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Öğe Alcoholic extract of Tarantula cubensis induces apoptosis in MCF-7 cell line.(ALLIED ACAD, 2017) Er, Ayse; Corum, Orhan; Corum, Duygu; Hitit, Mustafa; Donmez, Huseyin; Guzeloglu, AydinTarantula cubensis Alcoholic Extract (TCAE) is a homeopathic agent used for treating many disorders. This study aimed to define the effects of TCAE on the breast carcinoma cell line (MCF-7). After various concentrations (10, 20, 40, 80 and 160 mu l/ml) of TCAE were applied to MCF-7 cells and the human embryonic kidney cell line (HEK293), the cells were incubated for 1, 3, 6, 9, 12, 24 and 48 h, followed by analysis by MTT assays. According to the results of the MTT assays, cells treated with 20 or 40 mu l/ml TCAE for 6 h were applied to apoptosis analysis by flow cytometry. Secreted levels of tumor necrosis factor alpha (TNF alpha), interleukin (IL)-1 beta, IL-6, IL-10, Interferon-gamma (IFN gamma), Transforming Growth Factor beta (TGF beta), and Nuclear Factor-kappa B (NF-kappa B) were measured using ELISAs. TNF alpha and TGF beta levels increased while IL-6 and IL-10 levels fluctuated in MCF-7 cells. In conclusion, our study suggests that TCAE may change the normal cancer physiology and lead to cell death by activating apoptosis in MCF-7 cells.Öğe A Cardioprotective Role of Nerium oleander with the Expression of Hypoxia Inducible Factor 2A mRNA by Increasing Antioxidant Enzymes in Rat Heart Tissue(UNIV FED RIO GRANDE DO SUL, 2018) Hitit, Mustafa; Corum, Orhan; Corum, Duygu Durna; Donmez, Huseyin; Cetin, Gul; Dik, Burak; Er, AyseBackground: Nerium oleander (NO) distillate is used to either protect heart cells against oxidative stress or reduce the risk of cardiovascular disease by regulating the production of reactive oxygen species. Hypoxia-inducible factors (HIFs) regulate cellular antioxidant defense mechanisms under hypoxic conditions in which heart cells survive; however, the key responsible mechanism of NO distillate for cardioprotection remains elusive. The objective of this study was to evaluate the effects on heart tissue at different time intervals after administering NO distillate intraperitoneally (IP) while considering the transcriptional regulation of HIFs and representative antioxidant enzymes. Materials, Methods & Results: The NO plant was chopped, and distillated water was added. The mixture was distilled, and the distillate separated and collected into tubes, after which it was lyophilized to obtain dry material. Twenty male Wistar albino rats (2-3 month-old, 250-300 g each) were used in the study. The rats were randomly divided into four groups. The control group (n = 5) received IP injections of saline; the remaining 15 rats received IP injections of a single dose of 7.5 mL NO distillate. The NO distillate injected rats were divided into three groups according to the time from injection to harvest the heart tissue samples. The tissues were collected at 0 h (control; n = 5), 2 h (group 2; n = 5), 4 h (group 3; n = 5), and 8 h (group 4; n = 5) after injection and under general anesthesia (60 mg/kg ketamine, IP + 10 mg/kg xylazine, IP). Quantitative polymerase chain reaction (qPCR) was used to assess the expression profiles of the genes of interest in the heart tissues. Hypoxanthine phosphoribosyltransferase was used as the reference gene. The expression of manganese superoxide dismutase (MnSOD) mRNA was in a steady state level between the control group and group 2 (P > 0.05); however, it significantly increased in group 3 and 4 compared with that in the control (P < 0.05). Expression of catalase (CAT) mRNA was significantly higher in group 2 than in the control group (P < 0.05) although it was lower in group 3 and 4 than in group 2 (P < 0.05); however, it appeared to be similar among the control group, group 3, and group 4 (P > 0.05). Copper (Cu) SOD mRNA was equally expressed in both the control group and group 2 (P > 0.05) but was lower in group 3 and 4 than in group 2 (P < 0.05). Expressions of HIF1A, HIF2A, and HIF3A mRNA were detected in the rat heart tissues in the control and 2, 4, and 8 h after administration of NO distillate. Expression of HIF1A mRNA was in a steady state and did not differ among groups 2, 3, and 4 (P > 0.05). Similarly, the expression of HIF2A mRNA did not change between the control group and group 2 (P > 0.05); however, it was higher in group 3 than in the control (P < 0.05) and tended to be higher in group 3 than in group 2 (P = 0.063). HIF3A mRNA expression did not change significantly in the heart tissue of any of the groups (P > 0.05). Discussion: The present study using rats determined that MnSOD, CAT, CuSOD, HIF1A, HIF2A, and HIF3A mRNA are expressed in the heart tissues after administration of NO distillate. The increased expression of HIF2A mRNA after 4 h in accordance with a rise in CAT mRNA after 2 h, and MnSOD mRNA after 4 and 8 h might confirm the role of HIF2A mRNA in oxidative stress defense by regulating antioxidant enzymes; consequently, this study may expand our understanding of uses of NO distillate with respect to molecular pathways.Öğe Investigation of autophagic effects of melatonin on breast cancer stem cells(ALLIED ACAD, 2017) Donmez, Huseyin; Kocak, Nadir; Yildirim, Ibrahim HalilAutophagy plays important roles in physiologic cellular events and also in cancers. It has been reported that cells escaped from death via autophagy and if autophagy inhibited, cells conducted to apoptosis. In some studies, protective effects of the melatonin on induced autophagy in cancer cells reported. In this study, we aimed to evaluate the autophagic effect of melatonin in cancer stem cells. For this purpose, CD44+/CD24-phenotype cells sorted from melatonin-treated and untreated MCF-7 and HEK293 cells. Effect of melatonin on autophagy was analysed by immunofluorescence and western blot analysis. Results showed that melatonin-induced LC3 (Microtubule-associated protein 1A/1B-light chain 3) aggregation and formation of autophagic vacuoles in MCF-7 derived cancer stem cells and also induced LC3-I to LC3-II conversion in these stem cells when compared to untreated control group. In conclusion, melatonin showed a pro-autophagic effect in CD44+/CD24-stem cells derived from MCF-7 cells and anti-autophagic effect in CD44+/CD24-stem cells obtained from HEK293 cells.Öğe Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24-phenotype breast cancer stem cells(ELSEVIER GMBH, 2016) Nami, Babak; Donmez, Huseyin; Kocak, NadirTunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycin-induced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor. (C) 2016 Elsevier GmbH. All rights reserved.
