Browsing by Author "Karabulut, B"

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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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    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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    Effect of apoptosis and response of extracellular matrix proteins after chemotherapy application on human breast cancer cell spheroids
    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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    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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    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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    Targeting apoptosis in the hormone- and drug-resistant prostate cancer cell line, DU-145, by gossypol/zoledronic acid combination
    Sanli, UA; Gorumlu, G; Erten, C; Gul, MK; Cengiz, E; Kucukzeybek, Y; Karaca, B; Atmaca, H; Uzunoglu, S; Karabulut, B; Uslu, R
    Possible synergistic cytotoxic and apoptotic effects of gossypol with zoledronic acid on DU-145 cells were explored, along with the rationale behind any observed synergism due to the different apoptotic proteins involved. XTT cell proliferation assay was used to assess the cytotoxicity, and DNA fragmentation and caspase 3/7 activity were measured to verify apoptosis. Human Apoptosis Array was used to evaluate apoptotic proteins. The synergistic cytotoxic combination treatment had a versatile effect on apoptotic proteins, through inhibition of anti-apoptotic proteins (including cIAP-1, cIAP-2, survivin, livin, claspin, p53, p21, PON-2 and heat shock proteins) and concurrently the induction of pro-apoptotic proteins (Bad, Bax, Fas, FADD, cleaved caspase-3 and p27). Both drugs had a minimal toxicity profile comparing to cytotoxic agents. Combination treatments targeting many pivotal apoptosis-related proteins may be a rationale option for treatment of prostate cancer. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
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    Combination of AT-101/cisplatin overcomes chemoresistance by inducing apoptosis and modulating epigenetics in human ovarian cancer cells
    Karaca, B; Atmaca, H; Bozkurt, E; Kisim, A; Uzunoglu, S; Karabulut, B; Sezgin, C; Sanli, UA; Uslu, R
    We investigated the effects of AT-101/cisplatin combination treatment on the expression levels of apoptotic proteins and epigenetic events such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) enzyme activities in OVCAR-3 and MDAH-2774 ovarian cancer cells. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were performed. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array. DNMT and HDAC activities were evaluated by ELISA assay and mRNA levels of DNMT1 and HDAC1 genes were quantified by qRT-PCR. Combination of AT-101/cisplatin resulted in strong synergistic cytotoxicity and apoptosis in human ovarian cancer cells. Combination treatment reduced some pivotal anti-apoptotic proteins such as Bcl-2, HIF-1A, cIAP-1, XIAP in OVCAR-3 cells, whereas p21, Bcl-2, cIAP-1, HSP27, Clusterin and XIAP in MDAH-2774 cells. Among the pro-apoptotic proteins, Bad, Bax, Fas, phospho-p53 (S46), Cleaved caspase-3, SMAC/Diablo, TNFR1 and Cytochrome c were induced in OVCAR-3 cells, whereas, Bax, TRAILR2, FADD, p27, phospho-p53 (S46), Cleaved caspase-3, Cytochrome c, SMAC/Diablo and TNFR1 were induced in MDAH-2774 cells. Combination treatment also inhibited both DNMT and HDAC activities and also mRNA levels in both ovarian cancer cells. AT-101 exhibits great potential in sensitization of human ovarian cancer cells to cisplatin treatment in vitro, suggesting that the combination of AT-101 with cisplatin may hold great promise for development as a novel chemotherapeutic approach to overcome platinum-resistance in human ovarian cancer.
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    Radiosensitization of hormone-refractory prostate cancer cells by gossypol treatment
    Akagunduz, O; Karaca, B; Atmaca, H; Uzunoglu, S; Karabulut, B; Sanli, UA; Haydaroglu, A; Uslu, R
    Purpose: Many drugs have been tested to increase the sensitivity of prostate cancer cells to radiotherapy Gossypol, a natural polyphenolic compound extracted from the cotton plant, is one of the agents the efficacy of which has been investigated in the treatment of prostate cancer for this purpose. The main aim of this study was to investigate the best gossypol application with irradiation, when gossypol was applied either sequentially (24 h before and after irradiation) or concurrently in PC-3 hormone-refractory and radioresistant prostate cancer cells. Methods: The XTT viability assay was used to evaluate the cytotoxicity of different concentrations of gossypol in PC-3 cells. Irradiation was applied to PC-3 cells via 6 MV photon linear accelerator and delivered 24 h before, 24 h after radiation or at the same time with gossypol administration. Results: Gossypol caused radiosensitization of PC-3 cells that are known to be radioresistant, with high Bcl-2 levels. Among different applications of gossypol and irradiation (before, after and concurrent) in prostate cancer cells, the best results were observed by the application of gossypol 24 h before irradiation. Conclusion: Our study suggests that gossypol represents a promising novel anticancer treatment for radiosensitization of human hormone-refractory prostate cancer cells.
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    Targeting Bcl-2 Protein to Enhance Chemosensitivity of Hormone Refractory Prostate Cancer Cell Line, DU-145 by a Synergistic Combination of Docetaxel and Gossypol
    Uzunoglu, S; Karaca, B; Atmaca, H; Kisim, A; Karabulut, B; Uslu, R
    Objective: Docetaxel has become the standard of care for hormone-refractory prostate cancer (HRPC), however drug resistance and toxicity are still challenging in daily cancer practice. The anti-apoptotic pathway centered around the Bcl-2 protein might be one of the responsible pathways. Adding a second drug to docetaxel treatment is one of the most common approaches to solve this problem. Gossypol was reported to have potent anticancer activities in prostate cancer. In this study, we searched for the possible synergistic cytotoxic/apoptotic effects of this combination treatment in hormone- and drug resistant prostate cancer cell line, DU-145 via inhibition of Bcl-2 protein. Material and Methods: XTT cell viability assay was used to assess cytotoxicity of the drugs alone and in combination. For verifying apoptosis, Cell Death Detection Elisa Plus Kit and Caspase-Glo 3/7 assay were used. Western Blot analysis for Bcl-2 protein was carried out. Results: A novel drug combination of docetaxel and gossypol resulted in a significant synergistic cytotoxic activity and apoptosis as compared to any single agent alone, in a dose- and time dependent manner, and also significantly reduced Bcl-2 protein levels in DU-145, in doses that can be used clinically. Condusion: Adding gossypol to docetaxel as a combination treatment in HRPC patients might be a solution for taxane resistant patients. Inhibition of Bcl-2 might be one of the underlying routes of activity for this combination.
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    OCTREOTIDE IN COMBINATION WITH DOCETAXEL TRIGGERS APOPTOSIS BY INDUCING SSTR2 AND SSTR5 LEVELS IN HUMAN BREAST CANCER CELLS, MCF-7 AND MDA-MB-231
    Karaca, B; Muslu, U; Surmeli, ZG; Cakar, B; Atmaca, H; Asli, K; Sezgin, C; Uzunoglu, S; Karabulut, B; Uslu, R
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    Enhancement of taxane-induced cytotoxicity and apoptosis by gossypol in human breast cancer cell line MCF-7
    Karaca, B; Atmaca, H; Uzunoglu, S; Karabulut, B; Sanli, UA; Uslu, R
    Purpose: Gossypol is a natural polyphenolic compound extracted from cotton plant (Gossypium species) which has shown potent inhibitory effect on cell growth of many types of cancers. In this study, we aimed to evaluate the interaction of gossypol with some conventional drugs known to be effective in the treatment of breast cancer like taxanes, doxorubicin, gemcitabine, cisplatin and vinorelbine, in MCF-7 breast cancer cells. Materials and methods: The XTT viability assay was used to evaluate the cytotoxicity of various cytotoxic agents alone and in combination with gossypol in MCF-7 breast cancer cells. The combination effect analysis of Chou and Talalay was used to identify the most synergistic drug combinations. The possible synergistic effects of the combination of drugs on apoptosis were also evaluated by using two different apoptosis assays. Results: We identified strong synergistic cytotoxic and apoplotic activity of gossypol with taxanes among all other studied cytotoxic drugs. Conclusion: This study provides proof that gossypol combined with taxanes may have potential as a novel future treatment for breast cancer.
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    Regulation of growth factors in hormone- and drug-resistant prostate cancer cells by synergistic combination of docetaxel and octreotide
    Erten, C; Karaca, B; Kucukzeybek, Y; Gorumlu, G; Cengiz, E; Gul, MK; Atmaca, H; Uzunoglu, S; Karabulut, B; Sanli, UA; Uslu, R
    OBJECTIVE To evaluate the effects of combined treatment with docetaxel and octreotide, a somatostatin analogue, on human hormone- and drug-refractory prostate cancer cell lines, PC-3 and DU-145, and on some growth factors related to tumour growth and angiogenesis in prostate cancer. MATERIALS AND METHODS A cell proliferation assay was used to assess the cytotoxicity of the drugs. To verify apoptosis, both DNA fragmentation (by enzyme-linked immunosorbent assay) and caspase 3/7 activity were measured. We also investigated the effect of combined docetaxel and octreotide on growth factors secreted from prostate cancer cells using a human growth factor antibody array. RESULTS The combination of docetaxel and octreotide resulted in significant synergistic cytotoxic activity and apoptosis, which was dose- and time-dependent. The combined treatment also resulted in significantly less secretion of stem cell factor and platelet-derived growth factor-AB in PC-3 cells, and transforming growth factor-beta and basic fibroblast growth factor in DU-145 cells, than in untreated controls. CONCLUSION Octreotide, a somatostatin analogue, combined with docetaxel might provide a rationale treatment option for hormone-refractory prostate cancer cells, not only by direct inhibition of cell proliferation but also by inhibiting the secretion of growth factors.
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    A potent enantiomer of gossypol, AT-101: Screening of anti-angiogenic protein targets in glioblastoma multiforme cells
    Surmeli, Z; Bozkurt, E; Karaca, B; Karabulut, B; Sezgin, C; Sanli, UA; Uslu, R