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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.

reside in endosomes and lysosomes, where they accomplish the extensive or limited proteolysis of substrates at acid pH, thus contributing to cell protein turnover and homeostasis and activation or degradation of biologically active molecules [18]. Recently, these lysosomal cathepsins have been shown to actively participate in the cell death program following their translocation from lysosome into the cytosol [19]. For instance, CB has been shown to mediate caspase-independent cell death by microtubule stabilizing agent in lung cancer cells [20] and CD has been shown to mediate the caspase-dependent death pathway triggered by various DNA-damaging agents including etoposide [21,22]. These findings underscore the importance of a lysosome-mitochondrion axis in the control of the death signal triggered by cytotoxic stress [23,24]. Therefore, the function of these two organelles should also be considered when assessing the sensitivity or resistance towards apoptosis-inducing chemotherapy drugs in cancer cells. In this respect, it is to note that resistance to cis-platinum in ovarian cancer cells was found associated with abnormalities of the lysosomal apparatus [9]. The present study was aimed at elucidating the potential role and the mechanism of action of CB, CL and CD in the cytotoxic response to etoposide (VePesid, VP16, Sigma-Aldrich Corp., St Louis, MO, USA) and Paclitaxel (Taxol, Bristol-Myers Squibb, New York, NY, USA), two drugs that have a welldocumented clinical efficacy against ovarian cancers that manifest resistance to platinum-based therapy [4,25]. We analysed the cytotoxic pathways of these drugs in two human ovarian cancer cell lines, namely A2780 and NIHOVCAR3 (OVCAR), that have been reported to express normal or mutated p53, respectively [26,27]. Both VP16 and taxol turned out to be toxic to A2780, not to OVCAR cells. We found that chemosensitivity is associated with Bax-mediated permeabilization of lysosomes and mitochondria and that CD activity is mandatory for Bax activation and translocation onto lysosomes and mitochondria. Our data also indicate that Bax deficiency may result in the inability to permeabilize the lysosomes under toxic conditions, thus representing a primary defect that confers resistance towards chemotherapy drugs in cancer cells. We conclude that the CD-Bax cooperation is a critical determinant of chemosensitivity or chemoresistance in cancer cells.

Materials and methods
Chemicals were of analytical grade. Unless otherwise specified all reagents, including antibodies, were purchased from Sigma-Aldrich Corp.

Cell culture and treatments
The human ovarian cancer cell lines A2780 [28,29]. The same technology was employed for post-transcriptional silencing of Bax using the sequence 5Ј-GGAACUGAUCAGAACCAUCAUGGGCUG-3Ј for the sense strand of siRNA [29]. An inefficient control-duplex oligonucleotide was used as a negative control of transfection (sham

Cathepsin D activity
To assay CD activity an aliquot of cell homogenate was incubated for 12 hrs at 37°C in a formate-acetate buffer (12.5 mM, pH 3.65) with 1 g aminomethylcoumarine-haemoglobin substrate in 50 l final volume [30].

Statistical analysis
All experiments were independently replicated at least three times. Data are presented as means Ϯ S.D. The Microsoft Excel XLStats software was used. Significance was taken at a P-value less than 0.05.

Chemotherapy drugs activate a Bax-mediated intrinsic death pathway in A2780 cells not in OVCAR cells
Preliminary experiments in which the cells were exposed for 24 to 72 hrs to increasing doses of cis-platinum (0.5-5.0 M), resveratrol (10-200 M), VP16 (1-20 M) or taxol (10-100 nM) revealed that A2780 cells are sensitive whereas OVCAR cells are resistant towards all tested chemotherapy drugs. These drugs exert their cytotoxicity through the activation of the intrinsic apoptosis pathway [12,22,28,31,32]. To identify the molecular basis for the different sensitivity to chemotherapy drugs, we first explored the functionality of this pathway in the two cell lines. VP16, a drug widely used in the therapy of ovarian cancers that show resistance to cis-Platinum [25], was employed as a paradigm of anticancer drug. In some experiments we also included taxol, which also is employed in the second line therapy of drug resistant ovarian cancers [4]. For the cytotoxic treatments, we chose the lowest dose of VP16 and of taxol that revealed to be most effective in a 24-hr incubation, i.e. 10 M VP16 and 50 nM taxol, in the chemosensitive A2780 cell line. Both the treatments elicited an increased expression of p53 in the two cell lines, although this effect was prominent in A2780 cells (Fig. 1A). (Fig. 1B)

Bcl-2 and Bax are differently modulated in A2780 and OVCAR cells in response to cytotoxic treatments
Bcl-2 and Bax are members of the same family with opposite regulatory function in apoptosis, being bcl-2 anti-apoptotic and Bax pro-apoptotic [33]. The coordinate expression of these two proteins is considered a determinant factor in chemosensitivity and chemoresistance of ovarian cancer cells [34][35][36][37][38][39]. Western blotting assessment of the relative expression of bcl-2 and Bax in response to the cytotoxic drug VP16 shows that: (1) bcl-2 is highly expressed in OVCAR, and this expression is strongly reduced in cells exposed for 24 hrs to VP16, whereas A2780 basally express this protein at very low level ( Fig. 2A); (2) Bax, on the other hand, is expressed at fairly high level in A2780 cells whereas it is faintly detectable in OVCAR cells (Fig. 2B) and (3) [40]. Bcl-2 exerts its anti-apoptotic function by hetero-dimerization with Bax, thus preventing its oligomerization on mitochondria [41]. We looked at the localization of these proteins in A2780 and OVCAR cells exposed to VP16. The images in Fig. 2C show that in both control A2780 and OVCAR cells bcl-2 and Bax proteins are distributed throughout the cytoplasm and, apparently, do not form complexes. It is to note that Bax is revealed by the antibody only upon a conformational activation that exposes its N-terminus [15]. In A2780 cells, but not in OVCAR cells, the treatment with VP16 leads to the conformational activation of Bax that is revealed as macro-aggregates intensely positive (Fig. 2C). Apparently, bcl-2 does not co-localize within this macro-aggregates. Altogether, the present data indicate that expression and conformational activation of Bax, rather than bcl-2 expression, are the determinant factors in the response to chemotherapy drugs.

Inhibition of cathepsin D, not of cathepsins B or L, prevents cell death induced by chemotherapy drugs
Bax-mediated permeabilization of mitochondria and chromatin alterations in VP16-and taxol-induced cell death (Fig. 1) are suggestive of the involvement of lysosomal cathepsins [19]. To assess the possible participation of the cysteine proteases CB and CL and/or the aspartic protease CD in this process we first employed the inhibitors CA074Me and Pst, which are rather specific for CB and CL, and for CD, respectively [42][43][44]. With respect to cell density at time zero, cell loss (amounting to approximately 50%) was observed in A2780 cultures exposed for 24 hrs to either VP16 or taxol. This cell loss was completely prevented by Pst, not by CA074Me (Fig. 3A). Final cell density in CA074Me-treated A2780 cultures was reduced compared to that in control untreated or only Pst-treated counterparts, indicating that inhibition of CB and CL was itself cytotoxic to some extent to these cells (Fig. 3A). OVCAR cells showed once again resistance to both chemotherapy drugs and also non-sensitive to the growth-inhibitory effect of CA074Me (Fig. 3A) (Fig. 3B, lower panel). By contrast, in siRNAtransfected cultures the cytotoxic effect of both the chemotherapy drugs was negligible (Fig. 3B, lower panel). Cytofluorometry analysis of annexin V-labelled cells confirmed the protection by CD-siRNA against VP16 and taxol cytotoxicity (not shown). The fact that Pst and CD-siRNA elicited the same protective effect indicates that CD-mediated proteolysis is a determinant factor of the cytotoxic response to the above chemotherapy drugs.

Cathepsin D activity is essential for Bax-mediated permeabilization of mitochondria and apoptosis
Bax has been shown to promote mitochondrial permeabilization and relocation of pro-apoptotic proteins from mitochondria into the cytosol [46]. In various paradigms of cytotoxic treatments conformational changes and oligomerization on mitochondrial membrane of Bax were shown to depend on CD [15,28,29]. Fig. 4A and B). Caspase

3-mediated hydrolysis of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, results in the accumulation of nicked DNA in nuclei of apoptotic cells that can be demonstrated with the TUNEL technique. Thus, to definitely prove the involvement of CD in caspase 3 activation we checked for the presence of TUNEL-pos-
itive cells in A2780 cultures exposed to VP16 along with Pst. As expected in case of true caspase-dependent apoptosis, A2780 cells exposed to VP16 showed TUNEL positive (Fig. 4C). However, when the cytotoxic treatment was performed in the presence of Pst the cell monolayer appeared well preserved and nuclei did not stain for TUNEL (Fig. 4C), indicating that in these cells activation of caspase 3 was precluded. These data indicate that in chemotherapy drugsensitive cells activation of the CD-mediated proteolytic pathway precedes that of the caspase cascade.

Lysosome permeabilization, not cathepsin D expression, is the limiting factor for the activation of the intrinsic death pathway
Next, we investigated the molecular link between the proteolysis mediated by a lysosomal protease (i.e. CD) and the activation of a cytosolic protein (i.e. Bax). First, we considered the possibility that chemoresistance in OVCAR cells could arise from abnormally low expression of CD. Yet, when analysed by Western blotting, the basal expression level of CD was found much higher (~15 folds) in OVCAR than in A2780 cells (Fig. 5A). On treatment with VP16 the expression of CD did not change in OVCAR cells, whereas it greatly increased (~10 to 15 folds at 24 and 48 hrs, respectively) in chemosensitive A2780 cells (Fig. 5A). Based on previous findings [15,28,29,47,48], we hypothesized that Bax activation followed the cytosolic translocation of CD upon cytotoxic treatments. We thus looked at the compartmentalization of CD in the two cell lines under cytotoxic treatment. To better assess the possible relocation of CD from lysosomes into the cytosol, we labelled the

Fig. 3 Cathepsin D, not cathepsin B, mediates chemotherapy drug toxicity in A2780 cells. (A) A2780 cells and OVCAR cells plated on Petri dishes were exposed or not for 24 hrs to VP16 or taxol (Tax) in the absence or the presence of the CD inhibitor Pst or the CB inhibitor CA074Me (CA). At the end, cells were harvested and counted. The experiment was repeated four times in triplicate. Data are given as mean Ϯ S.D. In A2780 cells (top panel) both VP16 and taxol induced cell loss, which was prevented by Pst not by Ca074Me (compare final density at day 1 in treated versus initial density at d0 in cultures; note that Pst abrogated cell death by the chemotherapy drugs though it not completely rescued the cells from a growth block). Inhibition of CB and CL, on the other hand, did not protect from drug toxicity and, on itself, caused some cell loss in A2780 culture. In OVCAR cells (bottom panel) VP16 and taxol were not toxic and no effect were exerted by the cathepsin inhibitors either alone or in combination with the chemotherapy drugs. (B) A2780 cells plated on Petri dishes were transfected with a control duplex RNA (sham) or a CD specific siRNA and 24 hrs later incubated in fresh medium for further 24 hrs in the absence or in the presence of VP16 or taxol (Tax). Prior to start the treatment, CD activity was assayed in parallel homogenates to ascertain down-regulation of CD in siRNA-transfected cultures (upper panel). At the end of the incubation, cell survival was estimated counting the adherent trypan-blue excluding cells (lower panel). Data (mean Ϯ S.D.) are given as percentage of controls and arise from three independent experiments.
lysosomes with an antibody directed against Lamp1, an integral protein of lysosomal membrane. The images in Fig. 5B [49]. OVCAR and A2780 cells adherent on coverslip were exposed to VP16 and observed under the fluorescence microscope after AO staining. In control cells AO fluorescence appeared as intense red spots, indicating that it was confined within acid organelles (Fig. 5C). In OVCAR cells the AO fluorescence pattern was not affected by the cytotoxic treatments. By contrast, in treated A2780 cells a diffuse AO green-like fluorescence, indicative of cytosolic localization of the fluorochrome, was observed (Fig. 5C).

Bax mediates the permeabilization of lysosomes induced by VP16 in chemosensitive A2780 cells
How lysosomal membrane is permeabilized, thus allowing cathepsins to escape into the cytosol, following the treatment with a cytotoxic drug remains elusive. It has been proposed that members  of the bcl-2 family could also affect the stabilization of lysosomal membranes [50]. Consistently, Bax has been shown to insert onto lysosomal membranes in staurosporine-treated fibroblasts [51]. We found that on treatment with VP16, Bax in fact was activated and formed macro-aggregates positive also for Lamp1 in A2780 cells, but not in OVCAR cells (Fig. 6A). To see whether lysosomal relocation of Bax was functionally related to the permeabilization of lysosomes in VP16-treated A2780 cells, we specifically downregulated the expression of Bax in these cells and looked at the compartmentalization of CD and at lysosome integrity. Bax gene expression in A2780 cells was efficiently silenced (~85%) after a 24-hrs transfection with a specific siRNA (Fig. 6B). Under such conditions, VP16-induced cytosolic relocation of CD (Fig. 6C) and of AO (Fig. 6D) (Fig. 6E, upper panels). Bax oligomerization and mitochondria permeabilization, on the other hand, became apparent at 16 hrs (Fig. 6E, lower panels), indicating that apoptosis started at this time-point.

Cathepsin D triggers the translocation of Bax onto lysosome membrane upon VP16 treatment
Since Bax activation and oligomerization on mitochondria was proven to depend on CD activity (see Fig. 4A and B and [28,29]), we wondered whether CD was also implicated in Bax activation and relocation on lysosomal membranes. We thus looked at Bax localization in VP16-treated cells in which CD was either inhibited by pre-loading with Pst or post-transcriptionally down-regulated by siRNA transfection. The Bax-Lamp1 co-localization results shown in Fig. 7 clearly demonstrate that CD activity is essential to induce the activation of Bax and its oligomerization onto lysosomes under chemotherapy drug treatment. These data suggest the existence of a positive loop of activation between CD and Bax that amplifies the death signal and leads to a generalized permeabilization of lysosomes and mitochondria, which can be assumed as the point of no return in the apoptotic process.

Discussion
Resistance to chemotherapy drugs is the principal factor limiting long-term treatment success in ovarian cancer management [4]. The molecular dissection of the pathways activated by cytotoxic drugs may help to identify the molecular mechanisms implicated in multidrug resistance, and can positively reflect on designing new therapeutic approaches to circumvent this phenomenon [5]. Among the factors that concur to the resistant phenotype in cancer cells are the overexpression of ATP-dependent transporters that actively extrude the cytotoxic drugs [52] and defective activation of the apoptotic pathway due to altered expression of key regulators or executioners [12,21,31,34]. Mutation in the oncosuppressor p53 and deficient expression of Bax have been associated with chemoresistance and poor prognosis in ovarian  Fig. 4

(B). Parallel coverslips were pre-incubated or not with Pst (A). The cells were then exposed or not for 24 hrs to VP16 and thereafter processed for immunofluorescence staining of Bax (green fluorescence) and Lamp1 (red fluorescence). Bax staining is faintly detectable and diffused in the cytoplasm of untreated cells, whereas it appears as intense yellow spots, indicative of co-localization with Lamp1 (arrows), in sham-transfected and in Pst-non-preincubated A2780 cells exposed to VP16. Both siRNA-mediated downregulation or Pst-mediated inhibition of CD prevented the Bax translocation onto lysosomes in VP16-treated cells, as demonstrated by the lack of co-localization with Lamp1. In these cells Bax staining is faintly detectable and diffused in the cytoplasm as in control untreated cells.
cancers [31,37,53]. The present study focused on the cytotoxic mechanisms of VP16 and taxol, two drugs currently employed for the cure of ovarian cancer refractory to platinum-based chemotherapy [4,25]. We dissected the apoptotic pathway activated by these drugs in two ovarian cancer cell lines, namely the A2780 cell line known to express only the wild-type form of p53 [26] and the OVCAR cell line that express a functionally inactive mutant form of p53 [27]. A2780 cells were shown to be far more sensitive than OVCAR cells in response to both VP16 and taxol, and also to cis-platinum and resveratrol. The ratio of Bax to bcl-2 expression in the cell is a critical determinant of the threshold at which apoptosis is induced by chemotherapy drugs [33].  [55], and it is known that the cytosolic pH of cells undergoing apoptosis drops to 6.8-5.7 [56]. More recently, we have demonstrated that CD affects a limited proteolysis on prolactin at physiological pH, which results in the generation of bioactive peptides [57,58]. Therefore, it is conceivable that mature CD can act on small substrates at the cytosolic pH in cells undergoing apoptosis. It has been reported that cytosolic acidification is mandatory for mitochondrial translocation of Bax during drug-induced apoptosis of tumour cells [59]. We hypothesize that CD triggers the conformational activation of Bax by either affecting a limited proteolysis on Bax itself (though no changes in the apparent molecular weight of Bax was observed in our experiments) or, more likely, by proteolytic inactivation of a Bax-interacting chaperone that prevents Bax activation. One such candidate could be clusterin, which is highly expressed in malignant tumours and specifically binds to Bax in response to chemotherapeutic drugs [60]. Alternatively, CD proteolysis facilitates the binding of Bax with a chaperone (e.g. nucleophosmin) that promotes its translocation onto mitochondria [61].
Another important and original finding of the present study is that the permeabilization of lysosomes follows the activation and oligomerization of Bax onto lysosomal membranes and that CD activity is essential for such an event. This demonstrates the existence of an activation loop between CD and Bax that serves to recruit an increasing number of Bax molecules. With time, the number of active Bax molecules will exceed the number of bcl-2 and bcl-XL molecules available for heterodimerization, so that free active Bax molecules can start to oligomerize on mitochondrial membrane. This event will mark the 'point of no return', as it will determine the release of cytochrome c and other pro-apoptotic factors that will finally form the apoptosome. We propose the following model of toxicity of pro-apoptotic drugs: (i ) a CD-mediated proteolysis of a so far unknown substrate leads to the activation of Bax and in turn to Bax-mediated permeabilization of lysosomes and consequent cytosolic relocation of cathepsins; (ii ) with time, active Bax molecules reach a number sufficient to allow their dimerization on mitochondria and (iii ) the permeabilization of mitochondria allows the cytosolic relocation of factors needed for the formation of the apoptosome and consequent activation of the caspase cascade. It is to note that the chemotherapy drug treatment induced lysosomal permeabilization in A2780, not in OVCAR cells. Chemoresistance of p53-defective OVCAR cells could be due to: (i ) insufficient accumulation of the drug within the cell (possibly because of the overexpression of outward transporters) or (ii ) inability to rise up the cellular levels of Bax and/or CD that are, in fact, transcriptionally targeted by p53 [21,62] or (iii ) other unknown factors. However, it can be assumed that VP16 reached in OVCAR cells a concentration sufficient for signalling as it caused a marked up-regulation of p53 and a concomitant strong down-regulation of bcl-2 expression. Also, unlikely CD activity in OVCAR cells was not sufficient to start the apoptotic process, as the basal expression of CD in these cells was by far much higher than in A2780 cells. We rather suspect that the inability to initiate the permeabilization of lysosomes in chemoresistant OVCAR cells arises from insufficient Bax expression. In conclusion, we have shown that the cooperation between CD and Bax is essential to determine the permeabilization of lysosomes and that this represents the primary and limiting factor that determines the responsiveness to chemotherapy drugs. Forcing the leakage of cathepsins from lysosomes could be a strategy to overcome chemoresistance, provided that the down-stream apoptotic machinery is intact.