Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.     

Sex differences in human lymphocyte Na,K-ATPase as studied by labeled ouabain binding

Lymphocytes Na,K-ATPase is a plasma membrane enzyme that is up-regulated under lymphocytes activation. It is also studied as a model of brain cells Na,K-ATPase. Data about sex-related specificities of the enzyme are not available.The binding of tritium-labelled ouabain to lymphocyte plasma membrane Na,K-ATPase was studied in healthy volunteers of both sexes. The binding interactions were expressed in terms of K(D) and B(Max). The first parameter is related to the affinity of ouabain for the enzyme whereas the second one is related to its density on the cell membrane. Distinct sex-related differences were found. Whereas in males there is a significant direct correlation between the parameters K(D) and B(Max), in females this is not present. However, in females there is a significantly lower K(D) in the 25-37 age range. The latter result probably reflects the expression of subunit variants giving a greater affinity for ouabain. This circumstance may be relevant both to lymphocytes' ability to be activated and to brain function, if one admits that lymphocyte Na,K-ATPase faithfully represents the brain-borne one.     

Effects of prostaglandin E1alpha cyclodextrin [corrected] treatment on endothelial dysfunction in patients with systemic sclerosis

OBJECTIVE: Systemic sclerosis (SSc) is characterized by an altered nitric oxide (NO): endothelin I ratio and by endothelial dysfunction. AIMS: To verify the effects of prostaglandin E1 (PGE1) alpha-cyclodestrin treatment on endothelial function, quantified as flow-mediated dilation (FMD) of the radial artery. METHODS: In 16 women with SSc (age 57 +/- 2.7 years, means +/- SE) in whom a diagnosis of SSc had been made several years earlier (7.1 +/- 1.2 years), FMD was evaluated by an echotracking technique on the radial artery, using trinitroglycerin vasodilation as a non-endothelial measure of the vessel's ability to increase its diameter maximally. FMD was evaluated after 4 months washout period and after 4 months cyclic infusion of PGE1 alpha-cyclodestrin. Expired NO was measured at the same time. RESULTS: PGE1 alpha-cyclodestrin cyclic infusions did not modify systolic and diastolic blood pressure, heart rate or trinitroglycerin radial artery vasodilation. On the other hand, it induced a marked and significant increase in FMD of the radial artery, which was also accompanied by an increase in blood flow and expired NO. CONCLUSIONS: Endothelial dysfunction and reduced FMD associated with SSc are improved by cyclic treatment with PGE1 alpha-cyclodestrin. This effect occurs together with a concomitant increase in expired NO, suggesting its direct positive influence on endothelial function. It may also partly explain the clinical beneficial effect of the drug in SSc.     

Pathological animal models in the experimental evaluation of tumour microvasculature with magnetic resonance imaging

PURPOSE: The purpose of this study was to evaluate the applications of magnetic resonance imaging (MRI), and in particular, dynamic contrast-enhanced MRI (DCE-MRI), in the assessment of tumour microvasculature by means of animal tumour models evaluated before and after antiangiogenic treatment. MATERIALS AND METHODS: Forty-two MRI exams were performed with intravascular contrast media in 21 rats: tumours were induced by subcutaneous injection of colon carcinoma cells in 7 rats and mammary adenocarcinoma cells in 14 rats. Perfusion and permeability parameters of the implanted tumours were evaluated by using two contrast media (B22956/1 and Gd-DTPA37-albumin) to establish response to treatment with two different antiangiogenic drugs (tamoxifen and SU6668). These parameters were correlated with histology to obtain a radiological-histological map of tumour microvasculature. RESULTS: DCE-MRI revealed greater enhancement in the peripheral area than in the central area in all the examined animal models. In the mammary carcinoma experiment, vascular permeability measured by means of B22956/1 in the animals treated with the antiangiogenic drug (0.0043317+/-0.0040418 ml/min(-1)/ml(-1)) was significantly less than in untreated animals (0.0090460+/-0.0043680 ml/min(-1)/ml(-1)), whereas no significant difference was observed with Gd-DTPA-albumin (13.14+/-13.94 ml/min(-1)/ml(-1) in treated animals and 18.07+/-11.92 ml/min(-1)/ml(-1) in untreated animals). In the colon carcinoma experiment, mean permeability and perfusion decreased by 51% (from 5.2+/-1.1 to 2.5+/-0.8 ml/100 ml) and 59% (from 0.00165+/-5.1 to 0.0067+/-4.8 ml/min(-1)/ml(-1) of tissue), respectively, in all animals after antiangiogenic drug administration. CONCLUSIONS: DCE-MRI permits a noninvasive evaluation of tumour microcirculation and in particular of its dynamic characteristics and vascularity before and after antiangiogenic treatment.     

MHC-restricted cytotoxic T-lymphocyte assay: an improved method based on normal and SV40-immortalized rabbit epidermal target cells

Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous (51)Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.