Moleküler Biyoloji ve Genetik Bölümü / Department of Molecular Biology and Genetics
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Publication Atiprimod Triggered Apoptotic Cell Death Via Acting on PERK/eIF2 alpha/ATF4/CHOP and STAT3/NF-Kappa B axis in MDA-MB-231 and MDA-MB-468 Breast Cancer Cells(Springer, 2021) Çoker-Gürkan, Ajda; CAN, ESİN; ŞAHİN, SEMANUR; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANPurpose The constitutive activation of STAT3 through receptor tyrosine kinases triggered breast cancer cell growth and invasion-metastasis. Atiprimod impacts anti-proliferative, anti-carcinogenic effects in hepatocellular carcinoma, lymphoma, multiple myeloma via hindering the biological activity of STAT3. Dose-dependent atiprimod evokes first autophagy as a survival mechanism and then apoptosis due to prolonged ER stress in pituitary adenoma cells. The therapeutic efficiency and mechanistic action of atiprimod in breast cancer cells have not been investigated yet. Thus, we aimed to modulate the pivotal role of ER stress in atiprimod-triggered apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. Results Dose- and time-dependent atiprimod treatment inhibits cell viability and colony formation in MDA-MB-468 and MDA-MB-231 breast cancer cells. A moderate dose of atiprimod (2 mu M) inhibited STAT3 phosphorylation at Tyr705 residue and also suppressed the total expression level of p65. In addition, nuclear localization of STAT1, 3, and NF-kappa B was prevented by atiprimod exposure in MDA-MB-231 and MDA-MB-468 cells. Atiprimod evokes PERK, BiP, ATF-4, CHOP upregulation, and PERK (Thr980), eIF2 alpha (Ser51) phosphorylation's. However, atiprimod suppressed IRE1 alpha-mediated Atg-3, 5, 7, 12 protein expressions and no alteration was observed on Beclin-1, p62 expression levels. PERK/eIF2 alpha/ATF4/CHOP axis pivotal role in atiprimod-mediated G1/S arrest and apoptosis via Bak, Bax, Bim, and PUMA upregulation in MDA-MB-468 cells. Moreover, atiprimod renders MDA-MB-231 more vulnerable to type I programmed cell death by plasmid-mediated increased STAT3 expression. Conclusion Atiprimod induced prolonged ER stress-mediated apoptosis via both activating PERK/eIF2 alpha/ATF4/CHOP axis and suppressing STAT3/NF-kappa B transcription factors nuclear migration in TBNC cells.Publication Co-Chaperone Bag-1 Plays a Role in the Autophagy-Dependent Cell Survival Through Beclin 1 Interaction(MDPI, 2021) Türk, Miray; Tatlı, Özge; Alkan, Hamza Furkan; KILBAŞ, PELİN ÖZFİLİZ; Alkurt, Gizem; Dinler Doğanay, GizemExpression levels of the major mammalian autophagy regulator Beclin 1 and its interaction with Bcl-2 regulate the switch between autophagic cell survival and apoptotic cell death pathways. However, some of the regulators and the precise mechanisms of these processes still remain elusive. Bag-1 (Bcl-2 associated athanogene-1), a member of BAG family proteins, is a multifunctional pro-survival molecule that possesses critical functions in vital cellular pathways. Herein, we report the role of Bag-1 on Bcl-2/Beclin 1 crosstalk through indirectly interacting with Beclin 1. Pull-down experiments suggested a molecular interaction between Bag-1 and Beclin 1 in breast cancer cell lines. On the other hand, in vitro binding assays showed that Bag-1/Beclin 1 interaction does not occur directly but occurs through a mediator molecule. Bag-1 interaction with p-Beclin 1 (T119), indicator of early autophagy, is increased during nutrient starvation suggesting involvement of Bag-1 in the autophagic regulation. Furthermore, CRISPR/Cas9-mediated Bag-1 knock-out in MCF-7 cells hampered cell survival and proliferation and resulted in decreased levels of total LC3 under starvation. Collectively, we suggest that Bag-1 modulates cell survival/death decision through maintaining macroautophagy as a component of Beclin 1-associated complexes.Publication The Comparison of Differentially Expressed microRNAs in Bag-1 Deficient and Wild Type MCF-7 Breast Cancer Cells by Small RNA Sequencing(TUBITAK Scientific & Technical Research Council Turkey, 2022) KILBAŞ, PELİN ÖZFİLİZ; Alkurt, Gizem; Çoker Gürkan, Ajda; DİNLER DOĞANAY, GİZEM; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANThe multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1-mediated cell response, it has become essential to determine microRNA (miRNA) functions in breast cancer depending on Bag-1 expression level. This study aims to compare microRNA expression levels in wt and Bag-1 knockout (KO) MCF-7 breast cancer cells. hsa-miR-429 was selected as a potential miRNA in BAG-1KO MCF-7 cells because of the downregulation both in bioinformatics and validation qRT-PCR assay. According to predicted mRNA targets and functional enrichment analysis the ten hub proteins that are phosphatidylinositol4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA), kinase insert domain receptor (KDR), GRB2 associated binding protein 1 (GAB1), Rac family small GTPase1 (RAC1), vascular endothelial growth factor A (VEGFA), Cbl proto-oncogene (CBL), syndecan 2 (SDC2), phospholipase C gamma 1 (PLCG1), E1A binding protein p300 (EP300), and CRK like proto-oncogene, adaptor protein (CRKL) were identified as targets of hsa-miR-429. The functional enrichment analysis showed that the most significant proteins were enriched in PI3K/Akt, focal adhesion, cytoskeleton regulation, proteoglycans in cancer, and Ras signaling pathways. It was determined that hsa-miR-429 targeted these pathways in Bag-1 deficient conditions and could be used as a potential therapeutic target in future studies.Publication CRISPR/Cas9-Mediated Bag-1 Knockout Increased Mesenchymal Characteristics of MCF-7 Cells Via Akt Hyperactivation-Mediated Actin Cytoskeleton Remodeling(Public Library of Science, 2022) KILBAŞ, PELİN ÖZFİLİZ; Can, Nisan Denizce; Kızılboğa, Tuğba; Ezberci, Fikret; Doğanay, Hamdi Levent; Doğanay, Gizem Dinler; ARISAN, ELİF DAMLABag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation. © 2022 Kilbas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Publication Endoplasmic Reticulum Stress and Oncomir-Associated Chemotherapeutic Drug Resistance Mechanisms in Breast Cancer Tumors(TUBITAK - The Scientific and Technological Research Council of Turkey, 2021) Mehdizadehtapeh, Leila; YERLİKAYA, PINAR OBAKANBreast cancer, as a heterogenous malign disease among the top five leading causes of cancer death worldwide, is defined as by far the most common malignancy in women. It contributes to 25% of all cancer-associated deaths after menopause. Breast cancer is categorized based on the expression levels of cell surface and intracellular steroid receptors [estrogen, progesterone receptors, and human epidermal growth factor receptor (HER2)], and the treatment approaches frequently include antiestrogen, aromatase inhibitors, and Herceptin. However, the management and prevention strategies due to adverse side effects stress the patients. The unsuccessful treatments cause to raise the drug levels, leading to excessive toxic effects on healthy cells, and the development of multidrug-resistance (MDR) in the tumor cells against chemotherapeutic agents. MDR initially causes the tumor cells to gain a metastatic character, and subsequently, the patients do not respond adequately to treatment. Endoplasmic reticulum (ER) stress is one of the most important mechanisms supporting MDR development. ER stress-mediated chemotherapeutic resistance is very common in aggressive tumors. The in vitro and in vivo experiments on breast tumors indicate that ER stress-activated protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)- activating transcription factor (ATF4) signal axis plays an important role in the survival of tumors and metastasis. Besides, ER stress-associated oncogenic microRNAs (miRNAs) induce chemoresistance in breast tumors. We aimed to have a look at the development of resistance mechanisms due to ER stress as well as the involvement of ER stress-associated miRNA regulation following the chemotherapeutic regimen in the human breast tumors. We also aimed to draw attention to potential molecular markers and therapeutic targets.Publication Epibrassinolide Activates AKT to Trigger Autophagy With Polyamine Metabolism in SW480 and DLD-1 Colon Cancer Cell Lines(The Scientific and Technological Research Council of Turkey, 2020) Adacan, Kaan; YERLİKAYA, PINAR OBAKANEpibrassinolide (EBR), a plant-derived polyhydroxylated derivative of 5 alpha-cholestane, structurally shows similarities to animal steroid hormones. According to the present study, EBR treatment triggered a significant stress response via activating ER stress, autophagy, and apoptosis in cancer cells. EBR could also increase Akt phosphorylation in vitro. While the activation of Akt resulted in cellular metabolic activation in normal cells to proceed with cell survival, a rapid stress response was induced in cancer cells to reduce survival. Therefore, Akt as a mediator of cellular survival and death decision pathways is a crucial target in cancer cells. In this study, we determined that EBR induces stress responses through activating Akt, which reduced the mTOR complex I (mTORC1) activation in SW480 and DLD-1 colon cancer cells. As a consequence, EBR triggered macroautophagy and led to lipidation of LC3 most efficiently in SW480 cells. The cotreatment of spermidine (Spd) with EBR increased lipidation of LC3 synergistically in both cell lines. We also found that EBR promoted polyamine catabolism in SW480 cells. The retention of polyamine biosynthesis was remarkable following EBR treatment. We suggested that EBR-mediated Akt activation might determine the downstream cellular stress responses to induce autophagy related to polyamines.Publication Gemcitabine in Combination With Epibrassinolide Enhanced the Apoptotic Response in an ER Stress-Dependent Manner and Reduced the Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells(TUBITAK Scientific & Technical Research Council Turkey, 2022) Mehdizadehtapeh, Leila; RENCÜZOĞULLARI, ÖZGE; KURYAYEVA, FADINA; ÇEVİKLİ, SENA SEDEF; ÖZAGAR, SEVVAL; ODABAŞ, PINAR SİBEL; TUNÇKOL, SUDE; YETİM, HAKAN; Gürkan, Ajda Çoker; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANGemcitabine is a broad-spectrum antimetabolite and a deoxycytidine analog recognized as a standard therapy alone or in combination with other antineoplastic agents in the therapy of pancreas cancer. Drug resistance following gemcitabine treatment is a common phenomenon; therefore, combinational therapy models are usually preferred. Pancreatic ductal adenocarcinoma, or pancreas cancer, is the fourth leading cause of cancer-related deaths worldwide. With the increasing incidence of pancreatic cancer every year, the mortality rate is also rising significantly because of late diagnosis, and limited chemotherapy options. Adjuvant chemotherapy after surgical resection is the typical option for the treatment of early pancreatic cancer. Mostly, 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel is used for the prognosis of advanced pancreatic cancer; however, chemoresistance usually occurs limiting the effectiveness of the chemotherapy. Therefore, most of the studies are focused on gemcitabine combination with other drugs to overcome the situation.As an apoptotic agent and a member of brassinosteroids, epibrassinolide (EBR) induces endoplasmic reticulum (ER) stress-dependent cell death in different cancer cells, as shown by our group. In this study, we aimed to enhance the gemcitabine apoptotic effect by EBR combined treatment in pancreatic cancer cells. EBR treatment reduced cell viability and inhibited cell proliferation in PANC-1, MIA PaCa-2, and AsPC-1 cells. Each pancreatic cancer cell gave different responses to the EBR treatment because of different aggressiveness. However, EBR induced apoptosis through increasing ROS generation, which was associated with ER stress in PANC-1 and MIA PaCa-2 cells. Gemcitabine alone reduced the cell viability of each pancreatic cancer cell line; however, combination with EBR led to further induction of apoptotic cell death in each pancreatic cancer cell line. In addition, combined treatment of gemcitabine and EBR further decreased N-cadherin and vimentin expressions, suggesting that epithelial-mesenchymal transition of pancreatic cells is reduced. In conclusion, EBR had therapeutic potential to avoid the gemcitabine-induced side effects during the treatment of pancreatic cancer.Publication Gene Editing and RNAi Approaches for COVID-19 Diagnostics and Therapeutics(Springer Nature, 2021) Berber, Burak; Aydın, Cihan; Kocabaş, Fatih; Güney-Esken, Gülen; Yılancıoğlu, Kaan; Karadağ-Alpaslan, Medine; Çalıseki, Mehmet; Yüce, Melek; Demir, Sevda; Taştan, CihanThe novel coronavirus pneumonia (COVID-19) is a highly infectious acute respiratory disease caused by Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoV-2) (Prec Clin Med 2020;3:9-13, Lancet 2020;395:497-506, N. Engl J Med 2020a;382:1199-207, Nature 2020;579:270-3). SARS-CoV-2 surveillance is essential to controlling widespread transmission. However, there are several challenges associated with the diagnostic of the COVID-19 during the current outbreak (Liu and Li (2019), Nature 2020;579:265-9, N. Engl J Med 2020;382:727-33). Firstly, the high number of cases overwhelms diagnostic test capacity and proposes the need for a rapid solution for sample processing (Science 2018;360:444-8). Secondly, SARS-CoV-2 is closely related to other important coronavirus species and subspecies, so detection assays can give false-positive results if they are not efficiently specific to SARS-CoV-2. Thirdly, patients with suspected SARS-CoV-2 infection sometimes have a different respiratory viral infection or co-infections with SARS-CoV-2 and other respiratory viruses (MedRxiv 2020a;1-18). Confirmation of the COVID-19 is performed mainly by virus isolation followed by RT-PCR and sequencing (N. Engl J Med 2020;382:727-33, MedRxiv 2020a, Turkish J Biol 2020;44:192-202). The emergence and outbreak of the novel coronavirus highlighted the urgent need for new therapeutic technologies that are fast, precise, stable, easy to manufacture, and target-specific for surveillance and treatment. Molecular biology tools that include gene-editing approaches such as CRISPR-Cas12/13-based SHERLOCK, DETECTR, CARVER and PAC-MAN, antisense oligonucleotides, antisense peptide nucleic acids, ribozymes, aptamers, and RNAi silencing approaches produced with cutting-edge scientific advances compared to conventional diagnostic or treatment methods could be vital in COVID-19 and other future outbreaks. Thus, in this review, we will discuss potent the molecular biology approaches that can revolutionize diagnostic of viral infections and therapies to fight COVID-19 in a highly specific, stable, and efficient way.Publication In Vitro Investigations of miR-33a Expression in Estrogen Receptor-Targeting Therapies in Breast Cancer Cells(MDPI, 2021) KILBAŞ, PELİN ÖZFİLİZ; SÖNMEZ, ÖZLEM; Çoker-Gürkan, Ajda; Palavan-Ünsal, Narcin; Uysal-Onganer, Pınar; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANSimple Summary: Altered metabolic pathways determine the aggressivity of breast cancer cells. To highlight the potential markers gains importance to understand early molecular signatures of disease. microRNAs are the small non-coding RNAs found in different biological samples. Due to the dysregulation of metabolic pathways, the expression and secretion of microRNAs are modulated. (1) Background: Increased fatty acid synthesis leads to the aggressive phenotype of breast cancer and renders efficiency of therapeutics. Regulatory microRNAs (miRNAs) on lipid biosynthesis pathways as miR-33a have potential to clarify the exact mechanism. (2) Methods: We determined miR-33a expression levels following exposure of MCF-7 and MDA-MB-231 breast cancer cells to estrogen receptor (ER) activator (estradiol-17 beta, E2) or anti-estrogens (ICI 182,780, Fulvestrant, FUL) at non-cytotoxic concentrations. We related miR-33a expression levels in the cells to cellular lipid biosynthesis-related pathways through immunoblotting. (3) Results: miR-33a mimic treatment led to significantly downregulation of fatty acid synthase (FASN) in MCF-7 cells but not in MDA-MB-231 cells in the presence of estradiol-17 beta (E2) or Fulvestrant (FUL). In contrast to the miR-33a inhibitor effect, miR-33a mimic co-transfection with E2 or FUL led to diminished AMP-activated protein kinase a (AMPKa) activity in MCF-7 cells. E2 increases FASN levels in MDA-MB-231 cells regardless of miR-33a cellular levels. miR-33a inhibitor co-treatment suppressed E2-mediated AMPKa activity in MDA-MB-231 cells. (4) Conclusions: The cellular expression levels of miR-33a are critical to understanding differential responses which include cellular energy sensors such as AMPKa activation status in breast cancer cells.Publication Inhibition on JNK Mimics Silencing of Wnt-11 Mediated Cellular Response in Androgen-Independent Prostate Cancer Cells(MDPI, 2020) RENCÜZOĞULLARI, ÖZGE; Keskin, Buse; Grant, Guy H.; Uysal-Onganer, Pınar; ARISAN, ELİF DAMLAProstate cancer (PCa) is one of the most common cancers among men, and one of the leading causes of cancer death for men. The c-Jun N-terminal kinase (JNK) pathway is required for several cellular functions, such as survival, proliferation, differentiation, and migration. Wnt-11, a member of the Wnt family, has been identified for its upregulation in PCa; however, downstream signalling of Wnt-11 remains to be fully characterized. In this study, we investigated the role of the JNK pathway as a potential downstream factor for Wnt-11 signalling. For this purpose, LNCaP, DU145, and PC-3 PCa cells and normal epithelial PNT1A cells were treated with a specific JNK kinase inhibitor: JNKVIII. Our results showed that JNK inhibition decreased mitochondrial membrane potential and promoted cell death in a cell type-dependent manner. We found that JNK inhibition led to an increase in autophagy and prevented epithelial-mesenchymal transition (EMT) in independently growing androgen cells. JNK inhibition and the silencing of Wnt-11 showed similar responses in DU145 and PC-3 cells and decreased metastasis-related biomarkers, cell migration, and invasion. Overall, our results suggest that JNK signalling plays a significant role in the pathophysiology of PCa by mediating Wnt-11 induced signals. Our data highlights that both the JNK pathway and Wnt-11 could be a useful therapeutic target for the combinatory application of current PCa.Publication Interactome Analysis of Bag-1 Isoforms Reveals Novel Interaction Partners in Endoplasmic Reticulum-Associated Degradation(Public Library Science, 2021) Can, Nisan Denizce; Baştürk, Ezgi; Kızılboğa, Tuğba; Akçay, İzzet Mehmet; Dingiloğlu, Baran; Tatlı, Özge; Acar, Sevilay; KILBAŞ, PELİN ÖZFİLİZ; Elbeyli, Efe; Muratcioglu, Serena; Jannuzzi, Ayşe Tarbin; Gürsoy, Attila; Doğanay, Hamdi Levent; Yılmaz, Betül Karademir; Doğanay, Gizem DinlerBag-1 is a multifunctional protein that regulates Hsp70 chaperone activity, apoptosis, and proliferation. The three major Bag-1 isoforms have different subcellular localizations and partly non-overlapping functions. To identify the detailed interaction network of each isoform, we utilized mass spectrometry-based proteomics and found that interactomes of Bag-1 isoforms contained many common proteins, with variations in their abundances. Bag-1 interactomes were enriched with proteins involved in protein processing and degradation pathways. Novel interaction partners included VCP/p97; a transitional ER ATPase, Rad23B; a shuttling factor for ubiquitinated proteins, proteasome components, and ER-resident proteins, suggesting a role for Bag-1 also in ER-associated protein degradation (ERAD). Bag-1 pull-down from cells and tissues from breast cancer patients validated these interactions and showed cancer-related prominence. Using in silico predictions we detected hotspot residues of Bag-1. Mutations of these residues caused loss of binding to protein quality control elements and impaired proteasomal activity in MCF-7 cells. Following CD147 glycosylation pattern, we showed that Bag-1 downregulated VCP/p97-dependent ERAD. Overall, our data extends the interaction map of Bag-1, and broadens its role in protein homeostasis. Targeting the interaction surfaces revealed in this study might be an effective strategy in the treatment of cancer.Publication Mechanistic Approach on Melatonin-Induced Hormesis of Photosystem II Function in the Medicinal Plant Mentha spicata(MDPI, 2023) Moustakas, Michael; Sperdouli, Ilektra; Adamakis, Ioannis-Dimosthenis S.; Sas, Begüm; İŞGÖREN, SUMRUNAZ; Moustaka, Julietta; Morales, FerminMelatonin (MT) is considered a new plant hormone having a universal distribution from prokaryotic bacteria to higher plants. It has been characterized as an antistress molecule playing a positive role in the acclimation of plants to stress conditions, but its impact on plants under non-stressed conditions is not well understood. In the current research, we evaluated the impact of MT application (10 and 100 mu M) on photosystem II (PSII) function, reactive oxygen species (ROS) generation, and chlorophyll content on mint (Mentha spicata L.) plants in order to elucidate the molecular mechanism of MT action on the photosynthetic electron transport process that under non-stressed conditions is still unclear. Seventy-two hours after the foliar spray of mint plants with 100 mu M MT, the improved chlorophyll content imported a higher amount of light energy capture, which caused a 6% increase in the quantum yield of PSII photochemistry (Phi(PSII)) and electron transport rate (ETR). Nevertheless, the spray with 100 mu M MT reduced the efficiency of the oxygen-evolving complex (OEC), causing donor-side photoinhibition, with a simultaneous slight increase in ROS. Even so, the application of 100 mu M MT decreased the excess excitation energy at PSII implying superior PSII efficiency. The decreased excitation pressure at PSII, after 100 mu M MT foliar spray, suggests that MT induced stomatal closure through ROS production. The response of Phi(PSII) to MT spray corresponds to a J-shaped hormetic curve, with Phi(PSII) enhancement by 100 mu M MT. It is suggested that the hormetic stimulation of PSII functionality was triggered by the non-photochemical quenching (NPQ) mechanism that stimulated ROS production, which enhanced the photosynthetic function. It is concluded that MT molecules can be used under both stress and non-stressed conditions as photosynthetic biostimulants for enhancing crop yields.Publication Mechanistic Insights on Salicylic Acid Mediated Enhancement of Photosystem II Function in Oregano Seedlings Subjected to Moderate Drought Stress(MDPI, 2023) Moustakas, Michael; Sperdouli, Ilektra; Moustaka, Julietta; Şaş, Begüm; İŞGÖREN, SUMRUNAZ; Morales, FerminDramatic climate change has led to an increase in the intensity and frequency of drought episodes and, together with the high light conditions of the Mediterranean area, detrimentally influences crop production. Salicylic acid (SA) has been shown to supress phototoxicity, offering photosystem II (PSII) photoprotection. In the current study, we attempted to reveal the mechanism by which SA is improving PSII efficiency in oregano seedlings under moderate drought stress (MoDS). Foliar application of SA decreased chlorophyll content under normal growth conditions, but under MoDS increased chlorophyll content, compared to H2O-sprayed oregano seedlings. SA improved the PSII efficiency of oregano seedlings under normal growth conditions at high light (HL), and under MoDS, at both low light (LL) and HL. The mechanism by which, under normal growth conditions and HL, SA sprayed oregano seedlings compared to H2O-sprayed exhibited a more efficient PSII photochemistry, was the increased (17%) fraction of open PSII reaction centers (qp), and the increased (7%) efficiency of these open reaction centers (Fv '/Fm '), which resulted in an enhanced (24%) electron transport rate (ETR). SA application under MoDS, by modulating chlorophyll content, resulted in optimized antenna size and enhanced effective quantum yield of PSII photochemistry (phi(PSII)) under both LL (7%) and HL (25%), compared to non-SA-sprayed oregano seedlings. This increased effective quantum yield of PSII photochemistry (phi(PSII)) was due to the enhanced efficiency of the oxygen evolving complex (OEC), and the increased fraction of open PSII reaction centers (qp), which resulted in an increased electron transport rate (ETR) and a lower amount of singlet oxygen (O-1(2)) production with less excess excitation energy (EXC).Publication MiR-21 Is Required for the Epithelial-Mesenchymal Transition in MDA-MB-231 Breast Cancer Cells(MDPI, 2021) RENCÜZOĞULLARI, ÖZGE; Cieza-Borrella, Clara; Miralles Arenas, Francesc; Dwek, Miriam; Lange, Sigrun; Uysal-Onganer, Pınar; ARISAN, ELİF DAMLABreast cancer (BCa) is one of the leading health problems among women. Although significant achievements have led to advanced therapeutic success with targeted therapy options, more efforts are required for different subtypes of tumors and according to genomic, transcriptomic, and proteomic alterations. This study underlines the role of microRNA-21 (miR-21) in metastatic MDA-MB-231 breast cancer cells. Following the knockout of miR-21 from MDA-MB-231 cells, which have the highest miR-21 expression levels compared to MCF-7 and SK-BR-3 BCa cells, a decrease in epithelial-mesenchymal transition (EMT) via downregulation of mesenchymal markers was observed. Wnt-11 was a critical target for miR-21, and the Wnt-11 related signaling axis was altered in the stable miR-21 knockout cells. miR-21 expression was associated with a significant increase in mesenchymal markers in MDA-MB-231 BCa cells. Furthermore, the release of extracellular vesicles (EVs) was significantly reduced in the miR-21 KO cells, alongside a significant reduction in relative miR-21 export in EV cargo, compared with control cells. We conclude that miR-21 is a leading factor involved in mesenchymal transition in MDA-MB-231 BCa. Future therapeutic strategies could focus on its role in the treatment of metastatic breast cancer.Publication Palbociclib Suppresses the Cancer Stem Cell Properties and Cell Proliferation Through Increased Levels of miR-506 or miR-150 in Panc-1 and MiaPaCa-2 Cells(TUBITAK Scientific & Technical Research Council Turkey, 2022) RENCÜZOĞULLARI, ÖZGE; ARISAN, ELİF DAMLAThe prognostic characteristics of pancreatic cancer (PC) are determined by the contributing factors from the tumor microenvironment. Leptin is a critical oncogenic factor released by adipocytes as an adipokine into the tumor microenvironment, where it promotes tumor development by activating cancer stem cell (CSC) molecular regulators Notch, Hedgehog, and Wnt/(3-catenin signaling. One of the downstream targets of these pathways is CDK4/6 and cyclin D which is controlled by P16 INK4A that is highly mutated in PC. Therefore, the purpose of this study was to determine the effect of a CDK4/6 inhibitor, palbociclib, on Leptin-induced PC cells and to target the Notch, Hedgehog, and Wnt/(3-catenin signaling pathways via miR-150, miR-506, and miR-208 modulation. Leptin treatment increased the ability of Panc-1, MiaPaCa-2, and Capan-2 cells to proliferate and decreased the effect of palbociclib. Additionally, tumorspheres were generated from Leptin-treated (Leptin+) and Leptin-untreated (Leptin-) Panc-1 and MiaPaCa-2 cells and transfected with miR-506, miR-150 (tumorsuppressor miRNAs), or anti-miR-208 (oncomiR), followed by palbociclib treatment. Forced expression of miR-506 or miR-150 significantly increased the susceptibility of Leptin+ cells to palbociclib treatment by inhibiting colony and tumor spheroid formation, and CD44 expression in Panc-1 and MiaPaCa-2 cells. Additionally, the increased miR-150 expression is more effective at inhibiting N-cadherin, (3-catenin, p-GSK3(3, Notch, and Wnt5a/b expression in Leptin-/+ Panc-1 and MiaPaCa-2 cells. As a result, palbociclib suppressed the CSC profile induced by leptin treatment, inhibiting both tumorsphere forms and leptin-targeted signaling pathways, thereby disabling the Panc-1 and MiaPaCa-2 cells??? resistance mechanism. Increased expression of miR-506 or miR-150 and inhibition of miR-208 enhanced sensitivity of Panc-1 and MiaPaCa-2 Leptin-/+ cells to palbociclib treatment. As a result, this study proved that combining inhibitors of CSC molecular regulators with palbociclib improves the success rate of inhibition of PC cell proliferation.Publication The Protective Effects of Sodium Pentaborate Tetrahydrate Against UVB-induced Apoptosis in Human Keratinocytes(Hitit Üniversitesi, 2022) ABDİK, EZGİ AVŞARUltraviolet radiation (UV) is an environmental carcinogen causing human skin cancer. Exposure of the skin to UV produces apoptotic keratinocytes called sunburn cells within the epidermis. Boron, an essential element for plants, has several biological properties, such as anti-cancer, anti-microbial, and anti-oxidant. In the present study, the possible protective effects of sodium pentaborate tetrahydrate (SPT) against UVB-induced apoptosis in human keratinocyte cells, HaCaT, were investigated. They were treated with SPT at different concentrations (7.8-125 μg/mL) for 24h after UVB irradiation (20, 30 and 60mJ/cm2). Cell viability, annexin V assay, cell cycle analysis, and apoptosis-related gene levels were measured using RT-PCR. Treatment with SPT (15.6-31.25μg/mL) after 30 mJ/m2 UVB exposure significantly increased cell survival. Annexin V apoptosis analysis demonstrated a robust protective effect by treatment with SPT at concentrations of 15.6 and 31.25μg/mL after 30mJ/cm2 UVB irradiation. The cell cycle analysis revealed that UVB irradiation elevated the number of cells at the G0/G1 phase while SPT treatment after UVB irradiation increased the number of cells at G2/M phase, suggesting the changes were partially reversed. Furthermore, treatment with 15.6μg/mL SPT after 30 mJ/m2 UV irradiation blocked the activation of Caspase 3, Caspase 9, Bax, And P53. These results indicate that treatment with SPT exerts protective effects after UVB irradiation. Thus, treatment with SPT led to strong protection against UVB-induced apoptotic cell death in HaCaT cells.Publication The Protective Impact of Growth Hormone Against Rotenone-Induced Apoptotic Cell Death via Acting on Endoplasmic Reticulum Stress and Autophagy Axis(Scientific and Technological Research Council Turkey, 2023) RENCÜZOĞULLARI, ÖZGE; TORNACI, SELAY; ÇELİK, YAĞMUR; TAŞ, NAYAT NAROT; YERLİKAYA, PINAR OBAKAN; Arısan, Elif Damla; Gürkan, Ajda ÇokerHuman growth hormone (GH) is crucial modulator of cellular metabolisms, including cell proliferation and organ development, by stimulating insulin-like growth factor-1 (IGF-1), which has various functions such as cell proliferation, tissue growth, survival, or neuroprotection. Therefore, GH is implicated as a critical player in the cell and can enhance neurogenesis and provide neuroprotection during the treatment of neurological diseases such as Parkinson's disease (PD). In this study, the neuroprotective role of GH was investigated in rotenone-induced PD models for the first time. Both SH-SY5Y and SK-N-AS neuroblastoma cells were exposed to rotenone to mimic PD pathogenesis as stated in previous studies. Our data demonstrated that overexpression of GH led to the resistance of the SH-SY5Y and SK-N-AS cell lines to rotenone treatment. The levels of ER stress markers, CHOP, PERK, XBP-1, and ATF6, were higher in wt cells than GH+ SH-SY5Y cells. However, the level of autophagy markers LC3 increased and the levels of reactive oxygen species (ROS) decreased with the overexpression of GH. Furthermore, while rotenone significantly increased the SubG1 population in the cell cycle of SH-SY5Y wt cells, there was a minor alteration in GH+ cell population. Concomitantly, the levels of the proapoptotic marker, cleaved-PARP, and positive staining of Annexin V in SH-SY5Y wt cells were higher after rotenone treatment. Together, these results revealed that overexpression of GH enhanced the autophagy response by triggering the ER stress of SH-SY5Y cells to rotenone exposure and showed a neuroprotective effect in vitro PD models.Publication Riluzole: Anti-Invasive Effects on Rat Prostate Cancer Cells Under Normoxic and Hypoxic Conditions(Blackwell Publishing Ltd., 2020) Rizaner, Nahit; Uzun, Sercan; Fraser, Scott P.; Djamgoz, Mustafa B. A.; ALTUN, SEYHANAnti-invasive effects of riluzole and ranolazine, a neuro-protectant and an anti-anginal drug, respectively, on Mat-LyLu rat prostate cancer (PCa) cells were tested in vitro (a) at non-toxic doses and (b) under both normoxic and hypoxic conditions, the latter common to growing tumours. Tetrodotoxin (TTX) was used as a positive control. Hypoxia had no effect on cell viability but reduced growth at 48 hours. Riluzole (5 μmol/L) or ranolazine (20 μmol/L) had no effect on cell viability or growth under normoxia or hypoxia over 24 hours. Matrigel invasion was not affected by hypoxia but inhibited by TTX, ranolazine and riluzole under a range of conditions. The expression of Nav1.7 mRNA, the prevailing, pro-invasive voltage-gated sodium channel α-subunit (VGSCα), was up-regulated by hypoxia. Riluzole had no effect on Nav1.7 mRNA expression in normoxia but significantly reduced it in hypoxia. VGSCα protein expression in plasma membrane was reduced in hypoxia; riluzole increased it but only under hypoxia. It was concluded (a) that riluzole and ranolazine have anti-invasive effects on rat PCa cells and (b) that Nav1.7 mRNA and protein expression can be modulated by riluzole under hypoxia. Overall, therefore, riluzole and ranolazine may ultimately be "repurposed" as anti-metastatic drugs against PCa.Publication Specific c-Jun N-Terminal Kinase Inhibitor, JNK-IN-8 Suppresses Mesenchymal Profile of PTX-Resistant MCF-7 Cells through Modulating PI3K/Akt, MAPK and Wnt Signaling Pathways(MDPI, 2020) KILBAŞ, PELİN ÖZFİLİZ; Sönmez, Özlem; Uysal-Onganer, Pınar; Çoker Gürkan, Ajda; YERLİKAYA, PINAR OBAKAN; ARISAN, ELİF DAMLAPaclitaxel (PTX) is a widely used chemotherapeutic agent in the treatment of breast cancer, and resistance to PTX is a common failure of breast cancer therapy. Therefore, understanding the effective molecular targets in PTX-resistance gains importance in identifying novel strategies in successful breast cancer therapy approaches. The aim of the study was to investigate the functional role of PTX resistance on MCF-7 cell survival and proliferation related to PI3K/Akt and MAPK pathways. The generated PTX-resistant (PTX-res) MCF-7 cells showed enhanced cell survival, proliferation, and colony formation potential with decreased cell death compared to wt MCF-7 cells. PTX-res MCF-7 cells exhibited increased motility profile with EMT, PI3K/Akt, and MAPK pathway induction. According to the significant SAPK/JNK activation in PTX-res MCF-7 cells, specific c-Jun N-terminal kinase inhibitor, JNK-IN-8 is shown to suppress the migration potential of cells. Treatment of JNK inhibitor suppressed the p38 and SAPK/JNK and Vimentin expression. However, the JNK inhibitor further downregulated Wnt signaling members in PTX-res MCF-7 cells. Therefore, the JNK inhibitor JNK-IN-8 might be used as a potential therapy model to reverse PTX-resistance related to Wnt signaling.Publication Synthesis and Characterization of Novel ssDNA X-Aptamers Targeting Growth Hormone Releasing Hormone (GHRH)(Public Library Science, 2022) ŞAHİN, BURCU AYHAN; APAYDIN, ZEYNEP-ELİF; Çoker-Gürkan, Ajda; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANBackground Growth Hormone Releasing Hormone (GHRH), 44 amino acids containing hypothalamic hormone, retains the biological activity by its first 29 amino acids. GHRH (NH2 1-29) peptide antagonists inhibit the growth of prostate, breast, ovarian, renal, gastric, pancreatic cancer in vitro and in vivo. Aptamers, single-strand RNA, or DNA oligonucleotides are capable of binding to target molecules with high affinity. Our aim in this study is to synthesize and select X-aptamers against both GHRH NH2 (1-29) and GHRH NH2 (1-44) and demonstrate synthesized aptamers' target binding activity as well as serum stability. Methods and results Aptamers against GHRH NH2 (1-44) and NH2 (1-29) peptides were synthesized, and binding affinity (K-d) of 24 putative X-aptamers was determined by the dot-blot method, co-immunofluorescence staining and, SPR analysis. The serum stability of TKY.T1.08, TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers was 90-120 h, respectively. The dose-dependent binding of TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers on GHRHR in MIA PaCa-2 was approved by co-IF assay results. Moreover, SPR analysis indicated the Kd (4.75, 1.21, and 4.0 nM) levels of TKY2.T1.13, TKY.T2.08, TKY.T2.09 putative X-aptamers, respectively. Conclusion Our results illustrate the synthesis of 24 putative X-aptamers against both GHRH NH2 (1-44) and NH2 (1-29) peptides and TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers have high serum stability, high target binding potential with low K-d levels.