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Biochemistry and Biophysics |
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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Structurally, vinflunine and vinorelbine differ from vinblastine in the
velbanamine moiety ("top" portion) of the molecule (Fig. 1)
. Both drugs were synthesized by a novel method for coupling the
precursor alkaloids catharanthine and vindoline, which resulted in the
formation of an eight-membered rather than a nine-membered ring within
the velbanamine portion of the molecule (8
, 9)
. Vinflunine
was derived by further modification of vinorelbine, using superacidic
chemistry, which specifically introduced two fluorine atoms in the
velbanamine moiety (10)
.
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Microtubules display two types of unusual dynamic behavior, "dynamic instability" and "treadmilling," which appear to be important for progression through mitosis and the cell cycle. Dynamic instability is a stochastic switching of microtubule ends between phases of relatively slow growth and rapid shortening (17) . Treadmilling is a net addition of tubulin subunits at one end of a microtubule (the plus end) and the balanced net loss from the opposite (minus) end (18, 19, 20) . The dynamics of microtubules are coordinated with the actions of an undefined number of molecular motors to bring about the equi-partitioning of chromosomes to the two daughter cells by the mitotic spindle. For example, microtubules emanating from each of the spindle poles at prometaphase make vast growing and shortening excursions, probing the cytoplasm until they "find" and attach to the kinetochores of the chromosomes. During metaphase, chromosomes aligned at the metaphase plate oscillate back and forth under considerable tension, most likely produced by a combination of motor molecules working in concert with the growing and shortening of kinetochore-attached microtubules. Superimposed on the chromosome oscillations is microtubule treadmilling or flux (21) where tubulin is continuously added to microtubule plus ends at the kinetochores and lost from the minus ends at the spindle poles in balanced fashion. The dynamics and the forces produced by them appear to be important in cell cycle signaling at the metaphase/anaphase checkpoint (22) . Vinblastine has been shown to bind with high affinity to microtubule ends. This strongly suppresses both microtubule dynamic instability and treadmilling and appears to lead to a mitotic block at the metaphase/anaphase transition (12, 13, 14, 15 , 23 , 24) .
Vinflunine and vinorelbine share a number of properties with
vinblastine and other Vinca alkaloids. They inhibit
polymerization of purified microtubule protein into microtubules
(6)
. At high concentrations (
50
nM), they inhibit mitosis in tumor cells in
association with spindle microtubule depolymerization, and at very high
concentrations (
50 µM), they induce tubulin
paracrystal formation (6)
. Because of their superior
antitumor activities as compared with the classical Vinca
alkaloids, we wanted to determine the actions of these newer compounds
on microtubule dynamic instability and treadmilling. Here we report
that like vinblastine, both compounds suppress microtubule treadmilling
and dynamic instability. However, we found that the actions of
vinflunine and vinorelbine on microtubule dynamics are significantly
different from those of vinblastine. The differences may contribute to
the superior antitumor activities of these two semisynthetic drugs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of Microtubule Polymer Mass.
Purified tubulin (17 µM) was polymerized into
microtubules in the absence or presence of a range of vinflunine or
vinorelbine concentrations (35 min; 37°C) in 75 mM PIPES,
1.8 mM MgCl2, 1.0 mM
EGTA, and 1.5 mM GTP (pH 6.8) using sea urchin
(Strongylocentrotus purpuratus) axonemes as seeds for
assembly initiation (20)
. After incubation, polymerized
microtubules were separated from unpolymerized tubulin by
centrifugation (150,000 x g; 1 h;
35°C). The supernatant 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 µM) 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-µl 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, x63 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 Measurement (version 5.0) program (a kind gift from
Neal Gliksman and E. D. Salmon, University of North Carolina, Chapel
Hill, NC). From these length measurements, "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 >0.15 µm/min and its length was changed by
>0.2 µm. Similarly, a microtubule was considered to be in a
shortening phase when its shortening rate was >0.30 µm/min and its
length was changed by >0.2 µm. Length changes of 
0.2 µm were not
detectable; thus a change of 0.2 µm over a duration of 30 s was
considered to be a phase of attenuated dynamics, 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 the sum of the total time spent in the growth
and attenuation phases for all microtubules, whereas the rescue
frequency was calculated as the number of rescues divided by the sum of
the total time spent in the shortening phase for all microtubules.
Dynamicity is the total rate of measurable tubulin exchange at
microtubule ends (attributable to growth and shortening).
Determination of the Treadmilling Rate.
Microtubule protein (2.5 mg/ml) was thawed and resuspended in PEM
buffer [100 mM PIPES, 1 mM
MgCl2, 1 mM EGTA, 0.1 mM
GTP (pH 6.8)] containing a GTP-regenerating system consisting of 10
mM acetyl phosphate and 1 IU/ml acetate kinase. The
resuspended protein was polymerized into steady-state microtubules by
incubation for 30 min at 30°C. Drug was added at the specified
concentration, and incubation was continued for 30 min to allow
re-establishment of steady state. [3
H]GTP
(final specific activity, 167 mCi/mmol) was added, and 30 min later,
duplicate samples of each reaction were removed and stabilized in
microtubule 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 filters, which
were then washed three times with an equal volume of stabilizing buffer
to remove unincorporated [3
H]GTP. The amount of
[3
H]GDP incorporated into microtubules,
i.e., trapped on the glass fiber filters, was quantitated by
scintillation counting in Beckman Ready Protein scintillation cocktail
(26)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, vinflunine and vinorelbine reduced the microtubule polymer mass in a
concentration-dependent manner. The concentration at which
polymerization was inhibited by 50%, the IC50,
was 1.2 µM for vinflunine and 0.80 µM for
vinorelbine. We previously reported an IC50 of
0.54 µM for vinblastine under similar conditions (Ref.
15
; data also shown in Fig. 2
). Thus, the potencies of the
three drugs were in the same concentration range, with a 2-fold
difference between the strongest and the weakest. Hence, the relative
potencies can be ranked as vinblastine > vinorelbine > vinflunine.
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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 ± 0.02 events/min, and episodes of shortening
were "rescued" by the resumption of growth or pause at a frequency
of 3.1 ± 0.4 events/min (Table 2)
. Sometimes the
microtubules neither grew nor shortened detectably, but spent time in a
phase of attenuated dynamics or "pause" (segments of traces
between asterisks in Fig. 3
). Overall, control microtubules spent
71% of the time growing, 5% shortening, and 24% in an attenuated
(paused) state. Their dynamicity (the overall average rate of length
gain and loss at the plus ends) was 1.3 µm/min (Table 1)
.
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; Table 1
). The magnitude of their effects
and the parameters affected were similar for the two drugs. Their major
effects were a slowing of the microtubule growth rate, an increase in
the mean duration of a growth event, and an increase in the percentage
of time the microtubules spent growing. The rate of growth was slowed
by 29–32% from 0.49 ± 0.04 µm/min in controls to
0.35 ± 0.01 µm/min and 0.34 ± 0.01
µm/min for vinflunine and vinorelbine, respectively. The growth
duration was increased by 53–59% from 1.7 ± 0.2 min
in controls to 2.7 ± 0.2 min and 2.6 ± 0.3 min for vinflunine and vinorelbine, respectively. The percentage of
time the microtubules spent growing also increased by 16–20%, from
71% in controls to 85% and 82% with vinflunine and vinorelbine,
respectively. Interestingly, the drugs had little effect on the
shortening rate, but greatly reduced the duration of shortening by
30–36%, from a mean of 0.27 ± 0.05 min in controls to
a mean of 0.19 ± 0.03 min and 0.17 ± 0.02 min for vinflunine and vinorelbine, respectively.
Vinflunine and Vinorelbine Affect Dynamic Instability Parameters
Differently from Vinblastine.
Interestingly, 0.4 µM vinflunine, 0.4 µM
vinorelbine, and 0.4 µM vinblastine all reduced
dynamicity to similar degrees; by 21%, 32%, and 32%, respectively.
However, the three drugs affected several of the dynamic instability
parameters differently. As reported previously (15
, 24)
,
vinblastine suppressed dynamics primarily by reducing the rate and
extent of growth and shortening (Fig. 3
and Table 1
). However, unlike
vinblastine, vinflunine and vinorelbine did not reduce the shortening
rate (Table 1)
. The effects of vinflunine and vinorelbine on the
lengths grown and shortened differed significantly from those of
vinblastine as shown in the histograms in Fig. 4
. In the presence of vinflunine and vinorelbine, the percentage of
microtubules that underwent long growing excursions (2 µm; Fig. 4A
) was greater than or equal to that of control
microtubules, whereas with vinblastine, most of the growing changes
were short (<1.2 µm). Similarly, vinblastine reduced the lengths of
the shortening excursions much more strongly than vinorelbine or
vinflunine (Fig. 4B)
. Furthermore, vinblastine increased the
percentage of time microtubules spent in the attenuated state by 108%,
whereas in contrast, vinflunine and vinorelbine reduced it by 52% and
40%, respectively.
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. The catastrophe
frequency was virtually unchanged by any of the three drugs. In
summary, although all three Vinca alkaloids suppress dynamic
instability, the actions of vinflunine and vinorelbine differ from that
of vinblastine, indicating that the mechanisms of action of the two
newer drugs are different from that of vinblastine.
Vinflunine and Vinorelbine Suppressed Microtubule Treadmilling
in Vitro.
We examined the effects of vinflunine and vinorelbine on the
treadmilling rate in vitro by following
[3
H]GTP incorporation into MAP-rich
microtubules (26)
. The MAPs in this system suppress
dynamic instability so that treadmilling is the predominant dynamic
behavior (20
, 27) . As shown in Fig. 5
, vinflunine and vinorelbine, like vinblastine, suppressed the
treadmilling rate in a concentration-dependent manner. The
concentrations of each drug that inhibited the treadmilling rate by
50% differed greatly (over a 6-fold concentration range) with
IC50 values of 0.42 µM,
0.10 µM, and 0.066 µM
for vinflunine, vinorelbine, and vinblastine, respectively. Hence, the
relative potency of the three drugs is vinblastine > vinorelbine >
vinflunine.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, vinorelbine and vinflunine affected
other dynamic instability parameters differently than vinblastine,
suggesting that the mechanisms of action of vinflunine and vinorelbine
at microtubule ends differ from that of vinblastine. These differences
may contribute to the superior antitumor activity of the two newer
drugs as compared with that of vinblastine (5
, 7)
.
The most prominent effects of vinflunine and vinorelbine on dynamic
instability were a slowing of the microtubule growth rate, an increase
in growth duration, and an increase in the percentage of time the
microtubules spent growing. To illustrate these effects in comparison
with those of vinblastine, the average growth and shortening events in
the absence (control) and presence of 0.4 µM drug (from
Table 1
) are drawn in hypothetical life history plots in Fig. 6
. Here the microtubule length during a growth or shortening event,
relative to its length at the start of the event, is plotted against
time such that the mean rate of microtubule length change is
represented by the slope of each line. The X (horizontal)
component of each line segment represents the duration of the average
growth event (Fig. 6A)
or shortening event (Fig. 6B)
, and the Y (vertical) component represents
the length change in µm. For example, Fig. 6A
shows that
during an average growth event, a control microtubule grew 0.83 µm in
1.7 min. A comparison of growth events (Fig. 6A)
reveals
that although all three drugs slowed the growth rate to a similar
extent, vinflunine and vinorelbine did not significantly reduce the
length grown in a growth event, whereas vinblastine did. With regard to
the average shortening event (Fig. 6B)
, vinflunine and
vinorelbine, unlike vinblastine, had no effect on the shortening rate,
but all three drugs reduced the length shortened to 3–4 µm
compared with 5.4 µm observed for controls.
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Alternative mechanisms involving drug-tubulin oligomers also exist. 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. The equilibrium constants for Vinca alkaloid-tubulin self-association are larger than the equilibrium constants for Vinca alkaloid binding to tubulin, and they differ significantly among the three drugs with vinblastine > vinorelbine > 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, 32, 33) . Thus there will be different proportions of free drug and drug-tubulin oligomers present in suspensions of microtubules treated with the three drugs. Although the affinity of Vinca alkaloid-tubulin oligomers for microtubule ends is unknown, differences in the effects of the three drugs on dynamic instability could be attributable to differing affinities for binding of drug-tubulin oligomers to microtubule ends or to the effects of the different drug-bound oligomers at the ends.
Effects of Vinflunine and Vinorelbine on Treadmilling.
Treadmilling has been postulated to play a role in inducing tension on
the kinetochores during mitosis (18)
, a condition that may
be critical for signaling the passage from metaphase to anaphase
(22)
. A large number of antimitotic drugs that block
mitosis at the metaphase/anaphase transition inhibit treadmilling at
concentrations that have little effect on the mass of microtubule
polymer (14
, 23
, 34, 35, 36)
. In the present study, we found
that vinflunine inhibited the rate of treadmilling 4-fold less strongly
than vinorelbine and 7-fold less strongly than vinblastine (Fig. 5)
.
These potency differences on treadmilling may be an important
determinant of antitumor activity. We recently modeled how the
treadmilling rate can be greatly modified by small changes in the
dissociation rate constant at microtubule minus ends (20)
.
Vinblastine has been shown to stabilize microtubule plus ends and to
destabilize minus ends (15)
. The effects of vinflunine and
vinorelbine on dynamics at minus ends have not been determined, but it
seems possible that all three drugs might destabilize minus ends
differently. Such differential action at minus ends, which would result
in differences in the abilities of the three drugs to affect
treadmilling, may also be important in determining antitumor activity.
Possible Significance for the Antitumor Efficacy of Vinflunine and
Vinorelbine.
Vinflunine and vinorelbine have shown reduced toxicity in animal
studies, and vinorelbine has shown reduced toxicity in the clinic as
compared with vinblastine (4, 5, 6)
. Although differences in
pharmacokinetics may play an important role, there are several other
possible explanations for the reduced toxicity. One possibility
involves the roles of microtubule dynamics during mitosis and the
differences in the effects of the three drugs on the dynamics. Dynamic
instability is important for congression of the chromosomes to the
equatorial metaphase plate during prometaphase (37)
. It
also plays an important role in the tension-associated oscillations of
the chromosomes at prometaphase and metaphase (38)
.
Treadmilling or poleward tubulin flux (21)
may serve an
important function during metaphase by mediating transport of signaling
molecules from the kinetochores to the spindle poles, and also by
creating tension (18)
. Vinblastine suppresses treadmilling
more powerfully than vinflunine or vinorelbine (Table 1
; Fig. 6
), and
it strongly suppresses the rate of shortening, whereas vinflunine and
vinorelbine do not. These diverse actions of the three drugs are likely
to have different effects during mitosis, which may lead to different
effects on cell cycle progression and cell killing. Nontumor cells with
normal checkpoint proteins could conceivably tolerate the relatively
less powerful inhibitory effects of vinflunine and vinorelbine on
microtubule dynamics but not the relatively more powerful effects of
vinblastine. Furthermore, checkpoint mechanisms in tumor cells are
frequently faulty (39
, 40)
. Thus, cancer cells with
defective checkpoint proteins may be more susceptible than normal cells
to vinflunine and vinorelbine, yielding an overall effectiveness that
is superior to that of vinblastine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Institut de
Recherche Pierre Fabre and by USPHS Grants NS13560 and CA57291. 
2 To whom requests for reprints should be
addressed, at Department of Molecular, Cellular, and Developmental
Biology, University of California, Santa Barbara, CA 93106.
Phone: (805) 893-2819; Fax: (805) 893-8094.
3 The abbreviations used are: MAP,
microtubule-associated protein; PIPES,
piperazine-N,N'-bis-(2-ethanesulfonic acid).
Received 2/ 8/00. Accepted 7/19/00.
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) or vinflunine (
) concentrations. The total polymer
mass after 35 min of assembly was determined by sedimentation
("Materials and Methods"). The data for vinblastine (•),
determined under identical conditions, are from Panda et
al. 
) or presence of 0.4 µM
vinflunine (----
), vinorelbine (----
), or vinblastine
(
). These average events are depicted as hypothetical
microtubule life history traces where the length (in µm), relative to
microtubule length at the initiation of the growth or shortening event,
is plotted versus time. A, the average
microtubule growth rate, as indicated by the slope of the line, was
reduced to a similar extent by all three drugs. The mean duration of
growth was increased by 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, the shortening rate (slope) during an average
shortening event was reduced greatly 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 vinblastine. The mean length shortened was reduced by all three
Vinca alkaloids relative to the control.