bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of Autophagy Proteins in Response to a Compound Designed as a Sigma‐2 Receptor Agonist Derived from Tamoxifen
Authors: Stang Christopher R.T. , Adams Bryce R. , Faler Ashton D. , Schuler Brian R. , Kinder David H. , Sabol Patrick , Carlston Leah , Schneider Ryan A. 
Published: November 2023

The selective estrogen receptor modulator tamoxifen has been shown to have anti‐cancer effects in cells that lack estrogen receptor expression. It is thought that these effects are mediated through sigma‐2 receptor agonism. We previously reported the anti‐cancer effects of a novel tamoxifen derivative, SP‐1, against three diverse human cancer cell lines. SP‐1 was the first in a small library of compounds designed to target sigma‐2 receptors while lacking structural characteristics required for estrogen receptor binding. The cytotoxic effects of SP‐1 were determined to be caspase‐independent. Here we present further data on the anti‐cancer effects of SP‐1 in MCF7 breast adenocarcinoma cells along with further pharmacological investigation in HT‐29 colon adenocarcinoma, A375 melanoma, and A‐172 glioblastoma cells. The IC50 of SP‐1 in MCF7 cells was determined to be 31 μM using the XTT cell proliferation assay. This represents a fourth cancer cell line in which SP‐1 displays anti‐cancer activity with an IC50 in the 25 to 40 μM range. Western blotting using lysates from MCF7 cells was negative for cleavage of caspase‐7 and poly[ADP‐ribose]polymerase (PARP), suggesting yet again a caspase‐independent mechanism for SP‐1. Additionally, the pan‐caspase inhibitor, Z‐VAD‐FMK, failed to prevent cell death following SP‐1 treatment in all four cell lines.Given the lack of caspase involvement, we sought to determine whether autophagy was involved. Treatment with SP‐1 at 50 μM modulated several autophagy‐related proteins including phospho‐AKT (serine 473), light chain 3A/B (LC3A/B) and p62. Phosphorylation of the pro‐survival protein AKT was noted in the A‐172, HT‐29, and MCF7 cell lines at 24 hours following SP‐1 treatment. SP‐1 also elicited an increase in p62 expression in all four cell lines while conjugation of cytosolic LC3A/B I to autophagosome‐associated LC3A/B II was noted in three of the four cell lines. These results suggest the involvement of autophagy‐related machinery in the cell's response to SP‐1 treatment. Further investigations into autophagic flux and whether the autophagic response induced by SP‐1 is cytotoxic or cytoprotective are underway.
A constitutive androstane receptor (CAR) inhibitor CINPA1 as a tool to understand receptor structure and function
Authors: Cherian Milu T , Chai Sergio C , Casal Morgan A , Chen Taosheng 
Published: November 2023

This study aims to use CINPA1, a recently discovered small molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor), for understanding the binding modes of CAR and to guide CAR‐mediated gene expression profiling studies in human primary hepatocytes.CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo‐resistant cancer cells. CAR and PXR have many overlapping modulating ligands as well as many overlapping target genes which confounded attempts to understand and regulate receptor‐specific activity. Through a directed screening approach we previously identified a new CAR inhibitor, CINPA1, which is novel in its ability to inhibit CAR function without activating PXR.The cellular mechanisms by which CINPA1 inhibits CAR function were also extensively examined along with its pharmacokinetic properties. CINPA1 binding was shown to change CAR‐coregulator interactions as well as modify CAR recruitment at DNA response elements of regulated genes. CINPA1 was shown to be broken down in the liver to form two, mostly inactive, metabolites. We used the structure‐activity differences of CINPA1 and its metabolites to guide computational modeling using the CAR‐LBD structure. To rationalize how ligand binding may lead to different CAR pharmacology we performed an analysis of the docked poses of human CAR bound to an activator (CITCO) vs our inhibitor or the metabolites. From our modeling, we predicted strong hydrogen bonding of CINPA1 with N165 and H203 in the CAR‐LBD. We validated these residues to be important for CINPA1 binding using CAR single amino‐acid mutants in a functional reporter assay. Also predicted were residues making key hydrophobic interactions with CINPA1 but not the inactive metabolites. We identified some of these hydrophobic amino‐acids and additionally, determined the differential coregulator interactions of these mutants in mammalian two‐hybrid systems.We demonstrate that CINPA1 represents an excellent starting point for future optimization into highly relevant probe molecules to study the function of the CAR receptor in normal‐ and patho‐physiology, and possible development as therapeutics (e.g. for resensitizing chemoresistant neuroblastoma cells.)Support or Funding InformationSt. Jude Children's Research Hospital, ALSAC, and the NIH.
Examining the Role of ARRDC3 in Regulation of PAR1 Trafficking and Signaling in Invasive Breast Carcinoma
Authors: Arakaki Aleena 
Published: November 2023

The G protein‐coupled receptor (GPCR) protease‐activated receptor‐1 (PAR1) is activated by proteases secreted in the extracellular environment. Overexpression of PAR1 in breast cancer correlates with poor patient prognosis and increased breast cancer invasion and metastasis. However, the mechanism(s) by which PAR1 promotes breast cancer invasion and metastasis are not known. Activation of PAR1 occurs through an irreversible proteolytic cleavage event and thus, the precise regulation of receptor signaling is critical for proper cellular responses. The arrestin‐domain containing ARRDC3 protein functions as a tumor suppressor and mediates activated PAR1 lysosomal degradation in HeLa cells. We have previously shown that PAR1 trafficking is dysregulated in invasive breast carcinoma cells, in which ARRDC3 expression is low or absent, resulting in persistent signaling and invasion. We hypothesize that loss of ARRDC3 expression in invasive breast carcinoma results in the dysregulated PAR1 trafficking, persistent signaling and breast cancer invasion we observe. We used a tetracycline‐inducible lentiviral vector to re‐express ARRDC3 in the highly invasive breast carcinoma cell line MDA‐MB‐231. Using biochemical‐ and microscopy‐based approaches, we found that re‐expression of ARRDC3 was sufficient to rescue defective PAR1 trafficking and restored agonist‐induced receptor lysosomal degradation. In addition, activation of PAR1 resulted in a marked increase in Hippo‐YAP signaling in MDA‐MB‐231 cells that was attenuated upon ARRDC3 re‐expression. These findings suggest that ARRDC3 is a critical regulator of PAR1 signaling in invasive breast cancer. We will further investigate whether loss of ARRDC3 affects PAR1‐mediated Hippo‐YAP pathway activity and modulation of breast cancer migration, invasion and tumor growth and metastasis. These studies will be the first to link ARRDC3 to GPCR‐stimulated Hippo‐YAP signaling and will reveal an important pathway for dysregulation of Hippo‐YAP signaling induced by GPCRs in breast cancer.Support or Funding InformationFunding for this project was provided by the UCSD Tribal Membership Initiative Graduate Fellowship and UCSD Graduate Training Program in Cellular and Molecular Pharmacology through NIH General Medical Sciences, T32 GM007752 (Aleena Arakaki) and by the National Institute of Health NIGMS R01 GM090689 (JoAnn Trejo).
Muscarinic cholinergic receptors regulate oscillation of intracellular Ca2+ in steroid‐producing adrenocortical carcinoma cells
Authors: Sharthiya Harsh , Surachaicharn Nuntida , Shams Yara , Arshad Mohammad , Kopf Phillip G. , Malaiyandi Latha M. , Dineley Kirk E. 
Published: November 2023

Elevated circulating aldosterone levels are associated with hypertension, thrombosis formation, cardiac hypertrophy, and congestive heart failure. Aldosterone secretion is regulated by angiotensin II (Ang II), potassium, and adrenocorticotropic hormone (ACTH). Calcium is required for the stimulation of aldosterone secretion by these steroidogenic stimuli. Despite the importance of calcium in steroidogenesis, the relationship between intracellular Ca2+ homeostasis and steroid production by the adrenal cortex remains incompletely understood. We used the Ca2+‐sensitive fluorophore Fluo‐4 to study whole‐cell calcium in human adrenocortical carcinoma (HAC15) cells. We found that oscillation in intracellular Ca2+ could be triggered in a subset of cells by the cholinergic agonist carbachol. The oscillations were characterized by a relatively rapid periodicity, and often returned completely to baseline before spiking again. Removal of calcium from the extracellular perfusate suggested that oscillations resulted from repetitive release and recapture of calcium stored internally in the endoplasmic reticulum. Carbachol‐triggered oscillations were inhibited by muscarinic antagonists including atropine, pirenzepine, and darifenacin. Because darifenacin nearly completely abolished the carbachol effect, we concluded that regulation by M3 receptors is most important for the oscillations. Nicotinic cholinergic receptors were ruled out because nicotine did not alter intracellular calcium levels. Angiotensin II increased intracellular Ca2+, however these changes were relatively large and sustained, and not oscillatory in nature. Further experiments will investigate the relationship between muscarinic receptor activity and aldosterone production stimulated by angiotensin II.Support or Funding InformationSupported by funds from Midwestern University
Altered spinophilin interactions in the pancreas of a leptin receptor mutant (db/db) model of obesity
Authors: Stickel Kaitlyn C. , Watkins Darryl S. , Edler Michael C. , Dharmarajan Subramanian , Belecky‐Adams Teri L. , Berbari Nicolas F. , Mastracci Teresa L. , Baucum Anthony J 
Published: November 2023

Spinophilin is a multi‐domain scaffolding protein known to bind with multiple neuronal proteins, such as F‐actin and protein phosphatase 1 (PP1). Spinophilin modulation of glucose and M3 muscarinic receptor‐dependent insulin secretion has been recently evaluated. These studies established that spinophilin KO mice have decreased fat mass and increased lean mass. Yet spinophilin specific expression and the physiological and pathological functions of spinophilin within pancreatic beta cells have not been analyzed. Here we report that spinophilin is expressed in a rat INS‐1 pancreatic beta cell line and also human pancreatic islets. In isolated pancreatic mouse islets, spinophilin was detected in both pancreatic beta cells and non‐beta cell populations. Using proteomics‐based approaches we identified multiple putative spinophilin interacting proteins isolated from intact pancreas, including PP1, neurabin and myosin‐9. We are continuing to assess the interaction of spinophilin with these proteins in pancreata from mice that have a mutation in the leptin receptor (db/db), a well‐characterized mouse model of type 2 diabetes and obesity. We have found that the associations of PP1 and neurabin with spinophilin were decreased in db/db mice as early as 6 weeks. The association of myosin‐9 with spinophilin was substantially increased in this same population. We are currently working with a transgenic mouse population that expresses a tagged form of human spinophilin in pancreatic beta cells to evaluate if changes in the spinophilin interactions are occurring within the pancreatic beta cells. Ultimately, Our data demonstrate that spinophilin is expressed in pancreatic beta cells and that spinophilin protein interactions in the pancreas are modulated under pathological conditions such as obesity. Future studies will examine how these changes in the spinophilin interactome may contribute to pathological sequelae associated with obesity and diabetes.Support or Funding InformationSupport and funding provided by NIH ‐ K01NS073700, R21DA041876 and by Indiana University School of Medicine Center for Diabetes and Metabolic Diseases Pilot and Feasibility Program and by IUPUI Department of Biology.
The Aortic function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Potassium Channels
Authors: Akther Farjana , Shaligram Sonali , Karimi Mujibullah D , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak 
Published: November 2023

UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) rat is a validated model of type 2 diabetes mellitus. UCD‐T2DM is characterized by polygenic, adult‐onset obesity and spontaneous β‐cell failure and, as a result, more closely models the pathophysiology of type 2 diabetes in humans than other rodent models. The objectives of this study were to determine whether the aortic function is altered in male UCD‐T2DM rats, and to test the hypothesis that potassium (K+) channels contribute in modulating vascular reactivity of aorta in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10‐8 to 10‐5M) was measured in intact aortic rings pre‐contracted with phenylephrine (PE, 2μM) before and after pretreatment with tetraethylammonium (TEA, 2 mM), a non‐selective K+ channel blocker. Endothelium‐independent vasodilation to sodium nitroprusside (SNP, 10‐9 to 10‐5 M) was assessed in endothelium‐denuded rings pre‐contracted with PE (2μM). Furthermore, constrictor response curves to PE (10‐8 to 10‐5 M) were generated before and after incubation with L‐NAME (200μM), an endothelial nitric oxide synthase (eNOS) inhibitor.ACh responses were significantly potentiated in aortic rings of male UCD‐T2DM rats. Both maximal relaxation and sensitivity to ACh were significantly enhanced compared to those in controls. Incubation of aortic rings with TEA blunted the relaxation responses to ACh in both groups. However, the inhibitory effect of TEA on the ACh‐induced relaxation in UCD‐T2DM was greater than that in control rats. The smooth muscle sensitivity to NO as measured by SNP‐induced relaxation was not altered in aorta of UCD‐T2DM group. The responsiveness to PE was significantly enhanced in UCD‐T2DM rats. Accordingly, the basal nitric oxide (NO), as indicated by the potentiation of the response to PE after L‐NAME, was reduced in aorta of this group.These data, for the first time, show that the aortic function of male UCD‐T2DM rats is altered and that K+ channels may be involved. Further studies is required to document the underlying mechanisms of the altered vascular reactivity and to identify the contribution of specific subtypes of K+ channels in the potentiation of aortic endothelial function in this model.Support or Funding InformationSupported by NIHLBI, R15HL128988
Critical role for adenylyl cyclase 5 on L‐type CaV1.2 channel potentiation and vasoconstriction during diabetic hyperglycemia
Authors: Syed Arsalan U , Nystoriak Matthew A , Prada Maria Paz , Nieves‐Cintrón Madeline , Navedo Manuel F 
Published: November 2023

Diabetes mellitus is a devastating metabolic disease affecting millions worldwide that carries with it a major risk factor of cardiovascular disease. Foremost among the symptoms of uncontrolled diabetes is elevated blood glucose (i.e. hyperglycemia). The correlation between elevated glucose and hypertension has emerged as an important factor for cardiovascular disease. Chronic increase in blood pressure is a consequence of increased vasoconstriction (myogenic tone) in resistant arteries, resulting in impaired organ perfusion. Our lab has recently demonstrated that this pathophysiological increase in vasoconstriction as a result of elevated glucose is due to dysfunction of L‐type Ca2+ channels (LTCC) activity due to phosphorylation of CaV1.2 at S1928 by cAMP‐dependent protein kinase (PKA) in vascular smooth muscle (VSM). This novel result challenged long‐held views that associate PKA activity with VSM relaxation and vasodilation. In the present study we test the hypothesis that local PKA signaling associated with CaV1.2 phosphorylation and activation of LTCC in response to elevated glucose is regulated by adenylyl cyclase isoform‐5 (AC5) via A‐kinase anchor protein 150 (AKAP‐150). Proximity ligation assay (PLA) and ground state depletion (GSD) super‐resolution imaging suggest that AC5 co‐localizes and exit in a signaling complex along with AKAP150 and LTCC. Using patch clamp electrophysiology and pressurized arteriography, we observed decrease potentiation of LTCC and attenuated vasoconstriction of cerebral arteries in response to elevated glucose in vascular smooth muscle cells and arteries from wild type mice pre‐incubated with the general AC inhibitor 2,5‐DDA or from AC5 knockout mice. Taken together, these results suggest that AC5 is a critical component in the signaling cascade required to activate LTCC and induce vasoconstriction in response to elevated glucose.Support or Funding InformationT32 HL086350NIH RO1 HL098200NIH RO1 HL121059
Structure based drug design with Diabetic Retinopathy and Rage
Authors: Sriramoju Swetha , Evangelista Diana Evangelista , Goetz Kerry Goetz 
Published: November 2023

ObjectiveDiabetic Retinopathy is the leading cause of blindness, stimulating the rise in 20,000 cases in the United States per year, and leaving over 3 million individuals vulnerable at risk. During the early stages of Diabetic Retinopathy, inflammatory and innate immune responses are derivative from pattern‐recognition receptors (PRRs) expressed as Advanced Glycation End‐products (AGEs). It has been observed that AGEs increases the progression of retinopathy in patients enduring the early stages of retinopathy. The reason behind the complication progression induced by AGEs is the ligand ‐ receptor binding with the Receptor Advanced Glycation End‐products. Therefore, by instituting a drug to bind with RAGE, may prevent the ligand‐receptor binding between RAGEs and AGEs. The following study analyzed drug targets based on their hydrophobicity, as to whether they will be able to bind with RAGE.MethodsA docking program, AutoDock Tools and AutoDock Vina, were both used to determine the binding affinity, rmsd values, and Log P values of the drug targets.ResultsIt was found that Statin drugs and Fibric Acid drugs are the types of drugs that were able to bind with RAGE with a high hydrophobicity and affinity, while a very popularly marketed drug, Metformin, wasn't able to bind with RAGE.ConclusionRAGE may be of importance in the prevention and management of diabetes complications.Support or Funding Informationnone
Mid‐Term Exposure to Prednisone is Less Detrimental than Short‐Term Dexamethasone on Glucose Homeostasis in Rats
Authors: Ribeiro Nathalee Xavier Ribeiro , de Souza Melo Danylo Noleto , Sanches Jonas Rodrigues , França Lucas Martins , Cappelli Ana Paula Gameiro , Rafacho Alex , Abrau Iracelle Carvalho , de Andrade Paes Antonio Marcus 
Published: November 2023

Synthetic glucocorticoids (GCs) are pontent and widely used anti‐inflammatory and immunosuppressant agents. However, prolonged or high‐dose usage of GCs promote several side effects, especially on glucose and lipid metabolism. In spite of the abovementioned factors, there is scanty data on differential impact of distinct synthetic GC on metabolic targets, particularly glycolipidic homeostasis. Thus, this work aimed to assess the metabolic alterations caused by subchronic treatment with prednisone in comparison to acute dexamethasone. For this, 90‐days old Wistar rats were treated with dexamethasone (1 mg/kg/day, i.p., D5 group, n=8) for 5 consecutive days and compared to saline‐treated control rats (C5, n=9). Additionally, 60‐days old rats were treated with prednisone (80 mg/kg/day, orally) for 15 days (P15, n=7) and 30 days (P30, n=12) and compared to their respective controls (C15, n=7 and C30, n=12), which received vehicle solution. D5 rats showed decreased body weight (−12.3%) and lower retroperitoneal fat accumulation (−38%), increased fasting (+12%) and fed (+30%) glycemia, higher insulinemia (+80%), higher free fatty acids (+114%) and hypertriglyceridemia (+339%). Total fat and triglycerides content in the liver were 29% and 52% higher in D5 than in C5 rats, respectively. P15 rats weight gain was 61% lower, with reduction of retroperitoneal fat (−29%) and increased plasma triglyceride levels (+60%), as compared to C15 rats. 30 days treatment promoted similar but deeper effects on the same parameters. In vivo protocols revealed impaired glucose tolerance (oGTT) in D5 and P30 rats. However, P30 rats did not show reduced insulin sensitivity (ipITT, HOMA, TyG), as observed in D5 animals. Ex vivo assessment of glucose‐stimulated insulin secretion showed dysfunctional response in D5 but not P30 rats. In conclusion, our data suggest that mid‐term administration of prednisone promoted lesser metabolic side effects on glucose homeostasis, as compared to short‐term administration of dexamethasone, supporting its preferential use in order to minimize adverse metabolic effects associated to GCs treatment.Support or Funding InformationFAPEMA, UFMA and CAPES
Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: Evidence of covalent binding to C‐terminal CGHC redox motif
Authors: Gaspar Renato Simões , Sousa Hiran Reis , Sena Elyjany , Da Silva Samira Abdalla , de Lima Fontelles João Lucas , Araujo Thais , Fries Diana , Azevedo‐Santos Ana Paula , Laurindo Francisco , Trostchansky Andrés , de Andrade Paes Antonio Marcus 
Published: November 2023

Protein disulfide isomerase (PDI) plays a major role in platelet aggregation and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI‐modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized this peptide would also inhibit platelet aggregation by association to surface PDI. Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. We showed that CxxC decreased platelet aggregation in a dose‐dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. In thrombin‐activated platelets, surface expression of activated αIIbβ3 integrin, P‐selectin and granulophysin were analyzed by flow cytometry. CxxC decreased αIIbβ3 activation, but had no effect on the other markers. In addition, CxxC was the only peptide able to decrease cell‐surface PDI pull down, with no detectable unspecific binding to total free thiols. Finally, the capacity of CxxC to covalently bind to PDI active site was analyzed by ESI LC‐MS/MS. We detected the addition of one CxxC molecule to PDI through binding to Cys400, an important site for PDI prothrombotic activity. In summary, our data support an antiplatelet activity for CxxC through binding to Cys400 in PDI a′ domain, which can be further exploited as a model for site‐driven antithrombotic agents development.Support or Funding InformationFoundation for Research and Scientific Development of Maranhão ‐ FAPEMA, Nacional Counsel for Scientific Development and Technology ‐ CNPq, Coordination of Improvement of Higher Level Personnel ‐ CAPES and CSIC grupos I+D 2014.