bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Cholesterol Depletion on the Localization of CYP2D6
Authors: Fuchs Robert , Reed Robert , Backes Wayne L 
Published: June 2021

The human P450 system metabolizes most clinically relevant drugs. Hundreds of P450‐mediated drug‐drug interactions have been reported that attenuate therapeutic efficacy or result in dangerous toxicities. One member of this family, CYP2D6, is especially important for metabolism of psychiatric drugs. Co‐administration of CYP2D6‐dependent substrates has been shown to cause many drug‐drug interactions with deleterious consequences to patients. Psychotropics such as antidepressants are especially susceptible to P450 mediated drug‐drug interactions, as low CYP2D6 activities prevent bioactivation or elimination of these compounds from the body. The ability of P450s to metabolize drugs depends on environmental factors including reciprocal interactions with the phospholipid membrane. P450s compartmentalize into specific endoplasmic reticulum (ER) membrane microdomains – either ordered regions (“lipid rafts”) or disordered microdomains. Disruption of lipid rafts via cholesterol depletion suppresses the activity of raft‐bound proteins. Therefore, it is possible that cholesterol‐lowering drugs, such as statins, will suppress P450 activity via lipid raft disruption. However, it is unclear whether lipid raft depletion would affect P450‐dependent metabolism. We hypothesized that CYP2D6 and CYP2E1 localize into distinct ER microdomains based on specific amino acid motifs, and that microdomain disruption by statin‐mediated cholesterol depletion suppresses CYP2D6‐dependent drug metabolism. To test this, rats were treated with pyrazole, β‐naphthoflavone (BNF), pravastatin, or two of these three drugs in combination for one month. The purpose of this treatment was to deplete cholesterol through competitive inhibition of HMG CoA Reductase enzyme activity by pravastatin, or AhR‐mediated cholesterol depletion by pyrazole and BNF. Microsomes were isolated, followed by solubilization of non‐raft regions using Brij98. CYP2D6/2E1 localization was visualized via Western blotting, and microsomal cholesterol content was measured using a colorimetric assay. We found that CYP2D6 and CYP2E1, respectively, localize to ordered and disordered microdomains, and that treatment with pravastatin, pyrazole, BNF, or any combination of these drugs led to depletion of microsomal cholesterol content. Using proteomic mass spectrometry, we showed that BNF treatment caused CYP2D6 to segregate into disordered rather than ordered regions, suggesting that cholesterol depletion alters localization of this P450. Future studies will assess whether statin, pyrazole, or BNF treatments displace CYP2D6 from native rafts or attenuate its enzymatic activity on substrates used clinically. This work has importance for assessing whether statins may create adverse drug interactions with other substrates of CYP2D6.Support or Funding InformationSupported by GM123253, ES013648 & ES004344.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mithramycin analogues disrupt ETS transcription factor DNA binding
Authors: Hayden Reiya Cherron , Hou Caixia , Mandal Abhisek , Mitra Prithiba , Thorson Jon , Rohr Jurgen , Tsodikov Oleg , Leggas Markos 
Published: June 2021

IntroductionEwing sarcoma, prostate cancer, and leukemia are a few examples where ETS transcription factors drive tumorigenesis. The transcription factors EWS‐FLI1 and EWS‐ERG are common translocations in Ewing sarcoma and bind DNA at GGAA repeats leading to expression of genes that drive tumor growth. Pharmacologic inhibition of EWS‐FLI1 with mithramycin (MTM) was shown to inhibit expression of downstream genes and tumor growth in mice. But despite this specific inhibitory activity, MTM has a narrow therapeutic window with hematologic and hepatic toxicity attributed to displacement of the ubiquitously acting Sp1 transcription factor. Thus, a synthetic effort was initiated to develop MTM analogues with reduced toxicity and increased specificity for ETS binding sites. Structural studies informed the design of MTM analogues that may stabilize transcriptional complexes leading to the disruption of transcriptional activity and DNA damage. In vitro cytotoxicity assays demonstrated that MTM analogues have significantly higher cytotoxicity in EWS‐ETS expressing cell lines. Here we present mechanistic evidence for the differences in biochemical activity among MTM and its novel analogues.MethodsQualitative interactions between drug‐DNA‐protein were assessed and optimized by electrophoretic mobility shift assays (EMSA). Time‐resolved fluorescence energy transfer (TR‐FRET) assays were used to quantitatively determine ERG displacement from DNA in the presence of MTM and analogues. Expression of proteins indicating DNA damage (c‐PARP, γ‐H2AX) and phosphorylation at the C‐terminal domain (CTD) of RNAPII was determined by western blot following drug treatments in ETS and non ETS expressing cell lines.ResultsUsing TR‐FRET, we observed that MTM displaced DNA bound ERG more potently and in a concentration dependent manner as compared to MTM analogues. As compared to MTM, treatment with MTM analogues resulted in higher expression of DNA damage markers, γ‐H2AX and c‐PARP, specifically in cell lines containing EWS‐ETS translocations in a concentration dependent manner.ConclusionThese studies provide insights regarding differences among MTM and analogues in DNA binding and interactions with DNA associated proteins in the presence and absence of EWS‐ETS expression. Our results suggest that MTM analogues may bind and stabilize transcriptional complexes. These differences will provide the basis for structure activity relationships and for the development of analogues with decreased in vivo toxicity. Future work will incorporate co‐immunoprecipitation studies to determine if physical protein interactions are being disrupted by MTM and analogues and cellular thermal shift assays to directly probe drug interactions with EWS‐ETS proteins and with RNAPII.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Throughput Screening Identifies Novel Therapeutic Targets for Merkel Cell Carcinoma
Authors: Garman Khalid A. , Gelb Tara B. , Urban Daniel J. , Hall Matthew D. , Brownell Isaac F. 
Published: June 2021

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer. The majority of tumors are virus‐positive MCC (VP‐MCC) as they have Merkel cell polyomavirus integrated into the host genome. The remainder of tumors are virus‐negative (VN‐MCC) and are associated with ultraviolet light mutations. Primary MCC is treated by surgical resection and radiotherapy. Conventional chemotherapy is ineffective for metastatic MCC, but immune checkpoint inhibitors (ICI) can produce durable responses. However, some MCC patients cannot receive ICI and others have disease progression despite immunotherapy. To identify novel treatments for MCC, we performed high‐throughput small molecule screening of ~4,000 drugs for their ability to reduce MCC viability in VP‐MCC and VN‐MCC cell lines. Out of the compounds screened, 39 reduced viability in MCC cell lines relative to immortalized control cell lines. The 39 identified compounds were categorized into compounds reducing viability in all MCC cell lines (n=14); only in VP‐MCC cell lines (n=23); or only in VN‐MCC cell lines (n=2). The targets of high priority compounds were validated using RNA interference (RNAi) and in vivo efficacy was confirmed in preclinical xenograft mouse models. Collectively, our studies identify new potential pharmacological targets for treating VP‐MCC and VN‐MCC.Support or Funding InformationNIH Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of androgen receptor increases sensitivity of TRAIL‐induced apoptosis via upregulation of death receptor 5 in breast cancer
Authors: Jiang Haihong , Meissner Hannah , Xie Yan , Chen Linjie , Abel Peter W. , Luo Xu , Tu Yaping 
Published: June 2021

Background and objectiveTRAIL (TNF‐related apoptosis‐inducing ligand) is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors. However, most breast cancer cells are resistant to TRAIL‐induced apoptosis. Our objectives were to investigate the underlying molecular mechanisms of TRAIL‐resistance and to develop strategies to overcome such resistance.MethodsTo identify modulators of TRAIL‐induced apoptosis, we carried out a genomewide siRNA screen. To validate the screening result, we either silenced or overexpressed the identified genes in various breast cancer cells, and changes in TRAIL‐induced cell apoptosis were determined by flow cytometry and caspase‐dependent Poly (ADP‐ribose) polymerase (PARP) cleavage in vitro and in an orthotopic xenograft mouse model. Expression of TRAIL receptor, Death Receptor 5 (DR5) was analyzed by western blot. Finally, we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL‐therapy.ResultsWe unexpectedly identified androgen receptor (AR) to be responsible for TRAIL resistance. While AR is classically viewed as the key factor in prostate cancer progression, we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple‐negative breast cancers (TNBC) that are highly aggressive with poor prognosis. Importantly, breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance. AR overexpression in MDA‐MB‐231 and MDA‐MB‐436 cells suppressed the TRAIL sensitivity whereas knockdown of AR by siRNAs rendered MCF‐7 and MDA‐MB‐453 cells sensitive to TRAIL‐induced apoptosis. AR overexpression also induced TRAIL resistance in breast tumors in vivo. Further, we observed an upregulation of the DR5 expression in breast cancer cells following the silence or inhibition of AR by its antagonists Casodex and MDV3100. Treatment with AR antagonists increased TRAIL‐induced apoptosis and blocked MDA‐MB‐453 cell growth.ConclusionAR signaling suppresses TRAIL‐induced breast cancer cell apoptosis, in part, by suppressing DR5 expression. A combination of AR antagonists together with TRAIL may be a novel and effective therapy for breast cancer.Support or Funding InformationThe project supported by the National Institutes of Health Grants R21CA193271 and R01HL116849This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP‐dependent efflux transporter ABCC4 is a positive regulator of the Sonic Hedgehog signaling pathway
Authors: Wijaya Juwina , Vo BaoHan T , Wang Yao , Nourse Amanda , Janke Laura J , Orr Brent , Roussel Martine F , Schuetz John D 
Published: June 2021

The Sonic Hedgehog (Hh) signaling pathway is inappropriately activated in multiple malignancies, including basal cell carcinoma and medulloblastoma (MB). MB is the most common malignant pediatric brain tumor, accounting for 20% of all childhood brain tumors. While frontline therapy of MB, which includes tumor resection, radiation, and targeted chemotherapy yields 70% survival rate amongst average‐risk patients, radiation results in devastating neurocognitive impairments, and targeted therapy can give rise to drug‐resistant tumors. As such, further advances in therapy of MB will require identification of factors that modulate Hh pathway. We discovered that ABCC4 was, along with other Hh pathway genes, specifically over‐expressed in Hh‐driven MB. High ABCC4 expression was associated with significantly worse overall survival in Hh‐driven MB patients. In Hh pathway cellular model systems, ligand‐dependent pathway activation or loss of negative regulators upregulated ABCC4 expression, suggesting ABCC4 has a positive regulatory role and that blocking expression may impair signaling. Ablation of Abcc4 suppressed both normal, ligand‐dependent signaling as well as aberrant, cancer‐like, ligand‐independent signaling. Furthermore, targeted Abcc4 ablation using CRISPR technology in a murine model of Hh‐MB tumor extended survival of Hh tumor‐bearing mice. This study reveals ABCC4 as a critical regulator of the Hh pathway that contributes to MB pathogenesis and present a new molecular target and opportunity to improve therapy against Hh‐driven tumors.Support or Funding InformationThis work was supported by ALSAC and NIH P30CA021765 Cancer Center Support GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microtubule Targeting Agents Induce Cell Cycle Phase‐Specific Death Pathways in Primary Acute Lymphoblastic Leukemia Cells
Authors: Delgado Magdalena , Urbaniak Alicja , Butler Kaitlynn , Chambers Timothy C. 
Published: June 2021

Microtubule targeting agents (MTAs), such as taxanes (microtubule stabilizers) or vinca alkaloids (microtubule destabilizers), are important and widely used cancer chemotherapeutics which are thought to act primarily in mitosis. In addition to mitotic effects there is increasing evidence that MTAs also act in interphase which may be relevant to their clinical mode of action. Previously we discovered that vincristine (VCR), a microtubule destabilizer, is able to induce death in primary acute lymphoblastic leukemia (ALL) cells in G1 phase. In order to expand on this previous study, we sought to determine if other MTAs induced phase‐specific death and to investigate the associated cell death pathways. This was achieved through studies comparing primary ALL cells to HeLa cells after treatment with two microtubule destabilizers, vincristine and eribulin, and one microtubule stabilizer, paclitaxel, at varying concentrations. Consistent with previous studies, it was found that at high concentrations (>100 nM) of the microtubule destabilizers, death was induced in G1 phase of primary ALL cells. Interestingly, when treated with lower concentrations (<100 nM) of microtubule destabilizers, mitotic arrest followed by cell death occurred. However treatment with the microtubule stabilizer, paclitaxel, led to mitotic arrest followed by cell death at all tested concentrations. All three MTAs induced solely mitotic death in HeLa cells consistent with many other established cell lines. Furthermore, immunofluorescence studies indicated that G1 phase death in response to higher destabilizer concentrations was associated with complete loss of the microtubule network, while some intact microtubule structures remained after treatment with low destabilizer concentrations and paclitaxel. Finally preliminary experiments assessing phosphorylation and activation status of several Bcl‐2 proteins suggest that mitotic death is controlled through intrinsic apoptosis while death induced in G1 phase is not. These results suggest that microtubule destabilizers induce cell cycle phase specific‐death which is dependent on concentration while microtubule stabilizers do not and that the difference in these types of MTAs may arise from differential effects on the microtubule network. These findings advance our understanding of interphase microtubule function and have significant implications for the clinical use of MTAs particularly in the context of combination treatments.Support or Funding InformationThe present study was funded by a grant (to TCC) from the UAMS Chancellor's Circle.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Myocardial Contraction and Ca2+ Dynamics by the G Protein‐Coupled Estrogen Receptor
Authors: Whitcomb Victoria 
Published: June 2021

The G protein‐coupled estrogen receptor (GPER) mediates many cardioprotective effects. However, the underlying mechanisms are not clear.We investigated the role of GPER in cardiac contraction and signaling mediated by the cardiac b1 adrenergic receptor (β1AR). In anesthetized female rats, intrajugular administration of isoproterenol produces rapid and sustained increase in left ventricular systolic pressure (LVSBP) from 100±9 mmHg to 160±12 mmHg and a mild decrease in LV end diastolic pressure (LVEDP) from 10±5 mmHg to 5±5 mmHg. Administration of GPER agonist G‐1 during the plateau phase of the isoproterenol‐induced pressure rise rapidly reduces LVSBP to 90±5 mmHg and LVEDP to 0±5 mmHg. In freshly isolated primary murine cardiomyocytes, isoproterenol triggers oscillatory Ca2+ entry signals that are inhibited by β1AR antagonist metoprolol but not by b2‐AR antagonist ICI‐118551. GPER activation using G‐1 (Kd 11 nM) at 0.01, 0.1 and 1 mM suppresses the total Ca2+ signals by 0.5±0.3, 17.6±2 (p<0.05), and 30±2.1% (p<0.05), the amplitude of oscillations by 12.6±1.2, 36.6±7.1 (p<0.05), and 42.5±5.2% (p<0.05), and the frequency of oscillatory deflections by 0±0.8, 28.6±4.2 (p <0.05), and 30.3±5.4 (p<0.05), respectively. Isoproterenol promotes robust phosphorylation of the CaV1.2 channels at Ser1928 and phospholamban at Ser16/Thr17. G‐1 at 0.01, 0.1 and 1 mM prevents the former by 11.3±1.24, 28.4±2.3 (p<0.05) and 49.3±6.1%, and the latter by 5.3 ± 0.9, 22.3 ± 3.3 (p<0.05) and 42.5 ± 5.2%, respectively. Interestingly, GPER inhibition with G‐36 (IC50 112 nM) at 0.01, 0.1 and 1 mM increases isoproterenol‐induced total Ca2+ signals by 1±0.7, 26±2.5 (p<0.05) and 59.3±3.1% (p<0.05), oscillatory amplitude by 26±5.3, 129±21 (p<0.05) and 294±9.6% (p<0.05), and oscillatory frequency by 22.8±7.6, 59.1±8 (p<0.05) and 92±13% (p<0.05), respectively. G‐36 pretreatment at 0.01, 0.1 and 1 mM enhances isoproterenol‐stimulated Cav1.2 Ser1928 phosphorylation by 1.45±0.93, 27±9.6, and 47.8±7.1% (p<0.05), and phospholamban Ser16/Thr17 phosphorylation by 23.6±5.3, 53.3±17.5, and 32.2±5.6% (p<0.05), respectively.Taken together, the data suggest that GPER activation is an intrinsic component of β1AR‐mediated Ca2+ signaling in the myocardium and plays an important role in β1AR‐mediated cardiac contraction.Support or Funding InformationNational Institutes of Health Grant HL112184 and IOER 091707 to Quang‐Kim Tran; American Heart Association Grant 15SDG25090279 to Eric Wauson; IOER grant to Sarah ClaytonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Induces ATP Formation and Release by Regulating Acid‐Base Transition and Mitochondrial Oxidative Phosphorylation in the Heart
Authors: Wang Yongshun , Ballard Heather Janet 
Published: June 2021

ATP is released in the heart during hypoxia, ischaemia or catecholamine stimulation. Both ischaemia and hypoxia lower intracellular pH. However, little is known about the signaling pathways involved in CFTR regulated ATP formation and release in heart.Lactic acid infusion, the mimic of ischaemia, increased left ventricular wall interstitial ATP in anaesthetized rats, which was blocked by inhibitors of CFTR. The signaling mechanism was studied in cardiomyocytes isolated from adult rat heart: lactic acid increased intracellular cAMP, Ca in the near membrane area, and ATP release. Acidosis‐induced ATP release was abolished by CFTR inhibitors or CFTR siRNA. The CFTR potentiator, apigenin, or cAMP‐elevating agents, forskolin and IBMX (F&I), also increased ATP release, confirming the role of CFTR. Inhibition of either the Na+/H+ exchanger (NHE) with amiloride or the Na+/Ca2+ exchanger (NCX) with SN6 abolished acidosis‐induced ATP release. Acidosis or F&I failed to activate ATP release with either BAPTA, an intracellular calcium chelator, or calcium free incubation medium. Thus, both cAMP and calcium activated CFTR opening during acidosis.Forskolin or acidosis‐stimulated ATP release from cardiomyocytes was abolished in bicarbonate free incubation medium, suggesting that the role of CFTR is to permit bicarbonate entry. Bicarbonate increased mitochondrial cytochrome C expression and release of both ATP and cytochrome C from isolated mitochondria; these were inhibited by KH7, a soluble adenylyl cyclase (sAC)‐specific inhibitor, suggesting that mitochondrial cyclic AMP activates mitochondrial PKA, which phosphorylates mitochondrial proteins resulting in increased mitochondrial oxidative phosphorylation and accumulation of cytochrome‐C. Cyclosporin A or V5, mitochondrial permeability transition pore/Bax inhibitors, induced accumulation of cytochrome‐C in mitochondria, whereas they inhibited ATP release from cardiomyocytes, suggesting that cytochrome‐C leaves the mitochondria through mPTP.Pannexin1 inhibitors, caspase inhibitors or pannexin1 siRNA, attenuated the ATP release during acidosis or forskolin treatment, suggesting that Pannexin1, activated by caspase cleavage, functions as the ATP release channel. Immunofluorescence imaging suggested that CFTR was co‐localized with Pannexin1.Hence, we demonstrate for the first time that the CFTR channel is activated by calcium/cAMP signaling during acidosis, allowing the movement of bicarbonate into the cell. The bicarbonate activates the HCO3‐mito‐sAC‐cAMP‐PKA signaling cascade, which serves as a metabolic sensor modulating ATP generation, and subsequently modulates Pannexin1 gating (for ATP release) by activation of caspase.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Cardiac Progenitors Grown Under Microgravity Conditions Generate Myocytes With High Conduction Velocities
Authors: Prodan Nicole , Islas Jose , Iyer Dinakar , McConnell Bradley , Birla Ravi , Schwartz Robert J 
Published: June 2021

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSC) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiac myocytes or vasculature. Thus, reprogramming hADMSCs into mature myocytes may fare better in future investigations. To reprogram hAdMSCs into electrically conductive cardiac myocytes (CM), we pioneered a 3‐step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, beta 2‐adrenergic receptor (b2AR) and micro‐gravity signaling induced in 3D cardio‐spheroids. In stage 1, ETS2 and MESP1 activated Nkx2.5, Tbx5, Mef2C, dHAND and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells (CPCs). Next, in stage 2, b2AR activation repositioned CPCs into de novo myocytes, along with the appearance of RYR2, CAV2.1, CAV3.1, Nav1.5, SERCA2 and CX45 gene transcripts, myofibrils and action potentials. In stage 3, electrical conduction fostered by 3D cardio‐spheroids formed in a Synthecon®, Inc. rotating bioreactor that mimics micro‐gravity induced the appearance of hypoxic genes: HIF1a/b, PCG1a/b and NOS2 which coincided with the robust activation of adult contractile genes MYH6, MYH7 and TNNI3, ion channel genes and the appearance of hyperpolarization‐activated and cyclic nucleotide‐gated channels (HCN1‐4). Conduction velocities doubled to ~200mm/s after hypoxia, and doubled yet again after dissociation of the 3D cell clusters to ~400mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5–1m/s. Epinephrine stimulation of stage‐3 myocytes in patches resulted in an increase in amplitude of the electrical wave, indicative of mature CMs. Our efficient protocol converted hAdMSC into highly conductive CM and demonstrated the potential utilization of stage‐3 cells for tissue engineering applications for cardiac repair.Support or Funding InformationSupported by a grant from the Center for Advanced Science in SpaceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.