(-)-Epigallocatechin-3-gallate (EGCG) has been reported to affect many cellular regulatory pathways. This study aims to determine whether EGCG could target microRNA (miRNA), one of the mechanisms for cells to achieve subtle change in multiple targets. We found that, in both human and mouse lung cancer cells in culture, EGCG specifically upregulated the expression of miR-210, a major miRNA regulated by HIF-1α. Furthermore, we found that overexpression of miR-210 led to reduced cell proliferation rate and anchorage-independent growth as well as reduced sensitivity to EGCG. On the mechanisms of miR-210 regulation by EGCG, we demonstrated that the regulation was mediated through the hypoxia-response element in miR-210 promoter. Consistently, the upregulation of miR-210 was found to be correlated with the stabilized HIF-1α in lung cancer cell lines after EGCG treatment. This EGCG-induced stabilization of HIF-1α was further shown by the stabilization of HA-tagged HIF-1α but not the P402A/P564A-mutated HIF-1α by EGCG, suggesting that EGCG targets the oxygen-dependent degradation (ODD) domain. Direct evidence was obtained by affinity binding assay showing that EGCG specifically binds HIF-1α with a K(d) = 3.47 μM. This result suggests that EGCG binding interferes with the hydroxylation of key Pro residues in the ODD domain, preventing HIF-1α from the Pro hydroxylation-dependent ubiquitination and subsequent proteosome-mediated degradation. In summary, our results demonstrated, for the first time, the elevation of miR-210 by EGCG in lung cancer cell lines and this is mediated by the stabilization of HIF-1α. This event contributes to the anticancer activity of EGCG.

No abstract

Gap junctions play important roles in auditory function and skin biology; mutations in the Cx26 (connexin26) gene are the predominant cause of inherited non-syndromic deafness and cause disfiguring skin disorders. Mass spectrometry (MS) was used to identify PTMs (post-translational modifications) of Cx26 and to determine whether they occur at sites of disease-causing mutations. Cx26 was isolated from transfected HeLa cells by sequential immunoaffinity and metal chelate chromatography using a tandem C-terminal haemagglutinin epitope and a (His-Asn)6 sequence. In-gel and in-solution enzymatic digestions were carried out in parallel with trypsin, chymotrypsin and endoproteinase GluC. Peptides were fractionated using a reversed-phase matrix by stepwise elution with increasing concentrations of organic solvent. To improve detection of low-abundance peptides and to maximize sequence coverage, MALDI–TOF-MS (matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry; MS) and MALDI–TOF/TOF-MS/MS (matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry; MS/MS) spectra were acquired from each elution step using an Applied Biosystems 4800 tandem mass spectrometer. Acquisition, processing and interpretation parameters were optimized to improve ionization and fragmentation of hydrophobic peptides. MS and MS/MS coverage of Cx26 was significantly above that reported for other membrane proteins: 71.3 %by MS, with 29.9 %by MS/MS. MS coverage was 92.6 % if peptides resulting from in-source collisions and/or partial enzymatic cleavages were considered. A variety of putative PTMs of Cx26 were identified, including acetylation, hydroxylation, γ-carboxyglutamation, methylation and phosphorylation, some of which are at sites of deafness-causing mutations. Knowledge of the PTMs of Cx26 will be instrumental in understanding how alterations in the cellular mechanisms of Cx26 channel biogenesis and function lead to losses in auditory function and disfiguring skin disorders.

No abstract

Cocaine administration upregulates the levels of extracellular glutamate and dopamine in the striatum. Activation of the receptors alters calcium homeostasis in striatal neurons leading to the expression of the endoplasmic reticulum (ER) stress proteins. It was therefore hypothesized that cocaine upregulates the expression of the ER stress proteins, immunoglobulin heavy chain binding protein (BiP), Ire1alpha and perk via glutamate and dopamine receptor activation. A novel glutamate microbiosensor and Western immunoblot analyses were mainly performed to test the hypothesis in the rat dorsal striatum. The results showed that i.p. injection of repeated cocaine (20 mg/kg) for nine consecutive days significantly increased extracellular glutamate levels while acute cocaine injection did not. However, the immunoreactivities (IR) of the ER stress proteins in the dorsal striatum were significantly increased by either acute or repeated cocaine injections as compared with saline controls. Intrastriatal injection (i.s.) of the selective group I metabotropic glutamate receptor (mGluR) antagonist N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC; 25 nmol) or the mGluR5 subtype antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP; 2 and 25 nmol) significantly decreased repeated cocaine-induced increases in the IR of the ER stress proteins in the injected dorsal striatum. Similarly, the selective D1 antagonist (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390; 0.1 mg/kg, i.p.) or the N-methyl-d-aspartate antagonist dizocilpine/(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-ibenzo[a,d]cyclohepten-5,10-imine maleate (MK801; 2 nmol, i.s.) decreased acute or repeated cocaine-induced the IR of the ER stress proteins in the dorsal striatum. These data suggest that cocaine upregulates expression of the ER stress proteins in striatal neurons via a mechanism involving activation of glutamate and dopamine receptors.