Gökhan Kuş

Indomethacin based new triazolothiadiazine derivatives: Synthesis, evaluation of their anticancer effects on T98 human glioma cell line related to COX-2 inhibition and docking studies.

In the current work, new 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives (1-8) were synthesized via the ring closure reaction of 2-bromoacetophenone derivatives with 4-amino-5-[(5-methoxy-2-methyl-1-(4-chlorobenzoyl)-1H-indol-3-yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-thione, which was obtained via the solvent-free reaction of indomethacin with thiocarbohydrazide. MTT assay was carried out to determine the cytotoxic effects of the compounds on T98 human glioma cell line. Among these compounds, 3-[5-methoxy-2-methyl-1-(4-chlorobenzoyl)-1H-indole-3-yl)methyl]-6-(4-methylphenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine (8) was found to be the most effective compound and therefore flow cytometric method was performed to investigate the apoptotic effect of compound 8. The apoptosis stimulating percentages of compound 8 in comparison with the control group at 50 and 100 μM doses were calculated as 11% and 12%, respectively. Besides, real-time PCR assay was carried out to determine the effects of compound 8 on COX-2, caspase 3, 8 and 9, cytochrome c mRNA levels. According to the real-time PCR analysis, compound 8 reduced COX-2 mRNA levels significantly when compared with the control group, whereas the compound did not cause any significant change in other parameters (Caspase 3, 8, 9, cytochrome c). The docking study suggested that the COX-2 inhibitory effects of compound 8 and indomethacin were similar in the catalytic active site of COX-2. These results indicated that compound 8 showed dose-dependent anticancer activity via the inhibition of COX-2 pathway.

The role of hemostatic mechanisms in the development of thrombosis in Behcet's disease: an analysis by modified rotation thromboelastogram (ROTEM)

Behcets disease (BD) is a multisystemic disorder characterised by recurrent oral and genital ulcers. Vasculitis and thrombotic events are the most important causes of mortality. Thrombosis is the major clinical finding in patients with BD, but the cause of the thrombosis is still unclear. Thromboelastography is an alternative method to evaluate almost all steps of the hemostatic system. Today, the modified rotation thromboelastogram (ROTEM) is a newer coagulation test and a more powerful technique. Our aim in this study was to investigate whether hemostatic mechanisms play a role in the development of thrombosis in BD patients by using ROTEM. Thirty BD patients, 20 ankylosing spondylitis patients, and 14 healthy controls who are not taking anti-aggregant or anti-coagulant therapy were included in the study. Whole blood count, protrombin time, activated protrombin time, fibrinogen, D-dimer levels, and ROTEM parameters (clotting time, clot formation time (CFT), and maximum clot formation(MCF)) were determined by INTEM and EXTEM analysis. Of the 30 patients with BD, 19 were women and 11 were men, and mean age was 40.6 ± 11.2. Two of the BD patients had vascular involvement, but none of them were in active phase of the disease during the study. In INTEM assay, MCF (p < 0,001) value was significantly increased, and CFT (p>0.05) value was decreased in BD patients compared with the control group. In the EXTEM assay, there was a similar significant increase in MCF (p=0.002) value and a decrease in CFT (p=0.002) value in BD patients compared with the control group. The results of our study indicated that primary hemostatic mechanisms which can be detected by ROTEM may play a role in the development of thrombosis in patients with BD.

Examining impacts of ceranib-2 on the proliferation, morphology and ultrastructure of human breast cancer cells

Acid ceramidases are enzymes with a vital role in metabolizing ceramide to sphingosine-1-phosphate that is an antiproliferative metabolite in the ceramide pathway. Inhibition of exogenous ceramides with ceramidase inhibitors lead to augmented ceramide levels in cells and in turn lead to cell cycle arrest and apoptosis. Our study aimed at targeting ceramide metabolic pathway to induce apoptosis in human breast cancer cell line (MCF7) and we examined the antiproliferative and apoptotic activities of ceranib-2, an inhibitor of human ceramidase, on this cell line as well ultrastructural and mophological changes. Methods used for our examinations in this study were the colorimetric MTT assay, Annexin V/Propidium iodide and JC-1 staining, transmission electron microscopy and confocal microscopy. Ceranib-2 effectively inhibited the viability of MCF7 cells in 24 h in a dose dependent manner leading to apoptosis via the mitochondrial pathway by reducing the potential of mitochondrial membrane. Additionally, significant changes on cell morphology and ultrastructure were observed on MCF7 cells exposed to ceranib-2 indicating apoptotic cell death. Collectively, our data demonstrate that ceranib-2 exerts a great potential to be an antineoplastic compound and that the mechanism of its action rely on its apoptosis inducing ability.

Licofelone abolishes survival of carcinogenic fibroblasts by inducing apoptosis.

Abstract Dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX) pathways of arachidonic acid metabolism prevent cancer development and induce apoptosis. One of the most promising compounds that blocks both of these pathways is licofelone. We questioned whether licofelone affects the survival and/or promotes apoptosis of H-ras transformed rat embryonic fibroblast (5RP7) cells in vitro. Using 5-fluorouracil (5-FU) and colchicine as positive controls, we determined cell viability with 3-3-(4,5-D-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thyazolyl blue (MTT), apoptosis with flow cytometry and activity of caspase enzyme with real-time reverse transcription polymerase chain reaction (PCR). Compared to the control, all used six doses (10, 50, 100, 150, 250 and 250 µM) of 5-FU, colchicine and licofelone, which were cytotoxic and reduced the number of H-Ras transformed 5RP7 cells by as much as 78, 72 and 92%, respectively. In addition, we found that 150, 200 and 250 µM of licofelone induced apoptosis and necrosis of H-Ras transformed 5RP7 cells in a dose- and time-dependent manner. Each three tested drugs at 250 µM also increased the level of caspase-3 enzyme up to 5-fold. Although colchicine was effective in inducing early apoptosis, licofelone had much more capacity to induce the total of early plus late apoptosis by approximately 96% in cells after 48 hours. The present study reveals the possibility that licofelone posseses strong dose- and time-dependent anticancer and apoptotic properties on carcinogenic fibroblasts.

Cytotoxic and apoptotic functions of licofelone on rat glioma cells

Gliomas are the largest group of central nervous system tumors and despite of clinical treatments death rate is very high. Inhibition of both cyclooxygenase and lipoxygenase pathways that take role in arachidonic acid metabolism prevents cancer development and induces apoptosis. One of the most promising compounds that blocks both of these pathways is licofelone. Using colchicine and 5-fluorouracil as positive controls, we questioned whether licofelone affects the survival of rat glioma cell line (C6) and induces apoptosis in vitro. After growing the cells in culture, we determined viability with MT, apoptosis with flow cytometry and activity of caspase enzymes with real time PCR. All used doses of colchicine and 5-fluorouracil were cytotoxic and reduced the number of surviving C6 cells as much as 44% and 60%, respectively. Comparing to the control, treatments with 10, 50 and 100 μM licofelone for 24 or 48 h did not influence C6 survival, however, 150, 200 and 250 μM licofelone reduced the number of living cells by 58, 88 and 93%, respectively, and induced apoptosis of C6 cells in a dose and time dependent manner. Licofelone did not change the level of caspase-9, but increased the level of caspase-3. Comparing with 5-fluorouracil and colchicine, the present study reveals for the first time the possibility that licofelone possesses a strong dose and time dependent antiproliferative and proapoptotic properties on glioma cells.

Erythropoietin Protects the Kidney by Regulating the Effect of TNF-α in L-NAME-Induced Hypertensive Rats

Background/Aims: Hypertension is the leading cause of death worldwide. Chronic high blood pressure induces inflammation. Tumor necrosis factor (TNF)-α plays a major role in inflammation and also depresses the synthesis of erythropoietin, which exerts protective effects on tissue; however, the mechanism is still unclear. We investigated the protective effect of erythropoietin against tissue damage caused by hypertension in the kidney and whether this effect was suppressed by TNF-α. Methods: First, we detected the optimum chronic dose for darbepoetin-α (Depo), which is a long-acting erythropoietin analog for rats. We separated 60 female adult rats into 6 groups: control, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), L-NAME+Depo, L-NAME+Remicade (an anti-TNF-α antibody), L-NAME+Depo+Remicade, Depo, and control. After 1 month of treatment, we measured cardiovascular parameters, took blood samples, sacrificed the rats, and removed kidneys for analyses. Results: The apoptotic index and the plasma and kidney mRNA levels of TNF-α increased in the L-NAME group and decreased in all other treatment groups. Macrophage accumulation increased in the L-NAME and L-NAME+Remicade groups, while it decreased in the Depo group. The mRNA abundance of TNF receptor 1 (TNFR1) decreased slightly in the Depo group and TNFR2 increased significantly in the same group. Conclusion: Erythropoietin protects kidney tissue against hypertension by preventing the apoptotic effects of TNF-α by blocking macrophage accumulation, decreasing TNF-α levels, and switching the TNF-α receptors from the apoptotic receptor TNFR1 to the proliferative receptor TNFR2.