Novel Actions of the Antitumor Drugs Vinflunine and Vinorelbine on Microtubules1

[CANCER RESEARCH 60, 5045–5051, September 15, 2000] Novel Actions of the Antitumor Drugs Vinflunine and Vinorelbine on Microtubules1

Vivian K. Ngan, Krista Bellman, Dulal Panda, Bridget T. Hill, Mary Ann Jordan, and Leslie Wilson2 Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106 [V. K. N., K. B., D. P., M. A. J., L. W.], and Division de Cancerologie Experimentale, Centre de Recherche Pierre Fabre, 81106 Castres Cedex, France [B. T. H.]

ABSTRACT Structurally, vinflunine and vinorelbine differ from vinblastine in the velbanamine moiety (“top” portion) of the molecule (Fig. 1). Both Vinflunine is a novel Vinca alkaloid presently in Phase I clinical trials. drugs were synthesized by a novel method for coupling the precursor In preclinical studies, it exhibited superior antitumor activity to that of alkaloids catharanthine and vindoline, which resulted in the formation other Vinca alkaloids, including vinorelbine from which it was syntheti- cally derived. Vinca alkaloids appear to inhibit cell proliferation by af- of an eight-membered rather than a nine-membered ring within the fecting the dynamics of spindle microtubules. Here we have analyzed the velbanamine portion of the molecule (8, 9). Vinflunine was derived by effects of vinflunine and vinorelbine on microtubule dynamic instability further modification of vinorelbine, using superacidic chemistry, and treadmilling and found that these newer drugs exert effects on which specifically introduced two fluorine atoms in the velbanamine microtubule dynamics that differ significantly from those of the classic moiety (10). Vinca alkaloid, vinblastine. The major effects of vinflunine and vinorel- The characteristic block of cell proliferation during mitosis induced bine on dynamic instability were a slowing of the microtubule growth rate, by all five of the Vinca alkaloids appears to be attributable to a an increase in growth duration, and a reduction in shortening duration. In specific action on mitotic spindle microtubules. The mechanism of marked contrast to the action of vinblastine, they neither reduced the rate action of the Vinca alkaloids was initially thought to involve the of shortening nor increased the percentage of time the microtubules spent depolymerization of spindle microtubules and induction of paracrys- in an attenuated state, neither growing nor shortening detectably. In addition, vinflunine and vinorelbine suppressed treadmilling, but less talline tubulin-Vinca alkaloid arrays. At relatively high concentrations ␮ strongly than vinblastine. The diverse actions of these drugs on microtu- ( M levels), all five drugs inhibit microtubule polymerization in bules are likely to produce different effects on mitotic spindle function, reconstituted microtubule systems and in cells (6, 11, 12). However, leading to different effects on cell cycle progression and cell killing. recent evidence indicates that at low concentrations vinblastine and Nontumor cells with normal checkpoint proteins may tolerate the rela- vincristine exert a subtle but powerful action on microtubules; i.e., tively less powerful inhibitory effects of vinflunine and vinorelbine on they inhibit their dynamic behaviors at concentrations below those microtubule dynamics better than the more powerful effects of vinblas- required to significantly inhibit polymerization (13–15). For example, tine. Thus the unique constellation of effects of vinflunine and vinorelbine low vinblastine concentrations (nM levels) block mitosis in BSC-1 on dynamic instability and treadmilling may contribute to their superior cells in association with suppression of microtubule dynamics but in antitumor efficacies. the absence of appreciable changes in microtubule mass or spindle microtubule organization (13). The suppression of microtubule dy- INTRODUCTION namics by vinblastine and other antimitotic drugs, including taxol, Vinca alkaloids, including the natural products vincristine and estramustine, and colchicine, appears to cause mitotic block by acti- vinblastine and the semisynthetic derivatives vindesine and vi- vating the metaphase-anaphase checkpoint (12, 16). norelbine, are antimitotic drugs that are widely and successfully Microtubules display two types of unusual dynamic behavior, “dy- used in the treatment of cancer (1). Vinorelbine, the latest of the namic instability” and “treadmilling,” which appear to be important clinically approved Vinca alkaloids, has shown improved efficacy for progression through mitosis and the cell cycle. Dynamic instability and reduced toxicity. It is effective in the treatment of non-small is a stochastic switching of microtubule ends between phases of cell lung cancer, metastatic breast cancer, and ovarian cancer, and relatively slow growth and rapid shortening (17). Treadmilling is a net it shows promise in the management of lymphomas, esophageal addition of tubulin subunits at one end of a microtubule (the plus end) cancer, and prostatic carcinomas (2–4). A fifth new Vinca alkaloid, and the balanced net loss from the opposite (minus) end (18–20). The vinflunine, is a semisynthetic bifluorinated compound, which is dynamics of microtubules are coordinated with the actions of an now in Phase I clinical trials in Europe. Vinflunine has demon- undefined number of molecular motors to bring about the equi- strated superior activity over vinorelbine, vinblastine, and vincris- partitioning of chromosomes to the two daughter cells by the mitotic tine in a range of murine tumors and human tumor xenografts spindle. For example, microtubules emanating from each of the spin- (5–7). For example, vinflunine showed high activity against dle poles at prometaphase make vast growing and shortening excur- RXF944LX kidney cell and NCI-H69 small cell lung xenografts sions, probing the cytoplasm until they “find” and attach to the and moderate activity against PAXF546 pancreatic cell, PC-3 kinetochores of the chromosomes. During metaphase, chromosomes prostate cell, and TC37 colon cell human tumor xenografts, achiev- aligned at the metaphase plate oscillate back and forth under consid- ing an overall response of 64% (i.e., activity against 7 of 11 erable tension, most likely produced by a combination of motor xenografts tested). In contrast, only a 27% (3 of 11) response was molecules working in concert with the growing and shortening of observed for vinorelbine tested concurrently with only moderate kinetochore-attached microtubules. Superimposed on the chromo- activity against RXF944LX kidney cell and TC37 colon cell some oscillations is microtubule treadmilling or flux (21) where xenografts (5). tubulin is continuously added to microtubule plus ends at the kinetochores and lost from the minus ends at the spindle poles in balanced Received 2/8/00; accepted 7/19/00. fashion. The dynamics and the forces produced by them appear to be The costs of publication of this article were defrayed in part by the payment of page important in cell cycle signaling at the metaphase/anaphase check- charges. This article must therefore be hereby marked advertisement in accordance with point (22). Vinblastine has been shown to bind with high affinity to 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a grant from the Institut de Recherche Pierre Fabre and by USPHS microtubule ends. This strongly suppresses both microtubule dynamic Grants NS13560 and CA57291. instability and treadmilling and appears to lead to a mitotic block at 2 To whom requests for reprints should be addressed, at Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106. the metaphase/anaphase transition (12–15, 23, 24). Phone: (805) 893-2819; Fax: (805) 893-8094. Vinflunine and vinorelbine share a number of properties with 5045

polymerized tubulin by centrifugation (150,000 ϫ g; 1 h; 35°C). The super- natant was aspirated, the sedimented microtubules were depolymerized in assembly buffer by incubation on ice (2 h), and the protein content was determined. Analysis of Microtubule Dynamics by Quantitative Video Microscopy. Purified tubulin (15 ␮M) was polymerized into microtubules as described above except that incubation was carried out at 37°C in 87 mM PIPES, 36 mM

2-(N-morpholino)ethane sulfonic acid, 1.4 mM MgCl2, 1.0 mM EGTA, and 1.0 mM GTP (pH 6.8). Under the conditions used, the microtubules formed predominantly at the plus ends of the seeds, and little or no assembly occurred at the minus ends. The seed concentration was adjusted to yield three to eight seeds per microscope field. After 25 min, a 2–3-␮l sample was placed between two coverslips and mounted on a prewarmed glass microscope slide (24). Microtubules were imaged by video-enhanced differential interference contrast microscopy using a Zeiss IM35 inverted microscope with a Zeiss Planapo n.a.1.4, ϫ63 oil immersion objective and a stage maintained at 35–37°C. Samples were imaged for a maximum of 35 min. Microtubule images, from a minimum of 30 microtubules/condition, were captured in real time and recorded on video tape (24). Microtubule lengths were measured at 3–5-s intervals and analyzed using the Real Time Measure- ment (version 5.0) program (a kind gift from Neal Gliksman and E. D. Salmon, University of North Carolina, Chapel Hill, NC). From these length measure- ments, “life history” plots of microtubule length versus time were generated, and the rates of microtubule growth and shortening were calculated by least squares regression. We considered a microtubule to be in a growth phase when its rate of growth was Ͼ0.15 ␮m/min and its length was changed by Ͼ0.2 ␮m. Similarly, a microtubule was considered to be in a shortening phase when its shortening rate was Ͼ0.30 ␮m/min and its length was changed by Ͼ0.2 ␮m. Length changes of Յ0.2 ␮m were not detectable; thus a change of Յ0.2 ␮m over a duration of Ն30 s was considered to be a phase of attenuated dynamics, Fig. 1. Chemical structures of vinflunine, vinorelbine, and vinblastine. sometimes called a pause. A transition from a growth or attenuation phase to a shortening phase is termed a “catastrophe,” whereas a transition from shortening to a growth phase or to an attenuated state is called a “rescue.” The catastrophe frequency was calculated as the number of catastrophes divided by vinblastine and other Vinca alkaloids. They inhibit polymerization of the sum of the total time spent in the growth and attenuation phases for all purified microtubule protein into microtubules (6). At high concen- microtubules, whereas the rescue frequency was calculated as the number of Ն trations ( 50 nM), they inhibit mitosis in tumor cells in association rescues divided by the sum of the total time spent in the shortening phase for with spindle microtubule depolymerization, and at very high concen- all microtubules. Dynamicity is the total rate of measurable tubulin exchange trations (ϳ50 ␮M), they induce tubulin paracrystal formation (6). at microtubule ends (attributable to growth and shortening). Because of their superior antitumor activities as compared with the Determination of the Treadmilling Rate. Microtubule protein (2.5 mg/

classical Vinca alkaloids, we wanted to determine the actions of these ml) was thawed and resuspended in PEM buffer [100 mM PIPES, 1 mM MgCl2, newer compounds on microtubule dynamic instability and treadmill- 1mM EGTA, 0.1 mM GTP (pH 6.8)] containing a GTP-regenerating system ing. Here we report that like vinblastine, both compounds suppress consisting of 10 mM acetyl phosphate and 1 IU/ml acetate kinase. The resus- microtubule treadmilling and dynamic instability. However, we found pended protein was polymerized into steady-state microtubules by incubation for 30 min at 30°C. Drug was added at the specified concentration, and that the actions of vinflunine and vinorelbine on microtubule dynam- incubation was continued for 30 min to allow re-establishment of steady state. ics are significantly different from those of vinblastine. The differ- [3H]GTP (final specific activity, 167 mCi/mmol) was added, and 30 min later, ences may contribute to the superior antitumor activities of these two duplicate samples of each reaction were removed and stabilized in microtubule semisynthetic drugs. stabilizing buffer (30% glycerol, 10% DMSO, 5.6 mM ATP in PEM buffer; 30°C). Stabilized microtubule samples were filtered through GF/F glass fiber MATERIALS AND METHODS filters, which were then washed three times with an equal volume of stabilizing buffer to remove unincorporated [3H]GTP. The amount of [3H]GDP incorpo- Purification of Microtubule Protein and Tubulin. Microtubule protein rated into microtubules, i.e., trapped on the glass fiber filters, was quantitated preparations consisting of 70% tubulin and 30% MAPs3 were isolated from by scintillation counting in Beckman Ready Protein scintillation cocktail (26). bovine brain by three cycles of polymerization and depolymerization. Tubulin was purified from the microtubule protein by phosphocellulose chromatogra- RESULTS phy, drop-frozen in liquid nitrogen, and stored at Ϫ70°C (20). On the day of use, the tubulin was thawed on ice and then centrifuged (17,000 ϫ g; 20 min; Reduction of Microtubule Polymer Mass by Vinflunine and 4°C) to remove aggregated or denatured tubulin. Protein concentration was Vinorelbine. We wanted to analyze the effects of vinflunine and determined by the Bradford assay (25) using BSA as the standard. vinorelbine on the dynamic instability behavior of individual micro- ␮ Determination of Microtubule Polymer Mass. Purified tubulin (17 M) tubules polymerized from purified tubulin onto axoneme seeds. Thus, was polymerized into microtubules in the absence or presence of a range of we first determined the effects of vinflunine and vinorelbine on vinflunine or vinorelbine concentrations (35 min; 37°C) in 75 mM PIPES, 1.8 microtubule polymer mass in this system. Purified MAP-free tubulin mM MgCl , 1.0 mM EGTA, and 1.5 mM GTP (pH 6.8) using sea urchin 2 ␮ (Strongylocentrotus purpuratus) axonemes as seeds for assembly initiation (17 M) was polymerized to steady state at the ends of axoneme seeds (20). After incubation, polymerized microtubules were separated from un- in the absence or presence of a range of vinflunine or vinorelbine concentrations (0–4 ␮M). The assembled polymer was then collected 3 The abbreviations used are: MAP, microtubule-associated protein; PIPES, pipera- by centrifugation, and the protein content was determined (“Materials zine-N,NЈ-bis-(2-ethanesulfonic acid). and Methods”). As shown in Fig. 2, vinflunine and vinorelbine re- 5046

sufficiently high to reveal the effects of the drugs on the dynamic parameters by video microscopy, but not so high that it caused severe reduction of the microtubule polymer mass. An attempt was made to study the effects of the drugs at higher concentrations (0.6 ␮M), but for all three drugs, this concentration resulted in microtubules that were too few and too short for accurate analysis. As will be shown below, this concentration of all three drugs suppressed some parameters to the same extent, while affecting other parameters quite differently. Life history traces showing the changes in length with time of representative microtubules at their plus ends in the absence (controls) and presence of the three drugs are shown in Fig. 3. The dynamic instability parameters determined from large numbers of such traces were quantitated and are shown in Tables 1 and 2. Consistent with previous data (15, 24), the plus ends of control microtubules grew slowly for long periods of time and occasionally transitioned to brief phases of rapid shortening (Fig. 3). Transitions to rapid shortening (catastrophes) occurred at a frequency of 0.18 Ϯ 0.02 events/min, and Fig. 2. Effects of vinflunine, vinorelbine, and vinblastine on microtubule polymeriza- episodes of shortening were “rescued” by the resumption of growth or tion. Purified tubulin (17 ␮M) was assembled to steady state in the absence or presence of pause at a frequency of 3.1 Ϯ 0.4 events/min (Table 2). Sometimes the Œ f a range of vinorelbine ( ) or vinflunine ( ) concentrations. The total polymer mass after microtubules neither grew nor shortened detectably, but spent time in 35 min of assembly was determined by sedimentation (“Materials and Methods”). The data for vinblastine (F), determined under identical conditions, are from Panda et al. (15). a phase of attenuated dynamics or “pause” (segments of traces The apparent increase in polymer mass at 4 ␮M vinorelbine may result from aggregation between asterisks in Fig. 3). Overall, control microtubules spent 71% (30–32). Bars, SE. of the time growing, 5% shortening, and 24% in an attenuated (paused) state. Their dynamicity (the overall average rate of length duced the microtubule polymer mass in a concentration-dependent gain and loss at the plus ends) was 1.3 ␮m/min (Table 1). manner. The concentration at which polymerization was inhibited by Vinflunine and vinorelbine (0.4 ␮M) clearly suppressed dynamic ␮ ␮ 50%, the IC50, was 1.2 M for vinflunine and 0.80 M for vinorelbine. instability (Fig. 3; Table 1). The magnitude of their effects and the ␮ We previously reported an IC50 of 0.54 M for vinblastine under parameters affected were similar for the two drugs. Their major similar conditions (Ref. 15; data also shown in Fig. 2). Thus, the effects were a slowing of the microtubule growth rate, an increase in potencies of the three drugs were in the same concentration range, the mean duration of a growth event, and an increase in the percentage with a ϳ2-fold difference between the strongest and the weakest. of time the microtubules spent growing. The rate of growth was Hence, the relative potencies can be ranked as vinblastine Ͼ vinorel- slowed by 29–32% from 0.49 Ϯ 0.04 ␮m/min in controls to bine Ͼ vinflunine. 0.35 Ϯ 0.01 ␮m/min and 0.34 Ϯ 0.01 ␮m/min for vinflunine and Vinflunine and Vinorelbine Suppress the Dynamic Instability vinorelbine, respectively. The growth duration was increased by 53– Behavior of Reconstituted Microtubules in Vitro. We chose to 59% from 1.7 Ϯ 0.2 min in controls to 2.7 Ϯ 0.2 min and 2.6 Ϯ 0.3 compare the effects of vinflunine, vinorelbine, and vinblastine on min for vinflunine and vinorelbine, respectively. The percentage of dynamic instability of individual microtubules polymerized from 15 time the microtubules spent growing also increased by 16–20%, from ␮M tubulin at a concentration of 0.4 ␮M. This concentration was 71% in controls to 85% and 82% with vinflunine and vinorelbine,

Fig. 3. Life history plots of representative microtubules polymerized to steady state showing microtubule length changes at their plus ends with time in the absence (control) or presence of 0.4 ␮M vinblastine, 0.4 ␮M vinorelbine, or 0.4 ␮M vinflunine. No length change was detectable during periods of attenuated dynamics or pause, e.g., between the asterisks in the control and vinblastine panels.

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Table 1 Effects of 0.4 ␮M vinflunine, vinorelbine, and vinblastine on the dynamic instability of steady-state microtubules Behavioral parameter Control Vinflunine % changeb Vinorelbine % changeb Vinblastine % changeb Mean ratesa (␮m/min) Growth 0.49 Ϯ 0.04 0.35 Ϯ 0.01c Ϫ29 0.34 Ϯ 0.01c Ϫ32 0.34 Ϯ 0.04c Ϫ26 Shortening 20 Ϯ 221Ϯ 2d ϩ618Ϯ 2d Ϫ611Ϯ 1c Ϫ44 Mean durationa (min) Growth 1.7 Ϯ 0.2 2.7 Ϯ 0.2c,d ϩ59 2.6 Ϯ 0.3c,d ϩ53 1.8 Ϯ 0.2 ϩ6 Shortening 0.27 Ϯ 0.05 0.19 Ϯ 0.03d Ϫ30 0.17 Ϯ 0.02d Ϫ36 0.31 Ϯ 0.04 ϩ15 Attenuation 1.5 Ϯ 0.2 1.2 Ϯ 0.2d Ϫ20 1.5 Ϯ 0.2d Ϫ1 2.4 Ϯ 0.2c ϩ60 % time spent Growing 71 85c,d ϩ20 82d ϩ16 43c Ϫ39 Shortening 5.0 3.6 Ϫ28 3.4d Ϫ32 6.8 ϩ37 Attenuated 24 11c,d Ϫ52 14d Ϫ40 50c ϩ108 Dynamicity (␮m/min) 1.3 1.0 Ϫ21 0.90 Ϫ32 0.90 Ϫ32 a SE are shown. b The percentage difference relative to the corresponding behavioral parameter in the control (absence of drug). c Values that are statistically significant, at the Ն95% level, to the corresponding control value. d Values that are statistically significant, at the Ն95% level, to the corresponding vinblastine value.

respectively. Interestingly, the drugs had little effect on the shortening ration into MAP-rich microtubules (26). The MAPs in this system rate, but greatly reduced the duration of shortening by 30–36%, from suppress dynamic instability so that treadmilling is the predominant a mean of 0.27 Ϯ 0.05 min in controls to a mean of 0.19 Ϯ 0.03 min dynamic behavior (20, 27). As shown in Fig. 5, vinflunine and and 0.17 Ϯ 0.02 min for vinflunine and vinorelbine, respectively. vinorelbine, like vinblastine, suppressed the treadmilling rate in a Vinflunine and Vinorelbine Affect Dynamic Instability Param- concentration-dependent manner. The concentrations of each drug eters Differently from Vinblastine. Interestingly, 0.4 ␮M vinflunine, that inhibited the treadmilling rate by 50% differed greatly (over a ␮ ␮ ␮ ␮ 0.4 M vinorelbine, and 0.4 M vinblastine all reduced dynamicity to 6-fold concentration range) with IC50 values of 0.42 M, 0.10 M, and similar degrees; by 21%, 32%, and 32%, respectively. However, the 0.066 ␮M for vinflunine, vinorelbine, and vinblastine, respectively. three drugs affected several of the dynamic instability parameters Hence, the relative potency of the three drugs is vinblastine Ͼ differently. As reported previously (15, 24), vinblastine suppressed vinorelbine Ͼ vinflunine. dynamics primarily by reducing the rate and extent of growth and shortening (Fig. 3 and Table 1). However, unlike vinblastine, vin- DISCUSSION flunine and vinorelbine did not reduce the shortening rate (Table 1). The effects of vinflunine and vinorelbine on the lengths grown and We have found that vinflunine, vinorelbine, and vinblastine all shortened differed significantly from those of vinblastine as shown in inhibited microtubule polymerization and, at relatively low concen- the histograms in Fig. 4. In the presence of vinflunine and vinorelbine, trations, suppressed treadmilling, with vinblastine being the most the percentage of microtubules that underwent long growing excur- potent and vinflunine the least. Additionally, dynamicity as well as sions (Ն2 ␮m; Fig. 4A) was greater than or equal to that of control growth rates were suppressed by the three drugs at 0.4 ␮m to similar microtubules, whereas with vinblastine, most of the growing changes extents (Table 1). However, vinorelbine and vinflunine affected other were short (Ͻ1.2 ␮m). Similarly, vinblastine reduced the lengths of dynamic instability parameters differently than vinblastine, suggesting the shortening excursions much more strongly than vinorelbine or that the mechanisms of action of vinflunine and vinorelbine at micro- vinflunine (Fig. 4B). Furthermore, vinblastine increased the percent- tubule ends differ from that of vinblastine. These differences may age of time microtubules spent in the attenuated state by 108%, contribute to the superior antitumor activity of the two newer drugs as whereas in contrast, vinflunine and vinorelbine reduced it by 52% and compared with that of vinblastine (5, 7). 40%, respectively. The most prominent effects of vinflunine and vinorelbine on dy- The rescue and catastrophe frequencies and the lengths micro- namic instability were a slowing of the microtubule growth rate, an tubules grow and shorten are important in the formation of a bipolar increase in growth duration, and an increase in the percentage of time mitotic spindle. The two newer alkaloids affected the frequency of the microtubules spent growing. To illustrate these effects in compar- rescue quite differently from vinblastine at the conditions used. Vin- ison with those of vinblastine, the average growth and shortening flunine and vinorelbine increased the rescue frequency by 43% and events in the absence (control) and presence of 0.4 ␮M drug (from 60% respectively, as compared with controls, whereas vinblastine had Table 1) are drawn in hypothetical life history plots in Fig. 6. Here the either no effect or only a minimal one (Table 2). The catastrophe microtubule length during a growth or shortening event, relative to its frequency was virtually unchanged by any of the three drugs. In length at the start of the event, is plotted against time such that the summary, although all three Vinca alkaloids suppress dynamic insta- mean rate of microtubule length change is represented by the slope of bility, the actions of vinflunine and vinorelbine differ from that of each line. The X (horizontal) component of each line segment repre- vinblastine, indicating that the mechanisms of action of the two newer sents the duration of the average growth event (Fig. 6A) or shortening drugs are different from that of vinblastine. event (Fig. 6B), and the Y (vertical) component represents the length Vinflunine and Vinorelbine Suppressed Microtubule Tread- change in ␮m. For example, Fig. 6A shows that during an average milling in Vitro. We examined the effects of vinflunine and vinorel- growth event, a control microtubule grew 0.83 ␮m in 1.7 min. A bine on the treadmilling rate in vitro by following [3H]GTP incorpo- comparison of growth events (Fig. 6A) reveals that although all three

a Table 2 Effects of 0.4 ␮M vinflunine, vinorelbine, and vinblastine on steady-state microtubule transition frequencies Transition frequency (events/min) Control Vinflunine % change Vinorelbine % change Vinblastine % change Catastrophe 0.18 Ϯ 0.02 0.18 Ϯ 0.03 ϩ2 0.19 Ϯ 0.03 ϩ3 0.20 Ϯ 0.03 ϩ13 Rescue 3.1 Ϯ 0.40 4.4 Ϯ 0.67 ϩ43 4.9 Ϯ 0.69 ϩ60 2.6 Ϯ 0.32 Ϫ17 a Estimates of variation and error are shown and were calculated as transition frequency divided by the square root of the sample size (41). 5048

without becoming incorporated into the core of the microtubules (28). Furthermore, the binding of one or two vinblastine molecules per microtubule is sufficient to inhibit the treadmilling addition of tubulin subunits by 50% (29). The binding of vinblastine to tubulin at the end of a microtubule may stabilize the end by inducing a conformational change that increases the affinity of the tubulin for its neighboring tubulin molecules (15, 24, 28–30). Differences in the effects of vinflunine, vinorelbine, and vinblastine on microtubule dynamic instability could be attributable to differences in the binding of the three drugs to the microtubule ends or in the extent to which the drugs induce stabilizing effects. The binding of vinflunine and vinorelbine to microtubule ends has not yet been determined. However, it is reasonable to assume that these two drugs, like vinblastine, also bind to the ends. If so, the block of growth could be a result of drug- induced conformational changes in tubulin at the microtubule end, or by simple steric hindrance. As suggested above, vinflunine and vinorelbine may be less able than vinblastine to increase the affinity of tubulin for its neighbors at the microtubule ends. This idea is supported by the observation that vinblastine binds more strongly to tubulin polymers than vinflunine or vinorelbine (31–33), and it would explain how growth, and not shortening, is inhibited by vinflunine and vinorelbine when the drug is bound to the microtubule end. Alternative mechanisms involving drug-tubulin oligomers also ex- ist. It is known that the Vinca alkaloids bind to free tubulin and to tubulin spiral oligomers, as well as to microtubules. Vinflunine, vinorelbine, and vinblastine bind to GTP-tubulin subunits with similar affinities (31, 32). The drug-liganded tubulin subunits then undergo isodesmic self-association and form drug-tubulin spiral oligomers. Fig. 4. Effects of 0.4 ␮M vinflunine, vinorelbine, and vinblastine on the changes in microtubule length during growth events (A) and shortening events (B). The histograms The equilibrium constants for Vinca alkaloid-tubulin self-association show the distribution of length changes observed during the growth and shortening events are larger than the equilibrium constants for Vinca alkaloid binding to for all microtubules evaluated in all conditions. tubulin, and they differ significantly among the three drugs with vinblastine Ͼ vinorelbine Ͼ vinflunine (33). Also, the spiral oligomers formed with the three drugs are different (32) and the equilibrium constants for binding to polymers differ for the three drugs (31–33). Thus there will be different proportions of free drug and

Fig. 5. Inhibition of treadmilling by vinflunine (f), vinorelbine (Œ), and vinblastine (F). Treadmilling incorporation during a 30-min pulse with [3H]GTP was assayed as described under “Materials and Methods” at a microtubule protein concentration of 2.5 mg/ml and at the indicated drug concentrations. Results are presented as the percentage inhibition of incorporation into a non-drug-treated sample. Fig. 6. Diagrammatic representation of average microtubule growth (A) and shortening (B) events in the absence (control; ⅐⅐⅐⅐ࡗ) or presence of 0.4 ␮M vinflunine (----Ⅺ), drugs slowed the growth rate to a similar extent, vinflunine and vinorelbine (----‚), or vinblastine (OOOE). These average events are depicted as vinorelbine did not significantly reduce the length grown in a growth hypothetical microtubule life history traces where the length (in ␮m), relative to microtubule length at the initiation of the growth or shortening event, is plotted versus time. A, event, whereas vinblastine did. With regard to the average shortening the average microtubule growth rate, as indicated by the slope of the line, was reduced to event (Fig. 6B), vinflunine and vinorelbine, unlike vinblastine, had no a similar extent by all three drugs. The mean duration of growth was increased by effect on the shortening rate, but all three drugs reduced the length vinflunine and vinorelbine, but not altered significantly by vinblastine. The mean length grown was similar with vinflunine and vinorelbine but was decreased by vinblastine. B, shortened to ϳ3–4 ␮m compared with 5.4 ␮m observed for controls. the shortening rate (slope) during an average shortening event was reduced greatly by Possible Mechanisms for Suppression of Dynamic Instability by vinblastine, but not by vinflunine or vinorelbine. Also, the duration of the average shortening event was reduced by vinflunine and vinorelbine but increased slightly by Vinflunine and Vinorelbine. Free vinblastine appears to bind with vinblastine. The mean length shortened was reduced by all three Vinca alkaloids relative high affinity to a maximum of 16–17 sites at microtubule ends to the control. 5049

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2000 American Association for Cancer Research. ACTIONS OF VINFLUNINE AND VINORELBINE ON MICROTUBULES drug-tubulin oligomers present in suspensions of microtubules treated REFERENCES with the three drugs. Although the affinity of Vinca alkaloid-tubulin 1. Donehower, R. C., and Rowinsky, E. K. Anticancer drugs derived from plants. In: oligomers for microtubule ends is unknown, differences in the effects V. T. De Vita, S. Hellman, and S. A. Rosenberg (eds.), Cancer: Principles and of the three drugs on dynamic instability could be attributable to Practice of Oncology, pp. 409–417. Philadelphia: J B Lippincott Company, 1993. 2. Bunn, P. A., Jr., and Kelly, K. New chemotherapeutic agents prolong survival and differing affinities for binding of drug-tubulin oligomers to micro- improve quality of life in non-small cell lung cancer: a review of the literature and tubule ends or to the effects of the different drug-bound oligomers at future directions. Clin. Cancer Res., 4: 1087–1100, 1998. 3. Crown, J. Optimising treatment outcomes: a review of current management strategies the ends. in first-line chemotherapy of metastatic breast cancer. Eur. J. Cancer, 33: S15–S19, Effects of Vinflunine and Vinorelbine on Treadmilling. Tread- 1997. milling has been postulated to play a role in inducing tension on the 4. Johnson, S. A., Harper, P., Hortobagyi, G. N., and Pouillart, P. Vinorelbine: an overview. Cancer Treat. Rev., 22: 127–142, 1996. kinetochores during mitosis (18), a condition that may be critical for 5. Hill, B. T., Fiebig, H-H., Waud, W. R., Poupon, M-F., Colpaert, F., and signaling the passage from metaphase to anaphase (22). A large Kruczynski, A. Superior in vivo experimental antitumour activity of vinflunine, relative to vinorelbine, in a panel of human tumour xenografts. Eur. J. Cancer, 35: number of antimitotic drugs that block mitosis at the metaphase/ 512–520, 1999. anaphase transition inhibit treadmilling at concentrations that have 6. Kruczynski, A., Barret, J-M., Etievant, C., Colpaert, F., Fahy, J., and Hill, B. T. little effect on the mass of microtubule polymer (14, 23, 34–36). In Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid. Biochem. Pharmacol., 55: 635–648, 1998. the present study, we found that vinflunine inhibited the rate of 7. Kruczynski, A., Colpaert, F., Tarayre, J-P., Mouillard, P., Fahy, J., and Hill, B. T. treadmilling 4-fold less strongly than vinorelbine and 7-fold less Preclinical in vivo antitumor activity of vinflunine, a novel fluorinated Vinca alkaloid. strongly than vinblastine (Fig. 5). These potency differences on tread- Cancer Chemother. Pharmacol., 41: 437–447, 1998. 8. Langlois, N., Gueritte, F., Langlois, Y., and Potier, P. Application of the Polonovski milling may be an important determinant of antitumor activity. We reaction to the synthesis of vinblastine-type alkaloids. J. Am. Chem. Soc., 98: recently modeled how the treadmilling rate can be greatly modified by 7017–7024, 1976. 9. Mangeney, P., Andriamialisoa, R. Z., Langlois, N., Langlois, Y., and Potier, P. A new small changes in the dissociation rate constant at microtubule minus class of antitumor compounds: 5Ј-nor and 5Ј,6Ј-seco derivatives of vinblastine type ends (20). Vinblastine has been shown to stabilize microtubule plus alkaloids. J. Org. Chem., 44: 3765–3768, 1979. ends and to destabilize minus ends (15). The effects of vinflunine and 10. Fahy, J., Duflos, A., Ribet, J. P., Jacquesy, J. C., Berrier, C., Jouannetaud, M. P., and Zunino, F. Vinca alkaloids in superacidic media: a method for creating a new family vinorelbine on dynamics at minus ends have not been determined, but of antitumor derivatives. J. Am. Chem. Soc., 119: 8576–8577, 1997. it seems possible that all three drugs might destabilize minus ends 11. Binet, S., Chaineau, E., Fellous, A., Lataste, H., Krikorian, A., Couzinier, J. P., and Meininger, V. Immunofluorescence study of the action of navelbine, vincris- differently. Such differential action at minus ends, which would result tine and vinblastine on mitotic and axonal microtubules. Int. J. Cancer, 46: in differences in the abilities of the three drugs to affect treadmilling, 262–266, 1990. may also be important in determining antitumor activity. 12. Jordan, M. A., Thrower, D., and Wilson, L. Mechanism of cell proliferation by Vinca alkaloids. Cancer Res., 51: 2212–2222, 1991. Possible Significance for the Antitumor Efficacy of Vinflunine 13. Dhamodharan, R. I., Jordan, M. A., Thrower, D., Wilson, L., and Wadsworth, P. and Vinorelbine. Vinflunine and vinorelbine have shown reduced Vinblastine suppresses dynamics of individual microtubules in living cells. Mol. Biol. Cell, 6: 1215–1229, 1995. toxicity in animal studies, and vinorelbine has shown reduced 14. Jordan, M. A., Himes, R. H., and Wilson, L. Comparison of the effects of vinblastine, toxicity in the clinic as compared with vinblastine (4–6). Although vincristine, vindesine, and vinepidine on microtubule dynamics and cell proliferation differences in pharmacokinetics may play an important role, there in vitro. Cancer Res., 45: 2741–2747, 1985. 15. Panda, D., Jordan, M. A., Chu, K. C., and Wilson, L. Differential effects of vinblas- are several other possible explanations for the reduced toxicity. tine on polymerization and dynamics at opposite microtubule ends. J. Biol. Chem., One possibility involves the roles of microtubule dynamics during 271: 29807–29812, 1996. mitosis and the differences in the effects of the three drugs on the 16. Jordan, M. A., and Wilson, L. Microtubules and actin filaments: dynamic targets for cancer chemotherapy. Curr. Opin. Cell Biol., 10: 123–130, 1998. dynamics. Dynamic instability is important for congression of the 17. Mitchison, T. J., and Kirschner, M. Dynamic instability of microtubule growth. chromosomes to the equatorial metaphase plate during promet- Nature (Lond.), 312: 237–242, 1984. 18. Margolis, R. L., and Wilson, L. Microtubule treadmilling: what goes around comes aphase (37). It also plays an important role in the tension-associ- around. BioEssays, 20: 830–836, 1998. ated oscillations of the chromosomes at prometaphase and met- 19. Rodionov, V. I., Nadezhdina, E., and Borisy, G. G. Centrosomal control of micro- aphase (38). Treadmilling or poleward tubulin flux (21) may serve tubule dynamics. Proc. Natl. Acad. Sci. USA, 96: 115–120, 1999. 20. Panda, D., Miller, H. P., and Wilson, L. Rapid treadmilling of MAP-free brain an important function during metaphase by mediating transport of microtubules in vitro and its suppression by tau. Proc. Natl. Acad. Sci. USA, 96: signaling molecules from the kinetochores to the spindle poles, and 12459–12464, 1999. 21. Mitchison, T. J. Polewards microtubule flux in the mitotic spindle: evidence from also by creating tension (18). Vinblastine suppresses treadmilling photoactivation of fluorescence. J. Cell Biol., 109: 637–652, 1989. more powerfully than vinflunine or vinorelbine (Table 1; Fig. 6), 22. Nicklas, R. B., Ward, S. C., and Gorbsky, G. J. Kinetochore chemistry is sensitive to and it strongly suppresses the rate of shortening, whereas vin- tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Biol., 130: 929–939, 1995. flunine and vinorelbine do not. These diverse actions of the three 23. Jordan, M. A., Thrower, D., and Wilson, L. Effects of vinblastine, podophyllotoxin, drugs are likely to have different effects during mitosis, which may and nocodazole on microtubules in cells: implications for the role of microtubule dynamics in mitosis. J. Cell Sci., 102: 401–416, 1992. lead to different effects on cell cycle progression and cell killing. 24. Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., and Wilson, L. Kinetic Nontumor cells with normal checkpoint proteins could conceivably stabilization of microtubule dynamic instability in vitro by vinblastine. Biochemistry, tolerate the relatively less powerful inhibitory effects of vinflunine 32: 1285–1293, 1993. 25. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram and vinorelbine on microtubule dynamics but not the relatively quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., more powerful effects of vinblastine. Furthermore, checkpoint 72: 248–254, 1976. 26. Farrell, K. W., and Wilson, L. The differential kinetic stabilization of opposite mechanisms in tumor cells are frequently faulty (39, 40). Thus, microtubule ends by tubulin-colchicine complexes. Biochemistry, 23: 3741–3748, cancer cells with defective checkpoint proteins may be more sus- 1984. ceptible than normal cells to vinflunine and vinorelbine, yielding 27. Farrell, K. W., Jordan, M. A., Miller, H. P., and Wilson, L. Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling. an overall effectiveness that is superior to that of vinblastine. J. Cell Biol., 184: 1035–1046, 1987. 28. Wilson, L., Jordan, M. A., Morse, A., and Margolis, R. L. Interaction of vinblastine with steady-state microtubules in vitro. J. Mol. Biol., 159: 125–149, 1982. 29. Jordan, M. A., and Wilson, L. Kinetic analysis of tubulin exchange at microtubule ACKNOWLEDGMENTS ends at low vinblastine concentrations. Biochemistry, 29: 2730–2739, 1990. 30. Na, G. C., and Timasheff, S. N. Stoichiometry of the vinblastine-induced self- We thank Herb Miller for preparing the tubulin used in this work. association of calf brain tubulin. Biochemistry, 19: 1347–1354, 1980. 5050

31. Lobert, S., Vulevic, B., and Correia, J. J. Interaction of Vinca alkaloids with tubulin: 37. Hayden, J. J., Bowser, S. S., and Rieder, C. Kinetochores capture astral microtubules a comparison of vinblastine, vincristine, and vinorelbine. Biochemistry, 35: 6806– during chromosome attachment to the mitotic spindle: direct visualization in live newt 6814, 1996. cells. J. Cell Biol., 111: 1039–1045, 1990. 32. Lobert, S., Ingram, J. W., Hill, B. T., and Correia, J. J. A comparison of thermo- 38. Skibbens, R. V., Skeen, V. P., and Salmon, E. D. Directional instability of kineto- dynamic parameters for vinorelbine- and vinflunine-induced tubulin self-association chore mobility during chromosome congression and segregation in mitotic newt lung by sedimentation velocity. Mol. Pharmacol., 53: 908–915, 1998. cells: a push-pull mechanism. J. Cell Biol., 122: 859–875, 1993. 33. Lobert, S., and Correia, J. J. Energetics of Vinca alkaloid interactions with tubulin. 39. Lengauer, C., Kinzler, K. W., and Vogelstein, B. Genetic instabilities in human Methods Enzymol., in press, 2000. cancers. Nature (Lond.), 396: 643–649, 1998. 34. Wilson, L., Miller, H. P., Farrell, K. W., Snyder, K. B., Thompson, W. C., and Purich, 40. Cahill, D. P., da Costa, L. T., Carson-Walter, E. B., Kinzler, K. W., Vogelstein, B., D. L. Taxol stabilization of microtubules in vitro: dynamics of tubulin addition and loss at opposite microtubule ends. Biochemistry, 24: 5254–5262, 1995. and Lengauer, C. Characterization of MAD2B and other mitotic spindle checkpoint 35. Wilson, L., and Jordan, M. A. Pharmacological probes of microtubule function. In: genes. Genomics, 58: 181–187, 1999. J. S. Hyams and C. W. Lloyd (eds.), Microtubules, pp. 59–84. New York: Wiley- 41. Walker, R. A., O’Brien, E. T., Pryer, N. K., Soboeiro, M. F., Voter, W. A., Erickson, Liss, Inc., 1994. H. P., and Salmon, E. D. Dynamic instability of individual microtubules analyzed by 36. Wilson, L., and Jordan, M. A. Microtubule dynamics: taking aim at a moving target. video light microscopy: rate constants and transition frequencies. J. Cell Biol., 107: Curr. Biol., 2: 569–573, 1995. 1437–1448, 1988.

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