ABSTRACT
Stimulation of the serotonin 1A receptor (5-HT(1A)-R) causes activation of extracellular signal-regulated protein kinase (Erk) and protein kinase C alpha (PKCalpha) in both hippocampal HN2-5 cells and cultured hippocampal slices from postnatal day-15 (P15) mice. Our earlier studies demonstrated that PKCalpha is co-immunoprecipitated with Erk and the phosphorylation of PKCalpha in this Erk-PKCalpha complex is dependent on the Erk pathway. Furthermore, the T(638) residue, which must be phosphorylated for the complete activation of PKCalpha, is within an authentic Erk consensus domain (S/TP), and the PKCalpha protein also contains two docking sites for Erk such as KRGRIYL and KRGIIYRDLKL. Using Föster Resonance Energy Transfer (FRET) we have confirmed an association between Erk and PKCalpha. Employing PKCalpha and Erk mutants we next demonstrated that Erk causes direct phosphorylation and activation of PKCalpha. By mutating the phosphoinositide-dependent kinase-1 (PDK-1)-promoted phosphorylation site (S(497)) and the kinase site (K(368)) in PKCalpha, we observed that both of these autophosphorylation-deficient mutants are phosphorylated at T(638) in an Erk-dependent manner. To confirm that Erk indeed catalyzes phosphorylation of PKCalpha at T(638), we used a mutant Erk construct in which a relatively large amino acid residue in the ATP binding site (Q(103)) had been replaced with glycine, enabling this mutant to utilize a bulky analog of ATP, cyclopentyl ATP. An in vitro kinase assay using this mutant Erk protein, radiolabeled cyclopentyl ATP, and a synthetic oligopeptide containing the S/TP site of PKCalpha demonstrated phosphorylation of the peptide by Erk1/2. These results confirm the novel possibility that PKCalpha is a direct substrate of Erk1/2 in neuronal cells and help link two important signaling molecules that regulate maturation and protection of hippocampal neurons as well as many other cell types.
Subject(s)
Hippocampus/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase C-alpha/metabolism , Receptor, Serotonin, 5-HT1A/metabolism , Amino Acid Sequence , Animals , Cell Line , Fluorescence Resonance Energy Transfer , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Molecular Sequence Data , Neurons/metabolism , Phosphorylation , Protein Kinase C-alpha/genetics , Serotonin 5-HT1 Receptor Agonists , Signal Transduction , Threonine/genetics , Threonine/metabolismABSTRACT
We report a class of polysaccharide-based hybrid nanogels that can integrate the functional building blocks for optical pH-sensing, cancer cell imaging, and controlled drug release into a single nanoparticle system, which can offer broad opportunities for combined diagnosis and therapy. The hybrid nanogels were prepared by in-situ immobilization of CdSe quantum dots (QDs) in the interior of the pH and temperature dual responsive hydroxypropylcellulose-poly(acrylic acid) (HPC-PAA) semi-interpenetrating polymer networks. The-OH groups of the HPC chains are designed to sequester the precursor Cd(2+) ions into the nanogels as well as stabilize the in-situ formed CdSe QDs. The pH-sensitive PAA network chains are designed to induce a pH-responsive volume phase transition of the hybrid nanogels. The developed HPC-PAA-CdSe hybrid nanogels combine a strong trap emission at 741nm for sensing physicochemical environment in a pH dependent manner and a visible excitonic emission at 592nm for mouse melanoma B16F10 cell imaging. The hybrid nanogels also provide excellent stability as a drug carrier, which cannot only provide a high drug loading capacity for a model anticancer drug temozolomide, but also offer a pH-triggered sustained-release of the drug molecules in the gel network.
Subject(s)
Drug Carriers/chemistry , Gels/chemistry , Nanostructures/chemistry , Neoplasms/pathology , Polysaccharides/chemistry , Quantum Dots , Animals , Biocompatible Materials/chemistry , Cadmium/chemistry , Cell Line, Tumor , Drug Delivery Systems , Hydrogen-Ion Concentration , Materials Testing , Mice , Neoplasms/metabolism , Selenium/chemistry , Spectroscopy, Fourier Transform InfraredABSTRACT
Turmeric, an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound, named curcumin or diferuloylmethane, which eliminates cancer cells derived from a variety of peripheral tissues. Although in vitro experiments have addressed its anti-tumor property, no in vivo studies have explored its anti-cancer activity in the brain. Oral delivery of this food component has been less effective because of its low solubility in water.We show that a soluble formulation of curcumin crosses the bloodbrain barrier but does not suppress normal brain cell viability. Furthermore, tail vein injection, or more effectively, intracerebral injection through a cannula, blocks brain tumor formation in mice that had already received an intracerebral bolus of mouse melanoma cells (B16F10).While exploring the mechanism of its action in vitro we observed that the solubilized curcumin causes activation of proapoptotic enzymes caspase 3/7 in human oligodendroglioma (HOG) and lung carcinoma (A549) cells, and mouse tumor cells N18(neuroblastoma), GL261 (glioma), and B16F10. A simultaneous decrease in cell viability is also revealed by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]assays. Further examination of the B16F10 cells showed that curcumin effectively suppresses Cyclin D1, P-NF-kB, BclXL, P-Akt, and VEGF, which explains its efficacy in blocking proliferation, survival, and invasion of the B16F10 cells in the brain. Taken together,solubilized curcumin effectively blocks brain tumor formation and also eliminates brain tumor cells. Therefore, judicious application of such injectable formulations of curcumin could be developed into a safe therapeutic strategy for treating brain tumors.
