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    Differences and Similarities between Colorectal Cancer Cells and Colorectal Cancer Stem Cells: Molecular Insights and Implications
    (Amer Chemical Soc, 2023) Erisik, Derya; Özdil, Berrin; Açıkgöz, Eda; Asker Abdikan, Cemile Sinem; Yasin, Taha Kadir; Aktuğ, Huseyin
    Malignant tumors are formed by diverse groups of cancercells.Cancer stem cells (CSCs) are a subpopulation of heterogeneous cellsidentified in tumors that have the ability to self-renew and differentiate.Colorectal cancer (CRC), the third most frequent malignant tumor,is progressively being supported by evidence suggesting that CSCsare crucial in cancer development. We aim to identify molecular differencesbetween CRC cells and CRC CSCs, as well as the effects of those differenceson cell behavior in terms of migration, EMT, pluripotency, morphology,cell cycle/control, and epigenetic characteristics. The HT-29 cellline (human colorectal adenocarcinoma) and HT-29 CSCs (HT-29 CD133(+)/CD44(+) cells) were cultured for 72 h. The levelsof E-cadherin, KLF4, p53, p21, p16, cyclin D2, HDAC9, and P300 proteinexpression were determined using immunohistochemistry staining. Themigration of cells was assessed by employing the scratch assay technique.Additionally, the scanning electron microscopy method was used toexamine the morphological features of the cells, and their peripheral/centralelemental ratios were compared with the help of EDS. Furthermore,a Muse cell cycle kit was utilized to determine the cell cycle analysis.The HT-29 CSC group exhibited high levels of expression for E-cadherin,p53, p21, p16, cyclin D2, HDAC9, and P300, whereas KLF4 was foundto be high in the HT-29. The two groups did not exhibit any statisticallysignificant differences in the percentages of cell cycle phases. Theidentification of specific CSC characteristics will allow for earliercancer detection and the development of more effective precision oncologyoptions.
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    Differences and similarities in biophysical and biological characteristics between U87 MG glioblastoma and astrocyte cells
    (Springer, 2023) Özdil, Berrin; Çalık-Kocatürk, Duygu; Altunayar-Unsalan, Cisem; Acikgoz, Eda; Oltulu, Fatih; Görgülü, Volkan; Uysal, Ayşegül
    Current cancer studies focus on molecular-targeting diagnostics and interactions with surroundings; however, there are still gaps in characterization based on topological differences and elemental composition. Glioblastoma (GBM cells; GBMCs) is an astrocytic aggressive brain tumor. At the molecular level, GBMCs and astrocytes may differ, and cell elemental/topological analysis is critical for identifying potential new cancer targets. Here, we used U87 MG cells for GBMCS. U87 MG cell lines, which are frequently used in glioblastoma research, are an important tool for studying the various features and underlying mechanisms of this aggressive brain tumor. For the first time, atomic force microscopy (AFM), scanning electron microscopy (SEM) accompanied by energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS) are used to report the topology and chemistry of cancer (U87 MG) and healthy (SVG p12) cells. In addition, F-actin staining and cytoskeleton-based gene expression analyses were performed. The degree of gene expression for genes related to the cytoskeleton was similar; however, the intensity of F-actin, anisotropy values, and invasion-related genes were different. Morphologically, GBMCs were longer and narrower while astrocytes were shorter and more disseminated based on AFM. Furthermore, the roughness values of these cells differed slightly between the two call types. In contrast to the rougher astrocyte surfaces in the lamellipodial area, SEM-EDS analysis showed that elongated GBMCs displayed filopodial protrusions. Our investigation provides considerable further insight into rapid cancer cell characterization in terms of a combinatorial spectroscopic and microscopic approach.
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    The effects of PIKfyve inhibitor YM201636 on claudins and malignancy potential of nonsmall cell cancer cells
    (2021) Çetintaş, Vildan Bozok; Düzgün, Zekeriya; Doğan, Eda; Yıldırım, Zafer; Özdil, Berrin; Aktuğ, Hüseyin
    PIKfyve is an evolutionarily conserved lipid and protein kinase enzyme that has pleiotropic cellular functions. The aim of the present study was to investigate the effects of phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) inhibitor, YM201636, on nonsmall cell lung cancer (NSCLC) cells growth, tumorigenicity, and claudin (CLDN) expressions. Three NSCLC cell lines (Calu-1, H1299 and HCC827) were used to compare the effects of YM201636. Cytotoxic effects of YM201636 were analysed using XTT assay. Malignancy potential of cells assesses with wound healing and soft agar colony-forming assays. mRNA and protein expressions of claudins were analysed by qRT-PCR and immunofluorescence staining. Our results revealed that YM201636 inhibited the proliferation and malignancy potential of Calu-1, H1299, and HCC827 cells in a dose-dependent manner. After YM201636 treatment CLDN1, -3 and -5 expressions increased significantly in HCC827 cells. CLDN3 and -5 expressions also significantly increased in Calu-1 cell line. YM201636 treatment significantly reduced the CLDN1 and increased the CLDN5 expression in H1299 cells. Immunofluorescence staining of CLDN1, -3 and -5 proteins showed a significant increase after YM201636 treatment. Besides, YM201636 induced EGFR mRNA expression in all NSCLC cell lines. Our results have shown that YM201636 inhibits tumorigenicity of NSCLC cells. Furthermore, estimated glomerular filtration rate (EGFR) pathway is important signalling involved in the regulation of claudins. Understanding the mechanisms of PIKfyve inhibitors may improve cancer treatment particularly for EGFR overactivated NSCLC.
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    EMG, MAPK ve inflamasyona yakından bakış: Fare embriyonik kök hücre, somatik ve kanser hücrelerinde ne farklıdır?
    (2018) Oltulu, Fatih; Özdil, Berrin; Gürel, Çevik; Açıkgöz, Eda; Kocatürk, Duygu Çalık; Adalı, Yasemin; Yavaşoğlu, Altuğ
    Amaç: Yaşam; başlangıç, hayatta kalma ve ölümü içeren hücrelerin devamlı olan döngüsünden oluşmaktadır. Hücreler devamlı olarak bölünür, farklılaşır ve somatik hücreleri oluştururlar. Somatik hücreler de belli bir noktada kanser hücrelerini oluşturup kök hücreler ile benzer karakteristikleri paylaşabilmektedirler. Bu dönemlerdeki süreçler, somatik hücrelerde anormalliğe eğilimi ve kanser hücrelerine dönüşümü tanımlamak için önemlidir. Kanserin ortaya çıkışını ve ilerleyişini kontrol etmek için, hayatta kalma mekanizmaları özellikle de epitelyal-mezenkimal geçiş (EMG) ve iltihaplanma önemlidir. EMG, hem embriyonik gelişim aşamalarında, hem de kanser ilerlemesinde ortaya çıkar ve bu süreç özellikle moleküler temelli tedavi için terapötik hedef olabilir. Gereç ve Yöntem: EMG, MAP-Kinaz ve inflamasyon yolaklarının gen ekspresyon seviyesinde karşılaştırılmasında kanser hücreleri temsili için fare skuamöz akciğer kanseri hücreleri (SqLCCs), somatik kökenli hücre örneği olarak fare derisi fibroblastları (MSF'ler) ve embriyonik kök hücre olarak da fare embriyonik kök hücreleri (mEKH'ler) kullanılmıştır. ERK 1/2, Vimentin ve Twist'in immünofloresan boyama protein düzeyleri aynı hücrelerde karşılaştırılarak incelenmiştir. Bulgular: MSF'lerde ve SqLCC'lerde ERK1/2 protein ekspresyonu benzer iken mEKH’lerde ekspresyonu en düşük bulunmuştur. Ayrıca, Twist ve Vimentin ifadesi istatistiksel olarak üç hücre hattında farklı çıkmıştır. EMG'nin gen ekspresyon profiline ve MAPK sinyal yolağının desteklediği inflamasyona göre, özellikle Sparc, Vimentin, Mapksp1 ve Il24 gibi belirgin genlerde ekspresyon profilleri birbirinden çok farklı bulunmuştur. Sonuç: Üç farklı hücre hattı hem gen hem de protein ekspresyonunda farklı özellikler göstermiştir. Bu nedenle, bu moleküller terapötik hedef belirlenmesinde potansiyel itici güç olabilir.
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    Fare embriyonik kök hücrelerinin hücreler arası madde yoğunluğunun değiştirilerek farklılaştırılma süreçlerinin araştırılması
    (Ege Üniversitesi, Sağlık Bilimleri Enstitüsü, 2017) Özdil, Berrin; Aktuğ, Hüseyin
    Doku formunu oluşturan hücresel bileşenlerin sürekliliği ve özelleşmesi hem yetişkin hem de gelişmekte olan organizmalarda önemli işlemlerdir. Pluripotent embriyonik kök hücreler, primitif organizmanın iç hücre kütlesinden elde edilir. Bu hücrelerin farklılaşması kademeli olarak gerçekleşir ve hücrelerin pluripotent özellikleri giderek azalır. Hücreler, hem hücre içi hem de hücre dışı sinyalleri sürekli olarak alırlar, böylelikle iç metabolik aktivitelerini düzenlerler. Niş, kök hücrelerin farklılaşmanın en önemli belirleyicilerinden biri olarak bilinir ve in vitro koşullarda in vivo ekstra selüler matriks (ESM) modelini taklit etmek için kullanılan birçok hidrojel vardır. En yaygın olarak kullanılan hidrojellerden biri matrijeldir. Bununla birlikte, kök hücrenin farklılaşma kapasitesi için matrijelin katkısı ve bu hidrojelin protein konsantrasyonu hakkında literatürde sınırlı bilgi vardır. Farklılaşmayı moleküler seviyede tespit etmek ve ayırt etmek için, evre-spesifik embriyonik antijen 1 (stage specific embryonic antigen 1=SSEA-1) ve Octamer-Binding Transcription Factor ¾ (Oct3/4) olarak adlandırılan embriyonik kök hücre belirteçleri kullanılır. SSEA-1 belirteci, diferansiye olmamış fare embriyonik kök hücre (fEKH) tanımlamasında kullanılır. Osteopontin (OPN = SPP1), farklılaşmada osteojenik belirteçlerden biridir ve osteoblastik hücrelerde eksprese edildiği bilinmektedir. Çalışmamızda, hücre döngüsü ve immünofloresan (IF) boyamalar aracılığıyla fEKH kapasitesi ve farklılaşma kabiliyeti manipüle edilerek sürecin geliştirmesi amaçlandı. Üç farklı konsantrasyon (2, 4, 6 mg/ml) ve üç zaman noktasına odaklanıldı (gün 1, 3, 6). Kök hücre kimliğini ve osteoblastik farklılaşmayı gözlemlemek için sırayla SSEA-1 ve OPN proteinlerine özel boyamalar yapıldı. fEKH'lerin kök hücre özelliklerinin, hücre döngüsü ve IF ile gösterilen ekstraselüler matriks protein konsantrasyonuna bağlı olarak değiştiği sonucuna varıldı. Gelecekteki çalışmalarda, in vitro olarak kök hücre farklılaşmasındaki ilerlemeler ile rejeneratif tıp alanında hücre-doku bazında replasman altyapısı sağlanacaktır. Dahası, ekstraselüler matriks ile fEKH etkileşimleri, ortak sinyal yolları kullandıkları için karsinogenez, epitelyal mezenkimal geçiş (EMT) ve fibrotik bağ doku kökenli mekanizmaları da aydınlatabilir.
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    The morphological analysis of anaplastic thyroid cancer cell line
    (2022) Özışık, Hatice; Özdil, Berrin; Özdemir, Merve; Sipahi, Murat; Erdoğan, Mehmet; Çetinkalp, Şevki; Özgen, Gökhan
    Aim: Thyroid follicular cell derived cancers are classified into three groups such as papillary thyroid cancer (85%), follicular thyroid cancer (12%) and anaplastic (undifferentiated) thyroid cancer (ATC) (3%). ATCs have very rapid course, poor treatment outcomes and they are very aggressive. The aim of current study was to assess the analysis of the morphological differences of ATC cell line with the normal thyroid cell line (NTC). Materials and Methods: NTH and ATC cells were examined with haematoxylin and eosin, the nucleus: cytoplasm (N:C) ratios were detected, and cell cycles were investigated. These cell lines were compared according to their N:C ratio and their abundance in cell cycle phases. Results: The N:C ratio was higher in ATC than NTC. Both cell groups were mostly found in G0/G1 phase (68.4; 82.8) and have statistical difference in both G0/G1 and S phases. Conclusion: The rapid course and the rarity of ATC are significant barriers for clinical trials. Cultured cell lines are very important to explore the behaviour in the biology of ATC cells (such as the cell cycle), to understand the course of the disease, and to find an effective target for treatment.
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    Radiosensitivity of glioblastoma multiforme and astrocytic cell lines in cell signalling aspects
    (2023) Adalı, Yasemin; Öktem, Gülperi; Uysal, Ayşegül; Aktuğ, Hüseyin; Kocatürk, Duygu Çalık; Özdil, Berrin; Hoca, Sinan
    Objectives: The aim of this study is to investigate the radiosensitivity of Glioblastoma multiforme (GBM; U87 MG) and astrocyte (SVG p12) cell lines in vitro through the signalling pathways. Methods: GBM and astrocytes were treated with 2, 4, 6, and 8 gray of ionized radiation, followed by a clonogenic assay. The effective dose of radiation was determined as 2 gray. Immunofluorescence technics selected to analyse the macrophage migration inhibiting factor (MIF), nuclear factor of activated T-cells cytoplasmic 2 (NFATc2), osteopontin (OPN), mammalian target of rapamycin (mTOR) and stage-specific embryonic antigen-1 (SSEA-1). Additionally, p53 and cell cycle assays were performed. Results: On day 1, astrocytes showed decreased expression of MIF, OPN and mTOR and increased expression of SSEA-1 in the test group after 2 gray radiation. GBM showed decreased expression of p53 and mTOR, but increased expression of NFATc2. The results of MIF expression were found higher in GBM compared to astrocytes on day 1. Interestingly, on day 12, increased expression of SSEA-1, OPN and p53 were observed in both cell lines’ test groups. Further analysis showed that all control groups of GBM and astrocytes were significantly accumulated in the S phase. After radiotherapy application, percentage of GBM in G0/G1 phases and especially in G2/M phases increased; conversely, in the S phase it decreased. Moreover, percentage of astrocytes increased in the S phase and decreased in G0/G1 phases and in G2/M phases. Conclusions: This combination of findings suggests that as a result of the radiotherapy effect, GBM started to accumulate on check points. The central question in this study focused on changes in molecular protein expression in cancer cells after radiotherapy, particularly key signalling pathways of tumorigenesis and a new possible point of view for treating such diseases.