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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 Metadata only Inhibition of Polyamine Oxidase Prevented Cyclin-Dependent Kinase Inhibitor-Induced Apoptosis in HCT 116 Colon Carcinoma Cells(Springer, Van Godewijckstraat 30, 3311 Gz Dordrecht, Netherlands, 2013-12) Çoker Gürkan, Ajda; Palavan Ünsal, Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 113920; 125860; 156421; 6125Roscovitine and purvalanol are novel cyclin-dependent kinase (CDK) inhibitors that prevent cell proliferation and induce apoptotic cell death in various cancer cell lines. Although a number of studies have demonstrated the potential apoptotic role of roscovitine, there is limited data about the therapeutic efficiency of purvalanol on cancer cells. The natural polyamines (PAs) putrescine, spermidine, and spermine have essential roles in the regulation of cell differentiation, growth, and proliferation, and increased levels of these compounds have been associated with cancer progression. Recently, depletion of intracellular PA levels because of modulation of PA catabolic enzymes was shown to be an indicator of the efficacy of chemotherapeutic agents. In this study, our aim was to investigate the potential role of PA catabolic enzymes in CDK inhibitor-induced apoptosis in HCT 116 colon carcinoma cells. Exposure of cells to roscovitine or purvalanol decreased cell viability in a dose- and time-dependent manner. The selected concentrations of roscovitine and purvalanol inhibited cell viability by 50 % compared with control cells and induced apoptosis by activating the mitochondria-mediated pathway in a caspase-dependent manner. However, the apoptotic effect of purvalanol was stronger than that of roscovitine in HCT 116 cells. In addition, we found that CDK inhibitors decreased PA levels and significantly upregulated expression of key PA catabolic enzymes such as polyamine oxidase (PAO) and spermine oxidase (SMO). MDL-72,527, a specific inhibitor of PAO and SMO, decreased apoptotic potential of CDK inhibitors on HCT 116 cells. Moreover, transient silencing of PAO was also reduced prevented CDK inhibitor-induced apoptosis in HCT 116 cells. We conclude that the PA catabolic pathway, especially PAO, is a critical target for understanding the molecular mechanism of CDK inhibitor-induced apoptosis.Publication Embargo 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 Open Access Epibrassinolide Treatment Caused Autophagy or Apoptosis Decision in a Time-Dependent Manner through ER Stress in Colon Cancer Cells(2017) Adacan, Kaan; Çoker Gürkan, Ajda; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 156421; 113920; 125860; 6125Epibrassinolide (EBR) is a natural plant polyhydroxysteroid with structural similarity to steroid hormones. Lately we showed by SILAC assay that EBR treatment induces apoptosis by significantly altering the expressions of proteins having role in unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. ER stress level has been proposed as a critical step in cancer chemotherapy. Moderate ER stress was shown as an inducer of pro-survival machinery via autophagy, which is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. However chronic ER stress lead cells to apoptosis. EBR treatment induced ER stress in a time-dependent manner within 48 h in SW480 and DLD1 colon cancer cells. Downregulation of p62, LC3 and increase in ATGs expressions indicated that autophagy is induced in these cell lines after 12 h EBR exposure. The mammalian target of rapamycin (mTOR), a coordinator between nutritional stress and cellular growth machinery, is associated with ER stress. Our results indicated that short time EBR exposure induced mTOR expression accompanied by Ser2448 dephosphorylation. After 48 h EBR treatment, with prolonged ER stress, both cell lines undergo apoptosis. Therefore we conclude that time-dependent EBR treatment caused autophagy or apoptosis decision through ER stress in colon cancer cells.Publication Embargo 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 Metadata only Inhibition of extracellular signal-regulated kinase potentiates the apoptotic and antimetastatic effects of cyclin-dependent kinase inhibitors on metastatic DU145 and PC3 prostate cancer cells(Wiley Online Library, 2018-10-15) Çoker Gürkan, Ajda; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; RENCÜZOĞULLARI, ÖZGE; 222563; 113920; 156421; 125860; 6125Purvalanol and roscovitine are specific cyclin‐dependent kinase (CDK) inhibitors,which have antiproliferative and apoptotic effects on various types of cancer.Although, the apoptotic accomplishment of purvalanol and roscovitine waselucidated at the molecular level, the underlying exact of drug‐induced apoptosisthrough mitogen‐activated protein kinase (MAPK) signaling still speculative. Inaddition, the role of CDK inhibitors in thedownregulation of extracellular signal–regulated kinase 1/2 (ERK1/2)‐mediated epithelial‐mesenchymal transition (EMT)remains unclear. Here, we investigated the potential effect of each CDK inhibitors oncell proliferation, migration, and generation of reactive oxygen species due to theinhibition of MAPKs in metastatic DU145 and PC3 prostate cancer cells. Wereported that purvalanol and roscovitine induced mitochondria membrane potentialloss–dependent apoptotic cell death, which was also characterized by activation ofseveral caspases, cleavage of poly (ADP‐ribose) polymerase‐1 in DU145 and PC3cells. Cotreatment of either purvalanol or roscovitine with ERK1/2 inhibitor, U0126,synergistically suppressed cell proliferation, and induced apoptotic action. Also,ERK1/2 inhibition potentiated the effect of each CDK inhibitor on the down-regulation of EMT processes via increasing the epithelial marker and decreasingmesenchymal markers through reduction of Wnt signaling regulators in DU145 cells.This study provides biological evidence about purvalanol and roscovitine haveapoptotic and antimetastatic effects via MAPK signaling on prostate cancer cell byactivation of GSK3βsignaling and inhibition of phosphoinositide‐3‐kinase/AKT(PI3K/AKT) pathways involved in the EMT process.Publication Open Access Autocrine Growth Hormone-Triggered Curcumin Resistance Abolished by NF-κB Signaling Pathway Dependent on Inflammatory Cytokines and Active Polyamine Catabolic Machinery in MCF-7, MDA-MB-453 and MDA-MB-231 Breast Cancer Cells(2017) Çoker Gürkan, Ajda; Çelik, Merve; Uğur, Merve; Ünsal, Zeynep Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; 125860; 113920; 156421; 6125Autocrine Growth Hormone (GH) induces cell growth, proliferation metastasis in breast cancer. Curcumin is a promising therapeutic agent in cancer through affecting different molecular targets. Our aim was to demonstrate the molecular machinery of curcumin-mediated apoptosis in autocrine GH + MCF-7, MDA-MB-453 and MDA-MB-231 breast cancer cells (BCCs). Stable GH expressing BCCs were generated by GH gene insert PC3.1 plasmid transfection and Neomycin selection. Although GH + cells are resistant to curcumin treatment, dose-dependent drug exposure decreased cell viability, inhibited colony formation, invasion-metastasis via suppressing GH expression in each BCCs. Anti-hormonal concentration of curcumin (20 µM for MCF-7, MDA-MB-453 and 25 µM for MDA-MB-231) inhibited NF-κB p65 (Ser 536) phosphorylation and decreased DNA binding activity of NF-κB p65 in autocrine GH expressing BCCs. In addition, autocrine GH-mediated IL-1α, IL-6, IL-1β pro-inflammatory cytokine expressions downregulated by curcumin treatment. Moreover, curcumin overcome autocrine GH triggered drug resistant and induced caspase-mediated apoptotic cell death through activating Polyamine (PA) catabolic pathway enzymes which led to generation of toxic by-products such as H2O2 in MCF-7, MDA-MB-453 and MDA-MB-231 GH + BCCs. In conclusion, curcumin could overcome GH-mediated resistant phenotype via modulating NF-κB-mediated inflammatory cytokine expression and PA catabolic machinery activation in breast cancer cells.Publication Embargo Downregulation of c-Myc mediated ODC expression after purvalanol treatment is under control of upstream MAPK signaling axis in MCF-7 breast cancer cells(Tubitak Scientific & Technical Research Council Turkey, Ataturk Bulvarı No 221, Kavaklıdere, Ankara, 00000, Turkey, 2014) Alkurt, Gizem; Köse, Betsi; Çoker Gürkan, Ajda; Palavan Unsal, Narçin; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN; COŞKUN, DENİZ; 156421; 125860; 113920; 6125Roscovitine and purvalanol are specific cyclin-dependent kinase (CDK) inhibitors, which induce apoptosis by triggering cell cycle arrest in various cancer cells such as colon, prostate, and breast cancer cells. Although the apoptotic action of roscovitine was clarified at the molecular level, the exact mechanism of purvalanol-induced apoptosis is still under investigation. The mitogen-activated protein kinase (MAPK) signaling cascade is activated by different inducers related to growth, proliferation, differentiation processes, or environmental stress factors. Recent reports showed that modulation of MAPKs might lead to regulation of c-Myc, which is a transcription factor for the polyamine (PA) biosynthesis enzyme, ornithine decarboxylase (ODC). PAs are amine-derived cationic molecules that play crucial roles in cell proliferation, growth, and differentiation. In this study, we investigated the potential role of the MAPK signaling cascade in the purvalanol-induced apoptosis mechanism by comparing the results of roscovitine in MCF7 and MDA-MB-231 breast cancer cells. We found that CDK inhibitors decreased the cell viability in a dose-and time-dependent manner in MCF-7 and MDA-MB-231 cancer cells. Although both CDK inhibitors induced cell cycle arrest, which led to apoptosis by activating caspases and PARP cleavage in MCF-7 breast cancer cells, the apoptotic effect of purvalanol was less than that of roscovitine in MDA-MB-231 cells. Inhibition of MAPKs prevented CDK inhibitor-induced cell viability loss in both cell lines. We determined that purvalanol downregulated c-Myc and ODC expression levels, which led to sharp decrease in the PA pool in MCF-7 cells. On the contrary, purvalanol did not significantly alter c-Myc expression levels, which led to de novo biosynthesis of ODC in a time-dependent manner in MDA-MB-231 cells. Therefore, we suggest that a purvalanol-mediated resistance phenotype might be a possible outcome of c-Myc-mediated ODC expression level in MDA-MB-231 cells.