Identification of exosomal microRNAs and related hub genes associated with imatinib resistance in chronic myeloid leukemia.

Chemotherapy resistance is a major obstacle in cancer therapy, and identifying novel druggable targets to reverse this phenomenon is essential. The exosome-mediated transmittance of drug resistance has been shown in various cancer models including ovarian and prostate cancer models. In this study, we aimed to investigate the role of exosomal miRNA transfer in chronic myeloid leukemia drug resistance. For this purpose, firstly exosomes were isolated from imatinib sensitive (K562S) and resistant (K562R) chronic myeloid leukemia (CML) cells and named as Sexo and Rexo, respectively. Then, miRNA microarray was used to compare miRNA profiles of K562S, K562R, Sexo, Rexo, and Rexo-treated K562S cells. According to our results, miR-125b-5p and miR-99a-5p exhibited increased expression in resistant cells, their exosomes, and Rexo-treated sensitive cells compared to their sensitive counterparts. On the other hand, miR-210-3p and miR-193b-3p were determined to be the two miRNAs which exhibited decreased expression profile in resistant cells and their exosomes compared to their sensitive counterparts. Gene targets, signaling pathways, and enrichment analysis were performed for these miRNAs by TargetScan, KEGG, and DAVID. Potential interactions between gene candidates at the protein level were analyzed via STRING and Cytoscape software. Our findings revealed CCR5, GRK2, EDN1, ARRB1, P2RY2, LAMC2, PAK3, PAK4, and GIT2 as novel gene targets that may play roles in exosomal imatinib resistance transfer as well as mTOR, STAT3, MCL1, LAMC1, and KRAS which are already linked to imatinib resistance. MDR1 mRNA exhibited higher expression in Rexo compared to Sexo as well as in K562S cells treated with Rexo compared to K562S cells which may suggest exosomal transfer of MDR1 mRNA.

Effect of Probucol on Proliferation of Leukemia, Multiple Myeloma, Lymphoma, and Fibroblast Cells.

OBJECTIVES: Probucol is a bisphenol antioxidant with antiinflammatory, antilipidemic and antidiabetic effect. Development and progression of cancer is closely related to chronic inflammation and oxidative stress. Agents that target these processes have been shown to modulate cancer cell proliferation. In this regard, the effect of probucol on proliferation of different cancer cell lines was investigated. MATERIALS AND METHODS: Different concentrations of probucol solutions were prepared and applied to the following cancer cell lines: K562S (imatinib sensitive) and K562R (imatinib resistant) chronic myeloid leukemia (CML) cells; U937 histiocytic lymphoma cells; HL60 acute myeloid leukemia cells; U266, H929, and RPMI8226 multiple myeloma cells; and L929 fibroblast cells. Cell viability was conducted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: Significant toxicity was not exhibited due to probucol treatment (0.1-10 µM) in K562S and K562R CML cells, U937 histiocytic lymphoma cells, HL60 acute myeloid leukemia cells, U266 multiple myeloma cells, and L929 fibroblast cells. However, probucol treatment significantly inhibited the viability of H929 and RPMI8226 multiple myeloma cells at the concentration of 0.5-10 µM and 5-10 µM, respectively. CONCLUSION: Probucol treatment slightly inhibited the viability of other cancer cell lines, but significantly inhibited the viability of H929 and RPMI8226 multiple myeloma cells. However, its effect was not potent, since a 50% reduction in cell viability could not be achieved at the concentrations of probucol treatment administered.

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

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 ß-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 ß-catenin in imatinib resistance of CML and to test if any direct physical protein-protein interaction exists between NFkB and both ß-catenin and Akirin-2. METHODS: RT-PCR and western blot were performed to determine Akirin-2, NFκB-p65 and ß-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 ß-catenin proteins to MDR1 promoter region, respectively. RESULTS: ß-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 ß-catenin proteins. Nuclear ß-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 ß-catenin genes may provide an opportunity to overcome imatinib resistance in CML.

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.

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.

Inhibitory effects of indole α-lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages.

Alpha-lipoic acid (α-lipoic acid) is a potent antioxidant compound that has been shown to possess anti-inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL-1ß, IL-6 and TNF-alpha upon activation with LPS (Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α-lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α-lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I-4b, I-4e and II-3b. In conclusion, these indole α-lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production.

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.

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.

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.

Syntheses of novel indole lipoic acid derivatives and their antioxidant effects on lipid peroxidation.

The aim of the study was to examine antioxidant properties of conjugates based on indole and lipoic acid moieties. The design and syntheses of novel indole alpha-lipoic acid derivatives were performed. The antioxidant properties of target compounds were investigated using rat liver microsomal, NADPH-dependent lipid peroxidation inhibition. Some of the target compounds, especially those containing amide linker at position 5 of indole ring, proved to be highly effective in inhibiting lipid peroxidation as compared to alpha-lipoic acid.

Effect of cobalt on the oxidative status in heart and aorta of streptozotocin-induced diabetic rats.

Effects of cobalt on the antioxidant status of control and streptozotocin diabetic rat heart and aorta were examined at the second, fourth and sixth week of treatment. Rats were divided into four groups: control, diabetic, control treated with cobalt chloride and diabetic treated with cobalt chloride. Diabetes was induced by tail vein injection of streptozotocin (STZ). Cobalt treatment groups were given 0.5 mM of CoCl(2) in drinking water. The rats in both groups were further subdivided into three groups of six rats each. Rats in these subgroups were studied at 2-week intervals up to 6 weeks. At the end of the experiment, all animals were sacrificed by decapitation, heart and aorta samples were removed for determination of thiobarbituric acid reactive substance (TBARS) level and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities. It was found that lipid peroxidation levels and antioxidant enzyme activities were increased in the streptozotocin-induced diabetic rats at all times studied. Cobalt treatment of diabetic rats (0.5 mM in drinking water) resulted in attenuation of the increased levels of TBARS and antioxidant enzyme activities in heart and aorta. Thus, the effect of oral administration of cobalt at this dose during the early stage of experimental diabetes can be considered as a consequence of altered endogenous defence mechanisms in heart and aorta.

May nitric oxide molecule have a role in the pathogenesis of human cataract?

Recent studies have shown that nitric oxide molecule may have a role in the development of cataract. In this study, we measured the levels of a nitric oxide metabolite (nitrite) in the cataractous and normal human lenses. A modified Griess assay was used to determine the nitrite levels in the lenses as a measure of nitric oxide, based on the spectrophotometric method. Nitrite was detected in 26 (44.1%) cataractous lenses and was found below the detection limit in 33 (55.9%) cataractous lenses. Mean nitrite levels in cataractous lenses (2.77+/-5.26nmol/100mg) was found higher than the normal lenses (0.77+/-0.79nmol/100mg) but this increase was not statistically significant. Comparison of nitrite levels among lenses with various types of cataracts revealed higher levels in lenses with posterior subcapsular cataracts. Hypertensive patients had also significantly higher nitrite levels in their cataractous lenses. The increased levels in the cataractous lenses display a possible role of nitric oxide in the pathogenesis of cataract in human eyes.

Effects of supplementation with a combination of cobalt and ascorbic acid on antioxidant enzymes and lipid peroxidation levels in streptozocin-diabetic rat liver.

The effect of cobalt(II) chloride (CoCl2) and CoCl2 with ascorbic acid (AA) on components of the antioxidant defense system and lipid oxidative damage were studied in controls and streptozotocin-induced diabetic rat livers. Three days after injection, rats received either 0.5 mM CoCl2 or 0.5 mM CoCl2 with a combination of 1 g/L AA in drinking water up to 6 wk. The elevated blood glucose levels in diabetic rats were about 12% restored by oral administration of CoCl2 (0.05 mM) and were significant reduced (46%) following AA addition (1 g/L) to CoCl2. Cobalt therapy effectively decreased the increased activities of catalase (CAT), superoxide dismutase (SOD), and thiobarbituric acid reactant substances (TBARS) but could not restore the increased glutathione peroxidase (GSH-Px) in the liver of diabetic rats. Our findings suggest that cobalt therapy may prove effective in improving the impaired antioxidant status during the early state of diabetes, and ascorbic acid supplementation at this dose potentiates the effectiveness of cobalt action.