Overexpression of both catalytically active and -inactive cathepsin D by cancer cells enhances apoptosis-dependent chemo-sensitivity

Abstract

The aspartic protease cathepsin D (cath-D) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, cath-D is translocated to the cytosol. Because cath-D is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically active relative to the cysteine lysosomal enzymes such as cath-B and -L, it is therefore open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of wild-type cath-D and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of cath-D catalytic function. We demonstrate that wild-type or mutated catalytically inactive cath-D strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wild-type and mutated inactive cath-D are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and -3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly therefore, the stimulatory effect of cath-D on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic cath-D stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).

Cathepsin D—Many functions of one aspartic protease

“…Microinjection of wt or mutant CD induces apoptosis in human fibroblasts that was not inhibited by pepstatin A [120]. Both wild-type and mutant CD strongly enhances apoptotic response to etoposide, doxorubicin and 5-fluorouracil in rat tumor cell line [124]. These data highlight the possibility that CD pro-apoptotic effect may be mediated by interaction with some member(s) of apoptotic machinery.…”

“…This leads to mitochondrial dysfunction. The release of CD may cause mitochondrial dysfunction either directly [114]; by ability of CD to cleave Bcl-2 family member Bid, subsequent formation of active Bax conformation and insertion of active Bax in the outer mitochondrial membrane [69]; or by interaction with unknown member of apoptotic machinery where CD enzymatic activity is not involved [124]. CD-induced mitochondrial dysfunction results in release of cyt c from mitochondria followed by activation of caspase-9 and -3 [69,114].…”

“…While many functions of CD in the physiological and pathological processes could be attributed to its enzymatic activity, it is evident that some of its functions in the organism are independent on its protease activity and rely on the ability of pCD/CD to interact with other important molecules (Tab 2) [27,33,120,124,130,186,194,212,220]. It seems inevitable that searching for pCD/CD-interacting partners should be conducted in the next years to explore the mechanism of pCD/CD actions.…”

“…While some studies reported that CD can directly induce apoptosis [118][119][120], many studies demonstrated that CD is a mediator of apoptosis induced by several stimuli, such as staurosporine, IFN-gamma, Fas/CD95/APO-1, TNF, etoposide, 5-fluorouracil, cisplatin [118,121,122], adriamycin [122], resveratrol [123], doxorubicin [124], growth factor deprivation [125], oxidative stress [109,126,127], and sphingosine [113]. These data suggests that CD promote apoptosis induced by cytotoxic and stress agents.…”

Cathepsin D Primes Caspase-8 Activation by Multiple Intra-chain Proteolysis

“…2). Indeed, cathepsin D was shown to be inactivated at the cytoplasmic pH (40). Therefore, how can cathepsin D activate caspase-8 within a cell?…”