Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis

Despite expressing both Fas and Fas ligand, DU145 and LNCaP prostate cancer cells were resistant to anti-Fas-induced cell death. Resistance to Fas-mediated cytotoxicity could be overcome in DU145, but not in LNCaP, cells by pretreating cells with sublethal doses of cytotoxic drugs, such as camptothecin. Activated caspases were shown to be required for this cytotoxicity. Indeed, poly(ADP-Ribose) polymerase was shown to be proteolytically cleaved in cells treated with camptothecin plus anti-Fas, but not in cells treated with anti-Fas only. Moreover, pretreatment of cells with ZVAD completely blocked camptothecin-mediated Fas-induced apoptosis. Sensitization of cells to Fas-induced cell death did not involve up-regulation of Fas or FasL, and it was independent of alterations in the cell cycle. Reactive oxygen intermediates (ROI) have been shown to be important mediators of drug-induced apoptosis. Here, we demonstrate that treatment of DU145 cells with camptothecin, anti-Fas, or both, did not alter the intracellular levels of peroxide or superoxide anion. © 1999 Cancer Research Campaign

resistant to Fas remain to be fully elucidated. It seems that there is not a strict correlation between Fas and Fas ligand (FasL) expression and sensitivity to Fas-mediated cytotoxicity. In fact, activation of the Fas signalling pathway(s) in a variety of tumour cell lines and in activated lymphocytes does not always trigger apoptosis (Owen-Schaub et al, 1994). Caspases are thought to play a central role in apoptosis and de-regulation of some caspases may contribute to the development of some cancers. Furthermore, there is evidence that caspase-dependent cleavage of specific proteins may abrogate important survival pathways (Widmann et al, 1998). Importantly, commitment to Fas-induced apoptosis was shown to require caspase activity (Longthorne et al, 1997). Following Fas trimerization there seems to be sequential activation of caspase-1 (ICE)-and caspase-3 (CPP32)-like proteases (Enari et al, 1996). The activated caspases can then cleave various substrates, which include Poly(ADP-Ribose) polymerase (PARP), Lamin, rho-GDI, Actin, Ras GTPaseactivating protein, Raf-1 and Akt-1, eventually resulting in morphological changes to the cells and nuclei (Nagata, 1997;Widmann et al, 1998).
Previous work from our laboratory has shown the importance of reactive oxygen intermediates (ROI) during apoptosis in HL-60 and U937 leukaemic cell lines (McGowan et al, 1994). In particular, peroxide production has been reported in response to a variety of inducers of apoptosis (Gorman et al, 1997b;McGowan et al, 1998). The production of peroxide after cytotoxic insult and the ability of antioxidants to inhibit PARP cleavage and apoptosis, suggest that early activation of caspases may act as a common response to cytotoxic insult and that ROI may serve as common mediators of cell death in leukaemic cell lines (McGowan et al, 1996). In contrast to a well-established role in chemotherapeutic drug-induced apoptosis, the involvement of ROI in Fas-mediated signal transduction pathway(s) is still controversial. Schulze-Osthoff and colleagues (1994) have shown that neither inhibitors of the mitochondrial respiratory chain nor antioxidants protected murine fibrosarcoma cells from Fas-mediated cell death. In contrast, ROI have been reported to be involved in Fas-mediated signal transduction pathway and to be major mediators of apoptosis in neutrophils (Kasahara et al, 1997). In addition, Dobbelsteen and co-workers (1996) have shown that Fas ligation results in a rapid and specific efflux of glutathione (GSH). Severe depletion of GSH will lower the reducing capacity of a cell, enhancing oxidative stress independent of any increase in the production of ROI. Furthermore, an oxidative stress due to elevated intracellular superoxide levels has been shown to block Fas-mediated apoptosis in cells that were constitutively Fas-sensitive (Clément and Stamenkovic, 1996). These studies suggest that an imbalance in ROI levels may play a role in Fas-mediated apoptosis.
Due to the complexity of prostatic cancer, successful treatment of the disease is likely to require a multimodality therapy involving cytokines (immunotherapy), cytotoxic drugs (chemotherapy) and/or hormonal ablation. The present study was designed to examine possible ways of bypassing the resistance of prostate cancer cell lines to Fas-mediated apoptosis and to further elucidate the signalling pathways involved in Fas signal transduction.

Cell culture
Prostate cancer cells were cultured in RPMI-1640 medium containing 5% (v/v) fetal calf serum, 2 mM L-glutamine, supplemented with penicillin-streptomycin (10 IU ml Ϫ1 , 10 µg ml Ϫ1 ) and fungizone amphotericin B (2.5 µg ml Ϫ1 ). Cells were grown at 37ЊC in a humidified 5% carbon dioxide atmosphere and were routinely subcultured every 4 days. Cell density and viability were assessed by trypan blue exclusion, using a haemocytometer.

Morphological analysis
Briefly, supernatants were transferred to labelled test tubes and cells were washed once in HBSS. Cells in each well were then trypsinized using Trypsin/EDTA (TE) for 10 min at 37ЊC. TE was neutralized with complete media and cells were transferred to the appropriate test tube. Cells were then cytospun at 100 g (500 rpm) for 2 min. Slides were stained using the Rapi-Diff II kit (Langan Back Services, Ireland) and cell morphology was assessed by light microscopy, using previously defined criteria (Wyllie et al, 1980).

Cell cycle analysis
Freshly harvested cells (2.5 ϫ 10 5 ml Ϫ1 ) were fixed in ice-cold ethanol (70%, v/v), on ice for 15 min. Ethanol was removed by centrifugation (200 g ϫ 5 min) and cells were resuspended in 1 ml of a PBS/PI (50 µg ml Ϫ1 ) solution containing RNAase (0.5 mg ml Ϫ1 ). Following a 30 min incubation on ice, in the dark, cells were analysed by flow cytometry in FL2-H, on a FACScan equipped with Cell Quest software.

Detection of intracellular ROI and measurement of mitochondrial transmembrane potential (∆ψm)
Mitochondrial membrane depolarization, peroxide and superoxide levels were measured as previously described (Gorman et al, 1997a;Costa-Pereira and Cotter, 1999). Briefly, cells (2.5 ϫ 10 5 ) were harvested and incubated with 5 µg ml Ϫ1 JC-1 (∆ψm measurement), for 15 min; or with 5 µM DCFH-DA (peroxide level) for 1 h at 37ЊC; or with 5 µM DHE (superoxide anion) for 30 min at 37ЊC. Fluorescence due to JC-1 and oxidized DCFH-DA was measured on a FACScan (Becton Dickinson, Mountain View, CA, USA), equipped with Cell Quest software (Becton Dickinson), in FL-1, whereas the fluorescence due to oxidation of DHE was measured in FL-2. Cells were treated with the apoptosis-inducing agents either before or during the incubation period, depending on the time point at which ROI or ∆ψm were to be studied.

Prostate cancer cells are resistant to Fas-mediated cytotoxicity
Fas and FasL expression were assessed in LNCaP and in DU145 prostate cancer cells by flow cytometry ( Figure 1A). Although both cell lines expressed the ligand and the receptor, treatment of cells with anti-Fas IgM (200 ng ml Ϫ1 ; 24 h) induced only moderate levels of apoptosis in the androgen-responsive cell line, LNCaP, having no significant cytotoxic effects on the androgen-independent cell line, DU145 ( Figure 1B). LNCaP (but not DU145) cells were more susceptible to Fas-mediated apoptosis when treated concomitantly with anti-Fas and cyclohexamide (CHX, 0.25 µg ml Ϫ1 ), suggesting the presence of an endogenous protein inhibitor of the Fas signalling pathway in these cells. These results are in contrast to an article by Rokhlin et al (1997). However, their DU145 were only susceptible to treatment with 1 µg ml Ϫ1 anti-Fas at high concentrations of CHX (2.5-25 µg ml Ϫ1 ), and the resistance displayed by their LNCaP cells is uncharacteristic of our cell lines. The sensitization of LNCaP cells mediated by CHX was not due to up-regulation of Fas, as assessed by flow cytometry (results not shown). Dose-response curves for both LNCaP and DU145 cell lines treated with anti-Fas IgM showed that, even at high anti-Fas IgM concentrations (1.5 µg ml Ϫ1 ), DU145 cells were resistant to anti-Fas-induced apoptosis, indicating that there is a bona fide blockage in the Fas apoptotic pathway(s) in these cells. LNCaP cells were more susceptible to anti-Fas and there was, to a certain extent, a dependency on the dose of antibody used. The apoptosis levels, however, never surpassed those observed when LNCaP cells were treated with 0.25 µg ml Ϫ1 CHX (data not shown).

Sensitization of androgen-independent prostate cancer cells to anti-Fas-induced cell death
To address the possible mechanism(s) involved in the resistance displayed by prostate cancer cells to Fas-induced apoptosis, we have used a number of cytotoxic drugs known to have distinct intracellular targets. The chemotherapeutic drugs used were: actinomycin D (RNA transcription inhibitor, which can also induce topoisomerase I and II cleavable complexes), camptothecin (topoisomerase I inhibitor), cis-platinum(II)diamminedichloride (CPDD) (non-classical alkylating agent, which forms intrastrands linkages with DNA) and etoposide (topoisomerase II inhibitor).

Camptothecin-mediated sensitization of DU145 cells to anti-Fas-induced cell death does not involve up-regulation of Fas or FasL proteins
Recent reports (Friesen et al, 1996;Herr et al, 1997) suggested that apoptosis caused by certain anticancer drugs was mediated by the Fas/FasL system. In order to determine whether camptothecin was sensitizing DU145 cells to Fas-mediated apoptosis by up-regulating Fas and/or FasL, cells were treated with camptothecin and flow cytometric analysis of Fas and FasL expression was assessed at different time points (4 and 24 h post-treatment). Results show that there was no change, at the protein level, of Fas or FasL (Figure 3), suggesting that sensitization was not due to upregulation of Fas and/or FasL.

Camptothecin's effect is independent of alterations in the cell cycle
Camptothecin has been reported to cause a cell cycle arrest in S/G2 (Hsiang et al, 1989). Thus it was considered that the sensitization to Fas-mediated apoptosis could be a consequence of cell cycle arrest. Flow cytometric analysis of untreated cells, or cells treated with camptothecin (100 ng ml Ϫ1 ), anti-Fas IgM (200 ng ml Ϫ1 ), or camptothecin plus anti-Fas, for 8 h, showed no significant cell cycle abnormalities in the cell cycle profile of each sample. After a 24 h treatment with camptothecin plus anti-Fas IgM, only sub-G0/G1 population was slightly increased indicative of apoptosis ( Figure 4).

Activation of caspases is required for camptothecinmediated sensitization of DU145 cells to anti-Fasinduced apoptosis
There have been several reports showing that caspases play a pivotal role in Fas-induced apoptosis (Enari et al, 1996;Longthorne and Williams, 1997). Western blot analysis demonstrated that poly(ADP-ribose) polymerase was proteolytically cleaved in camptothecin-treated cells, but not in cells treated with anti-Fas only ( Figure 5A). A down-regulation of PARP, in the absence of cleavage product, was noted in cells treated with camptothecin only. This may be due to an inhibition of transcription. Apoptosis in cells treated with camptothecin plus anti-Fas could be completely blocked by pretreatment with ZVAD (50 µM) ( Figure  5B). Thus, camptothecin sensitization involves the activation of caspases analogous to apoptosis in Fas-sensitive cells.

Mitochondrial depolarization is a late and non-specific event in DU145 cells undergoing camptothecinmediated anti-Fas-induced apoptosis
Emerging evidence suggests a key role for mitochondria during apoptosis induced by a diverse range of stimuli. Discontinuity of the outer mitochondrial membrane results, in some instances, in redistribution of cytochrome c from the intermembrane space to the cytosol, followed by subsequent inner mitochondrial membrane depolarization (Kroemer, 1997). Disruption of the transmembrane potential (∆ψm) and generation of reactive oxygen intermediates (ROI) have been reported as early events in apoptosis (Lennon et al, 1991;Kroemer, 1997). ∆ψm depolarization was only significant (but not complete) at 24 h, when Ͼ 80% of cells were dead ( Figure  6). ∆ψm disruption was evidently a late event in our system. Taken together, results suggest that, although degradation of ∆ψm might contribute to the apoptosis of DU145 cells, it is unlikely that camptothecin causes sensitization to anti-Fas through this mechanism.

Camptothecin opens the Fas apoptotic pathway(s) in DU145 cells through an ROI-independent mechanism
Apoptotic cells are believed to be subject to some form of oxidative stress, as they accumulate oxidized proteins and lipids (Buttke and Sandstrom, 1994) and, in a number of cell lines, apoptosis induced by chemotherapeutic drugs is accompanied by rapid production of ROI (Lennon et al, 1991;McGowan et al, 1996;Gorman et al, 1997b). Furthermore, Fas-mediated cell death has been shown to be blocked by superoxide anion (Clément and Stamenkovic, 1996), suggesting that an imbalance in ROI levels may have a role to play in Fas-mediated cell death. The existence of a short-lived inhibitor protein of Fas signalling transduction pathway(s) has been widely suggested. However, since the protein synthesis inhibitor cyclohexamide did not sensitize DU145 cells to anti-Fas-mediated apoptosis, we sought to investigate alternative mechanism(s) which did not directly involve proteins. We hypothesized that camptothecin could be opening the Fas pathway in DU145 cells by either  Apoptosis levels were assessed as described in Figure 1B A B increasing peroxide levels ( Figure 7A) or by directly or indirectly causing a fall in superoxide levels ( Figure 7B). None of the treatment (anti-Fas, camptothecin, camptothecin plus anti-Fas), under our experimental conditions, caused a significant increase in peroxide or a decrease in superoxide anion levels, as measured by flow cytometry. Camptothecin, at higher concentrations (1-10 µg ml Ϫ1 ), did cause a rapid and marked increase in ROI levels (results not shown), but not at the concentrations used here (100 ng ml Ϫ1 , 24 h treatment). While it is possible that an alternative mechanism of oxidative stress contributes to camptothecin and anti-Fas cytotoxicities, the data presented in this study suggest that ROI are not significant players in camptothecin-mediated sensitization of DU145 cells to anti-Fas-induced apoptosis.

DISCUSSION
Although prostate cancer cells expressed both Fas and FasL, they were significantly resistant to Fas-induced cell death. This observation, and the fact that the Fas/FasL system is emerging as one of the key apoptotic pathways in eukaryotic cells, prompted us to investigate why Fas-mediated apoptotic pathway(s) were blocked in these cells. Understanding the mechanisms underlying this resistance may allow the development of strategies for effectively killing prostate cancer cells, using a combination of cytotoxic and immune approaches. In this study, two prostate cancer cell lines (LNCaP, DU145) were used. It was shown that LNCaP and DU145 cells expressed both Fas and FasL. Treatment of LNCaP and DU145 with anti-Fas IgM and CHX up to 24 h induced moderate levels of apoptosis in LNCaP, having no significant cytotoxic effects on DU145 cells. LNCaP were more susceptible to anti-Fas when pretreated with CHX, suggesting that there might be a short-lived inhibitor in these cells. The existence of a hypothetical suppressor of Fas-induced apoptosis ('Saf' -suppressor of apoptosis by Fas) has been postulated (Green, 1997). Such suppressor is likely to exist, as in many cells resistance to Fas can be overcome by treatment with cyclohexamide or actinomycin D, which are thought to eliminate such short-lived inhibitor. FLIP (for Flice inhibitory protein), a molecule recently identified by Irmler and co-workers (1997), seems to be an appropriate candidate for such a role. A number of cytotoxic drugs were shown to sensitize androgen-independent cells (DU145) to anti-Fas-induced apoptosis. Indeed, DU145 cells were sensitized by ~20-fold when treated with subtoxic doses of camptothecin and concomitantly incubated with anti-Fas. This sensitization was not particular to topoisomerase I inhibitors, as actinomycin D, CPDD and etoposide also sensitized cells to cell death. Interestingly, we were unable to chemosensitize the androgen-responsive cells (LNCaP) using the above mentioned cytotoxic drugs. It has been postulated that chemotherapeutic drugs induced apoptosis by activating the Fas/FasL apoptotic pathway (Friesen et al, 1996;Müller et al, 1997). There is an accumulating body of evidence which suggests that this is not always the case (Eischen et al, 1997;Villunger et al, 1997;McGahon et al, 1998). It has been suggested that lack of p53 expression might account for the lack of recruitment of Fas and FasL (Müller et al, 1997). Treatment of DU145 cells with camptothecin did not alter the levels of Fas, or FasL, as shown by flow cytometry. DU145 cells have been shown to possess a mutated p53 (Isaacs et al, 1991), which further suggests that DU145 cells might be unable to up-regulate Fas or FasL proteins. In addition, cell cycle analysis of cells treated with anti-Fas, camptothecin, or camptothecin plus anti-Fas, has shown that camptothecin was mediating Fas-induced cell death through a mechanism which was independent of cell cycle alterations. Camptothecin has been shown to induce single-strand DNA breaks in the presence of topoisomerase I, which is a major target for the anti-tumour effect (Hsiang et al, 1989). How this is translated into a cytotoxic effect is, however, currently unknown. Camptothecin sensitization was shown to involve activation of caspases analogous to apoptosis in Fas-sensitive cells. Indeed, poly(ADP-ribose) polymerase was proteolytically cleaved in cells treated with camptothecin plus anti-Fas, but not in cells treated with anti-Fas only. Furthermore, camptothecin-mediated sensitization of DU145 cells to Fas-induced apoptosis was completely blocked by the caspase inhibitor ZVAD.
A variety of stimuli have been shown to cause an accumulation of cytochrome c in the cytosol, cause disruption of mitochondria transmembrane potential and an increase in ROI, leading to activation of caspases, PARP cleavage, DNA degradation and subsequent apoptosis. We hypothesized that camptothecin could be sensitizing DU145 cells to anti-Fas-induced apoptosis through an ROI-mediated mechanism. Results, however, seem to negate this hypothesis: (1) an increase in peroxide, which is thought to induce apoptosis by increasing cellular oxidative stress beyond the tolerable threshold, could not be detected; (2) no decrease in superoxide anion was observed.
In summary, our results show that a combinatory treatment of androgen-independent DU145 cells with sublethal concentrations of camptothecin and very low concentrations of anti-Fas IgM, will kill nearly all cells (75-95%) in 24 h. The mechanism of this cooperation appears to be independent of alterations in the levels ROI (peroxide and superoxide anion). Because we were able to chemosensitize the androgen-independent cells (DU145), but not the androgen-responsive cells (LNCaP), it is tempting to speculate that the resistance of a prostate cell line to anti-Fas IgM treatment may be inversely proportional to its positive response to chemosensitization. This would have important implications for therapy since, although nearly all men with metastatic prostate cancer respond well initially to some sort of hormonal ablation therapy, prostate cancer cells invariably become androgen-independent. This is invariably fatal because no effective systemic therapy currently exists. Taken together, these results seem to indicate that a combination of chemotherapy and immunotherapy may present a novel approach for the elimination of androgen-independent cells.