bluuebunny/crossref_metadata_2025 huggingface dataset:
β2‐adrenergic receptor control of endosomal PTH receptor signaling via Gβγ
Authors: Jean‐Alphonse Frederic , Wehbi Vanessa , Chen Jingming , Taboas Juan , Xiao Kunhong , Vilardaga Jean‐Pierre
Published: November 2023

Cells express several G protein‐coupled receptors (GPCRs) at their surfaces transmitting simultaneous extracellular hormonal and chemical signals into cells. A comprehensive understanding of mechanisms underlying the integrated signaling response induced by distinct GPCRs is thus required. Here we found that the β2‐adrenergic receptor, which induces short cAMP response, prolongs nuclear cAMP and PKA activation by promoting endosomal cAMP production in PTH receptor signaling through the stimulatory action of Gβγ subunits on adenylate cyclase type 2.

Support or Funding Information

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDKK) of the US National Institutes of Health (NIH) under Award numbers R01 DK087688 and R01 DK102495, and K01AR062598, and the Costwold Foundation Fellowship Award.
Structural Basis for Regulation of GPR56 by its Alternatively Spliced Extracellular Domains
Authors: Arac Demet
Published: November 2023

Emerging evidence shows adhesion G‐protein‐coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates oligodendrocyte development. Accordingly, human GPR56 mutations cause myelination defects and reduced volume of white matter. Using biochemistry, protein engineering, structural biology and in vivo assays, we set out to identify the mechanisms underlying the physiological and pathophysiological functions of GPR56 in myelination. First, we engineered a small protein, termed monobody (Mb), which binds tightly and specifically to the ECR of GPR56. We solved the crystal structure of the ECR of GPR56 bound to this Mb at 2.5Å resolution, revealing for the first time the domain architecture and atomic structure of a complete aGPCR ECR. The structure reveals two domains in the ECR of GPR56: a previously unidentified domain at the N‐terminus and an unusually short, but autoproteolytically active GPCR‐Autoproteolysis INducing (GAIN) domain. Using in vitro and in vivo functional approaches, we observed the effects of several structure‐guided mutations and thereby identified a surface‐exposed conserved patch on the N‐terminal domain necessary to rescue CNS myelination in GPR56‐hypomorph zebrafish. Finally, we measured the effect of Mb binding on GPR56 signaling in vitro and observed a decrease in basal activity, classifying the Mb as an allosteric inverse‐agonist. Our results suggest that the ECR of GPR56 has an essential role in regulating GPR56 function in oligodendrocyte development and myelination in an intricate and multifaceted manner.

Support or Funding Information

NIGMS

Brain Research Foundation

Big Ideas Generator
Delta opioid receptor antagonism/inverse agonism and its effect on mu opioid receptor signaling in SH‐SY5Y cells
Authors: Griggs Nicholas , Mosberg Henry , Traynor John
Published: November 2023

Opioid analgesics are prescribed with success to combat chronic and severe pain, yet there are many clinical and public health problems associated with the use of these medications that include constipation, respiratory depression and physical dependence. Academic scientists to pharmaceutical companies are developing opioid drugs, G‐protein biased agonists and allosteric modulators to name a few, in an effort to identify safer and more effective analgesics. Previous studies have reported that the co‐administration of naltrindole (a delta opioid receptor antagonist) with morphine (mu opioid receptor agonist) in vivo maintains analgesia yet reduces the development of morphine tolerance and dependence. Following this observation, in collaboration with the Mosberg lab we have discovered and characterized several mu agonist/delta antagonist “bifunctional compounds” that lack tolerance and dependence liabilities in vivo. The goal of my studies is to use these bifunctional compounds, as well as selective mu and delta opioid receptor ligands, in an attempt to explain this phenomenon on a molecular level. The SH‐SY5Y human neuroblastoma cell line is an ideal system in which to assess mu and delta receptor cross‐talk because it endogenously expresses both receptor types. My first objective was to demonstrate mu opioid receptor tolerance at the level of the G protein (a decrease in GTPγ35S binding) using the selective agonist DAMGO. Compared to untreated cells, DAMGO (1μM, 6 hours) treated cells showed a 50% reduction in DAMGO‐mediated GTPγ35S binding and a slight decrease in basal GTPγ35S binding. I then found that the addition of the delta inverse agonist ICI 174,864 (1μM, 6 hours) prevented DAMGO‐mediated tolerance. To further study this response I used another delta antagonist TIPPΨ and three bifunctional compounds. Overall the results suggest that delta opioid receptor inhibition is advantageous for sustained mu opioid receptor function in a cellular model of opioid tolerance. Experiments are ongoing to decipher how delta blockade alters mu‐opioid receptor signaling.

Support or Funding Information

DA‐03910 and NIGMS‐GM007767
Aryl Hydrocarbon Receptor Activation Differentially Alters Cholera Toxin‐Specific IgA Levels in Feces and Serum
Authors: King Aspen , Gawronska Elleanour , Metten Madeline , DeKrey Gregory
Published: November 2023

Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some intestinal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) has been shown to suppress antigen‐specific systemic IgM and IgG responses, whereas the impact on mucosal IgA responses is less clear. The goal of this study was to determine if TCDD exposure could alter fecal levels of antigen‐specific IgA after oral immunization. Female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD (2.5, 10 or 40 μg/Kg) orally. On the following day, each animal was immunized with whole Cholera toxin (CT, 10 μg, po) with two subsequent immunizations being given at 10‐day intervals. Antibody levels were measured by ELISA in fecal extracts each week, and in serum at termination (week 4). We found that total serum IgA levels were significantly decreased (P < 0.05) in TCDD‐treated animals, relative to controls. In contrast, no change in total fecal IgA levels was observed in TCDD‐treated mice at any time point measured (weeks 1–4). A significant and dose‐dependent decrease in CT‐specific serum IgA, IgM and IgG1 was observed in TCDD‐treated animals (P ≤ 0.05). In feces, CT‐specific IgA levels were significantly decreased (P ≤ 0.05) in TCDD‐treated mice at week 2, but not at later time points (weeks 3 and 4). These results demonstrate that intestinal IgA responses can recover from AhR‐mediated suppression more rapidly than systemic responses.

Support or Funding Information

Supported by the University of Northern Colorado.
Calabash Clay (Nzu) Affects Pregnancy in Whistar Albino Rats
Authors: Aprioku Jonah Sydney , Ogwo‐ude Ezinne Margaret
Published: November 2023

Calabash clay (Nzu) is a natural product, locally prepared from clay, sand and other non‐nutritional components with or without salt. Nzu has been reported to contain heavy metals and is widely used as a traditional remedy for morning sickness during pregnancy and breastfeeding in most African countries, including Nigeria. The present study was carried out in female Whistar albino rats to explore its effect on pregnancy. Pregnant rats weighing 150–170 g were randomized into 3 groups (n=8 per group) and orally administered 0, 400 or 800 mg/kg body weight of Nzu. The treatments were started on the first day of pregnancy and continued till delivery. Rats were laprotomised after delivery under deep diethylether anaesthesia and the two horns of uteri were examined to determine the implantation sites and presence of resorption sites. Maternal body weight and number of pups delivered were recorded. Data obtained were analysed using students t Test where appropriate and expressed as mean±SEM. Animal experiments were conducted in accordance with our institutional ethical guidelines on animal research. There was maternal body weight gain over time in control rats (p < 0.0001;p = 0.0026), but in treated groups, body weight increased only between gestation day 0 (GD0) and GD10 (p = 0.0022;p = 0.0005) but not afterwards, p = 0.1479;p = 0.7497 (Fig. 1). Additionally, whereas, all control rats gave birth, only 6 (62.5 %) and 1 (12.5 %) of Nzu (400 and 800 mg/kg) treated rats gave birth. Besides, 2 (5 %) of the births that occurred in 400 mg/kg Nzu treated rats were still births (Table 1). Further, number of implantations in Nzu treated rats were lower (p = 0.0262; p = 0.0004) compared with control, as well as number of pups delivered, p = 0.0084; p < 0.0001 (Table 2). The number of pups delivered in Nzu treated rats were fewer than the number of implantations observed, whereas both were equal in control. The results demonstrate that maternal exposure to Nzu dose‐dependently affects pregnancy outcome by potentially inhibiting maternal weight gain, fetal growth, and causing resorption or loss of pregnancy.

Effect of Calabash clay (Nzu) on pregnancy outcome in Whistar albino rats

Dose (mg/kg)
No. of rats that gave birth
Total no. of implantations
Total no. of live pups delivered
No. of still births
No. of resorptions

Control
8
75
75
0
0

400
5
47
38
2
7

800
1
19
8
0
11

Calabash clay (Nzu) exposure during pregnancy reduces number of implantations and pups delivered in Whistar albino rats

Dose (mg/kg)
No. of implantations
No. of live pups

Control
9.38±0.46
9.38±0.46

400
5.88±1.59*
5.00±1.55**

800
2.38±1.56***
1.00±1.00***

P < 0.05,

P < 0.01,

P < 0.001, compared to control.

Values in mean±SEM, n = 8
In vivo and In Vitro Effects of Work Site Fracking Sand Dust (FSD) Inhalation on Rat Lung: Dose‐ and Time‐Response Evaluation
Authors: Fedan Jeffrey S. , Thompson Janet A. , Russ Kristen A. , Newcomer Dean , McKinney Walter , Cumpston Amy M. , Jackson Mark C. , Reynolds Jeffrey S.
Published: November 2023

A fine dust is generated when sand is manipulated during operations at drilling sites that utilize the hydraulic fracturing, or “fracking,” process, potentially putting workers at risk of inhalation. Previously we investigated the effects of inhalation of 10 and 30 mg/m3 FSD (mass median aerodynamic diameter,1.75 μm; count median aerodynamic diameter, 227nm) 1, 7 and 27 d after exposure (6 h/d for 4 d) and observed modest changes in airway reactivity (7 d; in terms of lung resistance and dynamic compliance) and inflammation (1 d). In the present investigation, additional animals were exposed to the same inhalation conditions to further study its effects on aspects of lung function. Pulmonary function was examined in anesthetized rats using the Flexivent system (single compartment and constant phase models). There were no changes in respiratory system resistance (Rrs), respiratory elastance (Ers), tissue damping (G), tissue elastance (H), Newtonian resistance (Rn) or hysteresivity (η) after any post‐exposure period in rats exposed to either dose of FSD. To investigate whether reactivity of airway smooth muscle or the modulatory effect of epithelium on airway reactivity was affected by FSD inhalation, methacholine (MCh) was applied to the intraluminalor extraluminal baths of the isolated, perfused trachea preparation (IPT). FSD inhalation had no effect on reactivity(EC50 values) or contractile responses to MCh at any FSD dose or post‐exposure time. The possible effects of FSD inhalation on epithelial ion transport was investigated in vitro using Ussing chambers. Neither dose of FSD affected transepithelial potential difference (Vt) or resistance (Rt), or short‐circuit current (Isc) at any time point. Whereas there was no effect of FSD on responses to apical amiloride (Na+ channel blocker), apical NPPB(Cl− channel blocker) or basolateral ouabain (Na+,K+‐pump inhibitor) at the 10 mg/m3 dose, responses to amiloride were significantly inhibited at all post‐exposure times in tracheas from rats exposed to 30 mg/m3 FSD, suggesting that epithelial Na+ transport was impaired by FSD. The results indicate that FSD is generally without appreciable biological effects on pulmonary function broadly speaking, either in vivo or in vitro; however, the dust does interfere with Na+ transport at the 30 mg/m3 dose in a manner that is sustained for at least one month. Such a change could result in hydration of the airway surface liquid.

Support or Funding Information

NIOSH
Differential gut microbiota modulates the susceptibility to acetaminophen‐induced hepatotoxicity
Authors: Cho Sung‐joon , Chlipala George , Green Stefan , Lee Hyunwoo , Jeong Hyunyoung
Published: November 2023

Acetaminophen (APAP) is a commonly used analgesic and antipyretic. APAP‐induced hepatotoxicity is the most frequent cause of acute liver failure in the US. Individual susceptibility to APAP hepatotoxicity is highly variable, and the risk factors for developing severe APAP hepatotoxicity are not well understood. The objective of this study is to investigate a potential role of gut microbiota in modulating susceptibility to APAP toxicity by using a mouse model. C57BL/6 mice from two different vendors [Jackson (JAX) and Taconic (TAC)] were cohoused with mice from the same or the other vendor for 4 weeks, creating the following 4 groups: JAX, TAC, CoJAX (i.e., JAX mice cohoused with TAC) and CoTAC (i.e., TAC mice cohoused with JAX). In parallel, separate 4 groups of mice were co‐housed (n=6–7/group) as untreated controls. Fecal samples were collected at the end of cohousing and subject to next‐generation sequencing analysis of bacterial 16S rRNA gene amplicons. The mice were treated with APAP (600 mg/kg, oral, n=19–21/group) and sacrificed at 24 h post‐dosing, followed by collection of the liver and serum samples. Hepatotoxicity was determined by measuring serum levels of liver enzymes (i.e., alanine aminotransferase, ALT). Results from gut microbiome analysis showed the differences in gut microbiota composition between JAX and TAC at the end of cohousing and indicated the transfer of gut microbiota between cohoused mice. After APAP dosing, JAX mice exhibited 16‐fold higher hepatotoxicity as compared to TAC mice. This difference was abrogated upon cohousing, suggesting that differential gut microbiota between JAX and TAC may modulate susceptibility to APAP hepatotoxicity. Correlation analysis between ALT and different gut microbiota taxa revealed that the relative abundance of bacteria from the genera Mucispirillum, Turicibacter and Ruminococcus in fecal samples prior to APAP dosing is associated with increased APAP hepatotoxicity. The basal mRNA expression levels of Cyp2e1 and Cyp1a2 in the liver did not differ among 4 groups of mice, and the basal hepatic glutathione level was 1.3‐fold higher in JAX compared to TAC, suggesting that neither differential glutathione nor Cyp2e1/Cyp1a2 level is responsible for the difference in APAP hepatotoxicity. Taken together, our data suggest that differential gut microbiota may modulate susceptibility to APAP‐induced hepatotoxicity. This finding may shed a light on identifying potential risk factors for APAP hepatotoxicity.

Support or Funding Information

This study was supported by Chicago Biomedical Consortium.
RNA sequencing analysis of commensal microbial regulation of host intestinal transcriptome
Authors: Fu Zidong Donna , Selwyn Felcy Pavithra , Cui Julia Yue
Published: November 2023

Commensal microbiota can regulate intestinal physiology by modifying host genes. To identify host responses induced by commensal microbiota along the intestine, transcriptomes of duodenum, jejunum, ileum, and large intestine (LI) were compared between germ‐free (GF) mice and conventional (CV) controls using RNA sequencing. Cuffdiff (FDR‐BH<0.05) revealed that jejunum had the highest number of differentially expressed genes (2137) between CV and GF mice, followed by LI (781), duodenum (392), and ileum (329). Pathway analysis (GSAA, FDR<0.05) identified that protein digestion and absorption was the most up‐regulated pathway in duodenum and LI, whereas ribosome (duodenum) and protein processing in endoplasmic reticulum (LI) ranked among the top most down‐regulated pathways in GF mice. Notably, caboxypeptidase a1 (Cpa1) and chymotrypsin‐like elastase family member 3b (Cela3b), which were previously thought to be pancreatic‐specific enzymes, ranked among the top 10 most up‐regulated genes within the entire transcriptomes in duodenum and LI of GF mice. Whereas Cpa1 is involved in cleaving C‐terminal branched‐chain and aromatic amino acid from dietary proteins, Cela3b preferably cleaves proteins after alanine residues and may also function in the intestinal transport and cholesterol metabolism. Xenobiotic metabolism by cytochrome P450s (Cyp) was down‐regulated in duodenum and ileum of GF mice. Notably, Cyp1a1 ranked among the top 10 most down‐regulated genes in duodenum and ileum of GF mice. Cyp3a cluster was down‐regulated in duodenum of GF mice. Conversely, enzymes involved in alcohol and aldehyde oxidation were up‐regulated in jejunum of GF mice. Although drug‐metabolizing enzymes were generally thought to be more important in small intestine, there was a consistent up‐regulation in Cyp2d and aldo‐keto reductase 1c (Akr1c) isoforms in LI of GF mice, suggesting the importance of LI in xenobiotic metabolism in absence of intestinal microbiota. For carbohydrate metabolism, glycolysis/gluconeogenesis was down‐regulated in ileum, whereas starch and sucrose metabolism was up‐regulated in LI of GF mice. For lipid metabolism, the cholesterol synthesis and triglyceride synthesis were up‐regulated in ileum and jejunum of GF mice, respectively. Fatty acid binding protein 6 (Fabp6/Ibabp) was the most down‐regulated gene in jejunum of GF mice. The lack of intestinal microbiota also alter the mRNA expression of genes involved in the absorption of water and ion, mineral, vitamin, nucleotides, and bile acids, immune response, and cell junction. Taken together, we have identified that segment‐specific host gene alterations were induced in GF conditions, and the intestinal microbiota may impact a lot more key metabolic pathways than previously anticipated.
Cyproterone Acetate Enhances TRAIL‐induced Androgen‐independent Prostate Cancer Cell Apoptosis via Upregulation of Death Receptor 5
Authors: Chen Linjie , Wolff Dennis W. , Xie Yan , Lin Ming‐Fong , Tu Yaping
Published: November 2023

Objective

Virtually all prostate cancer deaths occur due to obtaining the castration‐resistant phenotype after prostate cancer cells escaped from the apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF‐related apoptosis‐inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL‐induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL‐induced apoptosis of androgen receptor‐negative prostate cancer cells are reported.

Methods

Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP‐ribose) polymerase cleavage assays. Changes of gene expression were determined by western blot and RT‐PCR assays. The effect of cyproterone acetate on the gene promoter activity was determined by luciferase reporter assay.

Results

Cyproterone acetate dramatically increased the susceptibility of androgen receptor‐negative prostate cancer PC‐3 and DU145 cells to TRAIL‐induced apoptosis. Further investigation of the TRAIL‐induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) expression. Cyproterone acetate treatment increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT‐enhancer‐binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increased CHOP expression by inducing endoplasmic reticulum (ER) stress / unfolded protein response (UPR) and siRNA silencing of CHOP significantly reduced cyproterone acetate ‐induced DR5 upregulation and TRAIL sensitivity in prostate cancer cells.

Conclusion

Together, our study shows a novel effect of cyproterone acetate on prostate cancer cell apoptosis pathways and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration‐resistant prostate cancer.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (5P20GM103489 and R01HL116849), Nebraska State LB595 research program.
Pipoxolan exhibits antitumor activity toward oral squamous cell carcinoma cell through the reactive oxygen species mediated apoptosis
Authors: Hsiao Jun‐Han , Chou Pei‐Yu , Sheu Ming‐Jyh
Published: November 2023

Pipoxolan is a well‐known smooth muscle relaxant. However, its apoptotic effects and mechanism in oral squamous cell carcinoma (OSCC) have not been clarified. In the present study, we investigated whether pipoxolan could induce apoptosis in OSCC and explored its potential mechanisms. Cell cytotoxicity were evaluated by MTT assay and apoptosis were measured by Annexin V/PI double staining and flow cytometry analysis. Apoptotic‐related proteins were assessed by Western blotting. Our results demonstrated that pipoxolan treatment caused a time‐dependent decrease the protein expressions of Fas/CD95, cytosolic cytochrome c, active form of caspase‐8, ‐9 and ‐3, hydrolysis of PARP, and Bcl‐2. However, Bax was increased. Clearly, an apoptosis was associated with an increased Bax/Bcl‐2 ratio. Pipoxolan markedly suppressed the expressions of PI3K and phosphorylation of Akt. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Furthermore, following exposure of TW206 cells with pipoxolan, a time‐dependently decrease in the MMP and an increase in the ROS. Since high levels of ROS were produced early in the drug treatment, intracellular ROS seemed to play a key role in the pipoxolan‐induced apoptosis. Obviously, apoptosis was significantly abrogated by the free radical scavenger N‐acetyl‐L‐cysteine (NAC). Also, pipoxolan‐induced apoptosis can be blocked by pan‐caspase inhibitor (Z‐VAD‐FMK). The abovementioned data suggested that pipoxolan acted against TW206 and HSC‐3 in vitro via both intrinsic, extrinsic apoptotic signaling pathways, and exhibited in PI3K/Akt signaling pathway. Thus, pipoxolan might be a candidate therapeutic agent for OSCC.

Support or Funding Information

MOST104‐2320‐ B‐039‐ 007
Tackling liver cancer by a novel combination between OSU‐2S and sorafenib
Authors: Omar Hany A. , Tolba Mai F. , Hung Jui‐Hsiang , Al‐Tel Taleb H.
Published: November 2023

Background

Sorafenib is an FDA‐approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, the low efficacy and adverse effects at high doses limit the clinical application of sorafenib and strongly recommend its combination with other agents aiming at ameliorating its drawbacks. OSU‐2S, a PKCδ activator, was selected as a potential candidate anticancer agent to be combined with sorafenib to promote the anti‐cancer activity through synergistic interaction.

Methods

The antitumor effects of sorafenib, OSU‐2S and their combination were assessed by MTT assay, caspase activation, Western blotting, migration/invasion assays in four different HCC cell lines. The synergistic interactions were determined by Calcusyn analysis. PKCδ knockdown was used to elucidate the role of PKCδ activation as a mechanism for the synergy. The knockdown/over‐expression of p53 was used to explain the differential sensitivity of HCC cell lines to sorafenib and/or OSU‐2S.

Results

OSU‐2S synergistically enhanced the anti‐proliferative effects of sorafenib in the four used HCC cell lines with combination indices < 1. This effect was accompanied by parallel increases in caspase 3/7 activity, PARP cleavage, PKCδ activation and HCC cell migration/invasion. In addition, PKCδ knockdown abolished the synergy between sorafenib and OSU‐2S. Furthermore, p53 restoration in Hep3B cells through the over‐expression rendered them more sensitive to both agents while p53 knockdown from HepG2 cells increased their resistance to both agents.

Conclusions

OSU‐2S augments the anti‐proliferative effect of sorafenib in HCC cell lines, in part, through the activation of PKCδ. The p53 status in HCC cells predicts their sensitivity towards both sorafenib and OSU‐2S. The proposed combination represents a therapeutically relevant approach that can lead to a new HCC therapeutic protocol.

Support or Funding Information

This study was funded by grant number AJF201424 from Al Jalila Foundation, United Arab Emirates.
Etrasimod, a Novel Sphingosine‐1‐Phosphate Receptor (S1PR1) Modulator, Lacks Functional Activity at S1P2 Receptors
Authors: Adams John W , Unett David J. , Chen Xiaohua , Gaidarov Ibragim
Published: November 2023

The S1P1 receptor modulator FTY720 (Fingolimod), a prodrug that is phosphorylated in vivo, possesses potent immunomodulatory activities and has been approved for the treatment of multiple sclerosis. FTY720 phosphate also possesses functional activity at S1P3, 4, and 5 receptor subtypes but, until recently, was thought to lack activity at the S1P2 subtype. We sought to develop methods to reliably detect functional activity at S1PR2 and to investigate whether structurally diverse S1PR1 modulators, including Etrasimod (APD334) a novel S1P receptor modulator, possess activity at this receptor.

FTY720 phosphate and a panel of S1P receptor modulators were evaluated for their abilities to interact with S1PR2 using a broad panel of functional assays. Treatment of an S1PR2 expressing cell line with FTY720 phosphate produced rapid and robust receptor internalization. As observed with the S1P1 receptor, internalization was sustained following compound wash‐out. A number of additional S1PR modulators were found to induce the same internalization, while Etrasimod was inactive. Unlike the natural ligand S1P, these modulators failed to recruit b‐arrestin to S1PR2. However, label free Dynamic Mass Redistribution assays (Corning®, Epic®) readily detected S1PR2 mediated agonist like functional responses. Pretreatment of S1PR2 expressing cells with these modulators significantly blunted subsequent responses of S1PR2 toward its natural ligand S1P and reversed S1P‐mediated inhibition of fibronectin‐induced migration in S1P2 receptor expressing HEK293 cells.

These data demonstrate that, in addition to FTY720, a wide range of S1PR modulators can also modulate S1PR2 while Etrasimod lacked such activity. Dynamic Mass Redistribution and receptor internalization assays appear suitable for quickly assessing S1PR2 activity. While S1PR1 is a chemotactic receptor, our data support the prevailing view that S1PR2 is a chemorepellant receptor, through which S1P negatively regulates cell migration. S1PR2 also mediates the antiproliferative activity of S1P in normal and cancer cell lines. S1PR2 ablation has been shown to result in stimulation of tumor development and metastasis in various animal models of cancer. Our data suggest that some S1PR1 modulators may have a negative effect on signaling pathways involved in regulation of cancer cell growth and metastasis.

Support or Funding Information

Funding: Arena Pharmaceuticals, Inc.
Peptide Ligand of the Epidermal Growth Factor Receptor for Sensitizing Lung Cancer Cells to Gefitinib
Authors: ALHamar Zahrah Ali , Andey Terrick
Published: November 2023

Background

By the end of 2016, 13.3% and 26.5% of new lung cancer diagnoses and deaths respectively is anticipated in the United States. Effective treatment of lung cancer is, however, hampered by adverse effects and the rapid acquisition of resistance to therapies. The epidermal growth factor receptor (EGFR) is an important lung cancer target. However, resistance to EGFR‐targeted therapies such as the receptor tyrosine kinase inhibitors, RTKIs (e.g. gefitinib) limits favorable long‐term therapeutic outcomes. The current study investigates the sensitization of EGFR‐expressing, gefitinib‐resistant lung cancer cells by the lung surfactant protein D (SPD).

Methods

Resazurin reduction‐based cell viability assay was performed to validate resistance to gefitinib in a panel of seven non‐small cell lung cancer (NSCLC) cells. Sensitization studies with single and combination treatments of SPD and geftinib were carried out in H1573 cells. The effect of SPD‐mediated H1573 sensitization to gefitinib on EGFR signaling examined by western blot.

Results

Gefitinib inhibited lung cancer cell viability in HCC827, H661, A549, H460, H1299, H1975, and H1573 [IC50 = 0.06±0.01 μM, 4.82±0.77 μM, 5.44±0.16 μM, 5.88±1.02 μM, 7.38±0.75 μM, 7.65±0.58 μM, 23.61±2.21 μM respectively]. SPD (0.5 μg/mL) inhibited H1573 cell viability (43.78±1.72 %), and significantly sensitized the cells to 5.0 μM gefitinib (13.16±0.82 % cell viability). SPD and gefitinib mediated EGFR degradation (phospho‐EGFR Tyr 1045) and inhibited Ras signaling (phospho‐EGFR Tyr 1086) in H1573 cells.

Conclusion

The lung surfactant protein D (SPD) was effective in sensitizing highly resistant H1573 cells to gefitinib treatment by inhibiting EGFR activating signaling. SPD may represent a viable candidate for adjunctive therapy in chemo‐refractory lung cancer overexpressing EGFR.
The role of p90 ribosomal S6 kinase in lung adenocarcinoma and as a mediator of the cytotoxic effects induced by cisplatin
Authors: Mraiche Fatima , Abdulrahman Nabeel
Published: November 2023

p90 ribosomal S6 kinase (p90RSK) constitutes a family of serine/threonine kinases that have been shown to be involved in cell proliferation of various malignancies via direct or indirect effects on the cell‐cycle machinery. We investigated the role of p90RSK in lung adenocarcinomas and whether the inhibition of p90RSK diminishes cancer progression. Anti‐apoptotic and pro‐apoptotic markers including Bcl2 and Bax were measured by reverse transcription polymerase chain reaction. In addition, the A549 lung adenocarcinoma cell line was characterized for cell proliferation using the MTT assay and cell migration using the scratch migration assay. Our study revealed that total RSK1 protein expression is over expressed in the A549 human lung adenocarcinoma cell line, an effect which is significantly reduced upon pretreatment with BID (69.32 ± 12.41 % of control; P < 0.05). The inhibition of p90RSK also showed a significant suppression of cell proliferation (54.3 ± 6.73 % of control; P < 0.01) and cell migration (187.90 ± 16.10 % of control; P < 0.01). Treatment of the A549 cells with BID regressed the expression of Bcl2 mRNA (56.92 ± 6.07 % of control; P < 0.01). Clearly, p90RSK has an essential role in promoting tumor growth and proliferation in non‐small cell lung cancer (NSCLC). Cisplatin, one of the most widely used drug for NSCLC adjuvant chemotherapy, has been demonstrated to improve survival among patients with NSCLC. Unfortunately, the outcome of cisplatin therapy on NSCLC seems to have reached a plateau. To overcome the treatment plateau on NSCLC, the biological mechanisms of cisplatin action need to be understood. Interestingly, cisplatin decreased RSK1 expression in A549 cells. In addition, BID gave rise to enhanced cisplatin sensitivity as indicated by the decrease in gap closure. Our findings demonstrate that BID may serve as an alternative cancer treatment in NSCLC. In addition, RSK inhibition may increase the response of lung adenocarcinomas that have become resistant to platinum based chemotherapy.

Support or Funding Information

This work was supported by a Qatar University Internal Grant.
COMPARISON STUDY OF THE EFFECTS OF ZERUMBONE AND AG490 IN HUMAN RENAL CELL CARCINOMA ON THE ACTIVATION OF THE JANUS KINASE PATHWAY AND CELL SURVIVAL
Authors: Walker Zachary , Duncan Anthony , Bellomo Tiffany , Budd Kaitlin , Polina Aws , Hafron Jason , Banes‐Berceli Amy
Published: November 2023

In the United States, Renal Cell Carcinoma (RCC) accounts for 9 out of 10 cases of kidney cancer. Approximately 33% of the patients present with metastatic disease and of those initially treated by surgical resection 40–50% will develop recurrent metastatic disease.13,860 people are estimated to die from RCC in 2016 due to poor responsiveness to the current chemotherapy medications. Development of new therapies is limited because the molecular mechanisms of RCC and the chemoresistance are poorly understood. However, we and others have shown that alterations in the levels of the Janus Kinase (JAK2) and Signal transducers of activators of transcription (STAT) pathway may be involved as it has been implicated in invasiveness and cell survival in RCC cell lines.

We hypothesize that altered activation of the JAK/STAT/SHP‐1 pathway contributes to the development of RCC and the chemoresistance observed. To test this hypothesis we used the RCC cell line (ATCC) and we treated the cells for 24 hours with the JAK2 inhibitor AG490 and sunitinib (standard chemotherapy agent) alone and in combination. After 24 hrs we found a 20% decrease in cell viability in the AG490 treated cells but not in the sunitinib treated cells. Combination of AG490 and sunitinib for 24 hrs resulted in a 40% decrease in cell viability. We also treated cells for 24, and 48 hrs with AG490 to directly inhibit JAK2 and with zerumbone, an inducer of SHP‐1 activity, which will indirectly decrease JAK2 phosphorylation and activation. At 24 hrs we found significant decreases in JAK2 phosphorylation levels with both and almost undetectable levels by 48 hrs. The STAT1 and STAT3 levels were only decreased significantly after 48 hrs of treatment with both zerumbone and AG490. We also treated cells with these inhibitors and measured cell viability for 24, 48, 72 and 96 hrs. We found an 80% reduction in cell viability at 96 hrs of treatment with both the zerumbone and AG490. There data suggest that inhibition of JAK2 is a viable clinical target for future therapy as it enhanced the response to sunitinib as well as decreased cell viability on its own.

Support or Funding Information

Oakland University Center for Biomedical Research
Aryl Hydrocarbon Receptor/Cytochrome P4501 Pathway Mediates Breast Cancer Stem Cells Expansion through β‐Catenin/Akt Activation and Pten Inhibition
Authors: Al‐Dhfyan Abdullah O , Alhoshani Ali R. , Korashy Hesham M.
Published: November 2023

Breast cancer stem cells (CSCs) are small sub‐type of the whole cancer cells that drive tumor initiation, progression and metastasis. Recent studies have demonstrated a role for the aryl hydrocarbon receptor (AhR)/cytochrome P4501 pathway in CSCs expansion. However, the exact molecular mechanisms remain unclear. Thus the current study was designed to determine the effect of AhR activation and inhibition on breast CSCs development, maintenance, self‐renewal, and chemoresistance and to explore the role of β‐catenin, PI3K/Akt, and Pten signaling pathways. To test this hypothesis, CSCc characteristics in three human breast cancer cells SKBR‐3, MCF‐7, and MDA‐MB‐231, treated with AhR activators or inhibitors were determined using Aldefluor assay, side population, and mammosphere formation. The mRNA and protein expression of the target genes were determined by RT‐PCR and Western blot analysis respectively. Our results showed that the constitutive mRNA expression and cellular content of CYP1A1 and CYP1B1 were markedly higher in CSCs more than differentiating non‐CSCs of the three different human breast cancer cells. Activation of AhR/CYP1 in MCF‐7 cells by TCDD and DMBA, strong AhR activators, significantly increased CSC‐specific markers, mammosphere formation, aldehyde dehydrogenase (ALDH) activity, and percentage of Side Population (SP) cells, whereas inactivation of AhR/CYP1A1 using chemical inhibitor, α‐naphthoflavone (α‐NF), or by genetic shRNA knockdown, significantly inhibited the upregulation of ALDH activity and SP cells. Importantly, inactivation of the AhR/CYP1 significantly increased sensitization of CSCs to the chemotherapeutic agent doxorubicin. Mechanistically, Induction of AhR/CYP1A1 by TCDD and DMBA was associated with significant increase in β‐catenin mRNA and protein expression, nuclear translocation and its downstream target Cyclin D1, whereas AhR or CYP1A1 knockdown using shRNA dramatically inhibited β‐catenin cellular content and nuclear translocation. This was associated with significant inhibition of PTEN, and induction of total and phosphorylated Akt protein expressions. Importantly, inhibition of PI3K/Akt pathway by LY294002 completely blocked the TCDD‐induced SP cells expansion. In conclusions, the present study provides the first evidence that AhR/CYP1A1 signaling pathway is controlling breast CSCs proliferation, development, self‐renewal and chemoresistance through activation of β‐catenin and Akt and inhibition of the Pten pathways.

Support or Funding Information

National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (12‐MED3131‐02).
Functional Upregulation of STIM‐1/Orai‐1‐Mediated Store‐Operated Ca2+ Entry Contributing to the Aortic Remodeling and Hypertension Development by Chronic EtOH Consumption
Authors: Bomfim Guilherme Henrique Souza , Méndez‐López Iago , Arranz‐Tagarro Juan Alberto , Musial Diego Castro , Carbonel Adriana Aparecida Ferraz , Padín Juan Fernando , García Antonio García , Jurkiewicz Aron Aparecida Ferraz , Jurkiewicz Neide Hyppolito
Published: November 2023

Chronic ethanol (EtOH) consumption has been associated with deleterious effects on the cardiovascular system by abnormal calcium (Ca2+) handling. Store‐operated Ca2+ entry (SOCE) is related to cardiovascular remodeling which leads to the hypertension development, and the coupling between STIM‐1 (Ca2+ sensor) and Orai‐1 (channel pore) is a key mechanism to control SOCE through of store‐operated Ca2+ channels (SOCCs). However, the role of STIM‐1/Orai‐1‐mediated SOCE and its cross‐talk with EtOH‐triggered vascular remodeling and hypertension remain poorly understood. We address this subject in the present study by evaluating how chronic EtOH consumption induces alterations in Ca2+ handling via SOCE. Male Wistar Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats were subjected to the intake of increasing EtOH concentrations (5–20%, for 30 days). Systolic blood pressure (SBP) and EtOH concentration were measured; cardiovascular remodeling was assessed by histomorphometry; and function/expression of STIM‐1/Orai‐1‐mediated Ca2+ influx were evaluated by isometric contraction and western blot experiments. Compared to the WKY‐Control, our results show that: (1) chronic EtOH consumption caused a significant elevation of SBP in both strains; (2) cardiac hypertrophy and hypertrophic aortic wall remodeling much more pronounced in WKY‐EtOH; (3) decreased capacity of reticulum to store and release Ca2+; (4) increased STIM‐1/Orai‐1‐mediated SOCCs activation, which was selectively inhibited by YM‐58483; and (5) increased expression of STIM‐1 in WKY‐EtOH and SHR‐Control rats. These findings suggest that hypertension developed by EtOH consumption has as one of its origins the hypertrophic aortic remodeling and abnormal contraction triggered mainly by Ca2+ overload via STIM‐1/Orai‐1‐mediated SOCE through SOCCs.

Support or Funding Information

This work was supported by the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior‐CAPES (BEX 8477/13‐2); the Fundação de Amparo a Pesquisa do Estado de São Paulo‐FAPESP (2014/01569‐0); the Ministerio de Economía y Competitividad, España (SAF: 2013‐44108‐P and FPI BES‐2014‐069005).
Dantrolene and Nimodipine Reduce Arterial Tone Synergistically in Aortic Rings from Type‐1 Diabetic Rats
Authors: Roman Marie , Alemar Stephanie , Matias Jonathan , Torres‐Grajales Manuel , Zack Richard , Morales Myrna , Torres Hector , Crespo Maria J.
Published: November 2023

Diabetic patients have a high risk of developing cerebral vasospasms (CVSP). The current treatment of this condition is similar for diabetics and non‐diabetics, and includes the use of nimodipine and other calcium channel antagonists to reduce vasoconstriction. Concomitant administration of the ryanodine receptor (RyR) blocker dantrolene with these antagonists may be beneficial to diabetic patients, knowing that this combination reduces vasoconstriction in non‐diabetic patients. In this study, we evaluated the effects of dantrolene (50 μM), nimodipine (50 nM), and both drugs in combination, on the phenylephrine (PHE)‐induced contraction of aortic rings from streptozotocin (STZ)‐induced Type‐1 diabetic rats. Age‐matched non‐diabetic rats were used as controls (CT). PHE‐induced concentration response curves from 0.1 nM to 10 μM were performed on aortic rings in the presence and absence of dantrolene, nimodipine, or their combination. After a 30‐minute incubation period, the PHE‐induced contraction was reduced to the same percentages in diabetic and non‐diabetic rats by dantrolene (37 %; N=5, P<0.05) and nimodipine (about 50%; N=5, P<0.050). In contrast, the combination of these drugs reduced the PHE‐induced contraction by 81% in CT, but only by 69% in diabetic rats. In addition, endothelium‐independent relaxation with 10 μM sodium nitroprusside (SNP) was not affected by dantrolene or nimodipine in either diabetic or CT rats. Our results suggest that the combination of dantrolene and nimodipine may have beneficial effects in both diabetics and non‐diabetics with CVSP by decreasing arterial tone more than either drug alone. Supported by RCMI Grant G12‐RR03051.

Support or Funding Information

Supported by RCMI Grant G12‐RR03051.
Novel RAGE Antagonist, FPS‐ZM1, Reverses Mesenteric Arteriolar Remodeling in Type 2 Diabetic db/db Mice.
Authors: Labazi Hicham , McCallinhart Patricia , Sunyecz Ian , Trask Aaron
Published: November 2023

Type II diabetes (T2D) is a major risk factor for cardiovascular diseases. Recently, our laboratory established a differential effect of T2D on vascular remodeling that was dependent upon vessel size and location. In addition, our preliminary data showed an increase in circulating levels of advanced glycation end products (AGE) in T2D mice. Activation of AGE receptors (RAGE) was shown to be associated with vascular dysfunction and vascular smooth muscle proliferation, which may result in vascular remodeling. In the present study, we hypothesized that pharmacological RAGE blockade using a novel antagonist would reverse microvascular remodeling observed in T2D. Using a pressure myograph system, passive vascular mechanics were assessed in aortae and mesenteric arterioles (MA) isolated from 16 week‐old lean control mice (Db/db), obese T2D mice (db/db) and T2D mice (db/db+FPS) treated with RAGE antagonist (FPS‐ZM1, 1mg/Kg/day ip, 28 days). Body weight (BW) and fasting blood glucose (BG) were significantly increased in diabetic db/db compared to control Db/db mice (BW: 29.8±0.5 in Db/db vs. 47.3± 1.5 g db/db, p<0.05 and BG: 185 Db/db ± 13 vs 687 ± 20 mg/dL in db/db, p<0.05). FPS‐ZM‐1 treatment did not affect BW (46.3±1.5g) nor BG (679±17 mg/dL) in T2D mice. In aorta, neither diabetes nor RAGE antagonism in diabetic mice affected the macrovascular mechanical properties. In contrast, MAs isolated from T2D mice exhibited a significant increase in internal diameter (158±2 in Db/db vs. 208±13 μm in db/db at 100mmHg, p<0.05), external diameter (180±3 in Db/db vs. 233±14 μm in db/db at 100mmHg, p<0.05) and cross‐sectional area (CSA) (6066±597 in Db/db vs. 8753±881 μm2 in db/db at 100mmHg, p<0.05). RAGE antagonism in T2D significantly reduced MAs internal diameter (178±5 in db/db+FPS vs. 208±13 μm in db/db at 100mmHg, p<0.05), external diameter (200±5 in db/db+FPS vs. 233±14 μm in db/db at 100mmHg, p<0.05) and CSA (6453±400 in db/db+FPS vs. 8753±881 μm2 in db/db at 100mmHg, p<0.05). Together, our data showed a variation in macrovascular vs. microvascular remodeling in T2D as we have previously shown. Furthermore, our data suggest that AGE/RAGE signaling contributes to the microvascular remodeling observed in T2D and that RAGE blockade using a novel antagonist may present a potential therapeutic target to reduce or reverse diabetes‐mediated vascular complications.

Support or Funding Information

R00 HL116769 and Nationwide Children's Hospital
Chronic Deep Vein Thrombosis: Pentadecapeptide BPC 157 Effect on Vein Thrombi
Authors: Siroglavic Marko , Vukojevic Jaksa , Drmic Domagoj , Tvrdeic Ante , Seiwerth Sven , Sikiric Predrag
Published: November 2023

Aim

To determine the effect of stable gastric pentadecapeptide BPC 157 on vein thrombi in deep vein thrombosis in rats.

Materials and methods

Female Wistar Albino rats (200g b.w.) underwent an inferior vena cava ligation according to deep vein thrombosis model in rodents. Control groups received 1ml of saline intraperitoneally immediately after surgery. Treated groups received either 1ml of 10μg/kg or 10ng/kg BPC 157 immediately after surgery, respectively. Both control and treated groups were sacrificed either on 4th or 7th day after surgery. Blood samples for platelet count were taken and phlebography was performed prior to sacrifice. After sacrifice vein thrombi were extracted, evaluated macroscopically for length assessment and weighed on a microgram weighing scale.

Results

Control groups on 4th and 7th day had larger thrombi (weight on 4th day 0.096±0.005g and on 7th day 0.064±0.002g, length on 4th day 1.93±0.43cm and on 7th day 1.74±0.35cm) than either BPC 157 10μg/kg groups (weight on 4th day 0.043±0.004g and on 7th day 0.032±0.003g, length on 4th day 1.05±0.14cm and on 7th day 0.87±0.07cm) or BPC 157 10ng/kg groups (weight on 4th day 0.063±0.009g and on 7th day 0.049±0.005g, length on 4th day 1.35±0.16cm and on 7th day 1.17±0.08cm) respectively (Figure 1, Figure 2). Platelet count in control groups on 4th (427±53×109/L) and 7th day (774±63×109/L) was lower than either BPC 157 10μg/kg groups (on 4th day 711±83×109/L and on 7th day 1153±103×109/L) or BPC 157 10ng/kg groups (on 4th day 595±75×109/L and on 7th day 1045±89×109/L). All shown differences were statistically significant (P<0.05).

Conclusions

This study has shown a significant beneficial effect of pentadecapeptide BPC 157 in both decrease of thrombi length and mass as well as reduction of thrombocytopenia after a thrombotic incident.

Support or Funding Information

Ministry of Science, Education and Sports, Republic of Croatia (grant number 108‐1083570‐3635) University of Zagreb, Croatia (grant number BM099)