bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidant Activity of Ruthenium Complexes Containing Nitro‐imidazole Derivatives
Authors: DA SILVA ANA VIRGINIA LIMA , SASAHARA GREYCE LURI , GOUVEIA FLORÊNCIO SOUSA , RODRIGUES RAPHAEL OLIVEIRA , DE FRANÇA LOPES LUIZ GONZAGA , DE SOUSA EDUARDO HENRIQUE SILVA , SANTOS FLÁVIA ALMEIDA , NAGAO‐DIAS APARECIDA TIEMI
Published: June 2021

Ruthenium‐based coordination complexes are compounds with great pharmacological potential. Once their toxicity and pharmacokinetics properties can be easily modulated changing the ligands on the six coordination sites of the metal, those species can present a broad spectrum of applications, such as: antineoplastic, bactericide, antiparasitic, vasorelaxant, among others. However, antioxidant properties of that kind of substance is still poorly explored, even after recent works have shown evidence that nitrosyl ruthenium complexes can scavenger free radicals. In addition, nitro‐imidazole derivatives are organic compounds with antioxidant properties and are able to work as ligands in coordination complexes. Considering that, the compounds cis‐[Ru(NO2)(bpy)2(4NIMN)](PF6) (1) and cis‐[RuCl(bpy)2(MTZ)](PF6) (2), where bpy=2,2′‐bipyridine, 4NIMN=4‐nitroimidazole and MTZ=metronidazole, were selected for analysis. Thus, the aim of the present study was to investigate in vitro antioxidant activity of those ruthenium complexes. The complexes were synthesized using microwave‐assisted synthesis, adapting methods previously described. Structural characterization was held through spectroscopic and spectrometric techniques, confirming the proposed structures. Cytotoxicity of the compounds at 3.12 to 100.0 μM was evaluated on A549 human lung carcinoma, RAW 264.7 murine macrophages, AGS human gastric carcinoma cells and human neutrophils at 5×106 cells/mL. No significant cytotoxicity was observed at any concentration of the compounds. Antioxidant properties were evaluated by quantifying malondialdehyde (MDA) and reactive oxygen species (ROS) production, and by evaluating superoxide dismutase activity in LPS‐stimulated RAW 264.7 cells treated with 1 and 2 at 3 – 25 μM. All the concentrations of both complexes were able to significantly reduce the production of superoxide anion in relation to the vehicle group, similarly to the reference treatment (α – tocopherol) All concentrations could also reduce the amount of MDA and ROS produced. Finally, the ruthenium complexes 1 and 2 were not cytotoxic to human neutrophils, lung and gastric carcinomas, and presented potent antioxidant properties. Tests are being carried out in order to elucidate antioxidant mechanisms and to analyze possible anti‐inflammatory effects.

Support or Funding Information

CAPES; CNPq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Novel Inhibitor for the Serotonin 5HT2A Receptor
Authors: Alshbool Fatima Z , Karim Zubair A , Khasawneh Fadi T
Published: June 2021

The serotonin 5HT2A Receptor (5HT2AR) signaling pathway plays a vital role in platelet function in vivo, and has been implicated in the genesis of several forms of cardiovascular disorders. However, despite its clear involvement in such processes, there are no antagonists that are currently available for managing thrombotic disease which target this pathway. In light of the fact that the ligand binding of 5HT2AR is contained, at least in part, within the second extracellular loop (EL2), we investigated the function‐blocking activity of an antibody (abbreviated EL2Ab) that targets this domain. Our results revealed that 100 nM of EL2Ab significantly inhibited the “limited” human platelet aggregation and shape change induced by 15 μM 5HT. Moreover, 100 nM of EL2Ab also significantly reversed the ability of serotonin to enhance ADP‐induced platelet aggregation. This inhibitory effect was found to be dose dependent. Also, we found that EL2Ab (100 nM) did not have any detectable effects on aggregation by 10 μM of the agonist ADP alone. Similar results were also obtained under in vitro conditions in mouse platelets. In terms of ex vivo platelet aggregation, injections of 150 nM EL2Ab into live C57BL/6 mice before their blood was collected for testing, revealed complete blockade of (15 μM) serotonin‐induced aggregation/shape change, substantial‐ and dose‐dependent reversal‐ of serotonin‐enhanced, ADP‐induced aggregation; but no effect on aggregation by 1 μM U46619 control. As for its in vivo activity, it was found that injections of 150 nM EL2Ab prolonged the time for occlusion in a carotid artery thrombosis model, indicating that EL2Ab protects against thrombogenesis. Collectively, these results clearly demonstrate that EL2Ab exerts 5HT2AR‐specific anti‐platelet effects, thus making it the first function‐blocking antibody against these receptors and a potential therapeutic agent. Moreover, the identification of a functionally active 5HT2AR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical and Histological Studies on the Effect of African Iba, a Herbal Purgative, in Albino Rats
Authors: Ushedo Kelly , Adegbola Mosebolatan , Otuechere Chiagoziem
Published: June 2021

Limited data is available about the toxicity of herbal remedies used for self‐medication. Since a popular herbal remedy, African Iba Herbal Mixture (AFRIM), contains various bioactive molecules, the present study aimed to observe the biochemical and histological changes in the hepato‐renal system of albino rats. Four groups of six male rats per group were used for the study. To group A was administered distilled water and served as control, while groups B, C and D were respectively administered with 100, 200 and 400 mg/kg body weight of AFRIM via gastric intubation for 14 days. Thereafter, animals were anaesthetized and sacrificed by cervical dislocation; blood samples were collected for biochemical assays while the liver and kidney were processed for histopathological studies. While the administration of AFRIM did not significantly alter the levels of calcium, potassium and alanine aminotransferase across all treatment groups, aspartate aminotransferase, albumin, blood urea nitrogen were significantly (p < 0.05) reduced, especially at 200 and 400 mg/kg doses. Furthermore, there was a significant (p < 0.05) elevation in uric acid levels at the higher doses of AFRIM. Liver sections revealed mild to severe infiltration of inflammatory cells in zone 2 at 100 and 200 mg/kg of AFRIM. Kidney sections illustrated engorged glomeruli which appear shrunken with hyper‐cellularity of mesangial cells at the 200 mg/kg dose. In light of this, caution and adherence to dosage prescription is strongly advised. This study is a contribution to scientific knowledge on the safety/toxicity profile of African Iba Herbal Mixture among the growing list of Nigerian herbal remedies.

Support or Funding Information

This research did not receive any funding from any granting body.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions of Hydroxylated Tetrabromodiphenyl Ethers with Phase II Enzymes
Authors: Cisneros Katherine Viviana , Agarwal Vinayak , James Margaret O.
Published: June 2021

Polybrominated diphenyl ethers (BDEs) have been used as flame retardants since the 1970s, and some congeners are classified as persistent and bioaccumulative. There is evidence that hydroxy and hydroxy‐methyl tetra‐BDEs are also natural products, as their synthesis has been demonstrated in marine bacteria, sponges, and macroalgae. Despite the prevalence of BDEs in the environment, their biotransformation has not been extensively studied, however it is known that they can be debrominated or monooxygenated at varying rates and to varying degrees depending on degree of bromination and species of animal, thus producing metabolites that have proven to be more reactive or toxic, such as hydroxylated‐polybrominated diphenyl ethers (OH‐BDEs). The present studies examined the human hepatic phase II metabolism of three natural product OH‐BDEs (6‐OH‐BDE47, 2‐OH‐BDE68, and 2‐OH‐6′methoxy‐BDE68) and a synthetic OH‐BDE (4‐OH‐BDE68), which was synthesized to study structure‐metabolism activity. Human liver microsomes and cytosol were prepared from de‐identified tissue bank samples from two female and five male donors aged 31 to 75 under an exempt protocol approved by the University of Florida IRB. Recombinant human SULT1A1, 1B1 and 1E1 enzymes were expressed and purified from bacterial expression systems. Sulfonation and glucuronidation of each OH‐BDE were studied using radiolabeled co‐substrates, 3′phosphoadenosine‐5′phospho‐35S‐sulfate or uridine diphospho‐β‐D‐14C‐glucuronic acid. The sulfated or glucuronidated products were extracted as ion‐pairs into ethyl acetate and quantitated or were separated by TLC and quantified by imaging the radioactivity. The OH‐BDEs studied were somewhat more efficiently glucuronidated than sulfonated. Of the compounds studied, 2‐OH‐BDE68 was most readily conjugated, and exhibited an efficiency (Vmax/Km) of glucuronidation of 0.256 ± 0.095 ml/min/mg protein, mean ± S.D., n=3, while that for sulfonation was 0.179 ± 0.030 ml/min/mg protein. For both pathways, Km values were in the low μM range. Studies with human SULT enzymes showed that sulfonation of all four substrates was readily catalyzed by SULT1B1 and SULT1E1, but not SULT1A1. OH‐BDEs inhibited SULT1A1 activity at concentration ranges between 0.2 μM – 20 μM. These OH‐BDEs also inhibited SULT1E1 sulfonation activity with Ki constants ranging between 0.725 nM–219 nM. However, assuming that the glucuronide and sulfate conjugates are non‐toxic and readily excreted, as is the case for most such conjugates, these studies suggest that OH‐BDEs should not accumulate in people to the same extent as the parent BDEs.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystal Structure of CYP2C9*2 Genetic Variant in Complex with an Angiotensin II Receptor Blocker Drug Losartan
Authors: Shah Manish B. , Parikh Sonia
Published: June 2021

Cytochrome P450 (CYP)‐dependent monooxygenases are a superfamily of heme‐containing enzymes involved in the metabolism of various xenobiotics. The drug metabolizing CYP2C9 enzyme metabolizes up to 20% of the clinical drugs that include losartan, warfarin, ibuprofen, tolbutamide, and glimepiride. The CYP2C9*2 and *3 are the two prevalent genetic variants that demonstrate significant reduction of the catalytic activity compared to the wild‐type (WT) enzyme for many important CYP2C9 substrates. Despite the abundant structural information available regarding various CYPs, the structural information about the effect of genetic polymorphisms on drug binding has remained scant. The CYP2C9*2 variant represents an amino acid change from arginine to cysteine at position 144, also referred to as R144C. To understand how genetic variation in CYP2C9 affects drug binding and oxidation, we solved the crystal structure of CYP2C9*2 in complex with the antihypertensive drug losartan using X‐ray crystallography. The prodrug losartan is an angiotensin II receptor antagonist that is predominantly metabolized by CYP2C9 and CYP3A4. The overall structure of the CYP2C9*2‐losartan complex illustrate remarkable differences compared to the WT complex. These include altered conformation of important secondary structural elements and residue side chains previously shown to be responsible for binding interactions with different substrates. Furthermore, the new structure revealed significant changes in binding of losartan compared to the previously solved losartan complexes of the CYP2C9 WT, *3 and *30. The structure highlights how single nucleotide polymorphism affects drug binding and yield insights into altered metabolism. Overall, the structural complex provides a useful framework to study, computationally as well as biophysically, the binding of different substrates to genetic variants of CYP2C9 and understand the effect of amino acid substitution in decreased hydroxylation activity of the enzyme.

Support or Funding Information

The authors are grateful to Albany College of Pharmacy and Health Sciences for funding.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

Supported 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

Introduction

Ewing 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.

Methods

Qualitative 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.

Results

Using 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.

Conclusion

These 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 Information

NIH Intramural Research Program

This 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 objective

TRAIL (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.

Methods

To 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.

Results

We 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.

Conclusion

AR 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 Information

The project supported by the National Institutes of Health Grants R21CA193271 and R01HL116849

This 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 Information

This work was supported by ALSAC and NIH P30CA021765 Cancer Center Support Grant

This 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 Information

The 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 Information

National Institutes of Health Grant HL112184 and IOER 091707 to Quang‐Kim Tran; American Heart Association Grant 15SDG25090279 to Eric Wauson; IOER grant to Sarah Clayton

This 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 Information

Supported by a grant from the Center for Advanced Science in Space

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct Role of Endothelial Caveolae in Contraction and Relaxation Responses of Aortas from Pregnant Normotensive and Hypertensive Rats
Authors: Troiano Jéssica Antonini , Graton Murilo Eduardo , Potje Simone Regina , Gonçalves Emily Thalia , de Oliveira Fonseca Zanardo Jéssica Luiza , Benevides Priscila Scarpim , Antoniali Cristina
Published: June 2021

We evaluated the role of endothelial caveolae in contraction and relaxation of aortas of Wistar and spontaneously hypertensive rats (SHR) at the end of pregnancy. The quantification of the endothelial caveolae number in the absence or presence of methyl‐β‐ciclodextrin (DEX,10 mM/L) in aortas from non‐pregnant (NP) and pregnant (P) Wistar and SHR with 12 weeks old (n= 5) was assessed by electronic microscopy. Aorta rings from different groups (n= 5) were stimulated with cumulative concentrations (1 nM/L to 10 μM/L) of phenylephrine (PE) or acetylcholine (Ach) in the absence or presence of DEX (10 mM/L). We performed the analysis of caveolin‐1 (cav‐1) and p‐cav‐1 expression by Western Blot in aortic homogenates (n= 5). Ethics committee FOA‐UNESP proc. 00730/2015. The results were compared among groups (ANOVA, p <0.05). Hypertensive NP rats present a reduced number of caveolae in aortas when compared to aortas from Wistar NP. Wistar P has a lower number of caveolae in aortas when compared to aortas from Wistar NP, but the number of caveolae was not altered between aortas of SHR P and SHR NP. DEX was able to reduce the number of caveolae in aortas only in the NP groups of normotensive or hypertensive rats. Vascular reactivity assays showed that in the presence of DEX the aortas of the Wistar NP and SHR NP groups had an increase in the contractile response to PE as well as a reduction in the potency of ACh. However, in aortas of the Wistar P and SHR P groups had no change in the contractile response for PE and ACh. p‐cav‐1/cav‐1 expression is decreased in aortas of SHR NP when compared to Wistar NP. Pregnancy does not alter p‐cav‐1/cav‐1 expression in aortas of Wistar rats, but it increases cav‐1 phosphorylation in SHR aortas. These results showed that pregnancy does not alter the reduced number of caveolae in aortas of hypertensive rats. Due to the reduced number of caveolae in aortas of pregnant rats, DEX would have no effect on aortic reactivity to PE or ACh. Although the reduced number of caveolae, the increased p‐cav‐1 expression in aortas of SHR P could be associated with increased eNOS activity and consequently lower NO‐dependent reactivity, from aortas to PE.

Support or Funding Information

FAPESP 2015/09373‐0; 2016/22180‐9; 2018/10635‐7 and CAPES.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Calcitriol‐Mediated PTH Suppression is Attenuated by High Phosphate Diet in Experimental Model of CKD
Authors: Lee Lok Hang , Turner Mandy , Ward Emilie , Laverty Kim , Pruss Cynthia , Holden Rachel , Adams Michael
Published: June 2021

Background

Chronic kidney disease (CKD) patients have impaired phosphate and calcium excretion as well as vitamin D deficiency, which exacerbates secondary hyperparathyroidism (SHPT). A decrease in renal CYP27B1 conversion of calcifediol leads to reduced levels of calcitriol. SHPT leads to increased bone resorption and is a risk factor for vascular calcification and cardiovascular disease in the CKD population. The effectiveness of calcitriol in managing SHPT is problematic, as significant resistance develops with chronic therapy; a condition that can lead to parathyroidectomy.

Aim

Determine the time course and impact of increasing dietary phosphate on calcitriol‐induced suppression of PTH in experimental CKD.

Methods

In Sprague Dawley rats (N=10), a 0.25% adenine 0.5% phosphate diet was fed for 4–5 weeks to generate stable CKD. A single oral calcitriol dose (160ng/kg) was given pre‐CKD, and at 1, 3, and 5 (off adenine) weeks of CKD induction (0.5% dietary phosphate). From 7–8 weeks rats were given increased dietary phosphate (1.0%) and the response to calcitriol was re‐assessed. Changes in circulating PTH, fibroblast growth factor 23 (FGF‐23), calcium and phosphate in response to the biweekly sham or calcitriol dosing were assessed at 0, 4, 8 and 24 hours.

Results

In CKD rats, PTH increased from 80±25.8 pg/mL to 221±66.7 pg/mL (week 5) and FGF‐23 from 421±26.0 pg/mL to 6803±1510.2 pg/mL (week 5). The two week 1% phosphate markedly increased PTH to 1342±310 pg/mL (8 weeks) and FGF‐23 to 14490±4980.8 pg/mL (7 weeks). In CKD rats on a low phosphate diet, the biweekly calcitriol dose reduced PTH by up to 44% and raised FGF‐23 by 82% whereas in rats on high dietary phosphate PTH was no longer responsive to calcitriol suppression.

Conclusion

Responses to calcitriol therapy were attenuated with increasing dietary phosphate and increasing CKD severity. This study supports the importance of dietary phosphate restriction in the CKD population to limit the impact of PTH responsiveness and mineral bone disease in this population.

Significance

Characterization of calcitriol resistance will help to develop new dosing strategies for vitamin D receptor agonists in this population and prevent SHPT and parathyroidectomies.

Support or Funding Information

Funded by the Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased sodium transporters in distal nephron segments are associated with clozapineinduced hypertension
Authors: Ren Zhiyun , Yu Yanting , Zhu Gangyi , Asico Laureano , Jose Pedro , Wang Xiaoyan
Published: June 2021

Dopamine receptor subtype 4 (D4R) antagonists, such as clozapine, are used in the treatment of schizophrenia. A common side effect of clozapine is hypertension but the mechanism of the clozapine‐induced hypertension is unknown. D4R plays an important role in blood pressure (BP) regulation; deletion of the D4R gene in mice causes hypertension. To determine if renal mechanisms are involved in clozapine‐induced hypertension, we characterized the distribution of D4R in mouse kidney and measured BP, water and sodium balance, and renal protein expression of sodium transporters in clozapine‐ and vehicle‐treated mice. D4R (immunocytochemistry) was mainly located in thick ascending limb, distal convoluted tubule and cortical and medullary collecting ducts; there is slight staining of D4R in the proximal convoluted but not straight tubule. Clozapine (20mg/kg/day) or vehicle was delivered subcutaneously to adult male C57BL/6J mice for seven days via osmotic mini‐pump (n=5/group). In clozapine‐treated mice, systolic (110±0.5, mm Hg, under anesthesia) and diastolic BPs (76.6±1),) were elevated, relative to vehicle‐treated mice (SBP=97.6±1.6, DBP=69.5±2.3). Food and water intake, body weight, serum electrolytes and creatinine, urine volume, and creatinine clearance were similar in the two groups. The sodium excretion tended to decrease while sodium excretion and blood pressure plot in clozapine‐treated mice was shifted to the right of the vehicle‐treated mice. Renal protein expressions (immunoblotting) of sodium hydrogen exchanger 3 and sodium phosphate cotransporter 2 were similar in clozapine‐ and vehicle‐treated mice. However, the renal protein expressions of sodium potassium 2 chloride cotransporter (180±21, % of control) and sodium chloride cotransporter (171±22% ), and the three subunits of epithelial sodium channel, α (140±7%), β (166±21%) and γ (55 kDa=130±7%, 85kDa=114±10% ), were greater in clozapine‐than vehicle‐treated mice. Renal protein expression of a‐sodium potassium ATPase was also slightly increased (118±4%) in clozapine‐treated mice. These findings suggest that inhibition of D4R by clozapine increases sodium transporters in distal nephron segments. Furthermore, a similar increase in the protein expression of sodium chloride cotransporter was also found in the immortalized mouse renal distal convoluted tubule cells in vitro with inhibition of D4R with its siRNA and antagonists. Thus, inhibition of renal D4R may cause the impaired sodium excretion that may be responsible for the increased blood pressure in clozap‐inetreated mice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α7‐nAChR‐Mediated Therapeutic Angiogenesis Accounts for the Advantageous Effect of Low Nicotine Doses Against Myocardial Infarction in Rats
Authors: Youssef Mahmoud E , El‐Mas Mahmoud M , Abdelrazek Heba M , El‐Azab Mona F
Published: June 2021

Angiogenesis accelerates tissue regeneration in a variety of ischemic conditions including myocardial infarction (MI) and constitutes a valuable approach for post‐MI repair. Here we investigated whether angiogenesis promotion induced by α7‐nicotinic acetylcholine receptors (α7‐nAChRs) mitigates histopathological, electrocardiographic, and molecular consequences of MI in rats. These profiles were evaluated in rats with isoprenaline (85 mg/kg/day i.p. for 2 days)‐induced MI and co‐treated subcutaneously with nicotine (20 μg/kg/day s.c.), PHA‐543613 (PHA, selective α7‐nAChR agonist, 20 μg/kg/day), methyllycaconitine (MLA, selective α7‐nAChR antagonist, 40 μg/kg/day) plus nicotine, or the vehicle for 16 consecutive days. Isoprenaline‐insulted rats showed (i) ECG signs of MI such as significant ST‐segment elevations and prolonged QT‐intervals, (ii) deteriorated left ventricular histopathological scoring and elevated inflammatory cell infiltration, (iii) reduced immunohistochemical expression of cardiac CD34, a biomarker of capillary density, (iv) decreased cardiac expression of iNOS and α7‐nAChRs, and (v) adaptive increases in cardiac HO‐1 expression and plasma angiogenic markers such as vascular endothelial growth factor (VEGF) and nitric oxide (NO). These effects of isoprenaline, except cardiac iNOS and α7‐nAChRs downregulation, were ameliorated in rats co‐treated with nicotine or the selective α7‐nAChR agonist PHA for 16 consecutive days. We also show that concurrent α7‐nAChR blockade by MLA reversed the ECG, histopathological, and capillary density effects of nicotine, thereby reinforcing the advantageous cardioprotective and anti‐ischemic roles of α7‐nAChRs in this setting. In conclusion, the activation of α7‐nAChRs with PHA or doses of nicotine in the microgram scale promotes neovascularization and offers a promising therapeutic strategy for MI.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.