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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Microtubule-Destabilizing Agents Induce Focal Adhesion Structure Disorganization and Anoikis in Cancer Cells

Centre de Recherche, Unité des Biotechnologies et de Bioingénierie, Centre Hospitalier Universitaire de Québec, Hôpital Saint-François d'Assise, Québec, Canada (R.G.D, A.P., J.S.F., C.R., M.-F.C., R.C.-G., E.P.); IMOTEP Inc., Québec City, Québec, Canada (J.L., C.R.); and Le Centre de Recherche en Cancérologie de l'Université Laval, Québec, Canada (J.H.)

Received July 17, 2006; accepted November 7, 2006.

	Abstract

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Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH₂-terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.

A direct interaction between FA structures and MTs was demonstrated during cell spreading and migration, where MTs play an essential role for the development initiation and the maintenance of a directional cell movement (Brown and Turner, 2004). It was suggested that paxillin might capture MTs at FA sites to regulate the remodeling of cell/ECM contacts during spreading and motility. Interestingly, the growing MT protofilaments were shown to physically connect to FA sites to deliver "relaxing signals" opposed to the actin/myosin-dependent forces (Pletjushkina et al., 1998; Kaverina et al., 1999; Krylyshkina et al., 2003). The disruption of MT increases actin-myosin contractility and enlargement of actin stress fibers bundles (Danowski, 1989; Huang et al., 2004), and an initial increase of the size and the number of FA sites along with an increased cell adhesion (Pletjushkina et al., 1998; Kaverina et al., 1999). In others cell lines that were exposed to MT-disrupting agents, a decrease of cell adhesion was also reported (Dahlgren et al., 1987; Stracke et al., 1993).

A strong decrease of cell adhesion may in fact lead to cell detachment from the ECM, which often triggers anoikis. Anoikis defines the induction of apoptosis by the inappropriate or the loss of cell anchorage to ECM, following early inhibition of integrin signaling (Reddig and Juliano, 2005). All the features that characterize apoptosis are also observed during anoikis, such as caspases activation and nuclear fragmentation (Reddig and Juliano, 2005). Furthermore, it was observed that simultaneous pharmacological disruption of both MT and actin cytoskeleton networks amplify the cell rounding and enhance the level of apoptosis, suggesting that the disruption of structural elements or changes in cell shape might trigger anoikis (Flusberg et al., 2001). The molecular mechanisms of anoikis vary among cell types, modulated by a rapid and strong activation of MAP kinases such as JNK and p38 (Vachon et al., 2002; Harnois et al., 2004; Reddig and Juliano, 2005). Overexpression of oncogenes bypassing integrin-dependent survival signals such as Ras, Raf, Rac, and Src was also showed to generate anoikis-resistant cells (Reddig and Juliano, 2005). Moreover, overexpression of the functional FA signal transducers FAK or ILK (Attwell et al., 2000) blocked anoikis, despite the loss of cell anchorage (Reddig and Juliano, 2005).

In this study, we assessed the early and prolonged impacts of anti-MT drugs on 1) cytoplasmic MT disruption, 2) state of FA structures, 3) actin cytoskeleton, and 4) FA functions in relation with the kinetics that force cells into rounding, detachment from ECM, and death. We used anti-MT agents binding to the colchicine-binding site (colchicine) and the vinca domain (vinblastine) of -tubulin, respectively. We used also N-aryl-N'-(2-chloroethyl)ureas (CEUs) (Legault et al., 2000; Petitclerc et al., 2004) that are colchicine binding site-reacting agents forming covalent bonds with -tubulin, and cisplatin (cDDP) that is a strong DNA alkylator. For the first time, we report that MT disruption forces a biphasic sequence of events, starting with an increase of tumor cell adhesion to the ECM followed by a loss of cellular adhesion. Indeed, prolonged MT disruption leads to physical disorganization of FA architecture, paralleled with an irreversible loss of cell adhesion potential. Time-dependent analysis of the effects of anti-MT agents suggests that these important events initiate caspase-dependent cell death by anoikis. Altogether, this study provides new insights on the clinical usefulness of anti-MT agents and brings new concepts for potential combination therapy that could be foreseen for such agents that modulate cell adhesion.

	Materials and Methods

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Chemicals. Vinblastine sulfate, colchicine, paclitaxel, cisplatin, PD098059, SB203580, ML-7, and poly-HEMA were purchased from Sigma-Aldrich (St. Louis, MO). L-JNKI-1 was obtained from Alexis Biochemicals (San Diego, CA). All drugs were dissolved in DMSO and used at final concentrations lower than 0.25% (v/v). The chemicals for electrophoresis were purchased from Bio-Rad (Hercules, CA). CEUs were provided by IMOTEP Inc. (Quebec City, Canada) and prepared as published previously (Béchard et al., 1994).

Cell Lines and Culture. MDA-MB-231 breast carcinoma, HT1080 fibrosarcoma, and M21 human melanoma cells were purchased from American Type Culture Collection (Manassas, VA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and cultured in DMEM containing 2.2 g/l NaHCO₃, 4.5 g/l glucose, 100 µg/ml streptomycin sulfate A, 100 U of penicillin G, 292 µg/ml glutamine, and 5% bovine calf serum (Hyclone Laboratories, Logan, UT).

Antibodies. Mouse anti-FAK (clone 4.47), anti-vinculin (clone V284), anti-talin (clone TA205), and anti-ILK (clone 65.1.9) were obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit antibodies rose against cleaved caspase-3, -6, -7, and -9, and the mouse monoclonal anti-phosphotyrosine (clone PY-20) and anti-paxillin (clone 349) were purchased at BD Transduction Laboratories (Mississauga, ON, Canada). Mouse anti-caspase-8 and rabbit anti-paxillin (H-114) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-rabbit antibodies against the phosphorylated form of ERK1/2 (20G11), JNK (98F2), and p38 were all obtained from Cell Signaling Technology (Beverly, MA). The anti-mouse Alexa 594 and 488 and the anti-rabbit Alexa 488 were from Invitrogen (Carlsbad, CA), whereas the horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from GE Healthcare (Little Chalfont, Buckinghamshire, UK).

Confocal Fluorescence Microscopy. Cells were detached with phosphate-buffered saline (PBS) containing 1 mM EDTA and then plated on 10 µg/ml fibronectin-coated glass slides. After 90 min at 37°C in DMEM containing 0.5% BSA, 1 mM MgCl₂, and 0.2 M MnCl₂, cells were fixed 20 min with 3.7% formaldehyde. They were permeabilized using 0.1% Triton X-100/PBS for 3 min and blocked 30 min with 10% normal goat serum/PBS. Phosphotyrosines, FAK, paxillin, and ILK antibodies (1:100) were used in 10% normal goat serum/PBS for 1 h at 37°C. Cells were next incubated simultaneously with anti-mouse IgG Alexa 488-conjugated antibody (1:1000) and rhodamine-labeled phalloidin (1:1000) for 1 h at 37°C, and then they were examined by confocal microscopy with a Nikon D-Eclipse C1 imaging system mounted on a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) equipped with a 100x objective and driven by EZ-C1 version 1.7 software (Nikon).

Plasmids, Transfection, and Cell Viability Assay. The pcDNA-3.1 constructs for ILK-WT and the mutants S343A, S343D, and S359K were kindly provided by Dr. Shoukat Dedhar (Department of Advanced Therapeutics, BC Cancer Agency, Vancouver, BC, Canada). Cells (7500/well) were seeded in 96-well plates and incubated for 24 h at 37°C. They were next transfected or not with the FuGENE 6 reagent according to the manufacturer procedures (Roche Diagnostics, Indianapolis, IN). Twenty-four hours post-transfection, fresh medium containing DMSO or drugs tested was added, and cells were further incubated at 37°C for 30 h. Resazurin (25 µg/ml) was next added to the culture medium for 2 h at 37°C. The cell viability was calculated from fluorescence (excitation, 485 nm; emission, 590 nm) measured with a FL 600 Reader (Bio-Tek Instruments, Winooski, VT). The data from experiments conducted in triplicate were corrected for the background fluorescence of the medium and were expressed as the percentage of fluorescence obtained for control DMSO-treated cells.

Adhesion Assay. Treated cells were detached using PBS containing 1 mM EDTA and were resuspended in a DMEM adhesion medium containing 0.5% BSA, 1 mM MgCl₂, and 0.2 M MnCl₂. They were next plated in triplicates (1 x 10⁵ cells/well) onto fibronectin (10 µg/ml)-coated wells, previously blocked for 30 min with 1% BSA/PBS. Cells were allowed to attach without spreading (6–7 min). The adhesion medium was discarded, and the attached cells were washed before staining with crystal violet. Unbound dye was removed, and the plates were air-dried before addition of acetic acid (10% in PBS). The absorbance was read at 600 nm using a µQuant universal microplate spectrophotometer (Bio-Tek Instruments).

Western Blot Analysis. The pooled floating and adherent cells from DMSO or drug treatments were washed in ice-cold PBS, pooled, and then resuspended in SDS-PAGE buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.00125% bromphenol blue, and 5% -mercaptoethanol. The protein extracts (15 µg) were then sonicated, boiled for 5 min, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. All membranes were blocked for 1 h at 37°C with 5% (w/v) milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membranes were next incubated with the first antibody:1 h at 37°C with the -tubulin (1:500), ILK (1:1000), and paxillin (1:10,000) antibodies, diluted in 5% milk/TBST; overnight at 4°C with the vinculin (1:1000), talin (1:1000), phospho-p38 (1:1000), phospho-JNK (1:1000), phospho-ERK1/2 (1:1000), caspase-3 and -7 (1:1000), and caspase-6, -8, and -9 (1:500) antibodies diluted in 5% BSA/TBST. The antibody for FAK (1:2500) was diluted in 5% milk/TBST and incubated overnight at 4°C. All membranes were next incubated with a horseradish peroxidase-conjugated goat anti-mouse or -rabbit IgG antibody (1:2500) diluted in 5% milk/TBST, 1 h at room temperature, followed by chemiluminescent detection, using an ECL detection kit (GE Healthcare).

Two-Dimensional Isoelectric Focusing Electrophoresis. Isoelectric focusing was performed using the PROTEAN IEFCell apparatus according to the manufacturer's procedures (Bio-Rad). Treated cell pellets were incubated overnight at room temperature with rehydration buffer containing 8 M urea, 10 mM DTT, 4% CHAPS, and 0.2% (w/v) ampholytes, pH 3 to 10. This mixture was then applied to a ReadyStrip IPG (11 cm; pH 3–10). Proteins were isoelectrically focused with the following cycle steps: S1, 15 min/250 V; S2, slowly increase up to 8000 V/2 h 30 min; S3, 8000 V/4 h 20 min for a total of 35,000 Vh/gel; and S4, 500 V/until run stopped. After their equilibration with buffer I [6 M urea, 375 mM Tris, pH 8.8, 2% SDS, 20% glycerol, and 2% (w/v) DTT] for 10 min followed by buffer II [buffer I except that 2.5% (w/v) iodoacetamide was used instead of DTT] for 10 min, strips were next placed in melted agarose wells overlaying a 10% SDS-PAGE for second-dimension electrophoresis. Proteins were then transferred onto nitrocellulose membranes.

Paxillin Immunoprecipitation. Treated cells were extracted in standard radioimmunoprecipitation assay lysis buffer then centrifuged at 13,000 rpm for 15 min at 4°C. The clarified supernatants were precleared overnight with protein G-Sepharose (GE Healthcare) 50% (v/v) in radioimmunoprecipitation assay buffer (without SDS), centrifuged, and incubated overnight at 4°C with a limiting concentration of 2 µg of anti-paxillin antibody, before addition of protein G-Sepharose for 2 h. Beads were washed, resuspended in SDS-PAGE buffer, and boiled for 3 min before SDS-PAGE. To evaluate the effect of the alkaline phosphatase in vitro, beads were incubated 4 h at 37°C in buffer containing 10 mM Tris-HCl, pH 8, and 1 mM MgCl₂ and the absence or presence of 3.75 U/reaction of bacterial alkaline phosphatase (Invitrogen Canada Inc., (Burlington, ON, Canada).

Flow-Cytometric Analysis of Cell Cycle. Cells were plated in 100-mm Petri dish (1.5 x 10⁶ cells/well) and were left to adhere to the Petri dish for at least 24 h. The drugs were added 16 h before staining at the indicated concentrations. The adherent cells were trypsinized and pooled to the floating cells in ice-cold PBS. After centrifugation, the cells were resuspended in 200 µl of ice-cold PBS, and 70% ethanol was added slowly while vortexing for fixation. Cells were kept on ice for at least 30 min. Cells were diluted to 2 x 10⁶ cells/ml in PBS containing 50 µg/ml propidium iodide and 40 U/ml RNAase A. The cells were analyzed using a fluorescence-activated cell sorter (BD Biosciences PharMingen, San Diego, CA), and the results were integrated using the ModFit software (BD Biosciences PharMingen).

	Results

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MT-Disrupting Agents Vinblastine, Vincristine, and Paclitaxel and a Newly Described -Tubulin Alkylating CEU Induce Apoptosis. Classical MT-depolymerizing agents and -tubulin alkylating CEUs share similar cancer cell growth inhibition properties (Legault et al., 2000; Petitclerc et al., 2004; Mollinedo, 2005). Figure 1A illustrates that MT depolymerization occurs also in response to CEU-236, a newly discovered CEU compound having better growth inhibition (Moreau et al., 2005). In parallel, actin fibers seem to be reduced, confirming the cytoskeletal disorganization following anti-MT treatment. Compared with other -tubulin-alkylating CEUs such as CEU-022 and CEU-098, CEU-236 induced a faster apparition of the -tubulin alkylation by-product (<6 h; Fig. 1B), which can be distinguished from the native -tubulin (Legault et al., 2000) by its increased electrophoresis mobility. In contrast, CEU-091 did not alkylate -tubulin (Legault et al., 2000), induce the formation of the -tubulin alkylation by-product (Fig. 1B), or depolymerize the MT network (Fig. 1A).

View larger version (49K): [in this window] [in a new window]   Fig. 1. CEU-236 is potent -tubulin-alkylating CEU exhibiting MT-disrupting properties. A, MDA-MB-231 cells were exposed for 16 h with DMSO (0.1%, v/v); 30 µM CEU-022, CEU-098, CEU-236, and CEU-091; 10 and 30 µM cDDP; or 15 nM COL. After treatments, cells were detached using PBS-EDTA solution and then plated on fibronectin-coated wells before fixation, permeabilization, and staining with specific anti--tubulin and anti-F-actin antibodies, as described under Materials and Methods. Analyses were performed by confocal microscopy. B, MDA-MB-231 cells were treated for indicated time with 0.25% DMSO; 30 µM CEU-022, CEU-098, CEU-236, and CEU-091; 50 µM cDDP, COL, and paclitaxel; or 5 µM VINB. After treatments, protein extracts from pooled adherent and floating cells were separated by 10% SDS-PAGE and analyzed by Western blot analysis with an antibody raised against -tubulin. Similar results were obtained using M21 (Petitclerc et al., 2004) and HT-29 cancer cells (data not shown).

We next investigated whether -tubulin-alkylating CEU induces apoptosis in cancer cells. Cleavage-dependent activation of the initiator caspase-9 and caspase-8 constitutes hallmarks for the initiation of the intrinsic and extrinsic apoptosis program, respectively (Leist and Jaattela, 2001). We thus compared the kinetics of activation of these two major caspase-dependent apoptotic pathways along with those of major effector caspases such as caspase-3, -6, and -7 in response to CEU, COL, and VINB. Caspases activation was also analyzed in response to the DNA-alkylating agent cDDP, which is not an anti-MT agent (Fig. 2A). MDA-MB-231 cells were challenged for times ranging from 6 to 48 h. Drug concentrations used in this study yielded to at least 90% cell growth inhibition in response to all 48-h drug exposures (data not shown). DMSO or CEU-091 had no significant effect on the caspases studied (Fig. 2A). As expected, all drugs tested have triggered a sequence of caspases activation initiated by caspase-8 and -9 followed by the activation of the effector caspase-6, -7, and -3. However, the onset of caspase-8 activation occurred later in response CEU-022 or CEU-236. Similar patterns of caspases activation were observed using human melanoma (M21) and fibrosarcoma (HT1080) cell lines (data not shown). It is noteworthy that lower concentrations of MT-targeting agents used in this study (e.g., 50 nM for COL and VINB or 0.5 µM for CEU-236) also lead to caspases activation in HT1080 cells but after longer exposures (72 h; data not shown). Together, these results show that CEU induce apoptosis through sequential caspases activation. 4,6-Diamidino-2-phenylindole staining revealed that all MT-disrupting agents tested herein induced typical apoptotic DNA fragmentation 24-h post-treatment (data not shown). On the basis of caspase-9 and -8 sequences of activation, CEU-022 and CEU-236, conversely to others anti-MTs tested, initiate the intrinsic before the extrinsic apoptotic pathway. To further support the concept that caspases activation leads to cell death in our system, propidium iodide staining of cells after anti-MT agents was analyzed using fluorescence-activated cell sorting (FACS; Fig. 2B). MDA-MB-231 cells show an accumulation of cells in the G₂/M (area 3) and the noncycling section (area 4 of the graphs) when treated with CEU-022, CEU-098, or CEU-236. The nonalkylating molecule CEU-091 failed to influence the behavior of the cancer cells, and the CEU-236 showed the strongest effects, at much lower concentrations than the other CEUs.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Time-dependent proapoptotic caspases activation following MT disruption and induction of cell death. A, MDA-MB-231 cells were treated for escalating period of time with 0.25% DMSO; 30 µM CEU-022, CEU-098, CEU-236, and CEU-091; 50 µM cDDP and COL; or 5 µM VINB. After treatments, protein extracts from pooled adherent and floating cells were separated by 15% SDS-PAGE and analyzed by Western blot analysis with antibodies recognizing the activated forms (arrows) of caspase-9, -8, -6, -7, and -3. The anti-caspase-8 recognizes also the native 57-kDa form. B, FACS analysis of MDA-MB-231 cells after treatment with CEUs. The FACS was set to remove cell debris out of the evaluation, and the cell cycle was clearly delineated in 1, G0/G1; 2, S; 3, G2/M; and 4, DNA-fragmented cells (B, left).

MT-Disrupting Agents Induce a Biphasic Effect on the Tumor Cell Adhesion. As mentioned, MT-disrupting agents induce a biphasic response for adhesion to ECM of various cell lines. At first, there is an initial increase of cell adhesion to ECM followed by a sharp decrease of that cell adhesion (Dahlgren et al., 1987; Stracke et al., 1993; Pletjushkina et al., 1998; Kaverina et al., 1999). This is illustrated in Fig. 3, showing the time-dependent changes of cell adhesion in response to drugs and using fibronectin as the ECM. Interestingly, exposure to COL exhibited a dual effect on cell adhesion, characterized by an increase for up to 6 h post-treatment, followed by a sharp decrease of the cell adhesion to basal level after 30 h (Fig. 3). Similar changes were observed also in response to CEU-022 and CEU-236 when tested on M21 melanoma cells (data not shown). Hence, these responses are not specific to MDA-MB-231 epithelial tumor cells. CEU-091 induced comparable adhesion responses, whereas exposure to DMSO displayed no effect. cDDP induced no effect early on, but it displayed, between 16 and 30 h, a sharp decrease in cell adhesion. Together, our data strongly suggest that the increase of the cell adhesion to ECM observed is common to most MT-disrupting agents. It is noteworthy that the adhesion changes observed in this study occur before the onset of early steps of apoptosis, e.g., caspase-9 activation.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Biphasic effect of MT-disrupting agents on tumor cell adhesion potential. MDA-MB-231 cells were plated onto fibronectin-coated wells and allowed to attach for a short period (6–7 min) after the indicated drug treatments. Thirty-minute attachment, in the presence or absence of the anti-P4C10 antibody (10 µg/ml) (A) or with the indicated concentrations of CEU-091, CEU-022, CEU-098, and CEU-236, COL, or cDDP (B). Following the indicated attachment period, cells were fixed and stained with crystal violet for cell quantification, as described under Materials and Methods. Results are representative of four experiments and are expressed as the mean optical density (600 nm) ± S.E. of triplicates. The optical densities were normalized with the internal control (DMSO) for each experiments, and all the collected measurements of absorbance unit (a.u.) were below 1.0.

MT-Disrupting Agents Affect FA Structures. To assess whether distribution and integrity of FA structures reflect our observations on cell adhesion responses (Fig. 3), we performed confocal immunofluorescence analysis using an anti-phosphotyrosine antibody. This staining has been extensively used to detect rich tyrosinated-proteins (P-Y) of FA structures that seem punctated in attached cells (Mitra et al., 2005). Cells were costained using the F-actin-specific marker rhodamine-labeled phalloidin to study the structural cytoskeleton changes. MDA-MB-231 cells were challenged to reattach to a fibronectin matrix following short (3-h) and prolonged (12-h) drug exposures, matching times where significant changes were measured for the cell adhesion responses (Fig. 3). In comparison with DMSO, a dense punctated P-Y staining pattern was observed in response to 3-h exposure to COL, CEU-236, and CEU-022 (Fig. 4). In addition, a 3-h exposure of MT-targeting agents induced an increase in the number and size of actin-microfilament bundles (Fig. 4), a classic feature of increased cell contractility. These actin fibers are typically localized on cell periphery, and parallel bundles were oriented across cell length, as in normal attaching and spreading cells. In strict contrast to the 3-h exposure, the 12-h exposure with MT-disrupting agents used induced drastic scattering of P-Y staining, reminiscent of FA structures disorganization (Fig. 4). Furthermore, actin stress fibers seemed to reorganize in a faint structure directed toward cell periphery randomly orientated after 12 h, compared with 3-h exposure or DMSO treatment. The latter changes observed 12 h post-treatment coincided with cell rounding and detachment as observed using phase contrast microscopy (data not shown). No significant changes of -tubulin and actin protein levels were observed by Western blot analysis, supporting a reorganization of the cytoskeleton components rather than the modulation of their expression levels (data not shown). Exposure to CEU-091 induced no apparent changes on the actin stress fibers reorganization, compared with DMSO but, unexpectedly, it provoked enhanced FA structures number after 12 h (Fig. 4).

View larger version (60K): [in this window] [in a new window]   Fig. 4. Time-dependent analysis of MT-disrupting agents effects on FA structures and actin cytoskeleton integrity. MDA-MB-231 cells were treated for 3 or 12 h with 0.1% (v/v) DMSO; 30 µM CEU-022, CEU-098, and CEU-091; 1 µM CEU-236, 10 and 30 µM cDDP; or 15 nM COL. After these treatments, cells were detached, plated on fibronectin-coated wells, fixed, permeabilized, and then stained for detection of P-Y and F-actin, as described under Materials and Methods. Fluorescence of cells was analyzed by confocal microscopy. Data are representative of three independent experiments.

cDDP-treated cells for 3 or 12 h displayed similar FA structure staining patterns compared with DMSO treatment but an increased formation of F-actin fibers (Fig. 4), as reported previously (Huot et al., 1998). Distribution analysis of FAK, paxillin, and ILK were confirmed by our observations using the P-Y antibody that showed an increased cytoplasmic scattering staining in response to 16-h MT-disrupting agents exposure, compared with DMSO- or CEU-091-treated cells (Fig. 5). Furthermore, the FAK, paxillin, and ILK staining were less apparent at the tip of F-actin compared with untreated cells (data not shown). In contrast, MT-disrupting treatments did not induce apparent redistribution of vinculin, a major constituent of FA structures (data not shown). Thus, FA structures and cytoskeleton reorganizations correlate with the dual adhesion response induced by MT-disrupting agents but not in response to cDDP or CEU-091 (Fig. 3).

View larger version (62K): [in this window] [in a new window]   Fig. 5. A and B, effects of MT-disrupting agents and cDDP on FAK, paxillin, and ILK cellular distribution. MDA-MB-231 cells were treated for 16 h with 30 µM CEU-022 and CEU-091, 1 µM CEU-236, 10 and 30 µM cDDP, or 15 nM COL. After treatments, cells were detached and then plated on fibronectin-coated wells, before immunocytochemistry analysis of FAK, paxillin, and ILK proteins using specific monoclonal antibodies, as described under Materials and Methods. F-actin was revealed using rhodamine-labeled phalloidin. Fluorescence of cells was analyzed by confocal microscopy. Data are representative of three independent experiments.

View larger version (63K): [in this window] [in a new window]   Fig. 6. Kinetics analysis of biochemical and expression changes of FA-associated proteins in response to MT-disrupting agents and cDDP. MDA-MB-231 protein extracts used in Fig. 2 were separated by 10% SDS-PAGE to analyze by Western blot the expression and biochemical alterations induced by drug exposures of FAK, paxillin, talin, and vinculin, as described under Materials and Methods. Numbers on the top and right sides of the panels indicate the times exposure of drugs and the molecular masses in kilodaltons of native proteins, respectively. Brackets indicated the cleaved FAK or increased phosphorylation paxillin forms mentioned in the text. Similar results were obtained using M21 tumor cell lines (data not shown).

View larger version (96K): [in this window] [in a new window]   Fig. 7. CEU-022 increases paxillin phosphorylation. A, MDA-MB-231 or M21 cells were treated for the indicated period with 0.1% DMSO or 30 µM CEU-022. Protein extracts from these treated cells were separated by 2D isoelectric focusing/SDS-PAGE and then analyzed by Western blot using a paxillin antibody, as described under Materials and Methods. Numbers on the top and the right sides of the panels indicated the pI and kilodalton positions of the 2D gels, respectively. B, immunoprecipitated paxillin from protein extracts of 30 µM CEU-022-treated MDA-MB-21 cells for the indicated period were incubated in absence (left) or presence (right) of alkaline phosphatase (PPase). The immunoprecipitates were then separated by SDS-PAGE and analyzed by Western blot analysis for paxillin. Similar results were obtained using M21 cells (data not shown).

Moreover, we observed a time-dependent decrease of the levels of talin and vinculin in response to cDDP but not with anti-MT (Fig. 6). Similar results were observed in HT29 and M21 cells (data not shown). Altogether, these results further emphasize the differential responses induced by MT-disrupting agents on FA proteins remodeling and integrity compared with the DNA-alkylating agent cDDP.

MT-Disrupting Agents Induce ERK, JNK, and p38 Activation. ERK1/2 and the stress-activated JNK and p38 MAP kinases play important roles in the survival and/or cell death in response to stresses (Huot et al., 1998; Deschesnes et al., 2001; Fan and Chambers, 2001). These serine/threonine kinases phosphorylate several downstream substrates, including paxillin during cell migration (Huang et al., 2004). We appraised a potential correlation between the activation of these kinases and the sustained induction of paxillin phosphorylation observed in Figs. 6 and 7. We investigated the kinetics of ERK1/2, JNK, and p38 activation following MT-disrupting agent exposure. Figure 8 shows an acute activation of ERK1/2 and JNK occurring between 6 and 12 h post-treatment in response to most MT-disrupting agents tested, except for VINB, which rather induced phosphorylation of ERK after 48 h. This acute activation was then drastically decreased after 12 h. Unexpectedly DMSO and CEU-091 induced also the activation of ERK and JNK between 6 and 12 h, but not p38. Markedly, all MT-disrupting agents induced a time-dependent increase of p38 activation. Treatments with cDDP, which triggers ERK1/2 and JNK activation like MT-disrupting agents, rather activated p38 differently; its activation increased up to 12 h post-treatment and then progressively decreased to control level. Together, our data show a time-dependent correlation for the ERK1/2 and JNK peak activation and onset of increased paxillin phosphorylation, both observed after 12-h exposure with most of the MT-disrupting agents tested.

View larger version (62K): [in this window] [in a new window]   Fig. 8. Effects of MT-disrupting agents on the activation of ERK, JNK, and p38. The same MDA-MB-231-treated cell extracts used for the analysis of FA proteins in Fig. 2 were separated on a 10% SDS-PAGE for Western blot analysis of ERK, JNK, and p38 activation.

ERK1/2 and p38 Contribute to the Paxillin Phosphorylation Induced by MT-Disrupting Agents (Fig. 8). To determine the contribution of ERK1/2, JNK, and p38 activation on the increase of paxillin phosphorylation in response to MT-disrupting agents, cells were pretreated for 1 h with the following specific MAP kinase inhibitors: PD098059 (ERK), L-JNKI-1 (JNK), and SB203580 (p38). Interestingly, pretreatment with PD098059 decreased the paxillin phosphorylation induced by a 12- or 24-h cell exposure to COL, VINB, or CEU-236 (Fig. 9). SB203580 also decreased the level of paxillin phosphorylation induced by a 24-h exposure to MT-disrupting agent. In contrast, no significant decrease of paxillin phosphorylation from our Western analysis using the JNK inhibitor L-JNKI-1 in the conditions tested was observed. As expected, neither caspase nor calpain activation significantly contribute to increase paxillin phosphorylation following exposure to anti-MTs, as demonstrated using N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Fig. 9) or calpeptin pretreatments (data not shown), respectively. Interestingly, we observed that ML-7, a specific inhibitor of the myosin light chain (MLC) kinase, which activates actinmyosin contractility (Kolodney and Elson, 1995; Liu et al., 1998; Kirchner et al., 2003), decreased partially the paxillin phosphorylation induced after 24 h of exposure to MT-disrupting agents. Overall, these results support that ERK, p38 MAP, and MLC kinase-dependent mechanisms contribute to increased paxillin phosphorylation in response to pharmacological MT disruption.

View larger version (60K): [in this window] [in a new window]   Fig. 9. Effects of MAP kinase and caspases inhibitors on the induction of paxillin phosphorylation induced by MT-disrupting agents. PD098059 (50 µM; ERK), L-JNKI-1 (2 µM; JNK), SB203580 (5 µM; p38) and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) (50 µM; caspases) inhibitors were added to MDA-MB-231 cell cultures 1 h before the addition of anti-MT agents (COL, 50 µM; VINB, 5 µM; and CEU-236, 30 µM) or DMSO (0.1%, v/v) for 12 or 24 h, respectively. Cells were then harvested, and protein extracts (15 µg) were separated by 10% SDS-PAGE before analysis of paxillin by Western blot.

Overexpression of ILK Rescues the Loss of Cell Viability Induced by MT-Disrupting Agents. As mentioned, ILK is a crucial prosurvival FA-associated protein. ILK-homozygous deficient fibroblasts have impaired adhesion and spreading capacities, and they form fewer FA structures and actin stress fibers (Sakai et al., 2003). Interestingly, these defects can be rescued through ILK overexpression independently of the phosphorylation of PKB/Akt and GSK3- (Sakai et al., 2003). In addition, ILK overexpression was shown to protect cells against anoikis (Attwell et al., 2000). Our observations that early adhesion impairments occur before caspases activation bring up the hypothesis that MT-disrupting agents induce anoikis. We thus examined whether gain of function of ILK, an anoikis inhibitor, would confer cytoprotection against MT-disrupting agents. To that end, HT1080 tumor cells were transiently transfected with different constructs of the ILK protein and exposed to anti-MT agents before the resazurin viability assay (Legault et al., 2000; Mounetou et al., 2001; Mounetou et al., 2003). Wild-type form (WT), inactive (S343A), hyperactive (S343D), and dominant-negative forms of ILK (E359K) were overexpressed. Expression of these different hemagglutinin tag-ILK constructs were equivalent before drug treatments for all the transient transfections performed (Fig. 10A). In response to 30-h exposure to anti-MT agents, cell viability was partially restored by ILK-WT and also by its hyperactive form (S343D) (Fig. 10B). ILK-WT or S343D mutant overexpression also protected against cDDP treatment. In contrast, the tumor cell viability was similar in cells overexpressing the inactive (S343A) or dominant-negative forms of ILK (E359K), compared with control pcDNA3-transfected cells (Fig. 10B); thus, suggesting a protective action of ILK kinase activity in response to MT-disruption or DNA damages.

View larger version (46K): [in this window] [in a new window]   Fig. 10. ILK overexpression diminished the viability loss induced by MT-disrupting agents and cDDP. HT1080 cells were seeded and transfected transiently the following day for 24 h with the pcDNA3 control plasmid or expressing the WT hemagglutinin-ILK construct or the mutant forms S343A, S343D, and S359K. Transfected and untransfected HT1080 cells were next harvested for analysis of ILK expression by Western blot analysis with a specific antibody recognizing ILK (A) or treated for 30 h with 50 µM CEU-022, 2 µM CEU-236, 10 µM cDDP, 2 nM COL, or 0.1 nM VINB before viability tests using the resazurin assay (B), as described under Materials and Methods. The percentage of viability represents the percentage of fluorescence obtained in response to drug exposure compared with untreated cells. Analysis of variance was significant (p < 0.01) for each drug treatment, thus allowing the use of the Dunnett's test for multiple comparisons. For each treatment, the pcDNA3 clone was used as the internal control for comparison (*, p < 0.05; **, p < 0.01).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study aimed to understand the cell death mechanisms triggered by microtubule-disrupting agents. A key finding of this study is that regardless of the drug that depolymerized microtubules, they all lead to a similar sequence of molecular and cellular alterations that culminate by a cell death program reminiscent to anoikis. Anti-MT drugs produce, initially, a transient increase of the cell adhesion to the extracellular matrix that occurs 3 to 6 h postexposure to the drugs (Fig. 3B). Afterward, anti-MT drugs bring on a complete loss of the cell adhesion capability to the extracellular matrix. This biphasic change of adhesion was not observed in response to the DNA-alkylating agent cDDP, and it preceded cell detachment from ECM (data not shown) as well as the onset of caspase activation. Hence, the latter observations suggest that anti-MT agent induce de-adhesion and apoptosis, also known as anoikis. Our kinetics study supports that remodeling of focal adhesion structures is closely linked to the biphasic adhesion changes in response to MT-depolymerizing agents. Indeed, the increase and decrease of FA structure number match kinetically with the biphasic change of adherence.

We found that paxillin phosphorylation is induced in a sustained manner following exposure to anti-MT agent in contrast to the alkylating agent cDDP. The onset of this persistent signaling event coincides with the induction of an increased cell adhesion to the extracellular matrix after 6 h in HT29 cells and after 12 h in MDA-MB-231 and M21 cells (data not shown). Paxillin is subjected to a complex regulation of the phosphorylation of its tyrosine and serine/threonine residues; phosphorylation that has been shown to modulate cell adhesion and cell motility functions (Brown and Turner, 2004). In our study, the exact amino acid residues involved in paxillin phosphorylation remain to be identified. Nonetheless, taxoids and colchinoids were reported to induce tyrosine phosphorylation of paxillin, and this phosphorylation occurred concomitantly with early focal adhesion structure growth (Kaverina et al., 1999). Our results show that although p38, ERK1/2, and JNK are all activated, only ERK1/2 and p38 are relevant kinases to explain the increase of paxillin phosphorylation following anti-MT challenges (Fig. 9). ERK1/2 was previously reported to interact and to induce serine phosphorylation of paxillin in the formation/dissolution of focal contacts in migrating cells (Webb et al., 2004) and in response to phorbol ester, an agent promoting cell adhesion (Huang et al., 2004). Such serine phosphorylation is known to increase cell adhesion (Huang et al., 2004). p38 was also reported to bind to paxillin and to phosphorylate its serine residues on serine 85 (Vadlamudi et al., 1999; Huang et al., 2004). Rather, p38-dependent paxillin serine phosphorylation was linked to the detachment of paxillin from focal adhesion structures, leading to focal adhesion disorganization (Vadlamudi et al., 1999).

It is noteworthy that the level of actin cytoskeleton contractility seems important for the sustained paxillin phosphorylation, because the inhibition of MLC kinase using ML-7 decreased significantly the phosphorylation of paxillin. We think that the increase of actin contractility induced by the MT destabilization triggers an initial increase of paxillin phosphorylation. ERK1/2, whose activity was recently found to be dependent on the MLC kinase (Helfman and Pawlak, 2005), might be a good candidate. Next to ERK activation, the stress MAPK p38 sustained paxillin phosphorylation on serine/threonine residues. Phosphorylation regulation of some FA proteins such as FAK and paxillin is known to be of utmost importance in the control FA adhesion structure turnover, thereby regulating the capacity of a cell to adhere and detach from the ECM (Webb et al., 2004; Mitra et al., 2005). We suggest that lengthened microtubule disruption leads to sufficient p38 activation to maintain key serine residues of paxillin in a prolonged phosphorylated state that irreversibly impedes FA turnover machinery. For example, this sequence of events, which correspond to a persistent "low adhesion signal", might be a limiting step that irreversibly induces cell detachment and ultimately anoikis (Fig. 11). This interpretation can be further appreciated with the following observation: CEU-91, an N-aryl-N'-ethyl urea analog of CEU-022 devoid of -tubulin alkylation potency, has unexpectedly initiated the biphasic adhesion changes and ERK activation without triggering p38 activation and paxillin phosphorylation. The CEU-like structure of CEU-91 seems to act as a weak and reversible antagonist of -tubulin polymerization that is able to induce the early transient and reversible cell alterations (e.g., adherence changes and ERK1/2 activation) without triggering the activation of caspases and cell death.

View larger version (15K): [in this window] [in a new window]   Fig. 11. Model illustrating the signaling pathways underlying the induction of anoikis following sustained MT disruption. MT disruption triggers a rapid stress response due to the concomitant increase of the actin contractility. This link is presumably emerging from the release of the guanosine exchange factor (GEF) from perturbed MT integrity. This stress response leads to sustained paxillin phosphorylation, mainly through the p38 activation. Uncoupled phosphorylation regulation of paxillin lead to FA disorganization, which along with increased cytoskeletal rigidity lead to a low adhesion signal. The progressive loss of adhesion, which may be functionally inhibited by ILK overexpression, would occur possibly through integrin-mediated death signaling, leading to caspase activation, cleavage of FAK, and ultimately to anoikis.

FAK cleavage that is taking place simultaneously with early caspase activation but subsequently to focal adhesion rearrangement and adhesion loss (Fig. 10A) is possibly a determinant step consolidating the onset activation of anoikis in response to MT-disrupting agent. In some cases, FAK proteolysis correlates with FA disorganization and is associated to the disruption of the FA machinery and survival signaling pathways (Crouch et al., 1996; Reddig and Juliano, 2005). Accordingly our time-dependent observations show that FAK cleavage is a caspase-dependent process. However, our results suggest that FAK cleavage is not required for FA dismantlement in response to anti-MT, at least not at the onset of FA structure disorganization.

To further support the hypothesis that anoikis is involved in the anti-MT effects on tumor cells, we overexpressed ILK in tumor cells, a well known FA protein that protects cells from anoikis (Attwell et al., 2000). ILK overexpression in our study resulted in a protective effect of the cell against anti-MT agents, but it remains to be determined whether the protection conferred by ILK is consequential to the prevention of FA structure dismantlement and adhesion loss following cell exposure to MT disruptor, and/or results from downstream signaling events such as, for example, the direct stimulation of AKT activity. Interestingly, paxillin is among the known FA proteins interacting with ILK, whereas LD1 motif of paxillin is required for ILK recruitment to FA structures (Nikolopoulos and Turner, 2001, 2002). It is noteworthy that it remains to be established whether paxillin phosphorylation that is induced following MT-disrupting agents affects ILK-dependent survival function?

An important aspect that remains to be understood is the effect of MT disruption on the integrin-clustering ability, through the dismantlement of FA structures. Impairment of FA-dependent integrins clustering is not only an important aspect of anoikis signaling (Reddig and Juliano, 2005), through, for example, integrin-mediated death signaling, but also it is an underlying mechanism of cell adhesion mediated-drug resistance (CAM-DR) (Vachon et al., 2002; Harnois et al., 2004; Reddig and Juliano, 2005). In this context, it was shown that CEU, in contrast to cDDP, circumvent CAM-DR (Petitclerc et al., 2004). For example, we suggest that CEU and possibly other classical MT-disrupting agents interfere with CAM-DR-dependent survival signaling pathways by inducing the dismantlement of FA structures and thus blocking the integrins recruitment. In support of this, cDDP does not induce early FA dismantlement and significant changes in early adhesion.

In conclusion, we provide evidence that MT-destabilizing agents induce an anoikis-like program of cancer cells. As proposed in our model, the sustained MT disruption would initiate anoikis from a persistent paxillin phosphorylation state that triggers FA structures dismantlement (Fig. 11). Further analyses are needed on the stress signal-regulated phosphorylation cascades that may modulate the anti-MT activity. Our work shows an extension to the antitumor effects of microtubule inhibitors, because we demonstrate that they not only block the cell division-related events that are mediated through microtubule fibers but also the functional role of MTs in the cytoskeletal adhesion process. As a consequence for the clinical use of anti-MTs, drugs that could perturb further cell adhesion (such as disintegrins or other inhibitors of integrins functions; Cai and Chen, 2006; Meyer et al., 2006) may potentiate anti-MT effects on the anoikisprone cells.

	Acknowledgements

 
We thank Dr. Madeleine Carreau for providing access to confocal imaging system. We acknowledge Claude Marquis for excellent technical expertise.

	Footnotes

 
This work was supported by grants from the Canadian Institute of Health Research (to R.C.-G. and E.P.) and the Canadian Cancer Research Society, Inc. (to E.P.). E.P. is a scholar (Junior II) from the Fonds de la Recherche en Santé du Québec.

R.G.D. and A.P. contributed equally to the work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.110957.

ABBREVIATIONS: FA, focal adhesion; ECM, extracellular matrix; MT, microtubule; FAK, focal adhesion kinase; ILK, integrin-linked kinase; WT, wild type; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; CEU, N-aryl-N'-(2-chloroethyl)ureas; CEU-022, 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl; CEU-098, 4-iodo-[3-(2-chloroethyl) ureido]phenyl; CEU-091, 4-tert-butyl-[3-(2-ethyl)ureido]phenyl; CEU-236, [3-(5-hydroxypentyl)]-3-(2-chloroethyl)ureido] phenyl; cDDP, cisplatin, cis-platinum(II)diamine dichloride; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline/Tween 20; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; VINB, vinblastine; FACS, fluorescence-activated cell sorting; COL, colchicine; P-Y, tyrosinated-proteins; 2D, two-dimensional; PD098059, 2-(2-amino-3-methoxy-phenyl)chromen-4-one; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl; MLC, myosin light chain; L-JNKI-1, L-stereoisomer of a peptide inhibitor of c-Jun NH₂-terminal kinase; CAM-DR, cell adhesion-mediated drug resistance.

Address correspondence to: Dr. Eric Petitclerc, Centre de Recherche, Unité des Biotechnologies et de Bioingénierie, Centre Hospitalier Universitaire de Québec, Hôpital Saint-François d'Assise, 10 rue de l'Espinay, Québec, Québec, Canada G1L 3L5. E-mail: eric.petitclerc{at}crsfa.ulaval.ca

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