In this study we showed that FYAD binds to both cathepsins B and L and that complete inhibition of both of these enzymes is maintained in cultured neuroblastoma cells, creating a chemical knockout of both enzyme activities. The chemically related compound, ZFYD, bound only to cathepsin L and inhibition of this enzyme was also shown to be maintained in cultured neuroblastoma cells. The ability of the diazomethanes to penetrate cell membranes and to selectively bind only to active cathepsins in cells makes them unique tools for the complete inhibition of cellular cathepsins [17
]. The more widely used cathepsin B inhibitor, Ca074Me, was shown to be less effective at inhibiting cathepsin B and was not specific for this enzyme in cells. The un-esterified inhibitor, Ca074, has been shown to inhibit cathepsin B and not cathepsin L or calpain 1 [28
], but the charged carboxyl group of this compound impairs cellular penetration. While esterification enables the compound to enter cells as Ca074Me, this modified compound reacts with both cathepsins B and L and could react with additional enzymes and proteins [30
In our studies, complete inhibition of both cathepsins B and L induced cell death in the neuroblastoma cell lines (SK-N-SH and IMR-32) but not other tumor cells. Inhibitors that affected only one enzyme or failed to maintain inhibition of both cathepsins B and L did not induce apoptosis of these cells. This contrasts with other reports of inhibitors of either single or multiple cathepsins that cause cell death. Z-Phe-Gly-NHO-Bz is an inhibitor of both cathepsins B and L that causes death of many cell types [13
]. The specificity of this inhibitor in cellular systems is not well-established and in aqueous solution it hydrolyzes to an hydroxamate that can react with other enzymes [36
]. Inhibition of cathepsin B alone with Ca074Me has previously been proposed as a potential treatment for neuroblastoma, although the efficiency and specificity of cathepsin inhibition was not examined in this study [5
]. The specificity of Ca074Me in cellular systems has not been shown by direct protease labeling and it has been shown to have cellular effects unrelated to cathepsin B inhibition, making it less useful for in vivo studies of cathepsin function [37
]. Although many cathepsin inhibitors are commercially available, few have been shown to be specific in cellular systems.
Low-level inhibition of the proteases did not affect growth of SK-N-SH cells and 95 -100% enzyme inhibition was required to cause cell death. Lysosomal concentrations of cathepsins can be as high as 1 mM [17
], and consequently partial enzyme inhibition is unlikely to have a major impact on cellular proteolysis. We have not been able to generate siRNA techniques that stably knock-down expression of cathepsins B or L in neuroblastoma cells by more than 70% so are unable to confirm our inhibitor studies with this technology. The major targets of FYAD are clearly shown to be cathepsins B and L () and a related inhibitor that does not inhibit both cathepsins B and L does not cause cell death indicating that efficient inhibition of cathepsins B and L is most likely the cause of neuroblastoma cell death.
A direct role in apoptosis is becoming established as an important physiological role for cathepsin B, and stimulation of cathepsin B mediated apoptosis by lysosomal membrane disruption is proposed for cancer therapy [8
]. Cell death can also be stimulated by lysosomal rupture and release of cathepsin D [39
]. There was little evidence of lysosomal rupture in FYAD treated neuroblastoma cells and addition of pepstatin to treated cells did not prevent cell death in our studies. Pepstatin alone had little effect on growth of IMR-32 and SK-N-SH neuroblastoma cells and did not impair cell death induced by FYAD treatment. This indicates that it is lysosomal protease inhibition that initiates the cell death process and that release of other lysosomal enzymes such as cathepsin D into cytosol is not a significant cause of cell death after FYAD treatment of these neuroblastoma cells.
The mechanism by which cathepsin inhibition leads to cell death is not clear, but is likely to be due to accumulation of one or more proteins that are normally degraded by the concerted action of cathepsins B and L. Electron microscopy clearly shows that inhibition of cathepsins B and L caused major accumulation of dense granules in both IMR-32 and SK-N-SH neuroblastoma cell lines within 24 h. Although there is some evidence of apoptosis 24 h after treatment, it is likely that accumulation of undigested proteins precedes induction of apoptosis. Accumulation of autophagic-like vacuoles are seen in double knockout mice lacking both cathepsins B and L [42
], indicating that autophagy is probably induced prior to apoptosis. More detailed studies are required to elucidate the critical steps that lead to death of neuronal cells in the knock-out animal studies and death of neuroblastoma cells in our inhibitor studies.
Data from genetic deletion of cathepsins B and L in mice provide some support for the selective induction of apoptosis that we see for neuroblastoma cells. Deletion of cathepsin L in mice shows that this enzyme plays a role in endosomal processing of the MHC II invariant chain and turnover of EGF and its receptor but there is no evidence that deletion of this enzyme induces cellular apoptosis [44
]. A major phenotype is actually uncontrolled proliferation of keratinocytes, resulting in periodic hair loss [44
]. Apoptosis is not induced by genetic deletion of cathepsin B either, but TNF-induced apoptosis (a process mediated by cathepsin B activity) is blocked in these mice [46
]. However, double knockout mice lacking both cathepsins B and L show significant evidence of apoptosis in neuronal cells, with particularly significant loss of Purkinje cells [42
]. Other cell types are not significantly affected, supporting our observation that both enzymes must be inhibited and that apoptosis may be specifically induced in proliferating cells of neuronal origin. Current cancer therapies target abnormal growth characteristics of tumor cells while cathepsin inhibition may be targeting a normal function of proliferating cells of neuronal origin. The common defect of all neuroblastoma tumors is a failure in differentiation and they continue to proliferate after most normal cells have differentiated into cells of the peripheral nervous system.
Degeneration of the central nervous system by genetic deletion of both cathepsins B and L raises some concerns about potential use of inhibitors of these enzymes therapeutically. However, inhibitors of cathepsin B (and L) have been shown to have a neuroprotective effect in animal models of ischemia, preventing apoptosis [48
] and prolonged treatment of animals with cathepsin inhibitors do not produce any significant neurological toxic effects [19
]. The deleterious effects of protease loss in the cathepsin B and L double knockout mice are primarily due to early post-natal effects on rapidly proliferating neuronal cells whereas non-proliferating cells of the central nervous system of adult mice appears to be refractory to protease inhibition. Thus in children, particularly those over two years old, post-mitotic neuronal cells may not be affected by treatment with protease inhibitors, making such compounds potentially useful for development of treatments to eradicate neuroblastoma. Nevertheless, careful monitoring of potential adverse neurological effects of inhibitor treatment would need to be monitored and drugs that do not cross the blood-brain barrier may provide a less toxic treatment of this cancer of the peripheral nervous system. The unusual sensitivity of neuroblastoma cells to inhibitors of cathepsins B and L may provide a novel therapeutic approach to treat this particularly intractable childhood cancer.