Introduction

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Different chemical classes of anticancer drugs can induce tumor cell death by multiple mechanisms. Although the initial intracellular targets of different cytotoxic drugs may be heterogeneous, there is increasing evidence indicating that drug-induced cytotoxicity commonly converges on the induction of programmed cell death (apoptosis). As an example, topoisomerase II inhibitors, such as doxorubicin (DXR), etoposide, or amsacrine, which induce DNA strand breaks, activate genes involved both in DNA repair and in the apoptotic process (Avramis et al., 1998; Wang et al., 1999). Some of the genes activated by DNA-damaging agents are p53-responsive and can modulate cell cycle progression, such as WAF1/p21, proliferating cell nuclear antigen, and GADD45 (Harper et al., 1993) or are directly involved in apoptosis such as Fas-L (Mo and Beck, 1999; Wang et al., 1999).

In recent years there has been much interest in studying the role played by the Fas system in the induction of apoptosis of cancer cells following exposure to cytotoxic drugs [see Maggi (1998) for review]. Apoptotic cell death induced by activation of the Fas/Fas-L system requires a multistep cascade of biochemical events: the trimerization of Fas receptor induced by Fas-L stimulates the formation of a death-inducing signaling complex, which consists of the adapter protein FADD and the protease FLICE/caspase-8. The successive interaction of the FADD-death effector domain molecule with caspase-8 activates a caspase cascade, which ends with downstream activation of caspases (caspase-3 and -7) and cleavage of cellular substrates.

It has been reported that the exposure to cytotoxic agents as diverse as DXR, cis-platin, or methotrexate (Friesen et al., 1996; Herr et al., 1997) may promote apoptosis by inducing the expression of Fas/Fas-L in some cancer cell lines (e.g., leukemia), whereas in other instances the induction of apoptosis by cytotoxic drugs has been reported to be independent from the Fas system (Eischen et al., 1997; Gamen et al., 1997; Tolomeo et al., 1998). However, the final steps of Fas-independent apoptosis induced by cytotoxic drugs seem to proceed through the same downstream caspases (caspase-3 and -7) of the death receptors pathway (Eischen et al., 1997; Gamen et al., 1997; McGahon et al., 1998).

Another set of important effectors of apoptosis, involved in drug-induced cell death, is the bcl-2 family proteins, including a growing number of antiapoptotic (Bcl-2, Bcl-X_L) and pro-apoptotic (Bax, Bak, Bad, Bid) factors (Chittenden et al., 1995; Yang et al., 1995; Hsu et al., 1997; Luo et al., 1998). Bcl-2·Bax or Bcl-X_L·Bax complex forms a mitochondrial channel that regulates membrane permeability. Bax up-regulation described in human ovarian (Strobel et al., 1996), colon (Nita et al., 1998), and breast cancer cell lines (Sakakura et al., 1996) enhances the apoptotic response to antineoplastic drugs. The altered ratio between bax and the antiapoptotic components Bcl-2 or Bcl-Xl, modulate the successive formation of the "apoptosoma" and the final apoptotic caspase cascade (Green and Kroemer, 1998).

We recently reported that the new anthracycline MEN 10755, a topoisomerase II inhibitor (Arcamone et al., 1997), possesses a broader spectrum of antitumor activity than DXR in various human cancer cell lines, which suggests its possible use for treatment of DXR-resistant tumors (Arcamone et al., 1997; Pratesi et al., 1998).

In this study we used a human carcinoma cell line (A2780) to investigate the expression of genes related to the induction of apoptosis, with particular reference to a possible involvement of the Fas system. The A2780 cell line seems particularly suited for this purpose, since it is sensitive to Fas induction following drug treatment (Uslu et al., 1996), showing a low basal expression of Fas protein and absence of Fas-L expression (our data, see below).

Because MEN 10755 exhibits a reduced steady-state accumulation compared with DXR, despite a comparable extent of DNA double-strand breaks and cytotoxicity induced by both drugs (Arcamone et al., 1997), we also investigated whether a cytoplasmic site of action could be the target for apoptosis induced by MEN 10755 or DXR in this cell line. For this purpose we studied whether the two anthracyclines can induce apoptosis in A2780 cells deprived of the nucleus but retaining an intact mitochondrial function (cytoplasts).

	Materials and Methods

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Chemicals. Doxorubicin was purchased from Sigma Aldrich Chemical Co. (Oakville, Ontario, Canada), and stock solutions (5 mM) were prepared in distilled water, aliquoted, and stored at 20°C. Propidium iodide (PI), Nonidet P-40, bovine pancreatic RNase A, and cycloheximide (CHX) were purchased from Sigma Chemical Co. (St. Louis, MO). MEN 10755 (batch MB 77/15) was synthesized as previously described (Arcamone et al., 1997), and stock solutions (5 mM) were prepared in distilled water, aliquoted, and stored at 20°C. Agarose was purchased from Life Technologies, Inc. (Gaithersburg, MD). Human recombinant soluble Fas-L (sFas-L) and caspase-3 substrate were purchased from Alexis Corp. (San Diego, CA); sFas-L was always used in combination with an enhancer IgG antibody at a concentration of 1 µg/ml (Alexis Corp., product no. 804-034-C050). Caspase inhibitors ZVAD-fmk and DEVD-cho were purchased from Bachem AG (Switzerland), dissolved in water, and stored at 20°C. Alamar Blue dye, supplied as a ready-to-use solution, was purchased from Biosource International (Camarillo, CA).

Antibodies. Monoclonal anti-Fas APO-1-3 was purchased from Alexis Corp., FITC-conjugated goat anti-mouse IgG was from Sigma, mouse IGg3 isotype control was obtained from PharMingen (San Diego, CA), and ZB4 IGg1-blocking anti-Fas monoclonal antibody was from MBL (Nagoya, Japan).

Cell Culture and Drug Treatment. A2780 cells (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf serum (FCS, Life Technologies, Inc.), 2 mM glutamine, 100 units of penicillin, and 100 µg of streptomycin at 37°C in 5% CO₂ and 95% humidified air. Cells were plated in 6-well plates (5 × 10⁵ cells/well) and cultured for 16 to 18 h before treatment with various concentrations of drugs. Cells were detached from culture flasks by 0.05% trypsin and 0.02% EDTA treatment (Life Technologies, Inc.) for 5 min and processed for FACS analysis as described below. For experiments with caspase inhibitors, A2780 cells were preincubated with ZVAD-fmk (100 µM) or DEVD-cho (600 µM) for 3 h and then exposed to 1 µM MEN 10755 or DXR for an additional 48 h.

Cytotoxicity Assay. The cytotoxicity of DXR and MEN 10755 was determined using the sulforhodamine B (SRB) assay (Skehan et al., 1990). A2780 cells were plated in 96-well microtiter plates in 200 µl of medium. After 24 h, the cells were exposed to drugs at the appropriate dilutions and allowed to incubate additional 1 or 24 h. After drug treatment, cells were washed with saline, incubated in drug-free medium for 72 h, then the cellular viability was measured by the SRB assay. The LC₅₀ (the concentration used to obtain 50% cellular mortality) was reported as a function of the drug concentration used.

Determination of Apoptotic Cells by Flow Cytometry and Annexin V/PI Staining. Annexin V/PI staining was performed using the apoptosis detection kit purchased from R&D Systems (Minneapolis, MN). After trypsinization (we assessed that the use of trypsin/EDTA does not interfere with annexin V binding), cells (10⁶ cells) were washed twice with cold phosphate-buffered saline (PBS) pH 7.2, then resuspended in binding buffer (HEPES supplemented with 25 mM CaCl). Cells (10⁵) were incubated with 10 µl of fluorescein-conjugated annexin V (10 µg/ml) and 10 µl of PI (50 µg/ml) for 15 min at room temperature in the dark. At the end of the incubation time, 400 µl of binding buffer were added, and the samples were analyzed on a FACSort flow cytometer using Lysis II software (Becton Dickinson, Mountain View, CA). We use bivariate flow cytometry to simultaneously measure log green fluorescence (FL1-height) versus log red fluorescence (FL2-height). Proper flow cytometric analysis was performed using the following controls: unstained cells, cells stained with annexin V-FITC only, and cells stained with PI only. To exclude any overlap of the two emission spectra, we analyzed singly stained cells to adjust electronic compensation. Setting quadrant statistics for determination of the frequency of cells undergoing apoptosis was achieved on untreated cells stained with both annexin V and PI or on untreated cells unstained. After appropriate setting, annexin V-positive cells were identified in the lower right quadrant of the dot plot, whereas annexin V/PI double-stained cells were identified in the upper right panel (see Vermes et al., 1995).

Determination of Fas Expression by Flow Cytometry. Analysis of Fas expression by flow cytometry was performed by standard procedures. Briefly, cells (10⁶) were washed twice with PBS containing FCS 1% (PBS/FCS 1%), centrifuged at 1200 rpm for 10 min and then incubated for 1 h at 0°C with anti-Fas APO-1 to -3 (1 µg/10⁶ cells). The cells were washed twice with PBS/FCS 1% and stained for 1 h in the dark at 0°C with FITC-conjugated antibody. Cells were finally washed twice with PBS/FCS 1% and analyzed on a FACSort flow cytometer (Becton Dickinson). Fluorescence signals were collected in logarithmic mode while the relative cell numbers per channel were in linear mode.

RNase Protection Assay. Two human template sets, h-Apo-3 and h-Stress-1 (PharMingen), were used for the T7 polymerase-directed synthesis of high specific activity, ³²P-labeled, antisense RNA probes, which were hybridized with the target mRNAs (10 µg). mRNAs were extracted from A2780 cells using the TRIzol reagent (Life Technologies, Inc.), according to the method of Chomczynski and Sacchi (1987). Free probe and other single-stranded RNA molecules were digested with RNases. The remaining "RNase-protected" probes were purified, resolved on 7 M urea, 5% polyacrylamide gels, and imaged by autoradiography. Two housekeeping gene products, L32 and GAPDH, have been used to assess the total RNA level for normalizing sampling and technical errors. Autoradiographic signals were quantified by densitometry using STORM 840 (Molecular Dynamics, Sunnyvale, CA).

Cytoplasts Preparation. A2780 cells were enucleated as previously described (Wigler and Weinstein, 1975) except for modifications. Cells were detached from tissue culture plates by 0.05% trypsin and 0.02% EDTA treatment (Life Technologies, Inc. ) for 5 min and incubated (final concentration of 5 × 10⁶ to 5 × 10⁷ cells/ml) at 37°C for 45 min in RPMI medium containing 21 µM (10 µg/ml) cytochalasin B. The cell suspension was layered onto a previously prepared discontinuous Ficoll density gradient, consisting of 6 ml of 25%, 6 ml of 17%, 3 ml of 16%, 3 ml of 15%, and 6 ml of 12.5% Ficoll-400, all in RPMI medium. The gradients were centrifuged for 60 min in a prewarmed Beckman SW41 rotor at 25,000 rpm at 33°C. Cytoplasts were collected from the central interface between 12.5% and 15% Ficoll layers, diluted into RPMI 10% FCS, centrifuged, and plated. Enucleation efficiency was determined by staining with propidium iodide followed by evaluation under the fluorescence microscope. More than 90% of the cytochalasin-treated A2780 cells did not contain a nucleus.

Fluorogenic Substrate Assay for Caspase-3 Activity. A2780 cells were seeded in 6-well plates. After 18 to 20 h, DXR and MEN 10755 were added at a final concentration of 1 µM. In a set of experiments, after a 20-h period of incubation at 37°C, the cells were treated with Fas-L for 1.5, 3, and 6 h. The peak of stimulation was observed at 3 h of incubation, and this time point was chosen for the final experiments.

	Results

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Anthracycline-Induced Apoptosis in A2780 Cells. As a preliminary step for further studies on the involvement of the apoptotic pathway in anthracycline-induced cell death, we first compared the induction of apoptosis by MEN 10755 and DXR by using cytofluorimetry and annexin V/PI staining. Since the two anthracyclines have comparable cytotoxic potency on A2780 cell line (MEN 10755 LC₅₀ = 27± 11 nM, DXR LC₅₀ = 9 ± 2 nM after 24 h of treatment and successive 72 h in the absence of drug), we used equimolar concentrations of both compounds for further experiments. In particular the induction of apoptosis was measured at 1 and 0.1 µM, corresponding to the LC₉₀ after 48 and 72 h, respectively, of continuous drug exposure. These concentrations are comparable with drug levels observed after a single administration of 7 mg/kg, i.v., of DXR or MEN 10755 in A2780 tumor xenografts on nude mice (data not shown). After 48 h of exposure, MEN 10755 and DXR (both at 1 µM) induced apoptosis in 35.7± 2.1% and 53.8 ± 3.5% of cells. At 0.1 µM, the percentage was not significantly different from untreated cells. At 72 h, apoptosis became detectable even at 0.1 µM and was comparable for both drugs (Fig. 1). We also observed that anthracycline-induced apoptosis is partially inhibited following CHX treatment (1 µg/ml for 48 h): a 54% reduction of apoptotic cells for both MEN 10755 or DXR (0.1 µM) was observed (data not shown), indicating that a novel synthesis of cytoplasmic or nuclear factors is mandatory for the apoptotic pathway.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Apoptosis induction in A2780 cells treated separately with 0.1 and 1 µM MEN 10755 or DXR for 24, 48, or 72 h. Apoptotic cells were evaluated by annexin V/PI staining, as described under Materials and Methods. Statistical comparisons were made between DXR- and MEN 10755-treated and untreated cells at respective time points (*p < 0.05; **p < 0.01). , 24 h; , 48 h; , 72 h.

MEN 10755 Increases Transcription of Fas, bax, WAF1/p21, and GADD45 mRNAs but Does Not Induce Fas-L mRNA. We used an RNase protection assay to analyze the role of a panel of genes involved in the apoptotic pathway in anthracycline-induced cell death in A2780 cells.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   RPA assay for the evaluation of mRNA transcription modulation induced by drug treatment. A2780 cells were treated with 0.1 µM DXR () and 0.1 µM MEN 10755 () for 6, 12, 24, 36, and 48 h. Samples of total RNA were analyzed for distinct mRNA species using PharMingen's Riboquant ribonuclease protection assay system. A, autoradiograms with the APO-3 panel of [P32]mRNA probes. MEN 10755 treatment showed a significant up-regulation only of Fas mRNA starting from 6 h of incubation, and this effect was comparable with DXR treatment. B, autoradiograms with the Stress-1 panel of [P32]mRNA probes. Both anthracyclines increased transcription of bax, p21, and GADD45 genes. C, densitometric analysis of autoradiograms of ribonuclease protection assay has been plotted as the percentage of untreated controls. Data are representative for one of three separate experiments.

MEN 10755 Increases the Expression of a Functional Fas Receptor on A2780 Cell Membrane. The expression of Fas protein on the A2780 cell membrane was assessed through flow cytometry. We found that Fas expression correlates quite well with the increase in mRNA transcription.

Cytoplast Sensitivity to Anthracycline-Induced Cell Death. It has been demonstrated that enucleated cells (cytoplasts) are sensitive to Fas-mediated cytotoxicity with the same kinetics and cytoplasmic changes as their nucleated counterparts (Nakajima et al., 1995). We tested cytoplasts to confirm the hypothesis that the nucleus is the prominent target of MEN 10755-induced cytotoxicity and to further verify that the Fas/Fas-L system is not the effector of MEN 10755-induced apoptosis.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Cytoplasts prepared from A2780 cells or whole cells were treated with 10 µM MEN 10755 or DXR alone for 24 h or incubated with 1 µM MEN 10755 or DXR (24 h) and then stimulated with 100 ng/ml Fas-L alone or in combination with ZB4 2 µg/ml (18 h). Cellular viability was assayed by the Alamar Blue method. Data are representative for one of three separate experiments.

Caspases Activation Is Involved in Anthracycline-Induced Apoptosis in A2780 Cells. The above findings indicate that apoptosis induced by MEN 10755 is an active process requiring the nucleus as a target and active synthesis of proteins. We then examined the role of effector caspases, such as caspase-3 (Sun et al., 1999; Wesselborg et al., 1999), in MEN 10755-induced cell death. A2780 cells incubated with 1 µM MEN 10755 or DXR showed a consistent increase of caspase-3 activity after 18 to 24 h of treatment (Fig. 4), as previously described in other tumor cell lines (Datta et al., 1996; Los et al., 1997).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Kinetics of caspase-3 activation following treatment with 1 µM MEN 10755 or DXR. Caspase-3 activity was measured as described under Materials and Methods. Results shown are representative of three experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Caspase-3 activity in A2780 cells treated with 1 µM MEN 10755 or DXR. After 24 h of drug incubation, cells were stimulated for 3 h with Fas-L (100 ng/ml). Cell viability was assessed as described under Materials and Methods. Statistical comparisons were made between DXR + Fas-L- and MEN 10755 + Fas-L-treated cells and cells treated with DXR or MEN 10755 alone, respectively (**p < 0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Apoptosis inhibition following treatment with caspase inhibitors ZVAD-fmk or DEVD-cho. A2780 cells were preincubated for 3 h with 100 µM ZVAD-fmk or 600 µM DEVD-cho and then incubated also with 1 µM MEN 10755 or DXR for additional 48 h. The percentage of apoptotic cells was evaluated by cytofluorimetry using annexin V/PI staining. DXR + ZVAD-, DXR + DEVD-, MEN 10755 + ZVAD-, and MEN 10755 + DEVD-treated cells were statistically compared with untreated cells (*p < 0.05; **p < 0.01).

	Discussion

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MEN 10755 is a recently discovered disaccharide anthracycline, characterized by a potent antitumoral activity against a large number of human tumor xenografts, and it is currently undergoing phase I clinical studies. Previous studies (Arcamone et al., 1997) indicate that the enzyme topoisomerase II is the main target for the cytotoxic action of MEN 10755: the drug stimulates topoisomerase II cleavage of DNA and displays a remarkable ability to elicit DNA single- and double-strand breaks, transforming the enzyme into a DNA-damaging agent.

Although the events interposed between the stabilization of the cleavage complex and the loss of cell viability have not yet been identified, the net result is the initiation of cell death, occurring through cell cycle arrest, and induction of necrosis or apoptosis. The results of the present study indicate that apoptosis initiated by exposure of A2780 cells to either MEN 10755 or DXR: 1) proceeds through an essentially similar pathway, requiring the presence of the nucleus (as demonstrated by experiments with cytoplasts) and active protein synthesis (inhibition by CHX); 2) occurs concomitantly through the expression of a panel of genes related to the induction of apoptosis; and 3) impinges on the caspase effector pathway, which is pharmacologically similar (possibly involving caspase-3) to that activated through the Fas/Fas-L pathway. In the A2780 cell line, exposure to anthracyclines induces Fas expression, and this response is linked to a functional Fas-dependent apoptosis (induced by Fas-L), yet the Fas/Fas-L pathway is not involved in anthracycline-induced apoptosis.

Overexpression of Bax mRNA fits well with the apoptosis kinetics observed in MEN 10755-treated A2780 cells and could be one of the molecular mechanisms responsible of MEN 10755-induced apoptosis (Figs. 1 and 2C). Bax, a member of Bcl-2 family of proteins, forms channels in mitochondrial membranes and is able to induce apoptosis in some cancer cell lines, inducing directly cytochrome c release (Sakakura et al., 1996; Strobel et al., 1996): this leads to the activation of downstream caspases and consequently to apoptosis.

WAF1/p21 and GADD45 are p53-dependent genes associated with growth control and cell cycle checkpoints following DNA damage, causing a G₁ or G₂/M arrest to permit DNA repair before replication or mitosis (Wang et al., 1997; Medema et al., 1998). When DNA repair fails, apoptosis can occur to eliminate irreparably damaged cells. The increase of WAF1/p21 and particularly GADD45 mRNA in A2780 cells, could explain a G₂/M block that we observed by cytofluorimetric analysis of PI stained cells after 24 h of MEN 10755 treatment (data not shown).

Since a p53-sensitive region has been described in the gene promoter of the up-regulated pro-apoptotic factors (Chan and Owen-Schaub, 1995; Amundson et al., 1998), it is possible that, in this model of A2780 cells, the Fas up-regulation we have observed was caused by the activation of p53 tumor suppressor protein, as already described (Ruiz-Ruiz and Lopez-Rivas, 1999). We did not detect any increase in mRNA p53 transcription following drug treatment, but it has been published that p53 activation is not always related to an increase of p53 mRNA or protein (Caelles et al., 1994; Wagner et al., 1994).

In our study bcl-X was expressed at a steady-state level both in controls and MEN 10755-treated cells, in the absence of Bcl-2 expression. Given that Bcl-X may suppress cell death in the same way as Bcl-2, the functional redundancy between these apoptosis inhibitors may compensate for the absence of Bcl-2 (Nita et al., 1998).

The possibility of targets additional to nuclear topoisomerase II was suggested by the findings of Serafino et al. (1999), sustaining that anthracyclines may also act through mechanism(s) involving the cytoplasmic compartment. The reduced cellular uptake of MEN 10755 as compared with DXR (Arcamone et al., 1997), despite comparable cytotoxic effects, allowed us to investigate this hypothesis, further supported by the observation of a more relevant cytoplasmic localization of MEN 10755 (data not shown). In the present article, we demonstrated that enucleated cells were not sensitive to anthracycline treatment, suggesting that "cytoplasmic" mechanisms could not be "per se" sufficient to induce cell death, although we cannot exclude the possibility of there being additional targets to nuclear topoisomerase II.

On the basis of our data we hypothesize that MEN 10755, after induction of DNA damage-responsive genes, alters mitochondrial membrane through the synthesis of pro-apoptotic factors such as Bax (Chittenden et al., 1995; Yang et al.,1995; Hsu et al., 1997; Luo et al., 1998), which induce cytochrome c release, procaspase-9-Apaf-1 triggering, and finally, effector caspases activation. We also observed that CHX treatment blocks anthracycline-induced apoptosis, further supporting the hypothesis that new protein synthesis is required for MEN 10755 or DXR apoptotic activity.

When we evaluated the caspase inhibitors ZVAD-fmk and DEVD-cho, we found MEN 10755 or DXR apoptosis inhibited by both of them. Although it has been clearly demonstrated that the broad-spectrum caspase inhibitor ZVAD-fmk prevented drug-induced apoptosis (Tolomeo et al., 1998; Sun et al., 1999; Wesselborg et al., 1999), the effect of the inhibitor DEVD-cho, more specific for caspase-3, is not so clear (Tolomeo et al., 1998). In our hands, both peptides displayed a similar inhibitory effect on anthracycline-induced apoptosis, suggesting an important role for caspase-3 activation.

In conclusion, we demonstrated that MEN 10755-induced apoptosis is correlated to bax up-regulation in a cellular model of human ovarian cancer cells wild-type p53, at concentrations clinically significant, but is independent of Fas receptor triggering. However, the anthracycline-induced Fas is fully functional and the engagement of the receptor by exogenous Fas-L determines a further increase in the apoptotic response induced by drugs, thus enhancing the total cytotoxic effect.

On the basis of the findings presented in this article, the broad spectrum of antitumor activity exerted by MEN 10755 toward cancers, which are not responsive to DXR (Pratesi et al., 1996), cannot be explained on the basis of major qualitative differences in cellular targets relevant for cytotoxicity and induction of apoptosis.