bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipid‐derived electrophiles regulate isoform‐specific redox‐dependent kinase signaling
Authors: Surya Sanjna , Parvez Saba , Long Marcus John Curtis , Zhao Yi , Haegele Joseph , Huang Paul , Aye Yimon 
Published: November 2023

Akt is a serine/threonine‐specific kinase and a master regulator of metabolism, proliferation and apoptosis. Isoform‐specific deregulation of Akt plays a key role in distinct tumor phenotypes. Pan‐Akt inhibitors such as MK‐2206 are currently in clinical trials for cancer therapy, further highlighting the clinical significance of understanding Akt regulation and innovating novel tools to inhibit its activity. However, methods to develop isozyme‐specific therapeutics have remained elusive, due to large sequence homology and functional similarity among the three Akt isozymes, Akt1, 2 and 3. Recently, in a collaborative study with my graduate student and postdoc mentors (Long et al. Nature Chemical Biology 2016 accepted), we discovered that Akt3 is a privileged responder to 4‐hydroxynonenal (HNE), a bioactive lipid‐derived electrophilic signal. Target‐specific electrophilic modification of a cysteine residue (C119) unique to Akt3 resulted in downregulation of Akt3 activity in mammalian cells as well as in zebrafish. This modification alone was sufficient to upregulate endogenous downstream pro‐apoptotic genes. Akt1 and Akt2 were both unresponsive under otherwise identical conditions. My independent research will build on this discovery to investigate the functional impacts of HNE modification of a non‐cysteine residue, which we had recently discovered is also modified by HNE. This residue is also unique to Akt3. My preliminary data suggests that a functional mutation of this residue results in upregulation of Akt3 kinase activity. We are now making headway towards examining the effects of this non‐cysteine site‐specific electrophilic modification on downstream targets of Akt3 using chemical biology and genetic approaches in cultured cells and zebrafish that are well established in our laboratory. Further investigations in this area promises to reveal intricate regulatory mechanisms of kinase isozymes by endogenous lipid‐derived electrophiles, ultimately aiding in the development of isozyme‐specific drugs.Support or Funding InformationWe acknowledge the Hill Fellowship for Undergraduate Summer Research to Sanjna Surya, HHMI pre‐doctoral fellowship to Saba Parvez, and NIH New Innovator Award (1DP2GM114850), Beckman Young Investigator, NSF CAREER (CHE‐1351400), Burroughs Wellcome CRTG, and Sloan Fellowship programs to Yimon Aye for research support.
Gene Expression of SEB‐Induced C‐Jun N‐Terminal Kinase Apoptosis Pathway in Human PBMCs
Authors: Hendricks Michelle , Borgos Dylan , Butzen Jessica , Watson Alex , Mendis Chanaka , Jett Marti 
Published: November 2023

Staphylococcus aureus is a bacterium that produces Staphylococcus Enterotoxin B (SEB) which is known to cause apoptosis. The purpose of this study is to further investigate human PBMCs to better understand the SEB induced apoptosis events. It has been previously identified that SEB induces C‐Jun N‐Terminal Kinase (JNK), which is an interconnector of multiple different cascading signals. Since JNK has been identified as a central interconnector in SEB induced human PBMCs we believe that targeting JNK will also inhibit the effects of SEB induced apoptosis. This study used the JNK specific inhibitor SP600125 to assess the changes to the gene expression profile using RTPCR and PCR. Enzyme linked immunosorbent assays (ELISAs) were used to study the protein expression patterns of some genes of interest. We believe that our work will not only allow us to better understand the effect on apoptosis but also the complex interactions of multiple signal transduction pathways induced by SEB.
Eliza assay protein expression of caspase 3, 6, and 8.

Gene
SEBC
SEB 24
SEE SB5
SEB SB24
SEB SP5
SEB SP24

Caspase 3
1
1.056
0.902
2.784
0.745
0.706

Caspase 6
1
1.026
0.667
0.615
0.718
0.641

Caspase 8
1
1
1.24
1.04
0.96
0.92
Post‐Transcriptional Modulation of MCL1 by PTBP1 Regulates Cellular Apoptosis Induced by Antitubulin Chemotherapeutics
Authors: Cui Jia 
Published: November 2023

BackgroundMyeloid Cell Leukemia 1 (MCL1), an anti‐apoptotic Bcl‐2 family protein, is a key regulator of intrinsic apoptosis. Normal cells require strict control over MCL1 expression and aberrant MCL1 expression has been shown linked to the emergence of various diseases and chemoresistance. Previous studies have detailed how MCL1 expression is regulated by multiple mechanisms both transcriptionally and translationally. However, characterization of the post‐transcriptional regulators of MCL1 mRNA is limited. Polypyrimidine tract binding protein 1 (PTBP1) is a known regulator of post‐transcriptional gene expression that can control mRNA splicing, translation, stability, and localization. In the described study we characterize the impact that PTBP1 has on MCL1 expression, its molecular mechanisms, and its roles on cellular apoptosis.MethodsCross‐linking Immunoprecipitation (CLIP)‐seq data analysis and RNA immunoprecipitation (RIP) were performed to analyze protein interactions with RNA and map the RNA binding sites on the genes of interests. RNA half‐life was measured by treating cells with Actinomycin D and extracting total RNA at different time point. Cell viability was measured by the MTS assay and apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining followed by flow cytometry analysis.ResultsOur studies demonstrate that PTBP1 binds to MCL1 mRNA and that knockdown of PTBP1 up‐regulates MCL1 expression in cancer. Mechanistically, PTBP1 silencing stabilizes MCL1 mRNA. We further identify that this post‐transcriptional regulation is via the Argonaute2 (Ago2)‐mediated microRNA targeting to MCL1. Moreover, we show that PTBP1 down‐regulation is anti‐apoptotic to antitubulin chemotherapeutics in a MCL1‐dependent manner.ConclusionsOur findings suggest that PTBP1 is a novel post‐transcriptional regulator of MCL1 mRNA by which it controls apoptotic response to antitubulin chemotherapeutics.Support or Funding InformationThis research was funded by grants from the UAB Comprehensive Cancer Center Young Supporters Board, NIH grants R01 GM117391 (to WJP), and the UAB HHMI Med into Grad Fellowship (to JC).
Intracellular zinc trafficking and metallothionein gene activation in HUVEC treated with Crotalus atrox venom
Authors: Albrecht Eric , Garbar Veronica , Tomlins Scott , Williams Evan 
Published: November 2023

Hemorrhagic snake venom, such as Crotalus atrox, destroys local tissue and disrupts hemostasis. In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with C. atrox snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of C. atrox venom. HUVEC monolayers stimulated with 10 μg/ml C. atrox venom for 3 hours displayed cellular retraction, which coincided with 53.0±6.5 percent viability. In contrast, venom concentrations of 100 μg/ml produced a complete disruption of cellular adherence and viability decreased to 36.6±1.0. Venom induce cellular retraction involves cytoskeletal rearrangements. Therefore, we used the zinc probe FluorZin‐3AM to compare intracellular free zinc in HUVEC stimulated with 10 μg/ml C. atrox venom or 1 μg/ml cytochalasin D for 1 hour. Fluorescent intensity analysis returned values of 287.1±57.7 for C. atrox and 261.7±33.3 for cytochalasin D, demonstrating an increase in labile zinc compared to non‐stimulated controls. Labile intracellular zinc or other metals can induce the gene activation of intracellular metal binding proteins known as metallothioneins. Thus, we measured the gene and protein expression of metallothionein 1X (MT1X) and metallothionein 2A (MT2A) in HUVEC stimulated with C. atrox venom. MT2A gene expression increase by 4.4±0.41 fold compared to 96.1±3.0 for MT1X. This data suggest cell death from C. atrox venom involves intracellular zinc trafficking, and metallothionein gene activation.Support or Funding InformationCETL, OVPR Grant
The NPM1 Inhibitor NSC348884 Induces Apoptosis in Neuroblastoma Cells
Authors: Kristjansdottir Kolla , Akgul Nil , Vlcek Kelly 
Published: November 2023

Nucleophosmin‐1 (NPM1) is a nucleolar protein found to be upregulated in several cancers including prostate, colon, and breast cancer. NSC348884, a small molecule inhibitor of NPM1, has been shown to induce apoptosis in many of these cancers and its potential use in cancer therapy is currently being explored. We recently reported elevated levels of NPM1 in neuroblastoma cells, a solid tumor of the nervous system identified predominantly in infants and young children. However, the effects of the inhibitor NSC348884 on neuroblastoma had not been explored. Here, we report that treatment of WS and SY5Y neuroblastoma cells with 1–10 μM NSC348884 results in a concentration dependent decrease in metabolic activity. This was measured using several methods including phase‐contrast microscopy, live/dead fluorescence microscopy and WST‐1 assays. We further show that NSC348884 induces apoptosis in a dose‐dependent manner that correlates with appearance of apoptotic markers including cleaved poly(ADP‐ribose) polymerase (PARP) and Annexin V labeling. This induction of apoptosis was augmented by concurrent cytotoxic drug therapy in cell culture. Treatment using NSC348884 alone, or in combination with other cytotoxic drugs, has the potential to induce apoptosis in cancers that do not respond well to current chemotherapy treatments.Support or Funding InformationThis work was supported by the Department of Biomedical Sciences and the College of Health Sciences at Midwestern University.
Bcl‐2 Dependency in Cell Culture and Mouse Models of Sepsis‐Associated Acute Kidney Injury
Authors: Lynch Kyle Anthony , Del Gaizo Moore Victoria 
Published: November 2023

While sepsis is the third leading cause of death in the world, researchers admit that little is known about how the condition affects vital organs. One organ that is affected greatly in sepsis is the kidney, as up to half of septic patients in the United States develop Sepsis Associated Acute Kidney Injury (SA‐AKI). Prior evidence supports the notion that apoptosis plays an important role in decline of renal function. This project utilizes BH‐3 profiling, which measures changes in mitochondrial membrane permeability after treatment with different pro‐apoptotic peptides in order to predict dependence on specific Bcl‐2 family proteins for survival. As this study is the first to our knowledge to utilize BH‐3 profiling in HK‐2 cells, optimization of the technique are also presented. Using a cell line derived from proximal tubules (HK‐2), initial BH‐3 profiles showing the make‐up of apoptotic proteins have been conducted, which seem to indicate that these cells are dependent on BCL‐Xl for survival even in a non‐stressed state. Additional BH‐3 profiles utilizing HK‐2 cells stressed with a cytokine cocktail of recombinant TNFα, IL‐1β, and IFNγ have also been analyzed to show how dependency changes through the progression of sepsis. These cytokines were choosen since they are elevated clinically in septic patients, and the recombinant cocktail induces 30% apoptosis in HK‐2 cells as confirmed by Annexin‐V FITC staining quantitated via flow cytommetry. Additionally, Western Blot coupled with densiometry has also been used to analyze apoptotic regulatory proteins in in vitro and in vivo models of sepsis in order to adequately characterize the mechanism by which SA‐AKI occurs. In vivo models include kidney samples from a Cecal Ligation and Puncture (CLP) model of sepsis where increased expression of the pro‐apoptotic Bim occurs at 6 hours, as compared to sham controls. Further studies with these tissues as well kidney samples from a Staphylococcus aureus induced septic mouse model are underway.Support or Funding InformationThe authors would like to acknowledge the following funding sources: Elon University Honors Program (KL)Elon University Undergraduate Research Program (KL)Glen Raven Endowment for Undergraduate Research in the Sciences (KL)NSF MRI: CHE‐1229562 (VDGM)NCBI: 2013‐IDG‐1019 (VDGM)FR&D Elon University (VDGM). The authors would like to acknowledge the following for providing tissue samples: Hagir Suliman, DVM, PhD, Duke Cancer Institute, Charles McCall, MD, Wake Forest Translational Science Institute
A Flow Cytometry Analysis of Apoptosis Reversibility in Mammalian Lymphocytes Following Cytokine Deprivation
Authors: Parnes Andrew , Lauzon Robert 
Published: November 2023

Apoptosis is a form of programmed cell death that occurs in multicellular organisms in which cells are dismantled and removed in a controlled manner, avoiding harm to surrounding tissues. Apoptotic cell death involves a series of characteristic morphological and biochemical changes that occur in specific temporal fashion, and include early/intermediate (i.e.: caspase activation and phosphatidylserine externalization) and late (i.e.: internucleosomal DNA fragmentation) events. Previous studies carried out in cancer cell lines treated with various apoptotic inducers revealed that surprisingly, apoptosis was reversible and that cells could recover and resume growth following removal of the inducer. In this study, we have developed a mammalian model of apoptosis rescuability and reversibility using cultured mouse HT‐2 T lymphocytes deprived of the essential growth and survival factor interleukin 2 (IL2). Using quantitative flow cytometry analysis, we demonstrate that HT‐2 cells accumulate in the G1 phase of the cell cycle by 24 hours following IL2 deprivation prior to undergoing DNA fragmentation and death within 36–48 hours. Our findings further indicate that IL2‐deprived HT‐2 lymphocytes can be rescued from apoptosis by re‐addition of IL2 to the culture medium. We show that cellular morphology and cell cycle re‐entry is restored within 24–36 hours. Experiments are currently underway to determine which events along the apoptotic pathway (caspase activity, internucleosomal DNA fragmentation/TUNEL assay, and phosphatidylserine externalization/Annexin V binding) are congruent with reversibility.Support or Funding InformationAll funding provided by the Internal Education Fund (Union College).
Differential Phosphatidylserine sensing by TAM receptors regulates AKT dependent chemoresistance and PD‐L1 expression in epithelial cells
Authors: Kasikara Canan , Kumar Sushil , Birge Raymond 
Published: November 2023

Tyro3, Axl and Mertk (TAMs) are three homologous receptor tyrosine kinases that bind vitamin K‐dependent endogenous ligands, Protein S (ProS1) and Growth arrest specific factor 6 Gas6, and act as bridging molecules to promote PS‐mediated clearance of apoptotic cells (efferocytosis). In recent years, it has become appreciated that TAMs are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo and radio resistance to targeted therapeutics. TAMs have also been implicated as inhibitory receptors on infiltrating myeloid‐derived cells in the tumor microenvironment that can suppress anti‐tumor immunity. In addition to TAM overexpression, externalized PS is also concomitantly up‐regulated in the tumor microenvironment, suggesting a PS/TAM receptor axis operates in the tumor microenvironment. Previously, we developed chimeric TAM reporter cell lines comprised of the extracellular domains of each TAM fused to the intracellular domains of the IFNR1, and reported that each TAM receptor had a unique pattern of activation by Gas6 or ProS1, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In the present study, we leveraged this system to engineer epithelial cells that express WT TAMs, and show that while each TAM can promote PS‐mediated efferocytosis, AKT‐mediated chemo‐resistance, as well as up‐regulate the immune checkpoint inhibitory ligand PD‐L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM‐mediated efferocytosis could be partially blocked by PS‐targeted antibody 11.31 and Annexin V, demonstrating the existing of a PS/PS‐R (TAM‐receptor)/PD‐L1 axis that operates in epithelial cells that may drive immune escape. These studies provide a rationale that anti‐PS, anti‐TAM, and anti‐PD‐L1 based therapeutics may have therapeutic merit as combinatorial checkpoint inhibitors.Support or Funding InformationNIH CA 1650771New Jersey Health Foundation NJHF pilot grantRutgers Foundation grant
Overcoming Resistance to Anti‐EGFR Therapy in Breast Cancer with Rac Inhibitors
Authors: Borrero‐Garcia Luis Daniel , Troche‐Torres Angel Luis , Maldonado Maria del Mar , Dharmawardhane Suranganie 
Published: November 2023

Epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) targeted therapies have been successfully used in breast cancers that overexpress these receptors. Unfortunately, patients often develop intrinsic or acquired resistance to these therapeutics. Therefore, our goal is to determine the mechanisms of EGFR therapy resistance in breast cancer, and test new strategies to overcome therapy resistance. To this end, we created syngeneic gefitinib and lapatinib (EGFR and HER2 therapeutics) therapy resistant variants of the therapy sensitive SKBR3 human breast cancer cell line. Cells that demonstrated clinically relevant fold resistance were selected for further characterization and determination of mechanisms of resistance. Since resistance to EGFR‐targeted therapies often involve activation of downstream signaling, we evaluated AKT/protein kinase B (PKB) and Mitogen Activated Kinase (MAPK) pathways by western blotting for phospho (active) and total proteins, and the small GTPase Rac activity by pulldown assays. Western blot results showed no significant changes in AKT and MAPK pathways; however, Rac activation was elevated in the therapy resistant cells compared to the therapy sensitive cells. Since Rac is involved in regulating metastasis and epithelial‐to‐mesenchymal transition (EMT), and thus, implicated with cancer therapy resistance, we performed Real‐time PCR analysis for microRNAs involved in EMT. Real‐time PCR results revealed downregulation of miR‐200 and upregulation of miR‐221/222 families, which are known to regulate EMT. Moreover, western blot results confirmed the post‐transcriptional regulation of these miRNA targets in therapy resistant cells compared to their therapy sensitive cell variants. In addition to increased mesenchymal populations, EMT may also enhance cancer stem cell‐like properties. Results from mammosphere assays using ultra‐low attachment plates show that both gefitinib and lapatinib treatments reduced mammosphere formation in the parental cell lines, but not in the therapy resistant cell lines. Since Rac activity was higher in therapy resistant cells, we tested the role of EHop‐016, an anticancer small molecule inhibitor designed and developed by us that inhibits Vav/Rac/p21‐activated kinase (PAK) signaling downstream of EGFR. Cell viability assays showed that EHop‐016 (5 microM) reduced the viability of therapy sensitive, as well as gefitinib and lapatinib resistant cell variants by approximately 80%. In conclusion, this data demonstrates the relevance of targeting Rac to chemosensitize EGFR/HER2 therapy resistant cells. Therefore, Ehop‐016 and derivatives have potential in single or combination therapy to overcome therapy resistance in breast cancer.Support or Funding InformationSusan G Komen for the Cure and Puerto Rico Science, Technology, and Research Trust (PRSTRT) grants (to SD), UPR RCM NIH/NIMHHD grants G12MD007600, R25GM061838 (to LDB)
Targeting Both Aberrant Metabolism and Cell Proliferation in Cancer Therapy
Authors: Gibbs Bettine , Masamha Chioniso Patience 
Published: November 2023

Mantle Cell Lymphoma (MCL) is an aggressive malignancy that accounts for 5–10% of non‐Hodgkin lymphomas. Upon relapse, MCL is considered clinically incurable, hence it has the worst prognosis amongst all B‐cell lymphomas. Therefore, it is imperative to identify novel therapeutic strategies that effectively target deregulated pathways that contribute to MCL pathogenesis. The hallmark of MCL is the constitutive expression of the G1‐phase cell cycle regulatory oncogene cyclin D1 which is not normally expressed in B‐lymphocytes. This results in uncontrolled cell proliferation. This cyclin D1 driven hyper‐proliferative phenotype is associated with reduced survival in MCL patients. Uncontrolled cell growth results in metabolic reprogramming of the cells to support the energy and biosynthetic needs of the rapidly dividing cells. The changes in genes involved in metabolism result in increased glucose and glutamine uptake and metabolism in tumor cells. We hypothesize that targeting both hyper‐proliferation and altered cell metabolism would be a more effective way to induce cancer cell death. Western blot analysis shows that MCL cells overexpress both cyclin D1 and glutaminase, the enzyme that is critical for glutamine metabolism. Treatment targeting cell proliferation alone, or metabolism alone decreased the cell‐viability of MCL cells. However, greater cytotoxic activity was observed with combination treatment. Our preliminary data suggests that concurrent targeting of both metabolism and cell proliferation in MCL cells has a synergistic effect that enhances the induction of cancer cell death.Support or Funding InformationButler University Holcomb research award