Browsing by Author "Adan, Aysun"

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Cell Proliferation and Cytotoxicity Assays
(BENTHAM SCIENCE PUBL LTDEXECUTIVE STE Y-2, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATES, 2016) Adan, Aysun; Kiraz, Yagmur; Baran, Yusuf; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Adan, Aysun; Kiraz, Yagmur; Baran, Yusuf
Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.
Combined effect of midostaurin and sphingosine kinase-1 inhibitor on FMS-like tyrosine kinase 3 (FLT3) wild type acute myeloid leukemia cells
(De Gruyter, 2022) Adan, Aysun; Şahin, Hande Nur; 0000-0002-3747-8580; 0000-0002-2382-3160; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Adan, Aysun; Şahin, Hande Nur
Objectives Therapeutic potential of clinically approved FLT3 inhibitor midostaurin has been neglected in wild-type FLT3 positive acute myeloid leukemia (AML). Sphingosine kinase-1 (SK-1) having anti-proliferative functions is studied in various cancers, but not in FLT3 wild-type AML. We aimed to develop new therapeutic strategies to combat FLT3 wild-type AML by combining midostaurin with SK-1 inhibitor (SKI II) in THP1 cells. Methods The anti-proliferative effects of midostaurin, SKI II and in combination on THP1 cells were determined by MTT assay. The combination indexes were calculated using calcusyn software. SK-1 expression and PARP cleavage were checked by western blot. Cell cycle distributions (PI staining) and apoptosis (annexin-V/PI dual staining) were assessed by flow cytometry for each agent alone and in combinations. Results Midostaurin decreased SK-1 protein level. Midostaurin, SKI II and certain combinations decreased cell viability in a dose dependent manner. The combined anti-leukemic effects of the aforementioned drug combination afforded additive effect. Co-administration induced both necrosis and apoptosis via phosphatidylserine externalization, PARP cleavage and cell cycle arrest at G0/G1 and S phases. Conclusions Targeting sphingosine kinase-1 together with FLT3 inhibition could be a novel mechanism to increase limited clinic response to midostaurin in wild-type FLT3 overexpressing AML after further pre-clinical studies.
Computational analysis of microRNA-mediated interactions in SARS-CoV-2 infection
(PEERJ INC, 341-345 OLD ST, THIRD FLR, LONDON, EC1V 9LL, ENGLAND, 2020) Demirci, Muserref Duygu Sacar; Adan, Aysun; 0000-0003-2012-0598; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (MED1, MED9, MED 12L, MED 19), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STATI were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF lab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.
Differential in vitro anti-leukemic activity of resveratrol combined with serine palmitoyltransferase inhibitor myriocin in FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) carrying AML cells
(SPRINGER, 2022) Ersoz, Nur Sebnem; Adan, Aysun; 0000-0003-3343-9936; 0000-0002-3747-8580; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Ersoz, Nur Sebnem; Adan, Aysun
Treatment of FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) AML is restricted due to toxicity, drug resistance and relapse eventhough targeted therapies are clinically available. Resveratrol with its multi-targeted nature is a promising chemopreventive remaining limitedly studied in FLT3-ITD AML regarding to ceramide metabolism. Here, its cytotoxic, cytostatic and apoptotic effects are investigated in combination with serine palmitoyltransferase (SPT), the first enzyme of de novo pathway of ceramide production, inhibitor myriocin on MOLM-13 and MV4-11 cells. We assessed dose-dependent cell viability, flow cytometric cell death and cell cycle profiles of resveratrol in combination with myriocin by MTT assay, annexin-V/PI staining and PI staining respectively. Resveratrol's dose-dependent effect on SPT protein expression was also checked by western blot. Resveratrol decreased cell viability in a dose- dependent manner whereas myriocin did not affect cell proliferation effectively in both cell lines after 48h treatments. Although resveratrol induced both apoptosis and a significant S phase arrest in MV4-11 cells, it triggered apoptosis and non-significant S phase accumulation in MOLM-13 cells. Co-administrations reduced cell viability. Increased cytotoxic effect of co-treatments was further proved mechanistically through induction of apoptosis via phosphatidylserine relocalization. The cell cycle alteration in co-treatment was significant with an S phase arrest in MV4-11 cells, however, it was not effective on cell cycle progression of MOLM-13 cells. Resveratrol also increased SPT expression. Overall, modulation of SPT together with resveratrol might be the possible explanation for resveratrol's action. It could be an integrative medicine for FLT3-ITD AML after investigating its detailed mechanism of action in relation to de novo pathway of ceramide production.
Effects of cell-mediated osteoprotegerin gene transfer and mesenchymal stem cell applications on orthodontically induced root resorption of rat teeth
(OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND, 2017) Amuk, Nisa Gul; Kurt, Gokmen; Baran, Yusuf; Seyrantepe, Volkan; KARTAL YANDIM, Melis; Adan, Aysun; Akyildiz Demir, Secil; Kiraz, Yagmur; Sonmez, Mehmet Fatih; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü;
Aim: The aim of this study is to evaluate and compare therapeutic effects of mesenchymal stem cell (MSCs) and osteoprotegerin (OPG) gene transfer applications on inhibition and/or repair of orthodontically induced inflammatory root resorption (OIIRR). Materials and methods: Thirty Wistar rats were divided into four groups as untreated group (negative control), treated with orthodontic appliance group (positive control), MSCs injection group, and OPG transfected MSCs [gene therapy (GT) group]. About 100 g of orthodontic force was applied to upper first molar teeth of rats for 14 days. MSCs and transfected MSC injections were performed at 1st, 6th, and 11th days to the MSC and GT group rats. At the end of experiment, upper first molar teeth were prepared for genetical, scanning electron microscopy (SEM), fluorescent microscopy, and haematoxylin eosin-tartrate resistant acid phosphatase staining histological analyses. Number of total cells, number of osteoclastic cells, number of resorption lacunae, resorption area ratio, SEM resorption ratio, OPG, RANKL, Cox-2 gene expression levels at the periodontal ligament (PDL) were calculated. Paired t-test, Kruskal-Wallis, and chi-square tests were performed. Results: Transferred MSCs showed marked fluorescence in PDL. The results revealed that number of osteoclastic cells, resorption lacunae, resorption area ratio, RANKL, and Cox-2 were reduced after single MSC injections significantly (P < 0.05). GT group showed the lowest number of osteoclastic cells (P < 0.01), number of resorption lacunae, resorption area ratio, and highest OPG expression (P < 0.001). Conclusions: Taken together all these results, MSCs and GT showed marked inhibition and/or repair effects on OIIRR during orthodontic treatment on rats.
Emerging DNA Methylome Targets in FLT3-ITD-Positive Acute Myeloid Leukemia: Combination Therapy with Clinically Approved FLT3 Inhibitors
(SPRINGER LINK, 2024) Tecik, Melisa; Adan, Aysun; 0000-0002-3747-8580; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Biyomühendislik Bölümü; Tecik, Melisa; Adan, Aysun
The internal tandem duplication (ITD) mutation of the FMS-like receptor tyrosine kinase 3 (FLT3-ITD) is the most common mutation observed in approximately 30% of acute myeloid leukemia (AML) patients. It represents poor prognosis due to continuous activation of downstream growth-promoting signaling pathways such as STAT5 and PI3K/AKT. Hence, FLT3 is considered an attractive druggable target; selective small FLT3 inhibitors (FLT3Is), such as midostaurin and quizartinib, have been clinically approved. However, patients possess generally poor remission rates and acquired resistance when FLT3I used alone. Various factors in patients could cause these adverse effects including altered epigenetic regulation, causing mainly abnormal gene expression patterns. Epigenetic modifications are required for hematopoietic stem cell (HSC) self-renewal and differentiation; however, critical driver mutations have been identified in genes controlling DNA methylation (such as DNMT3A, TET2, IDH1/2). These regulators cause leukemia pathogenesis and affect disease diagnosis and prognosis when they co-occur with FLT3-ITD mutation. Therefore, understanding the role of different epigenetic alterations in FLT3-ITD AML pathogenesis and how they modulate FLT3I’s activity is important to rationalize combinational treatment approaches including FLT3Is and modulators of methylation regulators or pathways. Data from ongoing pre-clinical and clinical studies will further precisely define the potential use of epigenetic therapy together with FLT3Is especially after characterized patients’ mutational status in terms of FLT3 and DNA methlome regulators.
Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling
(SAGE PUBLICATIONS LTD1 OLIVERS YARD, 55 CITY ROAD, LONDON EC1Y 1SP, ENGLAND, 2016) Adan, Aysun; Baran, Yusuf; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Baran, Yusuf; Adan, Aysun
Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confirmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/Mcell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.
Flow cytometry: basic principles and applications
(TAYLOR & FRANCIS LTD2-4 PARK SQUARE, MILTON PARK, ABINGDON OR14 4RN, OXON, ENGLAND, 2017) Adan, Aysun; Alizada, Gunel; Kiraz, Yagmur; Baran, Yusuf; Nalbant, Ayten; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü
Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.
Involvement of Sphingolipid Metabolism Enzymes in Resveratrol-Mediated Cytotoxicity in Philadelphia-Positive Acute Lymphoblastic Leukemia
(ROUTLEDGE JOURNALS, TAYLOR & FRANCIS LTD2-4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLAND, 2021) Oguz, Osman; Adan, Aysun; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Oguz, Osman; Adan, Aysun
Targeting the key enzymes of sphingolipid metabolism including serine palmitoyltransferase (SPT), sphingosine kinase (SK) and glucosylceramide synthase (GCS) has a therapeutic importance. However, sphingolipid metabolism-mediated anti-leukemic actions of resveratrol in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) remain unknown. Therefore, we explored potential mechanisms behind resveratrol-mediated cytotoxicity in SD1 and SUP-B15 Ph + ALL cells in the context of sphingolipid metabolism and apoptosis induction. The anti-proliferative and apoptotic effects of resveratrol alone and in combination with SPT inhibitor (myriocin), SK inhibitor (SKI II), GCS inhibitor (PDMP) were determined by MTT cell proliferation assay and flow cytometry, respectively. The effects of resveratrol on PARP cleavage, SPT, SK and GCS protein levels were investigated by Western blot. Resveratrol inhibited proliferation and triggered apoptosis via PARP activation and externalization of phosphatidylserine (PS). Resveratrol increased the expression of SPT whereas it downregulated SK and GCS. Resveratrol's combinations with SKI II and PDMP intensified its anti-leukemic activity by increasing the relocalization of PS while its combination with myriocin suppressed apoptosis. Therefore, resveratrol inhibited cell proliferation and induced apoptosis through modulating SK, GCS and SPT expression, which may be considered as novel biomarkers of resveratrol-induced cytotoxicity in Ph + ALL.
Major apoptotic mechanisms and genes involved in apoptosis
(SAGE PUBLICATIONS LTD1 OLIVERS YARD, 55 CITY ROAD, LONDON EC1Y 1SP, ENGLAND, 2016) Kiraz, Yagmur; Adan, Aysun; Yandim, Melis Kartal; Baran, Yusuf; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Baran, Yusuf; Kiraz, Yagmur
As much as the cellular viability is important for the living organisms, the elimination of unnecessary or damaged cells has the opposite necessity for the maintenance of homeostasis in tissues, organs and the whole organism. Apoptosis, a type of cell death mechanism, is controlled by the interactions between several molecules and responsible for the elimination of unwanted cells from the body. Apoptosis can be triggered by intrinsically or extrinsically through death signals from the outside of the cell. Any abnormality in apoptosis process can cause various types of diseases from cancer to auto-immune diseases. Different gene families such as caspases, inhibitor of apoptosis proteins, B cell lymphoma (Bcl)-2 family of genes, tumor necrosis factor (TNF) receptor gene superfamily, or p53 gene are involved and/or collaborate in the process of apoptosis. In this review, we discuss the basic features of apoptosis and have focused on the gene families playing critical roles, activation/inactivation mechanisms, upstream/downstream effectors, and signaling pathways in apoptosis on the basis of cancer studies. In addition, novel apoptotic players such as miRNAs and sphingolipid family members in various kind of cancer are discussed.
A minimally invasive transfer method of mesenchymal stem cells to the intact periodontal ligament of rat teeth: a preliminary study
(TUBITAK SCIENTIFIC & TECHNICAL RESEARCH COUNCIL TURKEY, ATATURK BULVARI NO 221, KAVAKLIDERE, ANKARA, 00000, TURKEY, 2018) Amuk, Nisa Gul; Kurt, Gökmen; Kartal Yandım, Melis; Adan, Aysun; Baran, Yusuf; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü;
The aim of this study was to introduce a minimally invasive procedure for mesenchymal stem cell (MSC) transfer into the intact periodontal ligament (PDL) of the molar teeth in rats. Ten 12-week-old Wistar albino rats were used for this preliminary study. MSCs were obtained from bones of two animals and were labeled with green fluorescent protein (GFP). Four animals were randomly selected for MSC injection, while 4 animals served as a control group. Samples were prepared for histological analysis, Cox-2 mRNA expression polymerase chain reaction analysis, and fluorescent microscopy evaluation. The number of total cells, number of osteoclastic cells, and Cox-2 mRNA expression levels of the periodontal tissue of teeth were calculated. The number of total cells was increased with MSC injections in PDL significantly (P < 0.001). The number of osteoclastic cells and Cox-2 mRNA expression were found to be similar for the two groups. GFP-labeled MSCs were observed with an expected luminescence on the smear samples of the PDL with transferred MSCs. The results of this preliminary study demonstrate successful evidence of transferring MSCs to intact FIX in a nonsurgical way and offer a minimally invasive procedure for transfer of MSCs to periodontal tissues.
The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks
(SAGE PUBLICATIONS LTD1 OLIVERS YARD, 55 CITY ROAD, LONDON EC1Y 1SP, ENGLAND, 2016) Adan, Aysun; Baran, Yusuf; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Baran, Yusuf
Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated a parts per thousand yen2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.
Pleiotropic molecular effects of dietary polyphenols resveratrol and apigenin in Leukemia
(ELSEVIER, 2018) Adan, Aysun; Oğuz, Osman; 0000-0002-3747-8580; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Adan, Aysun; Oğuz, Osman
Resveratrol and apigenin are commonly found polyphenols in many fruits and vegetables and recognition of these dietary polyphenols for human health owing to their biological and pharmacological features including antiinflammatory and antioxidative functions has increased recently. In addition to direct antioxidative effects, they have impacts on various important signaling pathways dysregulated in cancer, genetic and epigenetic regulators, transcription factors and even on miRNAs as anticarcinogenic compounds. Numerous in vitro and in vivo studies have demonstrated their pleiotropic molecular mechanisms of action in cancer, particularly in solid tumors. However, in recent years, significant progress has been made in studying the antileukemic effects of resveratrol and apigenin at the cellular and molecular levels. Herein, we have critically discussed the main molecular targets of resveratrol and apigenin and their promising potential as chemopreventive agents as well as their limitations, with a special emphasis on leukemias.
Resveratrol triggers anti-proliferative and apoptotic effects in FLT3-ITD-positive acute myeloid leukemia cells via inhibiting ceramide catabolism enzymes
(HUMANA PRESS INC, 2022) Ersoz, Nur Sebnem; Adan, Aysun; 0000-0003-3343-9936; 0000-0002-3747-8580; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Biyomühendislik Bölümü; Ersoz, Nur Sebnem; Adan, Aysun
Resveratrol possesses well-defned anti-carcinogenic activities. However, how resveratrol exerts its anti-leukemic actions by modulating anti-apoptotic ceramide catabolism enzymes, mainly sphingosine kinase (SK-1) and glucosylceramide synthase (GCS), in FLT3-ITD AML remains unclear. Resveratrol, SKI II (SK inhibitor) and PDMP (GCS inhibitor) were evaluated alone or in combinations for their efect on cell proliferation (MTT assay), apoptosis (annexin V-FITC/PI staining by fow cytometry) and cell cycle progression (PI staining by fow cytometry) in MOLM-13 and MV4-11 cells. The combination indexes (CIs) were calculated based on cell proliferation data using CompuSyn software. Caspase-3 and PARP activation, changes in SK-1 and GCS levels by resveratrol alone or PARP cleavage in co-treatments were determined by western blot. Resveratrol and inhibitors alone inhibited cell proliferation in a dose- and time-dependent manner. Resveratrol downregulated SK-1 and GCS expression in both cell lines. It induced apoptosis by phosphatidylserine (PS) exposure together with caspase-3 and PARP cleavage and arrested the cell cycle slightly at the S phase. Co-administrations intensifed resveratrol’s efect by inhibiting cell proliferation synergistically (A CI of<1) or additively (A CI 1.0–1.1) and inducing apoptosis via PS relocalization and PARP cleavage. Resveratrol plus SKI II did not afect cell cycle progression signifcantly, however, resveratrol plus PDMP blocked cycle progression at G0/G1 and S phases for MOLM-13 cells and MV4-11 cells, respectively. Overall, resveratrol may inhibit FLT3-ITD AML cell proliferation by inhibiting ceramide catabolism and be evaluated as a chemopreventive after detailed analysis of the crosstalk between resveratrol and ceramide catabolism pathway.
Resveratrolün Ph+ Akut Lenfoblastik Lösemide Terapötik Potansiyeli ve Resveratrol Tarafından Tetiklenen Apoptozda Seramid Metabolizmasının Rolü
(TUBİTAK, 2019) Adan, Aysun; Baran, Yusuf; 0000-0002-3747-8580; 0000-0001-8354-780X; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Adan, Aysun
Proje ile resveratrolün, Ph+ ALL hücreleri üzerindeki büyümeyi inhibe edici etkisinin arkasında yatan mekanizmalar, seramid metabolizmasının hedeflenmesi ve BCR-ABL ifadesindeki değişimler ile ilişkilendirilerek araştırılmıştır. Resveratrol, SK inhibitörü (SKI II), GSS inhibitörü (PDMP), SPT inhibitörü (Miriosin, Myriocin) ve resveratrol: inhibitör kombinasyonlarının in vitro olarak Ph+ ALL SD1 ve SUP-B15 hücreleri üzerindeki büyümeyi durdurucu ve apoptotik etkileri MTT hücre çoğalması testi, Aneksin-V/PI boyaması, kaspaz3, PARP ifadelerinin ve sitokrom c salınımının belirlenmesi (western blot) ile, sitostatik etki (hücre döngüsü üzerindeki) ise akım sitometresi (PI boyaması) ile araştırılmıştır. Resveratrol ve sfingolipid metabolizması enzimlerini hedefleyen inhibitör kombinasyonlarının BCR-ABL protein ifadesi üzerine etkisi western blot ile belirlenmiştir. Ayrıca, resveratrolün SPT, SK-1/2, GSS protein ifadeleri üzerindeki etkisi western blot ile belirlenmiştir. Her iki hücre hattında resveratrol ve SKI II ve PDMP ile kombinasyonları hücre büyümesini baskılamış, apoptozu tetiklemiş ve hücre döngüsünü S fazında tutmuştur. Resveratrol:Miriosin kombinasyonu ise hücre büyümesi ve hücre döngüsü üzerinde hücreye özgü etkiler gösterirken apoptozu her iki hücrede tetiklemiştir. Her iki hücre tipinde resveratol ve kombinasyonları sitokrom-c salınımını, kaspaz-3 kesimini ve PARP kesimini genel olarak arttırmakla beraber hücreye özgü değişimler de saptanmıştır. Resveratrol her iki hücrede SK-1/SK2 ve GSS ifadesini azaltırken SPT ifadesini arttırmıştır. Resveratrol, SKI II ve PDMP BCR-ABL ifadesini azaltırken Miriosin arttırmıştır. Resveratrol: SKI II ve PDMP kombinasyonları BCR-ABL üzerinde artışlara neden olurken Miriosin ile kombinasyon BCR-ABL ifadesini azaltmıştır. Sonuç olarak, resveratrol seramid metabolizmasını ve BCR-ABL ifadesini düzenleyerek Ph+ ALL üzerinde hücre büyümesini baskılamış ve apoptozu tetiklemiştir.
Therapeutic Targeting of FLT3 in Acute Myeloid Leukemia: Current Status and Novel Approaches
(Dove Medical Press Ltd, 2022) Tecik, Melisa; Adan, Aysun; 0000-0002-3747-8580; AGÜ, Yaşam ve Doğa Bilimleri Fakültesi, Moleküler Biyoloji ve Genetik Bölümü; Adan, Aysun
FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients. The presence of FLT3-ITD (internal tandem duplication, 20–25%) mutation and, to a lesser extent, FLT3-TKD (tyrosine kinase domain, 5– 10%) mutation is associated with poorer diagnosis and therapy response since the leukemic cells become hyperproliferative and resistant to apoptosis after continuous activation of FLT3 signaling. Targeting FLT3 has been the focus of many pre-clinical and clinical studies. Hence, many small-molecule FLT3 inhibitors (FLT3is) have been developed, some of which are approved such as midostaurin and gilteritinib to be used in different clinical settings, either in combination with chemotherapy or alone. However, many questions regarding the best treatment strategy remain to be answered. On the other hand, various FLT3-dependent and-independent resistance mechanisms could be evolved during FLT3i therapy which limit their clinical impact. Therefore, identifying molecular mechanisms of resistance and developing novel strategies to overcome this obstacle is a current interest in the field. In this review, recent studies of approved FLT3i and knowledge about major resistance mechanisms of clinically approved FLT3i’s will be discussed together with novel treatment approaches such as designing novel FLT3i and dual FLT3i and combination strategies including approved FLT3i plus small-molecule agents targeting altered molecules in the resistant cells to abrogate resistance. Moreover, how to choose an appropriate FLT3i for the patients will be summarized based on what is currently known from available clinical data. In addition, strategies beyond FLT3i’s including immunotherapeutics, small-molecule FLT3 degraders, and flavonoids will be summarized to highlight potential alternatives in FLT3-mutated AML therapy.