Browsing by Author "Ensarioglu, HK"

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    Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm
    Ozgurbuz, U; Ensarioglu, HK; Celik, DA; Vatansever, HS
    Over the past years, inflammatory bowel disease (IBD) treatment has become more targeted, anticipating the use of immune-modifying therapies at an earlier stage. During the treatment process prevention and management of viral infections hold significant importance. The protective role of favipiravir on enterocytes which are affected by inflammation is still unknown. We aim to analyze the effects of favipiravir on enterocytes after an inflammatory condition. We conducted a 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to assess the cytotoxicity of favipiravir on intestinal epithelioid cells (IEC-6). To mimic the inflammation model in cell culture conditions, we exposed IEC-6 cells to tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). The cells were categorized into four groups: control, inflammation model, application of favipiravir before inflammation (prophylactic), and application of favipiravir after inflammation (treatment). We assessed the presence and distribution of caspase 1, caspase 3, interleukin 6 (IL6), interleukin 8 (IL8), mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase 1 (RIPK1), and TNF-alpha using indirect immunoperoxidase staining. TNF-alpha and IL8 levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in a culture medium. Caspase 1 was observed to be strong (+++) in the treatment group and weak (+) in the prophylactic group compared to the inflammation group. Caspase 3 was weak (+) in the inflammation group, and it was strong (+++) in the prophylactic and treatment group, the increase in the treatment group was significant. Therefore administering favipiravir before inducing inflammation appears to control the inflammatory caspase pathway in intestinal enterocytes, protecting them from inflammatory responses, while the caspase 3-dependent apoptotic pathway may not be active in enterocytes during inflammation. IL6 and IL8 were negative (-) in control, IL6 was weak (+) in inflammation and favipiravir treated groups; IL8 increased significantly in favipiravir groups compared to control and inflammation groups. Consequently, favipiravir may trigger IL6 release, initiating the inflammatory pathway and potentially enhancing IL8 interactions with other cytokines. TNF-alpha immunoreactivity was strong (+++) in the inflammation group, while it was moderate (++) in favipiravir-administered groups. MLKL immunoreactivity was strong (+++) in all groups, RIPK1 was weak (+) in control, strong (+++) in the inflammation and treatment group, moderate (++) in the prophylactic group, and the increase in inflammation and treatment group was significant compared to control. Our findings suggest that in the treatment group, necroptosis was triggered by increased MLKL and RIPK1, key players in inflammation and cell death. After immunocytochemical evaluation, our findings suggest that, after the onset of inflammation, favipiravir may play a role in cell death by increasing necroptosis rather than apoptosis.
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    Protective Effects of Citrus Flavonoid Hesperidin in Enterocytes After Induction with TNF-α and IFN-γ Which Mimic the COVID-19 Disease
    Özgürbüz, U; Vatansever, S; Becer, E; Ensarioglu, HK; Celik, DA
    BACKGROUND/AIMS: Coronavirus disease (COVID-19) is caused by a virus and exhibits various symptoms such as cough, fever, and chills. Flavonoids have a potential inhibitory effect on coronaviruses. In this study, we determined the effects of hesperidin on enterocyte cells (IEC) after tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma induction which mimics the severe acute respiratory therapy-coronavirus-2 (SARS-CoV-2) infection. MATERIALS AND METHODS: The IEC-6 were treated with 50 ng/mL of TNF-alpha and 100 ng/mL of IFN-y for 48 h to mimic inflammatory shock similar to COVID-19 disease. IEC-6 cells were cultured as control, COVID-19 disease mimic, hesperidin prophylactic, or treated groups. The cytotoxicity effect of hesperidin was analyzed using an MTT assay. Serum levels of TNF-alpha and interleukin (IL)8 were evaluated using ELISA. The distributions of TNF-alpha, IFN-y, IL-1ss, Insulin-like growth factor-I, and caspase-3 were analyzed by indirect immunoperoxidase staining. RESULTS: Both TNF-alpha and IL8 levels were higher in TNF-alpha and IFN-y induction of enterocyte culture medium than in the control. Lesser immunoreactivity of TNF-alpha was detected in the treatment group which hesperidin applicate after TNF-alpha and IFN-y combination. While IL-1 immunoreactivity was similar in both the hesperidin prophylactic and treatment groups, lesser immunoreactivity of TNF-alpha was observed in the hesperidin treatment group. Both IFN-y and vascular endothelial growth factor A immunoreactivities were also decreased in the hesperidin treatment group. CONCLUSION: We found that hesperidin had anti-inflammatory and cell protection effects in IEC after TNF-alpha and IFN-gamma induction which mimics the model of SARS-CoV-2 infection. Therefore, hesperidin could be used to reduce gastrointestinal system symptoms in COVID-19 disease.
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    A newly synthesized thiosemicarbazide derivative trigger apoptosis rather than necroptosis on HEPG2 cell line
    Basoglu-Uenal, F; Becer, E; Ensarioglu, HK; Ulusoy-Guezeldemirci, N; Kuran, ED; Vatansever, HS
    Thiosemicarbazide derivatives have been the focus of scientists owing to their broad biological activities such as anticancer, antimicrobial, and anti-inflammatory. Herein, we designed and synthesized a new thiosemicarbazide derivative (TS-1) and evaluated its antiproliferative potential against the human hepatocellular carcinoma cell line (HEPG2) and human umbilical vein endothelial cell line (ECV-304). Also, it was aimed to investigate the necroptotic and apoptotic cell death effects of TS-1 in HEPG2 cells, and these effects were supported by molecular docking. The new synthesized compound structure was characterized using various spectroscopic methods such as FT-IR, H-1-NMR, C-13-NMR, and elemental analysis. The cytotoxic activity of the tested compound was measured by the MTT assay. Apoptotic and necroptotic properties of the TS-1 were evaluated by indirect immunoperoxidase method using antibodies against Ki-67, Bax, Bcl-2, caspase-3, caspase-8, caspase-9, RIP3, and RIPK1. Apoptotic and necroptotic effects of TS-1 were supported by molecular docking. Compound TS-1 was synthesized as a pure compound with a high yield. The effective value of TS-1 was 10 mu M in HEPG2 cells. TS-1 did not show any cytotoxic effect on ECV-304. Caspase-3 and RIPK1 immunoreactivities were significantly increased in HEPG2 cells after being treated with TS-1. As the results of the molecular docking studies, the molecular docking showed that the TS-1 exhibits H-bond interaction with various significant amino acid residues in the active site of both RIPK1. It could be concluded that TS-1 could be a promising novel therapeutic agent by inducing apoptosis rather than necroptosis in HEPG2 cells.
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    Cell Death Mechanism of Organometallic Ruthenium(II) and Iridium(III) Arene Complexes on HepG2 and Vero Cells
    Kavukcu, SB; Ensarioglu, HK; Karabiyik, H; Vatansever, HS; Türkmen, H
    Due to side effects and toxicity associated with platinum-derived metal-based drugs, extensive research has been conducted on ruthenium (Ru) complexes. We aim to synthesize a highly oil soluble Ru(II)-p-cymene complex (Ru1) with an aliphatic chain group, a bimetallic Ru(II)-p-cymene complex (Ru2) with N,S,S triple-coordination and a bimetallic Ir(III)-pentamethylcyclopentadienyl complex (Ir1) with S,S double-coordination. Subsequently, we investigate the effects of these complexes on Vero and HepG2 cell lines, focusing on cell death mechanisms. Characterization of the complexes is performed through nuclear magnetic resonance spectroscopy (H-1 and C-13 NMR) and Fourier-transform infrared spectroscopy. The effective doses are determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay, applying different doses of the complexes to the two cell lines for 24 and 48 h, respectively. Immunoreactivities of Bax, Bcl2, caspase-3, RIP3, and RIPK1 are analyzed using the indirect immunoperoxidase technique. Notably, all the complexes (Ru1, Ru2, and Ir1) exhibit distinct cell death mechanisms, showing greater effectiveness than cisplatin. This study reveals the diverse mechanisms of action of Ru and Ir complexes based on different ligands. To the best of our knowledge, this study represents the first investigation of a novel RAED-type complex (Ru1) and unexpected bimetallic complexes (Ru2 and Ir1).
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    Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells
    Ozkasapoglu, S; Caglayan, MG; Akkurt, F; Ensarioglu, HK; Vatansever, HS; Celikkan, H
    Carbon nanodots havedrawn a great deal of attention due to theirgreen and expedient opportunities in biological and chemical sciences.Their high fluorescence capabilities and low toxicity for living cellsand tissues make them excellent imaging agents. In addition, theyhave a fluorimetric response against inorganic and organic species.Boron-doped carbon nanodots (B-CDs) with high fluorescence yield wereproduced from phenylboronic acid and glutamine as boron and carbonsources, respectively, by a hydrothermal method. First, the effectsof the temperature on their fluorescence yield and the structuralcharacteristics of B-CDs were investigated. Second, their cytotoxicityand cell death and proliferation behaviors were examined. The cytotoxicitywas evaluated by the MTT assay. The cellular properties were evaluatedwith the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After theMTT assay, 1:1 dilution of all applicants for 24 h was used in thestudy. After immunohistochemical analyses, the application of B-CDssynthesized at 230 & DEG;C did not change control cell (Vero) proliferation,and also apoptosis was not triggered. Colo 320 CD133+ and CD133-cell-triggered apoptosis and cellular senescence were found to besynthesis temperature dependent. In addition, Colo 320 CD133-cells were affected relatively more than CD133+ cells from B-CDs.While B-CDs did not affect the control cells, the colon cancer stemcells (Colo 320 CD133+) were affected in a time-dependent manner.Therefore, the use of the synthesized B-CD product may be an alternativemethod for controlling or eliminating cancer stem cells in the tumortissue.
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    Sensitive and reliable lab-on-paper biosensor for label-free detection of exosomes by electrochemical impedance spectroscopy
    Sazaklioglu, SA; Torul, H; Tamer, U; Ensarioglu, HK; Vatansever, HS; Gumus, BH; Çelikkan, H
    A new, sensitive, and cost-effective lab-on-paper-based immunosensor was designed based on electrochemical impedance spectroscopy (EIS) for the detection of exosomes. EIS was selected as the determination method since there was a surface blockage in electron transfer by binding the exosomes to the transducer. Briefly, the carbon working electrode (WE) on the paper electrode (PE) was modified with gold particles (AuPs@PE) and then conjugated with anti-CD9 (Anti-CD9/AuPs@PE) for the detection of exosomes. Variables involved in the biosensor design were optimized with the univariate mode. The developed method presents the limit of detection of 8.7 x 102 exosomes mL-1, which is lower than that of many other available methods under the best conditions. The biosensor was also tested with urine samples from cancer patients with high recoveries. Due to this a unique, low-cost, biodegradable technology is presented that can directly measure exosomes without labeling them for early cancer or metastasis detection.
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    Electrochemically generated paper SERS substrate for detection of exosome in urine samples
    Kayis, EÇ; Torul, H; Sazaklioglu, SA; Çelikkan, H; Ensarioglu, HK; Gumus, BH; Vatansever, HS; Tamer, U
    Here, a single-drop paper-based surface-enhanced Raman spectroscopy (SERS) immunoassay was developed to pave the way for monitoring exosome numbers for the early diagnosis of prostate cancer. Exosomes are nanosized (40-150 nm) membrane vesicles that provide intercellular communication. In our work, we offer a new paper SERS substrate for exosome detection in urine. We initially electrochemically deposited nanostar-shaped gold nanoparticles (AuNPs) on the working electrode to crate the paper SERS substrates. Then we functionalized them with 11-mercaptoundecanoic acid (11-MUA) and conjugated them with anti-CD9 antibodies. After capturing exosomes, the sandwich immunoassay structure was created by using gold nanorods (AuNRs) modified with 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) as a Raman tag. The SERS signal intensities of DTNB molecules at 1330 cm-1 were monitored to determine the exosome concentration. Each step occurred in only one drop of solution or sample. The developed single-drop paper-based SERS immunoassay exhibited a linear range from 1.0 x 103 to 1.0 x 109 exosome particles/mL with correlation coefficients (R2) of 0.9903. The limit of detection (LOD) was found as 9.9 x 101 exosome particles/mL. The developed system was tested with clinical urine samples from patients with benign prostatic hyperplasia, prostatitis, prostate cancer, and healthy individuals. The obtained results were compared with the exosome particle numbers in these samples determined by an enzyme-linked immunosorbent assay (ELISA) method and the accuracy of the system was evaluated with an average recovery value of 96.7 %. The developed biosensor system enables highly sensitive detection of exosomes in low-volume urine samples. The usage of a paper membrane as a SERS substrate, combined with the electrochemical deposition of gold nanoparticles, provides an eco-friendly and cost-effective solution, enabling wider use and applications.
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    Loss of Wasl improves pancreatic cancer outcome
    Hidalgo-Sastre, A; Desztics, J; Dantes, Z; Schulte, K; Ensarioglu, HK; Bassey-Archibong, B; Öllinger, R; Engleiter, T; Rayner, L; Einwächter, H; Daniel, JM; Altaee, ASA; Steiger, K; Lesina, M; Rad, R; Reichert, M; von Figura, G; Siveke, JT; Schmid, R; Lubeseder-Martellato, C
    Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study. we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3 beta, as well as YAP1 and phosphorylated beta-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/beta-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.