Egemen Dere

The effect of Dichlorvos on acetylcholinesterase activity in some tissues in rats

In this study, the changes with respect to time in the serum, brain, liver, kidney and small intestine acetylcholinesterase activities were investigated in both male and female rats administered dichlorvos intraperitoneally (i.p.). For this purpose, 4 mg kg-1 doses of dichlorvos were injected i.p. in the rats. The control groups, on the other hand, were administered physiological saline via the same route. Rats were killed by decapitation at 0, 2, 4, 8, 16, 32, 64 and 72 hours after administration of dichlorvos and tissues were harvested. Enzyme activities were determined following the necessary treatments. While a significant decrease in enzyme activities in the kidney and small intestine tissues with respect to time were not observed in either sex, a significant decrease in enzyme activities in the serum, as well as in the brain and liver tissues were observed. As a result of our study, acetylcholinesterase activity was found to be decreased compared to controls in both male and female rats from 2 and 4 hours. Enzyme inhibition continued for up to 72 hours.

Combination of fenretinide and indole-3-carbinol results in synergistic cytotoxic activity inducing apoptosis against human breast cancer cells in vitro.

The outcome in patients with breast cancer is not satisfactory to date, although new chemotherapy regimens have been introduced in clinics. Therefore, novel approaches are required for better management of patients with breast cancer. In this study, we tested the cytotoxic activity of a new combination of fenretinide, a synthetic retinoid, with indole-3-carbinol, a natural product present in vegetables such as broccoli and cabbage, against MCF-7 (estrogen receptor-positive) and MDA-MB-231 (estrogen receptor-negative) cell lines. It has been found that the combination resulted in more powerful cytotoxic activity, by induction of apoptosis, compared with that when they were used singly. In conclusion, this novel combination warrants in-vivo experiments to elucidate its possible use in the treatment of breast cancer.

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine.

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright

Cytotoxic and apoptotic effects of the combination of palladium (II) 5,5-diethylbarbiturate complex with bis(2-pyridylmethyl)amine and curcumin on non small lung cancer cell lines

Metal-based chemotherapeutics such as cisplatin are widely used treatment of lung cancer which is the major cause of cancer-related mortality worldwide. Recent studies demonstrated that novel metal-based compounds have strong cytotoxic activity in a similar way as cisplatin. Therefore, metal-based compounds have been synthesized and investigated in order to determine their cytotoxic activities. It has been also reported curcumin, which has been derived from turmeric plant, has powerful cytotoxic effect on various cancer cell lines. In the light of these data, it has been investigated the cytotoxic effects of combination of curcumin (0.78-100μM) and palladium (II) 5,5-diethylbarbiturate complex with bis(2-pyridylmethyl)amine [Pd(II) complex] (0.39-50μM) against non small lung cancer cell lines, A549 and H1299. It has been found that combination of Pd(II) complex and curcumin enhanced the cytotoxic activity and apoptotic cell death at 48h, compared to single use of each agent, only in H1299 cell line (combination index <1). Apoptosis was evident by annexin v staining positivity, increased caspase 3/7 activity and the presence of pyknotic nuclei. Pro-apoptotic genes of TNFRSF10A and HRK were found to be involved in apoptotic cell death. In conclusion, the application of this combination may be regarded as a novel and effective approach for the treatment of lung cancer due to its promising cytotoxic and apoptotic effect.

Enhanced cytotoxic activity of doxorubicin through the inhibition of autophagy in triple negative breast cancer cell line

BACKGROUND The outcome of triple negative breast cancer is still poor and requires improvement with better therapy options. Autophagy has recently been shown to play a role in anticancer drug resistance. Therefore, we investigated if the effectiveness of doxorubicin was augmented by the inhibition of autophagy. METHODS MDA-MB-231 was used as a model cell line for triple negative breast cancer and 3-methyladenine was used as an inhibitor of autophagy. Cells were treated with 0.46-1.84μM doxorubicin and 2.5-10μM 3-methyladenine for 48h. Cell death mode was examined with M30 and M65 ELISA assays. ROS level and LDH activity was examined and the cellular acidic compartment of cells was monitored by acridine orange staining. The expression of various autophagy and apoptosis related proteins/genes were evaluated with Western blotting and RT-qPCR respectively. RESULTS Synergism was observed between the compounds (CI value<1.0). RT-qPCR analysis revealed that the combination resulted in a down-regulation of autophagy-related genes. Moreover, the combination resulted in a different cell death modality, upregulating necroptosis-related genes. This suggests that the mode of cell death may switch from apoptosis to necroptosis, which is a more severe form of cell death, when autophagy is inhibited. These results were further confirmed at protein level by Western blotting. CONCLUSION Inhibition of autophagy seems to sensitize triple negative breast cancer cells to doxorubicin, warranting further in vivo studies for the proof of this concept. GENERAL SIGNIFICANCE Autophagy has a key role in drug resistance in MDA-MB-231 cells. Therefore combinatorial approaches may effectively overcome resistance.

Glutathione S-transferase activity in rats exposed to methyl parathion

Methyl parathion is an organophosphate insecticide that has been used in agriculture and the domestic sector for several years. This pesticide and others, arriving through different processes, exert significant effects on water quality with serious consequences for environmental and human health. The main objective of this study was to investigate the changes of Glutathione S-transferase enzyme activity in methyl parathion exposed rat tissues. For this purpose, wistar rats (Rattus norvegicus) were injected intraperitoneally with 7 mg kg−1 dose of methyl parathion, while corn oil was applied to control groups in the same way. The liver, kidneys, brain and small intestine were quickly removed after 0, 2, 4, 8, 16, 32, 64, and 72 hours of injection of methyl parathion and the glutathione S-transferase activity was determined in these tissues. As a result it was seen that glutathione S-transferase activity increases in all tissues in the group of male and female rats to which methyl parathion was given. The increase of glutathione S-transferase activity may be a result of methyl parathions toxic effect because it is one of the most important enzymes of detoxification metabolism.

PO-197 A possible link between the mitochondrial gatekeeper pyruvate dehydrogenase enzyme complex and emt

Introduction In recent years, mitochondria have attracted much attention in metastatic cancer research. The first finding of the importance of mitochondria in cancer was reported by Otto Warburg in 1920. In the following years,it was found that cancer is closely related to mitochondrial characteristics such as glucose metabolism and chemoresistance. In this context,epithelial mesenchymal transition (EMT), due to its potential of being a molecular marker for tumour metastasis, clarification of this process is essential. Although increments have been made in metastatic cancer research,there are no studies showing the relationship between pyruvate dehydrogenase enzyme complex (PDH) and EMT. Material and methods To inhibit PDH activity, pharmacological (PDH inhibitor, Cpi-613) and genomic approaches (stable cell lines were established by lentivirus) were used in A549 cells. The antiproliferative effect of Cpi-613 was investigated by xCELLigence System, SRB and ATP viability assays. After determining anti-proliferative doses of Cpi-613 and/or getting monoclonal cell lines (shCtrl and shPDHA1); wound healing, invasion and drug sensitivity tests were also applied. To demonstrate the EMT phenotype in cells, EMT-related protein expressions were analysed via western blotting in cell fractionations. Genes that regulate the activity of PDH, were also checked by qRt-PCR. Cell cycle specifity of Cpi-613 was also investigated. Glycolysis and/or survival-related proteins and cancer stem cell characteristics were also determined.SB431542 (TGF-βRI inhibitor) was also used to determine Cpi-613 induced EMT depends on TGF-β signalling. Results and discussions Inhibition of PDH by Cpi-613 and PDHA1 knockdown induced morphological changes which are characteristics of EMT. A more rapid wound healing, increased invasive potential and cemoresistance were also shown. The effect of Cpi-613 was non-specific for cell cycle. The possible inactivating kinase of PDH was found to be PDK4. SB431542 treatment also reversed the EMT phenotype. In shPDHA1 cells the expressions of GLUT1; MDR1 and MRP1; AKT, PI3K, MAPK; and OCT-4 proteins found to be increased. The proportion of CD133+ cells were also higher in shPDHA1 cells than those of shCtrl cells. Conclusion Knockdown of PDHA1 expression or inhibition of PDH activity induced the EMT phenotype and more importantly resulted in resistance to anticancer drugs. The induction of PDH enzyme activity could be a novel approach for the development of anti-metastatic cancer drugs.

PO-289 Dual inhibition of WNT/β-catenin signalling and histone deacetylation as a new strategy to eliminate breast cancer stem cells by augmentation of apoptosis

Introduction Epigenetic changes play a critical role in the regulation of cancer stem cell (CSC) properties and the development of drug resistance. Modulation of histone acetylation program is closely related to differentiation and apoptosis process. CSCs, a subset of tumour cells, are responsible for disease relapse because of an acquired resistance to apoptosis and the Wnt signalling which is associated with cell survival/self-renewal and differentiation, is re-activated in these cells. Therefore, in the present study, we focused on a possible cytotoxic/apoptotic effect of the combination of niclosamide (Wnt/β-catenin pathway inhibitor) and Valproic acid (VPA, histone deacetylase inhibitor) on breast CSCs. Material and methods The effect of niclosamide (1 µM, 24 hour pre-treatment) and VPA (0.63–5 mM) combination on the viability of MCF-7s cells (CSCs-enriched population) were demonstrated by the ATP assay. Acetylated histone H3 levels at selected doses for the combination were assessed by ELISA. Protein levels associated with the Wnt/β-catenin signalling pathway, EMT, and histone modifications were shown by western blotting. Cell death mode was investigated via Hoechst 33342/PI double staining, M30 ELISA, real-time PCR (gene levels associated with apoptosis and autophagy) and western blotting (protein levels associated with apoptosis, autophagy and ER stress). Results and discussions We found that combination therapy exhibited a marked decrease in cell viability by inducing extrinsic apoptosis along with the stronger Wnt inhibition and increased histone H3 acetylation in MCF-7s cells. Furthermore, it was found that mesenchymal markers (fibronectin, N-cadherin, and ZEB1) were decreased at 72 hour and cytokeratin 18 as an epithelial marker was re-expressed in which H3K9ac and H3K4me3 were also increased. In addition, ER stress and blockade of autophagic flux have also been shown to be involved in this process. Conclusion In conclusion, the future success of this combination approach in targeting CSCs and converted CSCs to non-CSCs may hold significant promise for successful treatment of breast cancer.

Pelargonium quercetorum Agnew induces apoptosis without PARP or cytokeratin 18 cleavage in non‑small cell lung cancer cell lines

Pelargonium species have various uses in folk medicine as traditional remedies, and several of them have been screened for their biological activity, including anticancer. Pelargonium quercetorum Agnew (P. quercetorum) is traditionally used for its anthelminthic activity. However, little is known about its biological activity or its effect on cancer cells. The aim of the present study was to determine the cytotoxic activity of P. quercetorum extract on lung cancer cell lines with varying properties. Following the analyses of its chemical composition, the cytotoxic activity was screened by the adenosine triphosphate viability test. M30-Apoptosense® and M65 EpiDeath® enzyme-linked immunosorbent assays were used to determine the cell death mode (apoptosis vs. necrosis). For apoptosis, additional methods, including Annexin-V-fluorescein isothiocyanate (FITC) and Hoechst 33342 staining, were employed. The cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) was assayed by western blotting to further dissect the apoptosis mechanism. The methanol extract of P. quercetorum caused cytotoxic activity in a dose-dependent manner. The mode of cell death was apoptosis, as evidenced by the positive staining of the cells for Annexin-V-FITC and the presence of pyknotic nuclei. Notably, neither PARP cleavage nor cytokeratin 18 fragmentation were observed. P.quercetorum caused cell death by an apoptosis mechanism that is slightly different from classical apoptosis. Therefore, future in vivo experiments are required for further understanding of the effect of this plant on cancer cells.