Several recent studies have established that DTX induces tumor cell death primarily through mitotic catastrophe and apoptosis [11–16]. However, the role of a lysosomal pathway in DTX-induced cell death has not been thoroughly investigated. DTX-induced apoptosis in melanoma and prostate cancer cells has been shown to involve activation of caspases-2 and -3, and changes in MMP [14, 16]. Mhaidat et al  showed that changes in MMP were almost completely inhibited after caspase-2 knockdown, suggesting that DTX-induced apoptosis is dependent on caspase-2 activation . Hernandez-Vargas et al  reported that the coupling of mitotic catastrophe with apoptosis occurs in breast cancer cells only at 0.1 μM (100 nM) concentrations, whereas very low concentrations of 2–4 nM induce mitotic catastrophe followed by a late necrosis. An earlier study by Schimming and colleagues  provided evidence that DTX is a potent inducer of mitotic arrest, apoptosis, and necrotic-like tumor cell lysis with pro-inflammatory properties in 15 syngeneic mouse xenografts.
Consistent with these previous findings, our present study demonstrates that DTX induced mitotic catastrophe as well as apoptosis associated with caspase-2 and -3 activation, loss of MMP, and DNA fragmentation in the HRPC cell line PC3. The loss of MMP and the DNA fragmentation were largely caspase-dependent since they were inhibited by Z-VAD-FMK. However, we noticed that although inhibition of caspases-2 and -3 with Ac-VDVAD-CHO and Z-VAD-FMK reduced DTX-induced cytotoxicity, it did not completely block cell death, as assessed by viability assays, morphological visualization, and analysis of LMP. These results indicated that DTX-induced cell death is not entirely dependent on caspase activation since, in addition to mitotic catastrophe and apoptosis, it involves a caspase-independent pathway associated with LMP.
Under our experimental conditions, DTX-induced caspase-independent cell death did not appear to involve necrosis since it was not associated with the generation of the necrotic signature cleavage fragments of PARP and topoisomerase 1 in cells treated with this drug in the presence of Z-VAD-FMK (Fig. 1B and data not shown). In previous reports we described the generation of necrosis-specific fragments of PARP and topoisomerase 1 in cancer cells undergoing primary necrosis, secondary necrosis, and caspase-independent cell death with necrotic morphology [45, 50, 51]. It is also unlikely that DTX-induced caspase-independent cell death occurs through autophagy, a stress response mechanism triggered by environmental stressors (i.e., starvation, oxidative stress, drugs) which often results in non-apoptotic cell death and involves cathepsin activation and lysosomal dysfunction . We reached this conclusion after immunoblotting analysis did not show a time-dependent upregulation in the expression of the autophagy markers LC3bII and Beclin in DTX-treated PC3 cells (data not shown).
LMP and cathepsin activation are events associated with chemotherapy-induced cell death [48, 49]. These events occur during both caspase-dependent and independent cell death, and involve the activation of cathepsins B, D, and L, [48, 49]. These proteases contribute to cell death by activating effectors such as mitochondria-associated proteins, caspases, apoptosis-inducing factor (AIF), or by directly cleaving nuclear and cytoplasmic factors [48, 49]. Our data suggest that DTX-induced caspase-independent cell death involves LMP associated with cathepsin B activity. Cathepsins D and L do not appear to contribute significantly to this process since their inhibition did not attenuate DTX-induced cell death. These results are consistent with a report implicating LMP and activation of cathepsin B, but not cathepsin D, in caspase-independent cell death induced by the microtubule stabilizing agents paclitaxel, epothilone B, and discodermolide in lung cancer cells .
The release of cathepsin B from lysosomes typically follows induction of LMP by agents such as reactive oxygen species, fatty acids, and microtubule toxins . Cathepsin B itself can induce LMP, although the mechanisms are unknown . Once released from the lysosomes, cathepsin B can induce both caspase-dependent and independent cell death, as well as nuclear fragmentation . Interestingly, our data show that inhibition of cathepsin B alone is not sufficient to completely block DTX-induced LMP and DNA fragmentation. However, inhibition of both cathepsin B and caspases led to a dramatic reduction of these events. These findings point to cathepsin B as a key mediator of lysosome-mediated cell death induced by microtubule-stabilizing drugs. They also suggest that DTX-induced cytotoxicity involves the concerted play of caspases and cathepsin B.
Several endogenous inhibitors of lysosome-mediated cell death have been identified [48, 49]. Among these, heat-shock protein (Hsp) 70 family members and LEDGF/p75 have recently attracted attention due to their frequent overexpression in tumors, and cytoprotective and chemoresistance properties. Daugaard et al  showed that ectopic expression of LEDGF/p75 protected cancer cells against the LMP-inducing agents siramesine and doxorubicin . Our data showed that ectopic overexpression of LEDGF/p75 attenuated DTX-induced cytotoxicity and LMP but had no effect on TRAIL-induced cell death. Overexpression of LEDGF/p75 also attenuated DTX-induced death in RWPE-2 cells but was not effective against TRAIL and STS (data not shown). This suggested that LEDGF/p75 could be a selective inhibitor of cell death, caspase-dependent or independent, associated with LMP. Interestingly, LEDGF/p75 overexpression in PC3 cells failed to prevent DTX-induced multinucleation, suggesting that DTX-induced mitotic catastrophe is not coupled to LMP.
The mechanisms by which LEDGF/p75 promotes lysosomal stability are presently unknown, although the available evidence suggests that this protein may protect cells against oxidative stress by transcriptionally activating stress and antioxidant genes that reduce intracellular ROS [24–26, 40, 41]. A recent study showed that LEDGF/p75 physically interacts with the oncogenic transcription factor menin and the MLL histone methyltransferase to activate cancer associated genes and promote leukemic transformation . Our group is currently exploring interactions between LEDGF/p75 and various transcription factors to determine if this protein's ability to promote chemoresistance is associated with its capacity to form protein complexes required for transcriptional activation of protective genes.