ABSTRACT
P21 Activated Kinase 1 (Pak1), an oncogenic serine/threonine kinase, is known to have a significant role in the regulation of cytoskeleton and cellular morphology. Runx3 was initially known for its role in tumor suppressor function, but recent studies have reported the oncogenic role of Runx3 in various cancers. However, the mechanism that controls the paradoxical functions of Runx3 still remains unclear. In this study, we show that Runx3 is a physiologically interacting substrate of Pak1. We identified the site of phosphorylation in Runx3 as Threonine 209 by mass spectrometry analysis and site-directed mutagenesis, and further confirmed the same with a site-specific antibody. Results from our functional studies showed that Threonine 209 phosphorylation in Runx3 alters its subcellular localization by protein mislocalization from the nucleus to the cytoplasm and subsequently converses its biological functions. This was further supported by in vivo tumor xenograft studies in nude mouse models which clearly demonstrated that PANC-28 cells transfected with the Runx3-T209E clone showed high tumorigenic potential as compared with other clones. Our results from clinical samples also suggest that Threonine 209 phosphorylation by Pak1 could be a potential therapeutic target and of great clinical relevance with implications for Runx3 inactivation in cancer cells where Runx3 is known to be oncogenic. The findings presented in this study provide evidence of Runx3-Threonine 209 phosphorylation as a molecular switch in dictating the tissue-specific dualistic functions of Runx3 for the first time.
Subject(s)
Biomarkers, Tumor/genetics , Core Binding Factor Alpha 3 Subunit/genetics , Intracellular Signaling Peptides and Proteins/genetics , Neoplasms/genetics , Animals , Cell Line, Tumor , Cell Nucleus/genetics , Cytoplasm , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Mice , Mutagenesis, Site-Directed , Neoplasms/drug therapy , Neoplasms/pathology , Phosphorylation , Threonine/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Fifty-two combinations of nitrofurantoin were developed to assess the effect of dosage form type, particle size, diluent, and process on in vitro availability. With the official procedure and conditions, dissolution rates fell in a 66-fold range. Statistical analysis of the dissolution rates indicated no significant differences as a result of particle size, processing method, or compression force. The diluent choice and dosage form type significantly influenced the dissolution rate. Based on in vitro screening, six formulations presenting a broad range of dissolution rates were selected for further study relating to human bioavailability and bioequivalence.
Subject(s)
Nitrofurantoin/metabolism , Capsules , Drug Compounding , Excipients , Hardness , Nitrofurantoin/administration & dosage , Particle Size , Solubility , Tablets , Therapeutic EquivalencyABSTRACT
Based on preliminary in vitro evaluation, six formulations presenting a broad range of dissolution rates were selected for bioequivalency determination in a randomized complete block crossover. In vitro-in vivo correlations were developed relating cumulative percent dissolved to cumulative percent excreted. These correlations appear to be useful for comparing different formulations as well as different batches of the same formulation.
