Cathepsin B Acts as a Dominant Execution Protease in Tumor Cell Apoptosis Induced by Tumor Necrosis Factor

Death receptors can trigger cell demise dependent or independent of caspases. In WEHI-S fibrosarcoma cells, tumor necrosis factor (TNF) induced an increase in cytosolic cathepsin B activity followed by death with apoptotic features. Surprisingly, this process was enhanced by low, but effectively inhibiting, concentrations of pan-caspase inhibitors. Contrary to caspase inhibitors, a panel of pharmacological cathepsin B inhibitors, the endogenous cathepsin inhibitor cystatin A as well as antisense-mediated depletion of cathepsin B rescued WEHI-S cells from apoptosis triggered by TNF or TNF-related apoptosis-inducing ligand. Thus, cathepsin B can take over the role of the dominant execution protease in death receptor-induced apoptosis. The conservation of this alternative execution pathway was further examined in other tumor cell lines. Here, cathepsin B acted as an essential downstream mediator of TNF-triggered and caspase-initiated apoptosis cascade, whereas apoptosis of primary cells was only minimally dependent on cathepsin B. These data imply that cathepsin B, which is commonly overexpressed in human primary tumors, may have two opposing roles in malignancy, reducing it by its proapoptotic features and enhancing it by its known facilitation of invasion

Heat Shock Protein 70 Promotes Cell Survival by Inhibiting Lysosomal Membrane Permeabilization

Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c–induced, apoptosome-dependent caspase activation in vitro and Fas ligand–induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes

Plasminogen Activator Inhibitor 1 Protects Fibrosarcoma Cells from Etoposide-Induced Apoptosis through Activation of the PI3K/Akt Cell Survival Pathway1

High levels of plasminogen activator inhibitor (PAI-1) in tumors are associated with poor prognosis in several cancer types, and the reason for this association is not fully understood. Plasminogen activator inhibitor 1 has been suggested to contribute to tumor growth by protecting cancer cells from apoptosis, and we have previously shown that wild type murine fibrosarcoma cells are significantly more resistant to apoptosis induced by chemotherapy than PAI-1-deficient fibrosarcoma cells. Here, we further investigated the molecular mechanisms underlying the antiapoptotic function of PAI-1 focusing on the phosphatidylinositol 3-phosphate kinase (PI3K)/Akt cell survival pathway. We demonstrate that the activation level of the Akt cell survival pathway is reduced in PAI-1-deficient cells. Inhibition of either PI3K or Akt by synthetic inhibitors sensitized the wild type but not the PAI-1-deficient cells to etoposide-induced cell death. More importantly, reintroduction of PAI-1 expression in PAI-1-deficient cells induced an increase in Akt activity and protection against etoposide-induced apoptosis. Concordantly, silencing of PAI-1 by RNA interference in wild type fibrosarcoma cells decreased the level of active Akt, and this was accompanied by a sensitization of the cells to etoposide-induced cell death. Altogether, our data suggest that PAI-1 influences sensitivity to etoposide-induced apoptosis through the PI3K/Akt cell survival pathway by acting upstream of PI3K and Akt. This points to PAI-1 as a possible therapeutic target in cancer diseases where PAI-1 inhibits chemotherapy-induced apoptosis