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    Differential effects of doxorubicin and docetaxel on nitric oxide production and inducible nitric oxide synthase expression in MCF-7 human breast cancer cells
    (Cognizant Communication Corp, 2004) Oktem, G; Karabulut, B; Selvi, N; Sezgin, C; Sanli, UA; Uslu, R; Yurtseven, ME; Omay, SB
    Anthracyclines and docetaxel are frequently used agents in the chemotherapy of breast cancer. In this study we examined the role of inducible nitric oxide synthase (iNOS) expression during the cytotoxicity of these drugs in the MCF-7 breast cancer cell line. Cell viability and cytotoxicity were evaluated by the trypan blue dye exclusion method. Apoptosis and necrosis were determined by the acridine orange/ethidium bromide dye method. The percentage of apoptotic cells was significantly higher with doxorubicin. However, total cytotoxic cell numbers were higher in the docetaxel group compared with doxorubicin, with respect to the control. Most of the cells were seen to be necrotic with the dye method. Cell extracts during the apoptotic process were applied to immunoblot by anti-iNOS monoclonal antibodies. While there was an increase in iNOS expression during docetaxel induced-cytotoxicity, a significant decrease in iNOS expression was detected during doxorubicin-induced cytotoxicity. The Griess method was used for detection of nitrate levels. It was compatible with immunoblot results. These data open a window for further studies to understand the mechanism underlining the cytotoxicity of docetaxel and doxorubicin.
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    Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids
    (Professor D A Spandidos, 2006) Oktem, G; Vatansever, S; Ayla, S; Uysal, A; Aktas, S; Karabulut, B; Bilir, A
    Multicellular Tumor Spheroid (NITS) represents a three-dimentional structural form of tumors in laboratory conditions, and it has the characteristics of avascular micrometastases or intervascular spaces of big tumors. Recent studies indicate that extracellular matrix (ECM) proteins play a critical role in tumor metastasis, therefore normal and cancer cells require an ECM for survival, proliferation and differentiation. Doxorubicin and Docetaxel are widely used in the therapy of breast cancer, as well as in in vivo and in vitro studies. In this study, we examined the effect of apoptosis and proliferation of cells on the human breast cancer cell line, MCF-7, by using p53, bcl-2 and Ki67 gene expression, and the tendency to metastasis with extracellular matrix proteins, laminin and type IV collagen after chemotherapy in the spheroid model. The apoptotic cell death in situ was detected by TUNEL method. TUNEL-positive cells and positive immunoreactivities of laminin, type IV collagen, p53 and, bel-2 were detected in the control group. There was no laminin and type IV collagen immunoreactivities in spheroids of drug groups. While TUNEL-positive cells and p53 immunoreactivity were detected in Docetaxel, Doxorubicin and Docetaxel/Doxorubicin groups, p53 immunoreactivity was not observed in the Docetaxel group. There was no bcl-2 immunoreactivity in either drug group. In addition, we did not detect Ki67 immunoreactivity in both control and drug treatment groups. However, the absence of Ki67 protein in MCF-7 breast multicellular tumor spheroids is possibly related to the cells in GO or S phase. These agents may affect the presence of ECM proteins in this in vitro model of micrometastasis of spheroids. These findings suggest that the possible mechanism of cell death in Doxorubicin and Docetaxel/Doxorubicin treatment groups is related to apoptosis through the p53 pathway. However, we considered the possiblity that there is another control mechanism for the Docetaxel group.
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    The effect of exogenous melatonin administration on trabecular width, ligament thickness and TGF-beta(1) expression in degenerated intervertebral disk tissue in the rat
    (Churchill Livingstone, 2006) Turgut, M; Oktem, G; Uslu, S; Yurtseven, ME; Aktug, H; Uysal, A
    Intervertebral disk (IVD) degeneration, a complex pathological condition of varying origins, causes low back pain. Degenerative changes in IVD tissue affect the adjacent vertebral structure, resulting in a decreased vertebral trabecular width. It has been suggested that transforming growth factor-beta 1 (TGF-beta(1)) may have a role in the repair of connective tissue, as it occurs in the IVD degeneration process. In this study, we investigated the effects of exogenous melatonin (MEL) administration on vertebral trabecular width, ligament thickness and TGF-beta(1) expression in degenerated IVD tissue. Fifteen adult male Swiss Albino rats were divided randomly into three groups; nonoperated control, operated degeneration, and MEL treatment groups. In the operated degeneration and MEL treatment groups, cuts were made parallel to the end plates in the posterior annulus fibrosus at the fifth and tenth vertebral segments of the tail to induce IVD degeneration. In each group, TGF-beta(1) immunoreactivity and morphometry of vertebral trabecular width and anterior and posterior ligament thickness were evaluated. Histologically, disorganisation and irregularity of collagen fibres was seen in the degenerated (operated) IVD. Increased TGF-beta(1) expression in multinuclear chondrocytes was also observed as was decreased vertebral trabecular width. Importantly, the reduction of trabecular width observed in the operated degenerated group was reversed after MEL administration (p<0.0001). Similarly, TGF-beta(1) expression in multinuclear chondrocytes was dramatically increased after exogenous MEL application. Thus, there was a regression in histopathological changes after MEL treatment, with disk appearances similar to those of the control group. Based on our findings. we suggest that MEL activates the recovery process in the degenerated IVD tissue, possibly by stimulating TGF-beta(1) activity. This is the first report investigating the involvement of the pineal hormone MEL in the repair of rat IVD. (C) 2006 Elsevier Ltd. All rights reserved.
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    Evaluation of the relationship between inducible nitric oxide synthase (iNOS) activity and effects of melatonin in experimental osteoporosis in the rat
    (Springer France, 2006) Oktem, G; Uslu, S; Vatansever, SH; Aktug, H; Yurtseven, ME; Uysal, A
    Inducible nitric oxide synthase (iNOS) plays a critical role in the pathogenesis of osteoporosis. iNOS generates nitric oxide (NO), a free radical contributing to the imbalance between bone formation and resorption caused by estrogen depletion. Melatonin is the major product of the pineal gland which is known to diminish iNOS expression and NO production significantly. The aim of this study was to determine the distribution of iNOS and the amount of apoptotic cells after melatonin treatment in ovariectomized rats. Since previous studies have shown that constitution of bone formation is primarily sustained in nucleus pulposus and epiphyseal cartilage, experiments were carried out on nucleus pulposus and epiphyseal cartilage; additional quantitation of osteoblasts and osteoclasts were evaluated on vertebral area as well. Vertebral sections of ovariectomized rats were obtained from formalin-fixed and parafin-embedded blocks. iNOS expression and quantitation of apoptotic cells in nucleus pulposus and epiphyseal cartilage were evaluated using indirect immunoperoxidase and TUNEL techniques, respectively. The number of osteoclasts and osteoblasts in trabecular bone was determined using histomorphometry. Ovariectomy increased iNOS expression and the number of apoptotic cells in nucleus pulposus and epiphyseal cartilage, whereas a 4-week treatment with melatonin (10 mg/kg/day) resulted in the reduction of both effects. These data indicate that there is strong influence of melatonin application on expression of iNOS, apoptosis, osteoclast and osteoblast numbers after ovariectomy. In conclusion, melatonin besides its usual use as an antiaging hormone, may also be an effective hormone in treatment of bone changes in estrogen deficiency states.
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    Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells
    (Pergamon-Elsevier Science Ltd, 2003) Saydam, G.; Aydin, HH; Sahin, F; Selvi, N; Oktem, G; Terzioglu, E; Buyukkececi, F; Omay, SB
    Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC50 of IFN-alpha-2b on K562 cells was found to be 600 IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore. (C) 2003 Elsevier Science Ltd. All rights reserved.
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    Involvement of protein phosphatase 2A in interferon-alpha-2b-induced apoptosis in K562 human chronic myelogenous leukaemia cells
    (Pergamon-Elsevier Science Ltd, 2003) Saydam, G.; Aydin, HH; Sahin, F; Selvi, N; Oktem, G; Terzioglu, E; Buyukkececi, F; Omay, SB
    Interferon-alpha (IFN-alpha)-2b is known to have antiproliferative effects on hematological malignant cells, especially chronic myelogenous leukaemia (CML). However, it can induce cytogenetical remissions in a very small percentage of the patients. Also during interferon therapy, resistance can emerge in the CML clones. K562 is an in vitro model cell line transformed from a Ph positive CML patient. It can be induced to differentiate to granulocytic and/or monocytic lineages with certain molecules. IFN-alpha-2b generally exerts its effects on CML cells by Janus family kinases (Jak/Stat) pathway, mostly through tyrosine kinase system. However, there is almost no data on the relevance of serine/threonine (Ser/Thr) protein phosphatase (PP) system in the interferon induced signal transduction pathways. In this study, we investigated serine/threonine protein phosphatases in the IFN-alpha-2b induced K562 cytotoxicity. Trypan blue dye exclusion test and MTT assay were utilised for determining cytotoxicity. IC50 of IFN-alpha-2b on K562 cells was found to be 600 IU/ml. However, no differentiation was determined by analysis of cell surface antigen expressions. Serine/threonine protein phosphatase inhibitors calyculin A (Cal A) and okadaic acid (OKA) augmented the IFN-alpha-2b induced cytotoxicity. Apoptosis assay by the mono-oligonucleosome detection and acridine orange/propidium iodide dye revealed marked apoptosis underlying cytotoxicity. Phosphatase enzyme assay revealed a gradual increase in protein phosphatase 2A (PP2A) activity during interferon induced cytotoxicity. Conversely, immunoblots showed no change in the expression of PP2A catalytic and regulatory subunits. In conclusion, PP2A plays a role in IFN-alpha-2b induced apoptosis of K562 cells and should be investigated as a new window furthermore. (C) 2003 Elsevier Science Ltd. All rights reserved.