Browsing by Author "Oktem, Gulperi"

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Age-Related Characterization of Dental Pulp Mesenchymal Stem Cells
(Wiley, 2023) Kaya, Egemen; Acikgoz, Eda; Aydogdu, Ilkay; Dindaroglu, Funda Cagirir; Atalayin, Cigdem; Oktem, Gulperi
Biomolecular Fingerprints of the Effect of Zoledronic Acid on Prostate Cancer Stem Cells: Comparison of 2d and 3d Cell Culture Models
(Elsevier Science inc, 2024) Guler, Gunnur; Acikgoz, Eda; Mukhtarova, Guenel; Oktem, Gulperi
Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.
Comparison of Cell Cycle Components, Apoptosis and Cytoskeleton-Related Molecules and Therapeutic Effects of Flavopiridol and Geldanamycin on the Mouse Fibroblast, Lung Cancer and Embryonic Stem Cells
(Sage Publications Ltd, 2016) Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Cetintas, Vildan Bozok
Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.
Deneysel Diabet Modeli Oluşturulan Farelerde Tirozin Kinaz İnhibitör Uygulanımının Testis Dokusu Üzerine Olan Etkilerinin Pluripotensi Kapasitesi ve Hücre Adezyonu Özelinde Araştırılması
(2017) Oktem, Gulperi; Demir, Kenan; Aktug, Huseyin; Yigitturk, Gurkan; Yavaşoğlu, Altuğ; Özdedeli, Kaan; Açıkgöz, Eda
Amaç: Tirozin kinaz inhibisyonunun diyabet etkisi altındaki testis dokusu üzerine göstereceği etkileri araştırmaktır. Gereç ve Yöntem: Çalışmamızda 31 adet CD1 türü erkek fare kullanıldı ve dört gruba ayrıldı: Grup 1'de (kontrol grubu) 7, Grup 2'de tirozin kinaz inhibitörü uygulanan 7, Grup 3'te diyabetik ve SF uygulanan 8, Grup 4'te diyabet + tirozin kinaz inhibitörü uygulanan 9 denek hayvanı yer aldı. Grup 1'de herhangi bir uygulama yapılmadı. Grup 2'deki farelere 3 hafta boyunca tirozin kinaz inhibitörü verildi. Diyabet oluşturulması için 0.1mol/L tek doz streptozotosinin intraperitoneal olarak verildi. 250 mg/dL ve üzeri kan glikoz seviyesi diyabetik olarak kabul edildi. Deneysel diyabet modeli oluşturulan farelere 1 hafta beklendikten sonra, Grup 3'e SF, Grup 4'e 3 hafta boyunca tirozin kinaz inhibitörü verildi. Sonunda tüm denek hayvanları anestezi altında sakrifiye edilerek histopatolojik inceleme için testis dokuları alındı. İstatistiksel analiz için tek yönlü varyans analizi (ANOVA) testi yapıldı, 0.05'ten küçük p değerleri, istatistiksel olarak anlamlı kabul edildi. Bulgular: Testis dokusu histopatolojik olarak incelendiğinde deneysel diyabete bağlı olarak seminifer tübülün germ hücre serilerinde kayıp, hücre bütünlüklerinde ise bozulma saptandı. Sonuç: Bu çalışma, diyabetin testiste germ hücre serilerinde sayısal olarak azalmaya ve hücre adezyon mekanizmasında bozulmaya yol açtığını göstermektedir. Tirozin kinaz inhibitörü uygulamasının, bu hasarlanmada tamir edici etkisinin olduğu düşünülmektedir. Bu hasarın tedavisinin derecesi, uygulanan tirozin kinaz inhibitörünün dozu ve süresine bağlı olarak farklılık gösterebilmektedir. Ancak, klinik diyabet uygulamalarında tirozin kinaz inhibitörü kullanılabilmesi için bu konuda moleküler çalışma sayılarının artışına ihtiyaç vardır.
Double Hit Strategy: Removal of Sialic Acid From the Dendritic Cell Surface and Loading With Cd44+/Cd24- Cell Lysate Inhibits Tumor Growth and Metastasis by Targeting Breast Cancer Stem Cells
(Elsevier, 2022) Acikgoz, Eda; Duzagac, Fahriye; Guven, Ummu; Yigitturk, Gurkan; Kose, Timur; Oktem, Gulperi
Cancer stem cells (CSCs), which represent the root cause of resistance to conventional treatments, recurrence, and metastasis, constitute the critical point of failure in cancer treatments. Targeting CSCs with dendritic cell (DC)-based vaccines have been an effective strategy, but sialic acids on the surface of DCs limit the interaction with loaded antigens. We hypothesized that removal of sialic acid moieties on immature DCs (iDCs) could significantly affect DC-CSC-antigen loading, thereby leading to DC maturation and improving immune recognition and activity. The lysate of CD44+/CD24-/low breast CSCs (BCSCs) was pulsed with sialidase-treated DCs to obtain mature dendritic cells (mDCs). The roles of cytoskeletal elements in antigen uptake and dendritic cell maturation were determined by immunofluorescence staining, flow cytometry, and cytokine measurement, respectively. To test the efficacy of the vaccine in vivo, CSCs tumor-bearing mice were immunized with iDC or mDC. Pulsing DCs with antigen increased the expression levels of actin, gelsolin, talin, WASp, and Arp2, especially in podosome-like regions. Compared with iDCs, mDCs expressed high levels of CD40, CD80, CD86 costimulatory molecules and increased IL-12 production. Vaccination with mDC: i) increased CD8+ and CD4 + T-cell numbers, ii) prevented tumor growth with anti-mitotic activity and apoptotic induction, iii) suppressed metastasis by decreasing Snail, Slug, and Twist expressions. This study reveals for the first time that sialic acid removal and loading with CSC antigens induces significant molecular, morphological, and functional changes in DCs and that this new DC identity may be considered for future combined immunotherapy strategies against breast tumors.
Doxorubicin-Induced Senescence Promotes Resistance To Cell Death by Modulating Genes Associated With Apoptotic and Necrotic Pathways in Prostate Cancer Du145 Cd133+/Cd44+cells
(Academic Press inc Elsevier Science, 2023) Tatar, Cansu; Avci, Cigir Biray; Acikgoz, Eda; Oktem, Gulperi
Cancer stem cells (CSCs) are the most important cause of cancer treatment failure. Traditional cancer treatments, such as chemotherapy and radiotherapy, damage healthy cells alongside malignant cells, leading to severe adverse effects. Therefore, inducing cellular senescence without triggering apoptosis, which further damages healthy cells, may be an alternative strategy. However, there is insufficient knowledge regarding senescence induction in CSCs that show resistance to treatment and stemness properties. The present study aims to elucidate the effects of senescence induction on proliferation, cell cycle, and apoptosis in prostate CSCs and non-CSCs. Prostate CSCs were isolated from DU145 cancer cells using the FACS method. Subsequently, senescence induction was performed in RWPE-1, DU145, prostate CSCs, and non-CSCs by using different concentrations of Doxorubicin (DOX). Cellular senescence was detected using the senescence markers SA-beta-gal, Ki67, and senescence-associated heterochromatin foci (SAHF). The effects of senescence on cell cycle and apoptosis were evaluated using the Muse Cell Analyzer, and genes in signaling pathways associated with the apoptotic/necrotic pathway were analyzed by real-time PCR. Prostate CSCs were isolated with 95.6 +/- 1.4% purity according to CD133+/CD44+ characteristics, and spheroid formation belonging to stem cells was observed. After DOXinduced senescence, we observed morphological changes, SA-beta-gal positivity, SAHF, and the lack of Ki67 in senescent cells. Furthermore; we detected G2/M cell cycle arrest and downregulation of various apoptosis-related genes in senescent prostate CSCs. Our results showed that DOX is a potent inducer of senescence for prostate CSCs, inhibits proliferation by arresting the cell cycle, and senescent prostate CSCs develop resistance to apoptosis.
Effect of Flavopiridol on Cell Cycle, Apoptosis and Biomolecule Structure Changes in Breast Cancer Stem Cells
(Galenos Publ House, 2020) Acikgoz, Eda; Guler, Gunnur; Oktem, Gulperi
Objective: Cancer stem cells (CSCs) are a small population in cancer, which are responsible for therapeutic resistance, relapse and metastasis. Flavopiridol has antitumor activity against various types of cancer cells. The mechanism of action of flavopiridol on CD44+/CD24- breast CSCs has not yet been fully elucidated. The aim of this study was to evaluate the mechanism of action of flavopiridol on breast CSCs (BCSC) in terms of apoptosis, cell cycle and biomolecular changes. Methods: In human breast cancer, cells with CD44+/CD24-markers were isolated from MCF-7 cell line using flow cytometry. The induction of apoptosis was investigated by Annexin-V. The effect of flavopiridol on cell cycle arrest was determined and the percent of cell populations at G0/G1, S and G2/M cycles were identified. The effect of the drug on three-dimensional cell cultures was investigated using a multicellular tumor spheroid model. In addition, the effect of flavopiridol on biomolecules has been evaluated using Fourier transform infrared (FTIR) spectroscopy, which has recently been used effectively in various scientific fields. Results: Flavopiridol especially induced early apoptosis. Cell cycle analyses revealed that flavopiridol induced cell cycle arrest in G0/G1 phase. Decreased number and diameter of spheroids was observed following flavopiridol treatment. ATR-FTIR data showed that treatment with flavopiridol led to significant changes in nucleic acids. Conclusion: According to the data obtained in this study, flavopiridol exhibits anticancer effects by altering the structure/expression level of nucleic acids and changing cell cycle progression and inducing apoptosis. These finding reveals that flavopiridol can be an effective antitumor agent for the treatment of breast cancer after in vivo and phase studies are completed.
Effects of Flavopiridol on Critical Regulation Pathways of Cd133high Lung Cancer Stem Cells
(Lippincott Williams & Wilkins, 2016) Cetintas, Vildan Bozok; Acikgoz, Eda; Yigitturk, Gurkan; Demir, Kenan; Oktem, Gulperi; Kaymaz, Burcin Tezcanli; Aktug, Huseyin
Background:Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs.Methods:The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR.Results:Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs.Conclusion:Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.
Embryonic Microenvironment Suppresses Yy1 and Yy1-Related Genes in Prostate Cancer Stem Cells
(Elsevier Gmbh, 2024) Taskiran, Aysegul; Oktem, Gulperi; Demir, Aleyna; Oltulu, Fatih; Ozcinar, Emine; Duzagac, Fahriye; Acikgoz, Eda
Yin yang 1 (YY1), a transcription factor, plays crucial roles in cell fate specification, differentiation, and pluripotency during embryonic development. It is also involved in tumorigenesis, drug resistance, metastasis, and relapse caused by cancer stem cells (CSCs), particularly in prostate cancer (PCa). Targeting YY1 could potentially eliminate prostate CSCs (PCSCs) and provide novel therapeutic approaches. PCa tissues often exhibit elevated YY1 expression levels, especially in high-grade cases. Notably, high-grade PCa tissues from 58 PCa patients and CD133high/CD44high high /CD44 high PCSCs isolated from DU145 PCa cell line by FACS both showed significantly increased YY1 expression as observed through immunofluorescence staining, respectively. To investigate the embryonic microenvironment impact on YY1 expression in CSC populations, firstly PCSCs were microinjected into the inner cell mass of blastocysts and then PCSCs were co-cultured with blastocysts. Next Generation Sequencing was used to analyze alterations in YY1 and related gene expressions. Interestingly, exposure to the embryonic microenvironment significantly reduced the expressions of YY1, YY2, and other relevant genes in PCSCs. These findings emphasize the tumor-suppressing effects of the embryonic environment by downregulating YY1 and YY1-related genes in PCSCs, thus providing promising strategies for PCa therapy. Through elucidating the mechanisms involved in embryonic reprogramming and its effects on YY1 expression, this research offers opportunities for further investigation into focused therapies directed against PCSCs, therefore enhancing the outcomes of PCa therapy. As a result, PCa tumors may benefit from YY1 and associated genes as a novel therapeutic target.
Enhanced G2/M Arrest, Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells
(Public Library Science, 2015) Acikgoz, Eda; Guven, Ummu; Duzagac, Fahriye; Uslu, Ruchan; Kara, Mikail; Soner, Burak Cem; Oktem, Gulperi
Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133(+high)/CD44(+high) prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependentmanner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.
Fourier Dönüşümü Kızılötesi Spektroskopisi ile Cd133+/cd44+ Prostat Kanser Kök Hücrelerinin İki Boyutlu ve Üç Boyutlu Ortamdaki Hücresel Farklılıklarının Belirlemesi
(2019) Güler, Günnur; Açıkgöz, Eda; Oktem, Gulperi
Amaç: Sferoid kültürleri, hücrelerin kendi iç dinamikleri ve diğer hücrelerle olanetkileşimleri açısından tek tabakalı kültürlere kıyasla tümör dokusununözelliklerini daha iyi yansıtmaktadır. Bu çalışmanın amacı, üç boyutlu (3D) ve ikiboyutlu (2D) kültür ortamlarında üretilen CD133+/CD44+ prostat kanser kökhücrelerinin (KKH) makromoleküllerindeki benzerlik ve farklılıklarınınaraştırılmasıdır.Gereç ve Yöntem: DU-145 prostat kanser hücre hattı içerisindekiCD133+/CD44+ yüzey belirteç özelliklerine sahip KKH’leri akış sitometrisi(FACS) kullanılarak izole edilmiştir. Agarla kaplı kültür kapları ile sferoidyapıları oluşturulmuştur. 2D ve 3D kültür ortamlarındaki KHK hücreleri Fourierdönüşümü kızılötesi (FTIR) spektroskopisi ile karşılaştırılmıştır.Bulgular: CD133+/CD44+ hücrelerin birinci haftada agarlı kültür ortamlarındamikro-agregatlar oluşturduğu gözlenmiştir. İkinci haftada ise, olgun sferoidyapıların oluştuğu saptanmıştır. 2D ve 3D (multisellüler tümör sferoidleri) kültürortamlarında üretilen hücreler ile yapılan FTIR analizleri KHK hücre yapısındakiproteinler, lipitler, karbonhidratlar ve nükleik asitlerde (DNA, RNA) önemliderecede farklanmalar olduğunu göstermiştir. Membran lipit açil zinciruzunluğu ve hücre zarı kalınlığı, proteinlerin sekonder yapıları ve DNAoligonükleotitlerin baz sekanslarında veya fonksiyonel gruplarında önemlifarklanmaların olduğu tespit edilmiştir. Elde edilen sonuçlar, 3D kültürortamında üretilen sfreoid yapılarının in vivo tümör dokusu ile benzer özelliklersergilediğini göstermiştir.Sonuç: Hücre ortam koşulları ile yaratılmış olan fiziksel, kimyasal ve biyolojiközellikler hücrelerin kendi iç dinamiğini ve mikroçevresi içerisindekietkileşimlerini önemli derecede etkilemektedir. 2D kültür ortamları ile hücreleritek boyutta indirgemek hücrelerin gerçek özelliklerini yansıtmamaktadır. Bunedenle, 3D kültür ortamları ile hücre dinamiklerinin incelenmesigerekmektedir. Bu çalışmadan elde edilen sonuçlar, kanserde önemli bir hücrepopülasyonunu oluşturan KKH’lerin membran yapısı, lipitler, proteinlerin sekonder yapıları ve DNA oligonükleotit yapılarının terapötik hedefolabileceğini göstermektedir.
Glycogen Synthase Kinase-3 Inhibition in Glioblastoma Multiforme Cells Induces Apoptosis, Cell Cycle Arrest and Changing Biomolecular Structure
(Pergamon-elsevier Science Ltd, 2019) Acikgoz, Eda; Guler, Gunnur; Camlar, Mahmut; Oktem, Gulperi; Aktug, Huseyin
Glioblastoma multiforme (GBM) is the most malignant and aggressive primary human brain tumors. The regulatory pathways of apoptosis are altered in GBMs, leading to a survival advantage of the tumor cells. Thus, identification of target molecules, which are effective in triggering of the cell death mechanisms in GBM, is an essential strategy for therapeutic purposes. Glycogen synthase kinase-3 (GSK-3) plays an important role in apoptosis, proliferation and cell cycle. This study focused on the effect of GSK-3 inhibitor IX in the GBM cells. Apoptosis induction was determined by Annexin-V assay, multicaspase activity and immunofluorescence analyses. Concentration-dependent effects of GSK-3 inhibitor IX on the cell cycle were also evaluated. Moreover, the effect of GSK inhibitor on the cellular biomolecules was assessed by using ATR-FTIR spectroscopy. Our assay results indicated that GSK-3 inhibitor IX induces apoptosis, resulting in a significant increase in the expression of caspase-3 and caspase-8 proteins. Cell cycle analyses revealed that GSK-3 inhibitor IX leads to dose -dependent G2/M-phase cell cycle arrest. Based on the FTIR data, treatment of GBM cells causes dysregulation in the carbohydrate metabolism and induces apoptotic cell death which was characterized by the spectral alterations in nucleic acids, an increment in the lipid amount with disordering state and compositional changes in the cellular proteins. These findings suggest that GSK-3 inhibitor IX exhibits anti-cancer effects by inducing apoptosis and changing biomolecular structure of membrane lipids, carbohydrates, nucleic acids and proteins, and thus, may be further evaluated as a potential effective candidate agent for the GBM combination therapies. (C) 2018 Elsevier B.V. All rights reserved.
Ir Spektroskopi Kullanılarak İn Vitro Meme Kanser Kök Hücrelerinin Araştırılması
(2020) Güler, Günnur; Acikgoz, Eda; Oktem, Gulperi; Guven, Ummu
Amaç: Kanser kök hücreleri (KKH), tümör içinde kendi kendilerini yenileme ve diğer hücre tiplerinefarklılaşabilme kapasitesi sebebiyle tümörün başlaması, ilerlemesi, nüksetmesi, metastaz ve terapötikdirence yol açmaktadır. Bu nedenle, meme kanser kök hücrelerinin (MKKH) karakteristik özelliklerininbelirlenmesi gerekmektedir. Bu çalışmanın amacı, MKKH‟lerin akış sitometrisi ile izole edildikten sonraFourier dönüşümlü kızılötesi (FTIR) spektroskopisi kullanarak hücre biyokimyasındakifarklılaşmalarının moleküler seviyede araştırılmasıdır.Gereç ve Yöntem: MCF-7 meme kanser hücre hattındaki CD44+/CD24- yüzey belirteç özelliğigösteren MKKH‟ler akış sitometrisi ile izole edilmiştir. MCF10A, MCF-7 kanser hücre (KH) hattı ve buhattan izole edilen CD44+/CD24- yüzey belirteç özelliklerine sahip MKKH‟'ler %0,9 NaCI içerisineresuspanse edildikten sonra FTIR spektrometre ile ölçülmüştür.Bulgular: MCF-7 içerisindeki CD44+/CD24- yüzey belirteç özelliğine sahip KKH‟lerinin sort oranı%2,0-2,3 olarak belirlenmiştir. Elde edilen FTIR spektrumlarında, MKKH, meme kanser hücreleri (KH,non-KKH, bulk populasyon) ve sağlıklı hücreler arasında spektral benzerlikler ve farklılıklar tespitedilmiştir. MKKH‟lerde lipit ve protein sinyalleri daha güçlü olup hücre zarı akışkanlığı ve dinamiğifazladır. Sağlıklı hücreler ile kıyaslandığında, KH‟lerde α-helikal proteinler ve DNA sinyallerindeazalmaya karşın negatif yüklü karboksil gruplarından kaynaklanan sinyallerde artış gözlenmektedir. Buveriler, MKKH‟lerin, sağlıklı ve KH‟lere kıyasla yapı, içerik ve dinamiği bakımından oldukça farklı birprofil sergilediğini göstermektedir.Sonuç: Bu çalışma, MKKH‟lerinin moleküler yapısı ve içeriğindeki değişikliklerin incelemesi vasıtasıylaterapötik hedefli ilaç çalışmaları yapılabileceğini ortaya koymaktadır. FTIR spektroskopisi boyar maddegerektirmeden, hassas ve hızlı ölçüm alınması, örnek hazırlamada kolaylık ve az miktarda örnekgerektirmesi sebebiyle ileri hücre çalışmalarında ve medikal alanda biyolojik örneklerin analizlerindekullanılabileceği de gösterilmiştir.
Metformin Eliminates Cd133high Prostate Cancer Stem Cells Via Cell Cycle Arrest and Apoptosis
(Kare Publ, 2021) Acikgoz, Eda; Cakir, Mustafa; Guven, Mustafa; Oktem, Gulperi
Objectives: Cancer stem cells (CSCs), a small subpopulation of tumors, are responsible for chemo-radioresistance, metastasis, and cancer recurrence. The main aim of the present study is to investigate the potential effects of metformin on prostate CSCs (PCSCs). Methods: Flow cytometry was used to isolate cells with co-expression of CD133 and CD44. Sorted PCSCs were treated with different concentrations of metformin to determine the effects of metformin on cell viability using MTT assay. The association of cells exposure to metformin with apoptotic cell death and caspase activity, as well as cell cycle, were performed using the Muse Cell Analyzer. Results: In our study, for the first time we demonstrated the anti-cancer effects of metformin on PCSCs. Our results revealed that treatment with metformin reduced cell viability in CD133(high)/CD44(high) cells in a dose- and time-dependent manner. Metformin significantly induced early apoptosis and triggered the activity of several caspases associated with the apoptotic process. Metformin significantly altered the cell cycle distribution in CD133(high)/CD44(high) cells, leading to G0/G1 phase arrest. Conclusion: The results of the study revealed that metformin triggers cell death and apoptosis and modulates cell cycle distribution in CD133(high)/CD44(high) PCSCS. The present study raises the possibility that metformin is a potential anti-cancer agent for targeting PCSCs.
Neuroprotective Effect of Intrastriatal Caffeic Acid Phenethyl Ester Treatment in 6-Oh Dopamine Model of Parkinson's Disease in Rats
(Hindawi Ltd, 2021) Soner, Burak Cem; Acikgoz, Eda; Inan, Salim Yalcin; Ayla, Sule; Sahin, Ayse Saide; Oktem, Gulperi
Parkinson's disease (PD) is the second most common neurodegenerative disorder, and the main cause of PD is still not known. Until now, no cure for Parkinson's disease is yet in sight. Caffeic acid phenethyl ester (CAPE) is a polyphenolic component of the propolis, which can be derived from honeybee hive propolis. We aimed to determine the effect of intrastriatal CAPE administration as a neuroprotective agent on 6-hydroxydopamine (6-OHDA)-induced PD model. Adult male Wistar rats weighing 280-320 g were used. The PD model was induced with unilateral intrastriatal 6-OHDA injection. Treatment groups received 20 mu mol/5 mu L/4 day and 80 mu mol/5 mu L/4 day CAPE 24 h after 6-OHDA injection. Eight days after 6-OHDA application, behavioral studies (adhesive tape removal test, open-field test, cylinder test, and apomorphine-induced asymmetric rotational behavior) were performed once more to compare the effects of CAPE on behavior tests. Striatal histological verifications, immunohistochemistry, and stereological quantitation were performed. Our results for the first time showed that, besides improving the motor performance, CAPE treatment also prevents 6-OHDA-induced loss of TH-positive neurons. From our results, CAPE may be a promising clinical agent in the treatment of PD.
Optimized Method for Using Embryonic Microenvironment To Reprogram Cancer Stem Cells
(Dokuz Eylul Univ inst Health Sciences, 2023) Soner, Burak Cem; Oltulu, Fatih; Ozcinar, Emine; Taskiran, Aysegul; Demir, Aleyna; Acikgoz, Eda; Oktem, Gulperi
Purpose: The embryonic microenvironment contains many properties that have not yet been fully explored. Our aim in this study is to report an optimized and efficient method that enables investigating the effects of the secretome of pluripotent embryonic stem cells on cancer stem cells.Material and Methods: The study is performed with a chimeric model consisted of mouse blastocysts, non cancer stem cells and human prostate cancer stem cells. Ovulation induced mice were used for blastocyst collection. DU145 prostate cancer cell line was separated into non cancer and cancer stem cells using cancer stem cell biomarker expressions by fluorescent activated cell sorting. Human prostate cancer stem cells and non cancer stem cells were microinjected into 4-day blastocyst culture in vitro by intracytoplasmic sperm injection.Results: Chimeric models provide us great convenience in basic oncological studies. In this study, using a chimeric model, we were able to study the secretome of mouse embryonic stem cells and their effect on cancer stem cells. The method is efficient and yield promising result; and could be used to study the effects on other cells as well.Conclusion: The embryonic stem cell microenvironment is suggested to have a great regenerative capacity, nowadays, the center of attraction for cancer research studies. Ethical issues restrict the human embryo studies, however, mimicking the in vivo human microenvironment with 3D cell cultures or bioprinting are now possible. Finally, optimization of new methods including 3D cell cultures with human cell lines will be a great opportunity for better understanding the reprogramming notion.
Repression of the Notch Pathway Prevents Liver Damage in Streptozotocin-Induced Diabetic Mice
(Via Medica, 2017) Acikgoz, Eda; Aktug, Huseyin; Yigitturk, Gurkan; Demir, Kenan; Guven, Ummu; Duzagac, Fahriye; Oktem, Gulperi
Introduction. Sunitinib is an oral inhibitor of vascular endothelial growth factor that is used to treat a variety of cancer. There are limited data regarding the effect of sunitinib on diabetes. In the liver, Notch signaling plays an important role in liver tissue development and homeostasis and its dysfunction is associated with liver pathologies. The aim of the present study is to investigate the effects of sunitinib on streptozotocin (STZ)-induced diabetic liver in mice models. Material and methods. An experimental diabetes mellitus (DM) model was created in 28 male CD-1 mice. Twenty-eight male CD-1 mice divided in four groups (n = 7 each) were used; control mice (C), control mice treated with sunitinib (C + S), diabetic mice (DM), and diabetic mice treated with sunitinib (DM + S) for four weeks. The histopathological changes in the liver were examined by histochemistry and immunohistochemistry. Immunoreactivity of Notch1, Jagged1, DLL-1 and VEGF were evaluated in control and diabetic mice after sunitinib treatment. Results. The significant morphological changes in the liver were mostly seen in hepatocytes that were hypertrophied in the DM mice, with an increased amount of eosinophilic granules; moreover, some hepatocytes contained empty vacuole-like structures. The livers of the DM mice revealed increased deposition of collagen fibers. After sunitinib treatment the hepatocytes and hepatic lobules had almost similar morphology to control mice. The immunoreactivities of Notch1, Jagged1, DLL-1 and VEGF in hepatocytes were significantly lower in the DM group when compared with the C, DM + S and C + S group treated with sunitinib. Conclusions. These results suggest that sunitinib effectively protects the liver from diabetes-induced damage through the inhibition of the Notch pathway.
Ribosome Biogenesis Mediates Antitumor Activity of Flavopiridol in Cd44+ Breast Cancer Stem Cells
(Spandidos Publ Ltd, 2017) Erol, Ayse; Acikgoz, Eda; Guven, Ummu; Duzagac, Fahriye; Turkkani, Ayten; Colcimen, Nese; Oktem, Gulperi
Flavopiridol is a synthetically produced flavonoid that potently inhibits the proliferation of human tumor cell lines. Flavopiridol exerts strong antitumor activity via several mechanisms, including the induction of cell cycle arrest and apoptosis, and the modulation of transcriptional regulation. The aim of the present study was to determine the effect of flavopiridol on a subpopulation of cluster of differentiation (CD)44(+)/CD24(-) human breast cancer MCF7 stem cells. The CD44(+)/CD24(-) cells were isolated from the MCF7 cell line by fluorescence-activated cell sorting and treated with 100, 300, 500, 750 and 1,000 nM flavopiridol for 24, 48 and 72 h. Cell viability and proliferation assays were performed to determine the inhibitory effect of flavopiridol. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarray. According to the results, the half maximal inhibitory concentration (IC50) value of flavopiridol was 500 nM in monolayer cells. Flavopiridol induced growth inhibition and cytotoxicity in breast cancer stem cells (BCSCs) at the IC50 dose. The present study revealed several differentially regulated genes between flavopiridol-treated and untreated cells. The result of the pathway analysis revealed that flavopiridol serves an important role in translation, the ribosome biogenesis pathway, oxidative phosphorylation, the electron transport chain pathway, carbon metabolism and cell cycle. A notable result from the present study is that ribosome-associated gene expression is significantly affected by flavopiridol treatment. The data of the present study indicate that flavopiridol exhibits antitumor activity against CD44(+)/CD24(-) MCF7 BCSCs through different mechanisms, mainly by inhibiting translation and the ribosome biogenesis pathway, and could be an effective chemotherapeutic molecule to target and kill BCSCs.
Sonic Hedgehog Signaling Is Associated With Resistance To Zoledronic Acid in Cd133high/Cd44high Prostate Cancer Stem Cells
(Springer, 2021) Acikgoz, Eda; Mukhtarova, Gunel; Alpay, Araz; Avci, Cigir Biray; Bagca, Bakiye Goker; Oktem, Gulperi
Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133(high)/ CD44(high) PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mechanisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be considered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance.
Tgf-β1 Uygulaması Sonrasında Prostat Kanseri Hücrelerinde Versicanın Farklı Mrna ve Protein Ekspresyonu
(2022) Acikgoz, Eda; Soner, Burak; Taskiran, Aysegul; Caggia, Silvia; Khan, Shafıq; Oktem, Gulperi
Amaç: Tümörün hücresel heterojenliği, kusurlu genetik ve epigenetik ağlar nedeniyle bir dizi eşsiz protein eksprese eden hücre popülasyonlarından kaynaklanır. Versicanın (VCAN), çeşitli kanser hücre tiplerinde transforme edici büyüme faktör-beta (TGF-β1) tarafından up regüle edildiği gösterilmiştir. Daha önceki çalışmalarımızda, kanser kök hücre (CSC’ler) tek katmanlı kültürlerinde TGF-β1 ekspresyonunun yükseldiğini ve CSC’lerden oluşan sferoidlerde VCAN ekspresyonlarının da önemli ölçüde arttığını ortaya koydu. Yöntem: Bu sonuçlar dahilinde monolayer yüksek TGF-β1 ekspresyonunun VCAN ekspresyonunu uyararak üç boyutlu yapılanmasını tetikleyebileceği varsayılmıştır. Bu çalışma, primer ve sekonder tümör derive hücre dizilerinde VCAN'ın ekspresyon profilini ve bu hücrelerde TGF-β1 sinyalleşmesinin etkisini araştırdı.Bulgular: PC3 insan prostat hücre dizisindeki VCAN gen ekspresyon düzeyinin western blot analizinde VCAN protein ekspresyonu ile korele olduğunu gösterdi. TGF-β1’in VCAN protein ekspresyonu üzerinde hiçbir indükleyici veya baskılayıcı etkisi yoktu. Sonuç: TGF-β1’in bu çalışmada kullanılan dozlarda prostat kanseri hücrelerinde VCAN ekspresyonu üzerinde uyarıcı ya da engelleyici etkileri yoktur. Metastatik yüksek VCAN protein seviyeleri gözlenirken, artmış protein stabilitesi ve/veya prostat karsinomunda sekonder tümör hücrelerinde önemli bir rol oynayabilecek farklı VCAN izoformlarının ekspresyonu gibi transkripsiyon sonrası değişikliklerden kaynaklanabilecek hiçbir mRNA tespit edilmedi.