AplidinTM Induces Apoptosis in Human Cancer Cells via Glutathione Depletion and Sustained Activation of the Epidermal Growth Factor Receptor, Src, JNK, and p38 MAPK*

Ana CuadradoDagger §, Luis F. García-FernándezDagger §, Laura González§||, Yajaira Suárez§, Alejandro LosadaDagger , Victoria Alcaide§**, Teresa Martínez§, José María Fernández-SousaDagger , José María Sánchez-PuellesDagger , and Alberto Muñoz§DaggerDagger

From the Dagger  Drug Discovery Department, PharmaMar S. A., Tres Cantos, E-28760 Madrid, Spain and the § Instituto de Investigaciones Biomédicas "Alberto Sols," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, E-28029 Madrid, Spain

Received for publication, January 30, 2002, and in revised form, October 21, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

We report that AplidinTM, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. AplidinTM induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. AplidinTM-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in AplidinTM action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by AplidinTM. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected AplidinTM action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, AplidinTM also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of AplidinTM.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

AplidinTM (a cyclic depsipeptide, C57H89N7O15, Mr = 1112) is a novel antitumor agent isolated in 1990 from the Mediterranean tunicate Aplidium albicans (1). In vitro, AplidinTM has potent cytotoxic activity against breast, colon, non-small cell lung, and prostate carcinoma and melanoma cells (2, 3). It has also shown strong antitumor activity in various xenograft models (4). AplidinTM has recently entered Phase II clinical trials in a variety of solid tumors upon showing promising toxicity and pharmacological properties in Phase I studies (5).

Little is known about the mechanism of action of AplidinTM. Early studies indicated that it exerts an antiproliferative effect through the inhibition of protein synthesis and reduction of ornithine decarboxylase activity (6, 7). In addition, AplidinTM inhibits vascular endothelial factor secretion and autocrine stimulation in a human leukemia cell line (8).

Many anticancer drugs elicit apoptosis in cancer cells (9-11). Several studies have reported the activation of one or more members of the MAPK1 family of intracellular signaling kinases by cytotoxic agents. Among them, JNK and p38 MAPK and, less frequently, ERK1/2 play important roles in these apoptotic processes (12-18). However, JNK and p38 MAPK are not always involved in apoptosis. They also promote proliferation, differentiation, or survival depending on the cell type and external stimulus (19-22). Interestingly, JNK is also activated by chemopreventive agents such as the isothiocyanates present in cruciferous vegetables (23) and has been shown to lie upstream of the caspase proteases that constitute the effector system in the apoptotic pathway (14). However, in other cases, such as apoptosis induced by doxorubicin or gamma -irradiation, caspase activation is independent of JNK (24).

To study the mechanism of action of AplidinTM in human cancer cells, we chose the MDA-MB-231 breast cell line, which contains mutated p53 and ras genes and is highly invasive and proliferative. Here we report that AplidinTM induced rapid and sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor tyrosine kinase Src, JNK, and p38 MAPK. SB203580, an inhibitor of both JNK and p38 MAPK in these cells, substantially inhibited the cytotoxic effect of AplidinTM, suggesting an important role of these kinases in AplidinTM action. These effects depend on a decrease in intracellular GSH. In addition, we found that EGFR activation by AplidinTM was partially mediated by Src, but was somehow abnormal because it did not lead to the same binding of Shc and Grb2 adaptor proteins as that induced by EGF. In view of the promising effects observed in patients in Phase I studies, we extended the study to two lines, ACHN and A-498 cells (both wild-type p53), of renal cancer, a neoplasia highly refractory to chemotherapy. In these cells, AplidinTM also activated EGFR, JNK, and p38 MAPK, but did not affect the high endogenous level of active Src.

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Cell Lines, Antibodies, and Reagents-- MDA-MB-231, ACHN, and A-498 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1 mM glutamine (all from Invitrogen). Wild-type and EGFR-deficient (egfr-/-) mouse embryo fibroblasts (MEFs) were obtained from Dr. E. Wagner (Institut für Molekulare Pathologie, Vienna, Austria). AplidinTM is manufactured by PharmaMar S. A. (Madrid, Spain). Stock solutions were freshly prepared in dimethyl sulfoxide and diluted in the cell culture to final concentrations as indicated. Ascorbic acid, diphenyleneiodonium (DPI), Tiron, vitamin E ((+)-alpha -tocopherol acid succinate), catalase, H2O2, GSH, N-acetylcysteine, ebselen, L-buthionine (SR)-sulfoximine (BSO), and 4,6-diamidino-2-phenylindole were from Sigma. PP2 (4-amino- 5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine), tyrphostin AG1478,SB203580, and benzyloxycarbonyl-VAD-fluoromethyl ketone caspase inhibitor were from Calbiochem. Human EGF was from PreproTech Ltd.

Anti-JNK1, anti-p38 MAPK, anti-ERK, anti-human EGFR, anti-Grb2, and anti-Src antibodies (for Western blotting) were from Santa Cruz Biotechnology. Anti-Src antibody (for immunoprecipitation) was generously donated by Dr. J. Brugge. Anti-phospho-JNK1, anti-phospho-p38 MAPK, anti-phospho-ERK, anti-phospho-Src Tyr418, anti-phospho-Akt Ser348, anti-caspase-3 (active form), and anti-beta -actin antibodies were from New England Biolabs Inc. Anti-platelet-derived growth factor receptor (PDGFR) antibody was from Upstate Biotechnology, Inc. Anti-phosphotyrosine and anti-Shc antibodies were from Transduction Laboratories. Anti-phospho-EGFR Tyr1068/Tyr1086/Tyr1148/Tyr1173 antibodies were from BioSource, International.

Nuclear Staining Assays-- After treatment, cells were washed once with phosphate-buffered saline (PBS) and fixed in a solution of chilled methanol/acetic acid (1:1) for 2 min. The fixed cells were washed with PBS, placed on slides, and stained with 2 µg/ml 4,6-diamidino-2-phenylindole for 15 min. Excess dye was washed off with PBS. Nuclear morphology was observed under a fluorescence microscope (Zeiss Axiophot).

Flow Cytometry Analysis-- Cells after different treatments with AplidinTM and/or inhibitors were stained with propidium iodide (Sigma) and analyzed by flow cytometry (FACScan, BD Biosciences). For staining, cells (1 × 106) were harvested, washed with PBS, and then fixed in 70% ethanol. Fixed cells were treated with DNase-free RNase for 30 min at 37 °C, washed with PBS, centrifuged, and incubated in PBS-containing propidium iodide (25 µg/ml). Forward light scatter characteristics were used to exclude the cell debris from the analysis. Apoptotic cells were determined by their hypochromic subdiploid staining profiles. To estimate early apoptotic cells, Alexa 488-conjugated annexin V (Molecular Probes, Inc.) was used together with propidium iodide dead cell counterstain following the manufacturer's recommendations.

DNA Synthesis Measurements-- Cells (3.5 × 104) were seeded in 24-well dishes (Nunc). One day later, cells were treated or not with AplidinTM in normal growth medium and pulsed with 5 µCi/ml [3H]thymidine (Amersham Biosciences) for 30 min at the indicated times post-treatment. At the end of the labeling period, the medium was removed, and the cells were rinsed twice in PBS and fixed with chilled 10% trichloroacetic acid for 10 min. Trichloroacetic acid was then removed, and the monolayers were washed with ethanol and air-dried at room temperature for 20 min. Thereafter, precipitated macromolecules were dissolved in 500 µl of 0.5 N NaOH and 0.1% SDS, and 450 µl of each sample was diluted in 5 ml of OptiPhase HighSafe scintillation solution (Wallac). Radioactivity was measured on a 1209 RackBeta counter (Wallac).

Crystal Violet Staining Method-- To estimate cell mass, the medium was removed, and 24-well dishes were washed with PBS, fixed with 1% glutaraldehyde for 15 min, washed twice with PBS, and stained with 100 µl of 0.1% aqueous crystal violet for 20 min. Dishes were rinsed four times in tap water and allowed to dry. One-hundred µl of 10% acetic acid was added, and the content of each well was mixed before reading the absorbance at 595 nm.

Immunoprecipitation and Western Blot Analysis-- To study the effect of AplidinTM on the activity of different kinases (Src, p38 MAPK, JNK, ERK, and Akt), cells were preincubated for 24 h in serum-free medium. For immunoprecipitation, cells were lysed in modified radioimmune precipitation assay buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM beta -glycerophosphate, 100 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were precleared by incubation with protein G-Sepharose at 4 °C for 30 min and then incubated overnight with the corresponding antibody (1 µg/ml). After a 1-h incubation with protein G-Sepharose, immune complexes were washed three times with the same radioimmune precipitation assay buffer lacking deoxycholic acid and SDS and three times with 50 mM Hepes (pH 7.4), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 25 mM beta -glycerophosphate, 100 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride and electrophoresed on 8% (EGFR and PDGFR) or 12% (Src) acrylamide gels. For Western blot analysis, cell protein extracts were prepared following standard procedures (25). Protein extracts were electrophoresed on 8 or 15% polyacrylamide gels and transferred to nylon membranes (Immobilon P, Millipore Corp.). The filters were washed, blocked with 5% bovine serum albumin in Tris-buffered saline (25 mM Tris (pH 7.4), 136 mM NaCl, 2.6 mM KCl, and 0.5% Tween 20), and incubated overnight at 4 °C with the appropriate antibody (1:1000 dilution). Blots were washed three times for 10 min with PBS + 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat antibody for 1 h at room temperature. Blots were developed by a peroxidase reaction using the ECL detection system (Amersham Biosciences).

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AplidinTM Induces Apoptosis in MDA-MB-231 Breast Cancer Cells-- AplidinTM showed high activity against MDA-MB-231 cells (Fig. 1A), with an antiproliferative IC50 of 5 nM at 48 h post-treatment (data not shown). This concentration is pharmacologically relevant, as the circulating plasma levels of AplidinTM in patients exposed to the maximum tolerated dose in Phase I clinical studies is 7.1 nM (26). Dose-dependent inhibition of DNA synthesis was detected 1-3 h after drug addition (Fig. 1B). By flow cytometry analysis, AplidinTM was found to induce the subdiploid profile, which is a hallmark of apoptosis (Fig. 1C). At 24 h, 20-25% apoptotic cells were found in cultures growing in normal medium treated with 50-500 nM AplidinTM, and ~10% apoptotic cells were found in cultures treated with 5 nM AplidinTM. In line with this, AplidinTM-treated cells showed time- and dose-dependent nuclear condensation and fragmentation in comparison with the homogeneous staining of untreated cells as assessed by 4,6-diamidino-2-phenylindole staining (Fig. 1D, upper panels) and the generation of the active form of caspase-3 (lower panels). When added simultaneously to the drug, benzyloxycarbonyl-VAD-fluoromethyl ketone, a general caspase inhibitor, decreased the cytotoxic activity of AplidinTM by ~50% as assessed by crystal violet staining (data not shown). This indicates that AplidinTM-induced apoptosis is mediated by caspase-dependent and -independent mechanisms. The cytotoxic action of AplidinTM is triggered rapidly, as a pulse treatment of 5 min (using 500 nM) gave the maximum antiproliferative effect at 48 h (data not shown).


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Fig. 1.   Effect of AplidinTM on MDA-MB-231 cell growth and viability. A, phase-contrast micrographs of cells treated with 50 nM AplidinTM (APL) or vehicle (Control) for 24 h. Scale bar = 25 µM. B, dose-dependent kinetics of the inhibition of DNA synthesis by AplidinTM. DNA synthesis was calculated by measuring thymidine incorporation as described under "Experimental Procedures" at the indicated times after treatment with 5 nM (open circle ), 50 nM (triangle ), or 500 nM () AplidinTM. C, flow cytometry analysis of DNA content in cultures incubated in the absence (Control) or presence of the indicated doses of AplidinTM in normal growth medium for 24 h. Percentages of apoptotic cells corresponding to the subdiploid population are shown. D, upper panels, fluorescence microscope image of cultures treated with vehicle or AplidinTM (500 nM) for 24 h after nuclear staining with 4,6-diamidino-2-phenylindole. Arrows indicate cells at an advanced stage of nuclear condensation and degradation. Lower panels, Western blot analysis showing the activation of caspase-3 upon AplidinTM treatment. An antibody that specifically recognizes the active truncated caspase-3 polypeptide was used. As a control, filters were reprobed with anti-beta -actin antibody.

AplidinTM-induced Apoptosis Relies on Sustained JNK and p38 MAPK Activation-- Doses 10-100-fold higher than the antiproliferative IC50 were used to identify the primary targets and early mechanism of action of AplidinTM. To examine the role of MAPKs in AplidinTM-induced apoptosis, we studied the effects of AplidinTM on the activity of ERK1/2, JNK, and p38 MAPK using antibodies specifically recognizing the active phosphorylated forms. AplidinTM caused rapid, strong, and sustained activation of p38 MAPK and JNK without altering their concentrations (Fig. 2A). The same result was obtained in immune complex kinase assays (data not shown). Addition of AplidinTM in vitro to immune complexes from untreated cells did not affect kinase activity (data not shown), suggesting that p38 MAPK/JNK activation in vivo is due to effects on their at least 11 upstream regulators (22). In agreement with the expression of an activated K-ras oncogene (27), MDA-MB-231 cells displayed a high basal level of phosphorylated ERK1/2 under unstimulated conditions, which was mostly unaffected by AplidinTM treatment (Fig. 2A). Likewise, AplidinTM did not change the cellular content of phosphorylated Akt (Fig. 2A), an enzyme that prevents cell death by multiple apoptosis inducers (see Ref. 28 for review).


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Fig. 2.   AplidinTM induces apoptosis in MDA-MB-231 cells through the sustained activation of p38 MAPK and JNK. A, levels of activated p38 MAPK, JNK, ERK, and Akt in cells treated with AplidinTM (500 nM) for the indicated times or left untreated (control (C)) were measured by Western blotting using phospho (P)-specific p38 MAPK, JNK, ERK, and Akt antibodies. Antibodies against total protein (p38 and JNK) were used to rule out effects at the expression level. B, SB203580 inhibited p38 MAPK and JNK activation by AplidinTM. Cells were pretreated with SB203580 (30 µM) or vehicle for 1 h before AplidinTM addition as indicated. Western blot analysis was carried out as described for A. C, the results from flow cytometry analysis showed that AplidinTM-induced apoptosis was inhibited by SB203580. Cells were treated with vehicle (CONTROL), AplidinTM (APL; 500 nM), SB203580 (30 µm) or AplidinTM plus SB203580 (same doses) for 24 h in serum-free medium. D, shown are phase-contrast micrographs of cultures treated for 48 h with vehicle, AplidinTM, SB203580, or AplidinTM plus SB203580 as described for C. Scale bar = 35 µM. Values shown correspond to a representative experiment. The experiments shown in A and B were performed three times, and those in C and D were performed twice.

To examine the importance of these effects in the induction of apoptosis, we used SB203580, a pyridinylimidazole that inhibits some p38 MAPK isoforms (29, 30). However, control experiments showed that, in MDA-MB-231 cells, SB203580 inhibited both JNK and p38 MAPK at 5-30 µM (Fig. 2B) (data not shown). This correlated with the inhibition of AplidinTM-induced apoptosis. As determined by flow cytometry, SB203580 (30 µM) decreased the number of apoptotic cells from 41 to 16% after 24 h of AplidinTM treatment in serum-free medium (Fig. 2C), an effect clearly visible by phase-contrast microscopy (Fig. 2D). These data indicate that the activation of JNK and/or p38 MAPK plays a critical role in the induction of MDA-MB-231 cell apoptosis by AplidinTM. Moreover, the activation of these kinases was unaffected by benzyloxycarbonyl-VAD-fluoromethyl ketone (data not shown), suggesting that these events lie upstream of caspase activation in the apoptosis cascade.

AplidinTM Induces Src-dependent EGFR Phosphorylation-- Because JNK activation by some stimuli involves Src-mediated EGFR phosphorylation (31), we studied whether AplidinTM could have this mode of action. We found that AplidinTM caused EGFR phosphorylation, which was partially blocked by pretreatment with PP2 (Fig. 3A), a specific inhibitor of Src (32, 33). AplidinTM induced a more sustained EGFR phosphorylation than EGF (Fig. 3B, upper panels); it peaked at 1-4 h and remained above the base line for 24 h (middle panels). Accordingly, the activation-linked down-regulation of EGFR content was delayed in AplidinTM-treated cells with respect to EGF-treated cells (Fig. 3B, middle panels). Combined treatment of MDA-MB-231 cells with EGF and AplidinTM resulted in cellular toxicity and persistent EGFR phosphorylation, indicating a dominant and probably aberrant effect of AplidinTM (Fig. 3B, lower panels). This was further suggested by analysis of the binding of the adaptor proteins Grb2 and Shc to EGFR. In contrast to what happened upon EGF addition, EGFR activation by AplidinTM did not cause binding of Grb2 or Shc to the receptor (Fig. 3C). Moreover, in cells treated with both agents, Grb2 binding was also inhibited, as was binding of Shc at early times (2 and 30 min post-treatment), although it strongly increased later (60 min) (Fig. 3C).


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Fig. 3.   AplidinTM induces Src-dependent EGFR phosphorylation. A, MDA-MB-231 cells were either pretreated or not for 1 h with PP2 (25 µM) and then treated with EGF (20 ng/ml, 5 min) or AplidinTM (APL; 500 nM, 1 h) as indicated. Phosphorylated (P) EGFR levels were analyzed by immunoprecipitation with anti-EGFR antibody followed by Western blotting using anti-phosphotyrosine antibody. Total EGFR levels were measured by reprobing the filters with anti-EGFR antibody. B, shown is the time course of induction of EGFR phosphorylation by EGF (20 ng/ml; upper panels), AplidinTM (500 nM; middle panels), or both (same doses; lower panels) analyzed as described for A. C, shown is the time course of Grb2 and Shc binding to EGFR upon treatment with EGF (20 ng/ml), AplidinTM (500 nM), or both. Binding was analyzed by immunoprecipitation (IP) with anti-EGFR antibody followed by Western blotting (WB) using anti-Grb2 or anti-Shc antibody. Western blotting using these antibodies was done to estimate total Grb2 and Shc content. D, cells were treated for the indicated times with either EGF (20 ng/ml) or AplidinTM (500 nM). Western blotting (20 µg/lane) was performed using each of the four residue-specific anti-phosphotyrosine (P-Y) antibodies shown. E, PDGFR was not phosphorylated upon AplidinTM treatment. NIH 3T3 cells were treated with AplidinTM (500 nM) or platelet-derived growth factor (PDGF; 10 ng/ml) for the indicated times. Phosphorylated and total PDGFRs were measured by immunoprecipitation and Western blotting using anti-PDGFR and anti-phosphotyrosine antibodies. F, AplidinTM induced rapid and sustained Src phosphorylation. Cells were treated with vehicle (control (C)) or AplidinTM (500 nM) for the indicated times, and the amount of activation-linked phosphorylated Src was measured by immunoprecipitation with anti-Src antibody followed by Western blotting using anti-phospho-Tyr418 antibody as described under "Experimental Procedures." The amount of total Src in the immunoprecipitates is shown below. Results shown correspond to a representative experiment. The experiments shown in A and B were performed three times, and those in C-F were performed twice.

EGFR is subjected to inhibitory and activating phosphorylation of specific residues by several kinases. Five sites of in vivo autophosphorylation have been identified in EGFR: Tyr992, Tyr1068, Tyr1086, Tyr1148, and Tyr1173 (34-36). To make sure that AplidinTM has a stimulatory effect on EGFR, we used residue-specific anti-phosphotyrosine antibodies. At least four of these five amino acids became phosphorylated upon AplidinTM addition (Fig. 3D). To assess the specificity of EGFR activation, the effect of AplidinTM on PDGFR was studied in mouse NIH 3T3 fibroblasts. AplidinTM did not induce phosphorylation of this receptor (Fig. 3E). These cells were sensitive to AplidinTM, although they lack EGFR expression, indicating that EGFR transactivation is not required for AplidinTM-induced apoptosis. Additionally, given that PP2 significantly reduces AplidinTM-induced EGFR phosphorylation and that Src transactivates EGFR in some cell systems (31, 37, 38), we studied the effect of AplidinTM on Src expression and activity. By means of an antibody that specifically recognizes the activation-linked phospho-Tyr418 of Src, AplidinTM rapidly activated Src without altering the amount of protein (Fig. 3F). Because MDA-MB-231 cells express Src and Fyn, but not Yes (data not shown), which are the three ubiquitously expressed members of the Src kinase family that have at least partially overlapping functions (39), the possibility that AplidinTM may also affect Fyn cannot be ruled out.

The role of EGFR activation in AplidinTM action was studied in egfr-/- MEFs. In both the presence and absence of serum, egfr-/- MEFs underwent apoptosis with delayed kinetics in comparison with wild-type MEFs (Fig. 4A) (data not shown). At late times after treatment, however, no differences were found in cell proliferation or viability (data not shown). Consistent with this, the activation of p38 MAPK and JNK egfr-/- MEFs was reduced and delayed, respectively, compared with wild-type MEFs (Fig. 4B, upper panels). To examine the contribution of EGFR and Src to the activation of p38 MAPK/JNK by AplidinTM, we used PP2. Pretreatment with PP2 and also with GSH caused only partial reduction of p38 MAPK and JNK activation in egfr-/- and wild-type MEFs. SB203580 had a stronger effect than PP2 and GSH in egfr-/- cells. In addition, we studied the contribution of the activation of Src and p38 MAPK/JNK to the cytotoxic action of AplidinTM by measuring the viability of MDA-MB-231 cells and wild-type and egfr-/- MEFs treated with the drug in the presence of PP2 or SB203580. In all three cell types, SB203580 significantly inhibited AplidinTM action. In contrast, PP2 was effective only in MDA-MB-231 cells, but not in MEFs (Fig. 4C). In agreement with the differential activation of p38 MAPK/JNK, these experiments also confirmed that egfr-/- MEFs are less sensitive to the drug than wild-type MEFs. Together, these results indicate that the activation of EGFR, Src, and p38 MAPK/JNK is involved in AplidinTM action in MDA-MB-231 cells, whereas only EGFR and p38 MAPK/JNK (but not Src) seem to be involved in the cytotoxic effect of AplidinTM in MEFs. EGFR activation participates in, but is not sufficient for, the induction of apoptosis by AplidinTM.


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Fig. 4.   Involvement of EGFR, Src, and p38 MAPK/JNK in AplidinTM action. A, EGFR was involved in AplidinTM-induced apoptosis. Shown are dot-plot diagrams of Alexa 488-conjugated annexin V/propidium iodide flow cytometry of egfr-/- and wild-type MEFs after treatment with AplidinTM. Cells were incubated for 5 h in the absence (Control) or presence of 500 nM AplidinTM (APL) in serum-free medium. The cells were labeled with Alexa 488-conjugated annexin V and propidium iodide and analyzed by flow cytometry. The lower left quadrants of each panel show the viable cells (Alexa 488-/propidium iodide-); the upper right quadrants contain the nonviable, necrotic, and/or late apoptotic cells (Alexa 488+/propidium iodide+); and the lower right quadrants represent the early apoptotic cells (Alexa 488+/propidium iodide-). The numbers in each quadrant represent the percentage of cells. The results are representative of one of two experiments, which gave similar results. B, upper panels, the levels of activated p38 MAPK and JNK in egfr-/- and wild-type MEFs treated with AplidinTM (500 nM) for the indicated times or left untreated (control (C)) were measured by Western blotting using phospho (P)-specific antibodies as described in the legend to Fig. 2. Antibodies against total p38 MAPK or JNK were used as controls. To reduce basal enzyme activities, cells were incubated overnight in serum-free medium. Three independent experiments were performed. Values from a representative experiment are shown. Lower panels, shown are the effects of SB203580, PP2, and GSH on p38 MAPK and JNK activation by AplidinTM. Cells were pretreated with SB203580 (SB; 30 µM), PP2 (25 µM), GSH (15 mM), or vehicle (control (C)) for 1 h before AplidinTM addition (500 nM, 1 h) as indicated. Conditions were as reported for A. C, shown is the inhibition of AplidinTM cytotoxicity in human MDA-MB-231 cells and wild-type and egfr-/- MEFs by SB203580 (30 µM) or PP2 (25 µM). Cultures were pretreated with the inhibitors for 2 h before AplidinTM (500 nM) addition, and the total cell mass in the cultures was measured 16 h (MDA-MB-231 and egfr-/- fibroblasts) or 4 h (wild-type fibroblasts) later by the crystal violet staining method. Means ± S.D. of values obtained in AplidinTM-untreated controls are shown. ***, p < 0.001.

Glutathione Depletion Is Critical for Activation of Src, EGFR, JNK, and p38 MAPK by AplidinTM-- Reactive oxygen species mediate JNK activation by pro-inflammatory cytokines (interleukin-1 and tumor necrosis factor-alpha ) (40), an effect that, in the case of H2O2, requires Src-dependent EGFR transactivation (31). These data led us to investigate the putative role of reactive oxygen species in AplidinTM action. As shown in Fig. 5 (A and B) (data not shown), pretreatment of MDA-MB-231 cells with various antioxidants such as ascorbic acid, DPI (an inhibitor of flavin-containing enzymes such as NAD(P)H oxidase), Tiron (a scavenger of superoxide ions), and vitamin E or addition of catalase (H2O2-degrading enzyme) did not affect the induction of EGFR phosphorylation by AplidinTM. In contrast, two compounds that raise the cellular level of GSH, N-acetylcysteine (a GSH precursor) and ebselen (a GSH peroxidase mimetic), reduced EGFR phosphorylation by AplidinTM (Fig. 5C). Confirming the involvement of GSH in AplidinTM action, treatment of cells with exogenous GSH diminished, whereas BSO (a specific inhibitor of gamma -glutamylcysteine synthetase) increased, AplidinTM-induced EGFR phosphorylation (Fig. 5C). None of the agents tested (N-acetylcysteine, ebselen, DPI, and Tiron) modified EGFR activation by its EGF ligand (Fig. 5D).


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Fig. 5.   AplidinTM-induced EGFR phosphorylation relies on cellular GSH depletion. Phosphorylated (P) and total EGFRs were measured by immunoprecipitation and Western blotting as described in the legend to Fig. 3. A, some antioxidants (N-acetylcysteine (NAC), 10 mM; and ebselen (EBS), 40 µM), but not others (DPI, 10 µM; and Tiron (Tir), 10 mM), inhibit AplidinTM-induced EGFR phosphorylation. Where indicated, MDA-MB-231 cells were pretreated with the antioxidants 1 h before addition of AplidinTM, and extracts were prepared 1 h later. B, exogenous catalase does not affect EGFR phosphorylation by AplidinTM. Cells were incubated with catalase (Cat; 1000 units/ml) for 1 h as indicated before AplidinTM (APL) treatment. As a control, we used cells treated with H2O2 (10 mM) plus or minus catalase pretreatment. C, cellular GSH content modulates EGFR phosphorylation by AplidinTM. Treatment of cells with exogenous GSH (15 mM, 1 h) or BSO (1 mM, 24 h) before AplidinTM had opposite effects on the induction of EGFR phosphorylation. In A-C, AplidinTM was used at 500 nM for 1 h. D, antioxidants do not affect EGF-induced EGFR phosphorylation. Cells were pretreated with DPI, Tiron, N-acetylcysteine, or ebselen for 1 h at the doses indicated for A before addition of EGF (20 ng/ml, 10 min). Results shown correspond to a representative experiment. The experiments shown in A and C were performed three times, and those in B and D were performed twice. C, control.

We next analyzed the possible link between GSH content, Src and EGFR, and the activation of JNK and p38 MAPK. Exogenously added GSH and N-acetylcysteine (but not BSO) reduced JNK and p38 MAPK activation by AplidinTM (Fig. 6A). Likewise, GSH also blocked Src activation by AplidinTM, whereas BSO had no effect (Fig. 6B). In addition, PP2 and tyrphostin AG1478 (an EGFR inhibitor) also inhibited the activation of these kinases by AplidinTM (Fig. 6C). Together, these data indicate that the depletion of GSH activates Src and that the activation of Src and EGFR is partially responsible for the activation of JNK and p38 MAPK. EGF alone did not induce p38 MAPK and induced JNK only weakly and did not affect the activation of these two kinases by AplidinTM (Fig. 6D).


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Fig. 6.   Sustained activation of p38 MAPK and JNK by AplidinTM is partially dependent on GSH depletion and Src and EGFR activation. A, intracellular GSH levels modulate the activation of p38 MAPK and JNK by AplidinTM. MDA-MB-231 cells were treated with the indicated agents that modulate GSH levels at the doses indicated in the legend to Fig. 4 for 1 h (GSH and N-acetylcysteine (NAC)) or 24 h (BSO) and thereafter were treated with AplidinTM (500 nM) or left untreated (control (C)). Levels of activated p38 MAPK or JNK were measured by Western blotting using phospho (P)-specific antibodies as described in the legend to Fig. 2. Antibodies against total p38 MAPK or JNK were used as controls. Three independent experiments were done. Values from a representative experiment are shown. B, Src activation by AplidinTM depends on GSH depletion. Cells were treated with exogenous GSH (15 mM, 1 h) or BSO (1 mM, 24 h) and thereafter treated with AplidinTM (500 nM). Activated Src was measured 45 min later by Western blotting using anti-phospho-Tyr418 antibody. Antibody against total Src was used as a control. C, inhibition of Src and EGFR reduces p38 MAPK and JNK activation by AplidinTM. Cells were pretreated with PP2 (25 µM) or AG1478 (20 µM) before addition of AplidinTM (500 nM). Phospho-p38 MAPK and phospho-JNK were measured as described for A in extracts prepared 1 h after AplidinTM treatment. The experiment was performed three times. Values from a representative experiment are shown. D, AplidinTM has a dominant effect over EGF on p38 MAPK and JNK activity. Cells were treated with EGF (20 ng/ml), AplidinTM (500 nM), or both for 1 h, and the levels of phospho-p38 MAPK and phospho-JNK were measured as described for A. Two independent experiments gave the same results.

AplidinTM Induces Cytotoxicity in Renal Cancer Cells-- To examine whether the mechanism of action of AplidinTM in MDA-MB-231 cells could be extrapolated to human renal cancer cells, we used the A-498 and ACHN cell lines. Both were sensitive to the antiproliferative (IC50 = 5 and 15 nM, respectively) and cytotoxic action of AplidinTM (Fig. 7A). As in MDA-MB-231 cells, we found that AplidinTM induced EGFR phosphorylation in A-498 cells (Fig. 7B). The effect on Src was also investigated. In contrast to breast cancer cells, both A-498 and ACHN cells showed a high level of Src activity, which was not affected by AplidinTM (Fig. 7C). Because the drug activated JNK and p38 MAPK in A-498 and ACHN cells (Fig. 7D), these results indicate that, in these two renal cancer cell lines, the activation of these kinases by AplidinTM does not require further Src activation. SB203580 reduced the activation of both kinases (Fig. 7D). The contribution of EGFR, p38 MAPK, and JNK activation and also of Src to the cytotoxic effect of AplidinTM in A-498 and ACHN cells was studied by flow cytometry using AG1478, PP2, and SB203580. In ACHN cells, all three inhibitors reduced the population of hypodiploid cells with the following range of potency: SB203580 > AG1478 > PP2 (Table I). Double AG1478 + SB203580 treatment was more effective than either agent alone, whereas combined treatments including PP2 inhibited also AplidinTM, but showed high toxicity in untreated cells. A possible explanation may be that inhibition of the basal activity of more than one of the target enzymes of these agents is deleterious, whereas cells treated with AplidinTM and combinations of inhibitors may display kinase activities similar to those of untreated cells. A-498 cells were very sensitive to the absence of serum (data not shown); and unexpectedly, in low serum, neither of the inhibitors reduced the induction of apoptosis by AplidinTM (Table I). In these cells, only the double AG1478 + SB203580 treatment had a small inhibitory effect, whereas the triple AG1478 + SB203580 + PP2 treatment had a higher effect, although it was toxic in the absence of AplidinTM.


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Fig. 7.   Human renal cancer cells are sensitive to AplidinTM. A, phase-contrast micrographs of A-498 and ACHN cell cultures treated with vehicle (Control, C) or AplidinTM for 24 h in normal growth medium. Scale bar = 40 µM. B, time course of the induction of EGFR phosphorylation by AplidinTM in A-498 cells. Total and phosphorylated (P) EGFRs were measured by immunoprecipitation and Western blotting as described in the legend to Fig. 3. C, effect of AplidinTM on Src activity in A-498 (upper panels) and ACHN (lower panels) cells. Total and activated Src proteins were measured by Western blotting using anti-Src antibody and the activation-linked anti-phospho-Tyr418 antibody, respectively, as described under "Experimental Procedures." D, activation of p38 MAPK (upper panels) and JNK (lower panels) by AplidinTM in A-498 (left panels) and ACHN (right panels) cells. The inhibitory effect of SB203580 (30 µM) is shown. Total and activated forms of both kinases were measured by Western blotting using appropriate antibodies as described in the legend to Fig. 2. In all experiments, AplidinTM was used at 500 nM. Results shown correspond to a representative experiment. The experiments shown in A-C were performed three times, and those in D were performed twice.

                              
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Table I
Role of EGFR, Src, and p38 MAPK/JNK in AplidinTM-induced apoptosis in renal ACHN and A-498 cells
Shown are the results from flow cytometry analysis of DNA content using propidium iodide in cultures incubated in the absence or presence of AplidinTM (500 nM) in serum-free medium (ACHN) or in medium supplemented with 1% serum (A-498) for 16 h. Percentages of apoptotic cells corresponding to the subdiploid population are shown. Cells were treated with the indicated inhibitor(s) from 2 h before AplidinTM addition to the end of the experiment or were left untreated (control). The results are representative of one of two experiments with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have examined the effect of the novel antitumor agent AplidinTM in human breast and renal cancer cells. AplidinTM shows cytotoxicity at pharmacologically relevant concentrations linked to both inhibition of cell proliferation and apoptosis. It induces apoptosis via caspase-dependent and -independent mechanisms irrespective of the cellular p53 status through the sustained activation of the serine/threonine kinases JNK and p38 MAPK. These events are triggered rapidly in part by the induction of GSH depletion and Src tyrosine kinase.

AplidinTM activates EGFR by a mechanism that is partly dependent on Src activation. Several findings indicate that EGFR activation by AplidinTM is aberrant. First, it is weaker and more prolonged than that induced by EGF; second, it is accompanied by a delay in the down-regulation of the receptor; and third, it does not induce binding to the receptor of adaptor proteins such as Grb2 and Shc, suggesting differences in signaling with respect to EGF. Our data obtained with human MDA-MB-231 and renal ACHN cancer cells and with mouse fibroblasts indicate that EGFR activation is involved in, but is not sufficient for, AplidinTM-induced apoptosis. Apart from its common physiological ligands EGF and transforming growth factor-alpha , multiple physiological and nonphysiological stimuli and agents transactivate EGFR, including redox stress, activation of various G-protein-coupled receptors and voltage-gated Ca2+ channels, cytokine receptors, osmotic stress, and UV and gamma -radiation (41, 42). The intensity and duration of EGFR transactivation, in addition to substrate availability and other signals, are critical for the cellular response, either proliferation or alternative pathways. Remarkably, AplidinTM has a dominant effect on EGF, altering the binding of adaptor proteins and inducing stronger activation of JNK and p38 MAPK and apoptosis also in EGF-treated MDA-MB-231 cells. Moreover, these cells harbor mutated p53 and ras genes and high levels of activated ERK and Akt, two enzymes that promote cell survival and proliferation. Together, these data emphasize the potent cytotoxic activity of AplidinTM.

Src has been proposed to transactivate EGFR upon several stimuli such as oxidative stress and G-protein-coupled receptor activation, in the latter case by forming a complex with Pyk2 that directly phosphorylates EGFR (38). In other systems, EGFR phosphorylation by Src results in hyperactivation of receptor kinase activity (37, 43) and in the enhancement of the mitogenic response of cells to EGF (44). Because Src binds to and is a substrate of EGFR (45), an activation loop resulting from this mutual catalytic regulation between both kinases (46) can amplify the AplidinTM signal causing the abnormally long activation and half-life of EGFR. To what extent this activation loop is responsible for the sustained activation of JNK and p38 MAPK is unknown, but the partial inhibition by tyrphostin AG1478 supports this idea. Although direct binding of AplidinTM to EGFR or indirect induction of receptor dimerization cannot be ruled out, our data support that AplidinTM causes EGFR activation through the previous activation of Src. In line with our data showing the activation of Src by AplidinTM, deregulation of Src activity is involved in the caspase-independent apoptosis induced by the adenoviral early region 4 open reading frame-4 protein (47). Thus, like c-Myc (see Ref. 48 for review), c-Src may transduce either proliferative or apoptotic signals.

AplidinTM action is inhibited by N-acetylcysteine, a potent antioxidant that can be a source of sulfhydryl metabolites, stimulate GSH synthesis, enhance glutathione S-transferase activity, promote detoxification, and act directly on reactive oxygen species (49). Together with the inhibitory effects of exogenous GSH and the stimulation by BSO of EGFR and Src activation by AplidinTM and the activation of Src by reactive oxygen species in many cell systems, these results strongly suggest that the activation of Src by AplidinTM depends on the depletion of cellular GSH. N-Acetylcysteine has been found to inhibit receptor kinase signaling in many cell systems, suppressing the activation of ERK by H2O2 (50); of ERK and JNK by interleukin-1 (51); and of ERK, JNK, and p38 MAPK by arsenite (52). Therefore, AplidinTM might act by inducing oxidative stress at the plasma membrane, leading to a reduction in GSH levels and Src activation. Alternatively, AplidinTM may directly modulate GSH synthesis and/or metabolism. Early GSH depletion is suggestive of an oxidative process and renders the cells more sensitive or precedes apoptotic cell death induced by various agents (Ref. 53 and references therein), although this is not the case for others such as camptothecin and etoposide (54). GSH regulates JNK and p38 MAPK (55). Similar to our results, the level of intracellular GSH is a critical regulator of the induction of JNK and p38 MAPK by alkylating agents such as methyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine (56).

JNK is involved in many cellular responses such as proliferation, differentiation, and apoptosis. It is thought that the duration of its activation may be crucial in the signaling decision; sustained (but not transient) JNK activation participates in the apoptosis induced by several stress stimuli (12, 57, 58). This indicates that the sustained JNK activation reported in this work is crucial for the pro-apoptotic effect of AplidinTM. Like JNK, p38 MAPK is also involved in the apoptotic response of many cells to cytotoxic agents (16, 59). Our results show that the activation of both p38 MAPK and JNK by AplidinTM depends in part on Src activation, as does that caused by changes in the redox status in some systems (33).

Data obtained using inhibitors indicate that, in human breast cancer cells, activation of EGFR, Src, and p38 MAPK/JNK is involved in AplidinTM action, whereas Src activation is dispensable in MEFs. Compared with breast cells, the inhibitory effect of AG1478, PP2, and SB203580 is variable in human renal cancer cells (similar in ACHN cells, but lower in A-498 cells), suggesting that other signaling pathways may be involved in AplidinTM action in this cell line. Therefore, AplidinTM-induced apoptosis in carcinoma cells is partly the result of Src activation, perhaps in combination with additional effects of GSH depletion, which leads to the sustained activation of JNK and p38 MAPK. Because N-acetylcysteine and PP2 inhibit but do not completely block p38 MAPK/JNK activation by AplidinTM and are cytotoxic in long incubations, the contribution of GSH depletion and Src activation cannot be precisely determined, and other upstream regulators of p38 MAPK/JNK might be involved in AplidinTM action. The lack of appropriate specific inhibitors of JNK or p38 MAPK has hampered the elucidation of the role of each individual kinase.

Several anticancer drugs in clinical use modulate JNK activity. JNK mediates the apoptosis induced by DNA-damaging drugs such as etoposide (VP-16) and camptothecin in human myeloid leukemia cells (14) and of vinblastine in KB3 carcinoma cells (18). In MDA-MB-231 cells, paclitaxel-induced apoptosis is mediated by the induction of JNK, which causes inactivation by phosphorylation of the anti-apoptotic Bcl-2 protein (17). Taxol has also been shown to increase p38 MAPK, ERK, and (to lesser extent) JNK activity in human breast cancer cells (59). Strikingly, tamoxifen, a partial agonist/antagonist of the estrogen receptor that has a cytostatic action by inhibiting estrogen action, also has a cytotoxic effect in the estrogen receptor-negative MDA-MB-231 and BT-20 cell lines via the induction of apoptosis (61). This effect is mediated by JNK activation and inhibited by vitamin E, but not by N-acetylcysteine or GSH (60). In HepG2 hepatoma cells, 5-fluorouracil-induced apoptosis is mediated by JNK activation (61). Also, cisplatin induces apoptosis through both JNK and p38 MAPK (16).

In summary, we report that AplidinTM induces apoptosis in human breast and renal cancer cells via the sustained activation of the signaling kinases JNK and p38 MAPK. We show that, in MDA-MB-231 cells, this is in part a consequence of Src activation, which is preceded by cellular GSH depletion. In addition, AplidinTM causes aberrant EGFR activation, which is also involved in the activation of p38 MAPK and JNK in MDA-MB-231 cells and mouse fibroblasts and in the cytotoxic effect in the latter and in renal ACHN cells. To establish the contribution of the abnormal EGFR activation to the anticancer activity of AplidinTM will require the correlation of EGFR expression and tumor sensitivity in patients. Our results indicate that there must be additional AplidinTM targets leading to p38 MAPK/JNK activation. In view of the promising clinical results of AplidinTM, the identification of these targets and also the elucidation of the signaling from GSH, EGFR, and Src to JNK and p38 MAPK merit further investigation.

    ACKNOWLEDGEMENTS

We thank Dr. E. Wagner for providing the wild-type and egfr-/- MEFs, Dr. J. Martín for help with the Src experiments and Dr. J. Brugge for anti-Src antibody. We are also grateful to Robin Rycroft for valuable assistance in the preparation of the English manuscript.

    FOOTNOTES

* This work was supported in part by Grant SAF2001-2291 from the Plan Nacional de Investigación y Desarrollo of Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

|| Recipient of a postdoctoral fellowship from the Consejo Superior de Investigaciones Científicas.

** Recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura of Spain.

Dagger Dagger To whom correspondence should be addressed: Inst. de Investigaciones Biomédicas "Alberto Sols," Arturo Duperier, 4, E-28029 Madrid, Spain. Tel.: 34-91-585-4640; Fax: 34-91-585-4587; E-mail: amunoz@iib.uam.es.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M201010200

    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MEFs, mouse embryo fibroblasts; DPI, diphenyleneiodonium; BSO, L-buthionine (SR)-sulfoximine; PDGFR, platelet-derived growth factor receptor; PBS, phosphate-buffered saline.

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