bluuebunny/crossref_metadata_2025 huggingface dataset:
GPR56 is a Novel Platelet Collagen Receptor that Mediates Platelet Activation
Authors: Yeung Jennifer , Rosselli Luciana Kauer , Vizurraga Alexander , Holinstat Michael , Tall Gregory
Published: June 2021

The adhesion G protein‐coupled receptor (AGPCR) GPR56 (ADGRG1) is required for brain development and nerve myelination. GPR56 is expressed in other tissues, suggesting additional physiological roles. We pharmacologically identified GPR56 in human and mouse platelets, corroborated its presence by immunoblotting, and showed that stimulation of GPR56 with a synthetic peptide‐ or small molecule‐agonist activates platelets. Currently there is an enigma regarding the complete repertoire of collagen receptors that mediate initial platelet interaction with the vessel wound‐exposed collagen sub‐endothelium that fully accounts for platelet activation. GPR56 is a known collagen receptor. We hypothesize that GPR56‐mediated platelet activation is dependent on blood flow shear forced dissociation of the non‐covalently bound GPR56 N‐terminal fragment (NTF) and the C‐terminal fragment (CTF) to expose the GPR56 tethered‐peptide‐agonist. As previously demonstrated, operative dissociation of these GPR56 fragments led to enhanced G13 and Gi activation. Thus, shear force‐induced shedding of the NTF onto collagen is a consequence of GPR56 activation during platelet translocation that leads to platelet shape change and activation via G13 and/or Gi activity. To establish the functional role of GPR56 in platelet activation, human or GPCR deficent (Gpr56−/−) and wildtype (WT) mouse platelets were stimulated with varying concentrations of synthetic GPR56 peptides or 3‐a‐DOG agonist and platelet activation was assessed via a number of tests. Both GPR56 peptide‐ and small molecule‐agonists potently induced platelet aggregation and spreading comparably to the established platelet agonist PAR4 AP. Additionally, dihydromunduletone (DHM), a GPR56 antagonist, inhibited platelet activation mediated by the GPR56 agonists, but not by PAR4 AP. In the case of hemostasis, high shear blood flow rate over collagen coated slides were performed with WT and Gpr56−/−. Gpr56−/− showed decreased platelet adhesion on collagen compared to WT. Gpr56−/− mice also had prolonged hemostatic tail bleeding times compared to the WT. Overall, these functional assays establish GPR56 as an additional collagen receptor that is critical for platelet adhesion and activation in order to maintain hemostasis.

Support or Funding Information

NIH RO1 GM120110 (G.G.T), RO1 NS103946 (G.G.T), T32‐HL007853 (CVC), and Michigan Life Sciences Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automated High‐Throughput Drug Discovery in Peptide Hydrogel‐Based 3D Cell Cultures
Authors: Langhans Sigrid A , Worthington Peter , Drake Kathleen , Li Zhiqin , Napper Andrew , Pochan Darrin
Published: June 2021

Automated cell‐based high‐throughput screening (HTS) is a powerful tool in drug discovery and currently, there is a push to combine complex cell culture systems with HTS to provide more clinically applicable results. Despite the increasing recognition that three‐dimensional (3D) models provide more in vivo‐like cell culture environments, there are mechanistic requirements inherent to HTS as well as material limitations that make this integration for high‐throughput drug discovery challenging. We used the peptide‐based hydrogel MAX8 tagged with the RGDS sequence to create a synthetic extracellular scaffold to culture cells in 3D, and show a preliminary implementation of the scaffold for 3D HTS with a pilot drug screen targeting the pediatric brain cancer medulloblastoma. MAX8 undergoes assembly under physiological conditions and ambient temperature into a hydrogel with a well‐defined, nanofibrillar matrix, desired porosity and stiffness to provide a biocompatible cell culture matrix. Due to the physical gel properties such as shear‐thinning and the fast gelation kinetics, cells can be homogenously encapsulated into evenly dispensed gel‐cell constructs using automatic distribution of cell‐peptide mixtures into 384‐well assay plates with standard liquid handling equipment. 2,202 compounds were screened in the 384‐well format against cells encapsulated in the hydrogel as well as cells growing on traditional 2D plastic. 82 compounds passed the first round of screening at a single point of concentration. 16‐point dose response was done on those 82 compounds, of which 17 compounds validated. Three‐dimensional cell‐based HTS could be a powerful screening tool that allows researchers to finely tune the cell microenvironment getting more clinically applicable data as a result. Here, we have shown the successful integration of a peptide‐based hydrogel into the high‐throughput format.

Support or Funding Information

This work was supported by funds from the NIH IDeA program, with grants from the National Institute of General Medical Sciences NIGMS (P20‐GM103464, P30‐GM114736, and U54‐GM104941), the DO Believe Foundation and the Nemours Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allotopic Gene Therapy as a Treatment for Mitochondrial Disease
Authors: Paul Jeremiah , Palladino Michael J
Published: June 2021

Mitochondrial encephalomyopathies (MEs) result from mutations in mitochondrial genes and have been implicated in the aging process as well as several neurological and neuromuscular diseases. Developing genetic methods to knock‐down (KD) or express (rescue) endogenous mitochondrial genes would enable the development of novel therapies for numerous diseases such as neurodegenerative diseases and mitochondrial disorders, including primary encephalomyopathies such as NARP, MLS, and FBSN.

We have shown that RNAs can be targeting to mitochondria including long RNAs capable of coding proteins. Our preliminary data suggest these can be expressed by mitoribosomes but expression appears to be inefficient. We hypothesize that removing 5′UTRs and the targeting elements will dramatically improve the efficiency of mitoribosome translation. One approach we are testing is to engineer mitochondrial‐targeted RNA endonucleases (mtREs); RNA endonuclease, which don't exist in nature, could increase the efficiency of translation in the mitochondria by removing interfering 5′ ribonucleotides. We are testing this hypothesis by engineering clones for studies in HELA cells and transgenic animals for studies in Drosophila. We will examine expression efficiency using confocal microscopy and Western blotting.

Support or Funding Information

University of Pittsburgh School of Medicine, Summer Undergraduate Research Program (SURP), Molecular Pharmacology Department, Summer 2018 American Society of Pharmacology and Experimental Therapeutics

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 adiponectin‐expressing cells in thymus
Authors: Zhang Yiwei , Cao Handi , Xu Aimin , Wang Yu
Published: June 2021

Objective

Adiponectin, a protein considered as the most abundant adipokine, was found to be expressed in the thymus of wild type mice. The present study characterized the identity and function of adiponectin‐expressing cells in thymus.

Methods

Thymus was collected from mice with (WT) or without (AKO) adiponectin for isolation and enrichment of thymic nurse cell complexes (TNCs). Different populations of lymphocytes were analyzed by flow cytometry and confocal microscopy. Protein expressions were evaluated by immunofluorescence staining and/or Western blotting.

Results

Adiponectin‐expressing cells were present in TNCs, the specific epithelial cell complexes which engulf thymocytes and provide microenvironment for T lymphocyte development. Compared with age‐matched WT mice, the number and size of TNCs in thymus of AKO mice were significantly reduced. The percentage composition of CD3+ double positive (DP) lymphocytes was significantly decreased, whereas the total amount of double negative (DN) thymocytes significantly increased in TNCs of AKO mice. Adiponectin‐expressing cells were derived from a subpopulation of early T progenitors, which facilitated the formation and function of TNCs. Treatment with adiponectin in vivo or ex vivo could promote the formation and functions of TNCs.

Conclusions

Adiponectin‐expressing cells play important roles in the positive and negative selection process of TNCs, thus regulating T‐cell maturation and development in thymus. The results have important implications on the anti‐inflammatory, anti‐diabetic and anti‐tumorigenic properties of adiponectin.

Support or Funding Information

This work was supported in part by grants from Seeding Funds for basic research of the University of Hong Kong, Research Council Grants (17153016) and Hong Kong Health and Medical Research Fund (04151796), Collaborative Research Fund of Hong Kong (C7055‐14G) and the National Basic Research Program of China (973 Program 2015CB553603).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐Specific Differences of Steroid Receptors Following Exposure to Environmentally Relevant Concentrations of Phenothiazine in Fundulus heteroclitus
Authors: Chiari John B , Laperche Jacob M , Patel Roshni , March Nicole , Calvitto Gabriella , Marek Sarah , McGinnis Courtney L.
Published: June 2021

Agricultural runoff and industrial discharge are known to contaminate ground and surface waters and are not typically removed during water purification. These compounds may have significant effects on biological systems through the disruption of normal endocrine function. This study aims to determine the sex specific effects of phenothiazine (PTZ), a potential endocrine disruptor, on markers for endocrine disruption and stress in Fundulus heteroclitus. The following steroid hormone receptors were assessed: glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR), estrogen related receptor alpha (ERα), estrogen receptor beta A (ERβA), and estrogen receptor beta B (ERβB). Phenothiazine was detected at levels between 1–2ppm via gas chromatography‐mass spectrophotometry (GC‐MS) in river water sampling conducted in southern Connecticut, but the biological impact of the compound is unknown. Phenothiazine has been used previously as an antipsychotic medication, as well as, industrially in plastics to add softness and flexibility. To assess the impact of phenothiazine, female and male fish were exposed to environmentally relevant concentrations of phenothiazine for one week and sacrificed. Brain, liver, and gonad tissues were removed and pooled from each treatment group. Quantitative PCR (qPCR) was conducted on tissues and analysis showed significant changes in mRNA expression and both a sex‐specific and tissue specific response. There were no changes in the testis and the female liver. In male brain there was a decrease in GR at 1ppm (p<0.05), and an increase in ERβB at 2ppm (p<0.01); in comparison to female brain which had an increase in PR (p< 0.01), AR (p<0.05), ERα (p< 0.0001) and ERβB (p<0.05). In male liver there was an upregulation in GR (p<0.05), and AR (p<0.05) mRNA expression and a down regulation of ERβB (p<0.0001). Lastly, within the ovary there is an increase in AR (p<0.05) and ERβA (p< 0.01) mRNA expression and a decrease in GR (p<0.05) mRNA expression. These findings indicate that phenothiazine may alter endocrine function through the steroid hormone receptors. Collectively, these tissue specific results demonstrate how phenothiazine disrupts normal fish physiology. Additionally, serum cortisol levels were assayed via enzyme linked immunosorbent assay (ELISA) and no significant changes in cortisol levels following phenothiazine treatment were observed.

Support or Funding Information

Connecticut Community Foundation, Quinnipiac River Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NG291: A Stable B2 Kinin Receptor Agonist that Increases the Permeability of Normal Blood Brain Barrier: Possible Role in Organophosphate Poisoning Treatment
Authors: Rodriguez Sergio Rafael , Martinez Solianne , Martins Henrique Antonio
Published: June 2021

Organophosphates are phosphorus containing compounds that inhibit acetylcholinesterase and generate a cholinergic crisis that is fatal if not treated. The classical antidotes prescribed are atropine and pralidoxime. They can protect peripherally, but cannot protect the central nervous system. Pralidoxime is the only FDA approved drug that re‐activates the acetylcholinesterase inhibited by organophosphate. Pralidoxime has limited neuroprotective potential because it is not able to cross the blood brain barrier (BBB) effectively. In order to increase this potential, our group is using a stable B2 kinin agonist NG291. This peptide can transiently open the normal blood brain barrier and increase the pralidoxime delivery to the brain after organophosphate poisoning. Sprague‐Dawley (SD) rats were subjected to NG291 treatment (50μg/Kg & 100μg/Kg) via intravenous (IV) route followed by 2% Evans Blue dye injection. 24 hours after the peptide injection the animals were perfused, and the amount of albumin‐Evans blue that reached the brain was qualitatively analyzed. To verify if the transient opening of blood brain barrier causes increase in the brain water content, controls (saline) and rats injected with the peptide NG291 were euthanized 24 hours after the peptide injection and the percentage of water content was measured. To assure that this transient opening is not causing damage to the brain, control and tests were submitted to analysis by histology using Fluoro Jade to verify the presence of dead neurons and glial fibrillary acidic protein (GFAP) to evaluate the astrocyte activation 72 hours after the injection of NG291. NG291‐treated animals have shown infiltration of Evans Blue to the brain parenchyma and accumulation in the ventricles showing increase of blood brain barrier permeation 24 hours after the injection. The water content of the brain did not increase with NG291 treatment. 72 hours after the treatment the histology using Fluoro Jade and GFAP did not show significant changes when compared to controls. We also determined for how long the BBB remains permeable following NG291 administration. In future studies, this peptide will be used to facilitate the delivery of pralidoxime into the brain and therefore increase the overall effect of this drug. This is done in hope to provide those currently exposed to organophosphates with an immediate treatment strategy.

Support or Funding Information

University of Puerto Rico Medical Sciences Campus RCMI‐8G12MD007600 Dr. Pedro Ferchmin and Dr. Vesna Eterovic, Universidad Central del Caribe

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simultaneous Spatial Mapping of Emtricitabine, Tenofovir, and Efavirenz in Mouse Tissue Using MALDI Mass Spectrometry Imaging
Authors: Seneviratne Herana Kamal , Hamlin Allyson N , Bumpus Namandjé N
Published: June 2021

Emtricitabine (FTC), tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug) and efavirenz (EFV) inhibit human immunodeficiency virus (HIV) replication and are currently used in combination for HIV treatment. FTC and TFV are nucleotide analog reverse transcriptase inhibitors whereas EFV is a non‐nucleoside analog. Despite the clinical importance of these drugs as commonly used antiretrovirals, their spatial distributions within tissues including those susceptible to HIV infection are poorly understood. Therefore, in this study, we employed a Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) method to detect, identify and localize FTC, TFV and EFV in mouse tissues following in vivo dosing. For this study, brain and liver were obtained from mice (n=3) 24 h following oral administration of FTC‐TFV‐EFV. Tissue samples were embedded with OCT medium and 20‐micron tissue sections were utilized for tissue imaging experiments. MALDI matrix α‐cyano‐4‐hydroxycinnamic acid was employed to facilitate the ionization and detection of all three compounds, FTC, TFV and EFV. FTC and EFV were detected as their sodium adducts, [M+Na]+ at m/z 270.0319 ± 5 ppm and 338.0166 ± 5 ppm, respectively; while, TFV yielded its protonated molecular ion, [M+H]+ at m/z 288.0856 ± 5 ppm. When collision‐induced dissociation fragmentation experiments were performed using an FTC‐TFV‐EFV mixture, FTC, TFV and EFV exhibited major fragment ions at m/z 130.0409, 270.0746, and 272.0430 corresponding to C4H5FN3O+, C9H13N5O3P+, and C13H10ClF3N+ structural moieties, respectively. Tissue spatial distribution profiles of FTC, TFV and EFV generated using their corresponding ions (sodium adducts and molecular ions) exhibited differences in their distribution across tissue sections. Interestingly, ion intensity of TFV was greater in liver tissue sections than FTC and EFV. In addition, relative homogeneity in distribution of an endogenous lipid species, PC (16:0/OH) indicated that the distribution patterns observed for above molecules were distinct. In order to correlate the tissue distributions of FTC, TFV, and EFV with their lipophilicities, Log P values were calculated. In conclusion, the above results demonstrate the differential spatial distribution of FTC, TFV and EFV in mouse brain and liver following in vivo dosing. Further, results from this study provide important insights into the disposition of FTC, TFV and EFV.

Support or Funding Information

This work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781

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 Vitamin A Metabolome in Healthy and Diseased Human Liver Using LC‐MS/MS Analysis
Authors: Zhong Guo , Kirkwood Jay , Won Kyound‐Jae , Tjota Natalie , Jeong Hyun‐Young , Isoherranen Nina
Published: June 2021

Retinoids are important endogenous compounds responsible for regulating multiple biological events. Currently, the qualitative and quantitative information about retinoid concentrations in human tissues is limited. This prevents a deeper understanding how retinoid homeostasis is regulated in vivo. In this study the hepatic vitamin A metabolome in 50 normal human liver samples was quantified using novel LC‐MS/MS methods and isotope‐labeled internal standards. Measured retinoids included retinyl palmitate (RP; median, 276 nmols/g; range, 35.1–4,282 nmols/g), retinol (ROL; median, 4.9 nmols/g; range, 0.8–39 nomls/g), all‐trans retinoic acid (atRA; median, 39.8 pmols/g; range, 14.9–670.5 pmols/g), 13‐cis‐RA (median, 8.3 pmols/g; range, 1.4–43.5 pmols/g), 4‐oxo‐atRA (median, 18.3 pmols/g; range, 1.8–376.3 pmols/g) and 4‐oxo‐13‐cis‐RA (median, 13.0 pmols/g; range, 5.3–64.0 pmols/g). Two 4‐oxo‐RA isomers were, for the first time, detected in human tissues and their concentrations were comparable to RA isomers. As 4‐oxo‐atRA also activates retinoic acid receptors (RARs), its presence in human liver suggests that 4‐oxo‐atRA is essential in the regulation of retinoid homeostasis. Additionally, atRA concentration was positively correlated with that of other retinoids in normal livers, but not with the relative mRNA expression of LRAT, ALDH1A1, CYP26A1, RARα and RARβ, all of which are retinoid‐related genes. Finally, the vitamin A metabolome was also measured in livers from subjects with non‐alcoholic fatty liver disease (NAFLD) (steatosis, n=9; nonalcoholic steatohepatitis (NASH), n=13) to explore whether the development of NAFLD is associated with disturbed vitamin A homeostasis. The data showed a decrease of hepatic concentrations of RP, atRA, 13‐cis‐RA, 4‐oxo‐atRA and 4‐oxo‐13‐cis‐RA in NAFLD liver samples when compared to normal liver samples, while the hepatic ROL level remained unchanged, indicating that hepatic vitamin A homeostasis is disrupted in NAFLD.

Support or Funding Information

This work was supported in part by grants from the National Institutes of Health R01 GM111772 and T32 DK 007247.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SOD2 confers development of resistance to PI3K pathway inhibition associated to enhanced stemness features in head and neck cancer
Authors: Chang Kwang‐Yu , Hsueh Wei‐Ting , Chu Jui‐Mei , Chou Shao‐Wen , Chang Wen‐Chang , Chuang Jian‐Ying
Published: June 2021

Background

Recent development of targeting therapies in head and neck cancer (HNC) focuses on the phosphoinositide 3‐kinase (PI3K) pathway, which is known to be a crucial factor in cancer development and resistance to multiple drugs. The effect of the specific inhibitors, however, is often overshadowed by the development of resistance which the mechanism remains to be determined.

Method

BEZ235, a dual inhibitor targeting against PI3K and the mammalian target of rapamycin (mTOR), was used to develop resistant HNC lines by long‐term co‐incubation and selection of the single cell clones. Cellular profiles of reactive oxidative stress (ROS) and the stemness features were explored to analyze the PI3K related functions.

Results

The resistant cells showed higher IC50 of the proliferation in presence of the PI3K/mTOR dual inhibitors BEZ235 and BGT226, the specific PI3K inhibitor LY294002, and the mTOR inhibitor rapamycin. They exhibited cross resistance to an EGFR inhibitor gefitinib and a platium chemotherapy cisplatin. Examination of the signaling in presence of BEZ235 showed persistent PI3K activation with expression of phosphorylated AKT at threonine 308 in the resistant cells. The activated AKT signaling was also noted when the cells were treated with cisplatin. the stemness features were assessed by flow cytometry with increased ALDH level noted in the resistant cells. This was supported by increased associated biomarkers in qPCR and higher Bmi1, Sox2 expression in Western blot. Functional studies indicated increased expression of ROS in the resistant cell lines. Analysis of the ROS scavengers revealed higher SOD2 mRNA level by qPCR but not SOD1. Notably from a publicly available cancer microarray database Oncomine, higher SOD2 expression was predominantly shown comparing to the other scavengers in the HNC tissues than the normal ones. Finally, the association of SOD2 in the stemness‐featured cells were shown by higher gene expression in the Alderfluor‐stained ALDH‐positive parental cells. Since treatment with BEZ235 further augmented SOD2 expression, co‐incubation of SOD inhibitors and BEZ235 reduced development of spheroid cells and showed advantage in successful inhibition of these stemness‐featured cells.

Conclusion

Acquisition of resistance may involve alterations of cell population which has more dominant survival mechanism. This may contribute to the cross‐resistance of the other anti‐tumor drug. Intracellular ROS modulation has crucial role in the process. Early intervention may be needed for keeping cells from developing resistance.

Support or Funding Information

This study was supported by the grant from National Health Research Institutes, Taiwan (CA‐107‐PP‐08)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Primary cilia in cancer cells
Authors: Jamal Maha Hassan , Nauli Surya
Published: June 2021

Primary cilia are sensory organelles that exist on the surface of most cells. Since primary cilia play an important role in cell cycle and cilia‐related signaling pathways (e.g. Hedghog and Wnt signaling pathways), ciliary dysfunction has been proposed as an essential factor of cancer development. In our current study, we characterized the expression of the primary cilia in two breast cancer (MCF7 and MDA132), two prostate cancer (PC3 and DU145), and one lung cancer (NL20‐TA) cell lines by counting number of cilia formation and measuring cilia length. We also examined the effect of shear stress on the cilia number and cilia length. Moreover, we investigated the correlation between primary cilia and cilia‐related signaling pathways. We found that the frequency and the length of the primary cilia were reduced in MCF7 and MDA‐MB‐231 breast cancer cells and in PC3 prostate cancer. Primary cilia were absent in DU‐145 prostate cancer and NL20‐TA lung cancer. The β‐catenin of Wnt signaling pathway was elevated in the prostate and lung cancers, while Gli1 and smoothened of Hedghog pathway remained the same. Our results suggested an association between primary cilia and Wnt signaling in cancer cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bisdemethoxycurcumin potentiates gemcitabine to induce mitochondrial dysfunctions and apoptosis in human pancreatic adenocarcinoma cells
Authors: Li Xuejun
Published: June 2021

Pancreatic cancer is a highly aggressive malignancy, gemcitabine is indicated as a standard single agent for the treatment of pancreatic cancer, however it only extends survival by a mere 3–6 weeks. In this present study we investigate whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, potentiates gemcitabine in human pancreatic cancer cells. The result suggested that BDMC produced a synergistic effects with gemcitabine by inducing mitochondrial dysfunctions and apoptosis in PANC‐1 and MiaPaCa‐2 pancreatic cancer cells. By using 2D‐MS, we found 13 essential proteins were significantly changed in response to gemcitabine alone or combined with BDMC. Pharmacological network analysis revealed that glucose‐regulated protein 78 (GRP78) as the hub activated by BDMC. We then demonstrated that BDMC upregulates GRP78 and facilitated apoptosis through eIF2α/CHOP pathway. In addition, DJ‐1 and prohibitin, two identified markers of chemoresistance, were increased by gemcitabine in PANC‐1 cells. This could be reversed by BDMC, suggesting that BDMC partially offsets the chemoresistance induced by gemcitabine. In conclusion, our study shows that BDMC promotes apoptosis through a GRP78‐dependent pathway and mitochondrial dysfunctions, and potentiates the antitumor effect of gemcitabine in human pancreatic cancer cells.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (No. 81874318, 81673453, 81473235).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory effects of caffeic acid phenethyl ester (CAPE) derivatives on multiple myeloma cell growth
Authors: Jackson My Lien , Wang Xinyu
Published: June 2021

Multiple myeloma (MM) is a rare blood cancer that affects thousands of people yearly in the U.S. It stems from monoclonal plasma cells and can cause symptoms such as bone loss and fatigue. Recent studies have shown that the bone marrow microenvironment may mediate the tumor proliferation, drug resistance, and migration of the MM cells. Caffeic acid phenethyl ester (CAPE) has been studied in research due to their anti‐cancer and anti‐inflammatory effects. The objective of this study is to examine the inhibitory effects of CAPE derivatives on MM cell growth and explore the structure‐activity relationship of CAPE. Selected CAPE derivatives include ferulic acid methyl ester (FAME), caffeic acid methyl ester (CAME), caffeic acid (CA), N‐octyl caffeate (NOC), phenethyl dimethyl caffeate (PDC), phenylethyl‐4‐methylcaffeate (P4MC), and fluorinated CAPE (FCAPE). These seven CAPE derivatives with a dose range from 1.0 to 50 μM were tested on the growth of MM RPMI 8226 cells within 72 hours. Our current results show that FCAPE, P4MC, and NOC decreased cell viability and proliferation of the MM 8226 cells in a dose and time‐dependent manner. These derivatives were selected to further study whether its inhibitory effect on MM cell growth is due to the induction of apoptosis. Western blotting was used to evaluate the protein expression of apoptosis‐related factors such as caspase 3, caspase 8, caspase 9 and poly‐(ADP‐ribose) polymerase (PARP). To evaluate the effect of these CAPE derivatives on MM 8226 cells in the presence of the microenvironment, a Transwell (TW) model using MM cells co‐culturing with bone marrow stromal cells will be applied to mimic the bone marrow microenvironment in vivo. When treating the HS‐5 stromal cells with CAPE derivatives, no significant decrease in proliferation is seen after 24 hours. We expect that after co‐culturing the MM 8226 cells with HS‐5 stromal cells using the TW model, the growth of MM cells will still be prohibited by those CAPE derivatives to some extent if not the same as MM cells being treated alone. In conclusion, our data suggest that CAPE derivatives inhibit the growth of MM cells through induction of apoptosis, and this effect of CAPE requires the integrity of a catechol ring functional group in order to carry out its cytotoxic effects.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ethylacetate fractional extract of Mentha spicata attenuates irinotecan‐induced mucositis in mice
Authors: Kathem Sarmed H , Abdu‐Jabbar Aliaa A , Ataimish Ahmed H , zalzala Munaf H
Published: June 2021

Irinotecan induced‐mucositis is an inflammatory event of intestine that has negative impact on patient health and affect treatment efficacy. Irinotecan first metabolized in the liver by a carboxylesterase (CES) to the active cytotoxic metabolite (SN‐38), which subsequently glucuronidated by hepatic enzymes to the inactive metabolite SN38G. The SN‐38G undergo deconjugation in the intestine liberating the active SN‐38 via β‐glucuronidase produced by the intestinal bacterial flora. Consequently, SN‐38 triggers inflammation events characterized by delayed intestinal mucositis and diarrhea. This study aims to ameliorate irinotecan‐induced mucositis and diarrhea in mice model using ethylacetate fractional extraction of Mentha spicata(EAEMS), an edible widley consumed plant. Mice model of irinotecan‐induced mucositis and diarrhea produced by I.P injection of irinotecan (75mg/Kg/day) for 4 days. EAEMS administered orally (160mg/kg) twice daily for 7 days starting one day before irinite can dose. Diarrhae scores and mice body weight variation measured to assess the severity of mucositis and diarrhea. Jejunal IL‐1β and MPO measured as markers of intestinal inflammation. β‐glucuronidase activity in fecal pelletes were measured also. Compared to model control mice, results revealed that animals received EAEMS presented with significantly lower diarrhea scores and body weight loss. Furthermore, jejunal IL‐1β and MPO significantly reduced and associated with significant inhibition of fecal β‐glucuronidase activity in animals received EAEMS compared to model control mice. In conclusion, we showed for the first time that EAEMS has a protective effect against irinotecan‐induced mucositis and diarrhea, an effect suggested to be mediated by inhibition of bacterial β‐glucuronidase activity. This promising result might have a therapeutic application in cancer patients suffering from delayed‐onset diarrhea due to irinotecan treatment.

Support or Funding Information

University of Baghdad, College of Pharmacy

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer Activity of Aqueous Extracts of Terminalia arjuna (TA) Bark
Authors: Liu Shi J , Liu Meeiyueh , Li Hong‐yu J , Simmen Rosalia CM , Johann Donald J
Published: June 2021

Our previous study demonstrated that an aqueous extract of TA bark (TAAqE) exerts cardioprotection against toxicity induced by doxorubicin (DOX), a commonly‐used anticancer chemotherapeutic agent. However, whether TAAqE has any effect on cancer cells remains unknown. Herein we investigated the effects of TAAqE on several human lung and breast cancer cell lines (ATCC) in vitro and on mice (Wnt‐transgenic model) bearing mammary tumors. Cell morphology and growth were monitored with phase‐contrast microscopy, while the colorimetric Cell Counting Kit (CCK)‐8 assay was used to evaluate cell viability. Fluorescent microscopy with Hoechst H33342, AnnexinV, propidium iodide (PI), and JC‐1 (a mitochondrial membrane potential dye) was used to detect cell membrane and mitochondrial integrity. Tumor growth in vivo was monitored with ultrasound biomicroscopy. We found that TAAqE, its fractions (designated as P faction and S fraction) and the ethanol extract of TA bark (TAEtOH) at 50 μg/ml inhibited growth of all cancer cells with negligible effect on non‐tumor lung fibroblasts (CCD‐19Lu) after 24‐h treatment. The order of the cytotoxic potency (at 50 μg/ml) was TAEtOH > P fraction > TAAqE ~ S fraction > gallic acid (10 μM), an active component of TAAqE, based on the cell viability assay. When TAAqE was separated with Microcon filter devices, the molecular mass of active components was greater than 10 kDa. Distinct AnnexinV/PI expression of these cancer cells suggested differential effects of TAAqE on apoptotic status. TAAqE and TAEtOH caused dose‐dependent cytotoxicity of human lung adenocarcinoma cells (A549) with 50 μg/ml showing more effective effects and different morphological changes such as increasing cytosolic vacuolization compared to DOX (1 μM). In non‐small cell lung cancer cells (H1975), TAAqE (5 μg/ml) caused a decrease in mitochondrial membrane potential in contrast to CCD‐19LU cells. In vivo treatment of Wnt‐transgenic mice with TAAqE (15 mg/kg body weight, daily in drinking water) for 3 days or 4 weeks reduced mammary tumor sizes with no recurrence noted 3.5 weeks and one month post‐treatment, respectively. In conclusion, TAAqE displays potential as an adjunct for cancer therapy in addition to its cardiotoxic protective effects. Mechanistically, TAAqE may induce cell membrane damage and reduction of mitochondrial function of cancer cells.

Support or Funding Information

UAMS Seeds of Science Funds to SJL

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of rat models with myocardial ischemia or anoxia and the expression of succinate receptor
Authors: Liu Yongxue , Zhang Fangyuan , Qi Yue , Yuan Meiru
Published: June 2021

Objective

To establish rat models with myocardial ischemia and anoxia and to detect the expression of succinate receptor (GPR91) in rat myocardium.

Methods

Total 48 Sprague‐Dawley (SD) rats used in the experiment. For the induction of myocardial ischemia, eighteen rats were randomly divided into three groups of sham operation (control), coronary artery ligation 12h and 24h, respectively. The rats were anaesthetized with sodium pentobarbital and ligated by the left anterior descending artery for 12h and 24h. The control rats were operated as the same except ligation. The electrocardiogram (ECG), the area of myocardial infarction and the serum contents of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin 1 (cTn‐1) in rats were recorded and detected at the setting time. For the induction of myocardial anoxia, thirty rats were divided into five groups of living in normal atmosphere (control), living in low atmosphere for 1d, 3d, 5d, and 7d. At the setting days, the serum contents of lactate LDH, CK and cTn‐1 in rats were detected. Real‐time fluorescent quantitative PCR and Western Blot were used for GPR91 mRNA and protein expression in rat myocardium.

Results

In the rats ligated by the left anterior descending artery for 12h and 24h, continuous ST segment elevations were recorded in ECG; the ratio of infarct size were 23.18±3.46 and 34.85±2.22 (P<0.05, compared with the control); the serum levels of LDH, CK and cTn‐1 were increased in some extent. In the rats with myocardial anoxia for 1d, 3 d, 5d, and 7d, elevation of LDH, CK and cTn‐1 contents in serum were also detected. Compared with that of the control rats, the expression of GPR91 mRNA and protein in myocardium of myocardial ischemia or anoxia rats were both up‐regulated.

Conclusion

The left anterior descending artery ligation or living in low atmosphere could induce myocardial ischemia or anoxia in rats. The expression of GPR91 mRNA and protein in myocardium of rats with myocardial ischemia or anoxia was increased. It implied that GPR91 could serve as a target for prevention and treatment of ischemic or anoxia myocardial injury.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Mechanism of Regulation of Sodium Channel Nav1.5 Expression in Hypoxic Cardiomyocytes
Authors: Wang Ning , Liu Yining , Li Jiamin , Yang Zhenyu , Cai Benzhi , Xu Haodong
Published: June 2021

Hypothesis

Because of the increased susceptibility of arrhythmias in patients with myocardial infarction (MI) in clinic, we hypothesized that Meis1, a critical developmental transcriptional factor, regulates NaV1.5 expression by affecting the SCN5a promoter activity, which leads to an alteration of cardiac electrical conduction and has a potential role in the regulation of NaV1.5 expression in hypoxic cardiomyocytes.

Methods

MI mouse model was induced by ligation of the left anterior descending artery (LAD) of mouse heart. Echocardiography and electrocardiogram (ECG) were performed to determine cardiac function and electrical activity, respectively. qRT‐PCR and western blot were used to determine the NaV1.5 and Meis1 expression levels in peri‐infarct (border zone) region of MI mouse hearts. In vitro, neonatal mouse ventricular cardiomyocytes were treatment with hypoxia (1% O2, 5% CO2, 94% N2) for 24 hours and further infected with Adeno‐Associated Viral‐Meis1 (AVV‐Meis1) and transfected with siRNA specifically targeting Meis1 (si‐Meis1). qRT‐PCR and Western blot were employed to determine if increased or decreased Meis1 had effects on NaV1.5 expression. In silico promoter analysis was performed to determine if the SCN5a promoter harbors Meis1 conservative binding sites in different species. Luciferase activity assay was performed to observe the influence of Meis1 on the SCN5a promoter activity in HeLa cells.

Results

Significantly prolongation of QTc interval was induced in mouse with MI operation. Both mRNA and protein levels of Meis1 and NaV1.5 were significantly decreased in infarcted hearts of the mice, compared to the sham group. In vitro, both mRNA and protein levels of NaV1.5 and Meis1 were significantly decreased in hypoxic ventricular cardiomyocytes of neonatal mouse, in comparison with a control group. Overexpression or knockdown of Meis1 was shown to enhance or reduce the expression of NaV1.5 mRNA and protein in ventricular cardiomyocytes of neonatal mouse. In silico promoter analysis revealed that the SCN5a promoter contains Meis1 conservative binding DNA sequences in humans, mice and rats. Luciferase reporter assay preformed with HeLa cells showed that Meis1 overexpression increased the SCN5a promoter activity.

Conclusion

Collectively, downregulation of Meis1 significantly contributes to NaV1.5 reduction after MI. The underlying molecular mechanism is that meis1 transcriptionally regulates the expression of the NaV1.5 protein by increasing SCN5a promoter activity. Our findings provide a new insight into the molecular mechanisms of sodium channel NaV1.5 expression in the development of ventricular arrhythmias related to MI.

Support or Funding Information

The study was supported by the National Nature Science Foundation of China (grants 81573425 and 81773733).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of CD4 T cells Contributes to Cardiac Fibrosis and Myocardial Dysfunction in T cell Specific S1P Receptor 1 Knockout Mice
Authors: Jin Zhuqiu , Abdullah Chowdhury
Published: June 2021

Sphingosine 1‐phosphate (S1P) is a bioactive small lipid molecule involved in apoptosis, barrier formation and function, cytoskeleton rearrangement, vascular maturation, wound healing, cell trafficking, and other functions via five G protein‐coupled S1P receptors or/and intracellular lipid signaling. T cell S1P receptor 1 (S1P1) is responsible for mature T cell exit from thymus to the blood and periphery. Nevertheless, effects of T lymphocyte S1P1 on cardiac function and fibrosis remains incomplete understood. To this purpose, we generated T cell S1P1 knockout mice (TS1P1KO) by crossing S1P1loxP/loxP mice with Lck‐Cre mice. Body weight (g) and glucose levels (mg/dl) in the blood were monitored. CD4 and CD8 T lymphocytes were counted by using flow cytometry. Pathological grading of heart histology was conducted in H&E stained sections. Masson Trichrome staining was utilized to evaluate cardiac fibrosis. Cardiac contractility was determined in Langendorff perfusion system. The TS1P1KO mice exhibited sustained deficiency of both CD4 and CD8 T cells in the blood compared with wild‐type littermate control. The TS1P1KO mouse heart featured an altered phenotype characterized by reduced cardiac contractility and increased cardiac fibrosis as well as morphological alteration, notably, loss of interstitial space between cardiac myocytes with thickening of vascular smooth muscle cells and perivascular edema compared with littermate vehicle mice. To explore the role of CD4 T cells in altered phenotype, naïve CD4 T cells were isolated by positive selection through MS column from CD4 magnetic microbeads‐labeled wild‐type littermate mouse splenocytes. Isolated CD4 T cells were adoptively transferred into TS1PKO mice intravenously at a dose of one million cells. TS1P1KO mice with reconstitution of CD4 T cells showed improved heart histology and less fibrosis as well as improved cardiac function compared with TS1P1KO mice without CD4 T cell transfer. In conclusion, deficiency of CD4 T cells in T cell S1P1 knockout mice contributes to cardiac fibrosis and myocardial dysfunction.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potency Adjusted Bovine Heparin is Comparable to Porcine Heparin in Patients Undergoing Open Heart Surgery
Authors: Gomes Walter , Braile Domingo , Leal Joao Carlos , Guizilini Solange , Regenga Marisa , Kouta Ahmed , Walenga Jeanine , Jeske Walter , Hoppensteadt Debra , Ramacciotti Eduardo , Nader Helena , Lima Marcelo , Fareed Jawed
Published: June 2021

Introduction

Heparin obtained from bovine intestinal mucosa is being considered for reintroduction in cardiovascular, surgical, and interventional procedures. Although the anticoagulant potency of bovine heparin is weaker (130–140 U/mg) than that of porcine heparin (180–200 U/mg), when potencies are adjusted to equivalent USP U/mL activity, both porcine and bovine heparin exhibit comparable anticoagulant activities in in‐vivo and in‐vitro settings.

Aim

The primary aim of this study was to compare bovine and porcine heparins at equivalent dosing in terms of anticoagulation and potential bleeding effects in patients undergoing coronary artery bypass graft (CABG) surgery.

Materials and Methods

Coagulation parameters and biomarkers of inflammation were measured in 32 patients included in a single center, blinded, controlled clinical trial, randomized to CABG surgery with either bovine or porcine heparin. Equivalent dosages of 28,500 units (potency adjusted to 10,000 U/mL) total dose heparin were used. Blood samples were collected at multiple times during and after surgery to measure global clotting activity (ACT, APTT) and amidolytic anti‐Xa levels. TNFa, IL‐6, and TFPI antigen were measured by ELISA; activated protein Cwas measued by a clot‐based assay. A preliminary report of these results has been published (Int J Cardiol 223:611–612, 2016).

Results

All coagulation parameters (ACT, APTT, anti‐Xa, residual bleeding) demonstrated comparable heparinization (p ≥ 0.05). The only difference between groups was an increased amount of protamine required to neutralize bovine heparin after surgery determined by ACT; expected due to the higher gravimetric quantity of bovine heparin used to adjust the U/mL potency. No differences were found between groups in levels of acute inflammatory markers (p ≥ 0.05).

Conclusion

This study shows that bovine and porcine heparins at potency adjusted dosages are equivalent anticoagulant drugs in CABG surgery resulting in comparable perioperative coagulation, bleeding, and anti‐inflammatory effects.

Support or Funding Information

None.

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 Differences in the Antiplatelet Effects of DT‐678 and Existing P2Y12 Antagonists
Authors: Henderlong Dawn Shirley , Deters Mackenzie Rae , Christian Barbara DaShawn , Lauver Dale Adam
Published: June 2021

Arterial thrombosis, leading to tissue ischemia, is the most common cause of death in the developed world. Dual antiplatelet therapy with low dose aspirin and a P2Y12 antagonist is widely used to prevent thrombus formation. The P2Y12 antagonist, clopidogrel, is a prodrug that undergoes a complex metabolism process involving cytochrome P450 enzymes (CYP450) to produce the biologically active metabolite (AM). The AM covalently binds to the P2Y12 receptor on platelets thereby inhibiting receptor activation by ADP resulting in the inhibition of platelet activation. Approximately 60–70% of Asians and 30% of Caucasians carry a genetic polymorphism in the CYP450 that prevents the production of the AM. Our laboratory has previously reported the development of DT‐678, a novel conjugate of the AM that does not require CYP450s. Instead, the drug is nonenzymatically activated through a glutathione exchange reaction.

Previous studies have demonstrated that DT‐678 is equally as effective as clopidogrel and ticagrelor at inhibiting platelet activation, while requiring a lower dose than clopidogrel. Additionally, we have shown that while clopidogrel and ticagrelor induce a 1‐ to 2‐fold increase in bleeding time, DT‐678 does not induce a significant increase in bleeding time. The purpose of the present study was to compare the antithrombotic effects of DT‐678 to clopidogrel and ticagrelor after oral administration. To do this, we used a New Zealand white rabbit model of FeCl3‐induced carotid artery injury. All three antiplatelet drugs showed an equal efficacy in prolonging the time to occlusion after FeCl3 application compared to vehicle. Further data suggest that DT‐678 differentially affects calcium mobilization in platelets and other cell types compared to clopidogrel and ticagrelor after application of 2MeSADP. Together, these data suggest mechanistic differences in the action of DT‐678 compared to existing P2Y12 antagonists and may explain differences in the bleeding times observed.

Support or Funding Information

National Institutes of Health National Heart, Lung and Blood Institute [1R43HL139380‐01]

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