Influence of morphine on TLR4/ NF-kB signaling pathway of MCF-7 cells.

Morphine affects the risk of metastasis in cancer. The TLR4 gene promotes migration in adenocarcinoma cells. We investigated the effect of morphine on TLR4, MyD88 and NF-κ B-expression and migration. Migration of estrogen receptor-positive MCF7 breast cancer cells was studied after 24 and 48 hours incubation with morphine, with boyden chamber method. Morphine effect on TLR4, MyD88 and NF-κB mRNA expression was determined by quantitative Real-Time polymerase chain reaction. Migration was reduced at the doses of 0.5 and 5 µM (p < 0.05). However, TLR4, MyD88 and NF-κBmRNA expression was decreased at the doses of 0.5, 5 and 500 µM. Morphine at the dose of 50 µM increased the expression of mentioned genes. MCF-7 cell line after 48 hours incubation with the dose of 0.5 µM morphine decreased the migration and at the dose of 0.5 µM down-regulated the mRNA expression of TLR4, MyD88 and NF-κB, however, the higher doses increased the expression of TLR4, MyD88 and NF-κB. Morphine affects TLR4expression in breast cancer cell, which depends on time and concentration (Tab. 1, Fig. 5, Ref. 24).

Effect of lipopolysaccharide on toll-like receptor-4 signals in mouse cancer cells.

BACKGROUND: Recent findings showed that activated TLR signals on cancer cells might promote cancer progression. This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on mouse melanoma and breast cancer cell proliferation and their TLR4 signalling. METHODS: Mouse melanoma cell line (B16F10) and breast cancer cell line (4T1) were taken as models. They were treated with LPS (0, 1.25, 2.5, 5, 7.5, 10 µg/ml) for 4, 16, 24, 48 h and MTT assay was done. The expression of TLR4, MyD88, NF-κB mRNA was detected by quantitative real time-polymerase chain reaction method quantitatively. RESULTS: Ultra-pure LPS at 5 µg/ml concentration increased significantly B16F10 cell viability 24 hour after stimulation, but not in 4T1 cell. The mRNA levels of TLR4, MyD88 and NF-κB were significantly up-regulated in both cell lines by stimulating the cells at 5 µg/ml LPS. CONCLUSIONS: Our data demonstrated that B16F10 and 4T1 cells are responsive to LPS, but their responses are time and dose dependent. These results provide new ways to understand the TLR4 signalling in tumour cells (Fig. 2, Ref. 24).

Gamma-secretase inhibitor does not modulate angiogenesis in colon adenocarcinoma in obese mice.

BACKGROUND: Notch is a signaling molecule which plays a role in angiogenesis and γ-secretase is required for processing of Notch. In this study, we investigated the effect of γ-secretase inhibitor (DAPT) on tumor angiogenesis in diet-induced obese mice. METHODS: 18 mice were divided into three groups; control, obese (diet-induced) and obese+DAPT. After 15 weeks, the obese mice were subjected for tumor induction of CT26 colon adenocarcinoma cells (5 x 105 cells). When the tumor size reached approximately 350 ± 50 mm3, half of the obese animals received DAPT (10mg/kg/day) subcutaneously. Blood samples were taken after 14 days and the tumors harvested for immunohistochemical staining and capillary density were reported as CD31 positive cells/mm2. RESULTS: The obese animals had higher serum leptin and NO concentrations, while, serum VEGF and VEGFR-1 concentrations were not different compare to control group. Administration of DAPT in obese mice significantly reduced serum VEGFR-1 and leptin concentrations and increased serum NO level (p < 0.05). Capillary density in the tumors of obese animals was not different compare to control groups. DAPT administration could not alter capillary density in the tumors. CONCLUSION: Administration of DAPT in obese mice altered serum angiogenic factors, however, it could not modulate tumor angiogenesis in diet-induced obese mice (Fig. 4, Ref. 26).

Impact of factor VIII and von Willebrand factor plasma levels on cerebral venous and sinus thrombosis: are they independent risk factors?

BACKGROUND: The aim of this study was to assess the plasma levels of factor VIII (FVIII), von Willebrand factor (vWF) and their association in patients with cerebral venous sinus thrombosis (CVST). METHODS: We prospectively included 25 CVST patients admitted to the university hospital and 53 voluntary subjects as a control group. FVIII and vWF were measured after 6 months when anticoagulant therapy was stopped. RESULTS: The mean FVIII and vWF levels were significantly higher in the CVST group compared to the control group (126.21 ± 54.69 vs. 91.9 ± 48.8 IU/dl; p = 0. 012; 157.05 ± 107.74 vs. 94 ± 84%; p = 0.01, respectively). Using analyses calculating the 95th percentile cut-off values, we found high levels of FVIII in patients compared to controls (29.2 vs. 5%; p = 0.01) and the odds ratio with 95% confidence interval (CI) was 7.82 (1.46, 41.6). After adjustment for vWF levels, sex and age, the risk remained significantly increased and the odds ratio with 95% CI was 10.5 (1.1, 101.4) (p = 0.041). CONCLUSION: FVIII is one of the most prevalent risk factors of CVST and may be associated with an approximately tenfold increased risk for developing CVST. This effect is independent of vWF levels; however, vWF is not an independent risk factor of CVST.

Shear-thinning and self-healing nanohybrid alginate-graphene oxide hydrogel based on guest-host assembly.

The study aims to develop a novel nanohybrid shear-thinning hydrogel with fast gelation, and variable mechanical and biological properties. This nanohybrid hydrogel was developed via self-assembly guest-host interaction between ß-cyclodextrin modified alginate (host macromere, Alg-CD) and adamantine modified graphene oxide (guest macromere, Ad-GO) and subsequent ionic crosslinking process. We found that the rheological and mechanical properties of hydrogels were controlled via macromere concentration and the host: guest macromere ratio, due to the modulation of crosslinking density and network structure. Noticeably, 12%(1:2) dual-crosslinked hydrogel (2DC12) significantly improved the strength (1.3-folds) and toughness compared to 10%(1:4) dual-crosslinked hydrogel (4DC10). Furthermore, the hydrogel erosion and cytocompatibility relied on the designed parameters. Remarkably, 2DC12 showed less than 20% weight loss after 20 days of incubation in physiological solution and more than 200% cell survival after five days. In conclusion, the nanohybrid Alg-GO hydrogel could be used as an injectable hydrogel for soft tissue engineering applications.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

BACKGROUND: The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, the mechanism underlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. METHODS: Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. RESULT: Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. CONCLUSION: Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).

Effect of Plasma-Derived Exosomes of Refractory/Relapsed or Responsive Patients with Diffuse Large B-Cell Lymphoma on Natural Killer Cells Functions.

OBJECTIVE: The purpose of this study was to investigate effect of plasma-derived exosomes of refractory/relapsed or responsive diffuse large B-cell lymphoma (DLBCL) patients on natural killer (NK) cell functions. MATERIALS AND METHODS: In this cross-sectional and experimental study, NK cells were purified from responsive patients (n=10) or refractory/relapsed patients (n=12) and healthy donors (n=12). NK cells were treated with plasma-derived exosomes of responsive or refractory/relapsed patients. We examined the expression levels of hsa-miR-155-5p, hsalet- 7g-5p, INPP5D(SHIP-1) and SOCS-1 in NK cells quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Percentages of NK cells expressing CD69, NKG2D and CD16, NK cell cytotoxicity and NK cell proliferation (using flow-cytometry) as well as interferon-gamma (IFN-γ) level in the supernatant of NK cells using ELISA were also investigated. RESULTS: We observed an increased level of hsa-miR-155-5p and a decreased level of SOCS-1 in NK cells treated with exosomes compared to untreated NK cell in healthy donors and DLBCL patients. An increase in hsa-miR-155-5p level was associated with an increased level of IFN-γ in healthy donors. The decreased levels of hsa-let-7g-5p were observed in NK cells treated with exosomes in comparison with untreated NK cells in DLBCL patients (P<0.05). There was no significant difference in the percentage of CD69+ NK cells and NKG2D+ NK cells in the absence or presence of exosomes of DLBCL patients in each group. Furthermore, we observed significant reduction of NK cell proliferation in DLBCL patients and healthy donors in the presence of exosomes of refractory/relapsed patients (P<0.05). A significant decrease was observed in cytotoxicity of NK cell in patients with DLBCL treated with exosomes of responsive patients. CONCLUSION: Our findings demonstrated adverse effect of plasma-derived exosomes of DLBCL patients on some functions of NK cell. It was also determined that low NK cell count might be associated with impaired response to R-CHOP and an increased recurrence risk of cancer.

PiggyBac as a novel vector in cancer gene therapy: current perspective.

Selection of suitable delivery system is one of the crucial aspects in gene therapy that determines the efficiency of gene therapy. The past two decades have witnessed extensive efforts for finding safe and efficient vectors to overcome the limitations of viral vectors. The utilization of DNA transposon-based vectors for gene therapy has emerged as a promising non-viral alternative. DNA 'cut-and-paste' is one of the main mechanisms of genome engineering by transposon elements. However, the lack of an efficient transposition system has limited the utilization of transposon vectors in mice and mammalian systems. PiggyBac (PB) is known as a highly efficient DNA transposon originally isolated from Trichoplusia ni as an alternative to Sleeping Beauty (SB). It has been shown that PB can be functional in various species including mammalian systems. This vector could overcome some limitations of other vectors in cancer gene therapy. Some advantages of PB include the capacity for integration into the genome and providing a stable expression, capacity to harbor 10 and 9.1 kb of foreign DNA into the host genome, without a significant reduction in their transposition activity and display non-overlapping targeting preferences. However, to advance PB to clinical applications, some obstacles still require to be overcome to improve its safety and efficiency. Hence, it seems that this vector could open new horizons in gene and cancer therapy.

Effect of Blocking of Neuropeptide Y Y2 Receptor on Tumor Angiogenesis and Progression in Normal and Diet-Induced Obese C57BL/6 Mice.

BACKGROUND: Obesity is a risk factor for some types of cancers. Angiogenesis is a necessary step in the multistage progression of tumors such as melanoma. Previous studies reported that neuropeptide Y (NPY) regulates angiogenesis by activating the Y2 receptor on endothelial cells. The present study examined the effects of the NPY Y2 receptor antagonist on tumor weight, angiogenesis and serum levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGF-R1), and nitric oxide (NO). METHODS: Twenty four male C57BL/6 mice were divided into control and obese groups. The control group was fed a normal diet whereas the obese group was fed a high fat diet. After 16 weeks, 2×10(6) B16F10 melanoma cells were injected subcutaneously into all animals. Half of the control and the obese animals received 1 µM, 100 µL/kg NPY Y2 receptor antagonist (BIIE 0246) intraperitoneally. After two weeks, the animals were sacrificed, and angiogenic factors and tumor weights and angiogenesis were analyzed. RESULTS: Tumor weight in the obese mice was higher than in the control (p<0.05). Treatment with BIIE 0246 reduced tumor weight in the obese animals (p<0.05), without effect on control group (p>0.05). Administration of an NPY Y2 receptor antagonist decreased tumor angiogenesis (evaluated as capillary density/mm2) and serum VEGF concentration in the obese group without altering serum VEGF-R1 and NO concentrations. CONCLUSIONS: Blockade of the NPY Y2 receptor suppressed tumor growth in obese mice by affecting tumor angiogenesis. Thus, it seems that NPY and its Y2 receptor antagonist might be new targets in melanoma tumor therapy.

Synthesis and pharmacology of site-specific cocaine abuse treatment agents: 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes.

A series of 2-substituted-6-amino-5-phenylbicyclo[2.2.2]octanes was synthesized and tested for inhibitor potency in [(3)H]WIN 35,428 (WIN) binding at the dopamine (DA) transporter and [(3)H]DA uptake assays. To demonstrate transporter selectivity for the compounds, inhibitor potency was also tested using [(3)H]nisoxetine and [(3)H]paroxetine binding assays for the norepinephrine (NE) and serotonin (5-HT) transporters, respectively. Synthesis was accomplished by bisannulation of the enamine derived from phenylacetaldehyde and dimethylamine with 2-cyclohexenone to give a mixture of endo- and exo-trans-6-amino-5-phenylbicyclo[2.2. 2]octan-2-ones. The separated ketones were reduced to the four diastereomeric alcohols which were converted to acetyl and benzoyl esters. The ketones, alcohols, and acetyl esters generally have low affinity for the three transporters and do not effectively inhibit the uptake of [(3)H]DA. In all cases, the benzoates show significantly greater inhibition of WIN binding compared to the corresponding ketones, alcohols, or acetate esters. One compound, (1R/S,4R/S)-6R/S-(N,N-dimethylamino)-5R/S-phenylbicyclo[2.2. 2]oct-2S/R-yl benzoate, is almost as potent as cocaine in binding to the DA transporter (IC(50) = 270 nM versus 159 nM for cocaine). The C-2 epimer, (1R/S,4R/S)-6R/S-(N, N-dimethylamino)-5R/S-phenylbicyclo[2.2.2]oct-2R/S-yl benzoate, was selective and potent in binding to the 5-HT transporter (IC(50) = 53 nM versus 1050 nM for cocaine) as compared to the DA transporter (IC(50) = 358 nM). A preliminary molecular modeling study of the benzoyl esters indicates that their relative potencies in the WIN binding assay are not correlated to the nitrogen to benzoate phenyl (centroid) distance or to the deviation of the nitrogen from the plane defined by the benzoate ring.

Role of peroxisome proliferator-activated receptor ß agonist on angiogenesis in hindlimb ischemic diabetic rats.

INTRODUCTION: Studies indicated that PPARß agonists play a role in modulation of angiogenesis. In this study, we evaluated the effect of specific PPARß agonist, GW0742, on angiogenesis and serum vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and nitrite concentrations in hindlimb ischemia in normal and diabetic rats. METHODS: Hindlimb ischemic rats were divided into four groups: control, diabetic, control, and diabetic treated with GW0742 (n=7 each). Diabetes was induced by injection of streptozotocin (55mg/kg, ip). GW0742 was injected 1day after surgery (1mg/kg, sc). After 21days, blood samples were taken, and gastrocnemius muscles were harvested for immunohistochemistry. RESULTS: GW0742 significantly increased serum nitrite and VEGFR-2 concentrations and VEGF-to-VEGFR-2 ratio in control and diabetic rats. Capillary density was lower in diabetic animals compared to the control, and GW0742 significantly restored the capillary density in the control and diabetic hindlimb ischemic rats. CONCLUSION: PPARß agonists restore skeletal muscle angiogenesis and can be considered for prevention and/or treatment of peripheral vascular complications in diabetic subjects.

Air pollution and hospitalization for respiratory diseases among children in isfahan, iran.

BACKGROUND: Adverse effects of urban air pollution on human health notably the paediatric age group is of great importance. Limited data exist from developing countries. This study investigates the hospitalization of children because of respiratory diseases and air pollution levels in Isfahan, the second large city in Iran. METHODS: Hospital admission data were collected retrospectively from 120 randomly selected respiratory patients in Pediatric wards from the main referral hospital in Isfahan from March 2005-2006, and simultaneous air pollution data were collected from two monitoring stations located in south and north parts of the city. RESULTS: The result of statistical modeling using generalized linear Poisson regression showed that PM(10) and sulfur dioxide (SO(2)) concentrations had statistically significant positive association with number of respiratory admissions of children. CONCLUSION: This study confirms the findings of previous studies about the association of air pollutants' levels with hospitalization because of respiratory diseases in young children. Air pollution continues to pose a threat to public health notably in the paediatric age group, and underscores the need to re-examine national environmental health policies and standards in developing countries.