22 research outputs found
Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase
<p>Abstract</p> <p>Background</p> <p>Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.</p> <p>Methods</p> <p>Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (<sup>3</sup>H-naloxone) binding assays were performed in wild type and gp91<it><sup>phox-/- </sup></it>neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47<it><sup>phox </sup></it>to plasma membrane was assessed by western blot.</p> <p>Results</p> <p>Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 ÎŒM, respectively. Competitive binding of <sup>3</sup>H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 ÎŒM, respectively. <sup>3</sup>H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91<sup><it>phox</it></sup>; in contrast, reduced <sup>3</sup>H-naloxone binding was found in neutrophils deficient in gp91<sup><it>phox </it></sup>or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of <sup>3</sup>H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91<sup><it>phox </it></sup>antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47<sup><it>phox </it></sup>to the membrane, leading to NOX2 inactivation.</p> <p>Conclusions</p> <p>Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.</p
Metallothionein â overexpression as a highly significant prognostic factor in melanoma: a prospective study on 1270 patients
Metallothioneins (MT) are ubiquitous, intracellular small proteins with high affinity for heavy metal ions. In the last decades, it was shown that MT overexpression in a variety of cancers is associated with resistance to anticancer drugs and is combined with a poor prognosis. In this prospective study, we examined the role of MT overexpression in melanoma patients as a prognostic factor for progression and survival. Between 1993 and 2004, 3386 patients with primary cutaneous melanoma were investigated by using a monoclonal antibody against MT on routinely fixed, paraffin-embedded tissues. In all, 1270 patients could be followed up for further statistical analysis (Fisher's exact test, MantelâHaenszel Ï2 test, KaplanâMeier curves). The MT data of disease-free interval and overall survival were compared univariately and multivariately in Cox regression analysis. Immunohistochemical overexpression of MT in tumour cells of patients with primary melanoma (310 of 1270; 24.4%) was associated with a higher risk for progression (117 of 167; 70.1%) and reduced survival (80 of 110; 72.7%) of the disease (P<0.0001). Similarly, KaplanâMeier curves gave highly significant disadvantages for the MT-positive group. Univariate analysis (relative risk 7.4; 95% confidence interval (CI) 5.2â10.2; P<0.0001 for progression; relative risk 7.1; 95% CI 4.7â10.9; P<0.0001 for survival), as well as multivariate analysis with other prognostic markers resulted in MT overexpression as a highly significant and independent factor for prognosis in primary melanoma