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Age-Related Characterization of Dental Pulp Mesenchymal Stem Cells
(Wiley, 2023) Kaya, Egemen; Acikgoz, Eda; Aydogdu, Ilkay; Dindaroglu, Funda Cagirir; Atalayin, Cigdem; Oktem, Gulperi
[No abstract available]
Biomolecular fingerprints of the effect of zoledronic acid on prostate cancer stem cells: Comparison of 2D and 3D cell culture models
(Elsevier Science Inc, 2024) Guler, Gunnur; Acikgoz, Eda; Mukhtarova, Guenel; Oktem, Gulperi
Revealing the potential of candidate drugs against different cancer types without disrupting normal cells depends on the drug mode of action. In the current study, the drug response of prostate cancer stem cells (PCSCs) to zoledronic acid (ZOL) grown in two-dimensional (2D) and three-dimensional (3D) culture systems was compared using Fourier transform-infrared (FT-IR) spectroscopy which is a vibrational spectroscopic technique, supporting by biochemical assays and imaging techniques. Based on our data, in 2D cell culture conditions, the ZOL treatment of PCSCs isolated according to both C133 and CD44 cell surface properties induced early/late apoptosis and suppressed migration ability. The CD133 gene expression and protein levels were altered, depending on culture systems. CD133 expression was significantly reduced in 2D cells upon ZOL treatment. FT-IR data revealed that the integrity, fluidity, and ordering/disordering states of the cell membrane and nucleic acid content were altered in both 2D and 3D cells after ZOL treatment. Regular protein structures decrease in 2D cells while glycogen and protein contents increase in 3D cells, indicating a more pronounced cytotoxic effect of ZOL for 2D cells. Untreated 3D PCSCs exhibited an even different spectral profile associated with IR signals of lipids, proteins, nucleic acids, and glycogen in comparison to untreated 2D cells. Our study revealed significant differences in the drug response and cellular constituents between 2D and 3D cells. Exploring molecular targets and/or drug-action mechanisms is significant in cancer treatment approaches; thus, FT-IR spectroscopy can be successfully applied as a novel drug-screening method in clinical research.
Cancer Stem Cell and Embryonic Development-Associated Molecules Contribute to Prognostic Significance in Ovarian Cancer
(Lippincott Williams & Wilkins, 2012) Oktem, Gulperi; Sanci, Muzaffer; Bilir, Ayhan; Yildirim, Yusuf; Kececi, Sibel D.; Ayla, Sule; Inan, Sevinc
Objectives: Embryonic molecules and cancer stem cell signaling resemble each other, and they organize cancer modality. We hypothesized that similar immunohistochemical expressions between tumor spheroids and patients' samples compared with clinical relevance would give an important clue in patients' prognosis. Methods: Immunohistochemical expression of c-kit, Notch1, Jagged1, and Delta1 in 50 cases of primary ovarian tumors (10 endometrioid, 10 serous, 10 mucinous adenocarcinoma, 10 borderline serous, and 10 borderline mucinous tumors) and MDAH-2774 spheroids were investigated. Results were compared in both spheroids and tumor samples with morphologic parameters (histological grade) and clinical data (age, stage, tumor size, and metastasis). Results: High c-kit and Notch1 immunoreactivity was shown in spheroids, but interestingly immunoreactivity of these molecules in tumor samples was different from patients' clinicopathological characteristics. In serous carcinoma, metastasis correlated with Notch1 immunoexpression; in mucinous carcinoma, Jagged1 immunohistochemistry correlated with grade, stage, and metastasis of tumor; in borderline serous and mucinous tumors, Jagged1 correlated with high grade. Moreover, Jagged1 correlated with stage and Notch1 with size in borderline mucinous tumor. Endometrioid carcinoma statistics showed that there was a correlation between age and Notch1 expression. Conclusion: Notch1, Jagged1, and Delta1 expressions might be useful markers for clinical prognosis of ovarian carcinomas; and Notch pathway, one of the most intensively studied putative therapeutic targets, may be a useful marker for cancer. Consequently, Jagged1 could be a marker for tumor grades and Notch1 as a marker for metastases.
Carvedilol in glioma treatment alone and with imatinib in vitro
(Spandidos Publ Ltd, 2010) Erguven, Mine; Yazihan, Nuray; Aktas, Esin; Sabanci, Akin; Li, Chiang J.; Oktem, Gulperi; Bilir, Ayhan
The purpose of the study was to investigate whether carvedilol has an antiproliferative effect alone and whether carvedilol provides an additive, synergistic or antagonistic effect on imatinib mesylate-induced cytotoxicity in both C6 glioma monolayer and spheroid culture. The C6 rat glioma chemoresistant experimental brain tumour cell line, that is notoriously difficult to treat with combination chemotherapy, was used both in monolayer and spheroid Cultures. We treated C6 glioma cells with carvedilol alone and a combination of carvedilol and imatinib mesylate at a concentration of 10 mu M. Following treatment, we evaluated cell proliferation index. bromodeoxyuridine labelling index (BrDU-LI), cell cycle distributions, apoptotic cell percentages, cAMP levels and three dimensional cell morphology at monolayer cultures. In addition BrDU-LI, volume and morphology of spheroids were also assessed. Carvedilol and imatinib mesylate alone reduced cell number. BrDU-LI cAMP levels and spheroid volume. Carvedilol and imatinib mesylate arrested cells at G0/G1 phase in a time-dependent manner and time-independent manner, respectively. Carvedilol increased apoptosis rate only at the 24th h but imatinib mesylate did for all time intervals. Interestingly carvedilol, drug with well-known protective effect on mitochondria, induced severe mitochondria damage, and imatinib mesylate induced autophagy confirmed only by transmission electron microscopy. These results suggest that carvedilol showed antitumour activity against rat C6 glioma cells and a combination of carvedilol with imatinib mesylate resulted in enhanced in vitro antitumour activity.
Characterization of CD133(+)/CD44(+) human prostate cancer stem cells with ATR-FTIR spectroscopy
(Royal Soc Chemistry, 2019) Guler, Gunnur; Guven, Ummu; Oktem, Gulperi
Current cancer treatments destroy the tumor mass but cannot prevent the recurrence of cancer. The heterogeneous structure of the tumor mass includes cancer stem cells that are responsible for tumor relapse, treatment resistance, invasion and metastasis. The biology of these cells is still not fully understood; therefore, effective treatments cannot be developed sufficiently. Herein, attenuated total reflection- Fourier transform infrared (ATR-FTIR) spectroscopy, combined with unsupervised multivariate analysis, was applied to prostate cancer stem cells (CSCs), non-stem cancer cells (non-CSCs) and normal prostate epithelial cells to elucidate the molecular mechanisms and features of CSCs, which are crucial to improving the target specific therapies. This work revealed the spectral differences in the cellular mechanisms and biochemical structures among three different cell types. Particularly, prostate CSCs exhibit differences in the lipid composition and dynamics when compared to other cell types. CSCs also harbor pronounced differences in their major cellular macromolecules, including differences in the protein amount and content (mainly a-helices), the abundance of nucleic acids (DNA/RNA), altered nucleic acid conformation and carbohydrate composition. Interestingly, macromolecules containing the CvO groups and negatively charged molecules having the COO-groups are abundant in prostate CSCs in comparison to prostate non-CSCs and normal prostate cells. Overall, this study demonstrates the potential use of ATR-FTIR spectroscopy as a powerful tool to obtain new insights into the understanding of the CSC features, which may provide new strategies for cancer treatment by selectively targeting the CSCs.
Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells
(Sage Publications Ltd, 2016) Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Demir, Kenan; Yavasoglu, Altug; Cetintas, Vildan Bozok
Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.
Determination of nitric oxide synthase activity and apoptosis of germ cells in different obstruction models
(Elsevier Gmbh, Urban & Fischer Verlag, 2009) Oktem, Gulperi; Altay, Baris; Turna, Burak; Aktug, Huseyin; Yavasoglu, Altug; Yilmaz, Zlem; Semerci, Buelent
We aimed to determine the changes of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) immunoreactivity and apoptosis after proximal and distal obstruction models on ipsilateral and contralateral testicular tissues. Mate albino Wistar rats were randomly divided into three groups (n = 30): a control group which underwent sham operations (n = 10), a unilateral vasal ligation (n = 10) and a unilateral epididymal ligation group (n = 10). iNOS and eNOS distribution and apoptosis were studied in both ipsilateral and contralateral testes using quantitative immunohistochemistry. Nitric oxide synthase activity was significantly affected in ipsilateral and contralateral testes cells after vasal and epididymal ligation. eNOS immunoreactivity increased markedly after ipsilateral vasal ligation (ILVL). Degeneration-related changes were also associated with changes in apoptotic rate. Analysis using the terminal dUTP nick end-labeling TUNEL method revealed that apoptotic cell numbers significantly increased after ILVL. p53 and bcl-2 immunoreactivity increased in both experimental groups compared with the sham-operated group. Changes in iNOS and eNOS immunolocalisation were strongly associated with cell damage, because germ cell degeneration was more prominent in the ILVL group. Altered p53 immunolocalisation was also associated with cell degeneration, and a rise in bcl-2 immunoreactivity might be considered to reflect a protective mechanism in the testis. These cellular changes could enlighten understanding of the interaction between testicular functioning and damage. (c) 2008 Elsevier GmbH. All. rights reserved.
The Detrimental Effects of Diabetes on Pluripotency Determined by KLF4, SOX2, C-MYC and OCT4 Immunoreactivity in Rat Testes
(Wroclaw Medical Univ, 2013) Aktug, Huseyin; Uysal, Aysegul; Yavasoglu, Altug; Oltulu, Fatih; Akarca, Saadet O.; Yilmaz-Dilsiz, Ozlem; Oktem, Gulperi
Background. Diabetes mellitus (DM) is a multisystem disorder. Type 1 DM can be experimentally induced in rats with streptozotocin (STZ). Diabetic conditions result in testicular oxidative stress and suppressed male reproductive activity as well as decreases in both testicular organ weights and subject weights. Objectives. The purpose of this study was to investigate immunohistochemical differences in testicular tissue due to STZ induced diabetes regarding pluripotency via transcription factors like Klf4, Sox2, c-Myc and Oct4, and to determine weight changes in both the subjects and the testes during the experiment. Material and Methods. Diabetes was induced in male adult rats for this study. A healthy control group and a diabetic group were observed for one month. Blood glucose levels over 250 mg/dL were considered diabetic. Results. On days 0, 3, 15 and 30, the subjects' weights and testicular organ weights were determined and analyzed. The results revealed statistically significant decreases (p < 0.05 and p < 0.001, respectively). Semiquantitative immunohistochemical analyses of Klf4, Sox2, c-Myc and Oct4 were studied in testes paraffin sections via light microscopy. Decreased immunoreactivity of Klf4 was observed in the diabetic group in comparison to the controls. Spermatogonial cells and Sertoli cells showed increased immunostaining for Sox2 and c-Myc, while decreased immunoreactivity of Oct4 was noted for both spermatogenic and Sertoli cells compared to the control group. Conclusions. This study clearly demonstrated that Klf4, Sox2 and Oct4 immunopositive cells in adult male rat testes manifested sustainable pluripotency and that diabetes has dramatically detrimental effects on this trait
Different doses of radiation on agar colony forming development in C6 glioma cells: Assessment by thymidine labeling index, and bromodeoxyuridine labeling index
(Ortadogu Ad Pres & Publ Co, 2007) Ozmen, Tolga; Oktem, Gulperi; Tuna, Sevilcan; Biltekin, Burcu; Denir, Secnur; Bilir, Ayhan
Objective: Gliomas are relatively frequent in adults, and are among the most malignant primary brain tumors. Glioblastoma. multiforme, like many other tumors that exhibit radiation sensitivity in vitro, seems to be very resistant to radiation in vivo, thus suggesting that irradiation may not be a rate-limiting factor for malignant glioma tumor growth. In this study, we aimed to determine the optimal dose of radiation in C6 glioma colony forming assay, which is a valuable tool for antitumor treatment screening. Material and Methods: 10(5) cell/lamella colony forming cells were radiated with 200 cGy, 400 cGy, 800 cGy and 1600 cGy for 10 minutes. Radiosensitivity was measured systematically 24, 48, 72 and 96 hours after the radiation by three methods: soft-agar bilayer assay, thymidinE incorporation, and bromodeoxyuridine (BrdU) incorporation. Results: The soft-agar bilayer assay, which assessed the colony-forming units, showed that the number of colonies in the control group (609, 3 +/- 86.8) were decreased after 200 cGy (8.3 +/- 3.6) and 400 cGy (7.2 +/- 4.3). No colony was detected in 800 cGy and 1600 cGy irradiated cells [3H] Thymidine incorporation was more prominent with higher doses of radiation. BrdU incorporation revealed that even at low doses (200 cGy) of radiation there was a significant decrease of cell proliferation. On higher doses like 1600 cGy it was more prominent. Conclusion: Cell survival, doubling time, and cell phases are parameters of growth kinetics, and the results suggest that C6 glioma cells are radiosensitive and are virtually affected by all radiation doses in our experiment even 200 cGy at 24 hours. Besides, colony forming assay with thymidine labeling index, and BrdU labeling index may be used as new methods for determining radiotherapy doses in clinical applications.
Double hit strategy: Removal of sialic acid from the dendritic cell surface and loading with CD44+/CD24-/low cell lysate inhibits tumor growth and metastasis by targeting breast cancer stem cells
(Elsevier, 2022) Acikgoz, Eda; Duzagac, Fahriye; Guven, Ummu; Yigitturk, Gurkan; Kose, Timur; Oktem, Gulperi
Cancer stem cells (CSCs), which represent the root cause of resistance to conventional treatments, recurrence, and metastasis, constitute the critical point of failure in cancer treatments. Targeting CSCs with dendritic cell (DC)-based vaccines have been an effective strategy, but sialic acids on the surface of DCs limit the interaction with loaded antigens. We hypothesized that removal of sialic acid moieties on immature DCs (iDCs) could significantly affect DC-CSC-antigen loading, thereby leading to DC maturation and improving immune recognition and activity. The lysate of CD44+/CD24-/low breast CSCs (BCSCs) was pulsed with sialidase-treated DCs to obtain mature dendritic cells (mDCs). The roles of cytoskeletal elements in antigen uptake and dendritic cell maturation were determined by immunofluorescence staining, flow cytometry, and cytokine measurement, respectively. To test the efficacy of the vaccine in vivo, CSCs tumor-bearing mice were immunized with iDC or mDC. Pulsing DCs with antigen increased the expression levels of actin, gelsolin, talin, WASp, and Arp2, especially in podosome-like regions. Compared with iDCs, mDCs expressed high levels of CD40, CD80, CD86 costimulatory molecules and increased IL-12 production. Vaccination with mDC: i) increased CD8+ and CD4 + T-cell numbers, ii) prevented tumor growth with anti-mitotic activity and apoptotic induction, iii) suppressed metastasis by decreasing Snail, Slug, and Twist expressions. This study reveals for the first time that sialic acid removal and loading with CSC antigens induces significant molecular, morphological, and functional changes in DCs and that this new DC identity may be considered for future combined immunotherapy strategies against breast tumors.
Effect of SIRT1 activators and inhibitors on CD44(+)/CD133(+)-enriched non-small cell lung cancer cells
(Spandidos Publ Ltd, 2020) Eroglu, Zuhal; Erdem, Ceren; Oktem, Gulperi; Cetintas, Vildan Bozok; Duzgun, Zekeriya
Lung cancer is one of the most commonly diagnosed cancers and it is associated with high rates of morbidity and mortality. Metastasis and relapse of the tumor depend on the survival and proliferation of lung cancer stem cells (LCSCs). the ability to identify CSCs may prevent recurrence and lead to more effective treatments. Sirtuins are a group of deacetylases that include seven variants (SIRT1-7), with sirtuin 1 (SIRT1) being the most intensively investigated. Evidence suggests thatSIRT1is both a tumor-suppressor gene and an oncogene. SIRT1 can deacetylate the tumor-suppressor protein p53 to decrease its activity. SIRT1 activators increase the deacetylation of p53, whereas SIRT1 inhibitors can stimulate p53 by inhibiting deacetylation. in the present study, CD44(+)and CD133(+)-enriched A549 (non-small cell lung cancer) cells collected using the CD44 and CD133 CSC surface markers by fluorescence-activated cell sorting method were treated with SIRT1 inhibitors (tenovin-6 and sirtinol) and SIRT1 activators (resveratrol and SRT1720), and their effects on apoptosis, as well as the mRNA and protein expression of SIRT1 and p53 were investigated. of these agents, it was found that resveratrol increased p53 expression by 4.1-fold, decreased SIRT1 expression by 0.2-fold, and it was the most potent inducer of apoptosis.
Effects of different drug treatments on the proliferation of human ovarian carcinoma cell line MDAH-2774
(Tubitak Scientific & Technical Research Council Turkey, 2018) Ayla, Sule; Bilir, Ayhan; Erturkoglu, Senol; Tanriverdi, Gamze; Soner, B. Cem; Sofuoglu, Kenan; Ghisolfi, Laura; Oktem, Gulperi
Background/aim: In this study, the effects of resveratrol as a natural polyphenol compound, gemcitabine as an antimetabolite that has nucleoside structure analogous to deoxycytidine, and para-aminophenol-derived paracetamol were investigated with single and combined applications in monolayers of the MDAH-2774 human ovarian cancer cell line. Materials and methods: Drugs were evaluated in cell culture with respect to cell proliferation, cell cytotoxicity (trypan blue dye exclusion test), synthesis phase of cell cycle, and cell structure in 24, 48, 72, and 96 h. Result: Resveratrol and gemcitabine diminished both cell proliferation and cell cycle synthesis phase indication in monolayer cell cultures (P < 0.05). All combination groups showed similar effects that were mainly more effective in respect to single usage of resveratrol and gemcitabine in monolayer cell cultures. Conclusion: The effects of gemcitabine, resveratrol, and paracetamol were investigated in monolayers of the MDAH-2774 human ovarian cancer cell line and a decrease in cell number in cell cycle synthesis phase, prevention of cell proliferation, and destruction of cell structure were observed.
Effects of flavopiridol on critical regulation pathways of CD133(high)/CD44(high) lung cancer stem cells
(Lippincott Williams & Wilkins, 2016) Cetintas, Vildan Bozok; Acikgoz, Eda; Yigitturk, Gurkan; Demir, Kenan; Oktem, Gulperi; Kaymaz, Burcin Tezcanli; Oltulu, Fatih; Aktug, Huseyin
Background:Flavopiridol a semisynthetic flavone that inhibits cyclin-dependent kinases (CDKs) and has growth-inhibitory activity and induces a blockade of cell-cycle progression at G1-phase and apoptosis in numerous human tumor cell lines and is currently under investigation in phase II clinical trials. Cancer stem cells (CSCs) are comprised of subpopulation of cells in tumors that have been proposed to be responsible for recurrence and resistance to chemotherapy. The aim of the present study was to investigate the effects of flavopiridol in cancer stem cell cytoskeleton, cell adhesion, and epithelial to mesenchymal transition in CSCs.Methods:The cells were treated with flavopiridol to determine the inhibitory effect. Cell viability and proliferation were determined by using the WST-1 assay. Caspase activity and immunofluorescence analyses were performed for the evaluation of apoptosis, cell cytoskeleton, and epithelial-mesenchymal transition (EMT) markers. The effects of flavopiridol on the cell cycle were also evaluated. Flow cytometric analysis was used to detect the percentages of CSCs subpopulation. We analyzed the gene expression patterns to predict cell cycle and cell cytoskeleton in CSCs by RT-PCR.Results:Flavopiridol-induced cytotoxicity and apoptosis at the IC50 dose, resulting in a significant increase expression of caspases activity. Cell cycle analyses revealed that flavopiridol induces G1 phase cell cycle arrest. Flavopiridol significantly decreased the mRNA expressions of the genes that regulate the cell cytoskeleton and cell cycle components and cell motility in CSCs.Conclusion:Our results suggest that Flavopiridol has activity against lung CSCs and may be effective chemotherapeutic molecule for lung cancer treatment.
Embryonic microenvironment suppresses YY1 and YY1-related genes in prostate cancer stem cells
(Elsevier Gmbh, 2024) Taskiran, Aysegul; Oktem, Gulperi; Demir, Aleyna; Oltulu, Fatih; Ozcinar, Emine; Duzagac, Fahriye; Guven, Ummu
Yin yang 1 (YY1), a transcription factor, plays crucial roles in cell fate specification, differentiation, and pluripotency during embryonic development. It is also involved in tumorigenesis, drug resistance, metastasis, and relapse caused by cancer stem cells (CSCs), particularly in prostate cancer (PCa). Targeting YY1 could potentially eliminate prostate CSCs (PCSCs) and provide novel therapeutic approaches. PCa tissues often exhibit elevated YY1 expression levels, especially in high-grade cases. Notably, high-grade PCa tissues from 58 PCa patients and CD133high/CD44high high /CD44 high PCSCs isolated from DU145 PCa cell line by FACS both showed significantly increased YY1 expression as observed through immunofluorescence staining, respectively. To investigate the embryonic microenvironment impact on YY1 expression in CSC populations, firstly PCSCs were microinjected into the inner cell mass of blastocysts and then PCSCs were co-cultured with blastocysts. Next Generation Sequencing was used to analyze alterations in YY1 and related gene expressions. Interestingly, exposure to the embryonic microenvironment significantly reduced the expressions of YY1, YY2, and other relevant genes in PCSCs. These findings emphasize the tumor-suppressing effects of the embryonic environment by downregulating YY1 and YY1-related genes in PCSCs, thus providing promising strategies for PCa therapy. Through elucidating the mechanisms involved in embryonic reprogramming and its effects on YY1 expression, this research offers opportunities for further investigation into focused therapies directed against PCSCs, therefore enhancing the outcomes of PCa therapy. As a result, PCa tumors may benefit from YY1 and associated genes as a novel therapeutic target.
Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y
(Springer, 2010) Bilir, Ayhan; Erguven, Mine; Yazihan, Nuray; Aktas, Esin; Oktem, Gulperi; Sabanci, Akin
The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.
Evaluation and Comparison of In Vitro Biocompatibility of Poly (Glycolic Acid) and Poly (Lactide-Co-Glycolide Acid) on Mature Spheroids of Tumorigenic and Non-Tumorigenic Cell Lines
(Ortadogu Ad Pres & Publ Co, 2011) Bilir, Ayhan; Erguven, Mine; Zilan, Aydin; Oktem, Gulperi; Korkmaz, Seval; Korkmaz, Mevlut
Objective: The aim of this study was to evaluate and compare in vitro biocompatibility and poly(glycolic acid) (PGA) and poly(lactide-co-glycolide acid) (PLGA) on tumorigenic and non-tumorigenic mature spheroids. Material and Methods: This is an in vitro experimental study. Tumorigenic (C6 glioma, SH-SY5Y, MDAH2774, MCF-7) and non-tumorigenic cells [CRL11372, primary osteoblasts (MCPO)] as well as their mature spheroids were cultured alone as a control group as well as in combination with PGA and PLGA. Total cell numbers, bromodeoxyuridine labeling index (BrDU-LI), apoptosis, morphology, and ultrastructure were evaluated. Results: PGA and PLGA significantly decreased the number of SH-SY5Y and C6 glioma cells; MDAH 2774 cells also decreased, but not significantly (p> 0.05). Low BrDU-LI (p< 0.05) with a high level of apoptosis (p< 0.05) at C6 glioma and a high level of BrDU-LI (p< 0.05) with a low level of apoptosis at MDAH2774 (p< 0.01) were noted. These biopolymers mostly decreased the number of CRL-11372 cells (p< 0.05), but indicated an increased apoptosis (p< 0.01) and significant BrDU-LI (p< 0.05). Biopolymers induced chromatin condensation (typical apoptotic ultrastructure) and vacuolization primarily at SH-SY5Y spheroids but rarely at MDAH-2774 spheroids. This apoptotic ultrastucture was most often observed at MCPO spheroids. PLGA and PGA induced similar BrDU-LI decreases among tumorigenic spheroids (p< 0.05), although this decrease was greater at MCF-7 (p< 0.05) in the PGA group. PGA primarily decreased BrDU-LI at CRL 11372 (p< 0.05), although the decrease was almost identical to that at MCPO for the two biopolymers (p< 0.05). A significant attachment affinity was determined at MDAH -2774 and C6 glioma spheroids. Conclusion: This study demonstrated the biocompatibility of PGA and PLGA at mature spheroids of tumorigenic and non-tumorigenic cell lines, which changed according to the cell type.
Experimental Repair of Rabbit Segmental Bone Defects by Using Autologous Bone Marrow and Electrical Stimulation
(Ortadogu Ad Pres & Publ Co, 2010) Gurbuz, Yusuf; Sahin, Fahri; Ozdedeli, Selcen; Oktem, Gulperi; Avci, Cigir Biray; Saydam, Güray; Ozdemir, Oguz
Objective: In this study, we have aimed to investigate the potential role of autologous bone marrow cell injection and muscular electrical stimulation as a separate and concomitant application on bone healing in experimental rabbit ulnar segmental bone defect model. Material and Methods: Forty New-Zealand rabbits, all over three months of age and weighing between 2500 and 3500 grams were divided into four groups. Four groups of rabbits were the control group (I), electrical stimulation group (II), bone marrow cells injection group (III) and bone marrow cells injection with electrical stimulation group (IV). Bone defect healing was evaluated radiologically according to the modified Lane and Sandhu scoring system and at the end of the sixth week, rabbits were sacrificed and their forearms were sampled for histopathological investigation. Results: When one-to-one comparison between all groups was performed, defect healing was found to be better in Groups II, III, and IV compared to Group I based on the radiological and histopathological parameters evaluated. This evaluation revealed that the healing was better in groups treated with bone marrow cell injection with or without electrical stimulation as well as in group treated with electrical stimulation compared to the control group with no treatment. Conclusion: Autologous bone marrow cells with or without electrical stimulation, would be used in healing of segmental bone defect with an adequate efficacy. Future stem cell studies combined with electrical current are required to demonstrate and to confirm that electrical current enhances in vivo cellular differentiation.
Expression profiling of stem cell signaling alters with spheroid formation in CD133(high)/CD44(high) prostate cancer stem cells
(Spandidos Publ Ltd, 2014) Oktem, Gulperi; Bilir, Ayhan; Uslu, Ruchan; Inan, Sevinc V.; Demiray, Sirin B.; Atmaca, Harika; Ayla, Sule; Sercan, Ogun; Uysal, Aysegul
Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133(high)/CD44(high) DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133(high)/CD44(high) population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 a1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.
Glycogen synthase kinase-3 inhibition in glioblastoma multiforme cells induces apoptosis, cell cycle arrest and changing biomolecular structure
(Pergamon-Elsevier Science Ltd, 2019) Acikgoz, Eda; Guler, Gunnur; Camlar, Mahmut; Oktem, Gulperi; Aktug, Huseyin
Glioblastoma multiforme (GBM) is the most malignant and aggressive primary human brain tumors. The regulatory pathways of apoptosis are altered in GBMs, leading to a survival advantage of the tumor cells. Thus, identification of target molecules, which are effective in triggering of the cell death mechanisms in GBM, is an essential strategy for therapeutic purposes. Glycogen synthase kinase-3 (GSK-3) plays an important role in apoptosis, proliferation and cell cycle. This study focused on the effect of GSK-3 inhibitor IX in the GBM cells. Apoptosis induction was determined by Annexin-V assay, multicaspase activity and immunofluorescence analyses. Concentration-dependent effects of GSK-3 inhibitor IX on the cell cycle were also evaluated. Moreover, the effect of GSK inhibitor on the cellular biomolecules was assessed by using ATR-FTIR spectroscopy. Our assay results indicated that GSK-3 inhibitor IX induces apoptosis, resulting in a significant increase in the expression of caspase-3 and caspase-8 proteins. Cell cycle analyses revealed that GSK-3 inhibitor IX leads to dose -dependent G2/M-phase cell cycle arrest. Based on the FTIR data, treatment of GBM cells causes dysregulation in the carbohydrate metabolism and induces apoptotic cell death which was characterized by the spectral alterations in nucleic acids, an increment in the lipid amount with disordering state and compositional changes in the cellular proteins. These findings suggest that GSK-3 inhibitor IX exhibits anti-cancer effects by inducing apoptosis and changing biomolecular structure of membrane lipids, carbohydrates, nucleic acids and proteins, and thus, may be further evaluated as a potential effective candidate agent for the GBM combination therapies. (C) 2018 Elsevier B.V. All rights reserved.
Immunohistochemical determination of mTOR pathway molecules in ovaries and uterus in rat estrous cycle stages
(F Hernandez, 2020) Ekizceli, Gulcin; Inan, Sevinc; Oktem, Gulperi; Onur, Ece; Ozbilgin, Kemal
mTOR is a member of the PI3K/Akt/mTOR signaling pathway that participates in cell growth, proliferation, protein synthesis, transcription, angiogenesis, apoptosis and autophagy. mTOR and MAPK pahways are two important key signal pathways which are related to each other. We investigated the role of mTOR and other signaling molecules in rat ovaries and uteruses in stages of the estrous cycle. Young adult female rats were divided into four groups as proestrous, estrous, metestrous and diestrous according to vaginal smears. Immunohistochemical staining of mTORC1, IGF1, PI3K, pAKT1/2/3, ERK1 and pERK1/2 was performed and pAKT1/2/3 and ERK1 were also analyzed using western blotting on ovarian and uterine tissue samples. According to our results, PI3K/Akt/ mTOR and ERK/pERK showed an increase in the rat ovulation period. When all the groups were evaluated the immunoreactivities for all of the antibodies were detected in the oocytes, granulosa and theca cells, corpus luteum and stroma of ovary and lamina propria, surface and glandular epithelium of uterus with the strongest observed with anti-ERK1 antibody and then with a decreasing trend with anti-mTORC1, anti-pAkt1/2/3, anti-IGF1, anti-PI3K and anti-pERK1/2 antibodies in the proestrus and estrus stages. Differently from other parts of the ovary, highest antibody expression in the corpus luteum was observed in the metestrous stage. Moreover, the existence of pAKT1/2/3 and ERK1 proteins was confirmed with the Western blotting technique. We suggest that mTOR and mTOR-related ERK signaling molecules may participate in the rat ovulation process.

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