Bcl-2 Down-Regulation and Tubulin Subtype Composition Are Involved in Resistance of Ovarian Cancer Cells to Vinflunine

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Bcl-2 down-regulation and tubulin subtype composition are involved in resistance of ovarian cancer cells to vinflunine

Marie-Anne Este`ve,1 Manon Carre´,1 potential was only disrupted by vinflunine in cells Ve´ronique Bourgarel-Rey,1 Anna Kruczynski,2 expressing Bcl-2. Altogether, our results indicate that Giuseppina Raspaglio,3 Cristiano Ferlini,3 modifications acquired during treatment (i.e., paclitaxel) and Diane Braguer1 have significant consequences on cell response to the following drug (i.e., vinflunine). Especially, this study 1Centre National de la Recherche Scientifique, Formation de shows that a specific pool of tubulin subtypes and a Recherche en Evolution 2737, Universite´delaMe´diterrane´e, down-regulation of Bcl-2 are associated with resistance of 2 Marseille, France; Pierre Fabre Oncology Research Institute, ovarian cancer cells to vinflunine. [Mol Cancer Ther Toulouse, France;and 3Department of Obstetrics and Gynaecology, Universita`Cattolica Sacro Cuore, Rome, Italy 2006;5(11):2824–33]

Introduction Abstract Vinflunine (JAVLOR), a new anticancer agent derived from Vinflunine, a new microtubule-targeting drug, has a Vinca alkaloids, has a marked antitumor activity in a panel in vitro in vivo marked antitumor activity and . Here, we of cancer cell lines (1), murine tumors, and human tumor studied the mechanisms mediating resistance to vinflu- xenografts (2, 3). Interesting antivascular and antiangio- nine. We investigated the response to vinflunine of ovarian genic properties have also been highlighted for vinflunine cancer cells initially selected as paclitaxel-resistant cells (4, 5). This drug is currently in phase III clinical trials in (A2780-TC1 cells). By comparison with A2780-wild- lung and bladder cancers, as well as in phase II clinical type (wt) cells, we showed that A2780-TC1 cells were trials in breast and ovarian cancers. highly resistant to vinflunine, with resistance factors The binding site of Vinca alkaloids is at the interface reaching 800 and 1,830 for IC50 and IC70, respectively. between tubulin heterodimers, towards the microtubule We showed that P-glycoprotein minimally participated in inner lumen (6). High concentrations of vinflunine cause this cell resistance. The examination of tubulin composi- the dissolution of the microtubule network and formation A tion revealed increased levels of acetylated -tubulin, of paracrystals by self-association of tubulin dimers (7). B B II-tubulin, and III-tubulin in A2780-TC1 cells before Lower concentrations induce microtubule depolymeriza- vinflunine treatment. As a consequence, vinflunine un- tion and the lowest effective concentrations suppress equally affected microtubule network organization and microtubule dynamics in cancer cells. Alterations of mitotic function in A2780-wt and A2780-TC1 cells. Whereas the spindle functions by vinflunine can lead to mitotic block drug depolymerized microtubules and induced a mitotic (8), maybe by suppressing the kinetochore-microtubule block in A2780-wt cells, it did not depolymerize micro- dynamics (9), but a postmitotic G1 arrest can also occur (10). tubules and induced a G2 block in A2780-TC1 cells. Vinflunine-induced cell death has been characterized as Elsewhere, the mitochondrial protein Bcl-2 was down- apoptosis but the signaling pathways are poorly under- regulated in A2780-TC1 cells. This down-regulation was stood. Vinflunine has been shown to cause c-jun NH2- related to resistance, as A2780-TC1 cells stably trans- terminal kinase-1 activation and caspase-3/7 cleavage (11). fected with a Bcl-2 construct recovered a partial sensitiv- Recently, we have shown that mitochondria centralized ity to vinflunine. Lastly, we confirmed the role played by the apoptotic signals triggered by vinflunine (10). These Bcl-2 by showing that the mitochondrial membrane organelles play a key role in the cytotoxicity of various microtubule-targeting drugs (MTD) by releasing apoptogenic factors, which lead to apoptosome formation fol- Received 5/15/06; revised 8/31/06; accepted 9/11/06. lowed by caspase activation (10, 12, 13). This process is Grant support: Association pour la Recherche contre le Cancer tightly regulated by the relative levels of proapoptotic and (grant 3220). antiapoptotic members of the Bcl-2 family that control the The costs of publication of this article were defrayed in part by the mitochondrial membrane permeability, thus determining payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to the cell susceptibility to apoptosis (14). indicate this fact. Despite their shown effectiveness, the clinical success of Requests for reprints: Diane Braguer, Centre National de la Recherche the MTDs has been severely hindered by the emergence of Scientifique, Formation de Recherche en Evolution 2737, UFR Pharmacie, resistant tumor cells. Interestingly, vinflunine has dimin- 27 Boulevard Jean Moulin, 13005 Marseille, France. Phone: 33-4-91-83-56-35; Fax: 33-4-91-83-56-35. ished drug resistance–inducing properties as compared E-mail: [email protected] with vinorelbine (15). Copyright C 2006 American Association for Cancer Research. One common cause of cancer cell resistance to MTDs doi:10.1158/1535-7163.MCT-06-0277 is the enhanced expression of the P-glycoprotein (P-gp).

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This transmembrane drug effluxpump rapidly extrudesa paclitaxel (A2780-TC1) were achieved as described (31). variety of hydrophobic drugs, including vinflunine (16), Briefly, A2780-wt cells were exposed to stepwise increasing from the targeted cells. Although the presence of P-gp has concentrations of cyclosporin A (up to 3 Amol/L) to limit been correlated with a poor drug response, P-gp-mediated P-gp overexpression and of paclitaxel (up to 100 nmol/L). resistance is difficult to circumvent in the clinics (17). Doubling times, determined by the trypan blue exclusion Then, it is important to understand and control the other method (32), were 24 and 33 hours for A2780-wt and mechanisms involved in anticancer drug resistance. A2780-TC1 cells, respectively. Another common mechanism of anticancer drug resis- Human full-length Bcl-2 was obtained by reverse tran- tance is mediated by the modification of their cellular scription-PCR using the primers 5-TTAAGCTTAT- target. Resistance to MTDs has been closely associated with GGCGCACGCTGGGAGAACAGGGT-3 (forward) and tubulin mutations and/or changes in microtubule compo- 5-CCTCTAGATTCACTTGTGGCCCAGATAGGCACC-3 sition (i.e., tubulin isotypes and posttranslational modifi- (reverse) and cloned into pUSE (Upstate Biotechnology, cations; refs. 18–21). For example, high levels of acetylated Lake Placid, NY). A construct (pUSE-Bcl2D) was made in and detyrosinated a-tubulin, markers of microtubule which 49 amino acids (32–80) in the loop domain were stability (22), have been observed in cells resistant to MTDs deleted by inverse PCR and replaced with a linker of (20, 21, 23, 24). Increase in expression of hIII-tubulin, the four alanines. A2780-TC1 subclone was then stably trans- main studied tubulin isotype, has been related to paclitaxel fected with pUSE-empty vector (A2780-TC1-pUSE cells), resistance, whereas its decrease has been observed during pUSE-Bcl2 vector (A2780-TC1-Bcl2 cells), or pUSE-Bcl2D resistance acquisition to Vinca alkaloids, including vin- vector (A2780-TC1-Bcl2D cells) by electroporation as de- flunine (15, 20, 25–29). With regard to the other tubulin scribed (31). isotypes, there is less evidence for their involvement in Exponentially growing cells (3 Â 104/cm2) were seeded resistance to MTDs (20, 25, 30), especially in resistance to 24 hours before drug treatment. For experiments on A2780- microtubule-depolymerizing agents. TC1 cells, cyclosporin A was maintained in the medium at Lastly, resistance to anticancer drugs can be mediated by 3 Amol/L. alterations in apoptotic signaling pathways. In this sense, Growth Experiments overexpression of the proapoptotic Bcl-2 family proteins, Cells were seeded in 96-well plates and incubated with the such as Baxor Bad, sensitizes cancer cells to MTDs. On the drugs during 72 hours. Number of viable cells was estimated other hand, up-regulation of the prosurvival Bcl-2 family with the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- proteins, including Bcl-2 itself, has largely been described nyltetrazolium bromide (Sigma) assay or the ATPlite kit as a mechanism by which tumor cells resist to MTDs (14). (Perkin-Elmer, Boston, MA) according to our previous However, decreased Bcl-2 has recently been associated works (10, 31). Absorbance was measured at 550 nm with a with resistance of human ovarian cancer cells to paclitaxel MR 7-000 plate reader (Dynatech, Denkendorf, Germany) (31). Thus, the role of Bcl-2 in cancer cell resistance to MTDs for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium remains unclear and not as simple as initially thought. bromide assays and with a Topcount automated luminom- In this work, we studied the mechanisms mediating eter (Perkin-Elmer) for ATPlite experiments. resistance of human ovarian cancer cells to vinflunine. First, P-gp Expression we showed that overexpression of the efflux pump P-gp was Cells were washed in PBS-1% bovine serum albumin only responsible for a small part of the resistance to before incubation with the monoclonal phycoerythrin- vinflunine. Second, a specific tubulin subtype composition conjugated anti-P-gp-antibody (clone UIC2, Coulter was observed in resistant cells. Third, we also found that Immunotech, Hamburg, Germany) for 30 minutes in the sensitive and resistant cells differ greatly in vinflunine- dark. Negative control was done with a phycoerythrin- induced modifications of the microtubule network structure conjugated secondary antibody (Jackson ImmunoResearch, and function. Lastly, we showed that Bcl-2 down-regulation Baltimore, MD). Finally, cells were washed twice in PBS-1% played a role in the resistance to vinflunine. Our results bovine serum albumin before analysis by flow cytometry strongly suggest that both microtubules and mitochondria (FACScan, BD Biosciences, San Jose, CA). have key roles in human cancer cell sensitivity to vinflunine. Rhodamine 123Uptake Cells were incubated for 10 minutes at 37jC with various Materials and Methods concentrations of verapamil or cyclosporin A before Drugs incubation with 0.1 Amol/L rhodamine 123 in PBS-0.2% Stock solutions of vinflunine (Pierre Fabre Oncologie, bovine serum albumin for 1 hour at 37jC. Cells were then Toulouse, France), vinblastine (Lilly, Saint Cloud, France), transferred onto ice, washed twice at 4jC, and fluorescence doxorubicin (Dakota, Cre´teil, France), and verapamil of accumulated rhodamine 123 was measured by flow (Sigma, St. Louis, MO) were prepared in distilled water. Stock cytometry (FACScan, BD Biosciences). solutions of paclitaxel (Sigma) and cyclosporin A (Novartis, Western Blotting Basel, Swizterland) were prepared in DMSO (Sigma). Cell lysis, protein separation, and visualization were Cell Culture done as described (13). The primary antibodies were Culture of human ovarian cancer A2780-wild-type (wt) directed against Bcl-2 (clone 124, DakoCytomation, cells and generation of the A2780 subclone resistant to Glostrup, Denmark), actin (clone 1A4, Sigma), a-tubulin

Mol Cancer Ther 2006;5(11). November 2006

(clone DM1A, Sigma), h-tubulin (clone TUB 2.1, Sigma), Isolation of Mitochondria tyrosinated a-tubulin (clone TUB 1A2, Sigma), detyrosi- Cells were harvested and suspended in a sucrose buffer nated a-tubulin (clone AA12, Abcam, Cambridge, United as detailed in previous studies (34, 35). Briefly, cells were Kingdom), acetylated a-tubulin (clone 6-11B-1, Sigma), homogenized with 50 strokes in a glass homogenizer class I h-tubulin isotype (clone SAP.4G5, Sigma), class II (Kontes, Vineland, NJ) and mitochondria were obtained h-tubulin isotype (clone MU176-UC, Biogenex, San Ramon, after successive centrifugations. The mitochondrial lysate CA; clone 7B9, Covance, Berkeley, CA), class III h-tubulin was prepared as described above for whole cells. isotype (clone SDL.3D10, Sigma), and class IV h-tubulin 3,3¶-DihexyloxacarbocyanineIodideUptake isotype (clone MU178-UC, Biogenex; clone ONS-1A6, For analysis of mitochondrial membrane potential DW Sigma). Peroxidase-conjugated goat anti-mouse antibody ( m), cells were incubated with vinflunine for 24 or was used as secondary antibody (Jackson ImmunoRe- 48 hours. Cells were then incubated with 25 nmol/L 3,3¶- search). Densitometric quantitation was done using Image dihexyloxacarbocyanine iodide (Molecular Probes, Leiden, J software; a-tubulin posttranslational modifications and the Netherlands) for 30 minutes in the dark and analyzed h-tubulin isotypes expression were normalized to total by flow cytometry (FACScan, BD Biosciences). To ensure a-tubulin and h-tubulin, respectively. that 3,3¶-dihexyloxacarbocyanine iodide uptake was specif- DW ReverseTranscription-PCR Analysis ic for mitochondrial m, we also treated cells with Sequences of primers to amplify hII-tubulin, hIII-tubulin, 50 Amol/L carbonyl cyanide m-chlorophenylhydrazone, h and 2-microglobulin have been detailed in a previous which is a protonophore that dissipates the mitochondrial DW work (33). The method used was slightly modified. Briefly, m (13). it included an initial cycle of denaturation at 94jC for h 5 minutes, followed by 22 (for 2-microglobulin) and 27 (for hII-tubulin and hIII-tubulin) cycles of denaturation at Results 94jC for 30 seconds, annealing at 55jC for 30 seconds, and A2780-TC1Cells Are Highly Resistant toVinflunine j extension at 72 C for 30 seconds, and one final cycle of In A2780-TC1 cells, the IC50 for paclitaxel was 2,000-fold extension at 72jC for 7 minutes. The amplification was in higher than in wt cells (5 and 10,000 nmol/L for A2780-wt h the linear range for 22 cycles for 2-microglobulin and 27 and A2780-TC1 cells, respectively), as previously deter- h for -tubulin isotypes. Separation and staining of the DNA mined (31). For vinflunine, IC20,IC50, and IC70 values bands were done as described (33). hII-Tubulin and hIII- reached 500, 20,000, and 55,000 nmol/L, respectively, in h tubulin expressions were normalized to 2-microglobulin A2780-TC1 cells, whereas they were 20, 25, and 30 nmol/L transcript. in A2780-wt cells. These results clearly indicated a lower Visualization of the Microtubular Network by Immu- sensitivity of A2780-TC1 cells to vinflunine, illustrated by nofluorescence Microscopy high resistance factors of 800 and 1,830 for IC50 and IC70, Cells were seeded on eight-well chamber slides (LabTek, respectively (Table 1). To assess whether this resistance was Naperville, IL). After incubation with vinflunine for 6 hours, specific to MTDs, we tested doxorubicin, a DNA-damaging cells were fixed, permeabilized, and a-tubulin was stained agent, and vinblastine. A2780-TC1 cells were characterized as described (10). Cells were observed under a DM-IRBE by resistance factors to vinblastine even higher than those microscope (Leica, Bensheim, Germany) coupled to a digital measured with vinflunine (Table 1). They also showed a camera (Coolsnap FX, Princeton Instruments, Trenton, NJ) decrease in sensitivity to doxorubicin, but much less than and analyzed with Metamorph software (Universal Imag- the one observed with MTDs (Table 1). ing Corp., Downingtown, PA). P-gp Overexpression Is Only Minimally Involved in Cell Cycle Analysis Vinflunine Resistance Cells were seeded on six-well plates. After vinflunine In our experimental conditions, A2780-TC1 cells were treatment for doubling time or 72 hours, cells were grown in the presence of 3 Amol/L cyclosporin A, the harvested, fixed, and incubated with propidium iodide broad-spectrum multidrug resistance inhibitor (36). Never- (Sigma) as previously described (14). DNA content was theless, their resistance to doxorubicin suggested that measured by flow cytometry (FACScan, BD Biosciences) a multidrug resistance mechanism may be involved. We and cytogram analysis was done with Mod Fit software therefore assessed expression and activity of the P-gp (BD Biosciences, Mississauga, Canada). pump. As expected, A2780-wt cells did not express P-gp, in 4¶,6-Diamidino-2-Phenylindole Staining contrast to A2780-TC1 cells (Fig. 1A). However, only 27% of Cells were seeded on eight-well chamber slides (LabTek) P-gp remained functional in working growth conditions of and treated for doubling time or 72 hours. After plate A2780-TC1 cells (Fig. 1B). centrifugation, cells were fixed in 3.7% formaldehyde and To determine if the remaining P-gp activity in A2780-TC1 incubated for 2 minutes with 0.25 Ag/mL 4¶,6-diamidino-2- cells was responsible for the resistance, we measured phenylindole (DAPI; Sigma) in the dark. Finally, cells were the vinflunine-induced inhibition of cell proliferation with observed under a DM-IRBE microscope as above. About 10 Amol/L of verapamil. As well as high concentrations of 500 cells were examined in each experiment; the percen- cyclosporin A (10 or 30 Amol/L), this high concentration of tages of cells in interphase, mitosis, and apoptosis were verapamil totally inhibited P-gp, as shown by the maximal determined. incorporation of rhodamine 123 (Fig. 1B). We observed that

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Table 1. A2780-TC1 cells are highly resistant to vinflunine, only increase in hII-tubulin and hIII-tubulin isotypes in resistant partially through overexpression of P-gp cells was associated to an increase at the transcriptional level (factors of 2.5 and 2.1, respectively; P < 0.05; Fig. 2D). A2780-wt A2780-TC1 Resistance Thus, the distribution pattern of tubulin subtypes is specific (nmol/L) (nmol/L) factor for A2780-TC1 cells, suggesting that modifications in MTD Vinflunine target could be involved in vinflunine resistance. F F Vinflunine Unequally Affects Microtubule Network IC20 20 1.73 500 19.1 25* F F IC50 25 2.18 20,000 2,135 800* Organization and Function in A2780-TC1 and A2780- F F IC70 30 1.73 55,000 3,940 1,830* wt Cells Vinflunine + verapamil Our observation that the cellular tubulin subset was F F c IC20 20 1.26 40 8.11 2 modified in resistant cells raised the possibility that the F F c IC50 25 0.70 2,000 879 80 microtubule network was also altered in response to F F IC70 30 2.48 50,000 4,200 1,670* vinflunine treatment. We observed that the microtubule Vinblastine F F c network morphology did not initially differ between the IC20 0.03 0.004 150 31.7 5,000 F F c two cell lines (Fig. 3). After 6 hours of treatment with a IC50 0.3 0.029 2,500 750 8,330 F F c high concentration of vinflunine (20,000 nmol/L), tubulin IC70 0.7 0.19 55,000 11,500 78,600 Doxorubicin aggregation in paracrystals appeared in both sensitive and F F c IC20 6 1.60 100 3.5 17 resistant cells (Fig. 3). In agreement with P-gp experiments, F F c IC50 25 2.08 400 37.9 16 it suggested that intracellular accumulation of vinflunine F F IC70 60 13.2 3,000 115 50* was similar in the two cell lines when high extracellular concentrations were used. Interestingly, 500 nmol/L NOTE: Exponentially growing cells were treated with vinflunine, vinblas- vinflunine induced microtubule depolymerization in tine, and doxorubicin with or without 10 Amol/L verapamil for 72 hours. F IC20,IC50, and IC70 values ( SE) were graphically determined from at least A2780-wt cells (Fig. 3A), whereas it did not modify the three independent experiments for each drug and each cell line. Resistance microtubule network in A2780-TC1 cells (Fig. 3B). Thus, factors were determined as ratio of IC values in A2780-TC1 cells / IC values in A2780-wt cells. differences in microtubule sensitivity to vinflunine *P < 0.01. appeared as early as 6 hours of treatment. cP < 0.05. Microtubule network modifications by MTDs generally lead to disturbances in cell cycle progression. The distribution of cells among the different phases of the cell cycle verapamil only partially restored A2780-TC1 cell sensitivity was not significantly modified in A2780-wt cells after a (Table 1). Moreover, the resistance factor changed from 24-hour treatment with IC50 and IC70 of vinflunine. At 1,830 to 1,670 for IC70 (Table 1), and it was not modified when higher concentrations were tested (data not shown). Thus, P-gp overexpression plays a role only for the lowest vinflunine concentrations, with a maximal 12-fold decrease in A2780-TC1 sensitivity. Even if the effluxpump participates in cancer cell resistance to vinflunine, it clearly appears that it was not the main cause. In short, other mechanisms are likely to be involved in drug resistance of A2780-TC1 cells. Tubulin Subtype Distribution Pattern Is Modified in A2780-TC1Cells Modifications of the drug target are an important source of drug resistance. In A2780-TC1 cells, the cross-resistance between taxanes and Vinca alkaloids suggested that tubulin can be altered. Therefore, we investigated tubulin posttranslational modifications and isotype expression levels. We first noticed that total a-tubulin and h-tubulin levels were identical in A2780-wt and A2780-TC1 cells (Fig. 2A). Similarly, there was no significant difference in hI and hIV isotypes (Fig. 2C), as well as in detyrosinated and tyrosinated a-tubulin (Fig. 2B). In sharp contrast, the a h h Figure 1. P-gp is overexpressed but only slightly active in A2780-TC1 acetylated -tubulin, II-tubulin, and III-tubulin expres- cells. A, cells were analyzed by flow cytometry with a phycoerythrin- sion levels were 2.24 F 0.69-fold, 2.47 F 0.54-fold, and conjugated antibody directed against P-gp. B, fluorescence values 1.69 F 0.29-fold increased in A2780-TC1 cells, respectively represent accumulation of rhodamine 123 in A2780-TC1 cells in the presence of P-gp inhibitors cyclosporin A or verapamil. The percentage of (Fig. 2B and C). These differences between A2780-wt and A P active P-gp in A2780-TC1 cells in our experimental conditions (3 mol/L A2780-TC1 cells were statistically significant ( < 0.05). cyclosporin A) is indicated. Representative of three independent Reverse transcription-PCR experiments showed that this experiments.

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revealed that this G2-M block still corresponded to a G2 arrest (Fig. 4D, DAPI). Altogether, the differences observed between the two cell lines are the expression of a smaller effect of vinflunine on microtubule organization and function in resistant cells. Even if P-gp is involved in the mechanism of resistance to low concentrations of vinflunine, the microtubule network function is differentially modified by the drug, leading to G2 arrest and mitotic arrest in A2780-TC1 and A2780-wt cells, respectively. Bcl-2 Down-Regulation Is Associated withVinflunine Resistance in A2780-TC1Cells The apoptosis regulator Bcl-2 is consistently down- regulated in A2780-TC1 cells with respect to A2780-wt cells (Fig. 5A). Interestingly, this phenomenon has been related to paclitaxel resistance of A2780-TC1 cells (31). In this study, we hypothesized that Bcl-2 down-regulation could participate in the strong resistance of A2780-TC1 cells to vinflunine. Therefore, A2780-TC1 cells were stably trans- fected with a pUSE-Bcl2 construct (A2780-TC1-Bcl2 cells), restoring the Bcl-2 expression level. It led to a significant increase in A2780-TC1 cell sensitivity to vinflunine (Fig. 5B) Figure 2. A2780-TC1 cells exhibit a specific distribution pattern of because the IC50 value decreased by 80% as compared with tubulin subtypes. Western blot analysis of tubulin subtypes was done on whole-cell lysates. Antibodies were directed against total a-tubulin and the pUSE-empty vector transformed cells. Interestingly, h-tubulin (A), a-tubulin posttranslational modifications (B), and h-tubulin expression of the loop-deleted Bcl-2 in A2780-TC1 cells was isotypes (C). Expression of actin was used as a loading control. not as effective as expression of the entire Bcl-2 in restoring h h Representative of three independent experiments. D, II-tubulin and III- vinflunine sensitivity (Fig. 5B). tubulin isotypes were analyzed by reverse transcription-PCR. h-Tubulin isotypes (mRNA) were normalized to h2-microglobulin transcript. Preferentially bound to mitochondria, Bcl-2 is absent Columns, mean of four independent experiments; bars, SE. *, P < 0.05. from mitochondria isolated from A2780-TC1 cells but strongly bound to mitochondria isolated from A2780-wt DW cells (Fig. 5C). As Bcl-2 regulates the mitochondrial m, 500 nmol/L, vinflunine caused a massive G2-M block we evaluated the eventual effect of such variations on (59%), corresponding to a mitotic block illustrated by the chromosome alignment at the metaphasic plate (Fig. 4A, DAPI). In contrast, in A2780-TC1 cells, 500 nmol/L vinflunine did not induce any modification in the cell cycle. At higher concentrations (IC50 and IC70), a G2-M block was observed in A2780-TC1 cells (Fig. 4B). Surpris- ingly, this blockage did not correspond to a mitotic block but to a G2 arrest, as cells contained intact nuclei with a noncondensed chromatin (Fig. 4B, DAPI). It could be attributed to tubulin paracrystal formation, induced at 6 hours of treatment (Fig. 3B) and persisting after 33 hours (data not shown), which probably hindered mitotic spindle formation. For longer time of treatment (72 hours), most of the A2780-TC1 cells was still blocked in G2 phase, whereas 20% of cells were in a polyploid state (Fig. 4C) and multinucleated (data not shown). Then, in the resistant cells, the antiproliferative effects of vinflunine are different from those observed in the sensitive cells. Lastly, we examined whether P-gp activity in resistant cells was implicated in these differences. Similar experiments were done after complete inhibition of P-gp A (10 mol/L verapamil) in A2780-TC1 cells treated with Figure 3. Vinflunine induces microtubule depolymerization only in 500 nmol/L vinflunine (IC20). In these conditions, cells A2780-wt cells, but formation of paracrystals in both subclones. A2780-wt cells (A) and A2780-TC1 cells (B) were treated for 6 h with accumulated in G2-M phase (Fig. 4D), indicating that P-gp vinflunine. Microtubules were visualized by fluorescent microscopy inhibition was sufficient to increase vinflunine effect on cell (bar, 10 Am). Higher magnifications (Â4) have been added for each cycle progression. Nevertheless, nuclear visualization condition. Representative of three independent experiments.

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Figure 4. Vinflunine induces mitotic block in A2780-wt cells and G2 block following by polyploidy in A2780-TC1 cells. A, B,andD, exponentially growing cells were incubated with vinflunine alone (A and B) or with 10 Amol/L verapamil (D) for 24 and 33 h for A2780- wt cells (A) and A2780-TC1 cells (B and D), respectively. Top, DNA content measured by flow cytometry. Percentages of G2-M arrested cells (G2-M) and polyploid (Poly) cells are indicated. Bottom, DAPI staining visualized by fluorescence microscopy (bar, 10 Am). Percentage of cells in mitosis (M) is indicated. C, exponentially growing A2780-TC1 cells were incubated with vinflunine for 72h. DNA content was measured by flow cytometry. Percentage of polyploid (Poly) cells is indicated. Repre- sentative of three independent experiments.

mitochondria integrity. After a 24-hour treatment with 48 hours and no depolarization occurred (Fig. 6B). Cyclo- DW vinflunine, we detected the hyperpolarization of mitochon- sporin A did not perturb the mitochondrial m as we drial membranes in A2780-wt cells (data not shown). obtained similar results on A2780-wt cells in presence of DW Because the m increase is transient and is generally the P-gp inhibitor (data not shown), ruling out the DW followed by a loss in m (37), we investigated whether possibility that cyclosporin A could be responsible for the it could be disrupted later. After 48 hours of treatment, differences observed between the two cell lines. hyperpolarization was emphasized in A2780-wt cells and, To further confirm the role of Bcl-2 in the effectiveness of in parallel, depolarization increased in a dose-dependent vinflunine, we did 3,3¶-dihexyloxacarbocyanine iodide manner,asshownbythemassivedecreasein3,3¶- measurements after a 48-hour treatment in A2780-TC1- dihexyloxacarbocyanine iodide uptake (Fig. 6A). In Bcl2 cells. We showed that restoration of Bcl-2 expression A2780-TC1 cells, we noticed a weak hyperpolarization levels allowed vinflunine to partially disturb mitochondrial after 24 hours of vinflunine treatment, only for highest membranes, as indicated by their massive hyperpolariza- concentrations (data not shown). Moreover, this hyperpo- tion for all the concentrations and their depolarization for larization of mitochondrial membranes was maintained at the highest (Fig. 6C).

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From all these data, we highlight the role of Bcl-2 down- regulation in ovarian cancer cell resistance to vinflunine. Integrity of mitochondria was affected by the drug only in cells expressing Bcl-2, and down-regulation of Bcl-2 inhibited vinflunine-induced permeabilization of mitochondria.

Discussion In the current study, we studied the response to vinflunine of ovarian cancer cells already resistant to paclitaxel (31). MTD effectiveness is generally correlated with a combina- tion of effects on microtubule network functions and cell signaling. We show that variations in tubulin subtype levels and Bcl-2 down-regulation were both involved in the acquired resistance of A2780-TC1 cells to paclitaxel. Interestingly, these modifications had important consequences on cell sensitivity to the following vinflunine treatment. Whereas vinflunine inhibited A2780-wt cell growth in a range of nanomolar concentrations, micromolar concentrations were required for induction of similar effects in A2780-TC1 cells. However, ovarian cancer cells were far less resistant to vinflunine than to vinblastine. These results support previous data on vinorelbine (15), indicating that vinflunine is a less potent inducer of resistance than are other Vinca alkaloids. Figure 6. Vinflunine induces mitochondrial membrane depolarization only in Bcl-2expressing cells. Exponentially growing A2780-wt cells ( A), A2780-TC1 cells (B), and A2780-TC1-Bcl2 cells (C) were incubated with vinflunine for 48 h. DW m was measured by flow cytometry. Representative of three independent experiments. Percentage of depolarized (Depo) and hyperpolarized (Hyper) mitochondria are indicated.

Vinflunine belongs to the P-gp-associated multidrug resistance group of antitumor agents (16). In our experimental conditions (3 Amol/L cyclosporin A), P-gp activity was considerably decreased (by 70%) and P-gp involvement was limited to low concentrations of vinflunine, allowing the study of the other mechanisms of resistance generally hidden behind P-gp overexpression. In contrast with previous studies describing modifications of tubulin induced during MTD treatment, our work associates vinflunine resistance to preexisting modifications in tubulin composition. Especially, we showed that acquisition of the resistance to paclitaxel (in A2780- TC1 cells) has been accompanied by an increase in the expression level of hIII-tubulin isotype. This observation is in agreement with various works that have correlated hIII isotype overexpression and decreased sensitivity to paclitaxel (20, 25, 27–29, 38), even in the clinics (39). Surpris- ingly, although high hIII expression has been related to microtubule destabilization in tumor cells (20, 28, 40), it has Figure 5. Bcl-2down-regulation is involved in cell sensitivity to been also described as predictive of resistance to vinor- vinflunine. Western blot analysis of Bcl-2protein was done on whole-cell elbine in patients (41). Similarly, the increase in hIII-tubulin a lysates (A) or lysates of isolated mitochondria (C). Expression of -tubulin may contribute to the resistance of A2780-TC1 cells to was used as a loading control. B, exponentially growing A2780-TC1-pUSE cells, A2780-TC1-Bcl2 cells, and A2780-TC1-Bcl2D cells were incubated vinflunine. This increase is associated to an increase at the with vinflunine for 72h and cell proliferation was assessed. Columns, transcriptional level, in the same range as that described in mean percentage of resistance of A2780-TC1-Bcl2 and A2780-TC1- the literature (20, 27, 30, 33). A2780-TC1 cells also contained Bcl2D cells, as compared with A2780-TC1-pUSE cells (arbitrary consid- a ered as 100% of resistance), from three independent experiments; bars, more acetylated -tubulin. As tubulin acetylation is SD. *, P < 0.05. associated with microtubule stability (22, 24), it could,

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more likely than hIII-tubulin increase, contribute to cytotoxicity in various ovarian cancer cell lines (31). resistance to the microtubule-depolymerizing agent vin- Similarly, paclitaxel induced apoptosis in prostate cancer flunine. This stabilization of microtubules is only related to cells expressing Bcl-2, whereas the cells lacking Bcl-2 were acetylation of a-tubulin as detyrosinated a-tubulin level is resistant to the drug (49). Low Bcl-2 expression levels in the same between sensitive and resistant cells. Lastly, the patients were also associated with resistance to paclitaxel most pronounced tubulin isotype modification, induced chemotherapy (31). The role of Bcl-2 in the anticancer by the paclitaxel resistance acquisition, concerns the hII activity of microtubule-depolymerizing agents had never isotype. Its increase in A2780-TC1 cells could participate in been fully evaluated. In this study, we provide evidence that cell resistance to vinflunine. However, whether changes in vinflunine effectiveness was largely decreased in the h expression levels of II-tubulin modulate cell sensitivity to absence of Bcl-2 whereas Bcl-xL and Baxexpressionwere MTDs remains unclear (30, 41). Altogether, our results unchanged (31), and that it was in part restored by indicate that the distribution pattern of tubulin subtypes is reintroducing Bcl-2. Accordingly, Bcl-2 down-regulation specific in A2780-TC1 cells. It should also be noticed that prevented the collapse of the mitochondrial potential other modifications of the tubulin/microtubule system induced by vinflunine. Maintained hyperpolarization of could have occurred during the acquisition of paclitaxel the mitochondrial membrane potential in A2780-TC1 cells resistance, and thus influenced vinflunine treatment. In could be responsible for inhibition of cell proliferation particular, tubulin mutations are a common mechanism of without apoptosis induction by vinflunine (data not shown), resistance to both microtubule-stabilizing and microtubule- as previously proposed for paclitaxel (13). In addition, the depolymerizing agents (18, 21, 42, 43). Such mutations increase in vinflunine effect on mitochondria in A2780- in tubulin can affect microtubule functions and binding of TC1-Bcl2 cells confirmed the key role of Bcl-2 in vinflunine microtubule-associated proteins, thus disturbing microtu- mechanism of action. Thus, differences in sensitivity to bule stability (18, 20, 21, 42, 43). vinflunine measured at the cellular level, depending on Bcl-2 Because tubulin isotype composition may be a determi- expression level, reflect the differences of action of vin- nant factor of microtubule functions (15, 20, 25–30, 38), it is flunine observed at the mitochondrial level. Added to the conceivable that the changes in expression levels of tubulin fact that Bcl-2 expression is correlated with paclitaxel subtypes in A2780-TC1 would alter the manner in which resistance, our results support the hypothesis that Bcl-2 is vinflunine targets microtubules. We showed that vinflu- necessary on mitochondria for maximal effectiveness of nine differently affects both microtubule network organi- MTDs. They could explain why the link between Bcl-2 and zation and function in A2780-TC1 and A2780-wt cells. A drug susceptibility remains controversial and strongly same concentration of vinflunine induced microtubule suggest that the role of Bcl-2 as a predictor of chemotherapy depolymerization and cell cycle arrest in A2780-wt cells response should be reexamined. while having no effect on A2780-TC1 cells. Thus, differ- Like other MTDs (32, 34, 50, 51), vinflunine directly ences in whole-cell sensitivity to vinflunine seem to reflect affects isolated mitochondria,4 leading to the release of the drug effect on the microtubule network. Interestingly, apoptogenic factors. Bcl-2 has been described as a when a G2-M blockage was triggered in A2780-TC1 cells, it mitochondrial target for paclitaxel (31, 52), and a model did not correspond to a classic mitotic arrest, as in A2780- for the interaction of paclitaxel with the Bcl-2 loop domain wt cells, but rather to an accumulation of cells in G2 phase. has been proposed (53). Whether other MTDs, including The following cell cycle led to the formation of multinu- vinflunine, can bind to Bcl-2 remains unknown. Consid- cleated cells, indicating that mitosis was not successfully ering the role played by Bcl-2 in cell sensitivity to completed. Thus, vinflunine exhibits antimitotic properties vinflunine, one may think that vinflunine could affect on A2780-wt cells as well as A2780-TC1 cells, but they do mitochondria by binding Bcl-2. Vinflunine might interact not lead to the same disturbances of cell cycle progression. with Bcl-2, in part, through the loop domain, as A2780- Because tubulin composition modulates microtubule dy- TC1-Bcl2D are less sensitive than A2780-TC1-Bcl2, whereas namics (20, 25, 28, 40), the specific distribution pattern of Bcl-2 association with mitochondria is the same in its tubulin subtypes in A2780-TC1 cells may be responsible for entire or loop-deleted form.4 Lastly, we showed that the specific effects of vinflunine on the mitotic spindle tubulin was present on mitochondrial membranes from function. Thus, even if it remains difficult to define the A2780 cells, and Bcl-2 has been shown to specifically bind participation of each particular subtype (44), the global both mitochondrial tubulin and voltage-dependent anion change in tubulin composition likely contributes to the channel (14, 31, 35). Such a protein complexcould regulate observed resistance profile. the direct initiation by MTDs of the mitochondrial Mitochondria are the crossroads for intracellular signaling signaling pathway. pathways induced by MTDs (10, 12–14, 45, 46). Inactivation To conclude, our results support a role for both target of Bcl-2, through hyperphosphorylation (47), leads to (microtubules) and signaling (mitochondria) in human mitochondrial membrane permeability and apoptosis trig- cancer cell sensitivity to vinflunine. Because diverse factors gering by MTDs, including vinflunine (10, 11). A relation- are generally responsible for the emergence of resistant ship has been established between Bcl-2 overexpression and clinical resistance to MTDs (48). Unexpectedly, Bcl-2 recently seemed to be required for paclitaxel-mediated 4 Personal data.

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