bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Extracellular Residues Critical to the Epithelial Sodium Channel
Authors: Dismuke Taylor Y , Booth Rachell
Published: November 2023

Disruption of homeostatic sodium reabsorption takes place in the kidney can result in various diseases such as osteoporosis, kidney stones, or hypertension. The protein channel found in the membrane of kidney cells that carries out sodium reabsorption is the epithelial sodium channel (ENaC). Identifying and characterizing residues that are critical to ENaC function are key to understanding ENaC's mechanism of action. Complete understanding of the structure‐function relationships within ENaC's extracellular loop are still rudimentary, but studies have demonstrated that it plays a role in channel regulation. Using random mutagenesis, mutations within the extracellular loop of βENaC were generated and incorporated into a yeast expression system. Qualitative screening using a survival pronging dilution assay with yeast expressing αβγENaC was used to monitor ENaC function. Yeast cells expressing αβMutγENaC demonstrated changes in growth compared to yeast expressing wild‐type αβγENaC. Future studies include sequencing to identify mutations and localization of αβMUTγENaC within yeast cells.

Support or Funding Information

This research was supported by National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number R15GM086798 (R. Booth and W. David) and R25GM10783 (K. Oyajobi, N. Blake, R. Booth, and R. Walter).
Hydrogen sulfide homeostasis and signaling in normal and neoplastic intestinal cells
Authors: Libiad Marouane , Sakamoto Naoya , Fearon Eric , Banerjee Ruma
Published: November 2023

Hydrogen sulfide (H2S) is an important gas signaling molecule with effects on multiple physiological processes including neuromodulation, inflammation and cardiac function. Maintaining healthy levels of H2S in mammalian cells requires tight control between its biosynthesis and catabolism. The biogenesis of H2S involves two enzymes from the transsulfuration (cystathionine β‐synthase (CBS) and γ‐cystathionase (CSE)) and cysteine catabolic (mercaptopyruvate sulfurtransferase) pathways. The sulfide oxidation pathway requires the concerted action of the mitochondrial enzymes: sulfide quinone reductase (SQR), a persulfide dioxygenase (ETHE1), rhodanese and sulfite oxidase. The dominant sulfide oxidation products vary in a tissue‐specific manner and are sulfate in liver and kidney and thiosulfate in colonic mucosa. The mechanism of sulfide‐based signaling remains unclear. Persulfidation of proteins is postulated to be a major mechanism by which sulfide‐based signals are relayed. However, it is unclear whether H2S or a downstream product is involved in persulfidation modification.

Herein, we report the analysis of human colon cancer biopsies and patient‐matched controls that reveal an up‐regulation at the protein level of SQR, ETHE1 and rhodanese in colon cancer tissues. We have also observed a differential distribution of these three proteins in colon cancer vs matched normal tissues. Colon cancer‐derived epithelial cell lines (HT‐29, LoVo, CaCo‐2, RKO and HCT116) also exhibited an increase in the protein levels of SQR, ETHE1 and rhodanese, compared with the nonmalignant colonic epithelial cells, HECE. Mercaptopyruvate sulfurtransferase is the major contributor to H2S production in the colon cells lines that were tested. Supplementation of the HT29 colon cancer cell line with exogenous thiosulfate enhances intracellular persulfide levels as visualized by SSP4 labelling.
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 Information

We 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

Background

Myeloid 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.

Methods

Cross‐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.

Results

Our 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.

Conclusions

Our 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 Information

This 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 Information

CETL, 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 Information

This 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 Information

The 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 Information

All funding provided by the Internal Education Fund (Union College).
Probing the Activation Mechanism of BAK in Mitochondrial Apoptosis
Authors: Tesney Michala , Singh Geetika , Guibao Cristina , Moldoveanu Tudor
Published: November 2023

Proteins of the B‐cell Lymphoma 2 (BCL‐2) family are the main regulators of mitochondrial outer membrane permeabilization (MOMP), the initiating event of the caspase cascade during mitochondrial apoptosis. Upon activation by BID binding to its trigger site, the BCL‐2 effector, Bcl‐2 Antagonist Killer (BAK) mediates MOMP. The mechanism of BAK oligomerization during MOMP remains poorly understood and controversial. It is postulated to involve sequential dimerization at an extensive interface overlapping the trigger site followed by weak affinity association of dimers at a poorly defined interface mediated by helix α6. Here we probed the oligomerization mechanism using disulfide‐bonded dimers of BAK engineered to trap the α6‐α6 interface. Using functional assays based on permeabilization of large unilamellar vesicles that mimic the outer mitochondrial membrane, and size exclusion chromatography multi‐angle light scattering, we probed and refined the current model for BAK‐mediated MOMP.
Interaction Between FAM129B, a Novel Adherent Junction protein, and KELCH‐like Associated Protein 1 (KEAP1) suppresses Apoptosis in Cancer Cells.
Authors: Hachem Fatme Ali , Chen Song Ali , Evans Hedeel Guy , Evans David R.
Published: November 2023

FAM129B, is a member of a family of novel proteins that include FAM129A and FAM129C. Little is known about the structure and functions of these proteins Preliminary data in our lab suggests that FAM129B plays a role in apoptosis suppression and promotes cancer cell invasion. Suppression of apoptosis is one of the mechanisms used by cancer cells to evade cell death and facilitate metastatic spread. FAM129B expression is highly elevated in cancer cell lines compared to non‐cancer cell lines as suggested by western blot analysis. Here, we have shown that FAM129B exclusively suppresses the TNFα induced apoptotic pathway and not the Fas‐mediated apoptosis and the mitochondrial pathway in cancer cells. We then investigated the effect of FAM129B on several components of the TNFα pathway: IkB kinase (IKKβ), the transcription factor NF‐kB, and the anti‐apoptotic proteins c‐FLIP, and cIAP. Silencing FAM129B expression using siRNA in can cells promoted IKKβ degradation and decreased NF‐kB translocation to the nucleus and the expression of c‐FLIP and cIAP following induction of apoptosis. We also show that KEAP1, a protein that promotes IKKβ degradation, forms a complex with FAM129B in vivo and in vitro. The ETGE motif in FAM129B and the KELCH repeats in KEAP1 are the sites of interaction between the two proteins. Using site directed mutagenesis, we changed the ETGE motif into ETAA. This mutation disrupted the interaction with KEAP1. Overexpression of recombinant FAM129B with the mutated ETGE motif in cancer cells, in contrast to the wild type protein, did not suppress apoptosis. We propose that FAM‐129B sequesters KEAP‐1, and blocks apoptosis by promoting the NF‐κB signaling pathway and the expression of antiapoptotic proteins
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 Information

NIH CA 1650771

New Jersey Health Foundation NJHF pilot grant

Rutgers 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 Information

Susan 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 Information

Butler University Holcomb research award
Activation of Cyr61 signaling in solid tumor cells diminishes response to a histone deacetylase inhibitor: Challenges for HDACIs
Authors: Ghosh Arnab , Ghosh Priyanka , Maity Gargi , Banerjee Sushanta K , Banerjee Snigdha
Published: November 2023

Background and Objective

Histone deacetylase (HDAC) inhibitors are clinically proven epigenetic‐based drugs for hematological cancers. However, the impact of these inhibitors on solid tumors is disappointing and debatable. Recent studies have shown that the solid tumor cell lines are less sensitive to a HDAC inhibitor Vorinostat (suberoylanilide hydroxamic acid; SAHA) as compared to hematological cell lines. However, it remains elusive why solid tumor cell lines responded weakly to HDAC inhibitors. Previously, it has been reported that CCN1/Cyr61 plays critical role in invasive front and drug resistance in breast and pancreatic cancer. Thus, our goal is to determine whether CCN1 signalling impair activity of SAHA in breast and pancreatic cancer cells.

Methods

To test this objective, various breast cancer cell lines (MCF‐7, ZR‐75‐1, MDA‐MB‐231, HCC‐70) and pancreatic cancer cell lines (BxPC‐3, AsPC‐1 and Panc‐1) were treated with SAHA with different does and times. Levels of CCN1 and its downstream signalling molecules were determined in SAHA‐treated or untreated cell extracts using Western blotting. In addition, cell viability, migration and sphere formation were examined in parental and CCN1‐depleted cells in the presence or absence of SAHA.

Results and Conclusions

Here we have shown that CCN1 was upregulated in various breast and pancreatic cancer cell lines following SAHA treatment via epigenetic mechanism. Distribution of CCN1 in the nucleus and cytoplasm was also drastically altered in SAHA‐treated cells. Normally, CCN1 expression was detected in the cytoplasm. However, following SAHA treatment, CCN1 expression was located predominantly in the nucleus. Functionally, CCN1 had dual outcome in these cells. The cell viability assay indicated that CCN1‐positve breast cancer or pancreatic cancer cells were more sensitive to SAHA as compared to CCN1‐negative cells. CCN1 ablation by neutralizing antibody treatment in MDA‐MB‐231 cells significantly suppressed the inhibitory effect of SAHA in MDA‐MB231 cells. However, aggressive phenotype such as migration towards SDF‐1 and sphere‐formation we remarkedly elevated in SAHA treated cells as compared to untreated cells. These effects of SAHA were rescued by CCN1‐depletion. Collectively, these studies indicate that CCN1 activation limits the response to SAHA in solid tumor cells and thus suggesting that combination therapy of SAHA andCCN1‐inhibitor could be an ideal therapeutic approach to make HADAC inhibitor sensitive to solid tumors.

Support or Funding Information

VA Merit Grant.
DDB2 Activates Rnf43 and Regulates Wnt/β‐catenin Signaling in Colorectal Cancer Cells
Authors: HUANG SHUO , Fantini Damiano , Merrill Bradley , Bagchi Srilata , Raychaudhuri Pradip
Published: November 2023

The Wnt/β‐catenin signaling pathway plays important roles in the development and progression of colorectal cancer. However, its de‐regulation in colorectal cancer is not clearly understood because, in addition to activating pro‐cancer genes, this signaling pathway activates expression of several tumor suppressor genes, including Rnf43. Previously, we showed that DDB2 suppresses metastasis of colorectal cancer cells harboring activating mutations in the Wnt/β‐catenin signaling pathway. Here, we show that DDB2 is critical for activation of Rnf43, which regulates Wnt‐signaling by inhibiting the levels of Wnt‐receptors. DDB2 recruits β‐catenin onto an upstream regulatory region in the Rnf43 gene, and is required for its expression by the Wnt‐signaling pathway. Cells lacking sequences in the regulatory region of Rnf43 involved in DDB2‐binding or DDB2‐deficient cells are impaired in expressing Rnf43 and are insufficient to ubiquitinate the Wnt‐receptor FZD5; consequently, those cells exhibit greater response to Wnt3a ligands. The results identify DDB2 as a novel partner as well as a negative regulator of Wnt‐signaling. The results also provide insights into how loss of DDB2 drives metastasis of colorectal cancer.

Support or Funding Information

NIH RO1‐CA175380
Modelling the Effects of Laser Photothermal Therapy on Proteins HSP70 and P53
Authors: Milcarek Andrew , Daus Kevin , Alpaugh Mary , Dobbins Tabbetha
Published: November 2023

Gold nanoparticles (AuNPs) and nanorods (AuNRs) are able to conjoin with biological molecules and efficiently absorb light for conversion into heat energy. Thus, they are being investigated for treatment of a variety of cancers using lasers to induce the conversion to heat in a technique known as laser photothermal therapy (LPTT). Since its first demonstration in 2005, LPTT has been increasingly researched and its efficacy has been demonstrated in mouse models. However, details which focus on the effect of heat on surrounding healthy cells is extremely limited. Likewise, study of whether intracellular vs. extracellular AuNP and AuNR uptake is most beneficial (and for which cancer types) is not undertaken. In this research, we examine these two aspects of laser photothermal therapy with the research questions: (i) how does heat exposure regulate proteins within the surrounding cells, specifically HSP70 and P53 and (ii) how do the particles distribute themselves at the surface of and/or within the cell. Specific to objective (i), the proteins HSP70 and P53 are examined. HSP70 is a heat shock protein responsible for wound healing, particularly in thermal burns. P53 is the “tumor repair protein” which is responsible for regulating cell transcription. Assuring maintenance of normal concentrations and the correct conformation of these proteins in the surrounding healthy cells will be important for implementing LPTT in patient care. Specific to objective (ii), benign planar cell models, human embryonic kidney cells (HEK‐293) and primary mouse embryonic fibroblast cells (3T3), are studied with quantitative monitoring of particle uptake concentration and distribution on cell viability during the therapy treatment. Size, surface charge, and surface treatment determine whether particles will undergo cellular uptake. These and other protein additives (such as chitosan coatings) determine whether the particles will diffuse across the cell wall. Key to studying cellular uptake is the comparative examination of planar tumor HeLa cells and the 3D Mary‐X tumor cell model. Objective (ii) informs the LPTT community of researchers as to which AuNPs and AuNRs conditions enhance cellular uptake for tumor cells—without selective uptake in benign cells.

Support or Funding Information

The work is being carried out in Dr. Tabbetha Dobbins lab at Rowan University. Funding at this time provided by Rowan University Department of Biomedical and Translational Sciences.
Pim‐1 signaling in drug‐resistant colon cancer cells promotes cell survival and chemoresistance through up‐regulation of lactate production
Authors: Park Ga Bin , Kim Daejin
Published: November 2023

Despite development of numerous target drugs, cancer drug resistance is still one of major obstacles to disturb anti‐cancer treatment in clinical field. Pim‐1 (Proviral integration site for Moloney murine leukemia virus‐1) kinase originally plays an essential role in cytokine‐induced signal transduction. However, overexpression of Pim‐1, as a proto‐oncogene, is also connected with cell proliferation, survival, and drug resistance. We found that the levels of Pim‐1 and mesenchymal markers in drug‐resistant colon cancer cells were highly expressed. To determine the effect of Pim‐1 on generating the chemoresistant cells, we investigated the influence of Pim‐1 on the expression of multi‐drug resistance gene. Targeted inhibition of Pim‐1 using small interference RNA (siRNA) significantly suppressed the up‐regulation of multiple drug resistance (MDR1), multidrug resistance protein 1 (MRP1), and cancer stem cell markers compared with the control group transfected with control siRNA. Silencing of Pim‐1 in chemoresistant HCT‐116 cells with siRNA resulted in downregulation of glycolysis‐associated enzymes, drug‐resistance related proteins, and a disintegrin and metalloproteinase (ADAM) family proteins. In addition, knockdown of Pim‐1 in OxR‐HCT‐116 cells triggered the ER stress‐mediated apoptosis through the loss of oxaliplatin or 5‐fluorouracil resistance. These finding suggest that Pim‐1‐associated signaling pathway plays a critical role in cancer progression and promoting chemoresistance of colon cancer cells.

Support or Funding Information

This study was supported by the Basic Science Research Program of Ministry of Education (NRF‐2015R1D1A1A01056672) and Ministry of Science, ICT & Future Planning (NRF‐2015R1C1A2A01053732) through the National Research Foundation (NRF) of Republic of Korea.
Genesis of Antibiotic Resistance XXIX: Clinical Perspectives of obdurate implementation of Antibiotics Time‐Out (ATO) in Mitigating Antibiotic Resistance Pandemic (ARP)
Authors: Anaya Edson , Martinez Juan , Calzoncit Melissa , Picazo Antonio , Blackmon Sarah , Jauregui Suzzette , Lechler Raul , Beautnagel Joanathan , Gutierrez Sonia , Johnson Dennie , Kannan Subburaj
Published: November 2023

Outcomes of a service learning project, the Strategies for Successful Implementation of ATO, along with its impact in mitigating ARP is presented in this work. ATO strategies were followed as suggested by CDC, http://www.cdc.gov/getsmart/healthcare/implementation/checklist.html with modification in accordance with hospital regulations. ATO strategies were adopted before each surgical procedure to ensure every patient received the appropriate prescribed antibiotic before being taken to the operating room, and prior to the start of surgery. This strategy was employed and directed toward the prevention of nosocomial infections and used as a measure to ensure that congruency in patient and corrective procedure. Accordant with the review of success rates among patients who had completed the surgical modality, ATO strategies generated higher success rate (data not presented in accordance with/per HIPAA guidelines). Reevaluating the prescription of antibiotics and dosage(s) of antibacterial regimen in the context of specific geographical location has enabled the providers to rule out antibiotics that are not effective in a specific community. Another contributing factor to higher success rates in surgical outcomes is vigilant and strict enforcement of the dialogue between the surgical staff (Medicos), pharmacy administration, and the antibiogram (data on file – medical records) of each patient from clinical antibiotic sensitivity assay has enhanced the positive outcome of ATO. The combined review of surgical medicos, pharmacy, Center for Disease Control and Prevention alerts concerning the spread of AR pathogen, and information gleaned from the Sanford Guide to Antimicrobial Therapy has enabled the prescription of appropriate and accurate dosages to aid patients in recovery in a more prudent manner, before and after the surgical modality. On a comparative basis, this practice is similar to the grand rounds undertaken on periodic basis in the teaching hospitals across the globe. Taken together, effective implementation of the ATO as a routine practice in the surgical units (across specialties, e.g. Cardiothoracic Surgery, Transplantation Units) would potentially mitigate the ARP. Supplementary data analysis were from the Continuing Medical Education activities completed by Subburaj Kannan, M.D.(A.M) Ph.D. Data analysis will be presented at EB 2017.

Support or Funding Information

Professional development funds to Subburaj Kannan
Isolation, Expression, and Characterization of a β–lactamase produced by Meiothermus ruber
Authors: Grulke Luther , Bergstrom Alexander , Groen Tara , Analitis Elaina , Scott Lori , Crawford Patrick
Published: November 2023

β‐lactamase enzymes are the most significant contributor to antibiotic resistance in bacteria. The pharmaceutical industry's response to antibiotic resistance has been to develop new antibiotics or β–lactamase inhibitors; these successes in the clinical setting require an understanding of the structure and function of the β–lactamases. A β–lactamase gene was identified in Meiothermus ruber, a thermophilic gram‐negative bacterium, with a bright red carotenoid pigment. M. ruber genomic DNA was utilized to clone the β–lactamase gene into the plasmid pKT1. The pKT1 plasmid contains a β–lactamase selection gene, which could potentially obfuscate kinetic assay data. Therefore, the β–lactamase gene from M. ruber was then amplified by PCR, inserted into pET‐26b, a protein‐expression vector containing a Kanamycin resistance selection gene, and transformed into E. coli. Restriction digestion and sequencing were performed on the successful transformants to verify the presence and correct orientation of the M.ruber β–lactamase gene. Activity and substrate preference of the gene product was determined by growing the bacteria in the presence of various β–lactam antibiotics. Preliminary tests suggest the M.ruber β–lactamase gene product can hydrolyze cephalosporin β–lactam antibiotics but not carbapenems. This result is supported by sequence comparison studies that suggest the gene product is a class C (AmpC‐type) β–lactamase. Further experiments are required to understand the structure and function of the β–lactamase from M.ruber.

Support or Funding Information

This work was funded by Augustana College Augustana College Summer Undergraduate Research Funds and the Department of Energy Joint Genome Institute's GEBA project.
Genesis of Antibiotic Resistance XXII: Mutagenic potential of the River Ganga (RG): Segment I, II, III‐a,b,c, vacate Hardy‐Weinberg‐Castle Equilibrium (HWCE), and concoct as a plausible contributor to an imminent Antibiotic Resistance Pandemic (ARP)
Authors: Francisco Martinez , Montoya Hector , Flores Jorge , Carbajal Carlos , Wickham Cheyenne , Lopez Christian , Johnson Dennie , Kannan Subburaj
Published: November 2023

A comparative study of the water quality of City of Eagle Pass, Texas, Water Works and the River Ganga was conducted to determine the induction of antibiotic resistance (AR) in bacteria such as fecal coliform bacteria (FC) that are known to cause antibiotic resistance infectious diseases. The correlation and regression analysis was conducted to determine the dependency among the following variables; i. Nephelometric Turbidity Units (NTU) of RG Segment‐I, II, III a, III b, and III c, ii. Saprobic index (SI=S), iii. Incidence of Fecal Coliform (MPN/100ml), iv. Morbidity and Mortality rate due to AR pathogen induced infectious diseases. A linear, (positive) correlation coefficient was observed between increases in NTU (~40NTU) paralleled with higher FC count (F.C. (MPN/100ml): ~ 160000). Regression coefficient analysis (where y= a + bx) showed a slope=1. An average Biological Oxygen Demand (BOD) ~ above 15 (min 19 to max 76 TPD) from the inflow of untreated sewage drains ranging (min 7 to max 14) draining minimum 550 million liter/ day (MLD) to maximum of 1780 MLD along with Grossly Polluting Industries (GPI) maximum of 504 (segment IIIB) pose unknown quantities of mutagenic agents. A cumulative average S (S=∑ (s × h)/∑h) where s=saprobic index value of indicator species, h‐frequency of occurrence of each species values of RG I, II, III a, b, and c showed strong pollution load correlating with FC count and NTU. Regression coefficient analysis on three variables; NTU, S index, and FC count showed slope =1, where y=a+bx. A comparison of RG I, II, III a, III b, and III c versus RG IV a, and IV b indicated that both segments of RG showed a similar trend in pollution load. Analysis has shown that a similar FC count is determined in both sections as 719 GPI ‐ releasing pollutants (~3000MLD) in RG I, II, III a, III b, and III c compare to only 36 GPI ~3000MLD releasing ~2360 MLD in to RG IV a, and IV b, with the later having the highest FC count. Data on mortality and morbidity (M&M) due to incidence of AR pathogen induced infectious diseases in the regions spanning RG segment 1 through III, were incomplete and incoherent. This is attributed to either the indifference of state and federal agencies in the collection of M&M data or the absence of adequate resources. Taken together, compare to the CEPWW, water quality criteria the RG Segment I – III a, III b, and III c is patently polluted and pose an abysmal concern as an ever increasing and accumulating mutagenic potency with the propensity of inducing an increased mutation frequency in the AR gene pool of FC, thus contributing to an ARP. Data analysis to be presented in EB 2017.

Support or Funding Information

Professional development funds to Subburaj Kannan
Stimulation of astrocyte fatty acid oxidation increases spare respiratory capacity under nutrient‐deprived conditions
Authors: Sifuentes Mikaela , Lechleiter James
Published: November 2023

Astrocytes offer increased resistance to toxic extracellular conditions and regulate the microenvironment surrounding neurons, presenting them as a key target for protective strategies against stroke and other metabolically‐intensive injuries. Our lab previously reported that thyroid hormone can increase astrocyte survival by stimulating fatty acid oxidation (FAO). We observe that under 24 hours of nutrient deprivation, the maximal oxygen consumption rate in astrocytes decreases significantly, but can be restored by thyroid hormone treatment in a dose‐dependent response after a 15 minute exposure. The rapid increase in spare respiratory capacity is blocked by inhibition of fatty acid transport into the mitochondria, indicating that prolonged glucose deprivation can be protected against using FAO as an alternative fuel source. Additionally, we find that starvation conditions induce the formation of large lipid droplets in astrocytes, possibly providing an endogenous source of fatty acids to be utilized during nutrient deprivation. Treatment of these astrocytes with thyroid hormone reduced lipid droplet size and concentration, in line with other reports that lipid droplets can be consumed through FAO. In conclusion, our observations support our hypothesis that thyroid hormone increases astrocyte viability by switching astrocyte mitochondrial function from a glucose‐dependent pathway to a FAO‐dependent one.

Support or Funding Information

Research supported by National Institutes of Health R01AG007218 and the American Heart Association Predoctoral Fellowship 16PRE27670002
Acute oxidative challenge induces glucose uptake through AMPK‐mediated NO production in skeletal muscle
Authors: Kellogg Dean L , McCammon Karen , Masters Bettie Sue , Roman Linda
Published: November 2023

We investigated the cellular pathways underlying glucose uptake in skeletal muscle (SkM) in response to reactive oxygen species (ROS). These underlying mechanisms are critical to understanding exercise‐induced oxidative stress and metabolic disease. Both AMP‐activated kinase (AMPK) and Nitric Oxide (NO) mediate the adaptive responses to oxidative stress. The goal of this study is to delineate the specific relationships between AMPK signaling and NO production and the resultant effects on GLUT4 translocation during acute oxidative stress. Utilizing an in vitro model of SkM (C2C12 myotubes), we demonstrate that exogenous ROS stimulate AMPK activity, NO production and phosphorylation of SkM‐specific neuronal NO synthase (nNOSμ). Dorsomorphin (an AMPK inhibitor) blocks nNOS phosphorylation, suggesting that the increase in NO is AMPK‐dependent. Furthermore, ROS induce translocation of GLUT4 to the cell membrane, which is blocked by L‐NAME (a NOS inhibitor), indicating that this process is dependent on NO. Taken together, these findings reveal that AMPK‐mediated phosphorylation of nNOS plays a central role in mediating the response of SkM to oxidative stress.

Support or Funding Information

ULT1TR001120 from the National Center for Advancing Translation Sciences to LJR