Inhibitory Effect of Apocynin on Proteasome Inhibition-Induced Apoptosis in Differentiated PC12 Cells

BACKGROUND: The dysfunction of the proteasome system has been implicated in neuronal degeneration. Apocynin, a specific inhibitor for nicotinamide adenine dinucleotide phosphate oxidase, has anti-inflammatory and anti-oxidant effects. However, the effect of apocynin on the neuronal cell death induced by proteasome inhibition has not been studied. METHODS: Using differentiated PC12 cells, in the respect of cell death process the suppressive effect of apocynin on the proteasome inhibition-mediated apoptosis was examined. RESULTS: The proteasome inhibitors MG132 and MG115 induced a decrease in Bid and Bcl-2 protein levels, an increase in Bax and p53 levels, mitochondrial depolarization, efflux of cytochrome c into cytosol and increase in caspases (-8, -9 and -3) activities. Treatment with apocynin attenuated the proteasome inhibitor-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, glutathione (GSH) depletion and cell death. CONCLUSIONS: Apocynin may attenuate the proteasome inhibitor-mediated apoptosis in differentiated PC12 cells by inhibiting the activation of the mitochondria-mediated pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of apocynin appears to be attributed to inhibition of the production of reactive oxygen species and the depletion of cellular GSH contents.

Protein Components of Lewy Bodies (LBs) Identified from Synthetic Proteasome Inhibitor (PSI)-induced Inclusions in PC12 Cells by MS Analysis / 中国生物化学与分子生物学报

Proteomic analysis is an effective way to identify protein constituent in Lewy bedy-like inclusions (or aggresome) in vitro. Exposure to synthetic proteasome inhibitor (PSI, 10 μmol/L) for 48 hours was used to induce the formation of cytoplasmic proteineous inclusions (termed as PSi-induced inclusions) in PC12 cells.The proteomic approaches of biochemical fractionation, two-dimensional electrophoresis (2-D) and identification via peptide mass fingerprints (PMF) were deployed, and 20 protein components of LBs were identified,i ncluding 2 proteins involved in the production of synaptic neurotransmitter, 6 subunits of the 26 S proteasome,2 cytoskeleton proteins, 2 subunits of mitochondrial complexes, 1 anti-oxidant protein, and 7 chaperone proteins and (or) chaperone-like proteins. The results suggested that these LB protein components might had been recruited in PSI-induced inclusions formed in PC12 cells under the condition of proteasome inhibition.

Eukaryotic Translation Factors Resided in PSI-induced Inclusions in PC12 / 中国生物化学与分子生物学报

Lewy body (LB), an eosinophilic inclusion localized in the neuronal perikaryon, consists of a wide range of proteins, including the consistent organization and the selective composition. Treatment of PC12 cells with synthetic proteasome inhibitor (PSI) at 10 μmol/L for 48 hours induced the formation of inclusions, which were detected by eosin staining and immunostaining for α-synuclein. To investigate the potential new components of PSI-induced inclusions in vitro, pure intact inclusions were successfully obtained by fractionation and subjected to two-dimensional electrophoresis (2-DE) then analyzed with unequivocal matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Eukaryotic translation initiation factor 3 subunit 5 (eIF-3ε), eukaryotic elongation factor 2 (eEF-2) and mitochondrial elongation factor Tu (EF-Tumt) were identified. The results suggest that 3 eukaryotic translation factors recruited in PSI-induced inclusions may influence formation of the intermediate organelles following the inhibition of proteasomes.