeting to Glioma

PubMed Central

Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

2015-01-01

Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications. PMID:28344260

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

Suppression by nimesulide of bombesin-enhanced peritoneal metastasis of intestinal adenocarcinomas induced by azoxymethane in Wistar rats.

PubMed

Iishi, Hiroyasu; Tatsuta, Masaharu; Baba, Miyako; Yano, Hiroyuki; Higashino, Koji; Mukai, Mutsuko; Akedo, Hitoshi

2003-01-01

The effects of the cyclooxygenase (COX)-2 inhibitor nimesulide on bombesin-enhanced peritoneal metastasis of azoxymethane (AOM)-induced intestinal adenocarcinomas were investigated in male Wistar rats. From the beginning of the study, the rats were given 10 weekly s.c. injections of AOM (7.4 mg/kg body weight) and s.c. injections of bombesin (40 microg/kg body weight) every other day. From week 16, the rats were given chow pellets containing 200 ppm or 400 ppm nimesulide ad libitum until termination of the study at week 45. Nimesulide at the higher dose significantly decreased the incidence of bombesin-enhanced metastasis to the peritoneum at week 45, although its administration had little or no effect on the location, histologic type, depth of involvement or infiltrating growth patterns of the tumors. Nimesulide also significantly decreased the incidence of bombesin-enhanced lymphatic vessel invasion by adenocarcinomas. Finally, it also inhibited bombesin-induced matrix metalloproteinase (MMP)-9 and pro-MMP-9 inductions. Our findings indicate that nimesulide may inhibit cancer metastasis through inhibition of pro-MMP-9 and MMP-9 inductions.

Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smetanina, Mariya A., E-mail: maria.smetanina@gmail.com; Laboratory of Gene Expression Control, Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 10, Novosibirsk 630090; Group of Pharmacogenomics, Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences, prospekt Lavrentyeva 8, Novosibirsk 630090

2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepaticmore » mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car{sup -/-}) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car{sup -/-} livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor. - Highlights: > The azo dye and mouse carcinogen OAT is a very effective mCAR activator. > OAT increases mCAR transactivation in a dose-dependent manner. > OAT CAR-dependently increases the expression of a specific subset of CAR target genes. > OAT induces an unexpectedly deferred, but CAR-dependent hepatocyte proliferation.« less

Effects of caffeine and Bombesin on ethanol and food intake

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dietze, M.A.; Kulkosky, P.J.

1991-01-01

The methylxanthine caffeine and ethyl alcohol are widely used and powerful psychotropic drugs, but their interactions are not well understood. Bombesin is a brain-gut neuropeptide which is thought to function as a neurochemical factor in the inhibitory control of voluntary alcohol ingestion. We assessed the effects of combinations of intraperitoneal doses of caffeine and bombesin on 5% w/v ethanol solution and food intake in deprived rats. Deprived male and female Wistar rats received access to 5% ethanol or Purina chow for 30 minutes after i.p. injections. In single doses, CAF and BBS significantly decreased both ethanol and food consumption, atmore » 50 mg/kg and 10 {mu}g/kg, respectively. CAF and BBS combinations produced infra-additive, or less-than-expected inhibitory effects on ethanol intake, but simple additive inhibitory effects on food intake. This experimental evidence suggests a reciprocal blocking of effects of CAF and BBS on ethanol intake but not food intake. Caffeine, when interacting and bombesin, increases alcohol consumption beyond expected values. Caffeine could affect the operation of endogenous satisfy signals for alcohol consumption.« less

Expression and purification of functional PDGF receptor beta.

PubMed

Shang, Qingbin; Zhao, Liang; Wang, Xiaojing; Wang, Meimei; Sui, Sen-Fang; Mi, Li-Zhi

2017-07-29

Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved. It is caused, at least in part, by the technical challenges in the expression and purification of the functional, full-length PDGF receptors. Herein, we reported our experimental details in expression and purification of the full-length PDGFRβ from mammalian cells. We found that purified PDGFRβ remained in two different oligomeric states, presumably the monomer and the dimer, with basal kinase activity in detergent micelles. Addition of PDGF-B promoted dimerization and elevated kinase activity of the receptor, suggesting that purified receptors were functional. Copyright © 2017 Elsevier Inc. All rights reserved.

High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

PubMed

Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

2014-02-01

β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Delta-opioid receptors as targets for migraine therapy.

PubMed

Charles, Andrew; Pradhan, Amynah A

2016-06-01

The purpose of this review is to contrast the properties of the δ-opioid receptor with those of the μ-opioid receptor, which is the primary target of most currently available opioid analgesics. We also discuss preclinical evidence that indicates the potential efficacy of δ-opioid receptor agonists as migraine therapy. The use of currently available opioid analgesics is highly problematic for patients with migraine. Delta-opioid receptors have key differences from μ receptors; these differences make the δ receptor an attractive therapeutic target for migraine. Delta-opioid receptors are expressed in both the peripheral and central nervous system in anatomical regions and cell types that are believed to play a role in migraine. Delta-receptor agonists have also shown promising effects in multiple migraine models, including nitroglycerin evoked hyperalgesia and conditioned place aversion, and cortical spreading depression. Evidence from animal models indicates that activation of δ receptors is less likely to cause tolerance and dependence, and less likely to cause hyperalgesia. In addition, δ receptors may have antidepressant and anxiolytic properties that are distinct from those of μ receptors. In human studies investigating other conditions, δ-receptor agonists have been generally safe and well tolerated. Delta-opioid receptor agonists have promising potential as acute and/or preventive migraine therapies, without the problems associated with currently used opioid analgesics.

Serum trypsin, alpha-amylase and lipase during bombesin stimulation in normal subjects and patients with pancreatic insufficiency.

PubMed

Hafkenscheid, J C; Hessels, M; Jansen, J B; Lamers, C B

1984-01-31

The effect of infusion of bombesin (60 pmol/kg 20 min) on pancreatic enzymes in serum was studied in 13 normal subjects and 12 patients with pancreatic insufficiency. In normal subjects administration of bombesin induced large increases in serum trypsin (p less than 0.01), while serum total alpha-amylase and pancreatic alpha-amylase did not change and serum lipase showed only a modest rise (0.01 less than p less than 0.05). Patients with pancreatic insufficiency had significantly lower serum concentrations of all enzymes studied (p less than 0.01) and in such patients bombesin did not change the concentrations of pancreatic enzymes in serum. It is concluded that determination of the serum trypsin response to bombesin may be of help in the diagnosis of pancreatic insufficiency.

Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits.

PubMed

Buchanan, Katherine A; Blackman, Arne V; Moreau, Alexandre W; Elgar, Dale; Costa, Rui P; Lalanne, Txomin; Tudor Jones, Adam A; Oyrer, Julia; Sjöström, P Jesper

2012-08-09

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium.

PubMed

Shabir, Saqib; Cross, William; Kirkwood, Lisa A; Pearson, Joanna F; Appleby, Peter A; Walker, Dawn; Eardley, Ian; Southgate, Jennifer

2013-08-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca²⁺. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca²⁺ and in a scratch repair assay. The results confirmed the functional expression of P2Y₄ receptors and excluded nonexpressed receptors/channels (P2X₁, P2X₃, P2X₆, P2Y₆, P2Y₁₁, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X₂, P2X₄, P2Y₁, P2Y₂, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca²⁺ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

Peptide receptor targeting in cancer: the somatostatin paradigm.

PubMed

Barbieri, Federica; Bajetto, Adriana; Pattarozzi, Alessandra; Gatti, Monica; Würth, Roberto; Thellung, Stefano; Corsaro, Alessandro; Villa, Valentina; Nizzari, Mario; Florio, Tullio

2013-01-01

Peptide receptors involved in pathophysiological processes represent promising therapeutic targets. Neuropeptide somatostatin (SST) is produced by specialized cells in a large number of human organs and tissues. SST primarily acts as inhibitor of endocrine and exocrine secretion via the activation of five G-protein-coupled receptors, named sst1-5, while in central nervous system, SST acts as a neurotransmitter/neuromodulator, regulating locomotory and cognitive functions. Critical points of SST/SST receptor biology, such as signaling pathways of individual receptor subtypes, homo- and heterodimerization, trafficking, and cross-talk with growth factor receptors, have been extensively studied, although functions associated with several pathological conditions, including cancer, are still not completely unraveled. Importantly, SST exerts antiproliferative and antiangiogenic effects on cancer cells in vitro, and on experimental tumors in vivo. Moreover, SST agonists are clinically effective as antitumor agents for pituitary adenomas and gastro-pancreatic neuroendocrine tumors. However, SST receptors being expressed by tumor cells of various tumor histotypes, their pharmacological use is potentially extendible to other cancer types, although to date no significant results have been obtained. In this paper the most recent findings on the expression and functional roles of SST and SST receptors in tumor cells are discussed.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

PubMed

Elshafae, Said M; Hassan, Bardes B; Supsavhad, Wachiraphan; Dirksen, Wessel P; Camiener, Rachael Y; Ding, Haiming; Tweedle, Michael F; Rosol, Thomas J

2016-06-01

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Methionine Deprivation Induces a Targetable Vulnerability in Triple-Negative Breast Cancer Cells by Enhancing TRAIL Receptor-2 Expression.

PubMed

Strekalova, Elena; Malin, Dmitry; Good, David M; Cryns, Vincent L

2015-06-15

Many neoplasms are vulnerable to methionine deficiency by mechanisms that are poorly understood. Because gene profiling studies have revealed that methionine depletion increases TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) mRNA, we postulated that methionine stress sensitizes breast cancer cells to proapoptotic TRAIL-R2 agonists. Human triple (ER/PR/HER2)-negative breast carcinoma cell lines were cultured in control or methionine-free media. The effects of methionine depletion on TRAIL receptor expression and sensitivity to chemotherapy or a humanized agonistic TRAIL-R2 monoclonal antibody (lexatumumab) were determined. The melanoma-associated antigen MAGED2 was silenced to delineate its functional role in sensitizing TNBC cells to methionine stress. An orthotopic TNBC model was utilized to evaluate the effects of dietary methionine deficiency, lexatumumab, or the combination. Methionine depletion sensitized TNBC cells to lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. MCF-10A cells transformed by oncogenic H-Ras, but not untransformed cells, and matrix-detached TNBC cells were highly sensitive to the combination of lexatumumab and methionine depletion. Proteomics analyses revealed that MAGED2, which has been reported to reduce TRAIL-R2 expression, was suppressed by methionine stress. Silencing MAGED2 recapitulated features of methionine deprivation, including enhanced mRNA and cell surface expression of TRAIL receptors and increased sensitivity to TRAIL receptor agonists. Dietary methionine deprivation enhanced the antitumor effects of lexatumumab in an orthotopic metastatic TNBC model. Methionine depletion exposes a targetable defect in TNBC cells by increasing TRAIL-R2 expression. Our findings provide the foundation for a clinical trial combining dietary methionine restriction and TRAIL-R2 agonists. Clin Cancer Res; 21(12); 2780-91. ©2015 AACR. ©2015 American Association for Cancer

Methionine Deprivation Induces a Targetable Vulnerability in Triple-negative Breast Cancer Cells by Enhancing TRAIL Receptor-2 Expression

PubMed Central

Strekalova, Elena; Malin, Dmitry; Good, David M.; Cryns, Vincent L.

2015-01-01

Purpose Many neoplasms are vulnerable to methionine deficiency by mechanisms that are poorly understood. Because gene profiling studies have revealed that methionine depletion increases TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) mRNA, we postulated that methionine stress sensitizes breast cancer cells to proapoptotic TRAIL-R2 agonists. Experimental Design Human triple (ER/PR/HER2)-negative breast carcinoma cell lines were cultured in control or methionine-free media. The effects of methionine depletion on TRAIL receptor expression and sensitivity to chemotherapy or a humanized agonistic TRAIL-R2 monoclonal antibody (lexatumumab) were determined. The melanoma-associated antigen MAGED2 was silenced to delineate its functional role in sensitizing TNBC cells to methionine stress. An orthotopic TNBC model was utilized to evaluate the effects of dietary methionine deficiency, lexatumumab or the combination. Results Methionine depletion sensitized TNBC cells to lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. MCF-10A cells transformed by oncogenic H-Ras, but not untransformed cells, and matrix-detached TNBC cells were highly sensitive to the combination of lexatumumab and methionine depletion. Proteomics analyses revealed that MAGED2, which has been reported to reduce TRAIL-R2 expression, was suppressed by methionine stress. Silencing MAGED2 recapitulated features of methionine deprivation, including enhanced mRNA and cell surface expression of TRAIL receptors and increased sensitivity to TRAIL receptor agonists. Dietary methionine deprivation enhanced the antitumor effects of lexatumumab in an orthotopic metastatic TNBC model. Conclusion Methionine depletion exposes a targetable defect in TNBC cells by increasing TRAIL-R2 expression. Our findings provide the foundation for a clinical trial combining dietary methionine restriction and TRAIL-R2 agonists. PMID:25724522

Ranatensin-HL: A Bombesin-Related Tridecapeptide from the Skin Secretion of the Broad-Folded Frog, Hylarana latouchii.

PubMed

Lin, Yan; Chen, Tianbao; Zhou, Mei; Wang, Lei; Su, Songkun; Shaw, Chris

2017-07-04

Bombesin-related peptides are a family of peptides whose prototype was discovered in amphibian skin and which exhibit a wide range of biological activities. Since the initial isolation of bombesin from Bombina bombina skin, diverse forms of bombesin-related peptides have been found in the skins across Anura. In this study, a novel bombesin-related peptide of the ranatensin subfamily, named ranatensin-HL, was structurally-characterised from the skin secretion of the broad-folded frog, Hylarana latouchii , through combination of molecular cloning and mass spectrometric methodologies. It is composed of 13 amino acid residues, pGlu-RAGNQWAIGHFM-NH₂, and resembles an N-terminally extended form of Xenopus neuromedin B. Ranatensin-HL and its C-terminal decapeptide (ranatensin-HL-10) were chemically synthesised and subjected to in vitro smooth muscle assays in which they were found to display moderate stimulatory effects on rat urinary bladder and uterus smooth muscles with EC 50 values in the range of 1-10 nM. The prepro-ranatensin-HL was highly homological to a bombesin-like peptide from Rana catesbeiana at both nucleotide and amino acid levels, which might provide a clue for the taxonomic classification of ranid frogs in the future.

Progesterone induces progesterone receptor gene (PGR) expression via rapid activation of protein kinase pathways required for cooperative estrogen receptor alpha (ER) and progesterone receptor (PR) genomic action at ER/PR target genes.

PubMed

Diep, Caroline H; Ahrendt, Hannah; Lange, Carol A

2016-10-01

Progesterone Receptors (PRs) are critical effectors of estrogen receptor (ER) signaling required for mammary gland development and reproductive proficiency. In breast and reproductive tract malignancies, PR expression is a clinical prognostic marker of ER action. While estrogens primarily regulate PR expression, other factors likely contribute to a dynamic range of receptor expression across diverse tissues. In this study, we identified estrogen-independent but progestin (R5020)-dependent regulation of ER target genes including PGR in ER+/PR+ cancer cell lines. R5020 (10nM-10μM range) induced dose-dependent PR mRNA and protein expression in the absence of estrogen but required both PR and ERα. Antagonists of either PR (RU486, onapristone) or ERα (ICI 182,780) attenuated R5020 induction of TFF1, CTSD, and PGR. Chromatin immunoprecipitation (ChIP) assays performed on ER+/PR+ cells demonstrated that both ERα and PR were recruited to the same ERE/Sp1 site-containing region of the PGR proximal promoter in response to high dose progestin (10μM). Recruitment of ERα and PR to chromatin and subsequent PR mRNA induction were dependent upon rapid activation of MAPK/ERK and AKT; inhibition of these kinase pathways via U0126 or LY294002 blocked these events. Overall, we have identified a novel mechanism of ERα activation initiated by rapid PR-dependent kinase pathway activation and associated with phosphorylation of ERα Ser118 for estrogen-independent but progestin-dependent ER/PR cross talk. These studies may provide insight into mechanisms of persistent ER-target gene expression during periods of hormone (i.e. estrogen) ablation and suggest caution following prolonged treatment with aromatase or CYP17 inhibitors (i.e. contexts when progesterone levels may be abnormally elevated). Copyright © 2016 Elsevier Inc. All rights reserved.

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

PubMed

Matsumoto, Y; Kawatani, M; Simizu, S; Tanaka, T; Takada, M; Imoto, M

2000-01-01

The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

Triggering receptor expressed on myeloid cells 2 (TREM2): a potential therapeutic target for Alzheimer disease?

PubMed

Deming, Yuetiva; Li, Zeran; Benitez, Bruno A; Cruchaga, Carlos

2018-06-20

There are currently no effective therapeutics for Alzheimer disease (AD). Clinical trials targeting amyloid beta thus far have shown very little benefit and only in the earliest stages of disease. These limitations have driven research to identify alternative therapeutic targets, one of the most promising is the triggering receptor expressed on myeloid cells 2 (TREM2). Areas covered: Here, we review the literature to-date and discuss the potentials and pitfalls for targeting TREM2 as a potential therapeutic for AD. We focus on research in animal and cell models for AD and central nervous system injury models which may help in understanding the role of TREM2 in disease. Expert opinion: Studies suggest TREM2 plays a key role in AD pathology; however, results have been conflicting about whether TREM2 is beneficial or harmful. More research is necessary before designing TREM2-targeting therapies. Successful therapeutics will most likely be administered early in disease.

Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Salman, Fadheela; Plant, Nick, E-mail: N.Plant@Surrey.ac.uk

The polychlorinated biphenyl group possesses high environmental persistence, leading to bioaccumulation and a number of adverse effects in mammals. Whilst coplanar PCBs elicit their toxic effects through agonism of the aryl hydrocarbon receptor; however, non-coplanar PCBs are not ligands for AhR, but may be ligands for members of the nuclear receptor family of proteins. To better understand the biological actions of non-coplanar PCBs, we have undertaken a systematic analysis of their ability to activate PXR and CAR-mediated effects. Cells were exposed to a range of non-coplanar PCBs (99, 138, 153, 180 and 194), or the coplanar PCB77: Direct activation ofmore » PXR and CAR was measured using a mammalian receptor activation assay in human liver cells, with rifampicin and CITCO used as positive controls ligands for PXR and CAR, respectively; activation of target gene expression was examined using reporter gene plasmids for CYP3A4 and MDR1 transfected into liver, intestine and lung cell lines. Several of the non-coplanar PCBs directly activated PXR and CAR, whilst the coplanar PCB77 did not. Non-coplanar PCBs were also able to activate PXR/CAR target gene expression in a substitution- and tissue-specific manner. Non-coplanar PCBs act as direct activators for the nuclear receptors PXR and CAR, and are able to elicit transcriptional activation of target genes in a substitution- and tissue-dependent manner. Chronic activation of PXR/CAR is linked to adverse effects and must be included in any risk assessment of PCBs. -- Highlights: ► Several Non-coplanar PCBs are able to directly activate both PXR and CAR in vitro. ► PCB153 is the most potent direct activator of PXR and CAR nuclear receptors. ► Non-coplanar PCB activation of CYP3A4/MDR1 reporter genes is structure-dependent. ► Non-coplanar PCB activate CYP3A4/MDR1 reporter genes in a tissue-dependent. ► PCB153 is the most potent activator of PXR/CAR target gene in all tissues.« less

[The effect of bombesin and its analogs on the secretion of gastric juice and its content of pepsin and hydrochloric acid].

PubMed

Barashkova, G M; Klimov, P K; Kuranova, I L; Churkina, S I; Filonova, E B

1990-07-01

I.V. Infusion of bombesine after eating raw meat inhibited for 30-60 min the secretion of gastric juice and hydrochloric acid in dogs. Within 90-120 min of simultaneous infusion of pentagastrin and bombesine, the amount of secreted juice and its acidity decreased and then the secretion of gastric parietal cells increased. Simultaneous infusion of histamine and bombesine increased the response of gastric parietal cells during the whole experiment as compared with the histamine effect alone. Microapplication of bombesine into cerebral structures also decreased the secretory response of the parietal cells.

Bile Acid Receptor Agonist GW4064 Regulates PPARγ Coactivator-1α Expression Through Estrogen Receptor-Related Receptor α

PubMed Central

Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D.

2011-01-01

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology. PMID:21493670

P2X Receptors as Drug Targets

PubMed Central

Jarvis, Michael F.

2013-01-01

The study of P2X receptors has long been handicapped by a poverty of small-molecule tools that serve as selective agonists and antagonists. There has been progress, particularly in the past 10 years, as cell-based high-throughput screening methods were applied, together with large chemical libraries. This has delivered some drug-like molecules in several chemical classes that selectively target P2X1, P2X3, or P2X7 receptors. Some of these are, or have been, in clinical trials for rheumatoid arthritis, pain, and cough. Current preclinical research programs are studying P2X receptor involvement in pain, inflammation, osteoporosis, multiple sclerosis, spinal cord injury, and bladder dysfunction. The determination of the atomic structure of P2X receptors in closed and open (ATP-bound) states by X-ray crystallography is now allowing new approaches by molecular modeling. This is supported by a large body of previous work using mutagenesis and functional expression, and is now being supplemented by molecular dynamic simulations and in silico ligand docking. These approaches should lead to P2X receptors soon taking their place alongside other ion channel proteins as therapeutically important drug targets. PMID:23253448

Origin of bombesin-like peptides in human fetal lung.

PubMed

Yoshizaki, K; de Bock, V; Solomon, S

1984-02-27

Four different forms of bombesin-like immunoreactive peaks were detected in extracts of human fetal lung by the use of reversed-phase high performance liquid chromatography (HPLC). Peaks I, II, III and IV, (increasing retention time), were eluted using a 14-38% of acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA). Peak II was the major material found in the extract of human fetal lung obtained at 16-20 weeks gestation. None of the four compounds contained in the eluted peaks had the same retention time as amphibian bombesin or porcine gastrin releasing peptide (GRP). On reversed-phase HPLC using two different solvent systems TFA or heptafluorobutyric acid (HFBA) as a hydrophobic counter ion, and in gel filtration chromatography, the chromatographic behavior of the main peak (peak II) was the same as that of the carboxyl terminal fragments of GRP, GRP18-27 or GRP19-27. This suggested that the peptide(s) in peak II resembled in composition the carboxy terminal 9 or 10 amino acids of porcine GRP. Following tryptic digestion the material in peak IV was converted to the more polar compound present in peak II. Two other peptide peaks were eluted close to peak II and these were presumed to be a modification of this main peak. One of the possible biosynthetic steps in the formation of bombesin-like peptides in human fetal lung could be a tryptic conversion of a less polar peptide to a more polar form (peak IV to II).

Comparative effect of chronic bombesin, gastrin-releasing peptide and caerulein on the rat pancreas.

PubMed

Damgé, C; Hajri, A; Lhoste, E; Aprahamian, M

1988-02-01

This study was designed to compare, on a molar basis, the effect of chronic bombesin, gastrin-releasing peptide (GRP) and caerulein on pancreatic growth in the rat. These 3 peptides were administered s.c. 3 times daily for 4 days at the following concentrations: 0.036, 0.36, 3.6 and 7.2 nmol/kg of body weight. Bombesin and GRP induced pancreatic growth in a dose-dependent manner from 3.6 nmol/kg. This growth was characterized by an increase in pancreatic weight, its protein and RNA contents but not in DNA content suggesting cellular hypertrophy. Caerulein exerted a biphasic effect on pancreatic growth, inducing cellular hypertrophy at low doses since 0.36 nmol/kg and atrophy with the highest dose (7.2 nmol/kg). Bombesin and caerulein (until 3.6 nmol/kg) increased the pancreatic content in chymotrypsin more than in amylase. The 7.2 nmol/kg caerulein treatment depressed all enzyme activities while the same dose of GRP increased pancreatic lipase content. It is concluded that (1) bombesin and GRP are equipotent trophic factors for the pancreas; (2) caerulein is the most potent factor and exerts a biphasic effect on pancreatic growth; (3) pancreatic growth and synthesis and/or secretion of enzymes are not regulated through the same mechanism.

Folate receptor‐targeted aminoglycoside‐derived polymers for transgene expression in cancer cells

PubMed Central

Godeshala, Sudhakar; Nitiyanandan, Rajeshwar; Thompson, Brian; Goklany, Sheba; Nielsen, David R.

2016-01-01

Abstract Targeted delivery of anticancer therapeutics can potentially overcome the limitations associated with current chemotherapeutic regimens. Folate receptors are overexpressed in several cancers, including ovarian, triple‐negative breast and bladder cancers, making them attractive for targeted delivery of nucleic acid therapeutics to these tumors. This work describes the synthesis, characterization and evaluation of folic acid‐conjugated, aminoglycoside‐derived polymers for targeted delivery of transgenes to breast and bladder cancer cell lines. Transgene expression was significantly higher with FA‐conjugated aminoglycoside‐derived polymers than with Lipofectamine, and these polymers demonstrated minimal cytotoxicty. Competitive inhibition using free folic acid significantly reduced transgene expression efficacy of folate‐targeted polymers, suggesting a role for folate receptor‐mediated uptake. High efficacy FA‐targeted polymers were employed to deliver a plasmid expressing the TRAIL protein, which induced death in cancer cells. These results indicate that FA‐conjugated aminoglycoside‐derived polymers are promising for targeted delivery of nucleic acids to cancer cells that overexpress folate receptors. PMID:29313013

Trophic effect of bombesin on the rat pancreas: is it mediated by the release of gastrin or cholecystokinin?

PubMed

Lhoste, E; Aprahamian, M; Pousse, A; Hoeltzel, A; Stock-Damge, C

1985-01-01

This work investigates the effect, on the rat pancreas, of a chronic administration of bombesin in function of the dose and duration of treatment and examines whether this effect may be mediated by the release of endogenous gastrin or cholecystokinin. Bombesin, administered three times daily for 5 or 15 days, induced a marked increase in pancreatic weight, its protein, RNA and enzyme contents with the dose of 10 micrograms/kg body weight; the ratios of pancreatic weight, protein and RNA contents to DNA contents increased significantly after a 5 day treatment, suggesting cellular hypertrophy. Pancreatic DNA content was markedly enhanced after a 15 day treatment, suggesting cellular hyperplasia. Antrectomy decreased plasma gastrin levels, but did not alter the pancreatico-trophic action of a 10 micrograms/kg bombesin treatment for 5 days. Proglumide, an inhibitor of cholecystokinin and gastrin in the pancreas, did not affect the growth of the pancreas induced by a 10 micrograms/kg bombesin treatment for 5 days. It is concluded that chronic bombesin induces, in the rat pancreas, cellular hypertrophy or hyperplasia depending on the duration of treatment. Pancreatic hypertrophy is not mediated by the release of endogenous gastrin or cholecystokinin.

Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

PubMed

Cabral-Marques, Otavio; Riemekasten, Gabriela

2017-11-01

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

P2X receptors in the cardiovascular system and their potential as therapeutic targets in disease.

PubMed

Ralevic, Vera

2015-01-01

This review considers the expression and roles of P2X receptors in the cardiovascular system in health and disease and their potential as therapeutic targets. P2X receptors are ligand gated ion channels which are activated by the endogenous ligand ATP. They are formed from the assembly of three P2X subunit proteins from the complement of seven (P2X1-7), which can associate to form homomeric or heteromeric P2X receptors. The P2X1 receptor is widely expressed in the cardiovascular system, being located in the heart, in the smooth muscle of the majority of blood vessels and in platelets. P2X1 receptors expressed in blood vessels can be activated by ATP coreleased with noradrenaline as a sympathetic neurotransmitter, leading to smooth muscle depolarisation and contraction. There is evidence that the purinergic component of sympathetic neurotransmission is increased in hypertension, identifying P2X1 receptors as a possible therapeutic target in this disorder. P2X3 and P2X2/3 receptors are expressed on cardiac sympathetic neurones and may, through positive feedback of neuronal ATP at this prejunctional site, amplify sympathetic neurotransmission. Activation of P2X receptors expressed in the heart increases cardiac myocyte contractility, and an important role of the P2X4 receptor in this has been identified. Deletion of P2X4 receptors in the heart depresses contractile performance in models of heart failure, while overexpression of P2X4 receptors has been shown to be cardioprotective, thus P2X4 receptors may be therapeutic targets in the treatment of heart disease. P2X receptors have been identified on endothelial cells. Although immunoreactivity for all P2X1-7 receptor proteins has been shown on the endothelium, relatively little is known about their function, with the exception of the endothelial P2X4 receptor, which has been shown to mediate endothelium-dependent vasodilatation to ATP released during shear stress. The potential of P2X receptors as therapeutic targets

Targeting of TAM Receptors Ameliorates Fibrotic Mechanisms in Idiopathic Pulmonary Fibrosis.

PubMed

Espindola, Milena S; Habiel, David M; Narayanan, Rohan; Jones, Isabelle; Coelho, Ana L; Murray, Lynne A; Jiang, Dianhua; Noble, Paul W; Hogaboam, Cory M

2018-06-01

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)-dependent manner. Gas6 (growth arrest-specific 6) ligand, Tyro3 (TYRO3 protein tyrosine kinase 3), and Axl (anexelekto) RTK expression and activity are increased in IPF. To determine if targeting these RTK pathways would inhibit fibroblast activation and the development of pulmonary fibrosis. Quantitative genomic, proteomic, and functional analyses were used to determine Gas6/TAM (Tyro3, Axl, and Mertk [MER proto-oncogene, tyrosine kinase]) RTK expression and activation in tissues and fibroblasts from normal and IPF lungs. The profibrotic impact of these RTK pathways were also examined in bleomycin-induced pulmonary fibrosis and in SCID/Bg mice that developed pulmonary fibrosis after the intravenous administration of primary IPF fibroblasts. Gas6, Axl, and Tyro3 were increased in both rapidly and slowly progressive IPF compared with normal lung samples and fibroblasts. Targeting these pathways with either specific antibodies directed at Gas6 or Axl, or with small-molecule TAM inhibitors indicated that the small molecule-mediated targeting approach was more efficacious in both in vitro and in vivo studies. Specifically, the TAM receptor inhibitor R428 (also known as BGB324) significantly inhibited the synthetic, migratory, and proliferative properties of IPF fibroblasts compared with the other Gas6/TAM receptor targeting agents. Finally, loss of Gas6 expression decreased lung fibrotic responses to bleomycin and treatment with R428 inhibited pulmonary fibrosis in humanized SCID/Bg mice. Gas6/TAM receptor activity contributes to the activation of pulmonary fibroblasts in IPF, suggesting that targeting this RTK pathway might be an effective antifibrotic strategy in this disease.

Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as therapeutic targets in breast-cancer.

PubMed

Garattini, Enrico; Bolis, Marco; Gianni', Maurizio; Paroni, Gabriela; Fratelli, Maddalena; Terao, Mineko

2016-07-05

Breast-cancer is heterogeneous and consists of various groups with different biological characteristics. Innovative pharmacological approaches accounting for this heterogeneity are needed. The forty eight human Nuclear-Hormone-Receptors are ligand-dependent transcription-factors and are classified into Endocrine-Receptors, Adopted-Orphan-Receptors (Lipid-sensors and Enigmatic-Orphans) and Orphan-receptors. Nuclear-Receptors represent ideal targets for the design/synthesis of pharmacological ligands. We provide an overview of the literature available on the expression and potential role played by Lipid-sensors, Enigmatic-Orphans and Orphan-Receptors in breast-cancer. The data are complemented by an analysis of the expression levels of each selected Nuclear-Receptor in the PAM50 breast-cancer groups, following re-elaboration of the data publicly available. The major aim is to support the idea that some of the Nuclear-Receptors represent largely unexploited therapeutic-targets in breast-cancer treatment/chemo-prevention. On the basis of our analysis, we conclude that the Lipid-Sensors, NR1C3, NR1H2 and NR1H3 are likely to be onco-suppressors in breast-cancer. The Enigmatic-Orphans, NR1F1 NR2A1 and NR3B3 as well as the Orphan-Receptors, NR0B1, NR0B2, NR1D1, NR2F1, NR2F2 and NR4A3 exert a similar action. These Nuclear-Receptors represent candidates for the development of therapeutic strategies aimed at increasing their expression or activating them in tumor cells. The group of Nuclear-Receptors endowed with potential oncogenic properties consists of the Lipid-Sensors, NR1C2 and NR1I2, the Enigmatic-Orphans, NR1F3, NR3B1 and NR5A2, as well as the Orphan-Receptors, NR2E1, NR2E3 and NR6A1. These oncogenic Nuclear-Receptors should be targeted with selective antagonists, reverse-agonists or agents/strategies capable of reducing their expression in breast-cancer cells.

Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

PubMed

Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

2017-11-01

Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

Prostaglandins and Their Receptors in Eosinophil Function and As Therapeutic Targets

PubMed Central

Peinhaupt, Miriam; Sturm, Eva M.; Heinemann, Akos

2017-01-01

Of the known prostanoid receptors, human eosinophils express the prostaglandin D2 (PGD2) receptors DP1 [also D-type prostanoid (DP)] and DP2 (also chemoattractant receptor homologous molecule, expressed on Th2 cells), the prostaglandin E2 receptors EP2 and EP4, and the prostacyclin (PGI2) receptor IP. Prostanoids can bind to either one or multiple receptors, characteristically have a short half-life in vivo, and are quickly degraded into metabolites with altered affinity and specificity for a given receptor subtype. Prostanoid receptors signal mainly through G proteins and naturally activate signal transduction pathways according to the G protein subtype that they preferentially interact with. This can lead to the activation of sometimes opposing signaling pathways. In addition, prostanoid signaling is often cell-type specific and also the combination of expressed receptors can influence the outcome of the prostanoid impulse. Accordingly, it is assumed that eosinophils and their (patho-)physiological functions are governed by a sensitive prostanoid signaling network. In this review, we specifically focus on the functions of PGD2, PGE2, and PGI2 and their receptors on eosinophils. We discuss their significance in allergic and non-allergic diseases and summarize potential targets for drug intervention. PMID:28770200

Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

NASA Astrophysics Data System (ADS)

Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.

2002-05-01

The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.

Expression of messenger molecules and receptors in rat and human sphenopalatine ganglion indicating therapeutic targets.

PubMed

Steinberg, Anna; Frederiksen, Simona D; Blixt, Frank W; Warfvinge, Karin; Edvinsson, Lars

2016-12-01

Migraine and Cluster Headache (CH) are two primary headaches with severe disease burden. The disease expression and the mechanisms involved are poorly known. In some attacks of migraine and in most attacks of CH, there is a release of vasoactive intestinal peptide (VIP) originating from parasympathetic cranial ganglia such as the sphenopalatine ganglion (SPG). Patients suffering from these diseases are often deprived of effective drugs. The aim of the study was to examine the localization of the botulinum toxin receptor element synaptic vesicle glycoprotein 2A (SV-2A) and the vesicular docking protein synaptosomal-associated protein 25 (SNAP25) in human and rat SPG. Additionally the expression of the neurotransmitters pituitary adenylate cyclase activating polypeptide (PACAP-38), nitric oxide synthase (nNOS), VIP and 5-hydroxttryptamine subtype receptors (5-HT1B,1D,1F) were examined. SPG from adult male rats and from humans, the later removed at autopsy, were prepared for immunohistochemistry using specific antibodies against neurotransmitters, 5-HT1B,1D,1F receptors, and botulinum toxin receptor elements. We found that the selected neurotransmitters and 5-HT receptors were expressed in rat and human SPG. In addition, we found SV2-A and SNAP25 expression in both rat and human SPG. We report that all three 5-HT receptors studied occur in neurons and satellite glial cells (SGCs) of the SPG. 5-HT1B receptors were in addition found in the walls of intraganglionic blood vessels. Recent focus on the SPG has emphasized the role of parasympathetic mechanisms in the pathophysiology of mainly CH. The development of next generation's drugs and treatment of cranial parasympathetic symptoms, mediated through the SPG, can be modulated by treatment with BoNT-A and 5-HT receptor agonists.

TAM Receptors in Leukemia: Expression, Signaling, and Therapeutic Implications

PubMed Central

Brandão, Luis; Migdall-Wilson, Justine; Eisenman, Kristen; Graham, Douglas K.

2016-01-01

In the past 30 years there has been remarkable progress in the treatment of leukemia and lymphoma. However, current treatments are largely ineffective against relapsed leukemia and, in the case of pediatric patients, are often associated with severe long-term toxicities. Thus, there continues to be a critical need for the development of effective biologically targeted therapies. The TAM family of receptor tyrosine kinases—Tyro3, Axl, and Mer—plays an important role in normal hematopoiesis, including natural killer cell maturation, macrophage function, and platelet activation and signaling. Furthermore, TAM receptor activation leads to upregulation of pro-survival and proliferation signaling pathways, and aberrant TAM receptor expression contributes to cancer development, including myeloid and lymphoid leukemia. This review summarizes the role of TAM receptors in leukemia. We outline TAM receptor expression patterns in different forms of leukemia, describe potential mechanisms leading to their overexpression, and delineate the signaling pathways downstream of receptor activation that have been implicated in leukemogenesis. Finally, we discuss the current research focused on inhibitors against these receptors in an effort to develop new therapeutic strategies for leukemia. PMID:22150307

Persistent interferon transgene expression by RNA interference-mediated silencing of interferon receptors.

PubMed

Takahashi, Yuki; Vikman, Elin; Nishikawa, Makiya; Ando, Mitsuru; Watanabe, Yoshihiko; Takakura, Yoshinobu

2010-09-01

The in vivo half-life of interferons (IFNs) is very short, and its extension would produce a better therapeutic outcome in IFN-based therapy. Delivery of IFN genes is one solution for providing a sustained supply. IFNs have a variety of functions, including the suppression of transgene expression, through interaction with IFN receptors (IFNRs). This suppression could prevent IFNs from being expressed from vectors delivered. Silencing the expression of IFNAR and IFNGR, the receptors for type I and II IFNs, respectively, in cells expressing IFNs may prolong transgene expression of IFNs. Mouse melanoma B16-BL6 cells or mouse liver were selected as a site expressing IFNs (not a target for IFN gene therapy) and IFN-expressing plasmid DNA was delivered with or without small interfering RNA (siRNA) targeting IFNRs. Transfection of B16-BL6 cells with siRNA targeting IFNAR1 subunit (IFNAR1) resulted in the reduced expression of IFNAR on the cell surface. This silencing significantly increased the IFN-beta production in cells that were transfected with IFN-beta-expressing plasmid DNA. Similar results were obtained with the combination of IFN-gamma and IFNGR. Co-injection of IFN-beta-expressing plasmid DNA with siRNA targeting IFNAR1 into mice resulted in sustained plasma concentration of IFN-beta. These results provide experimental evidence that the RNAi-mediated silencing of IFNRs in cells expressing IFN, such as hepatocytes, is an effective approach for improving transgene expression of IFNs when their therapeutic target comprises cells other than those expressing IFNs.

Increased arylhydrocarbon receptor expression offers a potential therapeutic target for pancreatic cancer.

PubMed

Koliopanos, Alexander; Kleeff, Jörg; Xiao, Yi; Safe, Stephen; Zimmermann, Arthur; Büchler, Markus W; Friess, Helmut

2002-09-05

The arylhydrocarbon receptor (AhR) was initially identified as a member of the adaptive metabolic and toxic response pathway to polycyclic aromatic hydrocarbons and to halogenated dibenzo-p-dioxins and dibenzofurans. In the present study, we sought to determine the functional significance of the AhR pathway in pancreatic carcinogenesis. AhR expression was analysed by Northern blotting. The exact site of AhR expression was analysed by in situ hybridization and immunohistochemistry. The effects of TCDD and four selective AhR agonists on pancreatic cancer cell lines were investigated by growth assays, apoptosis assays, and induction of the cyclin-dependent kinase inhibitor p21. There was strong AhR mRNA expression in 14 out of 15 pancreatic cancer samples, weak expression in chronic pancreatitis tissues, and faint expression in all normal pancreata. In pancreatic cancer tissues, AhR mRNA and protein expression were localized in the cytoplasm of pancreatic cancer cells. TCDD and the four AhR agonists inhibited pancreatic cancer cell growth in a dose-dependent manner, and decreased anchorage-independent cell growth. DAPI staining did not reveal nuclear fragmentation and CYP1A1 and was not induced by TCDD and AhR agonists. In contrast, TCDD and AhR agonists induced the expression of the cyclin-dependent kinase inhibitor p21. In conclusion, the relatively non-toxic AhR agonists caused growth inhibition in pancreatic cancer cells with high AhR expression levels via cell cycle arrest. In addition, almost all human pancreatic cancer tissues expressed this receptor at high levels, suggesting that these or related compounds may play a role in the therapy of pancreatic cancer in the future.

Stimulation of gastrin release and gastric secretion: effect of bombesin and a nonapeptide in fistula dogs with and without fundic vagotomy.

PubMed

Hirschowitz, B I; Gibson, R G

1978-01-01

Bombesin and a synthetic bombesin nonapeptide were studied by intravenous infusion at a dose of 0.5 microgram.kg-1.h-1 for 4 h in 7 dogs with esophagostomy and gastric fistula. In 3 of the dogs who had highly selective (fundic) vagotomy, mean integrated gastrin output over 4 h was double that in the 4 dogs with vagi intact during both nonapeptide (1,554 vs. 700 pg.ml-1.4 h-1) and bombesin infusion (2,442 vs. 1,440 pg.ml-1.4 h-1). Peak concentrations of serum gastrin reached during bombesin (490 +/- 100 vs. 320 +/- 90) were higher than those during nonapeptide infusion (270 +/- 40 vs. 160 +/- 28 pg/ml) in the vagotomized and intact dogs, respectively. The difference between vagotomized and vagally intact dogs suggests that the fundic vagotomy removed an inhibitor of gastrin release from the innervated antrum. Despite these differences in gastrin release, gastric acid output with the two peptides was the same (49--52 mEq/4 h) whether the fundus was denervated or innervated. This suggests that bombesin may stimulate gastric acid secretion by the release of an additional secretagogue which is not measured by the gastrin assay. Neither of the two inhibitors of gastrin release--antral acidification to pH 1.4 or less or atropine (100 microgram/kg)-- inhibited gastrin release by bombesin, even though the atropine reduced acid output by 80%. Bombesin is a potent gastric stimulus whose action is only partly explained by the measured gastrin release.

Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

PubMed Central

Pyo, Suhkneung; Kang, Chung Hyo; Lee, Chong Ock; Lee, Heung Kyoung; Choi, Sang Un; Park, Chi Hoon

2018-01-01

Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. PMID:29874279

Research Resource: Global Identification of Estrogen Receptor β Target Genes in Triple Negative Breast Cancer Cells

PubMed Central

Shanle, Erin K.; Zhao, Zibo; Hawse, John; Wisinski, Kari; Keles, Sunduz; Yuan, Ming

2013-01-01

Breast cancers that are negative for estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 are known as triple-negative breast cancers (TNBC). TNBCs are associated with an overall poor prognosis because they lack expression of therapeutic targets like ERα and are biologically more aggressive. A second estrogen receptor, ERβ, has been found to be expressed in 50% to 90% of ERα-negative breast cancers, and ERβ expression in TNBCs has been shown to correlate with improved disease-free survival and good prognosis. To elucidate the role of ERβ in regulating gene expression and cell proliferation in TNBC cells, the TNBC cell line MDA-MB-468 was engineered with inducible expression of full-length ERβ. In culture, ERβ expression inhibited cell growth by inducing a G1 cell cycle arrest, which was further enhanced by 17β-estradiol treatment. In xenografts, ERβ expression also inhibited tumor formation and growth, and 17β-estradiol treatment resulted in rapid tumor regression. Furthermore, genomic RNA sequencing identified both ligand-dependent and -independent ERβ target genes, some of which were also regulated by ERβ in other TNBC cell lines and correlated with ERβ expression in a cohort of TNBCs from the Cancer Genome Atlas Network. ERβ target genes were enriched in genes that regulate cell death and survival, cell movement, cell development, and growth and proliferation, as well as genes involved in the Wnt/β-catenin and the G1/S cell cycle phase checkpoint pathways. In addition to confirming the anti-proliferative effects of ERβ in TNBC cells, these data provide a comprehensive resource of ERβ target genes and suggest that ERβ may be targeted with ligands that can stimulate its growth inhibitory effects. PMID:23979844

Differential microRNA expression is associated with androgen receptor expression in breast cancer.

PubMed

Shi, Yaqin; Yang, Fang; Sun, Zijia; Zhang, Wenwen; Gu, Jun; Guan, Xiaoxiang

2017-01-01

The androgen receptor (AR) is frequently expressed in breast cancer; however, its prognostic value remains unclear. AR expression in breast cancer has been associated with improved outcomes in estrogen receptor (ER)‑positive breast cancer compared with ER‑negative disease. Eliminating AR function in breast cancer is critically important for breast cancer progression. However, the mechanism underlying AR regulation remains poorly understood. The study of microRNAs (miRNAs) has provided important insights into the pathogenesis of hormone‑dependent cancer. To determine whether miRNAs function in the AR regulation of breast cancer, the present study performed miRNA expression profiling in AR‑positive and ‑negative breast cancer cell lines. A total of 153 miRNAs were differentially expressed in AR‑positive compared with AR‑negative breast cancer cells; 52 were upregulated and 101 were downregulated. A number of these have been extensively associated with breast cancer cell functions, including proliferation, invasion and drug‑resistance. Furthermore, through pathway enrichment analysis, signaling pathways associated with the prediction targets of the miRNAs were characterized, including the vascular endothelial growth factor and mammalian target of rapamycin signaling pathways. In conclusion, the results of the present study indicated that the expression of miRNAs may be involved in the mechanism underlying AR regulation of breast cancer, and may improve understanding of the role of AR in breast cancer.

Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin.

PubMed

Rankin, S; Morii, N; Narumiya, S; Rozengurt, E

1994-11-14

In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows.

PubMed

Agrawal, Alea; Alharthi, Abdulrahman; Vailati-Riboni, Mario; Zhou, Zheng; Loor, Juan J

2017-01-01

G-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues. Quantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40 , MCFA receptor GPR84 , and niacin receptor HCAR2/3 . Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable. The data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression*

PubMed Central

Smith, Spenser S.; Dole, Neha S.; Franceschetti, Tiziana; Hrdlicka, Henry C.; Delany, Anne M.

2016-01-01

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3′-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling. PMID:27551048

Therapeutic antibody targeting of individual Notch receptors.

PubMed

Wu, Yan; Cain-Hom, Carol; Choy, Lisa; Hagenbeek, Thijs J; de Leon, Gladys P; Chen, Yongmei; Finkle, David; Venook, Rayna; Wu, Xiumin; Ridgway, John; Schahin-Reed, Dorreyah; Dow, Graham J; Shelton, Amy; Stawicki, Scott; Watts, Ryan J; Zhang, Jeff; Choy, Robert; Howard, Peter; Kadyk, Lisa; Yan, Minhong; Zha, Jiping; Callahan, Christopher A; Hymowitz, Sarah G; Siebel, Christian W

2010-04-15

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance.

PubMed

Knott, Thomas K; Madany, Pasil A; Faden, Ashley A; Xu, Mei; Strotmann, Jörg; Henion, Timothy R; Schwarting, Gerald A

2012-07-04

The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2(-/-) mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2(-/-) neurons. Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2(-/-) mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2(-/-) olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2(-/-) mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2(-/-) olfactory neurons. Results presented here show that many odorant receptors are under-expressed in β3GnT2(-/-) mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2(-/-) mice exhibit a relatively small deficit in

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins*

PubMed Central

Arévalo, Maria T.; Navarro, Ashley; Arico, Chenoa D.; Li, Junwei; Alkhatib, Omar; Chen, Shan; Diaz-Arévalo, Diana; Zeng, Mingtao

2014-01-01

Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax. PMID:24742682

Targeting the androgen receptor in triple-negative breast cancer.

PubMed

Gucalp, Ayca; Traina, Tiffany A

Triple-negative breast cancer represents approximately 15%-20% of all newly diagnosed breast cancers, but it accounts for a disproportionate number of breast cancer-related deaths each year. Owing to the lack of estrogen, progesterone, and human epidermal growth factor receptor 2 expression, patients with triple-negative breast cancer do not benefit from generally well-tolerated and effective therapies targeting the estrogen and human epidermal growth factor receptor 2 signaling pathways and are faced with an increased risk of disease progression and poorer overall survival. The heterogeneity of triple-negative breast cancer has been increasingly recognized and this may lead to therapeutic opportunities because of newly defined oncogenic drivers and targets. A subset of triple-negative breast tumors expresses the androgen receptor (AR) and this may benefit from treatments that inhibit the AR-signaling pathway. The first proof-of-concept trial established activity of the AR antagonist, bicalutamide, in patients with advanced AR+ triple-negative breast cancer. Since that time, evidence further supports the activity of other next-generation AR-targeted agents such as enzalutamide. Not unlike in estrogen receptor-positive breast cancer, mechanisms of resistance are being investigated and rationale exists for thoughtful, well-designed combination regimens such as AR antagonism with CDK4/6 pathway inhibitors or PI3K inhibitors. Furthermore, novel agents developed for the treatment of prostate cancer, which reduce androgen production such as abiraterone acetate and seviteronel, are being tested as well. This review summarizes the underlying biology of AR signaling in breast cancer development and the available clinical trial data for the use of anti-androgen therapy in the treatment of AR+ triple-negative breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

The immunohistochemical expression of calcitonin receptor-like receptor (CRLR) in human gliomas

PubMed Central

Benes, L; Kappus, C; McGregor, G P; Bertalanffy, H; Mennel, H D; Hagner, S

2004-01-01

Background: Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin. Aim: To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin. Methods: Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR. Results: All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels. Conclusions: This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours. PMID:14747444

The immunohistochemical expression of calcitonin receptor-like receptor (CRLR) in human gliomas.

PubMed

Benes, L; Kappus, C; McGregor, G P; Bertalanffy, H; Mennel, H D; Hagner, S

2004-02-01

Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin. To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin. Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR. All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels. This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours.

Dosimetry and first clinical evaluation of the new 18F-radiolabeled bombesin analogue BAY 864367 in patients with prostate cancer.

PubMed

Sah, Bert-Ram; Burger, Irene A; Schibli, Roger; Friebe, Matthias; Dinkelborg, Ludger; Graham, Keith; Borkowski, Sandra; Bacher-Stier, Claudia; Valencia, Ray; Srinivasan, Ananth; Hany, Thomas F; Mu, Linjing; Wild, Peter J; Schaefer, Niklaus G

2015-03-01

The aim of this first-in-man study was to demonstrate the feasibility, safety, and tolerability, as well as provide dosimetric data and evaluate the imaging properties, of the bombesin analogue BAY 864367 for PET/CT in a small group of patients with primary and recurrent prostate cancer (PCa). Ten patients with biopsy-proven PCa (5 with primary PCa and 5 with prostate-specific antigen recurrence after radical prostatectomy) were prospectively selected for this exploratory clinical trial with BAY 864367, a new (18)F-labeled bombesin analogue. PET scans were assessed at 6 time points, up to 110 min after intravenous administration of 302 ± 11 MBq of BAY 864367. Imaging results were compared with (18)F-fluorocholine PET/CT scans. Dosimetry was calculated using the OLINDA/EXM software. Three of 5 patients with primary disease showed positive tumor delineation in the prostate, and 2 of 5 patients with biochemical relapse showed a lesion suggestive of recurrence on the BAY 864367 scan. Tumor-to-background ratio averaged 12.9 ± 7.0. The ratio of malignant prostate tissue to normal prostate tissue was 4.4 ± 0.6 in 3 patients with tracer uptake in the primary PCa. Mean effective dose was 4.3 ± 0.3 mSv/patient (range, 3.7-4.9 mSv). BAY 864367, a novel (18)F-labeled bombesin tracer, was successfully investigated in a first-in-man clinical trial of PCa and showed favorable dosimetric values. Additionally, the application was safe and well tolerated. The tracer delineated tumors in a subset of patients, demonstrating the potential of gastrin-releasing-peptide receptor imaging. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Sex steroid receptor expression in idiopathic pulmonary fibrosis.

PubMed

Mehrad, Mitra; Trejo Bittar, Humberto E; Yousem, Samuel A

2017-08-01

Usual interstitial pneumonia (UIP) is characterized by progressive scarring of the lungs and is associated with high morbidity and mortality despite therapeutic interventions. Sex steroid receptors have been demonstrated to play an important role in chronic lung conditions; however, their significance is unknown in patients with UIP. We retrospectively reviewed 40 idiopathic UIP cases for the expression of hormonal receptors. Forty cases including 10 normal lung, 10 cryptogenic organizing pneumonia, 10 idiopathic organizing diffuse alveolar damage, 7 hypersensitivity pneumonitis, and 3 nonspecific interstitial pneumonitis served as controls. Immunohistochemistry for estrogen receptor α, progesterone receptor (PR), and androgen receptor was performed in all groups. Expression of these receptors was assessed in 4 anatomic/pathologic compartments: alveolar and bronchiolar epithelium, arteries/veins, fibroblastic foci/airspace organization, and old scar. All UIPs (100%) stained positive for PR in myofibroblasts in the scarred areas, whereas among the control cases, only 1 nonspecific interstitial pneumonitis case stained focally positive and the rest were negative. PR was positive in myocytes of the large-sized arteries within the fibrotic areas in 31 cases (77.5%). PR was negative within the alveolar and bronchial epithelium, airspace organization, and center of fibroblastic foci; however, weak PR positivity was noted in the peripheral fibroblasts of the fibroblastic foci where they merged with dense fibrous connective tissue scar. All UIP and control cases were negative for androgen receptor and estrogen receptor α. This is the first study to show the expression of PR within the established fibrotic areas of UIP, indicating that progesterone may have profibrotic effects in UIP patients. Hormonal therapy by targeting PR could be of potential benefit in patients with UIP/IPF. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of a new potent CCK antagonist, lorglumide, on caerulein- and bombesin-induced pancreatic secretion and growth in the rat.

PubMed Central

Scarpignato, C.; Varga, G.; Dobronyi, I.; Papp, M.

1989-01-01

1. The effect of lorglumide, a new potent cholecystokinin (CCK) antagonist, on pancreatic secretion and growth induced by caerulein and bombesin was studied in the rat. 2. Pancreatic exocrine secretion was studied both in vitro (isolated and perfused pancreatic segments) and in vivo (anaesthetized animals with cannulation of the common bile duct) whereas the trophic effect was investigated after short-term (5 days) administration of the peptides and/or lorglumide. 3. Both caerulein and bombesin stimulated amylase release from in vitro pancreatic segments in a concentration-dependent manner. Although the efficacy of both peptides was virtually identical, the potency of caerulein was higher than that of bombesin. Lorglumide displaced the concentration-response curves to caerulein to the right without affecting the maximum response, suggesting a competitive antagonism. The Schild plot analysis of data gave a straight line with a slope not significantly different from unity. The calculated pA2 for lorglumide was 7.31 +/- 0.45. The antagonist, however, was completely ineffective when tested against bombesin-induced amylase release. 4. In vivo experiments confirmed results from in vitro studies since lorglumide (5 and 10 mg kg-1) significantly reduced pancreatic exocrine secretion induced by caerulein without affecting the response to bombesin. 5. Administration of either peptide increased the weight of the pancreas, the total pancreatic protein and DNA, trypsin and amylase content. Lorglumide (10 mg kg-1), administered together with caerulein, reduced the peptide-induced increase in pancreatic weight, protein and enzyme content. On the contrary, when lorglumide was given together with bombesin, all the parameters that were examined were not altered by concomitant administration of the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2470456

Somatostatin Analogues for Receptor Targeted Photodynamic Therapy

PubMed Central

Kaščáková, Slávka; Hofland, Leo J.; De Bruijn, Henriette S.; Ye, Yunpeng; Achilefu, Samuel; van der Wansem, Katy; van der Ploeg-van den Heuvel, Angelique; van Koetsveld, Peter M.; Brugts, Michael P.; van der Lelij, Aart-Jan; Sterenborg, Henricus J. C. M.; ten Hagen, Timo L. M.; Robinson, Dominic J.; van Hagen, Martin P.

2014-01-01

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2 + AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate. PMID:25111655

MicroRNA and receptor mediated signaling pathways as potential therapeutic targets in heart failure.

PubMed

Tuttolomondo, Antonino; Simonetta, Irene; Pinto, Antonio

2016-11-01

Cardiac remodelling is a complex pathogenetic pathway involving genome expression, molecular, cellular, and interstitial changes that cause changes in size, shape and function of the heart after cardiac injury. Areas covered: We will review recent advances in understanding the role of several receptor-mediated signaling pathways and micro-RNAs, in addition to their potential as candidate target pathways in the pathogenesis of heart failure. The myocyte is the main target cell involved in the remodelling process via ischemia, cell necrosis and apoptosis (by means of various receptor pathways), and other mechanisms mediated by micro-RNAs. We will analyze the role of some receptor mediated signaling pathways such as natriuretic peptides, mediators of glycogen synthase kinase 3 and ERK1/2 pathways, beta-adrenergic receptor subtypes and relaxin receptor signaling mechanisms, TNF/TNF receptor family and TWEAK/Fn14 axis, and some micro-RNAs as candidate target pathways in pathogenesis of heart failure. These mediators of receptor-mediated pathways and micro-RNA are the most addressed targets of emerging therapies in modern heart failure treatment strategies. Expert opinion: Future treatment strategies should address mediators involved in multiple steps within heart failure pathogenetic pathways.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

PubMed

Smith, Spenser S; Dole, Neha S; Franceschetti, Tiziana; Hrdlicka, Henry C; Delany, Anne M

2016-10-07

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting the folate receptor: improving efficacy in inorganic medicinal chemistry.

PubMed

Carron, Pauraic Mc; Crowley, Aisling; O'Shea, Denis; McCann, Malachy; Howe, Orla; Hunt, Mary; Devereux, Michael

2018-02-09

The discovery of the high-affinity, high-specificity folate receptor in mamalian kidney cells, coupled with the ability of folate to enter cells by folate receptor-mediated endocytosis and the subsequent elucidation of the folate receptor's overexpression in specific cancer cell types; heralded the arrival of the area of chemotherapeutic folate targeting. The application of purely organic folate-based small-molecule drug conjugates that selectively target the folate receptor, which is over expressed in several diseases such as cancer, is well established. The application of inorganic folate-targeted drugs offers significant potential to expand and enhance this therapeutic approach. From the data made available to date, it is apparent that this aspect of inorganic medicinal chemistry is in its youth but has the capability to contribute greatly to cancer research, both in therapy and diagnosis. The union of folate-receptor targeting and inorganic medicine may also lead to the development of treatments for disorders such as chronic-inflammation, tuberculosis, neurodegenerative disease and leishmaniasis. In this review, we summarize what is known about the coordination chemistry of folic acid and the therapeutic potential of such complexes. We also describe approaches adopted to conjugate platinum drugs to folate- or folate-carrier- systems and their prospective ability to overcome problems associated with unwanted side-effects and resistance by improving their delivery and/or selectivity. The literature pertaining to non-platinum metal complex conjugates with folic acid is also reviewed revealing that this is an area that offers significant potential to develop targeted therapeutic approaches in areas such as chemotherapy and molecular imaging for diagnostics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Transcriptional targets shared by estrogen receptor- related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta.

PubMed Central

Vanacker, J M; Pettersson, K; Gustafsson, J A; Laudet, V

1999-01-01

The physiological activities of estrogens are thought to be mediated by specific nuclear receptors, ERalpha and ERbeta. However, certain tissues, such as the bone, that are highly responsive to estrogens only express a low level of these receptors. Starting from this apparent contradiction, we have evaluated the potentials of two related receptors ERRalpha and ERRbeta to intervene in estrogen signaling. ERalpha, ERRalpha and ERRbeta bind to and activate transcription through both the classical estrogen response element (ERE) and the SF-1 response element (SFRE). In contrast, ERbeta DNA-binding and transcriptional activity is restricted to the ERE. Accordingly, the osteopontin gene promoter is stimulated through SFRE sequences, by ERRalpha as well as by ERalpha, but not by ERbeta. Analysis of the cross-talk within the ER/ERR subgroup of nuclear receptors thus revealed common targets but also functional differences between the two ERs. PMID:10428965

Effect of bombesin and its mammalian counterpart, GRP, on exocrine pancreas in the rat.

PubMed

Varga, G; Papp, M; Dobronyi, I; Scarpignato, C

1988-01-01

The effect of equimolar doses (6 nmol/kg) of bombesin and its mammalian counterpart, GRP, on pancreatic growth and secretion was studied in adult rats. Both peptides were administered intraperitoneally three times a day for 5 consecutive days. Saline-treated rats were used as controls. At the end of the treatment, animals were anaesthetized and pancreatic juice was collected in basal conditions and after caerulein (0.75 nmol/kg i.p.) stimulation. Afterwards, the rats were sacrificed and growth and composition of the pancreatic tissue were determined. Compared with the control (saline) values, either basal or stimulated secretion was significantly increased after short-term treatment with both peptides. In addition, both bombesin and GRP increased pancreatic weight, total pancreatic protein, trypsin and amylase content. The DNA content was also increased by both peptides, although only the GRP effect proved to be significant. These results demonstrate that both bombesin and GRP have a growth-promoting effect on rat pancreas and concomitantly increase its secretory capacity. The mechanism of this peculiar biological action is likely to be connected with a direct stimulatory action on the gland.

Phosphatidylserine Sensing by TAM Receptors Regulates AKT-Dependent Chemoresistance and PD-L1 Expression.

PubMed

Kasikara, Canan; Kumar, Sushil; Kimani, Stanley; Tsou, Wen-I; Geng, Ke; Davra, Viralkumar; Sriram, Ganapathy; Devoe, Connor; Nguyen, Khanh-Quynh N; Antes, Anita; Krantz, Allen; Rymarczyk, Grzegorz; Wilczynski, Andrzej; Empig, Cyril; Freimark, Bruce; Gray, Michael; Schlunegger, Kyle; Hutchins, Jeff; Kotenko, Sergei V; Birge, Raymond B

2017-06-01

Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In addition, we leveraged this system to engineer epithelial cells that express wild-type TAM receptors and show that although each receptor can promote PS-mediated efferocytosis, AKT-mediated chemoresistance, as well as upregulate the immune checkpoint molecule PD-L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM receptor-mediated efferocytosis could be partially blocked by PS-targeting antibody 11.31 and Annexin V, demonstrating the existence of a PS/PS receptor (i.e., TAM receptor)/PD-L1 axis that operates in epithelial cells to foster immune escape. These data provide a rationale that PS-targeting, anti-TAM receptor, and anti-PD-L1-based therapeutics will have merit as combinatorial checkpoint inhibitors. Implications: Many tumor cells are known to upregulate the immune checkpoint inhibitor PD-L1. This study demonstrates a role for PS and TAM receptors in the regulation of PD-L1 on cancer cells. Mol Cancer Res; 15(6); 753-64. ©2017 AACR

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance

PubMed Central

2012-01-01

Background The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2−/− mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2−/− neurons. Results Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2−/− mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2−/− olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2−/− mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2−/− olfactory neurons. Conclusions Results presented here show that many odorant receptors are under-expressed in β3GnT2−/− mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2�

Bombesin functionalized 64Cu-copper sulfide nanoparticles for targeted imaging of orthotopic prostate cancer.

PubMed

Cai, Huawei; Xie, Fang; Mulgaonkar, Aditi; Chen, Lihong; Sun, Xiankai; Hsieh, Jer-Tsong; Peng, Fangyu; Tian, Rong; Li, Lin; Wu, Changqiang; Ai, Hua

2018-05-22

To synthesize and evaluate the imaging potential of Bom-PEG-[ 64 Cu]CuS nanoparticles (NPs) in orothotopic prostate tumor. [ 64 Cu]CuS NPs were synthesized in aqueous solution by 64 CuCl 2 and Na 2 S reaction. Then PEG linker with or without bombesin peptide were conjugated to the surface of [ 64 Cu]CuS NPs to produce Bom-PEG-[ 64 Cu]CuS and PEG-[ 64 Cu]CuS NPs. These two kinds of NPs were used for testing specific uptake in prostate cancer cells in vitro and imaging of orthotopic prostate tumor in vivo. Bom-PEG-[ 64 Cu]CuS and PEG-[ 64 Cu]CuS NPs were successfully synthesized with core diameter of approximately 5 nm. Radioactive cellular uptake revealed that Bom-PEG-[ 64 Cu]CuS was able to specifically bind to prostate cancer cells, and the microPET-CT imaging indicated clear visualization of orthotopic prostate tumors. Radiolabeled Bom-PEG-[ 64 Cu]CuS NPs have potential as an ideal agent for orthotopic prostate tumor imaging by microPET-CT.

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

PubMed

Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

1995-08-01

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

PDF Receptor Expression Reveals Direct Interactions between Circadian Oscillators in Drosophila

PubMed Central

Im, Seol Hee; Taghert, Paul H.

2010-01-01

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest the network is divisible into M and E oscillators which normally interact and synchronize. Sixteen oscillator neurons (the small and large LNvs) express a neuropeptide called pigment dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a ~70 kB PDF receptor (pdfr) transgene which does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. PMID:20394051

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Liver X receptor alpha regulates fatty acid synthase expression in chicken.

PubMed

Demeure, O; Duby, C; Desert, C; Assaf, S; Hazard, D; Guillou, H; Lagarrigue, S

2009-12-01

Liver X receptor alpha (LXRalpha), also referred to as nuclear receptor subfamily 1, group H, member 3 is a member of the nuclear hormone receptor superfamily, and has recently been shown to act as a master transcription factor governing hepatic lipogenesis in mammals. Liver X receptor alpha directly regulates both the expression of other lipogenic transcription factors and the expression of lipogenic enzymes, thereby enhancing hepatic fatty acid synthesis (FASN). In birds, like in humans, fatty acid synthesis primarily occurs in the liver. Whether LXRalpha is involved in hepatic regulation of lipogenic genes remained to be investigated in this species. Here we show that fatty acid synthase and the expression of other lipogenic genes (sterol regulatory element binding protein 1 and steroyl coenzyme A desaturase 1) are induced in chicken hepatoma cells in response to a pharmacological liver X receptor agonist, T0901317. A detailed analysis of the chicken FASN promoter revealed a functional liver X response element. These data define the chicken FASN gene as a direct target of LXRalpha and further expand the role of LXRalpha as a regulator of lipid metabolism in this species.

Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease.

PubMed

Chakraborty, Ashok; Hatzis, Christos; DiGiovanna, Michael P

2017-05-01

Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+ model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+ lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

Ethanol preconditioning of rat cerebellar cultures targets NMDA receptors to the synapse and enhances peroxiredoxin 2 expression.

PubMed

Mitchell, Robert M; Tajuddin, Nuzhath; Campbell, Edward M; Neafsey, Edward J; Collins, Michael A

2016-07-01

Epidemiological studies indicate that light-moderate alcohol (ethanol) consumers tend to have reduced risks of cognitive impairment and progression to dementia during aging. Exploring possible mechanisms, we previously found that moderate ethanol preconditioning (MEP, 20-30mM) of rat brain cultures for several days instigated neuroprotection against β-amyloid peptides. Our biochemical evidence implicated the NMDA receptor (NMDAR) as a potential neuroprotective "sensor", specifically via synaptic NMDAR signaling. It remains unclear how ethanol modulates the receptor and its downstream targets to engender neuroprotection. Here we confirm with deconvolution microscopy that MEP of rat mixed cerebellar cultures robustly increases synaptic NMDAR localization. Phospho-activation of the non-receptor tyrosine kinases Src and Pyk2, known to be linked to synaptic NMDAR, is also demonstrated. Additionally, the preconditioning enhances levels of an antioxidant protein, peroxiredoxin 2 (Prx2), reported to be downstream of synaptic NMDAR signaling, and NMDAR antagonism with memantine (earlier found to abrogate MEP neuroprotection) blocks the Prx2 elevations. To further link Prx2 with antioxidant-based neuroprotection, we circumvented the ethanol preconditioning-NMDAR pathway by pharmacologically increasing Prx2 with the naturally-occurring cruciferous compound, 3H-1,2-dithiole-3-thione (D3T). Thus, D3T pretreatment elevated Prx2 expression to a similar extent as MEP, while concomitantly preventing β-amyloid neurotoxicity; D3T also protected the cultures from hydrogen peroxide toxicity. The findings support a mechanism that couples synaptic NMDAR signaling, Prx2 expression and augmented antioxidant defenses in ethanol preconditioning-induced neuroprotection. That this mechanism can be emulated by a cruciferous vegetable constituent suggests that such naturally-occurring "neutraceuticals" may be useful in therapy for oxidative stress-related dementias. Copyright © 2016 Elsevier

Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice1, 2, 3

PubMed Central

Bonami, Rachel H.; Thomas, James W.

2015-01-01

Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or if this process is dysregulated in related autoimmunity. To resolve these issues, an editing-competent model was developed in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a non-autoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, as selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen-targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab’)2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy. PMID:26432895

Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics.

PubMed

Yonezawa, Tomo; Kurata, Riho; Yoshida, Kaori; Murayama, Masanori A; Cui, Xiaofeng; Hasegawa, Akihiko

2013-01-01

G protein-coupled receptor (GPCR) (also known as seven-transmembrane domain receptor) superfamily represents the largest protein family in the human genome. These receptors respond to various physiological ligands such as photons, odors, pheromones, hormones, ions, and small molecules including amines, amino acids to large peptides and steroids. Thus, GPCRs are involved in many diseases and the target of around half of all conventional drugs. The physiological roles of free fatty acids (FFAs), in particular, long-chain FFAs, are important for the development of many metabolic disease including obesity, diabetes, and atherosclerosis. In the past half decade, deorphanization of several GPCRs has revealed that GPR40, GPR41, GPR43, GPR84 and GPR120 sense concentration of extracellular FFAs with various carbon chain lengths. GPR40 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium- chain, but not long-chain, FFAs. GPR41 and GPR43 are activated by short-chain FFAs. GPR40 is highly expressed in pancreatic beta cells and plays a crucial role in FFAs-induced insulin secretion. GPR120 is mainly expressed in enteroendocrine cells and plays an important role for FFAs-induced glucagon-like peptide-1. GPR43 is abundant in leukocytes and adipose tissue, whilst GPR41 is highly expressed in adipose tissue, the pancreas and leukocytes. GPR84 is expressed in leukocytes and monocyte/macrophage. This review aims to shed light on the physiological roles and development of drugs targeting these receptors.

Targeting receptor-activator of nuclear kappaB ligand in aneurysmal bone cysts: verification of target and therapeutic response.

PubMed

Pelle, Dominic W; Ringler, Jonathan W; Peacock, Jacqueline D; Kampfschulte, Kevin; Scholten, Donald J; Davis, Mary M; Mitchell, Deanna S; Steensma, Matthew R

2014-08-01

Aneurysmal bone cyst (ABC) is a benign tumor of bone presenting as a cystic, expansile lesion in both the axial and appendicular skeleton. Axial lesions demand special consideration, because treatment-related morbidity can be devastating. In similar lesions, such as giant cell tumor of bone (GCTB), the receptor-activator of nuclear kappaB ligand (RANKL)-receptor-activator of nuclear kappaB (RANK) signaling axis is essential to tumor progression. Although ABC and GCTB are distinct entities, they both contain abundant multinucleated giant cells and are osteolytic characteristically. We hypothesize that ABCs express both RANKL and RANK similarly in a cell-type specific manner, and that targeted RANKL therapy will mitigate ABC tumor progression. Cellular expression of RANKL and RANK was determined in freshly harvested ABC samples using laser confocal microscopy. A consistent cell-type-specific pattern was observed: fibroblastlike stromal cells expressed RANKL strongly whereas monocyte/macrophage precursor and multinucleated giant cells expressed RANK. Relative RANKL expression was determined by quantitative real-time polymerase chain reaction in ABC and GCTB tissue samples; no difference in relative expression was observed (P > 0.05). In addition, we review the case of a 5-year-old boy with a large, aggressive sacral ABC. After 3 months of targeted RANKL inhibition with denosumab, magnetic resonance imaging demonstrated tumor shrinkage, bone reconstitution, and healing of a pathologic fracture. Ambulation, and bowel and bladder function were restored at 6 months. Denosumab treatment was well tolerated. Post hoc analysis demonstrated strong RANKL expression in the pretreatment tumor sample. These findings demonstrate that RANKL-RANK signal activation is essential to ABC tumor progression. RANKL-targeted therapy may be an effective alternative to surgery in select ABC presentations. Copyright © 2014 Mosby, Inc. All rights reserved.

PDF receptor expression reveals direct interactions between circadian oscillators in Drosophila.

PubMed

Im, Seol Hee; Taghert, Paul H

2010-06-01

Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LNvs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a approximately 70-kB PDF receptor (pdfr) transgene that does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye. (c) 2009 Wiley-Liss, Inc.

Fibroblast growth factor receptors in breast cancer: expression, downstream effects, and possible drug targets.

PubMed

Tenhagen, M; van Diest, P J; Ivanova, I A; van der Wall, E; van der Groep, P

2012-08-01

Cancer treatments are increasingly focusing on the molecular mechanisms underlying the oncogenic processes present in tumors of individual patients. Fibroblast growth factor receptors (FGFRs) are among the many molecules that are involved in oncogenesis and are currently under investigation for their potential as drug targets in breast cancer patients. These receptor tyrosine kinases play a role in several processes including proliferation, angiogenesis, and migration. Alterations in these basal processes can contribute to the development and progression of tumors. Among breast cancer patients, several subgroups have been shown to harbor genetic aberrations in FGFRs, including amplifications of FGFR1, FGFR2, and FGFR4 and mutations in FGFR2 and FGFR4. Here, we review in vitro and in vivo models that have partly elucidated the molecular implications of these different genetic aberrations, the resulting tumor characteristics, and the potential of FGFRs as therapeutic targets for breast cancer treatment.

G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

PubMed Central

2013-01-01

Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

PIM1 kinase inhibition as a targeted therapy against triple-negative breast tumors with elevated MYC expression.

PubMed

Horiuchi, Dai; Camarda, Roman; Zhou, Alicia Y; Yau, Christina; Momcilovic, Olga; Balakrishnan, Sanjeev; Corella, Alexandra N; Eyob, Henok; Kessenbrock, Kai; Lawson, Devon A; Marsh, Lindsey A; Anderton, Brittany N; Rohrberg, Julia; Kunder, Ratika; Bazarov, Alexey V; Yaswen, Paul; McManus, Michael T; Rugo, Hope S; Werb, Zena; Goga, Andrei

2016-11-01

Triple-negative breast cancer (TNBC), in which cells lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, is the breast cancer subtype with the poorest outcome. No targeted therapy is available against this subtype of cancer owing to a lack of validated molecular targets. We previously reported that signaling involving MYC-an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes-is disproportionally higher in triple-negative (TN) tumors than in receptor-positive (RP) tumors. Direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve. Here, by conducting a shRNA screen targeting the kinome, we identified PIM1, a non-essential serine-threonine kinase, in a synthetic lethal interaction with MYC. PIM1 expression was higher in TN tumors than in RP tumors and was associated with poor prognosis in patients with hormone- and HER2-negative tumors. Small-molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic mouse models of breast cancer by inhibiting the oncogenic transcriptional activity of MYC and restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that have elevated MYC expression.

Nicotinic ACh Receptors as Therapeutic Targets in CNS Disorders

PubMed Central

Dineley, Kelly T.; Pandya, Anshul A.; Yakel, Jerrel L.

2015-01-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor channels (nAChRs). These receptors are widely distributed throughout the central nervous system, being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in the mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer’s disease), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. PMID:25639674

Nicotinic ACh receptors as therapeutic targets in CNS disorders.

PubMed

Dineley, Kelly T; Pandya, Anshul A; Yakel, Jerrel L

2015-02-01

The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer's disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. Published by Elsevier Ltd.

TAM receptors Tyro3 and Mer as novel targets in colorectal cancer.

PubMed

Schmitz, Robin; Valls, Aida Freire; Yerbes, Rosario; von Richter, Sophie; Kahlert, Christoph; Loges, Sonja; Weitz, Jürgen; Schneider, Martin; Ruiz de Almodovar, Carmen; Ulrich, Alexis; Schmidt, Thomas

2016-08-30

CRC remains the third most common cancer worldwide with a high 5-year mortality rate in advanced cases. Combined with chemotherapy, targeted therapy is an additional treatment option. However as CRC still escapes targeted therapy the vigorous search for new targets is warranted to increase patients´ overall survival. In this study we describe a new role for Gas6/protein S-TAM receptor interaction in CRC. Gas6, expressed by tumor-infiltrating M2-like macrophages, enhances malignant properties of tumor cells including proliferation, invasion and colony formation. Upon chemotherapy macrophages increase Gas6 synthesis, which significantly attenuates the cytotoxic effect of 5-FU chemotherapy on tumor cells. The anti-coagulant protein S has similar effects as Gas6.In CRC patient samples Tyro3 was overexpressed within the tumor. In-vitro inhibition of Tyro3 and Mer reduces tumor cell proliferation and sensitizes tumor cells to chemotherapy. Moreover high expression of Tyro3 and Mer in tumor tissue significantly shortens CRC patients´ survival. Various in vitro models were used to investigate the role of Gas6 and its TAM receptors in human CRC cells, by stimulation (rhGas6) and knockdown (siRNA) of Axl, Tyro3 and Mer. In terms of a translational research, we additionally performed an expression analysis in human CRC tissue and analyzed the medical record of these patients. Tyro3 and Mer represent novel therapeutic targets in CRC and warrant further preclinical and clinical investigation in the future.

Active radar guides missile to its target: receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems.

PubMed

Yan, Jing-Jun; Liao, Jia-Zhi; Lin, Ju-Sheng; He, Xing-Xing

2015-01-01

Patients with hepatocellular carcinoma (HCC) usually present at advanced stages and do not benefit from surgical resection, so drug therapy should deserve a prominent place in unresectable HCC treatment. But chemotherapy agents, such as doxorubicin, cisplatin, and paclitaxel, frequently encounter important problems such as low specificity and non-selective biodistribution. Recently, the development of nanotechnology led to significant breakthroughs to overcome these problems. Decorating the surfaces of nanoparticulate-based drug carriers with homing devices has demonstrated its potential in concentrating chemotherapy agents specifically to HCC cells. In this paper, we reviewed the current status of active targeting strategies for nanoparticulate systems based on various receptors such as asialoglycoprotein receptor, transferrin receptor, epidermal growth factor receptor, folate receptor, integrin, and CD44, which are abundantly expressed on the surfaces of hepatocytes or liver cancer cells. Furthermore, we pointed out their merits and defects and provided theoretical references for further research.

Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

PubMed

Hauge-Evans, A C; Reers, C; Kerby, A; Franklin, Z; Amisten, S; King, A J; Hassan, Z; Vilches-Flores, A; Tippu, Z; Persaud, S J; Jones, P M

2014-10-01

Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes. © 2014 John Wiley & Sons Ltd.

Differential response of normal human fibroblasts to bombesin versus thrombin.

PubMed

Hendey, B; Mamrack, M D

1988-09-01

Normal human diploid fibroblasts (WS-1 cells) were growth-arrested under serum-free conditions for 48 hr. The addition of fetal bovine serum (10% final concentration) to these cells stimulated [3H]-thymidine incorporation into DNA and phosphoinositide breakdown over nine-fold. Thrombin, at concentrations above 0.1 unit/ml (u/ml), was also effective at stimulating DNA synthesis and phosphoinositide breakdown as well as causing a rise in intracellular pH. In contrast, the peptide bombesin (concentrations ranging from 1 nM to 100 nM) stimulated phosphoinositide breakdown but did not enhance DNA synthesis or cause an increase in cytoplasmic pH. The time course of accumulation of inositol phosphates differed in response to these agents. The thrombin effect peaked rapidly and leveled off after 5 min while the bombesin effect showed a constant increase for 30 min. Serum showed an intermediate response. The different rates of inositol phosphate accumulation observed with the two growth factors is viewed as representing a difference in the mechanism of phosphoinositide turnover. The relationship between the difference in phosphoinositide turnover and the initiation of DNA synthesis is also discussed.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

PubMed

Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C

2012-09-01

What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

PubMed

Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša

2013-03-01

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways

Muscarinic Receptors as Targets for Metronomic Therapy in Breast Cancer.

PubMed

Sales, María Elena

2016-01-01

It is actually known that acetylcholine works as a signaling molecule in non-neuronal cells and tissues, in addition to its neuronal function as neurotransmitter. It can act on two types of receptors nicotinic and muscarinic receptors (mAChRs). The latter belong to the G protein coupled receptor family and there are five subtypes genetically cloned. Their activation triggers classical and non-classical intracellular signals that could be linked to the proliferation of normal and/or transformed cells. The M3 subtype was identified in different types of tumors and its stimulation with agonists triggers cell proliferation, migration, invasion and metastasis. Our laboratory has extensively investigated the expression and function of mAChRs in breast tumors from animal and human origins. We found a profuse expression of mAChRs in breast tumors, but opposite to this, an absence of these receptors in normal breast cells and tissues. The stimulation of mAChRs with the cholinergic agonist carbachol for 20 h increased tumor cell death. Moreover, the combination of subthreshold concentrations of the agonist with paclitaxel potentiates cell death. The usage of low dose chemotherapy with short drug free intervals was named metronomic therapy and it has emerged as a novel regimen for cancer treatment with very low incidence of side effects. Our work and that of others indicate that mAChRs that are over-expressed in different types of tumor cells could be a useful target for metronomic therapy in cancer treatment.

Is androgen receptor targeting an emerging treatment strategy for triple negative breast cancer?

PubMed

Anestis, Aristomenis; Karamouzis, Michalis V; Dalagiorgou, Georgia; Papavassiliou, Athanasios G

2015-06-01

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. The absence of expression and/or amplification of estrogen and progesterone receptor as well as ERBB-2 prevent the use of currently available endocrine options and/or ERBB-2-directed drugs and indicates chemotherapy as the main current therapy. TNBC represents approximately 15% of breast cancer cases with high index of heterogeneity. Here, we review the role of androgen receptor in breast carcinogenesis and its association with alterations in the expression pattern and functional roles of regulatory molecules and signal transduction pathways in TNBC. Additionally, based on the so far preclinical and clinical published data, we evaluate the perspectives for using and/or developing androgen receptor targeting strategies for specific TNBC subtypes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Receptor-Targeted Nanoparticles for In Vivo Imaging of Breast Cancer

PubMed Central

Yang, Lily; Peng, Xiang-Hong; Wang, Y. Andrew; Wang, Xiaoxia; Cao, Zehong; Ni, Chunchun; Karna, Prasanthi; Zhang, Xinjian; Wood, William C.; Gao, Xiaohu; Nie, Shuming; Mao, Hui

2009-01-01

Purpose Cell surface receptor-targeted magnetic iron oxide (IO) nanoparticles provide molecular magnetic resonance imaging (MRI) contrast agents for improving specificity of the detection of human cancer. Experimental design The present study reports the development of a novel targeted IO nanoparticle using a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator conjugated to IO nanoparticles (ATF-IO). This nanoparticle targets urokinase plasminogen activator receptor (uPAR), which is overexpressed in breast cancer tissues. Results ATF-IO nanoparticles are able to specifically bind to and be internalized by uPAR-expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing subcutaneous and intraperitoneal mammary tumors leads to the accumulation of the particles in tumors, generating a strong MRI contrast detectable by a clinical MRI scanner at a field strength of 3 Tesla. Target specificity of ATF-IO nanoparticles demonstrated by in vivo MRI is further confirmed by near infrared (NIR) fluorescence imaging of the mammary tumors using NIR dye-labeled ATF peptides conjugated to IO nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared to those receiving non-targeted IO nanoparticles. Conclusions Our results suggest that uPAR-targeted ATF-IO nanoparticles have potential as molecularly-targeted, dual modality imaging agents for in vivo imaging of breast cancer. PMID:19584158

The sigma-2 receptor as a therapeutic target for drug delivery in triple negative breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makvandi, Mehran; Tilahun, Estifanos D.; Lieberman, Brian P.

Background: Triple-negative breast cancer (TNBC) is associated with high relapse rates and increased mortality when compared with other breast cancer subtypes. In contrast to receptor positive breast cancers, there are no approved targeted therapies for TNBC. Identifying biomarkers for TNBC is of high importance for the advancement of patient care. The sigma-2 receptor has been shown to be overexpressed in triple negative breast cancer in vivo and has been characterized as a marker of proliferation. The aim of the present study was to define the sigma-2 receptor as a target for therapeutic drug delivery and biomarker in TNBC. Methods: Three TNBCmore » cell lines were evaluated: MDA-MB-231, HCC1937 and HCC1806. Sigma-2 compounds were tested for pharmacological properties specific to the sigma-2 receptor through competitive inhibition assays. Sigma-2 receptor expression was measured through radioligand receptor saturation studies. Drug sensitivity for taxol was compared to a sigma-2 targeting compound conjugated to a cytotoxic payload, SW IV-134. Cell viability was assessed after treatments for 2 or 48 h. Sigma-2 blockade was assessed to define sigma-2 mediated cytotoxicity of SW IV-134. Caspase 3/7 activation induced by SW IV-134 was measured at corresponding treatment time points. Results: SW IV-134 was the most potent compound tested in two of the three cell lines and was similarly effective in all three. MDA-MB-231 displayed a statistically significant higher sigma-2 receptor expression and also was the most sensitive cell line evaluated to SW IV-134. Conclusion: Targeting the sigma-2 receptor with a cytotoxic payload was effective in all the three cell lines evaluated and provides the proof of concept for future development of a therapeutic platform for the treatment of TNBC. - Highlights: • TNBC cells are sensitive to sigma-2 receptor targeted drug conjugate SW IV-134. • MDA-MB-231 displayed the highest amount of sigma-2 receptors and corresponded well

Gene expression of ionotropic glutamate receptor subunits in the tectofugal pathway of the pigeon.

PubMed

Atoji, Y

2016-03-01

The tectofugal pathway in birds consists of four stations, the retina, optic tectum, rotundal nucleus, and entopallium, and it conveys visual information via three ascending pathways. These pathways consist of retino-tectal, tecto-rotundal and rotundo-entopallial cells, all of which are glutamatergic. The present study examined the localization of ionotropic glutamate receptors (iGluRs) to identify the target areas of glutamatergic projections in the tectofugal pathway in pigeons. Nine subunits of iGluRs were analyzed using in situ hybridization as follows: AMPA receptors (GluA1, GluA2, GluA3, and GluA4), kainate receptors (GluK1, GluK2, and GluK4), and NMDA receptors (GluN1 and GluN2A). Hybridization signals of subunits showed various intensities in different cells. In the optic tectum, a strong to moderate expression was observed in layer 10 (GluA2, GluA3, GluK4, and GluN1) and layer 13 (GluA2, GluK4, GluN1, and GluN2A). The rotundal nucleus intensely expressed GluA3, GluA4, GluK1, and GluK4. In the entopallium, an intense to moderate expression of GluK1 and GluK4, and a moderate to weak expression of AMPA and NMDA receptors were observed. Furthermore, the parvocellular and magnocellular parts of the isthmic nuclei showed a strong expression of GluA2, GluA3, GluK4, and GluN1. The present findings demonstrate the expression of iGluRs in glutamatergic projection targets of the tectofugal pathway in birds and suggest a diversity of iGluRs in the transmission of visual information. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Prostate cancer targeting motifs: expression of αν β3, neurotensin receptor 1, prostate specific membrane antigen, and prostate stem cell antigen in human prostate cancer cell lines and xenografts.

PubMed

Taylor, Robert M; Severns, Virginia; Brown, David C; Bisoffi, Marco; Sillerud, Laurel O

2012-04-01

Membrane receptors are frequent targets of cancer therapeutic and imaging agents. However, promising in vitro results often do not translate to in vivo clinical applications. To better understand this obstacle, we measured the expression differences in receptor signatures among several human prostate cancer cell lines and xenografts as a function of tumorigenicity. Messenger RNA and protein expression levels for integrin α(ν) β(3), neurotensin receptor 1 (NTSR1), prostate specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) were measured in LNCaP, C4-2, and PC-3 human prostate cancer cell lines and in murine xenografts using quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and immunohistochemistry. Stable expression patterns were observed for integrin α(ν) and PSMA in all cells and corresponding xenografts. Integrin β(3) mRNA expression was greatly reduced in C4-2 xenografts and greatly elevated in PC-3 xenografts compared with the corresponding cultured cells. NTSR1 mRNA expression was greatly elevated in LNCaP and PC-3 xenografts. PSCA mRNA expression was elevated in C4-2 xenografts when compared with C4-2 cells cultured in vitro. Furthermore, at the protein level, PSCA was re-expressed in all xenografts compared with cells in culture. The regulation of mRNA and protein expression of the cell-surface target proteins α(ν) β(3), NTSR1, PSMA, and PSCA, in prostate cancer cells with different tumorigenic potential, was influenced by factors of the microenvironment, differing between cell cultures and murine xenotransplants. Integrin α(ν) β(3), NTRS1 and PSCA mRNA expression increased with tumorigenic potential, but mRNA expression levels for these proteins do not translate directly to equivalent expression levels of membrane bound protein. Copyright © 2011 Wiley Periodicals, Inc.

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Diverse solid tumors expressing a restricted epitope of L1-CAM can be targeted by chimeric antigen receptor redirected T lymphocytes.

PubMed

Hong, Hao; Stastny, Michael; Brown, Christine; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2014-01-01

Adhesion molecule L1-CAM (CD171) was originally reported to be overexpressed on neuroblastoma and to play an important role during tumor progression. More recently, it has been shown to be overexpressed on many other solid tumors such as melanoma and carcinomas of the cervix, ovary, bladder, and others. Thus, there has been a growing interest in using this cell-surface molecule as a target for both antibody-based and cellular-based therapy-our group has previously examined the clinical utility of chimeric antigen receptor (CAR)-redirected cytolytic T cells that specifically target the CE7 epitope of L1-CAM on neuroblastoma patients. Here, we sought to determine whether this CE7 epitope is present on other recently identified L1-CAM tumors and whether it too can be targeted by CAR T cells. Our studies demonstrate that a diverse array of human tumor cell lines and primary solid tumors (ovarian, lung, and renal carcinoma, glioblastoma and neuroblastoma) do express the CE7 epitope and can efficiently stimulate CE7-specific CAR-redirected (CE7R) T-cell lytic activity and secretion of proinflamatory cytokines. L1-CAM was also detected on a limited number of normal tissues; however, L1-CAM expressed on normal human monocytes was not bound by the CE7 mAb nor was it targeted by CE7R T cells, suggesting that the CE7 epitope is more tumor restricted and not expressed on all L1-CAM tissues. Overall, the CE7 epitope of L1-CAM on a variety of tumors may be amenable to targeting by CE7R T cells, making it a promising target for adoptive immunotherapy.

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Noninternalizing Receptors.

PubMed

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J

2017-03-28

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ∼3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or noninternalizing receptor (CD45). When lymphocytes were preloaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell antitumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors.

Modes of Action, Resistance and Toxicity of Insecticides Targeting Nicotinic Acetylcholine Receptors.

PubMed

Ihara, Makoto; Buckingham, Steven D; Matsuda, Kazuhiko; Sattelle, David B

2017-01-01

Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Molecular analysis of nicotinic receptor expression in autism.

PubMed

Martin-Ruiz, C M; Lee, M; Perry, R H; Baumann, M; Court, J A; Perry, E K

2004-04-07

Autism is a developmental disorder of unknown aetiopathology and lacking any specific pharmacological therapeutic intervention. Neurotransmitters such as serotonin, gamma-aminobutyric acid (GABA) and acetylcholine have been implicated. Abnormalities in nicotinic acetylcholine receptors have been identified including cortical loss of binding to the alpha4/beta2 subtype and increase in cerebellar alpha7 binding. Receptor expression (mRNA) has not so far been systematically examined. This study aims to further explore the role of nicotinic receptors in autism by analysing nicotinic receptor subunit mRNA in conjunction with protein levels and receptor binding in different brain areas. Quantitative RT-PCR for alpha4, alpha7 and beta2 subunit mRNA expression levels; alpha3, alpha4, alpha7 and beta2 subunit protein expression immunochemistry and specific radioligand receptor binding were performed in adult autism and control brain samples from cerebral cortex and cerebellum. Alpha4 and beta2 protein expression and receptor binding density as well as alpha4 mRNA levels were lower in parietal cortex in autism, while alpha7 did not change for any of these parameters. In cerebellum, alpha4 mRNA expression was increased, whereas subunit protein and receptor levels were decreased. Alpha7 receptor binding in cerebellum was increased alongside non-significant elevations in mRNA and protein expression levels. No significant changes were found for beta2 in cerebellum. The data obtained, using complementary measures of receptor expression, indicate that reduced gene expression of the alpha4beta2 nicotinic receptor in the cerebral cortex is a major feature of the neurochemical pathology of autism, whilst post-transcriptional abnormalities of both this and the alpha7 subtype are apparent in the cerebellum. The findings point to dendritic and/or synaptic nicotinic receptor abnormalities that may relate to disruptions in cerebral circuitry development.

Targeting the androgen receptor in prostate and breast cancer – several new agents in development

PubMed Central

Proverbs-Singh, Tracy; Feldman, Jarett L.; Morris, Michael J.; Autio, Karen A.; Traina, Tiffany A.

2016-01-01

Prostate cancer and breast cancer share similarities as hormone-sensitive cancers with a wide heterogeneity of both phenotype and biology. The androgen receptor (AR) is a hormone receptor involved in both benign and malignant processes. Targeting androgen synthesis and the AR pathway has been and remains central to prostate cancer therapy. Recently, there is increased interest in the role of the AR in breast cancer development and growth, with data suggesting AR co-expression with estrogen, progesterone and human epidermal growth factor receptors, across all intrinsic subtypes of breast cancer. Targeting the AR axis is an evolving field with novel therapies in development which may ultimately be applicable for both tumor types. In this review, we offer an overview of available agents which target the AR axis in both prostate and breast cancer and provide insight into the novel drugs in development for targeting this signaling pathway. PMID:25722318

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Defective postnatal endochondral bone development by chondrocyte-specific targeted expression of parathyroid hormone type 2 receptor.

PubMed

Panda, Dibyendu Kumar; Goltzman, David; Karaplis, Andrew C

2012-12-15

The human parathyroid hormone type 2 receptor (PTH2R) is activated by PTH and by tuberoinfundibular peptide of 39 residues (TIP39), the latter likely acting as its natural ligand. Although the receptor is expressed at highest levels in the nervous system, we have observed that both PTH2R and TIP39 are expressed in the newborn mouse growth plate, with the receptor localizing in the resting zone and the ligand TIP39 localizing exclusively in prehypertrophic and hypertrophic chondrocytes. To address the role of PTH2R in postnatal skeletal growth and development, Col2a1-hPTH2R (PTH2R-Tg) transgenic mice were generated. The mice were viable and of nearly normal size at birth. Expression of the transgene in the growth plate was limited to chondrocytes. We found that chondrocyte proliferation was decreased, as determined by in vivo BrdU labeling of proliferating chondrocytes and CDK4 and p21 expression in the growth plate of Col2a1-hPTH2R transgenic mice. Similarly, the differentiation and maturation of chondrocytes was delayed, as characterized by decreased Sox9 expression and weaker immunostaining for the chondrocyte differentiation markers collagen type II and type X and proteoglycans. As well, there was altered expression of Gdf5, Wdr5, and β-catenin, factors implicated in chondrocyte maturation, proliferation, and differentiation.These effects impacted on the process of endochondral ossification, resulting in delayed formation of the secondary ossification center, and diminished trabecular bone volume. The findings substantiate a role for PTH2R signaling in postnatal growth plate development and subsequent bone mass acquisition.

Selective over-expression of fibroblast growth factor receptors 1 and 4 in clinical prostate cancer.

PubMed

Sahadevan, K; Darby, S; Leung, H Y; Mathers, M E; Robson, C N; Gnanapragasam, V J

2007-09-01

Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1-4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1-3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the

GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

PubMed

Cheung, Samantha K; Scott, Kristin

2017-01-01

Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

Nuclear receptor-mediated regulation of carboxylesterase expression and activity.

PubMed

Staudinger, Jeff L; Xu, Chenshu; Cui, Yue J; Klaassen, Curtis D

2010-03-01

Emerging evidence demonstrates that several nuclear receptor (NR) family members regulate drug-inducible expression and activity of several important carboxylesterase (CES) enzymes in mammalian liver and intestine. Numerous clinically prescribed anticancer prodrugs, carbamate and pyrethroid insecticides, environmental toxicants and procarcinogens are substrates for CES enzymes. Moreover, a key strategy used in rational drug design frequently utilizes an ester linkage methodology to selectively target a prodrug, or to improve the water solubility of a novel compound. This review summarizes the current state of knowledge regarding NR-mediated regulation of CES enzymes in mammals and highlights their importance in drug metabolism, drug-drug interactions and toxicology. New knowledge regarding the transcriptional regulation of CES enzymes by NR proteins pregnane x receptor (NR1I2) and constitutive androstane receptor (NR1I3) has recently come to light through the use of knockout and transgenic mouse models. Novel insights regarding the species-specific cross-regulation of glucocorticoid receptor (NR3C1) and PPAR-alpha (NR1C1) signaling and CES gene expression are discussed. Elucidation of the role of NR-mediated regulation of CES enzymes in liver and intestine will have a significant impact on rational drug design and the development of novel prodrugs, especially for patients on combination therapy.

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

PubMed Central

Mantilla, Carlos B.; Bailey, Jeffrey P.; Zhan, Wen-Zhi; Sieck, Gary C.

2012-01-01

Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability. PMID:22227062

Prostanoid receptors as possible targets for anti-allergic drugs: recent advances in prostanoids on allergy and immunology.

PubMed

Honda, Tetsuya; Tokura, Yoshiki; Miyachi, Yoshiki; Kabashima, Kenji

2010-12-01

Prostanoids, consisting of prostaglandins and thromboxane, are cyclooxygenase metabolites of arachidonic acid released in various pathophysiological conditions which exert a range of actions mediated through their respective receptors expressed on target cells. Although it has been difficult to analyze the physiological role of prostanoids, recent developments in both the disruption of the respective gene and receptor selective compounds have enabled us to investigate the physiological roles for each receptor. It has been demonstrated that each prostanoid receptor has multiple functions, and that their expression is regulated in a context-dependent manner that sometimes results in opposite, excitatory and inhibitory, outcomes. The balance of prostanoid production and receptor expression has been revealed to be important for homeostasis of the human body. Here, we review new findings on the roles of prostanoids in allergic and immune diseases, focusing on contact dermatitis, atopic dermatitis, asthma, rheumatoid arthritis, and encephalomyelitis, and also discuss the clinical potentials of receptor-selective drugs.

Contrasting skeletal phenotypes in mice with an identical mutation targeted to thyroid hormone receptor alpha1 or beta.

PubMed

O'Shea, Patrick J; Bassett, J H Duncan; Sriskantharajah, Srividya; Ying, Hao; Cheng, Sheue-yann; Williams, Graham R

2005-12-01

Thyroid hormone (T(3)) regulates bone turnover and mineralization in adults and is essential for skeletal development. Surprisingly, we identified a phenotype of skeletal thyrotoxicosis in T(3) receptor beta(PV) (TRbeta(PV)) mice in which a targeted frameshift mutation in TRbeta results in resistance to thyroid hormone. To characterize mechanisms underlying thyroid hormone action in bone, we analyzed skeletal development in TRalpha1(PV) mice in which the same PV mutation was targeted to TRalpha1. In contrast to TRbeta(PV) mice, TRalpha1(PV) mutants exhibited skeletal hypothyroidism with delayed endochondral and intramembranous ossification, severe postnatal growth retardation, diminished trabecular bone mineralization, reduced cortical bone deposition, and delayed closure of the skull sutures. Skeletal hypothyroidism in TRalpha1(PV) mutants was accompanied by impaired GH receptor and IGF-I receptor expression and signaling in the growth plate, whereas GH receptor and IGF-I receptor expression and signaling were increased in TRbeta(PV) mice. These data indicate that GH receptor and IGF-I receptor are physiological targets for T(3) action in bone in vivo. The divergent phenotypes observed in TRalpha1(PV) and TRbeta(PV) mice arise because the pituitary gland is a TRbeta-responsive tissue, whereas bone is TRalpha responsive. These studies provide a new understanding of the complex relationship between central and peripheral thyroid status.

REV-ERB and ROR nuclear receptors as drug targets

PubMed Central

Kojetin, Douglas J.; Burris, Thomas P.

2016-01-01

The nuclear receptors REV-ERB (consisting of REV-ERBα and REV-ERBβ) and retinoic acid receptor-related orphan receptors (RORs; consisting of RORα, RORβ and RORγ) are involved in many physiological processes, including regulation of metabolism, development and immunity as well as the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and opened up the possibility of targeting these receptors to treat several diseases, including diabetes, atherosclerosis, autoimmunity and cancer. This Review focuses on the latest developments in ROR and REV-ERB pharmacology indicating that these nuclear receptors are druggable targets and that ligands targeting these receptors may be useful in the treatment of several disorders. PMID:24577401

Glycoprotein CD98 as a receptor for colitis-targeted delivery of nanoparticle.

PubMed

Xiao, Bo; Yang, Yang; Viennois, Emilie; Zhang, Yuchen; Ayyadurai, Saravanan; Baker, Mark; Laroui, Hamed; Merlin, Didier

2014-03-21

Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, ex vivo studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.

Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

PubMed

Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

2016-11-01

To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D. Copyright © 2016 by The American Association of Immunologists, Inc.

Targeting G protein-coupled receptor kinases (GRKs) in Heart Failure

PubMed Central

Brinks, Henriette; Koch, Walter J

2010-01-01

In the human body, over 1000 different G protein-coupled receptors (GPCRs) mediate a broad spectrum of extracellular signals at the plasma membrane, transmitting vital physiological features such as pain, sight, smell, inflammation, heart rate and contractility of muscle cells. Signaling through these receptors is primarily controlled and regulated by a group of kinases, the GPCR kinases (GRKs), of which only seven are known and thus, interference with these common downstream GPCR regulators suggests a powerful therapeutic strategy. Molecular modulation of the kinases that are ubiquitously expressed in the heart has proven GRK2, and also GRK5, to be promising targets for prevention and reversal of one of the most severe pathologies in man, chronic heart failure (HF). In this article we will focus on the structural aspects of these GRKs important for their physiological and pathological regulation as well as well known and novel therapeutic approaches that target these GRKs in order to overcome the development of cardiac injury and progression of HF. PMID:21218155

Expression of Hormone Receptors and HER-2 in Benign and Malignant Salivary Gland Tumors.

PubMed

Can, Nhu Thuy; Lingen, Mark W; Mashek, Heather; McElherne, James; Briese, Renee; Fitzpatrick, Carrie; van Zante, Annemieke; Cipriani, Nicole A

2018-03-01

With the advent of targeted therapies, expression of sex hormone receptors and HER-2 in salivary gland tumors (SGTs) is of clinical interest. Previous reports of estrogen (ER) and progesterone (PR) receptor expression have varied. Androgen receptor (AR) and HER-2 overexpression are frequently reported in salivary duct carcinoma (SDC), but have not been studied systematically in other SGTs. This study examines ER, PR, AR, and HER-2 expression in SGTs. Immunohistochemistry for ER, PR, AR, and HER-2 was performed on 254 SGTs (134 malignant). ER, PR, and AR expression was scored using Allred system. HER-2 expression was scored using Dako HercepTest guidelines. FISH for HER-2 amplification was performed on select cases with HER-2 overexpression (2-3+). No SGT demonstrated strong expression of ER or PR. Combined strong AR and HER-2 expression was seen in 22 carcinomas: 14/25 SDC, 3/16 poorly differentiated, two oncocytic, and one each carcinoma ex pleomorphic adenoma, squamous cell, and intraductal carcinoma. Eighteen additional high grade carcinomas had HER-2 overexpression with absent, weak, or moderate AR expression; eight high grade carcinomas had isolated strong AR expression with 0-1+ HER-2 staining. Of 15 tested cases, six demonstrated HER-2 amplification by FISH, all of which had 3+ immunoreactivity. Neither benign nor malignant SGTs had strong expression of ER or PR. None of the benign SGTs overexpressed AR or HER-2. Coexpression of AR and HER-2 should not define SDC, but immunostaining should be considered in high grade salivary carcinomas, as some show overexpression and may benefit from targeted therapy.

Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy.

PubMed

Hu, Zhiwei; Cheng, Jijun; Xu, Jie; Ruf, Wolfram; Lockwood, Charles J

2017-02-01

Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

Emerging strategies for EphA2 receptor targeting for cancer therapeutics.

PubMed

Tandon, Manish; Vemula, Sai Vikram; Mittal, Suresh K

2011-01-01

High mortality rates with cancers warrant further development of earlier diagnostics and better treatment strategies. Membrane-bound erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is overexpressed in breast, prostate, urinary bladder, skin, lung, ovary and brain cancers. EphA2 overexpression in cancers, its signaling mechanisms and strategies to target its deregulation. High EphA2 expression in cancer cells is correlated with a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., ephrinA1) triggers events that are deregulated and implicated in carcinogenesis. EphrinA1-independent oncogenic activity and ephrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches. EphA2 has been tested as a drug target using multiple approaches such as agonist antibodies, RNA interference, immunotherapy, virus vector-mediated gene transfer, small-molecule inhibitors and nanoparticles. With over a decade of research, encouraging results with targeting of EphA2 expression in various pre-clinical cancer models necessitate further studies.

GPR48 Increases Mineralocorticoid Receptor Gene Expression

PubMed Central

Wang, Jiqiu; Li, Xiaoying; Ke, Yingying; Lu, Yan; Wang, Feng; Fan, Nengguang; Sun, Haiyan; Zhang, Huijie; Liu, Ruixin; Yang, Jun; Ye, Lei; Liu, Mingyao

2012-01-01

Aldosterone and the mineralocorticoid receptor (MR) are critical to the maintenance of electrolyte and BP homeostasis. Mutations in the MR cause aldosterone resistance known as pseudohypoaldosteronism type 1 (PHA1); however, some cases consistent with PHA1 do not exhibit known gene mutations, suggesting the possibility of alternative genetic variants. We observed that G protein–coupled receptor 48 (Gpr48/Lgr4) hypomorphic mutant (Gpr48m/m) mice had hyperkalemia and increased water loss and salt excretion despite elevated plasma aldosterone levels, suggesting aldosterone resistance. When we challenged the mice with a low-sodium diet, these features became more obvious; the mice also developed hyponatremia and increased renin expression and activity, resembling a mild state of PHA1. There was marked renal downregulation of MR and its downstream targets (e.g., the α-subunit of the amiloride-sensitive epithelial sodium channel), which could provide a mechanism for the aldosterone resistance. We identified a noncanonical cAMP-responsive element located in the MR promoter and demonstrated that GPR48 upregulates MR expression via the cAMP/protein kinase A pathway in vitro. Taken together, our data demonstrate that GPR48 enhances aldosterone responsiveness by activating MR expression, suggesting that GPR48 contributes to homeostasis of electrolytes and BP and may be a candidate gene for PHA1. PMID:22135314

Current drug treatments targeting dopamine D3 receptor.

PubMed

Leggio, Gian Marco; Bucolo, Claudio; Platania, Chiara Bianca Maria; Salomone, Salvatore; Drago, Filippo

2016-09-01

Dopamine receptors (DR) have been extensively studied, but only in recent years they became object of investigation to elucidate the specific role of different subtypes (D1R, D2R, D3R, D4R, D5R) in neural transmission and circuitry. D1-like receptors (D1R and D5R) and D2-like receptors (D2R, D2R and D4R) differ in signal transduction, binding profile, localization in the central nervous system and physiological effects. D3R is involved in a number of pathological conditions, including schizophrenia, Parkinson's disease, addiction, anxiety, depression and glaucoma. Development of selective D3R ligands has been so far challenging, due to the high sequence identity and homology shared by D2R and D3R. As a consequence, despite a rational design of selective DR ligands has been carried out, none of currently available medicines selectively target a given D2-like receptor subtype. The availability of the D3R ligand [(11)C]-(+)-PHNO for positron emission tomography studies in animal models as well as in humans, allows researchers to estimate the expression of D3R in vivo; displacement of [(11)C]-(+)-PHNO binding by concurrent drug treatments is used to estimate the in vivo occupancy of D3R. Here we provide an overview of studies indicating D3R as a target for pharmacological therapy, and a review of market approved drugs endowed with significant affinity at D3R that are used to treat disorders where D3R plays a relevant role. Copyright © 2016 Elsevier Inc. All rights reserved.

Androgen receptor expression in breast cancer in relation to molecular phenotype: results from the Nurses' Health Study.

PubMed

Collins, Laura C; Cole, Kimberly S; Marotti, Jonathan D; Hu, Rong; Schnitt, Stuart J; Tamimi, Rulla M

2011-07-01

Previous studies have demonstrated that androgen receptor is expressed in many breast cancers, but its expression in relation to the various breast cancer subtypes as defined by molecular profiling has not been studied in detail. We constructed tissue microarrays from 3093 breast cancers that developed in women enrolled in the Nurses' Health Study. Tissue microarray sections were immunostained for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6, epidermal growth factor receptor (EGFR) and androgen receptor (ER). Immunostain results were used to categorize each cancer as luminal A or B, HER2 and basal like. The relationships between androgen receptor expression and molecular subtype were analyzed. Overall, 77% of the invasive breast carcinomas were androgen receptor positive. Among 2171 invasive cancers, 64% were luminal A, 15% luminal B, 6% HER2 and 11% basal like. The frequency of androgen receptor expression varied significantly across the molecular phenotypes (P<0.0001). In particular, androgen receptor expression was commonly observed in luminal A (91%) and B (68%) cancers, but was less frequently seen in HER2 cancers (59%). Despite being defined by the absence of ER and PR expression and being considered hormonally unresponsive, 32% of basal-like cancers expressed androgen receptor. Among 246 cases of ductal carcinoma in situ, 86% were androgen receptor positive, but the frequency of androgen receptor expression differed significantly across the molecular phenotypes (P=0.001), and high nuclear grade lesions were less likely to be androgen receptor positive compared with lower-grade lesions. Androgen receptor expression is most commonly seen in luminal A and B invasive breast cancers. However, expression of androgen receptor is also seen in approximately one-third of basal-like cancers, providing further evidence that basal-like cancers represent a heterogeneous group. Our findings raise the

Enhancing Adoptive Cell Therapy of Cancer through Targeted Delivery of Small-Molecule Immunomodulators to Internalizing or Non-Internalizing Receptors

PubMed Central

Zheng, Yiran; Tang, Li; Mabardi, Llian; Kumari, Sudha; Irvine, Darrell J.

2017-01-01

Adoptive cell therapy (ACT) has achieved striking efficacy in B-cell leukemias, but less success treating other cancers, in part due to the rapid loss of ACT T-cell effector function in vivo due to immunosuppression in solid tumors. Transforming growth factor-β (TGF-β) signaling is an important mechanism of immune suppression in the tumor microenvironment, but systemic inhibition of TGF-β is toxic. Here we evaluated the potential of targeting a small molecule inhibitor of TGF-β to ACT T-cells using PEGylated immunoliposomes. Liposomes were prepared that released TGF-β inhibitor over ~3 days in vitro. We compared the impact of targeting these drug-loaded vesicles to T-cells via an internalizing receptor (CD90) or non-internalizing receptor (CD45). When lymphocytes were pre-loaded with immunoliposomes in vitro prior to adoptive therapy, vesicles targeted to both CD45 and CD90 promoted enhanced T-cell expression of granzymes relative to free systemic drug administration, but only targeting to CD45 enhanced accumulation of granzyme-expressing T-cells in tumors, which correlated with the greatest enhancement of T-cell anti-tumor activity. By contrast, when administered i.v. to target T-cells in vivo, only targeting of a CD90 isoform expressed exclusively by the donor T-cells led to greater tumor regression over equivalent doses of free systemic drug. These results suggest that in vivo, targeting of receptors uniquely expressed by donor T-cells is of paramount importance for maximal efficacy. This immunoliposome strategy should be broadly applicable to target exogenous or endogenous T-cells and defines parameters to optimize delivery of supporting (or suppressive) drugs to these important immune effectors. PMID:28231431

Early exposure to caffeine affects gene expression of adenosine receptors, DARPP-32 and BDNF without affecting sensibility and morphology of developing zebrafish (Danio rerio).

PubMed

Capiotti, Katiucia Marques; Menezes, Fabiano Peres; Nazario, Luiza Reali; Pohlmann, Julhana Bianchini; de Oliveira, Giovanna M T; Fazenda, Lidiane; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

2011-01-01

Adenosine receptors are the most important biochemical targets of caffeine, a common trimethylxanthine found in food and beverages. Adenosine plays modulatory action during the development through adenosine receptors and their intracellular pathways activation. In this study, we aimed to evaluate if caffeine gave to zebrafish in the very first steps of development is able to affect its direct targets, through the adenosine receptors mRNA expression evaluation, and latter indirect targets, through evaluation of the pattern of dopamine and cAMP-regulated phosphoprotein and brain-derived neurotrophic factor (BDNF) mRNA expression. Here, we demonstrate that zebrafish express adenosine receptor subtypes (A1, A2A1, A2A2 and A2B) since 24h post-fertilization (hpf) and that caffeine exposure is able to affect the expression of these receptors. Caffeine exposure from 1 hpf is able to increase A1 expression at 72-96 hpf and A2A1 expression at 72 hpf. No alterations occurred in A2A2 and A2B expression after caffeine treatment. DARPP-32, a phosphoprotein involved in adenosine intracellular pathway is also expressed since 24 hpf and early exposure to caffeine increased DARPP-32 expression at 168 hpf. We also evaluate the expression of BDNF as one of the targets of adenosine intracellular pathway activation. BDNF was also expressed since 24 hpf and caffeine treatment increased its expression at 48 and 72 hpf. No morphological alterations induced by caffeine treatment were registered by the check of general body features and total body length. Assessment of tactile sensibility also demonstrated no alterations by caffeine treatment. Altogether, these results suggest that caffeine is able to affect expression of its cellular targets since early phases of development in zebrafish without affect visible features. The up-regulation of direct and indirect targets of caffeine presents as a compensatory mechanism of maintenance of adenosinergic modulation during the developmental phase

Hapten-derivatized nanoparticle targeting and imaging of gene expression by multimodality imaging systems.

PubMed

Cheng, C-M; Chu, P-Y; Chuang, K-H; Roffler, S R; Kao, C-H; Tseng, W-L; Shiea, J; Chang, W-D; Su, Y-C; Chen, B-M; Wang, Y-M; Cheng, T-L

2009-01-01

Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

Engineering hepatitis B virus core particles for targeting HER2 receptors in vitro and in vivo.

PubMed

Mohamed Suffian, Izzat Fahimuddin Bin; Wang, Julie Tzu-Wen; Hodgins, Naomi O; Klippstein, Rebecca; Garcia-Maya, Mitla; Brown, Paul; Nishimura, Yuya; Heidari, Hamed; Bals, Sara; Sosabowski, Jane K; Ogino, Chiaki; Kondo, Akihiko; Al-Jamal, Khuloud T

2017-03-01

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30-34 nm diameter and 7 nm thick envelopes, consisting of 180-240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the Z HER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of Z HER2 -ΔHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of Z HER2 -ΔHBc particles in HER2-expressing tumours, compared to non-targeted ΔHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

Functional expression of ionotropic glutamate receptors in the rabbit retinal ganglion cells.

PubMed

Chen, Yin-Peng; Chiao, Chuan-Chin

2012-01-03

It has been known that retinal ganglion cells (RGCs) with distinct morphologies have different physiological properties. It was hypothesized that different functions of RGCs may in part result from various expressions of N-methyl-d-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-isoxazole-4-propinoic acid (AMPA), and kainic acid (KA) receptors on their dendrites. In the present study, we aimed to characterize the functional expression of AMPA and NMDA receptors of morphologically identified RGCs in the wholemount rabbit retina. The agmatine (AGB) activation assay was used to reveal functional expression of ionotropic glutamate receptors after the RGCs were targeted by injecting Neurobiotin. To examine the excitability of these glutamate receptors in an agonist specific manner, the lower concentrations of AMPA (2 μM) and NMDA (100 μM) were chosen to examine G7 (ON-OFF direction selective ganglion cells) and G11 (alpha ganglion cells) types of RGCs. We found that less than 40% of G7 type RGCs had salient AGB activation when incubated with 2 μM AMPA or 100 μM NMDA. The G11 type RGCs also showed similar activation frequencies, except that all of the OFF subtype examined had no AGB permeation under the same AMPA concentration. These results suggest that RGCs with large somata (G7 and G11 types) may express various heterogeneous functional ionotropic glutamate receptors, thus in part rendering their functional diversity. Copyright © 2011 Elsevier B.V. All rights reserved.

PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues

PubMed Central

2012-01-01

Background Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN2/gastrin-releasing peptide receptors are overexpressed. We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart. Methods Derivatisation of a stabilised DOTA-BN(7–14)[Cha13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy. Results The specificity and the binding affinity of the BN analogue for BN2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios (tumour-to-kidney, 0.6 vs. 0.4; tumour-to-liver, 8.8 vs. 5.9, 24 h p.i.). In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour

New target genes in endometrial tumors show a role for the estrogen-receptor pathway in microsatellite-unstable cancers.

PubMed

Ferreira, Ana M; Tuominen, Iina; Sousa, Sónia; Gerbens, Frans; van Dijk-Bos, Krista; Osinga, Jan; Kooi, Krista A; Sanjabi, Bahram; Esendam, Chris; Oliveira, Carla; Terpstra, Peter; Hardonk, Menno; van der Sluis, Tineke; Zazula, Monika; Stachura, Jerzy; van der Zee, Ate G; Hollema, Harry; Sijmons, Rolf H; Aaltonen, Lauri A; Seruca, Raquel; Hofstra, Robert M W; Westers, Helga

2014-12-01

Microsatellite instability (MSI) in tumors results in an accumulation of mutations in (target) genes. Previous studies suggest that the profile of target genes differs according to tumor type. This paper describes the first genome-wide search for target genes for mismatch repair-deficient endometrial cancers. Genes expressed in normal endometrium containing coding repeats were analyzed for mutations in tumors. We identified 44 possible genes of which seven are highly mutated (>15%). Some candidates were also found mutated in colorectal and gastric tumors. The most frequently mutated gene, NRIP1 encoding nuclear receptor-interacting protein 1, was silenced in an endometrial tumor cell line and expression microarray experiments were performed. Silencing of NRIP1 was associated with differences in the expression of several genes in the estrogen-receptor network. Furthermore, an enrichment of genes related to cell cycle (regulation) and replication was observed. We present a new profile of target genes, some of them tissue specific, whereas others seem to play a more general role in MSI tumors. The high-mutation frequency combined with the expression data suggest, for the first time, an involvement of NRIP1 in endometrial cancer development. © 2014 WILEY PERIODICALS, INC.

A compound chimeric antigen receptor strategy for targeting multiple myeloma.

PubMed

Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

2018-02-01

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Social information changes stress hormone receptor expression in the songbird brain.

PubMed

Cornelius, Jamie M; Perreau, Gillian; Bishop, Valerie R; Krause, Jesse S; Smith, Rachael; Hahn, Thomas P; Meddle, Simone L

2018-01-01

Social information is used by many vertebrate taxa to inform decision-making, including resource-mediated movements, yet the mechanisms whereby social information is integrated physiologically to affect such decisions remain unknown. Social information is known to influence the physiological response to food reduction in captive songbirds. Red crossbills (Loxia curvirostra) that were food reduced for several days showed significant elevations in circulating corticosterone (a "stress" hormone often responsive to food limitation) only if their neighbors were similarly food restricted. Physiological responses to glucocorticoid hormones are enacted through two receptors that may be expressed differentially in target tissues. Therefore, we investigated the influence of social information on the expression of the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA in captive red crossbill brains. Although the role of MR and GR in the response to social information may be highly complex, we specifically predicted social information from food-restricted individuals would reduce MR and GR expression in two brain regions known to regulate hypothalamic-pituitary-adrenal (HPA) activity - given that reduced receptor expression may lessen the efficacy of negative feedback and release inhibitory tone on the HPA. Our results support these predictions - offering one potential mechanism whereby social cues could increase or sustain HPA-activity during stress. The data further suggest different mechanisms by which metabolic stress versus social information influence HPA activity and behavioral outcomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Carbachol does not down-regulate substance P receptors in pancreatic acini.

PubMed

Patto, R J; Vinayek, R; Jensen, R T; Gardner, J D

1992-01-01

In a previous study, we found that first incubating guinea pig pancreatic acini with carbachol caused desensitization of the enzyme secretory response to cholecystokinin-octapeptide (CCK-8), bombesin, and carbachol but not that to substance P. This carbachol-induced desensitization could be accounted for by carbachol-induced down-regulation of receptors for CCK-8, bombesin, and carbachol. Although carbachol did not desensitize the enzyme secretory response to substance P, an effect of carbachol on substance P receptors was not examined. In the present study, in dispersed acini from guinea pig pancreas, substance P caused a twofold increase in amylase secretion. Stimulation was half-maximal at 0.7 nM and was maximal at 10 nM. Analysis of the ability of substance P to inhibit binding of 125I-substance P to substance P receptors indicated that acini possess a single class of receptors for substance P (Kd = 0.8 +/- 0.1 nM; Bmax = 1,037 +/- 145 fmol/mg of DNA). There was a close correlation between the relative potency with which substance P stimulated amylase secretion (0.7 nM) and the potency for inhibiting binding of 125I-substance P (Kd = 0.8 nM). First incubating pancreatic acini with carbachol did not alter either substance P-stimulated enzyme secretion or binding of 125I-substance P to substance P receptors, whereas in the same experiments, carbachol reduced binding of 125I-CCK-8 to cholecystokinin receptors by 50% and decreased in CCK-8-stimulated enzyme secretion by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed Central

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-01-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways. PMID:9276720

Bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by protein kinase C, and is enhanced by disruption of rho/cytoskeletal pathways.

PubMed

Seensalu, R; Avedian, D; Barbuti, R; Song, M; Slice, L; Walsh, J H

1997-09-01

Isolated canine G cells in primary culture have been used to study calcium, protein kinase C (PKC), and rho/cytoskeletal-dependent intracellular pathways involved in bombesin- stimulated gastrin release. A method to obtain highly purified G cells by culture (64% G cells) after flow cytometry on elutriated fractions of cells from digested canine gastric antral mucosa has been developed. Pretreatment of G cells with thapsigargin (10(-8)-10(-6) M) and release experiments in Ca2+-containing or -depleted media showed that influx of Ca2+ into the cells and not acute release from intracellular stores plays an important role in bombesin-stimulated gastrin release. Inhibition of PKC by the specific inhibitor GF 109 203X did not affect bombesin-stimulated release. Rho, a small GTP-binding protein that regulates the actin cytoskeleton, is specifically antagonized by Clostridium botulinum C3 exoenzyme. C3 (10 microg/ml) enhanced basal and bombesin-stimulated gastrin release by 315 and 266%, respectively. The importance of the cytoskeleton for regulation of gastrin release was emphasized by a more pronounced release of gastrin when the organization of the actin cytoskeleton was disrupted by cytochalasin D (5 x 10(-)7 and 10(-)6 M). Wortmannin, a potent inhibitor of phosphoinositide-3-kinase, did not alter bombesin-stimulated gastrin release. Thus, it is concluded that bombesin-induced gastrin release from canine G cells is stimulated by Ca2+ but not by PKC, and is enhanced by disruption of rho/cytoskeletal pathways.

Intradermal endothelin-1 excites bombesin-responsive superficial dorsal horn neurons in the mouse

PubMed Central

Akiyama, T.; Nagamine, M.; Davoodi, A.; Iodi Carstens, M.; Cevikbas, F.; Steinhoff, M.

2015-01-01

Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 μg/μl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 μg·ml−1·min−1), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans. PMID:26311187

A Subset of Mouse Colonic Goblet Cells Expresses the Bitter Taste Receptor Tas2r131

PubMed Central

Prandi, Simone; Bromke, Marta; Hübner, Sandra; Voigt, Anja; Boehm, Ulrich; Meyerhof, Wolfgang; Behrens, Maik

2013-01-01

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics. PMID:24367558

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

PubMed

Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam

2018-06-06

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.

Targeting Epidermal Growth Factor Receptor-Related Signaling Pathways in Pancreatic Cancer.

PubMed

Philip, Philip A; Lutz, Manfred P

2015-10-01

Pancreatic cancer is aggressive, chemoresistant, and characterized by complex and poorly understood molecular biology. The epidermal growth factor receptor (EGFR) pathway is frequently activated in pancreatic cancer; therefore, it is a rational target for new treatments. However, the EGFR tyrosine kinase inhibitor erlotinib is currently the only targeted therapy to demonstrate a very modest survival benefit when added to gemcitabine in the treatment of patients with advanced pancreatic cancer. There is no molecular biomarker to predict the outcome of erlotinib treatment, although rash may be predictive of improved survival; EGFR expression does not predict the biologic activity of anti-EGFR drugs in pancreatic cancer, and no EGFR mutations are identified as enabling the selection of patients likely to benefit from treatment. Here, we review clinical studies of EGFR-targeted therapies in combination with conventional cytotoxic regimens or multitargeted strategies in advanced pancreatic cancer, as well as research directed at molecules downstream of EGFR as alternatives or adjuncts to receptor targeting. Limitations of preclinical models, patient selection, and trial design, as well as the complex mechanisms underlying resistance to EGFR-targeted agents, are discussed. Future clinical trials must incorporate translational research end points to aid patient selection and circumvent resistance to EGFR inhibitors.

The Nicotinic Acetylcholine Receptors of the Parasitic Nematode Ascaris suum: Formation of Two Distinct Drug Targets by Varying the Relative Expression Levels of Two Subunits

PubMed Central

Williamson, Sally M.; Robertson, Alan P.; Brown, Laurence; Williams, Tracey; Woods, Debra J.; Martin, Richard J.; Sattelle, David B.; Wolstenholme, Adrian J.

2009-01-01

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite

CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria

PubMed Central

Beck, Christina M.; Willett, Julia L. E.; Kim, Jeff J.; Low, David A.; Hayes, Christopher S.

2016-01-01

Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI. PMID:27723824

Efficient Receptor Mediated siRNA Delivery in Vitro by Folic Acid Targeted Pentablock Copolymer-Based Micelleplexes.

PubMed

Lehner, Roman; Liu, Kegang; Wang, Xueya; Hunziker, Patrick

2017-08-14

Novel, biocompatible polyplexes, based on the combination of cationic pentablock copolymers with folic acid functionalized copolymers, were designed and developed for target-specific siRNA delivery. The resulting micelleplexes spontaneously formed polymeric micelles with a hydrophobic core surrounded directly by a cationic poly-2-(4-aminobutyl)-oxazole (PABOXA) and subsequently shielded by hydrophilic poly-2-methyl-oxazole (PMOXA) layer. The described micelleplexes form highly stable particles even in complete serum after 24 h compared with the highly cationic polymer PEI, which show aggregate formation in serum containing buffer solution. Targeted siRNA delivery and gene knockdown could be shown using green fluorescent protein (GFP) expressing HeLa cells, resulting in ∼31% and ∼8% suppression of the expression of GFP for targeted and nontargeted micelleplexes, respectively. Comparison studies of folic-receptor positive HeLa cells with normal folic-receptor-negative HEK293 cells revealed involvement of receptor mediated cellular uptake of fluorescently labeled siRNA. The new designed nanocarrier showed no cytotoxicity, having a potential application. The presented concept of shielding a nucleic-acid complexing cationic chains with a stealth layer and combining it with receptor ligand overcomes typical problems with undesired protein and cell interactions in delivery of nucleic acids using polymeric systems, opening new doors for application if RNA inhibition in the organism.

Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney.

PubMed

Yang, T; Michele, D E; Park, J; Smart, A M; Lin, Z; Brosius, F C; Schnermann, J B; Briggs, J P

1999-12-01

The discovery that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is a ligand for the gamma-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARalpha mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARgamma was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARbeta, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRalpha was localized to PCT and IMCD, whereas RXRbeta was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2 significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARalpha being involved in energy metabolism through regulating ACS in PCT and with PPARgamma being involved in modulating RMIC growth and differentiation.

Targeting breast to brain metastatic tumours with death receptor ligand expressing therapeutic stem cells

PubMed Central

Bagci-Onder, Tugba; Du, Wanlu; Figueiredo, Jose-Luiz; Martinez-Quintanilla, Jordi

2015-01-01

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study, we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging, we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next, we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain, we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective, potent and secretable variant of a TRAIL, S-TRAIL, and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore, the incorporation of pro-drug converting enzyme, herpes simplex virus thymidine kinase, into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. PMID:25910782

Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells

PubMed Central

Berger, Carolina; Sommermeyer, Daniel; Hudecek, Michael; Berger, Michael; Balakrishnan, Ashwini; Paszkiewicz, Paulina J.; Kosasih, Paula L.; Rader, Christoph; Riddell, Stanley R.

2014-01-01

Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) since it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors, and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1-expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates. PMID:25355068

Emerging strategies for EphA2 receptor targeting for cancer therapeutics

PubMed Central

Tandon, Manish; Vemula, Sai Vikram; Mittal, Suresh K.

2010-01-01

Importance of the field High mortality rates with cancers warrant further development of earlier diagnostics and better treatment strategies. Membrane-bound hepatocellular receptor tyrosine kinase class A2 (EphA2) is overexpressed in breast, prostate, urinary bladder, skin, lung, ovary and brain cancers. Areas covered in this review This review describes EphA2 overexpression in cancers, its signaling mechanisms and strategies to target its deregulation. What will the reader will gain High EphA2 expression in cancer cells is correlated to a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., EphrinA1) triggers events that are deregulated and implicated in carcinogenesis. Both EphrinA1-independent oncogenic activity and EphrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches. Take home message EphA2 has been tested as a drug target using multiple approaches such as agonist antibodies, RNA interference, immunotherapy, virus vectors-mediated gene transfer, small molecule inhibitors and nanoparticles. With over a decade of research, encouraging results with successful targeting of EphA2 expression in various pre-clinical cancer models necessitate further studies. PMID:21142802

Targeting xenobiotic receptors PXR and CAR in human diseases

PubMed Central

Banerjee, Monimoy; Robbins, Delira; Chen, Taosheng

2014-01-01

Nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenobiotic receptors regulating not only drug metabolism and disposition but also various human diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver diseases, suggesting that PXR and CAR are promising targets for drug discovery. Consequently, there is an urgent need to discover and develop small molecules that target these PXR- and/or CAR-mediated human-disease-related pathways for relevant therapeutic applications. This review proposes approaches to target PXR and CAR, either individually or simultaneously, in the context of various human diseases, taking into consideration the structural differences between PXR and CAR. PMID:25463033

Evaluation of somatostatin, CXCR4 chemokine and endothelin A receptor expression in a large set of paragangliomas.

PubMed

Kaemmerer, Daniel; Sänger, Jörg; Arsenic, Ruza; D'Haese, Jan G; Neumann, Jens; Schmitt-Graeff, Annette; Wirtz, Ralph Markus; Schulz, Stefan; Lupp, Amelie

2017-10-27

Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed. In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression. SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors. In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy.

Evaluation of somatostatin, CXCR4 chemokine and endothelin A receptor expression in a large set of paragangliomas

PubMed Central

Kaemmerer, Daniel; Sänger, Jörg; Arsenic, Ruza; D’Haese, Jan G.; Neumann, Jens; Schmitt-Graeff, Annette; Wirtz, Ralph Markus; Schulz, Stefan; Lupp, Amelie

2017-01-01

Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed. In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression. SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors. In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy. PMID:29163802

Targeting Discoidin Domain Receptors in Prostate Cancer

DTIC Science & Technology

2016-08-01

DDRs), a set of kinase receptors that signal in response to collagen . The project’s goal is to define the expression and therapeutic potential of...antibody, which blocks receptor activation by collagen I. Mice were inoculated with PC3 cells and anti-DDR1 or control antibody treatment. The study... collagen , the major organic component of the bone extracellular matrix. Purpose: To investigate the expression, therapeutic potential, and

Brain-Targeted (Pro)Renin Receptor Knockdown attenuates Angiotensin II-Dependent Hypertension

PubMed Central

Li, Wencheng; Peng, Hua; Cao, Theresa; Sato, Ryosuke; McDaniels, Sarah. J.; Kobori, Hiroyuki; Navar, L. Gabriel; Feng, Yumei

2012-01-01

The (pro)renin receptor is a newly discovered member of the brain renin-angiotensin system. To investigate the role of brain (pro)renin receptor in hypertension, adeno-associated virus-mediated (pro)renin receptor shRNA was used to knockdown (pro)renin receptor expression in the brain of non-transgenic normotensive and human renin-angiotensinogen double transgenic hypertensive mice. Blood pressure was monitored using implanted telemetric probes in conscious animals. Real-time PCR and immunostaining were performed to determine (pro)renin receptor, angiotensin II type 1 receptor and vasopressin mRNA levels. Plasma vasopressin levels were determined by Enzyme-Linked Immuno Sorbent Assay. Double transgenic mice exhibited higher blood pressure, elevated cardiac and vascular sympathetic tone, and impaired spontaneous baroreflex sensitivity. Intracerebroventricular delivery of (pro)renin receptor shRNA significantly reduced blood pressure, cardiac and vasomotor sympathetic tone, and improved baroreflex sensitivity compared to the control virus treatment in double transgenic mice. (Pro)renin receptor knockdown significantly reduced angiotensin II type 1 receptor and vasopressin levels in double transgenic mice. These data indicate that (pro)renin receptor knockdown in the brain attenuates angiotensin II-dependent hypertension and is associated with a decrease insympathetic tone and an improvement of the baroreflex sensitivity. In addition, brain-targeted (pro)renin receptor knockdown is associated with down-regulation of angiotensin II type 1 receptor and vasopressin levels. We conclude that central (pro)renin receptor contributes to the pathogenesis of hypertension in human renin-angiotensinogen transgenic mice. PMID:22526255

Targeting the androgen receptor in triple-negative breast cancer: current perspectives.

PubMed

Mina, Alain; Yoder, Rachel; Sharma, Priyanka

2017-01-01

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with frequent recurrence and metastasis. Unlike hormone receptor-positive subtypes, treatment of TNBC is currently limited by the lack of clinically available targeted therapies. Androgen signaling is necessary for normal breast development, and its dysregulation has been implicated in breast tumorigenesis. In recent years, gene expression studies have identified a subset of TNBC that is enriched for androgen receptor (AR) signaling. Interference with androgen signaling in TNBC is promising, and AR-inhibiting drugs have shown antitumorigenic activity in preclinical and proof of concept clinical studies. Recent advances in our understanding of androgenic signaling in TNBC, along with the identification of interacting pathways, are allowing development of the next generation of clinical trials with AR inhibitors. As novel AR-targeting agents are developed and evaluated in clinical trials, it is equally important to establish a robust set of biomarkers for identification of TNBC tumors that are most likely to respond to AR inhibition.

Neuromedin B and gastrin releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong renilla GFP in NPY neurons

PubMed Central

van den Pol, Anthony N.; Yao, Yang; Fu, Li-Ying; Foo, Kylie; Huang, Hao; Coppari, Roberto; Lowell, Brad; Broberger, Christian

2009-01-01

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright renilla GFP in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single cell RT-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin releasing peptide and neuromedin B which are found in axons in the arcuate nucleus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni2+, inclusion of BAPTA in the pipette, KB-R7943 and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around − 20 mV. Together, these data suggest two mechanisms, activation of non-selective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis. PMID:19357287

P2X receptors as targets for the treatment of status epilepticus

PubMed Central

Henshall, David C.; Diaz-Hernandez, Miguel; Miras-Portugal, M. Teresa; Engel, Tobias

2013-01-01

Prolonged seizures are amongst the most common neurological emergencies. Status epilepticus is a state of continuous seizures that is life-threatening and prompt termination of status epilepticus is critical to protect the brain from permanent damage. Frontline treatment comprises parenteral administration of anticonvulsants such as lorazepam that facilitate γ-amino butyric acid (GABA) transmission. Because status epilepticus can become refractory to anticonvulsants in a significant proportion of patients, drugs which act on different neurotransmitter systems may represent potential adjunctive treatments. P2X receptors are a class of ligand-gated ion channel activated by ATP that contributes to neuro- and glio-transmission. P2X receptors are expressed by both neurons and glia in various brain regions, including the hippocampus. Electrophysiology, pharmacology and genetic studies suggest certain P2X receptors are activated during pathologic brain activity. Expression of several members of the family including P2X2, P2X4, and P2X7 receptors has been reported to be altered in the hippocampus following status epilepticus. Recent studies have shown that ligands of the P2X7 receptor can have potent effects on seizure severity during status epilepticus and mice lacking this receptor display altered seizures in response to chemoconvulsants. Antagonists of the P2X7 receptor also modulate neuronal death, microglial responses and neuroinflammatory signaling. Recent work also found altered neuronal injury and inflammation after status epilepticus in mice lacking the P2X4 receptor. In summary, members of the P2X receptor family may serve important roles in the pathophysiology of status epilepticus and represent novel targets for seizure control and neuroprotection. PMID:24324404

P2X receptors as targets for the treatment of status epilepticus.

PubMed

Henshall, David C; Diaz-Hernandez, Miguel; Miras-Portugal, M Teresa; Engel, Tobias

2013-11-26

Prolonged seizures are amongst the most common neurological emergencies. Status epilepticus is a state of continuous seizures that is life-threatening and prompt termination of status epilepticus is critical to protect the brain from permanent damage. Frontline treatment comprises parenteral administration of anticonvulsants such as lorazepam that facilitate γ-amino butyric acid (GABA) transmission. Because status epilepticus can become refractory to anticonvulsants in a significant proportion of patients, drugs which act on different neurotransmitter systems may represent potential adjunctive treatments. P2X receptors are a class of ligand-gated ion channel activated by ATP that contributes to neuro- and glio-transmission. P2X receptors are expressed by both neurons and glia in various brain regions, including the hippocampus. Electrophysiology, pharmacology and genetic studies suggest certain P2X receptors are activated during pathologic brain activity. Expression of several members of the family including P2X2, P2X4, and P2X7 receptors has been reported to be altered in the hippocampus following status epilepticus. Recent studies have shown that ligands of the P2X7 receptor can have potent effects on seizure severity during status epilepticus and mice lacking this receptor display altered seizures in response to chemoconvulsants. Antagonists of the P2X7 receptor also modulate neuronal death, microglial responses and neuroinflammatory signaling. Recent work also found altered neuronal injury and inflammation after status epilepticus in mice lacking the P2X4 receptor. In summary, members of the P2X receptor family may serve important roles in the pathophysiology of status epilepticus and represent novel targets for seizure control and neuroprotection.

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Targeting C-type lectin receptors: a high-carbohydrate diet for dendritic cells to improve cancer vaccines

PubMed Central

van Dinther, Dieke; Stolk, Dorian A.; van de Ven, Rieneke; van Kooyk, Yvette; de Gruijl, Tanja D.; den Haan, Joke M. M.

2017-01-01

There is a growing understanding of why certain patients do or do not respond to checkpoint inhibition therapy. This opens new opportunities to reconsider and redevelop vaccine strategies to prime an anticancer immune response. Combination of such vaccines with checkpoint inhibitors will both provide the fuel and release the brake for an efficient anticancer response. Here, we discuss vaccine strategies that use C-type lectin receptor (CLR) targeting of APCs, such as dendritic cells and macrophages. APCs are a necessity for the priming of antigen-specific cytotoxic and helper T cells. Because CLRs are natural carbohydrate-recognition receptors highly expressed by multiple subsets of APCs and involved in uptake and processing of Ags for presentation, these receptors seem particularly interesting for targeting purposes. PMID:28729358

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells.

PubMed

Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A

2018-05-01

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Regulation of glucose transport by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts: Involvement of protein kinase C-dependent and -independent mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dettori, C.; Meldolesi, J.

1989-05-01

Glucose transport stimulation by insulin, bombesin, and bradykinin in Swiss 3T3 fibroblasts was compared with the phosphoinositide hydrolysis effects of the same stimulants in a variety of experimental paradigms known to affect generation and/or functioning of intracellular second messengers: short- and long-term treatments with phorbol dibutyrate, that cause activation and down-regulation of protein kinase C, respectively; cell loading with high (quin2), that causes clamping of (Ca{sup 2+}){sub i} near the resting level; poisoning with pertussis toxin, that affects the GTP binding proteins of the Go/Gi class; treatment with Ca{sup 2+} ionophores. ({sup 14}C) glucose transport stimulation by maximal (insulin) wasmore » affected by neither pertussis toxin nor protein kinase C down-regulation. This result correlates with the lack of effect of insulin on phosphoinositide hydrolysis. In contrast, part of the glucose transport responses induced by bombesin and bradykinin appeared to be mediated by protein kinase C in proportion with the stimulation induced by these peptides on the phosphoinositide hydrolysis. The protein kinase C-independent portion of the response to bradykinin was found to be inhibitable by pertussis toxin. This latter result might suggest an interaction between the bradykinin receptor and a glucose transporter, mediated by a protein of the Go/Gi class.« less

Host target modification as a strategy to counter pathogen hijacking of the jasmonate hormone receptor

DOE PAGES

Zhang, Li; Yao, Jian; Withers, John; ...

2015-11-02

In the past decade, characterization of the host targets of pathogen virulence factors took a center stage in the study of pathogenesis and disease susceptibility in plants and humans. However, the impressive knowledge of host targets has not been broadly exploited to inhibit pathogen infection. In this paper, we show that host target modification could be a promising new approach to “protect” the disease-vulnerable components of plants. In particular, recent studies have identified the plant hormone jasmonate (JA) receptor as one of the common targets of virulence factors from highly evolved biotrophic/hemibiotrophic pathogens. Strains of the bacterial pathogen Pseudomonas syringae,more » for example, produce proteinaceous effectors, as well as a JA-mimicking toxin, coronatine (COR), to activate JA signaling as a mechanism to promote disease susceptibility. Guided by the crystal structure of the JA receptor and evolutionary clues, we succeeded in modifying the JA receptor to allow for sufficient endogenous JA signaling but greatly reduced sensitivity to COR. Transgenic Arabidopsis expressing this modified receptor not only are fertile and maintain a high level of insect defense, but also gain the ability to resist COR-producing pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. Finally, our results provide a proof-of-concept demonstration that host target modification can be a promising new approach to prevent the virulence action of highly evolved pathogens.« less

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin.

PubMed

Burns, D M; Walker, B; Gray, J; Nelson, J

1999-01-01

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP(18-27), and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml(-1)). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth.

Expression and Functional Pathway Analysis of Nuclear Receptor NR2F2 in Ovarian Cancer

PubMed Central

Hawkins, Shannon M.; Loomans, Holli A.; Wan, Ying-Wooi; Ghosh-Choudhury, Triparna; Coffey, Donna; Xiao, Weimin; Liu, Zhandong; Sangi-Haghpeykar, Haleh

2013-01-01

Context: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers. Objective: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action. Design, Setting, and Patients: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers. Results: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer. Conclusions: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue. PMID:23690307

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

PubMed

Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

1992-12-01

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

Targeted anticancer therapy: overexpressed receptors and nanotechnology.

PubMed

Akhtar, Mohd Javed; Ahamed, Maqusood; Alhadlaq, Hisham A; Alrokayan, Salman A; Kumar, Sudhir

2014-09-25

Targeted delivery of anticancer drugs to cancer cells and tissues is a promising field due to its potential to spare unaffected cells and tissues, but it has been a major challenge to achieve success in these therapeutic approaches. Several innovative approaches to targeted drug delivery have been devised based on available knowledge in cancer biology and on technological advancements. To achieve the desired selectivity of drug delivery, nanotechnology has enabled researchers to design nanoparticles (NPs) to incorporate anticancer drugs and act as nanocarriers. Recently, many receptor molecules known to be overexpressed in cancer have been explored as docking sites for the targeting of anticancer drugs. In principle, anticancer drugs can be concentrated specifically in cancer cells and tissues by conjugating drug-containing nanocarriers with ligands against these receptors. Several mechanisms can be employed to induce triggered drug release in response to either endogenous trigger or exogenous trigger so that the anticancer drug is only released upon reaching and preferentially accumulating in the tumor tissue. This review focuses on overexpressed receptors exploited in targeting drugs to cancerous tissues and the tumor microenvironment. We briefly evaluate the structure and function of these receptor molecules, emphasizing the elegant mechanisms by which certain characteristics of cancer can be exploited in cancer treatment. After this discussion of receptors, we review their respective ligands and then the anticancer drugs delivered by nanotechnology in preclinical models of cancer. Ligand-functionalized nanocarriers have delivered significantly higher amounts of anticancer drugs in many in vitro and in vivo models of cancer compared to cancer models lacking such receptors or drug carrying nanocarriers devoid of ligand. This increased concentration of anticancer drug in the tumor site enabled by nanotechnology could have a major impact on the efficiency of cancer

Hypothyroidism Affects D2 Receptor-mediated Breathing without altering D2 Receptor Expression

PubMed Central

Schlenker, Evelyn H.; Rio, Rodrigo Del; Schultz, Harold D.

2015-01-01

Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age- matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. PMID:24434437

Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

PubMed

Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

2014-03-01

Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. Copyright © 2014 Elsevier B.V. All rights reserved.

A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors And Controls Bacterial Pathogenicity

PubMed Central

Swem, Lee R.; Swem, Danielle L.; O’Loughlin, Colleen T.; Gatmaitan, Raleene; Zhao, Bixiao; Ulrich, Scott M.; Bassler, Bonnie L.

2009-01-01

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development. PMID:19647512

Different expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in mouse tumor cells.

PubMed

Okabe, Kyoko; Hayashi, Mai; Wakabayashi, Naoko; Yamawaki, Yasuna; Teranishi, Miki; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2010-01-01

Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention. Copyright © 2011 S. Karger AG, Basel.

Selective targeting of a TNFR decoy receptor pharmaceutical to the primate brain as a receptor-specific IgG fusion protein.

PubMed

Boado, Ruben J; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Zhou, Qing-Hui; Pardridge, William M

2010-03-01

Decoy receptors, such as the human tumor necrosis factor receptor (TNFR), are potential new therapies for brain disorders. However, decoy receptors are large molecule drugs that are not transported across the blood-brain barrier (BBB). To enable BBB transport of a TNFR decoy receptor, the human TNFR-II extracellular domain was re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the TNFR therapeutic decoy receptor across the BBB. The HIRMAb-TNFR fusion protein was expressed in stably transfected CHO cells, and was analyzed with electrophoresis, Western blotting, size exclusion chromatography, and binding assays for the HIR and TNFalpha. The HIRMAb-TNFR fusion protein was radio-labeled by trititation, in parallel with the radio-iodination of recombinant TNFR:Fc fusion protein, and the proteins were co-injected in the adult Rhesus monkey. The TNFR:Fc fusion protein did not cross the primate BBB in vivo, but the uptake of the HIRMAb-TNFR fusion protein was high and 3% of the injected dose was taken up by the primate brain. The TNFR was selectively targeted to brain, relative to peripheral organs, following fusion to the HIRMAb. This study demonstrates that decoy receptors may be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse that selectively targets the brain, and enables penetration of the BBB in vivo. IgG-decoy receptor fusion proteins represent a new class of human neurotherapeutics. Copyright 2010 Elsevier B.V. All rights reserved.

TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

PubMed Central

Linger, Rachel M. A.; Keating, Amy K.; Earp, H. Shelton; Graham, Douglas K.

2011-01-01

Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs) characterized by a conserved sequence within the kinase domain and adhesion molecule-like extracellular domains. This small family of RTKs regulates an intriguing mix of processes, including cell proliferation/survival, cell adhesion and migration, blood clot stabilization, and regulation of inflammatory cytokine release. Genetic or experimental alteration of TAM receptor function can contribute to a number of disease states, including coagulopathy, autoimmune disease, retinitis pigmentosa, and cancer. In this chapter, we first provide a comprehensive review of the structure, regulation, biologic functions, and down-stream signaling pathways of these receptors. In addition, we discuss recent evidence which suggests a role for TAM receptors in oncogenic mechanisms as family members are over-expressed in a spectrum of human cancers and have prognostic significance in some. Possible strategies for targeted inhibition of the TAM family in the treatment of human cancer are described. Further research will be necessary to evaluate the full clinical implications of TAM family expression and activation in cancer. PMID:18620092

Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

PubMed Central

Yoon, J. Cliff; Chickering, Troy W.; Rosen, Evan D.; Dussault, Barry; Qin, Yubin; Soukas, Alexander; Friedman, Jeffrey M.; Holmes, William E.; Spiegelman, Bruce M.

2000-01-01

The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis. PMID:10866690

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

PubMed Central

Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

2017-01-01

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

Expression of CB2 cannabinoid receptor in Pichia pastoris.

PubMed

Feng, Wenke; Cai, Jian; Pierce, William M; Song, Zhao-Hui

2002-12-01

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.

Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons.

PubMed

van den Pol, Anthony N; Yao, Yang; Fu, Li-Ying; Foo, Kylie; Huang, Hao; Coppari, Roberto; Lowell, Bradford B; Broberger, Christian

2009-04-08

Neuropeptide Y (NPY) is one of the most widespread neuropeptides in the brain. Transgenic mice were generated that expressed bright Renilla green fluorescent protein (GFP) in most or all of the known NPY cells in the brain, which otherwise were not identifiable. GFP expression in NPY cells was confirmed with immunocytochemistry and single-cell reverse transcription-PCR. NPY neurons in the hypothalamic arcuate nucleus play an important role in energy homeostasis and endocrine control. Whole-cell patch clamp recording was used to study identified arcuate NPY cells. Primary agents that regulate energy balance include melanocortin receptor agonists, AgRP, and cannabinoids; none of these substances substantially influenced electrical properties of NPY neurons. In striking contrast, neuropeptides of the bombesin family, including gastrin-releasing peptide and neuromedin B, which are found in axons in the mediobasal hypothalamus and may also be released from the gut to signal the brain, showed strong direct excitatory actions at nanomolar levels on the NPY neurons, stronger than the actions of ghrelin and hypocretin/orexin. Bombesin-related peptides reduced input resistance and depolarized the membrane potential. The depolarization was attenuated by several factors: substitution of choline for sodium, extracellular Ni(2+), inclusion of BAPTA in the pipette, KB-R7943, and SKF96365. Reduced extracellular calcium enhanced the current, which reversed around -20 mV. Together, these data suggest two mechanisms, activation of nonselective cation channels and the sodium/calcium exchanger. Since both NPY and POMC neurons, which we also studied, are similarly directly excited by bombesin-like peptides, the peptides may function to initiate broad activation, rather than the cell-type selective activation or inhibition reported for many other compounds that modulate energy homeostasis.

Glucocorticoid and calcitonin receptor expression in central giant cell lesions: implications for therapy.

PubMed

Nogueira, R L M; Faria, M H G; Osterne, R L V; Cavalcante, R B; Ribeiro, R A; Rabenhorst, S H B

2012-08-01

Central giant cell lesion is an uncommon benign jaw lesion, with uncertain aetiology, and variable clinical behaviour. Studies of molecular markers may help to understand the nature and behaviour of this lesion, and eventually may represent a target for pharmacological approaches to treatment. The aim of this study was to analyse the expression of glucocorticoid and calcitonin receptors in central giant cell lesions before and after treatment with intralesional steroid. Paraffin-embedded blocks from patients who underwent treatment with intralesional triamcinolone hexacetonide injections were stained immunohistochemically. Biological material from patients who underwent a surgical procedure after treatment were tested immunohistochemically. 18 cases (9 aggressive and 9 non-aggressive) were included. The difference in calcitonin receptor expression was not statistically significant between the aggressive and non-aggressive lesions and between the patients with a good response and those with a moderate/negative response to treatment. Glucocorticoid receptor expression in the multinucleated giant cells was higher in patients with a good response. It can be postulated that immunohistochemical staining for glucocorticoid receptors may provide a tool for selecting the therapeutic strategy. An H-score greater than 48 for glucocorticoid receptors in multinucleated giant cells predicted a good response in this study. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Distribution of cellular HSV-1 receptor expression in human brain.

PubMed

Lathe, Richard; Haas, Juergen G

2017-06-01

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

Expression of early growth response factor-1 in rats with cerulein-induced acute pancreatitis and its significance

PubMed Central

Gong, Lan-Bo; He, Li; Liu, Yang; Chen, Xue-Qing; Jiang, Bo

2005-01-01

AIM: To observe the expressions of early growth response factor-1 (Egr-1) and tissue factor (TF) in rats with cerulein-induced acute pancreatitis and to explore its significance. METHODS: A large dose of cerulein was used to create the experimental acute pancreatitis model in rats. The changes of Egr-1 mRNA and protein in rats were observed during 30 min to 4 h after the treatment and immunohistochemical method was used to observe the localized expression of Egr-1 in tissues. In addition to the mRNA expression of Egr-1 target gene, TF was also observed. A blank control group, and a bombesin-administered group were used for comparison. RESULTS: After the stimulation of a large dose of cerulein, the rats showed typical inflammatory changes of acute pancreatitis. Thirty minutes after the stimulation, the mRNA expression of Egr-1 in the pancreatic tissue reached its peak and then declined, while the expression of Egr-1 protein reached its peak 2 h after the stimulation. Histologically, 2 h after the stimulation, almost all pancreatic acinar cells had the expression of Egr-1 protein, which was focused in the nuclei. The mRNA expression of TF occurred 1 h after the stimulation and gradually increased within 4 h. However, a large dose of bombesin only stimulated the pancreatic tissue to produce a little mRNA expression of Egr-1 and no mRNA expression of Egr-1 protein and TF. CONCLUSION: Egr-1 as a pro-inflammatory transcription factor may play an important role in the pathogenesis of acute pancreatitis by modulating the expression of TF. PMID:16124058

A Tupaia paramyxovirus vector system for targeting and transgene expression.

PubMed

Engeland, Christine E; Bossow, Sascha; Hudacek, Andrew W; Hoyler, Birgit; Förster, Judith; Veinalde, Rūta; Jäger, Dirk; Cattaneo, Roberto; Ungerechts, Guy; Springfeld, Christoph

2017-09-01

Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

Expression of anaesthetic and analgesic drug target genes in excised breast tumour tissue: Association with clinical disease recurrence or metastasis.

PubMed

Connolly, C; Madden, S F; Buggy, D J; Gallagher, H C

2017-01-01

Retrospective analyses suggest anaesthetic-analgesics technique during cancer surgery may affect recurrence/metastasis. This could involve direct effects of anaesthetic-analgesic drugs on cancer cells. While μ-opioid receptor over-expression in lung tumours is associated with greater metastasis, other anaesthetic-analgesic receptor targets in cancer recurrence/metastasis remain unexplored. Therefore, we evaluated the association between genetic expression of anaesthetic-analgesic receptor targets and recurrence/metastasis, using a repository of breast cancer gene expression and matching clinical data. A list of 23 genes encoding for the most prominent anaesthetic-analgesic receptor targets was compiled. This was processed through BreastMark- an algorithm integrating gene expression data from ~17,000 samples and clinical data from >4,500 breast cancer samples. Gene expression data was dichotomized using disease-free survival (survival without recurrence) and distant disease-free survival (survival without metastasis) as end points. Hazard ratios were calculated by Cox-regression analysis. Enrichment for prognostic markers was determined by randomly choosing 23-member gene lists from all available genes, calculating how often >5 significant markers were observed and adjusting p-values for multiple testing. This was repeated 10,000 times and an empirical p-value calculated. Of 23 selected genes, 9 were significantly associated with altered rates of metastasis and 4 with recurrence on univariate analysis. Adjusting for multiple testing, 5 of these 9 genes remained significantly associated with metastasis, non with recurrence. This ratio of genes (5/23) was not significantly enriched for markers of metastasis (p = 0.07). Several anaesthetic-analgesic receptor genes were associated with metastatic spread in breast cancer. Overall there was no significant enrichment in prognostic markers of metastasis, although a trend was observed.

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108].

PubMed

Szepeshazi, Karoly; Schally, Andrew V; Keller, Gunhild; Block, Norman L; Benten, Daniel; Halmos, Gabor; Szalontay, Luca; Vidaurre, Irving; Jaszberenyi, Miklos; Rick, Ferenc G

2012-07-01

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle.

PubMed

Mizuta, Kentaro; Zhang, Yi; Xu, Dingbang; Mizuta, Fumiko; D'Ovidio, Frank; Masaki, Eiji; Emala, Charles W

2013-09-02

stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576. These results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.

Increased expression of Toll-like receptors (TLRs) 7 and 9 and other cytokines in systemic lupus erythematosus (SLE) patients: ethnic differences and potential new targets for therapeutic drugs.

PubMed

Lyn-Cook, Beverly D; Xie, Chenghui; Oates, Jarren; Treadwell, Edward; Word, Beverly; Hammons, George; Wiley, Kenneth

2014-09-01

, IFN-γ and TNF-α in lupus patients compared to non-lupus patients. African American women with lupus had significantly higher serum levels of IL-6 and TNF-α. African American women with lupus demonstrated increased levels of specific pro-inflammatory cytokines and Toll-like receptors when compared to EA women. Increased expression in these lupus patients provides an opportunity for targeting with antagonist as a new therapy for systemic lupus erythematous. Published by Elsevier Ltd.

Enhanced Chemokine Receptor Expression on Leukocytes of Patients with Alzheimer's Disease.

PubMed

Goldeck, David; Larbi, Anis; Pellicanó, Mariavaleria; Alam, Iftikhar; Zerr, Inga; Schmidt, Christian; Fulop, Tamas; Pawelec, Graham

2013-01-01

Although primarily a neurological complaint, systemic inflammation is present in Alzheimer's Disease, with higher than normal levels of proinflammatory cytokines and chemokines in the periphery as well as the brain. A gradient of these factors may enhance recruitment of activated immune cells into the brain via chemotaxis. Here, we investigated the phenotypes of circulating immune cells in AD patients with multi-colour flow cytometry to determine whether their expression of chemokine receptors is consistent with this hypothesis. In this study, we confirmed our previously reported data on the shift of early- to late-differentiated CD4+ T-cells in AD patients. The percentage of cells expressing CD25, a marker of acute T-cell activation, was higher in patients than in age-matched controls, and percentages of CCR6+ cells were elevated. This chemokine receptor is primarily expressed on pro-inflammatory memory cells and Th17 cells. The proportion of cells expressing CCR4 (expressed on Th2 cells) and CCR5 (Th1 cells and dendritic cells) was also greater in patients, and was more pronounced on CD4+ than CD8+ T-cells. These findings allow a more detailed insight into the systemic immune status of patients with Alzheimer's disease and suggest possible novel targets for immune therapy.

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule.

PubMed

Dobosz, Michael; Haupt, Ute; Scheuer, Werner

2017-01-01

Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic "gold standard" for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1-3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of

Enhancing radiosensitization in EphB4 receptor-expressing Head and Neck Squamous Cell Carcinomas

PubMed Central

Bhatia, Shilpa; Hirsch, Kellen; Sharma, Jaspreet; Oweida, Ayman; Griego, Anastacia; Keysar, Stephen; Jimeno, Antonio; Raben, David; Krasnoperov, Valery; Gill, Parkash S.; Pasquale, Elena B.; Wang, Xiao-Jing; Karam, Sana D.

2016-01-01

Members of the Eph family of receptor tyrosine kinases have been implicated in a wide array of human cancers. The EphB4 receptor is ubiquitously expressed in head and neck squamous cell carcinoma (HNSCC) and has been shown to impart tumorigenic and invasive characteristics to these cancers. In this study, we investigated whether EphB4 receptor targeting can enhance the radiosensitization of HNSCC. Our data show that EphB4 is expressed at high to moderate levels in HNSCC cell lines and patient-derived xenograft (PDX) tumors. We observed decreased survival fractions in HNSCC cells following EphB4 knockdown in clonogenic assays. An enhanced G2 cell cycle arrest with activation of DNA damage response pathway and increased apoptosis was evident in HNSCC cells following combined EphB4 downregulation and radiation compared to EphB4 knockdown and radiation alone. Data using HNSCC PDX models showed significant reduction in tumor volume and enhanced delay in tumor regrowth following sEphB4-HSA administration with radiation compared to single agent treatment. sEphB4-HSA is a protein known to block the interaction between the EphB4 receptor and its ephrin-B2 ligand. Overall, our findings emphasize the therapeutic relevance of EphB4 targeting as a radiosensitizer that can be exploited for the treatment of human head and neck carcinomas. PMID:27941840

ck2-dependent phosphorylation of progesterone receptors (PR) on Ser81 regulates PR-B isoform-specific target gene expression in breast cancer cells.

PubMed

Hagan, Christy R; Regan, Tarah M; Dressing, Gwen E; Lange, Carol A

2011-06-01

Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G(1)/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments.

From Belly to Brain: Targeting the Ghrelin Receptor in Appetite and Food Intake Regulation.

PubMed

Howick, Ken; Griffin, Brendan T; Cryan, John F; Schellekens, Harriët

2017-01-27

Ghrelin is the only known peripherally-derived orexigenic hormone, increasing appetite and subsequent food intake. The ghrelinergic system has therefore received considerable attention as a therapeutic target to reduce appetite in obesity as well as to stimulate food intake in conditions of anorexia, malnutrition and cachexia. As the therapeutic potential of targeting this hormone becomes clearer, it is apparent that its pleiotropic actions span both the central nervous system and peripheral organs. Despite a wealth of research, a therapeutic compound specifically targeting the ghrelin system for appetite modulation remains elusive although some promising effects on metabolic function are emerging. This is due to many factors, ranging from the complexity of the ghrelin receptor (Growth Hormone Secretagogue Receptor, GHSR-1a) internalisation and heterodimerization, to biased ligand interactions and compensatory neuroendocrine outputs. Not least is the ubiquitous expression of the GHSR-1a, which makes it impossible to modulate centrallymediated appetite regulation without encroaching on the various peripheral functions attributable to ghrelin. It is becoming clear that ghrelin's central signalling is critical for its effects on appetite, body weight regulation and incentive salience of food. Improving the ability of ghrelin ligands to penetrate the blood brain barrier would enhance central delivery to GHSR-1a expressing brain regions, particularly within the mesolimbic reward circuitry.

Suppression of transient receptor potential melastatin 4 expression promotes conversion of endothelial cells into fibroblasts via transforming growth factor/activin receptor-like kinase 5 pathway.

PubMed

Echeverría, Cesar; Montorfano, Ignacio; Cabello-Verrugio, Claudio; Armisén, Ricardo; Varela, Diego; Simon, Felipe

2015-05-01

To study whether transient receptor potential melastatin 4 (TRPM4) participates in endothelial fibrosis and to investigate the underlying mechanism. Primary human endothelial cells were used and pharmacological and short interfering RNA-based approaches were used to test the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) pathway participation and contribution of TRPM7 ion channel. Suppression of TRPM4 expression leads to decreased endothelial protein expression and increased expression of fibrotic and extracellular matrix markers. Furthermore, TRPM4 downregulation increases intracellular Ca levels as a potential condition for fibrosis. The underlying mechanism of endothelial fibrosis shows that inhibition of TRPM4 expression induces TGF-β1 and TGF-β2 expression, which act through their receptor, ALK5, and the nuclear translocation of the profibrotic transcription factor smad4. TRPM4 acts to maintain endothelial features and its loss promotes fibrotic conversion via TGF-β production. The regulation of TRPM4 levels could be a target for preserving endothelial function during inflammatory diseases.

P2X7 Receptor as a Therapeutic Target.

PubMed

De Marchi, Elena; Orioli, Elisa; Dal Ben, Diego; Adinolfi, Elena

2016-01-01

P2X7 receptor is an ATP-gated cation channel that upon agonist interaction leads to cellular influx of Na(+) and Ca(2+) and efflux of K(+). P2X7 is expressed by a wide variety of cells and its activation mediates a large number of biological processes like inflammation, neuromodulation, cell death or cell proliferation and it has been associated to related pathological conditions including infectious, inflammatory, autoimmune, neurological, and musculoskeletal disorders and, in the last years, to cancer. This chapter describes structural features of P2X7, chemical properties of its agonist, antagonist, and allosteric modulators and summarizes recent advances on P2X7 receptor as therapeutic target in the aforementioned diseases. We also give an overview on recent literature suggesting that P2X7 single-nucleotide polymorphisms could be exploited as diagnostic biomarkers for the development of tailored therapies. © 2016 Elsevier Inc. All rights reserved.

Expression of plasma membrane receptor genes during megakaryocyte development

PubMed Central

Sun, Sijie; Wang, Wenjing; Latchman, Yvette; Gao, Dayong; Aronow, Bruce

2013-01-01

Megakaryocyte (MK) development is critically informed by plasma membrane-localized receptors that integrate a multiplicity of environmental cues. Given that the current understanding about receptors and ligands involved in megakaryocytopoiesis is based on single targets, we performed a genome-wide search to identify a plasma membrane receptome for developing MKs. We identified 40 transmembrane receptor genes as being upregulated during MK development. Seven of the 40 receptor-associated genes were selected to validate the dataset. These genes included: interleukin-9 receptor (IL9R), transforming growth factor, β receptor II (TGFBR2), interleukin-4 receptor (IL4R), colony stimulating factor-2 receptor-beta (CSFR2B), adiponectin receptor (ADIPOR2), thrombin receptor (F2R), and interleukin-21 receptor (IL21R). RNA and protein analyses confirmed their expression in primary human MKs. Matched ligands to IL9R, TGFBR2, IL4R, CSFR2B, and ADIPOR2 affected megakaryocytopoiesis. IL9 was unique in its ability to increase the number of MKs formed. In contrast, MK colony formation was inhibited by adiponectin, TGF-β, IL4, and GM-CSF. The thrombin-F2R axis affected platelet function, but not MK development, while IL21 had no apparent detectable effects. ADP-induced platelet aggregation was suppressed by IL9, TGF-β, IL4, and adiponectin. Overall, six of seven of the plasma membrane receptors were confirmed to have functional roles in MK and platelet biology. Also, results show for the first time that adiponectin plays a regulatory role in MK development. Together these data support a strong likelihood that the 40 transmembrane genes identified as being upregulated during MK development will be an important resource to the research community for deciphering the complex repertoire of environmental cues regulating megakaryocytopoiesis and/or platelet function. PMID:23321270

Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

PubMed Central

Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

2009-01-01

Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

Neurotrophin Receptors TrkA, p75NTR, and Sortilin Are Increased and Targetable in Thyroid Cancer.

PubMed

Faulkner, Sam; Jobling, Philip; Rowe, Christopher W; Rodrigues Oliveira, S M; Roselli, Severine; Thorne, Rick F; Oldmeadow, Christopher; Attia, John; Jiang, Chen Chen; Zhang, Xu Dong; Walker, Marjorie M; Hondermarck, Hubert

2018-01-01

Neurotrophin receptors are emerging targets in oncology, but their clinicopathologic significance in thyroid cancer is unclear. In this study, the neurotrophin tyrosine receptor kinase TrkA (also called NTRK1), the common neurotrophin receptor p75 NTR , and the proneurotrophin receptor sortilin were analyzed with immunohistochemistry in a cohort of thyroid cancers (n = 128) and compared with adenomas and normal thyroid tissues (n = 62). TrkA was detected in 20% of thyroid cancers, compared with none of the benign samples (P = 0.0007). TrkA expression was independent of histologic subtypes but associated with lymph node metastasis (P = 0.0148), suggesting the involvement of TrkA in tumor invasiveness. Nerves in the tumor microenvironment were positive for TrkA. p75 NTR was overexpressed in anaplastic thyroid cancers compared with papillary and follicular subtypes (P < 0.0001). Sortilin was overexpressed in thyroid cancers compared with benign thyroid tissues (P < 0.0001). Neurotrophin receptor expression was confirmed in a panel of thyroid cancer cell lines at the mRNA and protein levels. Functional investigations using the anaplastic thyroid cancer cell line CAL-62 found that siRNA against TrkA, p75 NTR , and sortilin decreased cell survival and cell migration through decreased SRC and ERK activation. Together, these data reveal TrkA, p75 NTR , and sortilin as potential therapeutic targets in thyroid cancer. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

PubMed

Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

2012-06-01

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

PIM kinase inhibition presents a novel targeted therapy against triple-negative breast tumors with elevated MYC expression

PubMed Central

Horiuchi, Dai; Camarda, Roman; Zhou, Alicia Y.; Yau, Christina; Momcilovic, Olga; Balakrishnan, Sanjeev; Corella, Alexandra N.; Eyob, Henok; Kessenbrock, Kai; Lawson, Devon A.; Marsh, Lindsey A.; Anderton, Brittany N.; Rohrberg, Julia; Kunder, Ratika; Bazarov, Alexey V.; Yaswen, Paul; McManus, Michael T.; Rugo, Hope S.; Werb, Zena; Goga, Andrei

2017-01-01

Triple-negative breast cancer (TNBC), which lacks the expression of the estrogen, progesterone, and HER2 receptors, represents the breast cancer subtype with the poorest outcome1. No targeted therapy is available against this subtype due to lack of validated molecular targets. We previously reported that MYC signaling is disproportionally elevated in triple-negative (TN) tumors compared to receptor-positive (RP) tumors2. MYC is an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes3. Direct inhibition of oncogenic MYC transcriptional activity has remained challenging4,5. The present study conducted an shRNA screen against all kinases to uncover novel MYC-dependent synthetic lethal combinations, and identified PIM1, a non-essential kinase. Here we demonstrate that PIM1 expression was elevated in TN tumors and was associated with poor prognosis in patients with hormone and HER2 receptor-negative tumors. Small molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic breast cancer models by inhibiting oncogenic transcriptional activity of MYC while simultaneously restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that exhibit elevated MYC expression. PMID:27775705

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin

PubMed Central

Burns, D M; Walker, B; Gray, J; Nelson, J

1999-01-01

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP18–27, and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml−;1). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth. © 1999 Cancer Research Campaign PMID:9888460

Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.

PubMed

Kanagawa, Naoko; Yanagawa, Tatsuya; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku

2010-03-26

Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Calcium-Sensing Receptor Tumor Expression and Lethal Prostate Cancer Progression.

PubMed

Ahearn, Thomas U; Tchrakian, Nairi; Wilson, Kathryn M; Lis, Rosina; Nuttall, Elizabeth; Sesso, Howard D; Loda, Massimo; Giovannucci, Edward; Mucci, Lorelei A; Finn, Stephen; Shui, Irene M

2016-06-01

Prostate cancer metastases preferentially target bone, and the calcium-sensing receptor (CaSR) may play a role in promoting this metastatic progression. We evaluated the association of prostate tumor CaSR expression with lethal prostate cancer. A validated CaSR immunohistochemistry assay was performed on tumor tissue microarrays. Vitamin D receptor (VDR) expression and phosphatase and tensin homolog tumor status were previously assessed in a subset of cases by immunohistochemistry. Cox proportional hazards models adjusting for age and body mass index at diagnosis, Gleason grade, and pathological tumor node metastasis stage were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association of CaSR expression with lethal prostate cancer. The investigation was conducted in the Health Professionals Follow-up Study and Physicians' Health Study. We studied 1241 incident prostate cancer cases diagnosed between 1983 and 2009. Participants were followed up or cancer-specific mortality or development of metastatic disease. On average, men were followed up 13.6 years, during which there were 83 lethal events. High CaSR expression was associated with lethal prostate cancer independent of clinical and pathological variables (HR 2.0; 95% CI 1.2-3.3). Additionally, there was evidence of effect modification by VDR expression; CaSR was associated with lethal progression among men with low tumor VDR expression (HR 3.2; 95% CI 1.4-7.3) but not in cases with high tumor VDR expression (HR 0.8; 95% CI 0.2-3.0). Tumor CaSR expression is associated with an increased risk of lethal prostate cancer, particularly in tumors with low VDR expression. These results support further investigating the mechanism linking CaSR with metastases.

Selective estrogen receptor modulators and betulinic acid act synergistically to target ERα and SP1 transcription factor dependent Pygopus expression in breast cancer.

PubMed

Tzenov, Youlian R; Andrews, Phillip; Voisey, Kim; Gai, Luis; Carter, Beverley; Whelan, Kathryn; Popadiuk, Catherine; Kao, Kenneth R

2016-06-01

Estrogen and progesterone hormone receptor (ER and PR) expression in invasive breast cancer predicts response to hormone disruptive therapy. Pygopus2 (hPYGO2) encodes a chromatin remodelling protein important for breast cancer growth and cell cycle progression. The aims of this study were to determine the mechanism of expression of hPYGO2 in breast cancer and to examine how this expression is affected therapeutically. hPYGO2 and ER protein expression was examined in a breast tumour microarray by immunohistochemistry. hPYGO2 RNA and protein expression was examined in ER+ and ER- breast cancer cell lines in the presence of selective estrogen hormone receptor modulator drugs and the specificity protein-1 (SP1) inhibitor, betulinic acid (BA). The effects of these drugs on the ability for ER and SP1 to bind the hPYGO2 promoter and affect cell cycle progression were studied using chromatin immunoprecipitation assays. hPYGO2 was expressed in seven of eight lines and in nuclei of 98% of 65 breast tumours, including 3 Ductal carcinoma in situ and 62 invasive specimens representing ER-negative (22%) and ER-positive (78%) cases. Treatment with either 4-Hydroxytamoxifen (OHT) or fulvestrant reduced hPYGO2 mRNA 10-fold and protein 5-10-fold within 4 h. Promoter analysis indicated an ER/SP1 binding site at nt -225 to -531 of hPYGO2. SP1 RNA interference and BA reduced hPYGO2 protein and RNA expression by fivefold in both ER- and ER+ cells. Further attenuation was achieved by combining BA and 4-OHT resulting in eightfold reduction in cell growth. Our findings reveal a mechanistic link between hormone signalling and the growth transcriptional programme. The activation of its expression by ERα and/or SP1 suggests hPYGO2 as a theranostic target for hormone therapy responsive and refractory breast cancer. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Laminar shear stress regulates endothelial kinin B1 receptor expression and function: potential implication in atherogenesis

PubMed Central

Duchene, Johan; Cayla, Cécile; Vessillier, Sandrine; Scotland, Ramona; Yamashiro, Kazuo; Lecomte, Florence; Syed, Irfan; Vo, Phuong; Marrelli, Alessandra; Pitzalis, Costantino; Cipollone, Francesco; Schanstra, Joost; Bascands, Jean-Loup; Hobbs, Adrian J; Perretti, Mauro; Ahluwalia, Amrita

2009-01-01

OBJECTIVE The pro-inflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many pro-inflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. METHODS AND RESULTS Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE-/- mice fed a high fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE-/- mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVEC) exposed to low LSS (0-2dyn/cm2) compared to physiological LSS (6-10dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFκB activation. CONCLUSION Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whilst physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma are associated with substantial upregulation, identifying this receptor as a potential therapeutic target. CONDENSED ABSTRACT Low laminar shear stress (LSS) underlies the pro-inflammatory processes in atherogenesis. Herein, we demonstrate that whilst physiological LSS represses inflammatory kinin B1 receptor (B1R) expression/function, low atherogenic LSS is associated with profound upregulation of both in atherosclerosis in both humans and animal

Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: A patch clamp analysis in gene-targeted mice

PubMed Central

Grosmaitre, Xavier; Vassalli, Anne; Mombaerts, Peter; Shepherd, Gordon M.; Ma, Minghong

2006-01-01

A glomerulus in the mammalian olfactory bulb receives axonal inputs from olfactory sensory neurons (OSNs) that express the same odorant receptor (OR). Glomeruli are generally thought to represent functional units of olfactory coding, but there are no data on the electrophysiological properties of OSNs that express the same endogenous OR. Here, using patch clamp recordings in an intact epithelial preparation, we directly measured the transduction currents and receptor potentials from the dendritic knobs of mouse OSNs that express the odorant receptor MOR23 along with the green fluorescent protein. All of the 53 cells examined responded to lyral, a known ligand for MOR23. There were profound differences in response kinetics, particularly in the deactivation phase. The cells were very sensitive to lyral, with some cells responding to as little as 10 nM. The dynamic range was unexpectedly broad, with threshold and saturation in individual cells often covering three log units of lyral concentration. The potential causes and biological significance of this cellular heterogeneity are discussed. Patch clamp recording from OSNs that express a defined OR provides a powerful approach to investigate the sensory inputs to individual glomeruli. PMID:16446455

Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.

PubMed

Grosmaitre, Xavier; Vassalli, Anne; Mombaerts, Peter; Shepherd, Gordon M; Ma, Minghong

2006-02-07

A glomerulus in the mammalian olfactory bulb receives axonal inputs from olfactory sensory neurons (OSNs) that express the same odorant receptor (OR). Glomeruli are generally thought to represent functional units of olfactory coding, but there are no data on the electrophysiological properties of OSNs that express the same endogenous OR. Here, using patch clamp recordings in an intact epithelial preparation, we directly measured the transduction currents and receptor potentials from the dendritic knobs of mouse OSNs that express the odorant receptor MOR23 along with the green fluorescent protein. All of the 53 cells examined responded to lyral, a known ligand for MOR23. There were profound differences in response kinetics, particularly in the deactivation phase. The cells were very sensitive to lyral, with some cells responding to as little as 10 nM. The dynamic range was unexpectedly broad, with threshold and saturation in individual cells often covering three log units of lyral concentration. The potential causes and biological significance of this cellular heterogeneity are discussed. Patch clamp recording from OSNs that express a defined OR provides a powerful approach to investigate the sensory inputs to individual glomeruli.

Transferrin receptors and the targeted delivery of therapeutic agents against cancer

PubMed Central

Daniels, Tracy R.; Bernabeu, Ezequiel; Rodríguez, José A.; Patel, Shabnum; Kozman, Maggie; Chiappetta, Diego A.; Holler, Eggehard; Ljubimova, Julia Y.; Helguera, Gustavo; Penichet, Manuel L.

2012-01-01

Background Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death. Scope of review In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses. Major conclusions Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo. General significance The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. PMID:21851850

Synthesis and Characterization of Tricarbonyl-Re/Tc(I) Chelate Probes Targeting the G Protein-Coupled Estrogen Receptor GPER/GPR30

PubMed Central

Burai, Ritwik; Ramesh, Chinnasamy; Nayak, Tapan K.; Dennis, Megan K.; Bryant, Bj K.; Prossnitz, Eric R.; Arterburn, Jeffrey B.

2012-01-01

The discovery of the G protein-coupled estrogen receptor GPER (also GPR30) and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer. PMID:23077529

Transient receptor potential ankyrin receptor 1 is a novel target for pro-tussive agents.

PubMed

Andrè, E; Gatti, R; Trevisani, M; Preti, D; Baraldi, P G; Patacchini, R; Geppetti, P

2009-11-01

The transient receptor potential ankyrin receptor 1 (TRPA1) is a cation channel, co-expressed with the pro-tussive transient receptor potential vanilloid type 1 (TRPV1) channel in primary sensory neurons. TRPA1 is activated by a series of irritant exogenous and endogenous alpha,beta-unsaturated aldehydes which seem to play a role in airway diseases. We investigated whether TRPA1 agonists provoke cough in guinea pigs and whether TRPA1 antagonists inhibit this response. Animals were placed in a Perspex box, and cough sounds were recorded and counted by observers unaware of the treatment used. Inhalation of two selective TRPA1 agonists, allyl isothiocyanate and cinnamaldehyde, dose-dependently caused cough in control guinea pigs, but not in those with airway sensory nerves desensitized by capsaicin. Coughs elicited by TRPA1 agonists were reduced by non-selective (camphor and gentamicin) and selective (HC-030031) TRPA1 antagonists, whereas they were unaffected by the TRPV1 antagonist, capsazepine. Acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes recently identified as TRPA1 stimulants and contained in cigarette smoke, air pollution or produced endogenously by oxidative stress, caused a remarkable tussive effect, a response that was selectively inhibited by HC-030031. Part of the cough response induced by cigarette smoke inhalation was inhibited by HC-030031, suggesting the involvement of TRPA1. A novel pro-tussive pathway involves the TRPA1 channel, expressed by capsaicin-sensitive airway sensory nerves and is activated by a series of exogenous (cigarette smoke) and endogenous irritants. These results suggest TRPA1 may be a novel target for anti-tussive medicines.

Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons

PubMed Central

Disney, Anita A.; Aoki, Chiye

2010-01-01

Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit. PMID:18265004

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

PubMed

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-02-15

To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

The gastrin/cholecystokinin-B receptor on prostate cells--a novel target for bifunctional prostate cancer imaging.

PubMed

Sturzu, Alexander; Klose, Uwe; Sheikh, Sumbla; Echner, Hartmut; Kalbacher, Hubert; Deeg, Martin; Nägele, Thomas; Schwentner, Christian; Ernemann, Ulrike; Heckl, Stefan

2014-02-14

The means of identifying prostate carcinoma and its metastases are limited. The contrast agents used in magnetic resonance imaging clinical diagnostics are not taken up into the tumor cells, but only accumulate in the interstitial space of the highly vasculated tumor. We examined the gastrin/cholecystokinin-B receptor as a possible target for prostate-specific detection using the C-terminal seven amino acid sequence of the gastrin peptide hormone. The correct sequence and a scrambled control sequence were coupled to the fluorescent dye rhodamine and the magnetic resonance imaging contrast agent gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Expression analysis of the gastrin receptor mRNA was performed by reverse transcriptase polymerase chain reaction on PC3 prostate carcinoma cells, U373 glioma, U2OS osteosarcoma and Colo205 colon carcinoma cells. After having confirmed elevated expression of gastrin receptor in PC3 cells and very low expression of the receptor in Colo205 cells, these two cell lines were used to create tumor xenografts on nude mice for in vivo experiments. Confocal lasers scanning microscopy and magnetic resonance imaging showed a high specificity of the correct conjugate for the PC3 xenografts. Staining of the PC3 xenografts was much weaker with the scrambled conjugate while the Colo205 xenografts showed no marked staining with any of the conjugates. In vitro experiments comparing the correct and scrambled conjugates on PC3 cells by magnetic resonance relaxometry and fluorescence-activated cell sorting confirmed markedly higher specificity of the correct conjugate. The investigations show that the gastrin receptor is a promising tumor cell surface target for future prostate-cancer-specific imaging applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

PubMed

Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

2016-06-01

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

Spatial pattern of receptor expression in the olfactory epithelium.

PubMed Central

Nef, P; Hermans-Borgmeyer, I; Artières-Pin, H; Beasley, L; Dionne, V E; Heinemann, S F

1992-01-01

A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system. Images PMID:1384038

P2X and P2Y receptors as possible targets of therapeutic manipulations in CNS illnesses.

PubMed

Köles, Laszlo; Furst, Susanna; Illes, Peter

2005-03-01

Adenine and/or uridine nucleotide-sensitive receptors are classified into two types belonging to the ligand-gated ionotropic family (P2X) and the metabotropic, G-protein-coupled family (P2Y). In humans, seven different P2X receptors (P2X(1-7)) and eight different P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11-14)) have been detected hitherto. All P2 receptors are expressed in the CNS, with the preferential expression of the P2X(2), P2X(4), P2X(6) and P2Y(1) receptors in neurons. In addition to the neurotransmitter and modulator functions, neurite outgrowth, proliferation of glial cells and the expression of transmitter receptors at target cells have also been suggested to be regulated by extracellular nucleotides in the nervous system. In spite of the expanding knowledge in the purinergic research field, the present therapeutic utilization of P2 receptor ligands is mostly related to peripheral diseases such as thromboembolic disorders and cystic fibrosis. In this review we provide some evidence that P2 receptors play an important role in the regulation of CNS functions related to hippocampal activity, the mesolimbic dopaminergic system and the nociceptive system. The role of purinergic receptors located on astrocytes/microglia and implications of these receptors for neurodegenerative/neuroinflammatory disorders, CNS injury and epilepsy will be highlighted as well. (c) 2005 Prous Science. All rights reserved.

Targeting Nuclear Receptors with Marine Natural Products

PubMed Central

Yang, Chunyan; Li, Qianrong; Li, Yong

2014-01-01

Nuclear receptors (NRs) are important pharmaceutical targets because they are key regulators of many metabolic and inflammatory diseases, including diabetes, dyslipidemia, cirrhosis, and fibrosis. As ligands play a pivotal role in modulating nuclear receptor activity, the discovery of novel ligands for nuclear receptors represents an interesting and promising therapeutic approach. The search for novel NR agonists and antagonists with enhanced selectivities prompted the exploration of the extraordinary chemical diversity associated with natural products. Recent studies involving nuclear receptors have disclosed a number of natural products as nuclear receptor ligands, serving to re-emphasize the translational possibilities of natural products in drug discovery. In this review, the natural ligands of nuclear receptors will be described with an emphasis on their mechanisms of action and their therapeutic potentials, as well as on strategies to determine potential marine natural products as nuclear receptor modulators. PMID:24473166

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

PubMed Central

2013-01-01

dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576. Conclusions These results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM. PMID:24004608

Peroxisome Proliferator Activated Receptors Alpha, Beta, and Gamma mRNA and protein expression in human fetal tissues

EPA Science Inventory

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study examine...

Activation of farnesoid X receptor induces RECK expression in mouse liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Xiaomin; Wu, Weibin; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032

2014-01-03

Highlights: •RECK is a novel transcriptional target gene of FXR in mouse liver. •The FXR response element is located within the intron 1 of RECK gene. •FXR agonist reverses the down-regulation of RECK in the liver in mouse NASH model. -- Abstract: Farnesoid X receptor (FXR) belongs to the ligand-activated nuclear receptor superfamily, and functions as a transcription factor regulating the transcription of numerous genes involved in bile acid homeostasis, lipoprotein and glucose metabolism. In the present study, we identified RECK, a membrane-anchored inhibitor of matrix metalloproteinases, as a novel target gene of FXR in mouse liver. We found thatmore » FXR agonist substantially augmented hepatic RECK mRNA and protein expression in vivo and in vitro. FXR regulated the transcription of RECK through directly binding to FXR response element located within intron 1 of the mouse RECK gene. Moreover, FXR agonist reversed the down-regulation of RECK in the livers from mice fed a methionine and choline deficient diet. In summary, our data suggest that RECK is a novel transcriptional target of FXR in mouse liver, and provide clues to better understanding the function of FXR in liver.« less

Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells.

PubMed

Park, Jae H; Brentjens, Renier J

2010-04-01

Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

ck2-Dependent Phosphorylation of Progesterone Receptors (PR) on Ser81 Regulates PR-B Isoform-Specific Target Gene Expression in Breast Cancer Cells ▿

PubMed Central

Hagan, Christy R.; Regan, Tarah M.; Dressing, Gwen E.; Lange, Carol A.

2011-01-01

Progesterone receptors (PR) are critical mediators of mammary gland development and contribute to breast cancer progression. Progestin-induced rapid activation of cytoplasmic protein kinases leads to selective regulation of growth-promoting genes by phospho-PR species. Herein, we show that phosphorylation of PR Ser81 is ck2 dependent and progestin regulated in intact cells but also occurs in the absence of PR ligands when cells enter the G1/S phase of the cell cycle. T47D breast cancer cells stably expressing a PR-B mutant receptor that cannot be phosphorylated at Ser79/81 (S79/81A) formed fewer soft agar colonies. Regulation of selected genes by PR-B, but not PR-A, also required Ser79/81 phosphorylation for basal and/or progestin-regulated (BIRC3, HSD11β2, and HbEGF) expression. Additionally, wild-type (wt) PR-B, but not S79/81A mutant PR, was robustly recruited to a progesterone response element (PRE)-containing transcriptional enhancer region of BIRC3; abundant ck2 also associated with this region in cells expressing wt but not S79/81A PR. We conclude that phospho-Ser81 PR provides a platform for ck2 recruitment and regulation of selected PR-B target genes. Understanding how ligand-independent PRs function in the context of high levels of kinase activities characteristic of breast cancer is critical to understanding the basis of tumor-specific changes in gene expression and will speed the development of highly selective treatments. PMID:21518957

Targeting striatal metabotropic glutamate receptor type 5 in Parkinson's disease: bridging molecular studies and clinical trials.

PubMed

Vallano, A; Fernandez-Duenas, V; Garcia-Negredo, G; Quijada, M A; Simon, C P; Cuffí, M L; Carbonell, L; Sanchez, S; Arnau, J M; Ciruela, F

2013-12-01

Metabotropic glutamate (mGlu) receptors are G protein-coupled receptors expressed primarily on neurons and glial cells modulating the effects of glutamatergic neurotransmission. The pharmacological manipulation of these receptors has been postulated to be valuable in the management of some neurological disorders. Accordingly, the targeting of mGlu5 receptors as a therapeutic approach for Parkinson's disease (PD) has been proposed, especially to manage the adverse symptoms associated to chronic treatment with classical PD drugs. Thus, the specific pharmacological blocking of mGlu5 receptors constitutes one of the most attractive non-dopaminergic-based strategies for PD management in general and for the L-DOPA-induced dyskinesia (LID) in particular. Overall, we provide here an update of the current state of the art of these mGlu5 receptor-based approaches that are under clinical study as agents devoted to alleviate PD symptoms.

Synthesis and characterization of iodinated tetrahydroquinolines targeting the G protein-coupled estrogen receptor GPR30.

PubMed

Ramesh, Chinnasamy; Nayak, Tapan K; Burai, Ritwik; Dennis, Megan K; Hathaway, Helen J; Sklar, Larry A; Prossnitz, Eric R; Arterburn, Jeffrey B

2010-02-11

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

EXPRESSION, PURIFICATION AND IN VITRO FUNCTIONAL RECONSTITUTION OF THE CHEMOKINE RECEPTOR CCR1

PubMed Central

Allen, Samantha J.; Ribeiro, Sofia; Horuk, Richard; Handel, Tracy M.

2009-01-01

Chemokine receptors are a specific class of G protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-β-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a Kd of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of ~ 0.1–0.2mgs of pure functional receptor per 109 cells will permit biophysical studies of this medically important receptor. PMID:19275940

From Belly to Brain: Targeting the Ghrelin Receptor in Appetite and Food Intake Regulation

PubMed Central

Howick, Ken; Griffin, Brendan T.; Cryan, John F.; Schellekens, Harriët

2017-01-01

Ghrelin is the only known peripherally-derived orexigenic hormone, increasing appetite and subsequent food intake. The ghrelinergic system has therefore received considerable attention as a therapeutic target to reduce appetite in obesity as well as to stimulate food intake in conditions of anorexia, malnutrition and cachexia. As the therapeutic potential of targeting this hormone becomes clearer, it is apparent that its pleiotropic actions span both the central nervous system and peripheral organs. Despite a wealth of research, a therapeutic compound specifically targeting the ghrelin system for appetite modulation remains elusive although some promising effects on metabolic function are emerging. This is due to many factors, ranging from the complexity of the ghrelin receptor (Growth Hormone Secretagogue Receptor, GHSR-1a) internalisation and heterodimerization, to biased ligand interactions and compensatory neuroendocrine outputs. Not least is the ubiquitous expression of the GHSR-1a, which makes it impossible to modulate centrally-mediated appetite regulation without encroaching on the various peripheral functions attributable to ghrelin. It is becoming clear that ghrelin’s central signalling is critical for its effects on appetite, body weight regulation and incentive salience of food. Improving the ability of ghrelin ligands to penetrate the blood brain barrier would enhance central delivery to GHSR-1a expressing brain regions, particularly within the mesolimbic reward circuitry. PMID:28134808

Prostanoid receptor EP2 as a therapeutic target.

PubMed

Ganesh, Thota

2014-06-12

Cycoloxygenase-2 (COX-2) induction is prevalent in a variety of (brain and peripheral) injury models where COX-2 levels correlate with disease progression. Thus, COX-2 has been widely explored for anti-inflammatory therapy with COX-2 inhibitors, which proved to be effective in reducing the pain and inflammation in patients with arthritis and menstrual cramps, but they have not provided any benefit to patients with chronic inflammatory neurodegenerative disease. Recently, two COX-2 drugs, rofecoxib and valdecoxib, were withdrawn from the United States market due to cardiovascular side effects. Thus, future anti-inflammatory therapy could be targeted through a specific prostanoid receptor downstream of COX-2. The PGE2 receptor EP2 is emerging as a pro-inflammatory target in a variety of CNS and peripheral diseases. Here we highlight the latest developments on the role of EP2 in diseases, mechanism of activation, and small molecule discovery targeted either to enhance or to block the function of this receptor.

Estrogen-related receptor alpha induces the expression of vascular endothelial growth factor in breast cancer cells

PubMed Central

Stein, Rebecca A.; Gaillard, Stéphanie; McDonnell, Donald P.

2009-01-01

Estrogen-related receptor alpha (ERRα) is an orphan member of the nuclear receptor family of transcription factors. In addition to its function as a metabolic regulator, ERRα has been implicated in the growth and progression of several malignancies. In the setting of breast cancer, not only is ERRα a putative negative prognostic factor, but we have recently found that knockdown of its expression retards tumor growth in a xenograft model of this disease. The specific aspects of ERRα function that are responsible for its actions in breast cancer, however, remain unclear. Using the coactivator PGC-1α as a protein ligand to regulate ERRα activity, we analyzed the effects of this receptor on gene expression in the ERα-positive MCF-7 cell line. This analysis led to the identification of a large number of potential ERRα target genes, many of which were subsequently validated in other breast cancer cell lines. Importantly, we demonstrate in this study that activation of ERRα in several different breast cancer cell lines leads to a significant increase in VEGF mRNA expression, an activity that translates into an increase in VEGF protein secretion. The induction of VEGF results from the interaction of ERRα with specific ERR-responsive elements within the VEGF promoter. These findings suggest that ERRα-dependent induction of VEGF may contribute to the overall negative phenotype observed in tumors in which ERRα is expressed and provide validation for its use as a therapeutic target in cancer. PMID:19429439

Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

PubMed

Schuster, M J; Wu, G Y; Walton, C M; Wu, C H

1999-01-01

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

Peroxisome Proliferator-Activated Receptor Alpha (PPARa), Beta (PPARI3), and Gamma (PPARy) Expression in Human Fetal Tissues.

EPA Science Inventory

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose homeostasis, are targets of pharmaceuticals, and are also activated by environmental contaminants. Almost nothing is known about expression of PPARs during human fetal development. This study uses qPCR...

Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

PubMed Central

Burch, R M; Connor, J R; Axelrod, J

1988-01-01

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

PubMed Central

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-01-01

AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

Immunolocalization of Leptin Receptor and mRNA Expression of Leptin and Estrogen Receptors as well as Caspases in the Chorioallantoic Membrane (CAM) of the Chicken Embryo.

PubMed

Grzegorzewska, Agnieszka K; Lis, Marcin W; Sechman, Andrzej

The chicken chorioallantoic membrane (CAM) is used as a model in tests of angiogenesis, the biocompatibility of materials as well as tumor invasive potential. To assess the properties of CAM tissue, the localization of leptin receptor in the CAM, and the mRNA expression of two leptin receptor isoforms, estrogen receptors (ERα and ERβ) and caspases (-1 and -3) in the CAM on embryonic days 12 (E12), 15 (E15) and 18 (E18) were investigated. The leptin receptor was immunolocalized in each structure of the CAM (chorionic epithelium, allantoic epithelium, mesodermal layer and the walls of blood vessels) and did not change among analyzed stages of embryonic development (E12, E15 and E18) and between sexes. Expression of mRNA of genes encoding leptin and estrogen receptors as well as caspases was detected in the CAM of female and male chicken embryos at all three analysed stages of development. The relative mRNA expression of the long form of leptin receptor exceeded that of its short isoform. The mRNA expression of ERβ was significantly higher than ERα as well as caspase-3 in comparison with caspase-1. There were no differences in mRNA expression of these genes between sexes and among analyzed developmental days. The results indicate that the CAM is a target tissue for leptin as well as for estrogens and that CAM development is partially regulated by caspase-1 and caspase-3 dependent cell death. These results should be taken into consideration in studies in which the CAM is used as an experimental model.

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

PubMed

Furness, John B; Cho, Hyun-Jung; Hunne, Billie; Hirayama, Haruko; Callaghan, Brid P; Lomax, Alan E; Brock, James A

2012-06-01

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with

Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes.

PubMed

Kruse, Rikke; Vienberg, Sara G; Vind, Birgitte F; Andersen, Birgitte; Højlund, Kurt

2017-10-01

Pharmacological doses of FGF21 improve glucose tolerance, lipid metabolism and energy expenditure in rodents. Induced expression and secretion of FGF21 from muscle may increase browning of white adipose tissue (WAT) in a myokine-like manner. Recent studies have reported that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. The effects of insulin during euglycaemic-hyperinsulinaemic clamps and 10 week endurance training on serum FGF21 were examined in individuals with type 2 diabetes and in glucose tolerant overweight/obese and lean individuals. Gene expression of FGF21, its receptors and target genes in muscle and WAT biopsies was evaluated by quantitative real-time PCR (qPCR). Insulin increased serum and muscle FGF21 independent of overweight/obesity or type 2 diabetes, and there were no effects associated with exercise training. The insulin-induced increases in serum FGF21 and muscle FGF21 expression correlated tightly (p < 0.001). In WAT, overweight/obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure adequate expression of most FGF21 target genes in WAT.

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo

PubMed Central

Lee, Justin B; Zhang, Kaixin; Tam, Yuen Yi C; Quick, Joslyn; Tam, Ying K; Lin, Paulo JC; Chen, Sam; Liu, Yan; Nair, Jayaprakash K; Zlatev, Ivan; Rajeev, Kallanthottathil G; Manoharan, Muthiah; Rennie, Paul S; Cullis, Pieter R

2016-01-01

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen–targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer. PMID:28131285

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

PubMed Central

Marini, Pietro; Cascio, Maria-Grazia; King, Angela; Pertwee, Roger G; Ross, Ruth A

2013-01-01

Background and Purpose Although cannabinoid CB2 receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2-transfected CHO cells. Experimental Approach We tested the ability of well-known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2-transfected CHO cell membranes. cAMP assays were also performed in hCB2-CHO cells. Key Results The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2-CHO cells, in both [35S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB2-CHO cells when tested in the [35S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB2-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB2-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues. Conclusion and Implications Our results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution. PMID:23711022

Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy.

PubMed

Rwibasira Rudinga, Gamariel; Khan, Ghulam Jilany; Kong, Yi

2018-02-14

Cardiovascular diseases (CVDs) are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs), including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR). Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

Lck is a relevant target in chronic lymphocytic leukaemia cells whose expression variance is unrelated to disease outcome.

PubMed

Till, Kathleen J; Allen, John C; Talab, Fatima; Lin, Ke; Allsup, David; Cawkwell, Lynn; Bentley, Alison; Ringshausen, Ingo; Duckworth, Andrew D; Pettitt, Andrew R; Kalakonda, Nagesh; Slupsky, Joseph R

2017-12-01

Pathogenesis of chronic lymphocytic leukaemia (CLL) is contingent upon antigen receptor (BCR) expressed by malignant cells of this disease. Studies on somatic hypermutation of the antigen binding region, receptor expression levels and signal capacity have all linked BCR on CLL cells to disease prognosis. Our previous work showed that the src-family kinase Lck is a targetable mediator of BCR signalling in CLL cells, and that variance in Lck expression associated with ability of BCR to induce signal upon engagement. This latter finding makes Lck similar to ZAP70, another T-cell kinase whose aberrant expression in CLL cells also associates with BCR signalling capacity, but also different because ZAP70 is not easily pharmacologically targetable. Here we describe a robust method of measuring Lck expression in CLL cells using flow cytometry. However, unlike ZAP70 whose expression in CLL cells predicts prognosis, we find Lck expression and disease outcome in CLL are unrelated despite observations that its inhibition produces effects that biologically resemble the egress phenotype taken on by CLL cells treated with idelalisib. Taken together, our findings provide insight into the pathobiology of CLL to suggest a more complex relationship between expression of molecules within the BCR signalling pathway and disease outcome.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

PubMed

Shoji, Mamoru; Sun, Aiming; Kisiel, Walter; Lu, Yang J; Shim, Hyunsuk; McCarey, Bernard E; Nichols, Christopher; Parker, Ernest T; Pohl, Jan; Mosley, Cara A; Alizadeh, Aaron R; Liotta, Dennis C; Snyder, James P

2008-04-01

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

IL-4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells

PubMed Central

Bauer, K.; Hadzijusufovic, E.; Cerny-Reiterer, S.; Hoermann, G.; Reifinger, M.; Pirker, A.; Valent, P.; Willmann, M.

2018-01-01

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this ‘comparative oncology’ study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI-1 and C2. Of all cytokines tested IL-4 was found to downregulate expression of CD30 in NI-1 and C2 cells. We also found that the CD30-targeting antibody-conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL-4-induced downregulation of CD30 interferes with brentuximab vedotin-effects. Indeed, pre-incubation of NI-1 cells with IL-4 decreased responsiveness towards brentuximab vedotin. To overcome IL-4-mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit-targeting drugs masitinib and PKC412 in inhibiting growth of NI-1 and C2 cells. In summary, CD30 is a new marker and IL-4-regulated target in neoplastic canine MC. PMID:27507155