Moleküler Biyoloji ve Genetik Bölümü / Department of Molecular Biology and Genetics
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Publication Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines(Springer Wien, Sachsenplatz 4-6, Po Box 89, A-1201 Wien, Austria, 2014-03) Palavan Unsal, Narçin; Calcabrini, Annarica; Agostinelli, Enzo; Bolkent, Şehnaz; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 113920; 6125; 3894Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR-) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the EBR-induced apoptosis only in LNCaP (AR+) cells. In a similar way, MDL 72,527, the specific PAO and SMO inhibitor, co-treatment with EBR during 24 h, reduced the formation of cleaved fragments of PARP in LNCaP (AR+) cells.Publication Anti-metastatic effect of ranolazine in an in vivo rat model of prostate cancer, and expression of voltage-gated sodium channel protein in human prostate(2019-03-20) Bugan, İlknur; Küçük, Selma; Karagöz, Zeynep; Fraser, SP; Kaya, Handan; Dodson; Foster, Cs; Djamgoz, Mba; ALTUN, SEYHAN; 4995Background Voltage-gated Na+ channels (VGSCs) are functionally upregulated in rat and human prostate cancer (PCa) where channel activity promotes cellular invasiveness in vitro and metastasis in vivo. Ranolazine is a clinically used VGSC inhibitor/anti-anginal drug, which has been shown previously to inhibit breast cancer metastasis in vivo. Methods Using the Dunning model of rat PCa, the effect of ranolazine applied systemically (by gavage) was tested on the development of primary tumours and metastases following subcutaneous inoculation of Mat-LyLu cells into Copenhagen rats. In addition, human prostate tissue microarrays were used to determine VGSC protein expression in cancerous versus non-cancerous tissue. Several public databases were searched to compare Nav1.7/ SCN9A expression levels in ‘normal’ vs. PCa tissues. Results Ranolazine (2.5 and 5 µM) decreased the number of lung metastases by up to 63%. In contrast, primary tumourigenesis was not affected. Ranolazine also reduced the percentage of cells in the metastases expressing Nav1.7, the main VGSC subtype expressed in PCa, but the expression level was higher. In prostate tissue microarrays, VGSC protein expression was significantly higher in cancerous versus non-cancerous tissue. There was no correlation between the VGSC expression and either prostate-specific antigen or Gleason score. In public databases, little information could be found on Nav1.7 protein expression in PCa. In addition, the database information on Nav1.7 mRNA (SCN9A) expression levels did not correlate with previously reported upregulation in PCa cells and tissues. Conclusions The main conclusions were (i) ranolazine inhibited metastasis and (ii) it was a subpopulation of cells with particularly high levels of Nav1.7 protein that reached the metastatic sites. These data extend earlier studies and suggest that Nav1.7 expression could serve as a functional biomarker of metastatic PCa and that VGSC blockers may be useful as antimetastatic agents.Publication Antioxidative defense system differences to drought stress of tomato cultivars(Elsevier Science Bv, Po Box 211, 1000 Ae Amsterdam, Netherlands, 2014-09) AYAN, ALP; ATAK, ÇİMEN; ÇELİK, ÖZGE; 185510; 113987; 6653The aim of this study is to investigate the enzymatic and non-enzymatic responses of two different industrial tomato varieties (X5671R and 5MX12956) against drought stress. Fourteen-day-old seedlings were subjected to drought stress by non-watering and the stress was continued for 7 days. The effects of drought stress on tomato varieties analysed due to the results of biochemical analysis (total protein content, lipid peroxidation, relative water content, chlorophyll contents) and enzymatic activity and isozyme analysis of SOD, CAT, APX and POX. The highest water loss was recorded in X5671R as 73.17%. 64% total chlorophyll decrease was observed in both varieties on 7th day. Total protein, proline, malondialdehyde concentrations and antioxidant enzymes presented increase with related to increasing drought stress for both varieties. Due to the results, we concluded that 5MX12956 variety is more drought tolerant then X5671R. Isozyme densities and band differences were recorded. The densities of POX isozymes were started to increase from 5th day of stress. Fe-SOD, Cu/Zn-SOD1-2 isozyme bands were identified in X5671R variety, while 5MX12956 showed only Cu/Zn-SOD isozyme band. Differences in APX isozymes were observed from the 4th day of drought stress in both varieties. The results indicated the differences in antioxidant metabolism of tomato plants.Publication Applications of Ionizing Radiation in Mutation Breeding(InTech, 2017-05) ATAK, ÇİMEN; ÇELİK, ÖZGE; 113987; 6653As a predicted result of increasing population worldwide, improvements in the breeding strategies in agriculture are valued as mandatory. The natural resources are limited, and due to the natural disasters like sudden and severe abiotic stress factors, excessive floods, etc., the production capacities are changed per year. In contrast, the yield potential should be significantly increased to cope with this problem. Despite rich genetic diversity, manipulation of the cultivars through alternative techniques such as mutation breeding becomes important. Radiation is proven as an effective method as a unique method to increase the genetic variability of the species. Gamma radiation is the most preferred physical mutagen by plant breeders. Several mutant varieties have been successfully introduced into commercial production by this method. Combinational use of in vitro tissue culture and mutation breeding methods makes a significant contribution to improve new crops. Large populations and the target mutations can be easily screened and identified by new methods. Marker assisted selection and advanced techniques such as microarray, next generation sequencing methods to detect a specific mutant in a large population will help to the plant breeders to use ionizing radiation efficiently in breeding programs.Publication Assessment of Interleukin-1 Gene Cluster Polymorphisms in Lone Atrial Fibrillation: New Insight into the Role of Inflammation in Atrial Fibrillation(Wiley-Blackwell, 111 River St, Hoboken 07030-5774, Nj USA, 2013-10) Çoker Gürkan, Ajda; Güngör, Barış; Ekmekçi, Ahmet; Arman, Ahmet; Özcan, Kazım S.; Üçer, Ekrem; Çalık, Nazmi; Yılmaz, Hale; Tezel, Tuna; Bolca, Osman; 125860; 173248BackgroundSystemic inflammation is accepted as one of the pathophysiological mechanisms of atrial fibrillation (AF). The role of inflammation has been shown previously. Interleukin (IL) system is the main modulator of the inflammatory responses and genetic polymorphisms of IL-1 cluster genes are associated with increased risk for inflammatory diseases. ObjectivesTo investigate the association between polymorphisms of IL-1 cluster genes and lone AF. Subjects and MethodsDNA samples were collected from 70 proven lone AF patients and 70 healthy subjects. Genomic DNA was typed for the variable number of the tandem repeat (VNTR) IL-1 receptor antagonist (RN) gene polymorphism, IL-1B -511 C > T(rs16944) promoter polymorphism, and +3953 C > T(rs1143634) polymorphism in exon 5 by polymerase chain reaction. ResultsIn lone AF group the frequency of IL-1RN2/2 and IL-1RN1/2 genotypes were higher than in the control group (7.2% vs 4.3% and 48.5% vs 22.8%, respectively; (2) = 14.1; P = 0.028). The frequency of allele 2 was significantly higher in the lone AF group (32.1% vs 15.7%; (2) = 10.7; P = 0.005). Allele and genotype distribution of IL-1B -511 C > T and +3953 C > T polymorphisms were not statistically different between the groups. C-reactive protein (CRP) levels were higher in lone AF patients compared to the control group (median = 1.25, interquartile range [IQR] = 0.85 vs median = 1.08, IQR 0.46 mg/L, respectively; P = 0.02). In multivariate regression analysis, presence of allele 2 of IL-1 VNTR polymorphism and elevated plasma high-sensitive-CRP levels were the independent predictors of lone AF. ConclusionPresence of allele 2 of VNTR polymorphism of IL-1RN gene may cause increased risk for lone AF probably due to the inadequate limitation of inflammatory reactions.Publication Association between IL-1RN VNTR, IL-1 beta -511 and IL-6 (-174, -572, -597) Gene Polymorphisms and Urolithiasis(Karger, Allschwilerstrasse 10, Ch-4009 Basel, Switzerland, 2013) Çoker Gürkan, Ajda; Palavan Unsal, Narçin; Arısan, Serdar; Sonmez, Nurettin Cem; ARISAN, ELİF DAMLA; 125860; 113920; 6125Urolithiasis is a common multifactorial urological disorder that is characterized by stone formation. Interleukin (IL)-1 and IL-6 are pro-inflammatory cytokines that might be linked with urolithiasis. The single nucleotide polymorphisms within the IL-1 and IL-6 cytokine genes altered the cytokine expression levels. Our aim was to investigate the potential of IL-1 beta (-511 C>T), IL-6 (-174 G>C, 572 G>C, 597 G>A) and IL-1 RN VNTR gene polymorphisms to be a genetic marker for urinary stone disease. The polymorphisms studied in the promoter regions of IL-1 beta and IL-6 genes did not reveal a strong association with urolithiasis when compared to the control group (p = 0.293, 0.871, 0.921, 0.536, respectively). However, a significant difference was observed between control and patient groups for IL-1 RN VNTR gene polymorphism (chi(2) = 6.131, d.f. = 2, p = 0.047). Our data provide evidence that IL-1RN VNTR gene polymorphism may be involved in the pathogenesis of urinary stone formation, contributing to genetic susceptibility for urolithiasis. (C) 2013 S. Karger AG, BaselPublication Association Between Sporadic Parkinson Disease And Interleukin-1 Beta-511 Gene Polymorphisms In The Turkish Population(John Libbey Eurotext Ltd, 127 Ave De La Republique, 92120 Montrouge, France, 2010-06) Arman, Ahmet; Işık, Nihal; Candan, Fatma; Becit, Kezban Serap; List, Edward O.; Çoker, Ajda; TR173248; TR217343; TR125860; TR198466The pathogenesis of Parkinson Disease (PD) remains poorly understood; however, inflammation is thought to play an important role in disease progression. Recent reports suggest that IL-1, a major proinflammatory cytokine, might play a role in PD progression. The purpose of this study was to determine the relationship between IL-1 gene family polymorphisms [IL-1 alpha (-889), IL-1Ra (VNTR) and IL-1 beta (-511, +3953)] and PD in the Turkish population. In this study, we examined the genotypes of IL-1 gene family polymorphisms in 365 individuals, of which 199 were healthy control subjects and 166 were PD patients. No significant differences were found in the genotype distribution or in the allele frequencies of IL-1 alpha (-889), IL-1Ra (VNTR) and IL-1 beta (+3953) between PD cases and control subjects. However, distribution of the IL-1 beta -511 2/2 (T/T) genotype was found to be significantly lower in PD patients than in healthy controls (p = 0.018, x(2): 8.242, OR: 2.211, 95% CI: 1.261-3.877). In addition, the IL-1 beta -511 allele 1 (C) frequency was significantly elevated in PD patients versus controls (p = 0.048, x(2): 3.87, OR: 1.178, 95% CI: 0.999-1.388). These results suggest that IL-1 alpha (-889), IL-1Ra and IL-1 beta (+3953) gene polymorphisms have no association with PD, while allele 1 (C) of IL-1 beta (-511) is associated with PD and may provide a susceptibility factor for this disease in the Turkish population. Furthermore, the 2/2 (T/T) genotype of IL-1 beta (-511) may protect individuals from PD.Publication Atiprimod induce apoptosis in pituitary adenoma: Endoplasmic reticulum stress and autophagy pathways(WILEY, 111 RIVER ST, HOBOKEN 07030-5774, NJ USA, 2019-12) Çoker Gürkan , Ajda; Keçeoğlu, Gizem; Palavan Unsal, Narcin; ARISAN, ELİF DAMLA; ŞAHİN, BURCU AYHAN; YERLİKAYA, PINAR OBAKANPituitary adenoma is the most common tumor with a high recurrence rate due to a hormone-dependent JAK/signal transducer and activator of transcriptions (STAT) signaling. Atiprimod, a novel compound belonging to the azaspirane class of cationic amphiphilic drugs, has antiproliferative, anticarcinogenic effects in multiple myeloma, breast, and hepatocellular carcinoma by blocking STAT3 activation. Therapeutic agents' efficiency depends on endoplasmic reticulum (ER) stress-autophagy regulation during drug-mediated apoptotic cell death decision. However, the molecular machinery of dose-dependent atiprimod treatment regarding ER stress-autophagy has not been investigated yet. Thus, our aim is to investigate the ER stress-autophagy axis in atiprimod-mediated apoptotic cell death in GH-secreting rat cell line (GH3) pituitary adenoma cells. Dose-dependent atiprimod treatment decreased GH3 cell viability, inhibited cell growth, and colony formation. Upregulation of Atg5, Atg12, Beclin-1 expressions, cleavage of LC-3II and formation of autophagy vacuoles were determined only after 1 mu M atiprimod exposure. In addition, atiprimod-triggered ER stress was evaluated by BiP, C/EBP-homologous protein (CHOP), p-PERK upregulation, and Ca+2 release after 1 mu M atiprimod exposure. Concomitantly, increasing concentration of atiprimod induced caspase-dependent apoptotic cell death via modulating Bcl(2) family members. Moreover, by N-acetyl cycteinc pretreatment, atiprimod triggered reactive oxygen species generation and prevented apoptotic induction. Concomitantly, dose-dependent atiprimod treatment decreased both GH and STAT3 expression in GH3 cells. In addition, overexpression of STAT3 increased atiprimod-mediated cell viability loss and apoptotic cell death through suppressing autophagy and ER stress key molecules expression profile. In conclusion, a low dose of atiprimod exposure triggers autophagy and mild-ER stress as a survival mechanism, but increased atiprimod dose induced caspase-dependent apoptotic cell death by targeting STAT3 in GH3 pituitary adenoma cells.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 Bag-1 promotes cell survival through c-Myc-mediated ODC upregulation that is not preferred under apoptotic stimuli in MCF-7 cells(Wiley-Blackwell, 111 River St, Hoboken 07030-5774, NJ USA, 2015-07) Kızılboğa, Tuğba; Demir, Salih; Palavan Ünsal, Zeynep Narçın; Dinler Doğanay, Gizem; ARISAN, ELİF DAMLA; KILBAŞ, PELİN ÖZFİLİZ; Alkurt, Gizem; 195744; 6125; 113920; 152975Bag-1, Bcl-2 associated athanogene-1, is a multifunctional protein that can regulate a wide variety of cellular processes: proliferation, cell survival, transcription, apoptosis and motility. Bag-1 interacts with various targets in the modulation of these pathways; yet molecular details of Bag-1's involvement in each cellular event are still unclear. We first showed that forced Bag-1 expression promotes cell survival and prevents drug-induced apoptosis in MCF-7 breast cancer cells. Increased mRNA expressions of c-myc protooncogene and ornithine decarboxylase (ODC), biosynthetic enzyme of polyamines, were detected in Bag-1L+ cells, and western blots against the protein product of c-Myc and ODC confirmed these findings. Once ODC, a c-Myc target, gets activated, polyamine biosynthesis increases. We observed enhanced polyamine content in the Bag-1L+ cells. On the contrary, when polyamine catabolic mechanisms were investigated, Bag-1 silencing suppressed biosynthesis of polyamines because of the downregulation of ODC and upregulation of PAO. Exposure of cells to apoptotic inducers enhances the cell death mechanism by producing toxic products such as H2O2 and aldehydes. Bag-1L+ cells prevented drug-induced PAO activation leading to a decrease in H2O2 production following cisplatin or paclitaxel treatment. In this line, our results suggested that Bag-1 indirectly affects cell survival through c-Myc activated signalling that causes elevation of ODC levels, leading to an increase of the polyamine content. Copyright (c) 2015 John Wiley & Sons, Ltd.Publication Bag-1 silencing enhanced apoptosis via altering the ubiquitination process of Foxo3a in MCP-7 breast cancer cells(Spandidos Publ Ltd, Pob 18179, Athens, 116 10, Greece, 2017) Dinler Doğanay, Gizem; Palavan Ünsal, Zeynep Narçın; ARISAN, ELİF DAMLA; KILBAŞ, PELİN ÖZFİLİZ; 195744; 113920; 152975; 6125Publication Bag-1 Silencing Enhanced Chemotherapeutic Drug-induced Apoptosis in MCF-7 Breast Cancer Cells Affecting PI3K/Akt/mTOR and MAPK Signaling Pathways(2019-01-19) Akçay, İzzet Mehmet; Dinler Doğanay, Gizem; ARISAN, ELİF DAMLA; KILBAŞ, PELİN ÖZFİLİZ; 195744; 152975; 113920The multifunctional anti-apoptotic Bag-1 protein has important roles in apoptosis, proteasome-mediated degradation, transcriptional regulation, and intracellular signaling. Bag-1 promotes cell survival and proliferation, and is overexpressed in breast cancer. Therefore, Bag-1-targeted therapy might be a promising strategy to treat breast cancer. However, the effects of Bag-1 silencing in combination with conventional chemotherapeutic drugs on cell viability and major signaling pathways have not yet been fully investigated in breast cancer cells. In this study, we investigated the cytotoxic effects of Bag-1 silencing, alone and in combination with cisplatin or paclitaxel treatment, in MCF-7 breast cancer cells. Bag-1 knockdown by shRNA or siRNA transfection sensitized MCF-7 cells to apoptosis induced by cisplatin or paclitaxel. Combination of Bag-1 silencing and drug treatment more potently downregulated the pro-survival PI3K/Akt/mTOR and p44/42 mitogen activated protein kinase (MAPK) pathways, and more potently upregulated the stress-activated p38 and SAPK/JNK MAPK pathways. Bag1-silenced drug-treated cells had also highly reduced proliferative capacity, downregulated cyclin–cyclin dependent kinase complexes and upregulated tumor suppressors p21 and Rb. These results overall indicated that Bag-1 silencing enhanced cisplatin- or paclitaxel-induced cytotoxicity through multiple pathways. In conclusion, Bag-1 targeted therapy might enhance the therapeutic potential of conventional anti-cancer drugs in the treatment of breast cancer.Publication Bag-1L is a Stress-withstand Molecule Prevents the Downregulation of Mcl-1 and c-Raf Under Control of Heat Shock Proteins in Cisplatin Treated HeLa Cervix Cancer Cells(Asian Pacific Organization Cancer Prevention, Apjcp Head Office, Korean Natl Cancer Center, 323 Ilan -Ro, Ilsandong-Gu, Goyang-Si, Gyeonggi-Do, 410-769, South Korea, 2014) Çoker Gürkan, Ajda; Eralp, Tuğçe Nur; Dinler Doğanay, Gizem; Palavan Unsal, Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; KILBAŞ, PELİN ÖZFİLİZ; 195744; 113920; 125860; 156421; 272026; 152975; 6125Background: Cisplatin, a DNA damaging agent, induces apoptosis through increasing DNA fragmentation. However, identification of intrinsic resistance molecules against Cisplatin is vital to estimate the success of therapy. Bag-1 (Bcl-2-associated anthanogene) is one anti-apoptotic protein involved in drug resistance impacting on therapeutic efficiency. Elevated levels of this protein are related with increase cell proliferation rates, motility and also cancer development. For this reason, we aimed to understand the role of Bag-1 expression in Cisplatin-induced apoptosis in HeLa cervix cancer cells. Cisplatin decreased cell viability in time-and dose-dependent manner in wt and Bag-1L+HeLa cells. Although, 10 mu M Cisplatin treatment induced cell death within 24h by activating caspases in wt cells, Bag-1L stable transfection protected cells against Cisplatin treatment. To assess the potential protective role of Bag-1, we first checked the expression profile of interacting anti-apoptotic partners of Bag-1. We found that forced Bag-1L expression prevented Cisplatin-induced apoptosis through acting on Mcl-1 expression, which was reduced after Cisplatin treatment in wt HeLa cells. This mechanism was also supported by the regulation of heat shock protein (Hsp) family members, Hsp90 and Hsp40, which were involved in the regulation Bag-1 interactome including several anti-apoptotic Bcl-2 family members and c-Raf.Publication Bag-1L mediated chemoresistance mechanism through preventing downregulation of Mcl-1 and c-Raf by heat shock proteins in HeLa cells(2014-11) Eralp, Tuğçe Nur; Çoker Gürkan, Ajda; Dinler Doğanay, Gizem; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; KILBAŞ, PELİN ÖZFİLİZ; 272026; 113920; 195744; 156421; 125860; 152975; 6125Background: Cisplatin, a DNA damaging agent, induces apoptosis through increasing DNA fragmentation. However, identification of intrinsic resistance molecules against Cisplatin is vital to estimate the success of therapy. Bag-1 (Bcl-2-associated anthanogene) is one anti-apoptotic protein involved in drug resistance impacting on therapeutic efficiency. Elevated levels of this protein are related with increase cell proliferation rates, motility and also cancer development. For this reason, we aimed to understand the role of Bag-1 expression in Cisplatininduced apoptosis in HeLa cervix cancer cells. Cisplatin decreased cell viability in time- and dose-dependent manner in wt and Bag-1L+HeLa cells. Although, 10μM Cisplatin treatment induced cell death within 24h by activating caspases in wt cells, Bag-1L stable transfection protected cells against Cisplatin treatment. To assess the potential protective role of Bag-1, we first checked the expression profile of interacting anti-apoptotic partners of Bag-1. We found that forced Bag-1L expression prevented Cisplatin-induced apoptosis through acting on Mcl-1 expression, which was reduced after Cisplatin treatment in wt HeLa cells. This mechanism was also supported by the regulation of heat shock protein (Hsp) family members, Hsp90 and Hsp40, which were involved in the regulation Bag-1 interactome including several anti-apoptotic Bcl-2 family members and c-Raf.Publication Biological responses of ultrafine grained pure titanium and their sand blasted surfaces(Elsevier Science Bv, Po Box 211, 1000 Ae Amsterdam, Netherlands, 2018-10-01) Günay Bulutsuz, Aslı; Berrak, Özge; Yeprem, H. Aygül; Yurci, Mehmet Emin; ARISAN, ELİF DAMLA; 173771; 122108; 113920; 9193The use of materials as implants has become vital for determining optimal product design to enhance the needs of usage and longevity in body. Ultrafine grained pure titanium offers advanced mechanical properties for medical applications for most adequate materials meso/micro scaled dental implants. Besides advanced mechanical properties, increased surface properties also offers enhance biocompatibility. In this experimental study, the effects of bulk structure on surface modification by sand blasting for coarse-grained and ultrafine-grained (UFG) commercially pure titanium reported. To determine the effects of bulk structure on the polished and modified surfaces the specimen groups are investigated using Optic Microscope (OM), Electron Back Scattering Diffraction (EBSD) and Confocal Laser-Scanning Microscope (CLSM). Surface roughness is determined with stylus profilometer (SP) and CLSM. Understanding the biocompatibility of titanium surfaces to cell-cell interactions and cell proliferation capacity of attached-cells were determined by cell viability assays and fluorescence microscopy techniques. According to our results, the titanium surfaces were highly available to cell attachment and cell proliferation. The ratios of cell proliferation of cells which are attached on different titanium surfaces were dependent on the grain size and the surface roughness. UFG and blasted surfaces are more suitable for cell proliferation of human gingival fibroblast cells.Publication Calpain inhibitor AK 295 inhibits calpain-induced apoptosis and improves neurologic function after traumatic spinal cord injury in rats(Soc Espanola Neurocirugia, C/O Dr Poza, Gran Via Salzillo 42, 30005 Murcia, Spain, 2009-06) Çolak, Ahmet; Kaya, Mehmet; Karaoğlan, Alper; Sağmanlıgil, Ayhan; Akdemir, Osman; Şahan, Elife; ÇELİK, ÖZGE; TR113987; TR110929Background. An increase in the level of intracellular calcium activates the calcium-dependent neutral protease calpain, which in turn leads to cellular dysfunction and cell death after an insult to the central nervous system. fit this study, we evaluated the effect of a calpain inhibitor, AK 295, on spinal cord structure, neurologic function, and apoptosis after spinal cord injury (SCI) in a murine model. Methods. Thirty albino Wistar rats were divided into 3 groups of 10 each: the sham-operated control group (group 1), the spinal cord trauma group (group 2), and the spinal cord trauma plus AK 295 treatment group (group 3). After having received a combination of ketamine 60 mg/kg and xylazine 9 mg/kg to induce anesthesia, the rats in groups 2 and 3 were subjected to thoracic trauma by the weight drop technique (40 g-cm). One hour after having been subjected to that trauma, the rats in groups 2 and 3 were treated with an intraperitoneal injection of either dimethyl sulfoxide 2 mg/kg or AK 295 2 mg/kg. The effects of the injury and the efficacy of AK 295 were determined by an assessment of the TUNEL technique and the results of examination with a light microscope. The neurologic performance of 5 rats from group 2 and 5 from group 3 was assessed by means of the inclined plane technique and the modified Tarlov's motor grading scale 1, 3, and 5 days after spinal cord trauma. Findings. Light-microscopic examination of spinal cord specimens from group 2 revealed hemorrhage, edema, necrosis, and vascular thrombi 24 : hours after trauma. Similar (but less prominent) features were seen in specimens obtained from group 3 rats. Twenty-four hours after injury, the mean apoptotie cell numbers in groups I and 2 were zero and 4.57 +/- 0.37 cells, respectively. In group 3, the mean apoptotic cell number was 2.30 +/- 0.34 cells, a value significantly lower than that in group 2 (P < .05). Five days after trauma, the injured rats in group 2 demonstrated significant motor dysfunction (P < .05). In comparison, the motor scores exhibited by group 3 rats were markedly better (P < .05). Conclusions. AK 295 inhibited apoptosis via calpain-dependent pathway's and provided neuroprotection and improved neurologic function in a rat model of SCI. To our knowledge, this is the first study to evaluate the use of AK 295, a calpain inhibitor, after SCI. Our data suggest that AK 295 might be a novel therapeutic compound for the neuroprotection of tissue and the recovery of function in patients with a SCI.Publication Calreticulin is a fine tuning molecule in epibrassinolide-induced apoptosis through activating endoplasmic reticulum stress in colon cancer cells(Wiley, 111 River St, Hoboken 07030-5774, NJ USA, 2017-06) Çoker Gürkan, Ajda; Adacan, Kaan; Özbey, Utku; Somuncu, Berna; Baran, Didem; Ünsal Palavan, Zeynep Narçın; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 113920; 125860; 6125Epibrassinolide (EBR), a member of brassinostreoids plant hormones with cell proliferation promoting role in plants, is a natural polyhydroxysteroid with structural similarity to steroid hormones of vertebrates. EBR has antiproliferative and apoptosis-inducing effect in various cancer cells. Although EBR has been shown to affect survival and mitochondria-mediated apoptosis pathways in a p53-independent manner, the exact molecular targets of EBR are still under investigation. Our recent SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) data showed that the most significantly altered protein after EBR treatment was calreticulin (CALR). CALR, a chaperone localized in endoplasmic reticulum (ER) lumen, plays role in protein folding and buffering Ca2+ ions. The alteration of CALR may cause ER stress and unfolded protein response correspondingly the induction of apoptosis. Unfolded proteins are conducted to 26S proteasomal degradation following ubiquitination. Our study revealed that EBR treatment caused ER stress and UPR by altering CALR expression causing caspase-dependent apoptosis in HCT 116, HT29, DLD-1, and SW480 colon cancer cells. Furthermore, 48 h EBR treatment did not caused UPR in Fetal Human Colon cells (FHC) and Mouse Embryonic Fibroblast cells (MEF). In addition our findings showed that HCT 116 colon cancer cells lacking Bax and Puma expression still undergo UPR and related apoptosis. CALR silencing and rapamycin co-treatment prevented EBR-induced UPR and apoptosis, whereas 26S proteasome inhibition further increased the effect of EBR in colon cancer cells. All these findings showed that EBR is an ER stress and apoptotic inducer in colon cancer cells without affecting nonmalignant cells.Publication Cathepsin K analysis in a pycnodysostosis cohort: demographic, genotypic and phenotypic features(Biomed Central Ltd, 236 Grays Inn Rd, Floor 6, London Wc1X 8Hl, England, 2014-04-26) Arman, Ahmet; Bereket, Abdullah; Çoker Gürkan, Ajda; Şimşek Kiper, Pelin Özlem; Güran, Tülay; Özkan, Behzat; Atay, Zeynep; Akçay, Teoman; Haliloğlu, Belma; Boduroğlu, Koray; Alanay, Yasemin; Turan, Serap; 173248; 9240; 125860; 169245; 183446; 171694; 201621; 218579; 206057; 28175; 125850Background: To characterize cathepsin K (CTSK) mutations in a group of patients with pycnodysostosis, who presented with either short stature or atypical fractures to pediatric endocrinology or dysmorphic features to pediatric genetics clinics. Methods: Seven exons and exon/intron boundaries of CTSK gene for the children and their families were amplified with PCR and sequenced. Sixteen patients from 14 families with pycnodysostosis, presenting with typical dysmorphic features, short stature, frequent fractures and osteosclerosis, were included in the study. Results: We identified five missense mutations (M1I, I249T, L7P, D80Y and D169N), one nonsense mutation (R312X) and one 301 bp insertion in intron 7, which is revealed as Alu sequence; among them, only L7P and I249 were described previously. The mutations were homozygous in all cases, and the families mostly originated from the region where consanguineous marriage rate is the highest. Patients with M1I mutation had fractures, at younger ages than the other pycnodysostosis cases in our cohort which were most probably related to the severity of mutation, since M1I initiates the translation, and mutation might lead to the complete absence of the protein. The typical finding of pycnodysostosis, acroosteolysis, could not be detected in two patients, although other patients carrying the same mutations had acroosteolysis. Additionally, none of the previously described hot spot mutations were seen in our cohort; indeed, L7P and R312X were the most frequently detected mutations. Conclusions: We described a large cohort of pycnodysostosis patients with genetic and phenotypic features, and, first Alu sequence insertion in pycnodysostosis.Publication CDK Inhibitors Induce Mitochondria-mediated Apoptosis Through the Activation of Polyamine Catabolic Pathway in LNCaP, DU145 and PC3 Prostate Cancer Cells(Bentham Science Publ Ltd, Executive Ste Y-2, Po Box 7917, Saif Zone, 1200 Br Sharjah, U Arab Emirates, 2014-01) Çoker Gürkan, Ajda; Calcabrini, Annarica; Agostinelli, Enzo; Palavan Unsal, Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 113920; 156421; 125860; 6125Androgen signaling is critical in prostate cancer development and progression. The co-existence of hormone responsive and irresponsive cells due to functional androgen receptor (AR) in prostate gland is the major obstacle in prostate cancer therapy models. Targeting aberrant cell cycle by novel cell cycle blocking agents is a promising strategy to treat various types of malignancies. Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors able to activate apoptotic cell death by inducing cell cycle arrest at G1/S and G2/M phases in cancer cells. Polyamines are unique cationic amine derivatives involved in the regulation of cell proliferation. Although the elevated intracellular level of polyamines (putrescine, spermidine and spermine) is typical for prostate gland, abnormal regulation of polyamine metabolism might result in rapid cell proliferation and, thus in prostate cancer progression. Therefore, treatment with drug-induced depletion of intracellular polyamine levels through the activated polyamine catabolism is critical to achieve successful strategies for prostate cancer. In this study we aimed to investigate the apoptotic efficiency of CDK inhibitors in three prostate cancer cell lines (LNCaP, DU145 and PC3), showing different AR expression profile. We found that both purvalanol and roscovitine were able to induce apoptosis at moderate cytotoxic concentrations by decreasing mitochondria membrane potential. The apoptotic effect of both CDK inhibitors was due to activation of caspases by modulating Bcl-2 family members. The efficiency of drugs was quite similar on the three prostate cell lines used in this study. However, DU145 cells were found the least sensitive against CDK inhibitors while purvalanol was more potent than roscovitine. Similarly to classical chemotherapeutic agents, both drugs could up-regulate polyamine catabolic enzymes (SSAT, SMO and PAO) in cell type dependent manner. Transient silencing of SSAT and/or inhibition of PAO/SMO with MDL72527 prevented CDK inhibitors-induced apoptotic cell death in DU145 and PC3 cells. Although roscovitine was less effective in DU145 cells, pre-treatment with alpha-difluoromethylornithine (DFMO), an inhibitor of ODC, enhanced the roscovitine-induced apoptotic cell death through the cleavage of caspase-9 and caspase-3. Therefore, we conclude that polyamine catabolism might have essential role in the cellular responses against CDK inhibitors in different androgen-responsive or irresponsive prostate cancer cells.Publication CDK inhibitors-induced SSAT expression requires NF kappa B and PPAR gamma in MCF-7 breast cancer cells(TUBİTAK Scientific & Technical Research Council Turkey, Ataturk Bulvarı No 221, Kavaklıdere, Ankara, 00000, Turkey, 2015) Yıldırım, Şeyma; Öztürk, Mert Burak; Berrak, Özge; Çoker Gürkan, Ajda; Ünsal Palavan, Zeynep Narçın; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 125860; 113920; 6125The cyclin-dependent kinase (CDK) inhibitors purvalanol and roscovitine are therapeutic agents that control cell proliferation through regulating cell-cycle machinery. They also affect polyamine (PA) metabolism, which is activated in malignant tissues. Therefore, PA catabolism became a remarkable target in cancer therapies. Induction of the PA catabolic enzyme spermidine/spermine N-1-acetyltransferase (SSAT) is under the control of transcription factors such as NF kappa B and PPAR gamma. The purpose of this study was to investigate the therapeutic potential of CDK inhibitors in combination with PAs in MCF-7 breast cancer cells. In order to understand the involvement of PA catabolic enzyme SSAT in this process we also checked its transcriptional regulation in the presence of CDK inhibitors. MCF-7 cells were exposed to CDK inhibitors in the absence or presence of Spd and Spm. Cell viability loss was evaluated by MTT assay. Apoptosis was determined by annexin-V/PI staining using FACS flow. The SSAT transcription level was measured by qRT-PCR. Intracellular PA pool was determined by HPLC. Protein expressions were assessed by western blotting. We found that CDK inhibitors decreased cell viability in a time-dependent manner and induced apoptosis. Co-treatment of Spd or Spm with CDK inhibitors prevented the apoptotic potential of both drugs. Purvalanol increased SSAT expression levels in a time-dependent manner. Although the induction of SSAT by purvalanol resulted in the activation of NF kappa B at early time points, induction was accomplished by PPAR gamma as a late response after purvalanol treatment. We concluded that both transcriptional control mechanisms could be responsible for SSAT regulation in a time-dependent manner.