Browsing by Author "Uzunoglu, S"

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    Augmentation of methylprednisolone-induced differentiation of myeloid leukemia cells by serine threonine protein phosphatase inhibitors
    Uzunoglu, S; Uslu, R; Tobu, M; Saydam, G; Terzioglu, E; Buyukkececi, F; Omay, SB
    To elucidate the roles of serine/threonine protein phosphatases type 1 (PP1) and type 2A (PP2A) in methylprednisolone-induced differentiation of HL60 cells into granulocytes and K562 cells into monocytes, we examined the effect of serine/threonine protein phosphatase inhibitors, okadaic acid and Gal-A on the proliferation/ differentiation of HL60 and K562 cells. Okadaic acid and Gal-A augmented methylprednisolone induced granulocytic differentiation and cell death of HL60 cells and monocytic differentiation and cell death of K562 cells in different dose ranges, respectively. These data suggest an important role of PP1 and PP2A in the mechanism leading to differentiation of leukemic cells. (C) 1999 Elsevier Science Ltd. AU rights reserved.
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    Anti-angiogenic effects of trabectedin (Yondelis; ET-743) on human breast cancer cells
    Atmaca, H; Uzunoglu, S
    Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata, has been shown to have antitumor effects. In this study, we assessed the possible anti-angiogenic effects of trabectedin on human umbilical vein endothelial cells (HUVECs) and breast cancer cell lines. An XTT cell viability assay was used to determine cytotoxicity. A scratch assay was used to detect the migration of cells after trabectedin treatment. Angiogenic cytokine profiles of breast cancer cell lines, before and after treatment with trabectedin, were investigated using an angiogenesis antibody array. Changes in mRNA expression levels of VEGF were evaluated using qRT-PCR. Trabectedin inhibited the viability of HUVECs and breast cancer cells in a concentration-and time-dependent manner. The migration of both HUVECs and breast cancer cells was suppressed by trabectedin treatment. Angiogenic cytokines which are known to regulate tumorigenicity and angiogenesis, such as GM-CSF, IGFBP-2, VEGF, and uPA, were inhibited, while several anti-angiogenic cytokines such as TIMP-1 and Serpin E1were induced in breast cancer cells. Furthermore, expression levels of VEGF mRNA were inhibited in all breast cancer cells tested. Although additional studies are needed to elucidate the molecular mechanisms underlying the anti-angiogenic activity of trabectedin, our results suggest that trabectedin may act as a potential anti-angiogenic agent in breast cancer cells.
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    Zoledronic Acid Effects Interleukin-6 Expression in Hormone-Independent Prostate Cancer Cell Lines
    Asbagh, LA; Uzunoglu, S; Cal, C
    Objective: To investigate the inhibitory effects of zoledronic acid (ZA) on tumor related growth factor IL-6 in hormone resistant prostate cancer cell lines. The association between apoptosis and IL-6 inhibition was also assessed. Materials and Methods: PC-3 and DU145 cell lines were treated with different concentrations of ZA (1-100 mu M) at various intervals (24-72 h.). The cell viability was investigated by XTT assay and apoptotic effect was evaluated by cell death detection ELISA kit. Caspase 3/7 activity assay was performed to confirm apoptosis. IL-6 levels were measured by ELISA in the supernatant, and these data were also confirmed by IL-6 mRNA analysis using RT-PCR. Results: PC-3 and DU145 cell lines were sensitive to ZA mediated cytotoxicity in a dose- and time-dependent manner. However, the apoptotic effect was significantly different among PC-3 and DU145 cells (p < 0.05). IL-6 secretion was significantly lower in both cell lines, compared to the untreated control cells (p < 0.05). Although the increased inhibition of IL-6 secretion was associated with increased apoptosis in DU 145 cells (p = 0.002), there was no similar association for PC-3 cell line (p = 0.347). When compared to the untreated controls, the number of cDNA copies was significantly lower in the ZA treated DU145 cell line at doses of 30 and 90 mu M (p < 0.05), suggesting a reduced expression of IL-6 mRNA. Conclusion: ZA exhibited a time- and dose-dependent apoptotic effect on PC-3 and DU 145 prostate cancer cell lines and this effect was associated with inhibited secretion of IL-6 in DU145 cell line.
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    Evaluation of the association between null genotypes of glutathione-S-transferases and Behcet's disease
    Uzunoglu, S; Acar, H; Okudan, N; Gökbel, H; Mevlitoglu, I; Sari, F
    Glutathione S-transferases (GST) play an important role in oxidative stress related syndromes. An imbalance of the oxidant and antioxidant systems is important in the pathogenesis of Behcet's disease (BD). The objective of this study was to evaluate the association of null genotypes of GST-M1 and GST-T1 with BD since some preliminary molecular genetic data were recently published. Ninety-four Turkish BD patients (42 male, 52 female, 37.1 +/- 10.4 years) and 140 healthy volunteers (70 male, 70 female, 36.8 +/- 11.7 years) matched for age and gender with the patients as the control group were included in the study. Distributions of GST-M1 and GST-T1 genotypes were determined by multiplexed PCR using three sets of primers for GST-M1, GST-T1, and beta-globulin genes. There was no association between BD and the frequencies of GST-M1 and GST-T1 null genotypes when compared to controls by separate analysis. However, by cross and pooled combination analysis there was a significant association between the frequencies of pooled GSTs with one or both null genotypes in BD and controls. This is the first evidence that the association between the frequencies of GST-M1 and GST-T1 null genotypes and BD might be dependent on the interaction of multiple null allele polymorphisms rather than a single null allele of GST-M1 and GST-T1.
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    Paclitaxel in combination with AT-101 induces apoptosis via supressing Bcl-2, bcl-XL, mcl-1 proteins in human breast cancer cells.
    Cakar, B; Gursoy, P; Atmaca, H; Kisim, A; Bozkurt, E; Uzunoglu, S; Sezgin, C; Sanli, UA; Karabulut, B; Uslu, R; Karaca, B
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    FIBROBLAST GROWTH FACTOR RECEPTOR 2 (FGFR2) POLYMORPHISM STATUS OF TURKISH BREAST CANCER PATIENTS
    Uslu, R; Karaca, B; Atmaca, H; Kisim, A; Cakar, B; Karabulut, B; Sezgin, C; Uzunoglu, S
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    SYNERGISTIC EFFECT OF GOSSYPOL, A COTTON-PLANT SEED, WITH CONVENTIONAL CHEMOTHERAPEUTICS IN MCF-7 CELLS
    Karaca, B; Atmaca, H; Eniseler, AG; Unuvar-Purcu, D; Kisim, A; Karabulut, B; Uzunoglu, S; Uslu, R
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    Trabectedin to induce mitochondrial membrane potential dissipation and reactive oxygen species generation in breast cancer cells.
    Atmaca, H; Bozkurt, E; Cakar, B; Surmeli, ZG; Uzunoglu, S; Uslu, R; Karaca, B
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    Overcoming drug resistance in hormone- and drug-refractory prostate cancer cell line, PC-3 by docetaxel and gossypol combination
    Cengiz, E; Karaca, B; Kucukzeybek, Y; Gorumlu, G; Gul, MK; Erten, C; Atmaca, H; Uzunoglu, S; Karabulut, B; Sanli, UA; Uslu, R
    Drug resistance is a significant challenge of daily oncology practice. Docetaxel and gossypol both have antitumoral activity in hormone-refractory prostate cancer (HRPC). Our results revealed that docetaxel and gossypol were synergistically cytotoxic and apoptotic in PC-3 cells in a dose- and time-dependent manner. We further investigated the expression profiles of genes involved in drug resistance and metabolism with a Human Cancer Drug Resistance and Metabolism PCR Array(A (R)) (SuperArray). Six of the 84 genes that are known to regulate drug resistance, metabolism, cell cycle, DNA repair and oncogenesis were downregulated a parts per thousand yen3-fold change by the combination treatment. These results may be important in devising mechanism-based and targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers.
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    Combined gossypol and zoledronic acid treatment results in synergistic induction of cell death and regulates angiogenic molecules in ovarian cancer cells
    Atmaca, H; Gorumlu, G; Karaca, B; Degirmenci, M; Tunali, D; Cirak, Y; Purcu, DU; Uzunoglu, S; Karabulut, B; Sanli, UA; Uslu, R
    In the present study, we aimed to evaluate the possible synergistic, cytotoxic effects of combination treatment of gossypol and zoledronic acid, in human ovarian cancer cell lines, OVCAR-3 and MDAH-2774, and to elucidate the role of this novel combination treatment on angiogenesis-related molecules in ovarian cancer. The XTT cell viability assay was used for showing cytotoxicity. Both DNA fragmentation by ELISA assay and caspase 3/7 activity measurement were used for demonstrating apoptosis. To elucidate the angiogenic molecules affected by combination treatment, mRNA levels of angiogenic molecules were measured using the Human Angiogenesis RT2 Profiler (TM) PCR Array (SuperArray, Frederick, MD) in ovarian cancer cell lines, OVCAR-3 and MDAH-2774. The combined treatment resulted in significant, synergistic cytotoxicity, and induced apoptosis. This effect was observed to happen in a dose- and time-dependent manner. Moreover, the combination treatment of 10 mu M gossypol and 5 mu M zoledronic acid resulted in significant down-regulation (>= thee-fold) in mRNA levels of some pivotal angiogenic molecules in OVCAR-3 and MDAH-2774 cells as compared to the untreated control. However, this effect was different in the two ovarian cancer cell lines observed. Gossypol, in combination with zoledronic acid, may provide a rational treatment option for ovarian cancer, not only by direct inhibition of cell proliferation, but also inhibition of angiogenesis-related molecules.
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    Enhancing cytotoxic and apoptotic effect in OVCAR-3 and MDAH-2774 cells with all-trans retinoic acid and zoledronic acid: a paradigm of synergistic molecular targeting treatment for ovarian cancer
    Karabulut, B; Karaca, B; Varol, U; Muslu, U; Cakar, B; Atmaca, H; Kisim, A; Uzunoglu, S; Uslu, R
    Background: Ovarian cancer is the most fatal gynecologic malignancies in the world. Although, platinum based treatments are widely used, the disease becomes treatment refractory within two years, and novel treatment options should be searched. All-trans retinoic acid (ATRA) induces growth arrest, differentiation and cell death in some types of cancer cells and its combination with various anticancer agents results in enhanced cytotoxicity. Zoledronic acid is a common bisphosphonate known for its anticancer effects beyond its current use in the treatment of cancer-induced bone disease. We aimed to investigate the possible additive/synergistic effect of both agents in OVCAR-3 and MDAH-2774 ovarian cancer cell lines, since both agents show superiority to conventional cytotoxics in terms of adverse events. Methods: XTT cell proliferation assay was used for showing cytotoxicity. For verifying apoptosis, both DNA Fragmentation by ELISA assay and caspase 3/7 activity measurement were used. OligoGeArray (R) which consists of 112 apoptosis related genes was used to elucidate the genetic changes within cancer cells. To validate our oligoarray results, quantitative real-time PCR was performed on four selected genes that were maximally effected by the combination treatment: lymphotoxin beta receptor (LTBR), myeloid cell leukemia-1 (MCL-1), tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), TNFRSF1A-associated death domain protein (TRADD). Results: We demonstrated that a novel combination of ATRA and zoledronic acid is a strong inducer of apoptotic related cell death in both ovarian cancer cells. While the combination therapy significantly induced proapoptotic genes such as tumor necrosis factor receptor superfamily (TNFRSF), TRADD and caspase 4, some of the antiapoptotic genes such as members of MCL-1, LTBR, BAG3 and Bcl-2 family members were inhibited. Conclusions: These are the preliminary molecular results of a novel combination treatment of ATRA and zoledronic acid, with fewer side effects as compared to conventional cytotoxic agents. With additional experimental analysis, it may serve as a good option for the treatment of refractory and elderly ovarian cancer patients, for whom there exists very limited choice of treatment.
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    Comparison of XTT and Alamar blue assays in the assessment of the viability of various human cancer cell lines by AT-101 (-/- gossypol)
    Uzunoglu, S; Karaca, B; Atmaca, H; Kisim, A; Sezgin, C; Karabulut, B; Uslu, R
    This study compared the two different commercially available in vitro viability assays: XTT and Alamar blue (AB), to detect anti-proliferative effects of AT-101, a cotton plant extract, on six different human carcinoma cell lines including: prostate (PC-3 and DU-145), breast (MCF-7 and MDA-MB-231), and ovary (OVCAR-3 and MDAH 2774) in a time- and dose-dependent manner. Cells were exposed to AT-101 in the concentration range of 2.5-40 mu M for 24, 48, and 72 h. The AB assay was slightly more sensitive than the XTT assay in the evaluation of AT-101 at 24 h, suggesting that the AB assay might be used for detecting early changes in cell viability as compared to the XTT assay. Moreover, the AB assay showed less intra-assay variability as compared to the XTT. The non-toxic, non-radioactive AB metabolism assay allows rapid assessment of large numbers of samples, with simple equipment and at reduced cost for continuous monitoring of cancer cell viability, and, thus, should be accepted as a suitable alternative viability method.
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    Zoledronic acid in combination with serine/threonine phosphatase inhibitors induces enhanced cytotoxicity and apoptosis in hormone-refractory prostate cancer cell lines by decreasing the activities of PP1 and PP2A
    Cirak, Y; Varol, U; Atmaca, H; Kisim, A; Sezgin, C; Karabulut, B; Uzunoglu, S; Uslu, R; Karaca, B
    OBJECTIVES To investigate if the cytotoxic and apoptotic effect of zoledronic acid (ZA) can be enhanced by the addition of the serine/threonine protein phosphatase inhibitors calyculin A (CA) and okadaic acid (OA) in hormone and drug refractory prostate cancer cells, PC-3 and DU-145. To discover the effect of these combination treatments on phosphatase 1 (PP1) and PP2A protein expression levels in prostate cancer cells. MATERIALS AND METHODS An XTT cell viability assay was used to determine cytotoxicity. Apoptosis was evaluated by enzyme-linked immunosorbent assay (ELISA) using a Cell Death Detection ELISA Plus Kit and verified by measuring caspase 3/7 enzyme activity. The PP1 and PP2A enzyme activities were evaluated by serine/threonine phosphatase ELISA and expression levels of PP1 and PP2A proteins were then re-assessed by Western blot analysis. RESULTS Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis compared with any agent alone in both PC-3 and DU-145 prostate cancer cells. The combination of ZA with phosphatase inhibitors resulted in enhanced suppression of both PP1 and PP2A enzyme activity and protein levels, which was more overt with the ZA/CA combination. CONCLUSION Results from our study increase the translational potential of our in vitro findings and offer the basic rationale for the design of new combinatory strategies with ZA and phosphatase inhibitors for the treatment of prostate cancer, which may become resistant to conventional therapy.
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    Lack of association between leptin levels and leptin gene polymorphism in obese women
    Okudan, N; Gökbel, H; Acar, H; Uzunoglu, S; Belviranli, M
    The aim of the present study was to evaluate the relationship between oligopolymorphism in the 25th codon of leptin gene and obesity. Eighty-seven obese women and 75 healthy women were constituted obese and control groups. Body fat percent, fat mass and lean body mass were determined by bioimpedance meter and leptin levels were determined. The presence of 25th codon oligopolymorphism in the leptin gene was done by PCR-RFLP technique. Mean leptin levels were 38.5 +/- 22.0 ng/ml, and 147.9 +/- 44.8 ng/ml in the control and obese groups, respectively. The correlations of serum leptin level to body fat percentage and fat mass in the control group were significant. The correlations in the obese group were not significant. This data implies that the difference of leptin levels between control and obese groups are more likely to be associated with alterations in the leptin gene other than 25th codon or alterations in the leptin receptor gene.
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    Pretreatment with AT-101 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of breast cancer cells by inducing death receptors 4 and 5 protein levels
    Kisim, A; Atmaca, H; Cakar, B; Karabulut, B; Sezgin, C; Uzunoglu, S; Uslu, R; Karaca, B
    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has been shown to induce extrinsic pathway of apoptosis in many types of cancer cells. AT-101, an (-)-enantiomer of gossypol, is a potent anticancer agent that is shown to be an inhibitor of Bcl-2/Bcl-XL. In this study, we searched whether pretreatment with either of these drugs would result in the enhancement of apoptosis through induction of death receptors and activation of mitochondrial pathways within breast cancer cells. Human breast cancer (MCF-7 and MDA-MB-231) and normal breast cells (MCF-10A) were treated with drugs alone/in combination/sequentially. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were done. ELISA and Western blot analysis were done to assess DR4 and DR5 protein levels. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. The sequential treatment of AT-101 followed by TRAIL resulted in significant synergistic cytotoxicity and apoptosis. Moreover, pretreatment of breast cancer cells with AT-101 and then with TRAIL caused enhancement of the expression levels of DR4 and DR5 in both cancer cell lines, suggesting that these cells were under strong apoptotic stimuli. These findings all together, strongly suggest that pretreatment with AT-101 enhances TRAIL-induced death-inducing signaling complex resulting in the engagement of the mitochondrial pathway to apoptosis in breast cancer cells. These promising, preliminary results make AT-101 and TRAIL a novel combination treatment candidate for breast cancer.
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    Up-regulation of serine/threonine protein phosphatase type 2A regulatory subunits during methylprednisolone-induced differentiation of leukaemic HL-60 cells
    Aydin, HH; Selvi, N; Saydam, G; Tobu, M; Uzunoglu, S; Uslu, R; Buyukkececi, F; Omay, SB
    Serine/threonine protein phosphatase 2A (PP2A) may play a role in leukaemic cell differentiation of the HL 60 myeloid leukaemic cell-line after methylprednisolone induction. We have investigated the specific enzyme activity and expression of catalytic and regulatory subunits of PP2A. The resulting specific enzyme activity and immunoblots showed an increase in enzyme activity and the expression of regulatory subunits after methylprednisolone treatment. There was no change in the expression of PP2A catalytic subunits. It is suggested that the effect of methylprednisolone on leukaemic differentiation may be the result of PP2A upregulation.