Hydrogen Peroxide Induces Lysosomal Protease Alterations in PC12 Cells

Abstract

Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H2O2. Furthermore, activation of CATD occurred following exposure to H2O2 and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H2O2 exposure, while inhibition of caspase-3 significantly increased cell viability compared to H2O2 administration alone. Collectively, our data suggest that H2O2 induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations.

Increased In Vitro Lysosomal Function in Oxidative Stress-Induced Cell Lines

“…Lysosomes contain a number of proteases and acid hydrolases that kill bacteria cells, including the aspartic protease cathepsin D and the cystein protease cathepsin B, which are the most abundant and the most often reported apoptosis-inducers [12]. Cathepsins in the lysosomes were activated and then translocated to the cytosol during apoptosis induced by oxidative stress [12,13].…”

“…Cathepsins in the lysosomes were activated and then translocated to the cytosol during apoptosis induced by oxidative stress [12,13]. Therefore, our study also aimed to evaluate apoptotic cell death caused by lysosomes isolated from HeLa cells subjected to oxidative stress.…”

“…Oxidative stress results from increased levels of reactive oxygen species (ROS) through oxidative metabolism or decreased clearance of ROS due to anti-oxidant deficiency, and apoptotic cell death can be induced under severe oxidative stress conditions [12]. Alteration of both lysosomal cysteine and aspartic proteases has been observed following oxidative stress-induced apoptosis [12], suggesting that lysosomal enzyme mediation is a key factor in inducing apoptotic cell death. A complete breakdown of the lysosomes with the release of high concentrations of lysosomal enzymes into the cytosol results in unregulated cell death when exposed to severe oxidative stresses [13].…”

Oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome

“…Prdx2 (increased 1.9 fold, p=0.016367) is produced in response to oxidative stress and is known to reduce intracellular H 2 O 2 [29–31]; an increase in expression of this gene is not surprising given the decrease in peroxidases seen in the LN BMNC from the NHD13 mice. Ctsb (1.8 fold, p=0.01396) was also increased; Ctsb has been linked to oxidative stress as its release from lysosomes is thought to contribute to oxidative stress induced by H 2 O 2 [32]. In addition, we noted Hbq1 to be increased 6.1-fold (p value = 0.049627) in the LN BMNC from the NHD13 mice.…”

Self‐evolving oxidative stress with identifiable pre‐ and postmitochondrial phases in PC12 cells

“…Here, in an attempt to model a self‐evolving oxidative stress in a cell lineage, we used differentiated cholinergic‐like phenotyped PC12 cells (dPC12) stressed by an acute and strong H 2 O 2 treatment (0.5 mM) for a short time period (30 min), followed by stressor elimination from the medium (Greene and Tischler, 1976; Greene, 1978). H 2 O 2 has been widely employed to induce stress in PC12 cells, but lower concentrations and continuous presence of the exogenous stressor in the culture medium are generally used (Lee et al, 2007). In our model, exogenous stressor elimination after stress induction allowed studies on the self‐evolving oxidative response of these cells.…”