Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis

Abstract

Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant proapoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 ) but not cathepsins B or D ) causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.

Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic

“…29,35 Thus, we suggest that lysosomal membrane disruption induced by oxidative stress can be inhibited by OTR4120 treatment, preventing the release of cathepsin D to the cytosol. The results of cell death protection by natural GAGs assessed by cytochrome c release and caspases activities indicate that endogenous GAGs are effectively able to regulate apoptosis but to a lower extent than their mimetic.…”

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…To explore this, we first analyzed the integrity of lysosomes in L929 cells exposed to acridine orange in the presence and absence of TNF␣/Z-VAD-FMK. Due to proton trapping, this vital dye accumulates mainly in the acidic vacuolar apparatus, preferentially in lysosomes (15). When excited by blue light, acridine orange emits red/orange fluorescence at high (lysosomal) concentrations and green fluorescence at low (nuclear and cytosolic) concentrations.…”

“…We hypothesized that cathepsins, which are released from lysosomes during both apoptosis and necrosis (15)(16)(17)(18)(19), might be involved in this cleavage. In the present study, we show that topo I is cleaved into fragments of approximately 70 kd and 45 kd in mouse L929 fibroblasts and human endothelial cells undergoing necrotic cell death, and that cathepsins, particularly cathepsin L, are capable of generating these fragments.…”

Downregulation of the antiapoptotic MCL‐1 protein and apoptosis in MA‐11 breast cancer cells induced by an anti‐epidermal growth factor receptor–Pseudomonas exotoxin a immunotoxin

“…This suggests that lysosomal and mitochondrial factors might sustain and augment each other. 25,26 The cell viability data also suggest a role of cathepsin B in IT-induced cell death, and the factor(s) responsible for cathepsin B/L-mediated facilitation of IT-induced cell death will be further examined. In our cell system, even the highest z-FA-FMK concentration (50 M) had no effect on caspase-3 processing or Mcl-1 downregulation, indicating that cathepsin B/L functions downstream of or in parallel with the Mcl-1 pathway.…”