Chemotherapy drug response in ovarian cancer cells strictly depends on a cathepsin D-Bax activation loop.

The ovarian cancer cell lines A2780 (wild-type p53) and NIHOVCAR3 (mutated p53) showed, respectively, sensitivity and resistance towards several chemotherapy drugs. We hypothesized that the two cell lines differ in their ability to activate the intrinsic death pathway and have, therefore, dissected the lysosome-mitochondrion signalling pathway by pharmacological inhibition or genetic manipulation of key regulators and executioners. Biochemical and morphological confocal fluorescence studies showed that: (1) In A2780 cells bcl-2 is expressed at an undetectable level, whereas Bax is expressed at a rather high level; by contrast, bcl-2 is highly expressed and Bax is expressed at extremely low levels in NIHOVCAR3 cells; (2) Chemotherapy treatment reduced the expression of bcl-2 in NIHOVCAR3 cells, yet these cells resisted to drug toxicity; (3) Cathepsin D (CD), not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; (4) Lysosome leakage and cytosolic relocation of CD occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; (5) Bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; (6) CD activity is mandatory for the oligomerization of Bax on both mitochondrial and lysosomal membranes; (7) Bax activation did not occur in the resistant NIHOVCAR3 cells despite their high content in CD. The present data are consistent with a model in which on treatment with a cytotoxic drug the activation of a CD-Bax loop leads to the generalized permeabilization of lysosomes and eventually of mitochondria, thus reaching the point of no return, and culminates with the activation of the caspase cascade. Our data also imply that dysfunctional permeabilization of lysosomes contributes to the development of chemoresistance.

Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini.

Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarized transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single-chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16-kDa form, at a physiological pH, was observed only in conditioned medium but not milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarized secretion being controlled by prolactin itself.

High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells.

We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal protease implicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant protein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precursor (proCDrec) that reacted with a polyclonal antibody against residues 7-21 of the propeptide sequence. In contrast to the control, in cells infected with the recombinant virus proCDrec was largely secreted into the culture medium, although it contained high-mannose oligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCDrec was processed into the 48 kDa intermediate single-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method based on Pepstatin A-affinity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on its latency in activity assay at acid pH and on its reactivity with antibodies specific for the N-terminus, the purified protein was judged to be in the inactive precursor form. During incubation at acid pH the purified proCDrec underwent autocatalytic processing and acquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCDrec specifically reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus-directed proCDrec displays structural and functional features resembling those of native human proCD. This system can therefore be exploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interesting protein.

Preconditioning-induced cytoprotection in hepatocytes requires Ca(2+)-dependent exocytosis of lysosomes.

A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na+ overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H+/ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [Ca2+]. Chelation of free cytosolic Ca2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.

Destination 'lysosome': a target organelle for tumour cell killing?

Lysosomes and lysosome-related organelles constitute a system of acid compartments that interconnect the inside of the cell with the extracellular environment via endocytosis, phagocytosis and exocytosis. In recent decades it has been recognized that lysosomes are not just wastebaskets for disposal of unused cellular constituents, but that they are involved in several cellular processes such as post-translational maturation of proteins, degradation of receptors and extracellular release of active enzymes. By complementing the autophagic process, lysosomes actively contribute to the maintenance of cellular homeostasis. Proteolysis by lysosomal cathepsins has been shown to mediate the death signal of cytotoxic drugs and cytokines, as well as the activation of pro-survival factors. Secreted lysosomal cathepsins have been shown to degrade protein components of the extracellular matrix, thus contributing actively to its re-modelling in physiological and pathological processes. The malfunction of lysosomes can, therefore, impact on cell behaviour and fate. Here we review the role of lysosomal hydrolases in several aspects of the malignant phenotype including loss of cell growth control, altered regulation of cell death, acquisition of chemoresistance and of metastatic potential. Based on these observations, the lysosome is proposed as a potential target organelle for the chemotherapy of tumours. We will also present some recent data concerning the technologies for delivering chemotherapeutic drugs to the endosomal-lysosomal compartment and the strategies to improve their efficacy.

Endosomal-lysosomal proteolysis mediates death signalling by TNFalpha, not by etoposide, in L929 fibrosarcoma cells: evidence for an active role of cathepsin D.

In several 'in vitro' models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFalpha, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase II-inhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFalpha. In contrast, pre-loading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFalpha cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFalpha or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFalpha and occurs within the endosomal-lysosomal compartment.

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas.

Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen.