bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) in renal elimination of meta‐iodobenzylguanidine (mIBG)
Authors: López Quiñones Antonio J , Wagner Dave J , Wang Joanne 
Published: June 2021

Radiolabeled meta‐iodobenzylguanidine (mIBG) is utilized as both a diagnostic imaging agent (123I‐mIBG) and targeted radiotherapy (131I‐mIBG) for neuroblastoma. mIBG is cleared predominantly by the kidney through glomerular filtration and active secretion. Whole‐body scintigraphy showed rapid uptake and extensive accumulation in non‐tumor tissues, which can interfere with mIBG tumor imaging quality and lead to peripheral toxicities. Although mIBG is known to be transported into neuroblastoma tumor cells by the norepinephrine transporter (NET), the molecular mechanisms underlying mIBG transport in the kidney and non‐tumor tissues are unclear. We hypothesize that human organic cation transporters 1–3 (hOCT1–3) as well as multidrug and toxin extrusion proteins (hMATE1/2‐K) are involved in mIBG renal secretion and uptake in non‐tumor tissues. To test this hypothesis, the uptake of mIBG was measured in HEK293 cell lines stably transfected with hNET, hOCT1, hOCT2, hOCT3, hMATE1 and hMATE2‐K followed by LC‐MS/MS quantification. The transepithelial transport of mIBG was further evaluated utilizing a MDCK cell line co‐transfected with hOCT2 and hMATE1 to model secretion in renal proximal tubule cells. Uptake studies in the HEK293 system demonstrated that mIBG is a universal substrate for hOCT1, 2, 3, hMATE1 and 2‐K. Kinetic analysis further revealed that hOCT1, hOCT2, and hOCT3 transport mIBG with similar apparent affinities (Km of 17.5 ± 5.4, 17.2 ± 2.8, 15.5 ± 6.6 μM, respectively) which are comparable to hNET (Km of 35.7 ± 8.9 μM). In the hOCT2/hMATE1 MDCK cells, the basal‐to‐apical (B‐to‐A) flux of mIBG is much greater than its A‐to‐B flux and increased by 20‐fold as compared to B‐to‐A flux in vector transfected control cells. The apparent B‐to‐A permeability of mIBG in the hOCT2/hMATE1 cells was 12.6 ± 2.7 cm/sec, which is 18‐fold greater than that in control cells. Interestingly, the intracellular accumulation in the hOCT2/hMATE1 cells was increased only by 1.9‐fold as compared to the control cells, suggesting that apical hMATE1 efficiently effluxes mIBG from renal cells after mIBG uptake via OCT2. Together, our data demonstrated that mIBG is efficiently transported by hOCTs and hMATEs and that the hOCT2/hMATEs pathway is involved in active secretion of mIBG in the kidney. Inhibition of hOCT2 and/or hMATEs by other drugs may alter mIBG renal clearance and/or intrarenal accumulation, leading to clinically significant drug‐drug interactions during 131I‐mIBG therapy.Support or Funding InformationThis work is supported by NIH Grants GM066233 and T32‐GM007750.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytochrome P450 Enzyme Contributions to the N‐oxide Detoxification Pathway of Tobacco‐Specific Nitrosamines; a Possible Role in Tobacco‐Related Cancer Risk
Authors: Perez‐Paramo Yadira X , Watson Christy J.W. , Xia Zuping , Chen Gang , Lazarus Philip 
Published: June 2021

Tobacco‐specific nitrosamines (TSNAs) including 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) are among the most potent carcinogens found in tobacco products. The major mode of metabolism of NNK is by reduction to form 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), which, like other TSNAs including N′‐nitrosonornicotine (NNN), N′‐nitrosoanabasine (NAB), and N′‐nitrosoanabatine (NAT), is detoxified primarily by UDP‐glucuronosyltransferases (UGTs) to their glucuronide products, but also by N‐oxidation of the common pyridine ring. TSNA‐N‐oxides were identified previously in the urine of rodents exposed to TSNAs, and NNAL‐N‐oxide was found in the urine of smokers. The enzymes responsible for this conversion have not yet been determined. To identify enzymes that catalyze TSNA‐N‐oxide formation, we used specific inhibitors (furafylline, tranylcypromine, clopidrogrel, montelukast, sulfaphenazole, quinidine, chlomethiazole, and ketoconazole) of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in oxidation reactions using pooled human liver microsomes. Inhibition of NNK‐N‐oxide and NNAL‐N‐oxide formation was observed when using inhibitors of CYPs 2A6, 2C8, 2C19, 2E1 and 3A4; no detectable levels of NNN‐, NAT‐ or NAB‐N‐oxide formation were observed in human liver microsomes. To confirm results from these inhibition studies, NNK‐N‐oxide and NNAL‐N‐oxide formation were examined using microsomal fractions from CYP over‐expressing HEK293 cell lines. CYPs 2A13, 2C8 and 2E1 exhibited the highest oxidation activity against NNK, with Vmax/KM values of 675, 610 and 559 nL•min−1•mg−1, respectively, and against NNAL, with Vmax/KM values of 105, 67 and 86 nL•min−1•mg−1, respectively. These data suggest that several cytochrome P450 enzymes play an important role in the in situ detoxification of NNK and NNAL and that inter‐individual variability in the activity of these enzymes could potentially be important in TSNA‐induced cancer risk assessment.Support or Funding InformationThis work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program (to Y. Perez‐Paramo), and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the interaction between KRAS and Argonaute 2
Authors: Waninger Jessica , Shankar Sunita , Siebenaler Ronald , Tesmer John , Chinnaiyan Arul 
Published: June 2021

KRAS is one of three members of the RAS family of small GTPases and nearly one third of all human cancers harbor mutations in the KRAS gene. These mutations lead to a constitutively active GTP‐bound form of KRAS which results in aberrant activation of MAPK and PI3K pathways that control cell differentiation, growth, and survival. While much has been learned about the effectors of KRAS and its complex signaling network, little progress has been made in successfully targeting it in the treatment of cancer. Recently, we identified a functional protein‐protein interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA‐induced silencing complex (RISC). Investigation of this interaction revealed that the switch II domain of KRAS (both wild type (WT) and mutant) binds to the N‐terminal (Nt) domain of AGO2. Mutant KRAS‐AGO2 binding results in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex (model 1). Analysis of this interaction revealed that KRAS‐AGO2 binding is direct through Tyr64 of KRAS and Lys112/Glu114 of AGO2.Recently, using genetically engineered mouse models (GEMM), we showed that mutant KRAS is dependent on AGO2 for the development of pancreatic ductal adenocarcinoma (PDAC) from early precursor lesions in the pancreas, suggesting a critical role for the KRAS‐AGO2 interaction in vivo. To further characterize KRAS‐AGO2 binding, we purified recombinant proteins individually, isolated complex via size exclusion chromatography (SEC), performed differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), and small angle x‐ray scattering (SAXS). Additionally, in order to evaluate endogenous regulators of the KRAS‐AGO2 interaction we investigated the role of the epidermal growth factor receptor (EGFR), which is known to regulate the RNAi function of AGO2 via phosphorylation at Tyr393. Interestingly, we observed that phosphorylation at this residue disrupts the interaction of AGO2 with WT but not mutant KRAS, providing a basis for differential regulation of the KRAS‐AGO2 interaction through growth factor activation. Finally, to examine structural changes induced by EGFR phosphorylation of AGO2 at Tyr393 that may affect KRAS binding, we performed parallel molecular dynamics simulations (MDS) of non‐phosphorylated and phospho‐Tyr393 AGO2. These studies revealed immediate structural reorganization of the RAS binding site and an outward rotation of the entire Nt domain of AGO2. Taken together these data shed light on a potential mechanism underlying the differential regulation of mutant vs WT KRAS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic Role of Cytochrome P450 (CYP) 1 Enzymes in Polycyclic Aromatic Hydrocarbon (PAH)‐Mediated Carcinogenesis
Authors: Moorthy Bhagavatula , Gastelum Grady , Veith Alex , Wang Lihua , Zhou Guodong , Jiang Weiwu 
Published: June 2021

Lung cancer is the leading cause of cancer‐related deaths in the United States. Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals that increase the risk of lung cancer in humans. PAHs induce phase I and II metabolism enzymes through aryl hydrocarbon receptor (AhR)‐ dependent and independent pathways. Cytochrome P450 (CYPs) enzymes are among these phase I enzymes and promote detoxification of PAHs. However, CYP1A1, 1A2, and 1B1, which belong to the CYP1A1 family of enzymes, are also involved in the bioactivation of PAHs, leading to the formation of reactive intermediates that can form mutagenic DNA adducts. In this study, we hypothesized that mice lacking all the genes of the Cyp1 family will be resistant to PAH‐mediated pulmonary carcinogenesis. To test this hypothesis, A/J (WT) and Cyp1a1/1a2/1b1‐triple knockout (Cyp1‐3ko) mice were exposed to 3‐methylcholanthrene (MC; 100 μmol/kg), benzo(a)pyrene (BP; 200 μmol/kg), or corn oil (vehicle) via a single intraperitoneal injection. Liver and lung tissues were harvested at 1, 8, and 15 days post PAH exposure for our short‐term studies. Mice in our tumor study were harvested 36 weeks post PAH exposure for tumor incidence and multiplicity analysis. We found that MC caused significant induction of hepatic CYP1A1/1A2 gene expression in WT but not CYP1‐3KO mice at each time point. MC treatment caused formation of DNA adducts in liver and lung for up to 15 days post MC treatment in both genotypes. However, compared to WT animals, Cyp1‐3ko mice had significantly reduced hepatic DNA adducts on day 15 and up to 95% less pulmonary DNA adducts at 1, 8 and 15 days post MC treatment. In addition to the DNA adduct inhibition, Cyp1‐3ko mice formed 93% less pulmonary tumors compared to their MC treated WT counterparts. The significant reduction in DNA adducts and pulmonary tumors in Cyp1‐3ko mice provide strong evidence of the mechanistic role these enzymes play in PAH‐mediated carcinogenesis. Future studies on the specific role of each of the enzymes of the CYP1A family will be important for the identification of specific targets that could be used in the prevention and/or treatment of lung carcinogenesis in humans exposed to PAHs and other environmental carcinogens.Support or Funding Information1R01ES029382‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FoxO1‐Induced Inflammation Contributes to the Development of Diabetic Vascular Remodeling
Authors: LIU Jingjin 
Published: June 2021

Complications from diabetes often result in poor outcomes for patients, even after optimized interventions. Forkhead transcription factor 1 (FoxO1) is a key angiogenic regulator that plays a pathologic role in the progression of diabetes. The present study was designed to investigate the involvement of FoxO1 in the development of diabetic vascular complications and elucidate its underlying mechanisms. Comparative studies were carried out in wild‐type (WT) versus FoxO1‐selective inhibitor AS1842856 (AS)‐gavaged rats at 8 weeks after STZ injection, as well as in human aortic arterial smooth muscle cells (HASMCs). We found that AS prevented both aortic wall morphological changes in STZ‐induced diabetic rats and recruited SMCs in carotid artery media. Treatment with pharmacological inhibition or knockdown of FoxO1 prevented increases in pro‐inflammatory factors and adhesion factors, and NLRP3 (NOD‐like receptor family protein 3) inflammasome activation. Furthermore, downregulation of FoxO1 attenuated diabetes‐induced phenotypic switch both in vivo in rat carotid artery, and in vitro in human smooth muscle cells (HASMCs). FoxO1 knockdown also reversed the synergic effects of TNF‐α and high glucose responsible for inducing cellular death, NLPR3 inflammasome and HASMC phenotype switching. Furthermore, FoxO1 pharmacological inhibition restored FoxO1‐3‐phosphoinositide‐dependent protein kinase 1 (Pdk1) binding, restraining FoxO1 nuclear translocation, which served as the basis for subsequent transcriptional regulation during hyperglycemia. Taken together, our data suggest that FoxO1 is a novel promoter of vascular remodeling in STZ‐induced diabetes, and thus may have important therapeutic implications for diabetes‐caused cardiovascular diseases.Support or Funding InformationThe research work of the authors was supported by the Research Grants Council (RGC)/GRF grants (17158616M and 17121315M).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mild Hypercaloric Intake is Associated with an Impaired Vascular Smooth Muscle Phenotype in Absence of Hyperglycemia: Potential Modulation by Anti‐diabetic Drugs
Authors: Abrahamian Carla F , Sleiman Fatima , El‐Yazbi Ahmed F. 
Published: June 2021

Cardiovascular disease is the leading cause of mortality and morbidity in diabetes. Diabetes‐induced vascular complications are associated with the contractile‐to‐synthetic phenotypic switch of the vascular smooth muscle cells (VSMCs). Emerging experimental and clinical data have shown that functionally impaired VSMCs contribute to the high incidence rates of macrovascular complications in diabetes. Moreover, a significant proportion of diabetic patients show signs of vascular impairment at initial presentation before prolonged exposure to hyperglycemia. The underlying mechanisms leading to VSMC deterioration, particularly in early stages of metabolic dysfunction, remain largely unknown. In this study, we have investigated VSMC phenotypic alterations in a model of early metabolic derangements. Rats fed a mild hypercaloric (MHC) diet were used at time points prior to the development of hyperglycemia. Aortic VSMCs were isolated from control rats, from MHC fed rats sacrificed after 4, 8, or 12 weeks of exposure. At twelve weeks, MHC‐fed rats did not show increased fasting or random blood glucose, impaired glucose tolerance, increased weight, or hypertension. The effect of a two‐week treatment with metformin and pioglitazone prior to sacrifice at the 12‐week time point was examined. A gradual reduction in VSMC activity in MTS assays was observed as a function of MHC‐feeding duration, with the sharpest decrease observed at 12 weeks. Cell cycle assays revealed no changes in cell proliferation indicating that the changes in MTS assays were rather a reduction in metabolic activity. Moreover, expression of VSMC contractile phenotype markers, a‐smooth muscle actin and calponin, decreased progressively with MHC feeding. Interestingly, in vivo treatment with non‐hypoglycemic doses of either metformin and pioglitazone, known to have positive vascular pleiotropic effects, restored VSMCs metabolic activity and contractile marker expression to control levels. Significantly, no changes were detected VSMC metabolic activity and contractile marker expression following in vitro exposure to either a range of concentrations of free fatty acids, to simulate the MHC diet effect, or to metformin or pioglitazone. The contrast between in vitro and in vivo results suggests a direct effect of lipids on VSMC phenotype and function is unlikely; however, the changes observed might be a result of a more complex interaction with more than one tissue type. We hypothesize that the observed effect of MHC diet is mediated by the induction of perivascular adipose inflammation. Studies are underway to verify this claim.Support or Funding InformationSupported by AUB MPP fund #320148This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Modulation by Adenosine A1 and A3 Receptors of Acute Endotoxemia‐Induced Hemodynamics, Cardiac Autonomic Impairment, and Oxidative Damage
Authors: El‐Yazbi Ahmed F. , Fouda Mohammed , Mroueh Ali , El‐Mas Mahmoud M 
Published: June 2021

We and others have previously reported that endotoxemia elicits detrimental cardiac autonomic and hemodynamic consequences. Despite the recognized roles of adenosine and its receptors in central and peripheral cardiovascular control, the contribution of adenosine receptors in the hemodynamic and autonomic sequalae of endotoxemia remains largely unknown. In this study, we examined whether selective A1 or A3 receptor stimulation would modify the hemodynamic and autonomic responses to acute exposure to lipopolysaccharide (LPS). Rats were instrumented for conscious invasive hemodynamic recording through femoral indwelling catheters. Rats were injected with LPS (5 mg/kg i.v.) and variation in blood pressure (BP), heart rate (HR), left ventricular systolic and diastolic functions (dP/dt max and tau, respectively), and frequency domain parameters (HF & LF power and LF/HF) of heart rate variability (HRV) were recorded over two hours. The modifying effect of two doses of A1‐ (CPA, 75 and 300 μg/Kg) or A3‐agonists (IB‐MECA 25 and 50 μg/Kg) were studied. As expected, LPS injection was associated with falls in BP, reflex tachycardia, and increased systolic contractility; however cardiac sympathetic impairment was evident as reduced LF power and sympathovagal balance. A3 receptor stimulation by IB‐MECA caused dose‐dependent counteraction of the hypotensive effect of LPS as well as the associated positive chronotropism and increased systolic contractility. The LPS‐evoked decreases in LF power of HRV and shift in sympathovagal balance towards parasympathetic dominance were also reversed by IB‐MECA. Alternatively, all the LPS actions, but the hypotension, disappeared upon concurrent stimulation of A1 receptor (CPA). We also show that except the improved sympathovagal balance, the favorable effects of CPA or IB‐MECA on LPS responses were eliminated after selective antagonism of their respective receptors. Significantly, signs of left ventricular focal ischemia and elevated myocardial oxidative stress triggered by LPS injection were reversed by selective stimulation of either A1 or A3 receptor subtypes in a dose‐dependent manner. Our results imply that while different adenosine receptors might have varying impacts on myocardial and hemodynamic function, stimulation of A3 receptor subtype ameliorates endotoxemia‐evoked cardiac autonomic and myocardial damage. This will have implications on selective targeting of adenosine receptors as a therapeutic approach for autonomic and hemodynamic rescue in sepsis.Support or Funding InformationSupported by the Science and Technology Development Fund, Egypt (STDF Grant No. 14895), and by AUB‐FM MPP grant #320148.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐181c‐5p enhances NFκB‐mediated inflammation via targeting PTPN4 in H9C2 cardiomyocytes during hypoxia/ reoxygenation
Authors: CAI YIN , GE LIANG , MA HAICHUN , IRWIN MICHAEL G. , XIA ZHENGYUAN 
Published: June 2021

BackgroundActivation of nuclear factor kappa B (NFκB) and subsequent inflammation occurs in various models of myocardial ischemia/reperfusion injury (I/RI). Thus, inhibition of NFκB‐mediated inflammation is of clinical significance for improving post‐ischemic recovery. miR‐181c‐5p was significantly up‐regulated in the early phase of myocardial infarction in patients. However, whether miR‐181c‐5p mediates cardiac I/RI through NFκB‐mediated inflammation is unknown. We aimed to explore the role and mechanism of miR‐181c‐5p in inflammation during I/RI in H9C2 cardiomyocytes under hypoxia/reoxygenation (H/R) injury.Methods and ResultsIn response to H/R (6 hours hypoxia followed by 6 hours reoxygenation), the expression of miR‐181c‐5p was significantly up‐regulated, suggestive of a potential role of miR‐181c‐5p in the pathology of myocardial I/RI. In addition, H/R significantly induced H9C2 cardiomyocytes injury, evidenced by increased release of lactic acid dehydrogenase (LDH, cell injury marker) and reduced cell viability, concomitantly with enhanced phosphorylation and degradation of IκBα, p65 nuclear translocation and increased production of NFκB‐mediated pro‐inflammatory cytokines tumor necrosis factor α (TNFα), interleukin (IL)6 and IL1β. To demonstrate the potential role of miR‐181c‐5p in H/R‐induced cell inflammation and injury, H9C2 cardiomyocytes were transfected with the miR‐181c‐5p agomir or its negative control. Overexpression of miR‐181c‐5p significantly aggravated H/R‐induced cell injury (increased LDH level and reduced cell viability) and exacerbated NFκB‐mediated inflammation [greater phosphorylation and degradation of IκBα, p65 nuclear translocation and increased levels of pro‐inflammatory cytokines (TNFα, IL6, IL1 β)]. By using computational prediction algorithms (TargetScan and miRDB), protein tyrosine phosphatase non‐receptor type 4 (PTPN4) was predicted as a potential target gene of miR‐181c‐5p, as also verified by the luciferase reporter assay. Indeed, overexpression of miR‐181c‐5p significantly attenuated the mRNA and protein expression of PTPN4 in H9C2 cardiomyocytes, while knockdown of PTPN4 significantly aggravated H/R‐induced enhancement of LDH level, phosphorylation and degradation of IκBα and pro‐inflammatory cytokines level, which mimicked the pro‐inflammatory effects of miR‐181c‐5p.ConclusionsmiR‐181c‐5p exacerbates H/R‐induced cardiomyocyte injury and NFκB‐mediated inflammation via targeting PTPN4, and the miR‐181c‐5p/PTPN4 signaling may be a novel target to combat myocardial I/RI.Support or Funding InformationThe work was supported by GRF (17117217M, Research Grants Council of Hong Kong).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.