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Publication Open Access Aging-related diseases and autophagy(2016) Çoker Gürkan , Ajda; Palavan Unsal, Narcin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANAutophagy is fundamental, evolutionary conserved physiological process at molecular level which targets long-lived cytosolic proteins and organelles to be recycled through lysosomal degradation. Diminished autophagic activity caused cellular stress in many organisms following aging, and inhibition of autophagy in model organisms causes degenerative changes and pathologic diseases observed with high incidence ratio generally in older ages. Consequently the delayed senescence or increased longevity in model organisms often stimulate autophagy, and autophagy inhibition compromises anti-aging effects. The cytoprotective function of autophagy is presented in various human diseases such as lung, liver, cardiovascular diseases, neurodegeneration, myopathies, cancer, stroke, infections and metabolic diseases which are found associated with autophagic targets. These pathologies are defined with their age-dependent characteristics, is not fully understood that how autophagy network regulates metabolism and may cause diseases in age-related manner. In this book chapter, we are going to discuss the autophagy and aging relationship in three different parts. In the first section autophagy and aging relationship is going to be presented through explaining responsible signalling network. The autophagy and age-related neurological disorders, genetic basis of age-dependent diseases and the functional role of autophagy is going to be discussed in the second and third part of the chapter.Publication Restricted The Role of the PI3K/AKT/mTOR Signaling Axis in the Decision of the Celastrol-Induced Cell Death Mechanism Related to the Lipid Regulatory Pathway in Prostate Cancer Cells(Elsevier, 2020) RENCÜZOĞULLARI, ÖZGE; Çoban, Mervenur; Sevgin, Bortecine; Çoker-Gürkan, Ajda; YERLİKAYA, PINAR OBAKAN; Palavan-Ünsal, Narcin; ARISAN, ELİF DAMLAProstate cancer is one of the leading cancer types among the male population worldwide, with high incidence and mortality rates. Celastrol is a promising bioactive component extracted from Tripterygium wilfordi. Celastrol is a multimodal agent with therapeutic advantages, with activities that affect cell proliferation, inflammation (through affecting proteotoxic stress) and cell death mechanisms such as autophagy, apoptosis and paraptosis. Celastrol alters the PI3K/AKT signaling axis to suppress the cell viability of cancer cells. Although a number of celastrol targets have been identified in cancer cells, the genomic differences in malignant cells prevent the efficacy of celastrol. Therefore, new studies are required to highlight its potential therapeutic effects in cancer cells. In this study, we investigated the therapeutic potential of celastrol in LNCaP, DU145 and PC3 prostate cancer cells. According to our results, celastrol decreased cell viability in a dose-dependent manner in the cells. LNCaP prostate cancer cells were more sensitive to celastrol treatment compared to DU145 and PC3 cells. We found that celastrol modulated PI3K/AKT/mTOR signaling to alter lipid regulatory pathways by affecting LAMP -1 and lipin-1 in PC3 cells. Although celastrol downregulated FASN in all cell lines, active mTOR signaling led to altered responses in prostate cancer cells by affecting the cellular fate decision pathways.Publication Open Access Celastrol Modulates Lipid Synthesis via PI3K/Akt/mTOR Signaling Axis to finalize Cell Death Response in Prostate Cancer Cells(2017) Çoker Gürkan, Ajda; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; RENCÜZOĞULLARI, ÖZGE; 113920; 222563; 125860; 156421; 6125FASN is key enzyme during lipid biogenesis is associated with prostate cancer. In this study, we aim to investigate the potential role of celastrol, root extracts of Tripterygium wilfordii on modulation of lipid biosynthesis-associated PI3K/Akt signaling. To determine the effect of celastrol on cell viability, prostate cancer cells were exposed with celastrol in dose dependent manner. AR (+) LNCaP and AR (−) DU145 and PC3 cell viability were inhibited by celastrol with IC50 in the range of 0.05–1 µM. To address the role of celastrol on cell death mechanism, celastrol-treated prostate cancer cells were evaluated with immunoblotting and flow cytometric analysis. Celastrol significantly upregulated PARP and caspase 9 cleavage also increased sub-G1 population. Celastrol also inhibited cell migration and invasion. These effects were associated with decreased PI3K/Akt signaling axis and downregulation of epithelial mesenchymal transition in prostate cancer cells. Likewise, lipid biosynthesis was downregulated with celastrol, however inhibition of PI3K/Akt signaling axis via LY294002 further decrease the cell migration and proliferation rate in prostate cancer cells. Our data suggest that, celastrol suppressed cell proliferation via inhibition of lipid biosynthesis through downregulation of PI3K/Akt signal axis. Targeting lipid metabolism-related enzymes in prostate cancer may offer new avenues for therapeutic approaches.Publication Embargo Epibrassinolide-induced apoptosis regardless of p53 expression via activating polyamine catabolic machinery, a common target for androgen sensitive and insensitive prostate cancer cells(Wiley-Blackwell, 111 River St, Hoboken 07030-5774, Nj Usa, 2014-12) Çoker Gürkan, Ajda; Palavan Unsal, Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 113920; 125860; 6125BACKGROUNDEpibrassinolide (EBR), is a member of the brassinosteroids (BR), has been shown as an apoptotic inducer in different cancer cell lines. We previously showed that EBR induced apoptosis by activating polyamine catabolic pathway, which lead to the accumulation of cytotoxic compounds such as hydrogen peroxide and aldehydes in LNCaP and DU 145 prostate cancer cells. However, we found that LNCaP prostate cancer cells expressing functional androgen receptor (AR) was found more sensitive to EBR than those with non-functional AR (DU 145 cells). RESULTSTo better understand the apoptotic effect of EBR, we aimed to investigate the cellular responses in p53 null, PC3 prostate cancer cells. We showed that EBR induced mitochondria-mediated and caspase-dependent apoptosis in wt and p53 stable transfected PC3 cells, which suggesting that EBR-induced apoptosis regardless of p53 expression. In addition, inhibition of p53 by pifithrin- orthe activation of Mdm2 by Nutlin-3 co-treatment did not alter EBR induced PARP cleavage. Furthermore, EBR treatment was also induced apoptosis in both LNCaP(wt p53) and DU 145 (mt p53)cells, respectively. These all findings verified that EBR-induced apoptosis regardless of p53 expression. The PA catabolic pathway was also altered in PC3 cells causing the generation of reactive oxygen species (ROS) and intracellular PA pool decrease. However, the silencing of spermidine-spermineacetyltransferase (SSAT), a key enzyme at polyamine catabolic machinery prevented the EBR-induced apoptosis. CONCLUSIONSTherefore, we concluded that EBR-induced apoptosis was mainly related with PA catabolic pathway and independent from p53 expression. Prostate 74: 1622-1633, 2014. (c) 2014 Wiley Periodicals, Inc.Publication Open Access 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 Restricted Palbociclib Negatively Regulates Fatty Acid Synthesis Due to Upregulation of AMPK Alpha and miR-33a Levels to Increase Apoptosis in Panc-1 and MiaPaCa-2 Cells(Wiley, 2022) RENCÜZOĞULLARI, ÖZGE; YERLİKAYA, PINAR OBAKAN; GÜRKAN, AJDA ÇOKER; Telci, Dilek; ARISAN, ELİF DAMLAFatty acids (FAs) synthesis mechanism has various regulators such as fatty acid synthase (FASN), AMP-regulated protein kinase (AMPK), or mammalian target of rapamycin (mTOR), which are aberrantly dysregulated in various pancreatic cancer cells. In this study, we aim to understand the regulatory role of palbociclib, a CDK4/6 inhibitor, on the cellular energy metabolism through regulation of AMPK/mTOR signaling by modulation of intracellular miR-33a levels in Panc-1 and MiaPaCa-2 cells. Palbociclib downregulated FAs metabolism more effectively in MiaPaCa-2 cells than Panc-1 cells. Moreover, palbociclib treatment increased the levels of miR-33a in each cell line albeit a higher increase was evident in MiaPaCa-2 cells. Stress-mediated activation of mTOR signaling axis was found associated with palbociclib-mediated AMPK alpha activation and miR33a upregulation. These findings provided that a deeper understanding about possible interactions of cell cycle activity and reduction of FAs synthesis may facilitate the enhancement of cell death mechanisms in pancreatic cancer cells.Publication Restricted Circulating MicroRNA Expression Profiles to Identify a Potential Link Between Prostate Cancer and Obesity(Elsevier, 2022) Arışan, Serdar; KILBAŞ, PELİN ÖZFİLİZ; RENCÜZOĞULLARI, ÖZGE; Ünsal, Narcin Palavan; Çoker-Gürkan, Ajda; Uysal-Onganer, Pınar; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKANEffective diagnostic methods are needed to apply appropriate treatment strategies in patients with aggressive prostate cancer. From this point of view, risk factors that cause prostate cancer or its aggressiveness should be considered. Obesity is a critical risk factor for triggering prostate cancer's metastatic properties. microRNAs are used as biomarkers in diagnosing cancer and obesity depending on their tissue-specific expression patterns. This study investigates the role of obesity in the metastatic profile of prostate cancer depending on the differential expression signatures of selected miRNAs in prostate cancer and obese patients. The roles of miR-100, miR-141 and miR-145 in prostate cancer and obesity are partially known. However, their potential to become circular biomarkers in the blood is not elucidated. There is no previous data on miR-4463 and miR-653 on prostate cancer and obesity association. In this study, the blood samples were taken and obtained serum from 69 patients of 6 subgroups that consisted of one healthy group and five unhealthy groups based on their different prostate cancer or obesity levels. Five selected miRNA expression analyses (miR-100, miR-141, miR-145, miR-4463, and miR-653) were performed through total RNA isolation, which was confirmed via synthetic cel-miR-39 miRNA. Quantitative Real-Time PCR analyzed the expression levels of selected miRNAs. Data analysis was performed via normalising target miRNA expression levels with cel-miR-39. In this study, we found that the relationship between prostate cancer and obesity was investigated at the molecular level. It was suggested that target miR-100 could be a promising biomarker for non-obese and aggressive prostate cancer patients. miR-145 is a more potential biomarker than miR-141 for non-aggressive and non-obese patients. miR-4463 can be used to predict more prostate cancer patients than obese patients. Lastly, miR-653 can be a biomarker for non-aggressive prostate cancer cells.Publication Restricted Epibrassinolide-Induced Autophagy Occurs in an Atg5-Independent Manner Due to Endoplasmic Stress Induction in MEF Cells(Springer, 2020) Adacan, Kaan; YERLİKAYA, PINAR OBAKAN; Çoker-Gürkan, Ajda; Kaya, Resul İsmail; Palavan-Ünsal, Narcin; ARISAN, ELİF DAMLAEpibrassinolide (EBR), a polyhydroxysteroid belongs to plant growth regulator family, brassinosteroids and has been shown to have a similar chemical structure to mammalian steroid hormones. Our findings indicated that EBR could trigger apoptosis in cancer cells via induction of endoplasmic reticulum (ER) stress, caused by protein folding disturbance in the ER. Normal cells exhibited a remarkable resistance to EBR treatment and avoid from apoptotic cell death. The unfolded protein response clears un/misfolded proteins and restore ER functions. When stress is chronic, cells tend to die due to improper cellular functions. To understand the effect of EBR in non-malign cells, mouse embryonic fibroblast (MEF) cells were investigated in detail for ER stress biomarkers, autophagy, and polyamine metabolism in this study. Evolutionary conserved autophagy mechanism is a crucial cellular process to clean damaged organelles and protein aggregates through lysosome under the control of autophagy-related genes (ATGs). Cells tend to activate autophagy to promote cell survival under stress conditions. Polyamines are polycationic molecules playing a role in the homeostasis of important cellular events such as cell survival, growth, and, proliferation. The administration of PAs has been markedly extended the lifespan of various organisms via inducing autophagy and inhibiting oxidative stress. Our data indicated that ER stress is induced following EBR treatment in MEF cells as well as MEF Atg5(-/-) cells. In addition, autophagy is activated following EBR treatment by targeting PI3K/Akt/mTOR in wildtype (wt) cells. However, EBR-induced autophagy targets ULK1 in MEF cells lacking Atg5 expression. Besides, EBR treatment depleted the PA pool in MEF cells through the alterations of metabolic enzymes. The administration of Spd with EBR further increased autophagic vacuole formation. In conclusion, EBR is an anticancer drug candidate with selective cytotoxicity for cancer cells, in addition the induction of autophagy and PA metabolism are critical for responses of normal cells against EBR.Publication Open Access Endoplasmic reticulum stress is activated after epibrassinolide treatment leading autophagy in wild type and Atg5/ mouse embryonic fibroblasts: a survival mechanism in nonmalignant cells.(2017) Adacan, Kaan; Çoker Gürkan, Ajda; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 113920; 125860; 6125Autophagy is a process of cellular selfdegradation during which macromolecules, damaged/aged organelles and proteins are delivered to the lysosome by engulfment within doublemembrane vesicles. The accumulation of un/misfolded proteins in the ER can activate ER stress leading unfolded protein response (UPR) resulting autophagy. Beclin1, LC3 and Autophagy Related Genes (Atg) play key roles in formation of autophagosome membrane, which are triggered by Ulk1 signaling through AMPK (AMPactivated protein kinase). Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cellgrowth regulator that integrates growth factor and nutrient signals. Epibrassinolide (EBR) is a member of brassinostreoids plant hormones with a structural similarity to mammalian steroids. Our previous work demostrated that EBR treatment caused ER stress and UPR which resulted in caspasedependent apoptosis in SW480, DLD1, HT29 and HCT116 colon cancinoma cell lines. Here, we demostrate that EBR treatment activates UPR by inducing ER stressrelated proteins in wild type (wt) MEF (mouse embryonic fibroblast) cells. In contrary to our previous results in malignant cells, MEF cells do not undergo apoptosis after EBR treatment in stead autophagy is activated causing cell survival. We used Atg5/ MEF cells to understand the correlation between ER stress and autophagy. Suprisingly, we observed a significant downregulation of p62/SQSTM1 protein, which is known as one of the autophagy marker, in Atg5/ MEF cells. ER stress was also triggered in these cells. Therefore, we conclude that autophagy is a key mechanism to promote cell survival after EBR treatment against ER stress in both wt and Atg5/ MEF cells. These findings suggested that EBR as an ER stress inducer, is able to activate autophagy in order to promote cell survival in nonmalignant cells.Publication Open Access 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.