Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

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[CANCER RESEARCH 64, 4957–4964, July 15, 2004] Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells

Ste´phane Honore,1 Kathy Kamath,2 Diane Braguer,1 Susan Band Horwitz,3 Leslie Wilson,2 Claudette Briand,1 and Mary Ann Jordan2 1FRE-CNRS 2737, Universite´delaMe´diterrane´e, Marseille, France; 2Department of Molecular, Cellular and Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, California; and 3Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York

ABSTRACT tumor cell lines. However, combinations of paclitaxel with epothilones or eleutherobin (drugs that also bind to the paclitaxel binding Discodermolide is a new microtubule-targeted antimitotic drug in site) did not inhibit cell proliferation synergistically (8). The mecha- Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics nism of discodermolide also differs from that of paclitaxel in that and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts discodermolide cannot substitute for paclitaxel in A549-T12 pacli- synergistically with paclitaxel to inhibit proliferation of A549 human lung taxel-resistant and paclitaxel-requiring cells, whereas epothilones and cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To eleutherobin can (8). understand their synergy, we examined the effects of the two drugs singly Microtubules are highly dynamic cytoskeletal structures that alter- and in combination in A549 cells and found that, surprisingly, their nate between phases of slow growth and rapid shortening, a process antiproliferative synergy is related to their ability to synergistically inhibit called dynamic instability (9–12). Dynamic instability is crucial for microtubule dynamic instability and mitosis. The combination of disco- the ability of microtubules to participate in spindle formation and dermolide and paclitaxel at their antiproliferative IC50s(7nM for disco- function during mitosis, including attachment of microtubules to the dermolide and 2 nM for paclitaxel) altered all of the parameters of kinetochores of each chromosome, congression of chromosomes to dynamic instability synergistically except the time-based rescue fre- the metaphase plate, signaling of the metaphase/anaphase transition, quency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by and anaphase chromosome separation (11). only 24%, giving a combination index (CI) of 0.23. Discodermolide and Because discodermolide and paclitaxel both suppress microtubule dynamics at concentrations that disrupt mitosis and inhibit cell pro- paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for liferation, we asked whether the synergistic antiproliferative actions of and they synergistically en- discodermolide and paclitaxel that have been observed in tumor cells ,(0.59 ؍ paclitaxel alone, respectively; CI Microtubules are unique receptors for might result from synergism in their effects on microtubule dynamic .(0.85 ؍ hanced apoptosis (CI drugs. The results suggest that ligands that bind to large numbers of instability. We examined the effects of the two drugs singly and in binding sites on an individual microtubule can interact in a poorly un- combination on microtubule dynamic instability in living A549 hu- derstood manner to synergistically suppress microtubule dynamic insta- man lung cancer cells during interphase at drug concentrations that bility and inhibit both mitosis and cell proliferation, with important inhibited proliferation. We found that the synergistic effects of dis- consequences for combination clinical therapy with microtubule-targeted drugs. codermolide and paclitaxel on cell proliferation can be explained, at least in part, by a synergistic inhibition of microtubule dynamic instability. We found that the synergistic inhibition of microtubule INTRODUCTION dynamic instability correlated with synergistic increases in the per-

Discodermolide (Fig. 1), currently in Phase I clinical trials, is a centage of G2-M-arrested cells and in apoptosis. The results indicate novel microtubule-targeted antimitotic drug isolated from the marine that drugs like discodermolide and paclitaxel that can bind to large sponge Discodermia dissoluta (1). Like paclitaxel, discodermolide numbers of binding sites on each individual microtubule target may increases microtubule polymer mass and induces cold- and calcium- act together in unique ways to alter microtubule architecture or tubulin stable tubulin polymers (1, 2). In cells, it induces microtubule bun- conformation. In addition, as discussed, the binding of one drug to the dling, cell cycle arrest at the G2-M phase, aneuploidy, and apoptosis microtubule may conceivably influence the binding of the other drug (1–4). Discodermolide competitively inhibits the binding of [3H]pac- to the microtubule. The results have important consequences for litaxel to microtubules, suggesting that discodermolide and paclitaxel clinical therapy with combinations of drugs that bind to the same or have common or overlapping binding sites (5, 6). As with other overlapping sites on microtubules. microtubule-targeted drugs, low concentrations of discodermolide suppress microtubule dynamics, an action that appears responsible for MATERIALS AND METHODS its ability to inhibit the cell cycle transition from metaphase to anaphase (7). Surprisingly, Martello et al. (8) found that discodermo- Agents. Discodermolide was provided as a gift from Frederick Kinder lide and paclitaxel synergistically inhibit proliferation of four human (Novartis Pharmaceuticals Corp., East Hanover, NJ), paclitaxel was from Sigma (St. Louis, MO), and [3H]paclitaxel (37 Ci/mmol) from Moravek (Brea, CA). Received 2/25/04; revised 5/5/04; accepted 5/19/04. Cell Culture and Proliferation. A549 human non-small cell lung carci- Grant support: Groupement des Entreprises Franc¸aises dans la Lutte contre le Cancer Marseille, Provence, Association pour la Recherche sur le Cancer, and USPHS Grants noma cells were maintained in RPMI 1640 containing 10% fetal bovine serum. CA57291 and NS13560. S. Honore is the recipient of an Association pour la Recherche After incubation with drug for 72 h, cell proliferation was analyzed as de- sur le Cancer fellowship. scribed previously (7) using the sulforhodamine B assay (13). The costs of publication of this article were defrayed in part by the payment of page Microinjection of Rhodamine-Labeled Tubulin, Time-Lapse Micros- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. copy, and Image Acquisition. Rhodamine-labeled tubulin was prepared, Note: Supplementary data for this article can be found at Cancer Research Online stored, and microinjected into the cells as described previously (7). Injected (http://cancerres.aacrjournals.org). cells were incubated for2hat37°C to allow incorporation of the rhodamine- Requests for reprints: Mary Ann Jordan, Department of Molecular, Cellular and tubulin into the cellular microtubules. Cells were then incubated with drug for Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, CA 93106. Phone: (805) 893-5317; Fax: (805) 893-4724; E-mail: 4 h to allow attainment of an equilibrium intracellular drug concentration. The [email protected]. rhodamine-labeled microtubules were observed using a Nikon Eclipse E800 4957

RESULTS To determine the concentrations of discodermolide and paclitaxel to use to examine their potential synergistic actions on microtubule dynamics, we first determined the effects of the two drugs individually on cell proliferation and mitotic arrest in A549 cells over a range of drug concentrations. A549 cell proliferation was inhibited by 50% Fig. 1. Structures of paclitaxel and discodermolide. at7nM discodermolide (7) and at 2 nM paclitaxel (data not shown; 72-h drug incubation; see “Materials and Methods”). As determined by flow cytometry of DNA content after staining of cells with PI (see “Materials and Methods”), each drug singly induced G -M arrest in a microscope maintained at 37 Ϯ 1°C, with a Nikon plan apochromat 1.4 N.A. 2 concentration-dependent manner, with half-maximal arrest occurring ϫ100 objective lens. Thirty to 41 images/cell were acquired at 3-s intervals using a Hamamatsu Orca II digital camera (Middlesex, NJ) driven by Meta- at 41.5 nM discodermolide and at 4 nM paclitaxel and maximal arrest morph software (Universal Imaging, Media, PA). occurring at 166 nM discodermolide and at 20 nM paclitaxel (Fig. 2). Analysis of Microtubule Dynamic Instability. The positions of the plus To determine whether discodermolide and paclitaxel act synergisti- ends of individual microtubules in interphase cells over time were recorded cally to suppress microtubule dynamic instability, we determined the and analyzed (7), and the lengths of individual microtubules were graphed as effects of each drug singly on microtubule dynamic instability over a function of time (life histories). Criteria for determination of rates, durations the concentration ranges, 7–166 nM for discodermolide and 2–20 nM and lengths of growth and shortening events, transition frequencies, and for paclitaxel. We then determined the effects of the combination of dynamicity were described in detail previously (7). As discussed previously the two drugs at their IC50s for inhibition of proliferation on the (7), in control cells 69% of the microtubules were dynamic during the period parameters of microtubule dynamic instability and calculated the CIs of observation, and the fraction of dynamic microtubules decreased with according to the method of Chou and Talalay (14). increasing drug concentration. To determine the mechanistic effects of the Effects of Discodermolide and Paclitaxel on Dynamic Instabil- drugs on the individual parameters of dynamic instability, only dynamic ity Parameters When Added Singly to A549 Cells. After rhoda- microtubules could be used for the measurements. Given the concentration- dependent nature of both suppression of dynamic instability and the degree of mine-tubulin was microinjected into cells and allowed to incorporate total stabilization, it is unlikely that adoption of this methodology significantly into microtubules, cells were incubated with a range of concentrations alters the conclusions concerning synergism at the level of microtubule dy- of either drug (7, 41.5, 83, and 166 nM discodermolide, or 2, 6, 12, and namics. 20 nM paclitaxel, or a combination of 7 nM discodermolide and 2 nM Drug effects on dynamic instability of microtubules in interphase cells were paclitaxel, or no drug). The plus ends of microtubules in the lamellar used as an indication of drug effects in mitosis, the stage at which the cell cycle regions of the cells were then imaged by time-lapse fluorescence is arrested. Although we cannot be sure, all of our studies to date indicate that microscopy (Fig. 3, A–C). Images were collected and analyzed as suppression of interphase microtubule dynamics that occurs at drug concen- described in “Materials and Methods,” and life history traces of the trations that arrest the cells in mitosis is accompanied by suppression of centromere dynamics in mitotic spindles (an indication of suppression of the dynamics of bundles of microtubules attached to the centromeres). In the one instance where mitotic arrest occurred in the absence of suppression of microtubule dynamics (by 2-methoxyestradiol),4 we also observed mitotic arrest in the absence of suppression of centromere dynamics. Thus, there appears to be a strong correlative relationship between drug effects on interphase microtubule dynamics and on mitotic microtubule dynamics. Analysis of Drug Synergism. The combination index (CI) was calculated to determine whether the drugs interacted synergistically, additively, or antag- ϭ onistically (14). The CI is defined by the following equation: CI (D)1/ ϩ (Dx)1 (D)2/(Dx)2, in which (D)1 is the concentration of drug necessary to achieve a particular effect in the combination; (Dx)1 is the concentration of the same drug that will produce the identical level of effect by itself; (D)2 is the concentration of the second drug that will produce a particular effect in the combination; and (Dx)2 is the concentration of the second drug, which will produce the same level of effect by itself. CI Ͼ1 indicates antagonism, CI Ͻ1 indicates synergy, and CI ϭ 1 indicates additivity.

G2-M Block and Apoptosis. After 48 h of drug treatment, adherent and floating cells were collected and incubated (15 min) with annexin-V-FITC (Euromedex, Souffelweyersheim, France) for determining surface exposure of phosphatidylserine in apoptotic cells (15) and propidium iodide (PI) for determination of DNA content by flow cytometry (Ref. 16; FACScan, Becton Dickinson, San Diego, CA). DNA distribution was modeled as a sum of cells in G1,G2-M, S phase, and an aneuploid population close to the G1 peak, using ModFitLT software (3). Intracellular Paclitaxel Concentration. The intracellular concentration of paclitaxel and its intracellular metabolites were determined chromatographi- cally (16). Briefly, cells were incubated with 2 nM [3H]paclitaxel Ϯ 7nM discodermolide (4 h). Cells were harvested, washed several times in phosphate buffer, and lysed in 60% methanol. Radioactivity of cell lysates was deter- f mined by liquid scintillation counting (LS1707; Beckman, Fullerton, CA). Fig. 2. Concentration-dependence for G2-M arrest ( ) by discodermolide and pacli- F taxel, also showing G2-M arrest ( ) induced by the combination of 7 nM discodermolide and2nM paclitaxel. Results are from three independent experiments; bars, ϮSE. The 4 K. Kamath, T. Okouneva, L. Wilson, and M. A. Jordan, unpublished observations. results were obtained from flow cytometry as shown in Fig. 5. 4958

Fig. 3. Time-lapse sequence of microtubules in A549 cells in the absence of drug (A–C), and life history graphs of changes in microtubule length over time in the presence and absence of drug(s): (D) control; (E)7nM discodermolide; (F)2nM paclitaxel; (G) combination of 7 nM discodermolide and2nM paclitaxel; (H)83nM discodermolide; and (I)6nM paclitaxel. Arrows in A–C indicate one dynamic microtubule that changed length over the course of 8 s. In the presence of both discodermolide and paclitaxel (G), microtubule dynamic instability were significantly more suppressed than with either drug added singly (E–F). In the presence of paclitaxel (I), the ends of microtubules “chattered” or underwent many small changes in length (arrows), whereas in the presence of discodermolide (H), the ends were more stable to minor changes in length.

changes in microtubule length versus time were constructed for 30 by discodermolide (7 nM), the mean shortening rate was reduced by individual microtubules for each condition (Fig. 3, D–I). These traces 20%, the mean shortening length was reduced by 42%, and the were used to determine the individual dynamic instability parameters dynamicity was reduced by 23%. In comparison, at the IC50 for (Tables 1 and 2; Supplementary Data). In controls (in the absence of inhibition of cell proliferation by paclitaxel (2 nM), the mean short- drug; Fig. 3, A–C), microtubules underwent episodes of slow growth ening rate was reduced by 14%, the mean shortening length was and rapid shortening, and sometimes underwent periods of pause or reduced by 30%, and the dynamicity was reduced by 25%. attenuated dynamic instability during which the changes in length Nearly all of the catastrophe and rescue frequencies were affected were below the resolution of the microscope (see “Materials and similarly by the two drugs at their individual IC50s for inhibition of Methods”). As determined by analysis of the life history traces, proliferation. The frequencies of catastrophe and rescue are the fre- control microtubules shortened at 24 Ϯ 2.1 ␮m/min and grew at quency of switching from periods of microtubule growth or pause to 13.8 Ϯ 1.4 ␮m/min. The mean growing and shortening lengths in shortening and from periods of shortening to growth or pause, respec- controls were 2.5 Ϯ 0.2 ␮m and 5.0 Ϯ 0.4 ␮m, respectively. Control tively. They can be calculated either on the basis of time or of distance microtubules spent 52% of their time in a paused state, neither grown or shortened. At 7 nM discodermolide, the only significant growing nor shortening to a detectable extent. effect was to greatly increase the length-based rescue frequency by Discodermolide and paclitaxel, when added singly to A549 cells, 93%. Similarly, at 2 nM paclitaxel, the only significant effect was to each suppressed microtubule dynamic instability in a concentration- greatly increase the length-based rescue frequency by 86%. At higher dependent manner (Fig. 3, E and F). They affected nearly all of the drug concentrations (Ն83 nM discodermolide or Ն6nM paclitaxel; parameters of dynamic instability to similar degrees with respect to Supplementary Data), both discodermolide and paclitaxel, when their abilities to inhibit cell proliferation (Tables 1 and 2; Supplemen- added singly, reduced the time-based catastrophe frequency and in- tary Data). For example, at the IC50 for inhibition of cell proliferation creased the time-based and length-based rescue frequencies. Interest- 4959

Table 1 Effects of discodermolide and paclitaxel, singly and in combination, on the parameters of microtubule dynamic instability in living A549 cells Parameters of microtubule dynamic instability in A549 cells incubated with 7 nM discodermolide or 2 nM paclitaxel, singly, or with the combination of 7 nM discodermolide and 2nM paclitaxel. The concentrations used singly are the IC50s for inhibition of A549 cell proliferation by each drug applied singly. The percentage change from control values is shown for the parameters that were significantly different from controls. Data for 7 nM discodermolide are from (6).

7nM discodermolide Parameters Control 7 nM discodermolide Change 2 nM paclitaxel Change ϩ2nM paclitaxel Change Combination Index Growth rate (␮m/min) 13.8 Ϯ 1.4 12.8 Ϯ 1.0 13.2 Ϯ 1.0 9.8 Ϯ 0.8a Ϫ28% 0.30 Shortening rate (␮m/min) 24 Ϯ 2.1 19.1 Ϯ 1.8a Ϫ20% 20.7 Ϯ 1.6a Ϫ14% 10.4 Ϯ 2.3a Ϫ58% 0.21 Growth duration (min) 0.18 Ϯ 0.02 0.19 Ϯ 0.01 0.17 Ϯ 0.01 0.17 Ϯ 0.01 Shortening duration (min) 0.21 Ϯ 0.02 0.15 Ϯ 0.01 0.17 Ϯ 0.01 0.18 Ϯ 0.01 Pause duration (min) 0.34 Ϯ 0.06 0.29 Ϯ 0.03 0.28 Ϯ 0.03 0.45 Ϯ 0.06b ϩ32% 0.26 Growth length (␮m) 2.5 Ϯ 0.2 2.5 Ϯ 0.2 2.2 Ϯ 0.2 1.7 Ϯ 0.1a Ϫ31% 0.41 Shortening length (␮m) 5 Ϯ 0.4 2.9 Ϯ 0.2c Ϫ42% 3.5 Ϯ 0.3c Ϫ30% 1.8 Ϯ 0.1c Ϫ65% 0.21 % time spent growing 20 26 24 12.7 Ϫ9% 0.23 % time spent shortening 28 20 20 14.3 Ϫ14% 0.30 % time spent in pause 52 54 56 73.0 ϩ21% 0.23 Dynamicity (␮m/min) 9.4 7.2 Ϫ23% 7.1 Ϫ25% 2.7 Ϫ71% 0.23 a P Ͻ 0.05, estimate of significance by Student’s t test. b P Ͻ 0.01 estimate of significance by Student’s t test. c P Ͻ 0.001 estimate of significance by Student’s t test.

Table 2 Effects of discodermolide and paclitaxel, singly and in combination, on the frequency of catastrophe and rescue of microtubule dynamic instability in living A549 cells Transition frequencies in A549 cells treated with 7 nM discodermolide or 2 nM paclitaxel, singly, or with the combination of 7 nM discodermolide and 2 nM paclitaxel. The concentrations used singly are the IC50s for inhibition of A549 cell proliferation by each drug applied singly. The percentage change from control values is shown for the parameters that were significantly different from controls. Data for 7 nM discodermolide are from (7).

7nM discodermolide Parameters Control 7 nM discodermolide Change 2 nM paclitaxel Change ϩ2nM paclitaxel Change Combination index Time-based catastrophe frequency 1.68 Ϯ 0.12 1.67 Ϯ 0.09 1.62 Ϯ 0.13 0.95 Ϯ 0.06a Ϫ43% 0.25 (minϪ1 Ϯ SE) Time-based rescue frequency 3.38 Ϯ 0.30 4.62 Ϯ 0.32 4.58 Ϯ 0.30 4.7 Ϯ 0.15 (minϪ1 Ϯ SE) Distance-based catastrophe frequency 0.48 Ϯ 0.03 0.44 Ϯ 0.02 0.41 Ϯ 0.01 0.75 Ϯ 0.05a ϩ56% 0.55 (␮mϪ1 Ϯ SE) Distance-based rescue frequency 0.14 Ϯ 0.01 0.27 Ϯ 0.01b ϩ93% 0.26 Ϯ 0.01b ϩ86% 0.51 Ϯ 0.02a ϩ265% 0.28 (␮mϪ1 Ϯ SE) a P Ͻ 0.01 estimate of significance by Student’s t test. b P Ͻ 0.001 estimate of significance by Student’s t test. ingly, the only exception to the similarity in the actions of disco- paclitaxel acted synergistically to suppress the dynamicity (a CI Ͻ1 dermolide and paclitaxel on the individual dynamic instability indicates synergy, whereas a CI Ͼ1 indicates antagonism). parameters was that the effects of discodermolide on the length-based Discodermolide and paclitaxel acted synergistically on nearly all of catastrophe frequency were distinct from those of paclitaxel. At the the parameters of dynamic instability, as shown in the right-hand concentration that induced 80% of maximal G2-M arrest (83 nM), columns of Tables 1 and 2. The two drugs together induced a dramatic discodermolide did not affect the length-based catastrophe frequency, and synergistic decrease in microtubule dynamicity (Ϫ71%) as com- whereas 6 nM paclitaxel (the concentration that induced 80% of pared with a reduction of only 23–25% for either drug alone ϭ maximal G2-M arrest) significantly increased the length-based catas- (CI 0.23). In addition, they synergistically inhibited both the trophe frequency by 80% (P Ͻ 0.001; Supplementary Data). Thus, growing and the shortening rates (CI ϭ 0.30 and 0.21, respectively), paclitaxel appears to not completely stabilize microtubule ends, whereas each drug singly inhibited only the shortening rate (Table 1). whereas discodermolide stabilizes the entire length of microtubules. The combination of the two drugs also reduced both the growing and Synergistic Inhibition of Microtubule Dynamic Instability by a the shortening lengths synergistically (CI ϭ 0.41 and 0.21, respec- Combination of Discodermolide and Paclitaxel at Their Antipro- tively), whereas each drug singly reduced only the shortening length liferative IC50s. When added together to A549 cells, the combination of7nM discodermolide and 2 nM paclitaxel (the concentrations of discodermolide and paclitaxel that inhibited proliferation by 50% at 72 h incubation) significantly suppressed microtubule dynamic instability (Fig. 3G). The traces in the life history graphs were analyzed, and the values of each parameter in Tables 1 and 2 and the Supple- mentary Data were graphed (data not shown). From these graphs, the concentration of each drug singly that produced an effect equal to the combination of 7 nM discodermolide and 2 nM paclitaxel was determined from the graphs. The CIs were calculated for each parameter as described in “Materials and Methods” according to the method of Chou and Talalay (14). For example, when cells are treated with both drugs at the same time, the dynamicity was reduced from 9.4 ␮m/min in controls to 2.7 ␮m/min (Table 1). In contrast, when the drugs were added singly, it required 132 nM discodermolide to suppress the Fig. 4. Bar graph showing the percentage changes in several parameters of microtubule dynamicity to 2.7 ␮m/min and 10.8 nM paclitaxel to suppress it to this dynamic instability that were altered synergistically by 7 nM discodermolide and 2 nM ϩ paclitaxel. Values were obtained from data on Tables 1 and 2. GR, growth rate; SR, same value (Supplementary Data). Thus, the CI is 2/10.8 7/ shortening rate; LBRF, length-based rescue frequency; TBCF, time-based catastrophe 132 ϭ 0.23, indicating that the combination of discodermolide and frequency; LBCF, length-based catastrophe frequency; OD, overall dynamicity. 4960

(Table 1). The combination synergistically increased the duration of pause (ϩ32%), the percentage of time spent paused (ϩ21%), the length-based catastrophe frequency (ϩ56%), and synergistically decreased the time-based catastrophe frequency (Ϫ43%), whereas each drug singly exerted no significant effect on these parameters (Tables 1 and 2). The length-based rescue frequency was strongly and synergistically increased by 265% (CI ϭ 0.28) by the combination, whereas each drug singly increased this parameter by only ϳ90% (Table 2). Fig. 4 illustrates the percentage changes in several parameters of microtubule dynamic instability that were altered synergistically by 7 nM discodermolide and 2 nM paclitaxel. The only parameter that was not synergistically altered was the time-based rescue frequency. (The CI was not calculated for this parameter because its value after incubation with the combination was not statistically different from its value with either drug alone.) Do Discodermolide and Paclitaxel Act Synergistically to Induce

G2-M Arrest and Apoptosis? To determine whether the synergistic inhibition of microtubule dynamic instability by the combination of paclitaxel and discodermolide was associated with a similar synergistic effect on G2-M block, we used flow cytometry to analyze the concentration dependence for the effects of each drug alone and for the combination of drugs on the DNA content of A549 cells. Cells were incubated for 20 h with 7–166 nM discodermolide or 2–20 nM paclitaxel or with the combination of 7 nM discodermolide ϩ 2nM paclitaxel. Fig. 5 shows representative flow cytograms for the distribution of DNA content in control cells and after incubation with 7 nM discodermolide, 2 nM paclitaxel, or the combination of both drugs. f Fig. 2 shows the concentration dependence for G2-M arrest ( )as determined from the data of Fig. 5 and similar flow cytograms for each drug concentration. Both drugs increased the percentage of cells arrested in G2-M in a concentration-dependent manner. The percentage of cells in G2-M after incubation with the combination (41 Ϯ 14%) was greater than after incubation with 7 nM discodermolide alone (16 Ϯ 10%) or 2 nM paclitaxel alone (9.6 Ϯ 4.5%). The CI (CI ϭ 0.59 Ϯ 0.04; 95% confidence interval) indicates that the combination of paclitaxel and discodermolide acts synergistically to induce a G2-M block. We examined the potential ability of discodermolide and paclitaxel to induce apoptosis after 72-h drug incubation using flow cytometry of live A549 cells doubly stained with both annexin-V-FITC and PI

Fig. 6. Induction of apoptosis in controls and after 48-h incubation with 7 nM discodermolide and 2 nM paclitaxel added singly and in combination. Apoptosis was determined by flow-cytometry for annexin-V-FITC and propidium iodide (PI) dual labeling. A, cytograms of annexin-V-FITC binding (abscissa) versus PI uptake (ordinate) show three distinct populations: (a) viable cells (low FITC and low PI signal) in gate R1; (b) early apoptotic cells (high FITC and low PI signal) in gate R3; and (c) cells that have lost membrane integrity as a result of very late apoptosis (high FITC and high PI signal) in gate R4. Percentages of apoptotic cells (gate R3 and gate R4) are indicated on each cytogram. B, discodermolide and paclitaxel concentration dependence for induction of apoptotic cells as determined from cytograms like those shown in A. The percentages of apoptotic cells (R3 ϩ R4) when the drugs were added individually are shown with F and when added as the combination of 7 nM discodermolide and 2 nM paclitaxel, the results are shown as f. Representative of four independent experiments; bars, ϮSE.

(15, 16). Cells were distributed into three populations (Fig. 6A): (a) viable cells (low annexin-V labeling and low PI, gate R1); (b) early apoptotic cells (high annexin-V labeling and low PI labeling, gate R3); (c) and cells that have lost membrane integrity as a result of very Fig. 5. Histograms from one representative experiment showing cellular DNA amounts late-stage apoptosis (high annexin-V labeling and high PI labeling, as determined by flow cytometry (see “Materials and Methods”) in control A549 cells and gate R4). Thus, cells in the quadrant R3 and R4 were apoptotic cells. after incubation with 7 nM discodermolide, 2 nM paclitaxel, or the combination of 2 nM Drug concentrations and the percentages of apoptotic cells are indi- paclitaxel and 7 nM discodermolide. The percentages of cells arrested in G2-M and aneuploid cells are indicated on each panel. cated on each cytogram. Each drug alone induced early apoptosis in 4961

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. SYNERGISTIC SUPPRESSION OF MICROTUBULE DYNAMICS a concentration-dependent manner (Fig. 6B). The percentage of ap- similar, there is an important distinction between the two drugs. The optotic cells produced by the combination of discodermolide and paclitaxel-induced increase in the length-based catastrophe frequency paclitaxel (53.8 Ϯ 5.8%) was the same as that produced by either 21 is initially counterintuitive, but it may result from selective stabiliza- nM discodermolide or 3.8 nM paclitaxel when used singly. The CI tion of the microtubule core without accompanying stabilization of the (0.85 Ϯ 0.05; 95% confidence interval) indicates that the combination microtubule ends. Discodermolide may bind equally well to the mi- acted with low synergy to induce apoptosis (discussed additionally crotubule core and the ends, whereas paclitaxel may bind less well to below). the extreme ends, thus, allowing “chatter” or frequent short catastro- Intracellular Paclitaxel Concentrations. The intracellular pacli- phes and rescues. Chatter was clearly visible in life history traces with taxel concentration was measured with and without coincubation with paclitaxel (Fig. 3I) but not discodermolide (Fig. 3H). Such chatter was discodermolide to determine whether the observed synergies might observed previously with epothilone B (20). Microtubule ends differ have resulted artifactually from an increase in the intracellular pacli- from their cores in that tubulin-GTP or tubulin-GDP-Pi (21) is located taxel concentration after coincubation. After incubation with 2 nM at the extreme ends, whereas tubulin-GDP makes up the core. Thus, [3H]paclitaxel (4 h), the intracellular concentrations of paclitaxel did differential actions of drugs on microtubule catastrophes might occur not differ statistically in the presence or absence of 7 nM discodermo- if, for example, paclitaxel and epothilone B, but not discodermolide, lide (2.1 Ϯ 0.4 ϫ 10Ϫ8 and 3.4 Ϯ 1.9 ϫ 10Ϫ8 mol/g protein, bind less well to tubulin-GTP than to tubulin-GDP. Reduced binding respectively; P ϭ 0.667). In addition, the intracellular inactive me- of paclitaxel to microtubule ends relative to the cores is suggested by tabolites of paclitaxel were Ͻ1% with or without discodermolide previous experiments performed in vitro (18, 22). (data not shown). Synergistic Inhibition of Microtubule Dynamic Instability by the Combination of Discodermolide and Paclitaxel. Competitive 3 DISCUSSION inhibition of [ H]paclitaxel binding to microtubules by discodermolide suggests that discodermolide and paclitaxel may share a common, We have examined the effects of discodermolide and paclitaxel on high-affinity binding site (5, 6). Recent evidence also indicates that microtubule dynamic instability in living A549 cells, both singly and there is a second, low-affinity, paclitaxel-binding site on microtubules in combination, to determine whether the synergistic antiproliferative and that discodermolide binds noncompetitively to this low affinity effects of the drugs observed previously (8) might be because of a site (6). Discodermolide may also bind to other distinct sites on synergistic suppression of microtubule dynamics. Surprisingly, in microtubules that would not be discernible by competition with view of the fact that both drugs appear to bind to the same or [3H]paclitaxel. The striking similarity in the effects of the two drugs overlapping sites on microtubules, when added together, the two on dynamic instability seems to favor a common binding site. Thus, it drugs synergistically suppressed nearly all of the dynamic instability is surprising that these two drugs can synergize when used simulta- parameters and synergistically induced mitotic block and apoptosis. neously. For example, other microtubule stabilizing agents that inhibit The drug concentrations used, 7 nM for discodermolide and 2 nM for competitively [3H]paclitaxel binding to microtubules such as eleuth- paclitaxel, were chosen because they half-maximally inhibited cell erobin and epothilones A and B (23, 24) do not act synergistically proliferation when added singly to the cells, and because Martello et with paclitaxel in cells (8). al. (8) found that they synergistically inhibited A549 cell proliferation Several possible mechanisms could explain the synergism between when combined at equipotent antiproliferative concentrations. discodermolide and paclitaxel. Microtubules are unusual receptors for Discodermolide Stabilizes Microtubule Dynamic Instability drugs in that an individual microtubule contains ϳ1690 tubulin Similarly to Paclitaxel with One Exception. It was first necessary to dimers/␮m of length and, thus, very large numbers of binding sites. In determine the effects of the drugs as single agents. Low concentra- addition, occupancy of just a few sites on an individual microtubule tions of discodermolide alone (7 nM) or paclitaxel alone (2 nM; the exerts powerful suppression of dynamics (17, 25). The two drugs did IC50s for proliferation) had nearly identical effects on dynamic insta- exert different effects on the length-based catastrophe frequency. One bility, qualitatively and quantitatively. Consistent with previous work possibility is that the two drugs, although they may bind to the same with paclitaxel (18, 19), the most potent actions of each drug were to site, induce subtle differences in the conformation of the tubulins to suppress the rate and extent of shortening and induce a 23–25% which they are bound, which, in turn, differentially influences the reduction in dynamicity (Table 1). No other parameters were signif- stability of the microtubule lattice. icantly affected except for the length-based rescue frequency (Table It is also conceivable that the binding of one of the drugs to a site 2), which is directly dependent on the length a microtubule shortens on the microtubule enhances the binding of the second drug in a during a shortening event. At higher drug concentrations (Ͼ7nM for cooperative manner. For example, although paclitaxel and the endog- discodermolide and Ͼ2nM for paclitaxel), each drug suppressed the enous microtubule-binding protein tau appear to compete for the same growing and shortening rates, they increased the percentage of time binding site on the interior of the microtubule (26), it was found spent in a paused state, they increased the durations of paused states, recently that tau induces paclitaxel to bind to microtubules coopera- they increased the time- and length-based rescue frequency, they tively in a tau concentration-dependent manner, indicating that tau decreased the time-based catastrophe frequency, and they strongly induces a conformational change that enhances paclitaxel binding.5 suppressed overall dynamicity. When analyzed individually, the two Similarly, the steep accelerating concentration dependence for pacli- drugs altered the dynamic instability parameters in similar ways, both taxel-induced G2-M arrest and induction of apoptosis compared with qualitatively and quantitatively, relative to their ability to inhibit the more linear curves for discodermolide (Figs. 2 and 6) suggest a proliferation and block mitosis, except for the length-based catastro- possible cooperative binding of paclitaxel. Thus, it is conceivable that phe frequency (Supplementary Tables 1 and 2; Ref. 7). paclitaxel might enhance positively the binding of discodermolide to The length-based catastrophe frequency was increased by 80% at microtubules or enhance the effects of discodermolide binding to the paclitaxel concentration that induced 80% (IC80) of maximal microtubules. In addition, calculation of the CI for dynamicity indi- G2-M arrest (6 nM paclitaxel), whereas discodermolide had no signif- cates that the major contribution (meaning smallest numerical contri- icant effect on this parameter at the IC80 for G2-M arrest (83 nM; Supplementary Table 2). These results indicate that, although the 5 J. L. Ross, C. D. Santangelo, V. Makrides, and D. K. Fygenson. Tau induces mechanisms of action of discodermolide and paclitaxel are very cooperative Taxol binding to microtubules, submitted for publication. 4962

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. SYNERGISTIC SUPPRESSION OF MICROTUBULE DYNAMICS bution) to the “less-than-one” CI comes not from the paclitaxel drug receptors, drugs that bind to the same or overlapping sites on component (2/10.8 ϭ 0.185) but rather from the discodermolide microtubules may offer an important new chemotherapeutic strategy. component (7/132 ϭ 0.053; see sample calculation of CI in “Re- sults”). In other words, paclitaxel has a much greater influence on the ACKNOWLEDGMENTS effects of discodermolide than vice versa. The synergy may also result from differential binding of the two We thank Charles Prevot (FRE-CNRS 2737) for the flow cytometry experiments, Dr. Joseph Ciccolini for intracellular paclitaxel measurements, and drugs to the various tubulin isotypes. 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Stéphane Honore, Kathy Kamath, Diane Braguer, et al.

Cancer Res 2004;64:4957-4964.

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