Arzu Zeynep Karabay

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

Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7 macrophage-like cells by targeting p53, Bax, Bcl-2, cytochrome c and PARP proteins

Abstract Methylsulfonylmethane (MSM) is a non-toxic, natural organosulfur compound, which is known to possess antioxidant and anti-inflammatory activities. In recent years, MSM has been widely used as a dietary supplement for its beneficial effects against various diseases, especially arthritis. Despite being a popular supplement product, the mechanism of action of MSM is not well known. This study was designed to investigate the effects of MSM on cytotoxic signals induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in RAW 264.7 macrophage-like cells. The results showed that MSM reversed apoptosis of RAW 264.7 macrophage-like cells at non-cytotoxic concentrations probably through the modulation of apoptotic proteins. After pre-treatment of cells with non-toxic doses of MSM; caspase-3 activation, p53 accumulation, cytochrome c release and Bax/Bcl-2 ratio were significantly decreased and full length poly ADP-ribose polymerase (PARP) was significantly increased. In addition, the loss of mitochondrial membrane potential was decreased with MSM pretreatment in activated macrophages. Since excess nitric oxide production causes apoptosis of macrophages, anti-apoptotic effects of MSM are thought to be mediated by its inhibitor effects on inducible nitric oxide synthase (iNOS) protein and nitric oxide levels. More interestingly, higher doses of MSM exhibited biphasic effects, inhibited cell viability, induced apoptosis of macrophages, increased caspase-3 activity and PARP cleavage. Thus, our results reveal the molecular mechanism of of MSM indicating that MSM supplementation may be beneficial for complications related to nitric oxide-dependent apoptosis in inflammatory conditions. However, the optimum concentration of MSM must be chosen carefully to elicit the desired effect.

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.

Synthesis and effects of some novel tetrahydronaphthalene derivatives on proliferation and nitric oxide production in lipopolysaccharide activated Raw 264.7 macrophages.

In this study, novel N-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene-2-yl)-carboxamide (6-15) and 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalene-2-carboxamide (16-32) derivatives were synthesized and their in vitro effects at 5 μM and 50 μM concentrations on proliferation and nitric oxide (NO*) production in lipopolysaccharide (LPS) activated RAW 264.7 macrophage cells were determined. Compounds 12, 17, 24 and 26 were found to decrease nitrite levels in a dose-dependent manner in LPS-activated cells. At the tested concentrations, these compounds did not exhibit cytotoxic effects. Interestingly, compound 27 which contains nitroxide free radical was the most active compound in this series showing 59.2% nitrite inhibition in LPS-activated macrophage cells.

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.

Food Additive Sodium Benzoate (NaB) Activates NFκB and Induces Apoptosis in HCT116 Cells

NaB, the metabolite of cinnamon and sodium salt of benzoic acid is a commonly used food and beverage preservative. Various studies have investigated NaB for its effects on different cellular models. However, the effects of NaB on cancer cell viability signaling is substantially unknown. In this study, the effects of NaB on viability parameters and NFκB, one of the most important regulators in apoptosis, were examined in HCT116 colon cancer cells. Cell culture, light microscopy, spectrophotometry, flow cytometry, and western blot were used as methods to determine cell viability, caspase-3 activity, NFκB, Bcl-xl, Bim, and PARP proteins, respectively. NaB (6.25 mM–50 mM) treatment inhibited cell viability by inducing apoptosis, which was evident with increased Annexin V-PE staining and caspase-3 activity. NFκB activation accompanied the induction of apoptosis in NaB treated cells. Inhibition of NFκB with BAY 11-7082 did not show a pronounced effect on cell viability but induced a more apoptotic profile, which was confirmed by increased PARP fragmentation and caspase-3 activity. This effect was mostly evident at 50 mM concentration of NaB. Bcl-xl levels were not affected by NaB or BAY 11-7082/NaB treatment; whereas, total Bim increased with NaB treatment. Inhibition of NFκB activity further increased Bim levels. Overall, these results suggest that NaB induces apoptosis and activates NFκB in HCT116 colon cancer cells. Activation of NFκB emerges as target in an attempt to protect cells against apoptosis.

Expression analysis of Akirin-2, NFκB-p65 and β-catenin proteins in imatinib resistance of chronic myeloid leukemia

ABSTRACT Objective: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and β-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and β-catenin in imatinib resistance of CML and to test if any direct physical protein–protein interaction exists between NFkB and both β-catenin and Akirin-2. Methods: RT–PCR and western blot were performed to determine Akirin-2, NFκB-p65 and β-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and β-catenin proteins to MDR1 promoter region, respectively. Results: β-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein–protein interaction could not be found between NFκB-p65 and Akirin-2 or β-catenin proteins. Nuclear β-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells. Discussion: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and β-catenin genes may provide an opportunity to overcome imatinib resistance in CML.

Effects of glucosamine on LPS/IFN-γ induced RAW 264.7 macrophage apoptosis

Amac: Apoptoz, doku homeostazisinin duzenlenmesi gibi normal fizyolojide ve ceșitli hastaliklarin patofizyolojisinde onemli rol oynayan genetik olarak programlanmiș bir hucre olum mekanizmasidir. Ceșitli hucre tiplerinde mikrobiyal ve viral patojenlere karși bir savunma mekanizmasi olarak ortaya cikan iNOS enzim aktivasyonu ve nitrik oksit sentezi, ateroskleroz, romatoid artrit, diyabet, septik șok, multipl sklerozis gibi inflamatuvar ve immun patolojilerde onemli rol oynamakta, kontrolsuz yuksek duzeydeki nitrik oksit uretimi hucre olumune yol acmaktadir. Yakin zamanda, osteoartrit gibi inflamatuvar bozukluklar icin yararli oldugu iddia edilen glukozamin gunumuzde klinik olarak kullanilmaktadir. Glukozaminin, bașlica immun sistem hucreleri olan makrofajlarin apoptozu uzerine olan etki mekanizmasi hakkinda sinirli bilgi bulunmaktadir. Bu calișmada, farkli konsantrasyonlardaki glukozaminin, LPS/IFNγ ile aktive edilmiș RAW 264.7 makrofajlar uzerindeki etkileri belirlenmiștir. Materyal ve metod: RAW 264.7 makrofaj hucre serisi, LPS/IFNγ ile uyarim oncesinde glukozamin varliginda ve yoklugunda kultur edildi. Nitrit duzeyleri, hucre canliligi, kaspaz-3 aktivitesi, mitokondri membran potansiyeli tayini ve Annexin V-PI hucre boyamasi ile akim sitometrik analizler gercekleștirilmiștir. Sonuclar ve tartișma: Glukozaminin, nitrit duzeylerini inhibe ettigi, mitokondri membran potansiyelini arttirdigi, kaspaz-3 enzim aktivitesini azalttigi ve anlamli olarak antiapoptotik etkiler sergiledigi (p