bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of cellular senescence mechanisms in alcoholic liver injury
Authors: Meng Fanyin , Lorenzo Sugeily Ramos , Francis Heather , Glaser Shannon , Alpini Gianfranco 
Published: November 2023

BackgroundCellular senescence is a stress‐responsive program limiting the proliferation of damaged cells and leading to stable cell‐cycle arrest. Accumulation of senescent hepatocytes may contribute to loss of functional hepatic mass and lead to liver decompensation and fibrosis. The current study aimed to characterize the functional role of cellular senescence during alcoholic‐induced hepatitis.MethodsSenescence related gene expression was assessed using a Cellular Senescence PCR Array and/or real‐time PCR analysis in ethanol‐ and LPS‐treated normal human hepatocytes (N‐Heps) and cholangiocytes (HiBEC), as well as in liver specimen from mice fed alcohol for 5 weeks relative to control liver tissue. Cellular senescence and viability were measured by SA‐β‐gal Activity and MTS assay. The upstream modulators of senescence were defined in ethanol/LPS treated hepatocytes and cholangiocytes in vitro, and in TLR‐4 knockout mice in vivo.ResultsWe identified that 5 week of ethanol feeding significantly increased total liver histopathology score and hepatocellular senescence by PCR array and SA‐β‐gal assay. The up‐regulation of hepatic senescence initiators, PAI‐1 and EGR1, were further verified by real‐time PCR assay. Treatment of N‐Heps and HiBECs with ethanol (86 mM) and LPS (20 ng/ml) for 72 hr significantly increased PAI‐1 and EGR1 expression, along with the enhanced SA‐β‐gal activity and reduced cellular viability detected by MTS assay. Silencing of PAI‐1 decreased ethanol and LPS‐induced senescence in both N‐Heps and HiBECs, whereas inhibition of EGR1 only reduced the senescence and increased viability in N‐Hep cells. Interestingly, silencing of PAI‐1 and EGR1 also reduced the pro‐fibrotic markers, α‐SMA and MMP‐9 in N‐Hep cells, whereas silencing of LPS receptor TLR4 decreased these markers in the same group of N‐Hep cells, suggesting LPS‐mediated cellular senescence during alcohol‐induced liver fibrosis. Furthermore, the expression of TLR4 and the verified LPS related senescence markers, including E2F1, ID1 and IGFBP3 were significantly altered in ethanol‐fed mouse liver specimens compared to controls. TLR4 knockout mice displayed less sensitivity to alcoholic injury, along with reduced PAI‐1 and EGR1 levels and recovered expressions of α‐SMA and MMP‐9.Summary and ConclusionOur results show that PAI‐1 and EGR1 are the critical regulator of LPS induced cellular senescence and alcoholic hepatitis. The findings provide new insight into the function of LPS regulated cellular senescence and increase opportunities for the development of novel treatment paradigms for the management of alcoholic liver diseases.Support or Funding InformationVA Merit Review Awards and NIH/NIDDK R01 Grants
Inhibition of Aggressive Natural Killer Cell Leukemia Cell Functions by Inhibitors of the Mevalonate Pathway and Its Products
Authors: Carlson Whitney , Pradhan Prajakta , Steele Timothy 
Published: November 2023

Aggressive natural killer cell leukemia is a rare form of leukemia characterized by an increase in proliferation of malignant natural killer cells. Aggressive natural killer cell leukemia is one of the deadliest cancers known. Survival time is normally weeks to a few months once diagnosed. Aggressive natural killer cell leukemia is highly resistant to treatment, making it important to find a treatment for natural killer cell leukemia. In this study, effects of a geranylgeranyl transferase inhibitor, a farnesyl transferase inhibitor, and Rho GTPase inhibitors were investigated on the aggressive natural killer cell line YT‐INDY. Cancer cells are capable of upregulating mevalonate metabolism for protein prenylation and cholesterol biosynthesis. The farnesyl transferase inhibitor Tipifarnib and geranylgeranyl transferase inhibitor GGTI‐286 inhibit mevalonate metabolism by blocking the farnesylation and geranylgeranylation of proteins, respectively. We investigated the effects of GGTI‐286 and Tipifarnib on cell proliferation, cell cycle progression, and activation of the ERK MAP kinase pathway. Rho GTPases are small molecules that, when active, regulate functions such as progression through the cell cycle, cell growth, and transcription. Upregulation of Rho GTPases have been found in certain cancers. We determined whether the Rac1 inhibitors NSC 23766 and EHT 1864, cdc42 inhibitors ZCL 278 and ML 141 and ROCK inhibitors GSK429286A and Y‐27632 could inhibit YT‐INDY cell proliferation and ERK MAP kinase activation. The results of our investigations showed that Tipifarnib and GGTI‐286 were potent inhibitors of YT‐INDY proliferation (Tipifarnib IC50 = 7.2 uM, and GGTI‐286 IC50 = 8.0 uM), cell cycle progression and ERK MAP kinase activation. Among the Rho GTPase inhibitors, we found that the ROCK inhibitors (GSK429286A and Y‐27632) and Rac1 inhibitors (NSC 23766 and EHT 1864) produced significant inhibition of YT‐INDY proliferation. We concluded from these investigations that inhibitors of the mevalonate pathway or products of the mevalonate pathway can interrupt YT‐INDY cell growth, suggesting that this approach could lead to more effective treatment of this devastating cancer.Support or Funding InformationOur laboratory gratefully acknowledges the financial support of this research by the Iowa Osteopathic Education and Research Foundation.
Neuropilin Expression in Podocytes: The Implications for SEMA3 Anti‐Cancer Therapy
Authors: Nzikoba Ellah Nifashe , Zessler Astrid , Li Xiaoran , Briscoe David , Adam Rosalyn M. , Schumacher Valerie , Bielenberg Diane R. 
Published: November 2023

Neuropilins (Nrp) are transmembrane receptors in endothelial and carcinoma cells that bind to angiogenic ligands including vascular endothelial growth factor (VEGF) to promote neovascularization, the sprouting of new blood capillaries providing oxygen and nutrients to the tumor. Additionally, Nrp binds to an alternate family of proteins, Class 3 Semaphorins (SEMA3), which compete with VEGF and inhibit angiogenesis. Targeting of either Nrp1 or Nrp2 with neutralizing antibodies or with exogenous SEMA3 proteins has successfully inhibited cancer progression in preclinical trials. Our goal was to investigate the expression, localization and function of Nrps within the kidney to examine potential therapeutic off‐target effects. We hypothesized that podocytes express Nrp1 but not Nrp2 and that the Nrp1 ligand, SEMA3A, would induce proteinuria while the Nrp2 ligand, SEMA3F, would not. Using immunoblotting, immunohistochemistry and X‐gal staining of kidneys from wildtype (Nrp2+/+) and transgenic mice (Nrp2+/lacz, Nrp2−/−), we determined that Nrp1 is highly expressed in the kidney and localized to tubular epithelial cells and glomerular podocytes. Nrp2, on the other hand, is weakly expressed in the kidney and localized to endothelial cells between tubules. Glomeruli are devoid of Nrp2 expression. In order to examine the effect of Nrp2 deletion on renal filtration function, we examined the protein content of urine from Nrp2+/+ and Nrp2−/− mice and found that both genotypes had similar protein filtration. Alternately, mice were treated intravenously with adenovirus encoding SEMA3 proteins. Livers from mice infected with these adenoviruses secreted SEMA3 proteins systemically. Mice exposed to SEMA3 proteins showed increased protein content in the urine. Taken together, these results may explain why NRP1 and VEGF combination antibody treatment caused severe proteinuria in cancer patients in a human Phase Ib clinical trial. Our data suggests that systemic therapies targeting NRP2 (anti‐NRP2 antibodies) may cause less toxicity than those targeting NRP1 yet still maintain anti‐tumor efficacy.Support or Funding InformationEllah Nzikoba was funded by the ASIP SROPP program and the study was funded by DK104641 (DRB, RMA).
HLA‐DR Positive Acute Promyelocytic Leukemia (APL)
Authors: Mendoza Alejandro S , Qing Xin , Dungo Maria , Lasky Joseph , Panosyan Eduard , Cai Junchao 
Published: November 2023

BACKGROUNDAcute promyelocytic leukemia (APL) with t(15;17)(q22;q21)/PML‐RARα is a subtype of acute myeloid leukemia(AML) with distinct morphologic and immunophenotypic characteristics. It is a highly aggressive disease that requiresrapid diagnosis and early intervention. Compared with other types of AML, APL typically displaysa triad of absent or weak CD34, absent HLA‐DR, and positive CD117. HLA‐DR positive APL is extremely rareand its clinical and pathological features have not been reported. A total of 45 cases of APL with t(15,17)/PMLRARα were diagnosed at Harbor‐UCLA Medical Center from year 2006 to 2015. Among them, only two cases were positive for HLA‐DR by flow cytometry immunophenotyping. Here we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of these two cases.RESULTS AND DISCUSSIONThe patients in both cases presented with anemia, thrombocytopenia, and decreased WBC count. In case A, the peripheralblood smear showed many myeloblasts and only occasional abnormalpromyelocytes. Although a thin Auer rod was found in rare blasts, neitherthe thick Auer rods that typically seen in APL nor faggot cells were identified. While case B showed characteristic morphologic features of hypergranular variant of APL in the bone marrow, no blasts orabnormal promyelocytes were identified in the peripheral bloodsmear. Therefore, APL should not be excluded even when abnormalpromyelocytes are not present in the peripheral blood smears. In addition, MPO cytochemical stain performed on the bone marrow aspiratesmear of case A did not show uniform strong staining pattern that is commonly observed in APL. Besides HLA‐DR, CD34 was also expressed on the blasts of case A. These atypical morphologic and immunophenotypic features may be attributed to the additional cytogenetic abnormalities found in case A.CONCLUSIONHLA‐DR‐positive APL is very rare and represents about 4% of all APL cases in our study. Due to its rare occurrence, continuous reporting of HLA‐DR positive APL will bring to the awareness of the broad clinical and pathological spectrum of APL, provide better clinicopathologic correlation, and facilitate rapid diagnosis and thus promptinitiation of appropriate management of this aggressive malignant tumor.Support or Funding InformationWe declare no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Use of Anchored Multiplex PCR Enrichment for Detection of Gene Fusions in Solid Tumors by Next Generation Sequencing
Authors: Green Donald C , Deharvengt Sophie J , de Abreu Francine B , Steinmetz Heather B , Peterson Jason D , Tsongalis Gregory J 
Published: November 2023

BackgroundThe known genomic complexities of human cancer are being used to combat the disease with novel targeted therapies. Next generation sequencing (NGS) technologies have been introduced to determine a human cancer profile that is then used to develop a therapeutic management strategy. To date most NGS assays assess SNVs, Indels and some CNVs in various cancer related genes. Gene fusions are another type of mutation that have recently been implicated in the diagnosis and treatment of human cancers. In this study we assessed the ability to detect gene fusions using an anchored multiplex PCR‐enriched NGS assay.MethodsRNA was extracted from 15 formalin fixed, paraffin embedded tissue samples and 10 cell lines using the AllPrep DNA/RNA FFPE kit. cDNA synthesis and library preparation were performed using 200 ng of RNA. RNA quantity and quality were evaluated using the Qubit HS RNA Assay and the Archer PreSeq RNA QC Kit, respectively. Libraries were quantified using the KAPA Library Quantification Kit and sequenced on the Illumina MiSeq System using the Archer FusionPlexTM Solid Tumor panel which detects fusions in 53 genes. Data analysis was performed using the Archer Analysis platform (v4.0.11). We examined the limit of detection (LOD) by varying the amount of total RNA input (200‐2.5 ng), precision in two sequencing runs using two different operators, and accuracy by comparison of results to results from independent FISH and NGS assays.ResultsWe established QC metrics that included: (1) tumor content, (2) RNA quantification, (3) RNA quality, (4) library quantification, and (5) post‐sequencing analysis. While NGS results could be obtained using 10 ng of input RNA, more robust results were obtained when we used higher RNA inputs. Accuracy was determined to be 100% with detection of for all known fusions in the samples used. The following fusions were present in these samples: EML4‐ALK, ETV6‐NTRK3, EWSR1‐FLI1, TPM3‐NTRK1, TNM4‐NRG1, FGFR2‐COL14A1, MBNL1‐RAF1, ETV6‐RUNX1, BRD4‐NUTM1, and TMPRSS2‐ERG. All positive (CCD6‐RET, EML4‐ALK, SLC34A2‐ROS1) and negative controls gave the expected results.ConclusionsThis study demonstrates the feasibility of using the Archer Solid Tumor FusionPlex assay for detection of gene fusions in human cancers. The assay detects fusions of any partner gene with the 53 genes in the panel and thus makes novel fusion discovery possible.
Anti‐proliferative Properties of Methanolic Extracts of Annona muricata in Colon, Lung and Skin Cancer Cell Lines
Authors: Robles Jose F , Muñoz Kevin , Rivera Nilka , Suarez Edu 
Published: November 2023

Annona muricata, commonly known as Soursop, is a tree that grows in tropical climates. Traditionally, different parts of A. muricata are used to prepare teas and juices, which have been used as homemade remedies for diseases such as diabetes and cancer. Previous studies in our laboratory, showed that extracts obtained from A. muricata's leaves had anti‐proliferative activity towards HT‐29 colorectal adenocarcinoma cell. Based on these previous results, in this study we evaluate the effect of the Soursop's fruit peel extract on the proliferation of colorectal adenocarcinoma at 24‐, 48‐ and 72‐hours. In addition, we explored if this extract affects the proliferation of non‐malignant CCD‐1074sk (epithelial) and MRC‐5 (fibroblast) cell lines using the same experimental design. We hypothesized that Soursop's fruit peel extracts will inhibit proliferation of HT‐29 colorectal adenocarcinoma but will not show affect the studied non‐malignant cell lines. We prepared our samples by performing methanolic extractions of the fruit's peel. For the proliferation assays we seeded the HT‐29, CCD‐1074 and MCR‐5 cells in 96‐well plates and after an overnight recovery period, we applied five treatments consisting of concentrations of the extracts: 400ug/mL, 300ug/mL, 200ug/mL, 100ug/mL, and 50ug/mL and DMSO at 1,43% final concentration as our control vehicle. Our preliminary results on the HT‐29 cells showed that at 24‐hours post treatment, only the highest concentration (400 ug/mL) presented a significant inhibition (p<0.05) at 48‐hours we observed inhibition from 50ug/mL to 400ug/mL and at 72‐hours we observed inhibition at all experimental concentrations. For the non‐malignant cell lines CCD‐1097and MRC‐5 the extract did not affect the proliferation of these when compared to their respective vehicle controlsOur preliminary data supports our hypothesis, suggesting that A. muricata fruit's peel extracts has a selective anti‐proliferative effect towards malignant cells.Future biological experiments include the confirmation of this preliminary data and to evaluate the effect of our extracts on cell migration and invasion. As well, we expect to perform separation and identification of the chemical components present in the extracts by using High Pressure Liquid Chromatography (HPLC‐MS) coupled with Mass Spectrometry to identify key components in the extracts observed effects.
MED28 Regulates Proinflammatory Cytokine‐induced Epithelial–Mesenchymal Transition and Invasion in Human Breast Cancer Cells
Authors: Hsieh Nien‐Tsu , Huang Chun‐Yin , Li Chun‐I , Weng Yu‐Ting , Lee Ming‐Fen 
Published: November 2023

MED28, one of the Mediator subunits, exhibits multiple cellular roles, including involved in transcriptional activation and as a merlin, Grb2, and cytoskeleton‐associated protein, which is highly expressed in several types of malignancy including breast and colorectal tumors. Chronic inflammation, as suggested as one of the hallmarks of cancer, may promote carcinogenesis. Inflammatory cytokines such as tumor necrosis factor‐alpha (TNFα) may mediate tumor initiation, promotion, and progression. Recently, we have identified that MED28 is involved in regulating cell growth, epithelial–mesenchymal transition (EMT), and migration in human breast cancer cells. The objective of this study is to investigate if MED28 is involved in the proinflammatory cytokine‐induced EMT in human breast cancer cells. In aggressive MDA‐MB‐231 cells, MED28 suppression attenuated the mesenchymal morphology and reduced the expression of several mesenchymal biomarkers; however, MED28 overexpression exhibited a reciprocal effect. In addition, TNFα upregulated the expression of the target genes of NFκB/p65 and enhanced an even more aggressive phenotype, whereas suppression of MED28 attenuated the effect. Moreover, pterostilbene, a resveratrol analogue, suppressed the expression of MED28, p‐p65, and p65 downstream target genes, which warrants further investigation for a putative translational application in breast cancer.Support or Funding InformationThis work was supported by the grants NSC102‐2320‐B‐309‐001‐MY3 and MOST105‐2320‐B‐309‐001 to M‐F Lee and MOST103‐2815‐C‐309‐012‐B to C‐I Li.
Internalization and trafficking of PD‐L1 in MDAMB231 breast cancer cells
Authors: Rudkouskaya Alena , Barroso Margarida 
Published: November 2023

Immunotherapy has emerged as a very promising type of cancer treatment. However, despite successful clinical use of several monoclonal antibodies developed to target immune checkpoint inhibitors present in tumor cells or tumor‐infiltrating lymphocytes, there is very little known about their endocytic pathway in cancer cells. To elucidate this process we studied programmed cell death‐ligand 1 (PD‐L1) uptake in MDAMB231 breast cancer cells using both live imaging and immunofluorescent staining of fixed cells. We found that five minute pulse with 10 ug/mL PD‐L1 AF488 and transferrin‐AF555 (Tf‐AF555), followed by wash and live imaging, results in significant accumulation of PD‐L1 both at the plasma membrane and in the endosomes often also containing Tf‐transferrin receptor complexes (Tf‐TfR). To further investigate PD‐L1 trafficking, we performed one hour uptake of both PD‐L1AF555 and Tf‐AF647, fixed the cells and stained with Early Endosome Antigen1 (EEA1). Airyscan imaging of these cells showed that many large vesicles, characteristic to MDAMB231 cells, are positive for all three markers suggesting again that even at late stages PD‐L1 is trafficking along the endocytic pathway with Tf‐TfR complexes. These findings may help to develop non‐invasive quantitative imaging approach to monitor the spatial distribution and accumulation of PD‐L1 in the tumor xenografts in preclinical studies to advance cancer immunotherapy.Support or Funding InformationThis work was funded by NIH grant R01 EB019443.
Autophagy and Glutamine Synthetase Induction Sustain ALL Blast Resistance to L‐Asparaginase in Human Bone Marrow Mesenchymal Stromal Cells
Authors: Chiu Martina , Bardelli Donatella , Taurino Giuseppe , Dander Erica , Bianchi Massimiliano G , Mirandola Prisco , Carubbi Cecilia , D'Amico Giovanna , Rizzari Carmelo , Bussolati Ovidio 
Published: November 2023

L‐Asparaginase (ASNase) is one of the cornerstones of acute lymphoblastic leukaemia (ALL) treatment. Its antileukemic effect mainly relies on its ability to hydrolyse blood asparagine and glutamine. Asparagine depletion causes a severe nutritional stress in ALL blasts exhibiting low expression of asparagine synthetase (ASNS), which cannot, therefore, synthesize asparagine de novo. Conversely, given the role of glutamine as obliged nitrogen donor for asparagine synthesis, the glutaminolytic activity of ASNase is of relevance in the therapy of ASNS‐positive ALL blasts. It has been proposed that bone marrow mesenchymal stromal cells (MSCs) may support the resistance of ALL blasts to ASNase through the release of asparagine and glutamine. Given that no data are yet available on ASNase effects on MSCs themselves, aim of this work was to ascertain whether and how MSCs adapt to ASNase and if the inhibition of this adaptation reverts nutritional support to ALL blasts.MSCs isolated from healthy donors and the ALL cell lines RS4;11 have been cultured in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FBS, 2 mM glutamine and antibiotics. Co‐cultures were established by adding RS4;11 cells to confluent monolayers of MSCs. ASNase from E. chrysanthemi was used at 1 U/ml, while glutamine synthetase (GS) was inhibited by 1 mM methionine‐L‐sulfoximine (MSO). Viability was measured through resazurin assay or flow cytometry. Autophagy was assessed by LC3 II detection in Western Blot.MSCs were poorly sensitive to ASNase (IC50 > 10 U/ml) and, once treated with the enzyme, recovered their growth after a transient proliferative arrest. In MSCs, ASNase increased GS expression and activated a nutritional stress response marked by the phosphorylation of eIF2α. Moreover, ASNase inhibited mTORC1 activity, detected as the phosphorylation of p70S6K1, and, consequently, triggered autophagy. When GS was inhibited by MSO, the autophagic response was delayed, while CHOP was steadily induced and MSC growth was not rescued. When co‐cultured with MSCs, ALL blasts were effectively protected from ASNase‐induced cytotoxicity. The protective effect was markedly hindered when MSCs had been pre‐treated with ASNase and MSO. Our data show that the adaptation of MSCs to ASNase treatment through autophagy and GS induction is needed for ALL blast survival and may constitute an important therapeutic target.
The Anti‐migraine Effects of Delta‐9‐tetrahydrocannabinol in Female Rats are Mediated by CB1 Receptors
Authors: Kandasamy Ram , Dawson Cole T , Lee Andrea T , Morgan Michael M 
Published: November 2023

Migraine affects 15% of the world's population, yet treatment options are limited by poor efficacy and adverse effects such as medication overuse headache. Anecdotal data suggest that marijuana may be an effective treatment for migraine. We tested this hypothesis by measuring whether administration of delta‐9‐tetrahydrocannabinol (THC), the psychoactive constituent of marijuana, has anti‐migraine effects in female rats. In addition, we administered cannabinoid receptor antagonists to determine whether these anti‐migraine effects are mediated by CB1 or CB2 receptors. Migraine pain was induced in female rats by microinjection of the TRPA1 agonist allyl isothiocyanate (AITC, 10%) onto the dura via a cannula implanted through the skull one week earlier. Migraine‐like pain was assessed by measuring changes in home cage wheel running. Rats were injected with either vehicle, the CB1 receptor antagonist SR141716 (1 mg/kg), or the CB2 receptor antagonist SR144528 (3.2 mg/kg) 30 min prior to simultaneous administration of AITC and vehicle or THC (0.32 mg/kg, i.p.). Microinjection of AITC caused a reduction in wheel running that lasted approximately 3 hours. Administration of THC attenuated the depression of wheel running caused by AITC administration. Administration of the CB1, but not the CB2, receptor antagonist attenuated the anti‐migraine effects of THC. Neither antagonist depressed wheel running in control animals. Taken together, these data indicate that the anti‐migraine effects of THC are mediated by CB1 receptors.Support or Funding InformationThis study was supported in part by State of Washington Initiative Measure No. 502 and NIH grant NS095097 (to MMM).