Tulin Ozkan

Effect of L-carnitine on the synthesis of nitric oxide in RAW 264·7 murine macrophage cell line

L‐Carnitine (β‐hydroxy‐γ‐trimethyl aminobutyric acid) plays a critical role in inflammatory diseases by modulating inflammatory cell functions. Inducible nitric oxide synthase (iNOS), a proinflammatory enzyme responsible for the generation of nitric oxide (NO), has been implicated in the pathogenesis of inflammatory diseases. Mechanism of action of L‐carnitine on inflammation via iNOS and nuclear factor κB (NF‐κB) is unclear. In this study, we aimed to investigate the effect of L‐carnitine on nitric oxide synthesis in lipopolysaccharide (LPS)‐stimulated RAW 264·7 macrophage cells. For this purpose, cells were pretreated with various concentrations of L‐carnitine and subsequently incubated with LPS (1 µg·ml−1). NO levels, iNOS protein expression, and NF‐κB activity were determined using colorimetric detection, Western blotting and transfection assays. Our results showed that treatment with L‐carnitine suppressed nitric oxide production, iNOS protein expression and NF‐κB activity. We demonstrated that inhibitory effect of L‐carnitine on iNOS protein expression is at transcriptional level. This study may contribute to understanding the anti‐inflammatory effect of L‐carnitine. Copyright

Methylation analysis of the DAPK1 gene in imatinib-resistant chronic myeloid leukemia patients

Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation-specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non-resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR-ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non-resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.

Effect of lomeguatrib–temozolomide combination on MGMT promoter methylation and expression in primary glioblastoma tumor cells

Temozolomide (TMZ) is commonly used in the treatment of glioblastoma (GBM). The MGMT repair enzyme (O6-methylguanine-DNA methyltransferase) is an important factor causing chemotherapeutic resistance. MGMT prevents the formation of toxic effects of alkyl adducts by removing them from the DNA. Therefore, MGMT inhibition is an interesting therapeutic approach to circumvent TMZ resistance. The aim of the study was to investigate the effect of the combination of lomeguatrib (an MGMT inactivator) with TMZ, on MGMT expression and methylation. Primary cell cultures were obtained from GBM tumor tissues. The sensitivity of primary GBM cell cultures and GBM cell lines to TMZ, and to the combination of TMZ and lomeguatrib, was determined by a cytotoxicity assay (MTT). MGMT and p53 expression, and MGMT methylation were investigated after drug application. In addition, the proportion of apoptotic cells and DNA fragmentation was analyzed. The combination of TMZ and lomeguatrib in primary GBM cell cultures and glioma cell lines decreased MGMT expression, increased p53 expression, and did not change MGMT methylation. Moreover, apoptosis was induced and DNA fragmentation was increased in cells. In addition, we also showed that lomeguatrib–TMZ combination did not have any effect on the cell cycle. Finally, we determined that the sensitivity of each primary GBM cells and glioma cell lines to the lomeguatrib–TMZ combination was different and significantly associated with the structure of MGMT methylation. Our study suggests that lomeguatrib can be used with TMZ for GBM treatment, although further clinical studies will be needed so as to determine the feasibility of this therapeutic approach.

Synthesis and anticancer effects of some novel 2-(4-phenoxyphenyl)-1H-benzimidazole derivatives on K562 cell line

A series of novel 2-(4-phenoxyphenyl)-1H-benzimidazole derivatives was synthesized and tested in vitro on human chronic myelogenous leukemia (CML) cell line K562. Benzimidazoles containing 5-amidino (10), 5-N-isopropylamidino (11), 5-bromo (13), and 5,6-dimethyl (14) derivatives exhibited remarkable cytotoxic activity. The quantitative analysis of apoptosis by flow-cytometry demonstrated that the percentages of early and late apoptotic K562 cells treated with these compounds were significantly higher than cells without treatment. We also investigated the effects of these compounds on the expression of apoptosis-related genes BAX, BCL-2, BAD and BIM. Treatment of K562 cells wih compounds 10–14 significantly increased the expression levels of the proapoptotic genes BAX, BAD and BIM, whereas compound 20 increased BAX and BAD.

Methylsulfonylmethane Induces p53 Independent Apoptosis in HCT-116 Colon Cancer Cells

Methylsulfonylmethane (MSM) is an organic sulfur-containing compound which has been used as a dietary supplement for osteoarthritis. MSM has been shown to reduce oxidative stress and inflammation, as well as exhibit apoptotic or anti-apoptotic effects depending on the cell type or activating stimuli. However, there are still a lot of unknowns about the mechanisms of actions of MSM. In this study, MSM was tested on colon cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis revealed that MSM inhibited cell viability and increased apoptotic markers in both HCT-116 p53 +/+ and HCT-116 p53 −/− colon cancer cells. Increased poly (ADP-ribose) polymerase (PARP) fragmentation and caspase-3 activity by MSM also supported these findings. MSM also modulated the expression of various apoptosis-related genes and proteins. Moreover, MSM was found to increase c-Jun N-terminal kinases (JNK) phosphorylation in both cell lines, dose-dependently. In conclusion, our results show for the first time that MSM induces apoptosis in HCT-116 colon cancer cells regardless of their p53 status. Since p53 is defective in >50% of tumors, the ability of MSM to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy. Moreover, the remarkable effect of MSM on Bim, an apoptotic protein, also suggests its potential use as a novel chemotherapeutic agent for Bim-targeted anti-cancer therapies.

The roles of epigenetic modifications of proapoptotic BID and BIM genes in imatinib-resistant chronic myeloid leukemia cells.

Abstract In chronic myeloid leukemia (CML), epigenetic modifications such as promoter hypermethylation and inactive histone modification are known mechanisms of drug resistance. In our study, we investigated the roles of promoter hypermethylation of BIM and BID genes and H3K27me3 histone modification on imatinib resistance. We detected higher expression levels of BIM and BID genes and lower expression levels of EZH2, EED2, SIRT1, and SUZ12 genes in imatinib-resistant K562/IMA-3 cells compared to imatinib-non-resistant K562 cells. While we determined the EZH2 and DNMT enzymes as bounded to the promoter of the BIM gene, we did not detect hypermethylation of this promoter. We also found the H3K27me3 histone modification promoter of BIM and BID genes in both cell lines. In conclusion, our results support the notion that DNA promoter methylation may be formed independently from EZH2-H3K27me3 and pro-apoptotic BIM and BID genes are not methyllated in the imatinib resistance of CML cells.

Synthesis and cytotoxic evaluation of novel N-substituted amidino-1-hydroxybenzimidazole derivatives.

A new class of N-substituted amidino-1-hydroxybenzimidazole derivatives (15–24) were synthesized and evaluated for their in vitro cytotoxic activities against human leukemia cell lines, HL-60 and K562. The preliminary results showed that compounds 16, 20, 21 and 23 had moderate antitumor activity against HL-60 cell line. Further investigation on the mechanism of the observed cytotoxic effects demonstrated that compound 21 increased the expression of autophagic and apoptotic genes and induced apoptosis of HL-60 cells.

Determination of the apoptotic effect and molecular docking of benzamide derivative XT5 in K562 cells

BACKGROUND The tyrosine kinase inhibitor, imatinib, used as a first line treatment in Chronic Myeloid Leukemia (CML) patients, may lead to resistance and failure to therapy. Novel combinations of imatinib with other drugs is a strategy to improve treatment efficiency. OBJECTIVE In this study, the antileukemic and apoptotic effects of a benzamide derivative XT5 and benzoxazole derivative XT2B and their combination with imatinib were investigated in imatinib-sensitive (K562S) and imatinib-resistant (K562R) CML cells. METHODS In vitro cytotoxicity was determined by MTT assay. Then, apoptotic effect of XT5 on CML cell lines was tested by Annexin V flow cytometry, caspase activation and RT-PCR. Docking calculation was performed using AutoDock Vina in PyMOL environment using AutoDock/Vina plugin for PyMOL. RESULTS According to our MTT assay data, XT5 indicated significant antiproliferative effect on cell lines, therefore we investigated apoptotic effects of XT5. Treatment of K562 cell lines with a combination of XT5 and imatinib-XT5 increased cytotoxicity, the Annexin V binding and caspase 3/7 activation. In addition to apoptosis assays, we observed an increase in the expression levels of the pro-apoptotic (BAX, BAD and BIM) genes in XT5 treated K562R and K562S cells. Molecular modelling experiments showed that XT5 showed hydrogenbonding interactions with important amino acids of BCR-ABL kinase receptor; however XT2B did not show any hydrogen bond interaction. CONCLUSION Our results indicate that XT5 could be a potential candidate to be used as a new anticancer drug and XT5 combination with imatinib as an alternate treatment strategy for overcoming imatinib resistance.

Apoptotic Effects of Some Tetrahydronaphthalene Derivatives on K562 Human Chronic Myelogenous Leukemia Cell Line

BACKGROUND Retinoids which are vitamin A (Retinol) derivatives have been suggested to mediate the inhibition of cancer cell growth and apoptosis. It has been reported that all trans retinoic acid (ATRA) exhibited suppressive effects on different types of leukemia including chronic myelogenous leukemia. OBJECTIVE In the present study, we aim to find out the effects of 6 synthetic N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalene-2-yl)-carboxamide derivatives (compound 6-12) on cell viability and apoptotic pathways in K562 human chronic myelogenous leukemia cell line. METHODS Cell viability and apoptosis were examined by spectrophotometric thiazolyl blue tetrazolium bromide (MTT) and caspase-3 assay, western blot, RT-PCR and flow cytometry. RESULTS Our results indicated that compound 6 (5-(1,2-Dithiolan-3-yl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)pentanamide), 8 (4-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)butanamide) and 11 (E-3-(4-Hydroxy-3-methoxyphenyl)-N-(3,5,5,8,8-pentamethyl- 5,6,7,8-tetrahydronaphthalen-2-yl)acrylamide) exhibited apoptotic effects in K562 human chronic myelogenous leukemia cell line and induced caspase 3, PARP cleavage, Bax/Bcl-2 ratio, Bad and Bim gene expressions. CONCLUSION Some retinoid derivatives tested in this study induced apoptosis of K562 cells which suggest that these compounds may serve as potential agents in the treatment of chronic myelogenous leukemia.

Synthesis and antiproliferative evaluation of novel 5-(4-methylpiperazin-1-yl)-2-phenyl- 1H-benzimidazole derivatives.

Abstract A series of novel 5-(4-methylpiperazin-1-yl)-2-phenyl-1H-benzimidazoles (5–14) were synthesized and evaluated for their in vitro antiproliferative activities against the human leukemia cell line HL-60. Compounds 5–7 and 10–12 exhibited potent antiproliferative activities against this cell line. The quantitative analysis of apoptosis by flow cytometry demonstrated that the percentages of apoptotic HL-60 cells treated with compounds 5 and 10–12 were significantly higher than in the control.