ABSTRACT
It has been recently reported that tea flavanols, including epigallocatechin gallate (EGCG), efficiently inhibit glucosidase II in liver microsomes. Since glucosidase II plays a central role in glycoprotein processing and quality control in the endoplasmic reticulum we investigated the possible contribution of endoplasmic reticulum stress and unfolded protein response (UPR) to the pro-apoptotic activity of EGCG in mouse hepatoma cells. The enzyme activity measurements using 4-methylumbelliferyl-alpha-d-glucopyranoside substrate confirmed the inhibition of glucosidase II in intact and alamethicin-permeabilized cells. EGCG treatment caused a progressive elevation of apoptotic activity as assessed by annexin staining. The induction of CHOP/GADD153, the cleavage of procaspase-12 and the increasing phosphorylation of eIF2alpha were revealed in these cells by Western blot analysis while the induction of endoplasmic reticulum chaperones and foldases was not observed. Time- and concentration-dependent depletion of the endoplasmic reticulum calcium stores was also demonstrated in the EGCG-treated cells by single-cell fluorescent detection. The massive alterations in the endoplasmic reticulum morphology revealed by fluorescent microscopy further supported the development of UPR. Collectively, our results indicate that EGCG interferes with protein processing in the endoplasmic reticulum presumably due to inhibition of glucosidase II and that the stress induces an incomplete unfolded protein response with dominantly pro-apoptotic components.
Subject(s)
Antineoplastic Agents/pharmacology , Catechin/analogs & derivatives , Glycoside Hydrolase Inhibitors , Liver Neoplasms, Experimental/enzymology , Transcription Factor CHOP/metabolism , Animals , Apoptosis/drug effects , Calcium/metabolism , Catechin/pharmacology , Dose-Response Relationship, Drug , Endoplasmic Reticulum/metabolism , Eukaryotic Initiation Factor-2/metabolism , Fluorescent Antibody Technique , Glucosides/metabolism , Hymecromone/analogs & derivatives , Hymecromone/metabolism , Liver Neoplasms, Experimental/pathology , Mice , Phosphorylation , Protein Folding , Stress, Physiological , Time Factors , Transcription Factor CHOP/genetics , alpha-GlucosidasesABSTRACT
Using a case-control sample we evaluated a possible involvement of the Vacuolar protein sorting 26 (VPS26) gene in the pathogenesis of AD. VPS26 located at 10q22.1 denotes a retromer subunit functionally involved in the cellular trafficking of Memapsin 2 (BACE). Genotyping of eight single nucleotide polymorphisms covering the complete VPS26 gene and haplotypic analysis revealed no association with AD. Thus, we conclude that VPS26 can be excluded as a major positional and functional candidate gene conferring risk to AD.
Subject(s)
Alzheimer Disease/genetics , Genetic Predisposition to Disease , Polymorphism, Genetic/genetics , Vesicular Transport Proteins/genetics , Aged , Alleles , Case-Control Studies , Chi-Square Distribution , Female , Gene Frequency , Humans , Male , Middle AgedABSTRACT
Several studies have reported conflicting results concerning the genetic association between Alzheimer's disease (AD) and the microsatellite marker D10S1423 on chromosome 10p12-14. In an ethnically homogeneous German population of 422 patients with AD and 254 cognitively healthy controls, the 238-bp allele of the D10S1423 marker showed a weak, but after correction for multiple testing no longer significant association with AD (p = 0.015, uncorrected; p = 0.11, corrected). These findings do not support the presence of a relevant susceptibility locus for AD on chromosome 10p12-14.
