Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions

Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

Introduction p53 plays important roles in regulating the metabolic reprogramming of cancer, such as aerobic glycolysis. Oroxylin A is a natural active flavonoid with strong anticancer effects both in vitro and in vivo. Methods wt-p53 (MCF-7 and HCT116 cells) cancer cells and p53-null H1299 cancer cells were used. The glucose uptake and lactate production were analyzed using Lactic Acid production Detection kit and the Amplex Red Glucose Assay Kit. Then, the protein levels and RNA levels of p53, mouse double minute 2 (MDM2), and p53-targeted glycolytic enzymes were quantified using Western blotting and quantitative polymerase chain reaction (PCR), respectively. Immunoprecipitation were performed to assess the binding between p53, MDM2, and sirtuin-3 (SIRT3), and the deacetylation of phosphatase and tensin homolog (PTEN). Reporter assays were performed to assess the transcriptional activity of PTEN. In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 cells. Results Here, we analyzed the underlying mechanisms that oroxylin A regulated p53 level and glycolytic metabolism in wt-p53 cancer cells, and found that oroxylin A inhibited glycolysis through upregulating p53 level. Oroxylin A did not directly affect the transcription of wt-p53, but suppressed the MDM2-mediated degradation of p53 via downregulating MDM2 transcription in wt-p53 cancer cells. In further studies, we found that oroxylin A induced a reduction in MDM2 transcription by promoting the lipid phosphatase activity of phosphatase and tensin homolog, which was upregulated via sirtuin3-mediated deacetylation. In vivo, oroxylin A inhibited the tumor growth of nude mice-inoculated MCF-7 or HCT116 cells. The expression of MDM2 protein in tumor tissue was downregulated by oroxylin A as well. Conclusions These results provide a p53-independent mechanism of MDM2 transcription and reveal the potential of oroxylin A on glycolytic regulation in both wt-p53 and mut-p53 cancer cells. The studies have important implications for the investigation on anticancer effects of oroxylin A, and provide the academic basis for the clinical trial of oroxylin A in cancer patients

Effectiveness and Safety of rhIGF-1 Therapy in Children: The European Increlex® Growth Forum Database Experience.

BACKGROUND/AIMS: We report data from the EU Increlex® Growth Forum Database (IGFD) Registry, an ongoing, open-label, observational study monitoring clinical practice use of recombinant human insulin-like growth factor-1 (rhIGF-1) therapy in children. METHODS: Safety and effectiveness data on rhIGF-1 treatment of 195 enrolled children with growth failure were collected from December 2008 to September 2013. RESULTS: Mean ± SD (95% CI) height velocity during first year of rhIGF-1 treatment was 6.9 ± 2.2 cm/year (6.5; 7.2) (n = 144); in prepubertal patients naïve to treatment, this was 7.3 ± 2.0 cm/year (6.8; 7.7) (n = 81). Female sex, younger age at start of rhIGF-1 therapy, and lower baseline height SDS predicted first-year change in height SDS. The most frequent targeted treatment-emergent adverse events (% patients) were hypoglycemia (17.6%, predictors: young age, diagnosis of Laron syndrome, but not rhIGF-1 dose), lipohypertrophy (10.6%), tonsillar hypertrophy (7.4%), injection site reactions (6.4%), and headache (5.9%). Sixty-one serious adverse events (37 related to rhIGF-1 therapy) were reported in 31 patients (16.5%). CONCLUSION: Safety and effectiveness data on use of rhIGF-1 in a 'real-world' setting were similar to those from controlled randomized trials. Severe growth phenotype and early start of rhIGF-1 improved height response and predicted risk of hypoglycemia

Shape and cost optimization of cold formed "Z " purlin using genetic algorithms

Cold formed "Z " purlins are widely used in civil engineering structures. An important advantage is the great flexibility of producing different geometry of this profile. In cold formed structures it is difficult (practically impossible) to change the shape along the element, so it is recommended to have "economic " shape of cross section of such purlin. The optimization process was incorporated for “Z ” purlin that works as simple beam under continuous load. Purlins were optimized for different spans and different loads. Optimization process was solved with use of genetic algorithms. This method was chosen because it is the most suitable for discreet problems with many nonlinear parameters. In addition, methods supported by genetic algorithms predominate other methods (like one direction searching method). Moreover this method was successfully exploited by us in other problems like cost and shape optimization of portal frame made of welded I beams/columns. The shape optimization were taken into consideration. Design variables were geometry of cross section, and grade of steel. Constraints were based on European national standards (Eurocode – 3, for cold formed structures). Ultimate and serviceability limit states were taken into account for elements undergoing bending. Results of analysis are presented in tables (optimal dimensions of cross sections, and grade of steel) for different spans and continuous loads. The main objective of this paper is an implementation of these results in industry (especially by producers an

Concentrated Flow Paths in Riparian Buffer Zones of Southern Illinois

Riparian buffers in agricultural landscapes should be designed to trap pollutants in overland flow by slowing, filtering, and infiltrating surface runoff entering the buffer via sheet flow. However, observational evidence suggests that concentrated flow is prevalent from agricultural fields. Over time sediment can accumulate in riparian buffers forming berms that restrict sheet flow; these berms ultimately back up surface runoff, resulting in an eventual breakthrough that concentrates overland flow. This study examines the occurrence of concentrated flow paths (CFPs) in riparian buffers at both the field and watershed scale. At the field scale, intensive topographic surveys were conducted at ten field sites in southern Illinois. To assess the prevalence of CFPs at the watershed scale, three watersheds in southern Illinois were selected for walking stream surveys along randomly selected 1,000 m reaches. CFPs were identified in all topographic surveys and all walking stream surveys. Among field sites, concentrated flow accounted for 82.5–100% of the drainage leaving the agricultural fields. Sediment berm accumulation was identified at all field sites and was positively correlated with CFP size. At the watershed scale, CFPs were more abundant in agricultural areas compared to forested land. Results from this study indicate that concentrated flow was prevalent across all study sites at both the field and watershed scale. Thus, surface water quality may suffer in areas with poorly functioning buffers, and managers must consider the occurrence of CFPs when designing and maintaining riparian buffers to protect stream water quality