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Vol. 60, Issue 1, 225-232, July 2001
Department of Molecular, Cellular, and Developmental Biology and Neuroscience Research Institute, University of California, Santa Barbara, California (V.K.N., K.B., L.W., M.A.J.) and Division de Cancerologie Experimentale, Centre de Recherche Pierre Fabre, Castres, France (B.T.H.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The two second-generation Vinca alkaloids, vinorelbine and vinflunine, affect microtubule dynamics very differently from vinblastine, a first generation Vinca alkaloid. For example, vinblastine strongly suppresses the rate and extent of microtubule shortening in vitro, whereas vinorelbine and vinflunine suppress the rate and extent of microtubule growing events. We asked whether these differences result in differences in mitotic spindle organization that might be responsible for the superior antitumor activities of the two second-generation Vinca alkaloids. IC50 values for inhibition of HeLa cell proliferation for vinflunine, vinorelbine, and vinblastine were 18, 1.25, and 0.45 nM, respectively, similar to the concentrations that induced mitotic block at the metaphase/anaphase transition (38, 3.8, and 1.1 nM, respectively), indicating that mitotic block is a major contributor to antiproliferative action for all three drugs. Mitotically blocked cells exhibited aberrant spindles, consistent with induction of block by suppression of microtubule dynamics. Despite differences in their actions on individual dynamic instability parameters, morphologically detectable differences in spindle effects among the three drugs were minimal, indicating that overall suppression of dynamics may be more important in blocking mitosis than specific effects on growth or shortening. We also found that the peak intracellular drug concentration at the mitotic IC50 value was highest for vinflunine (4.2 ± 0.2 µM), intermediate for vinorelbine (1.3 ± 0.1 µM), and more than 10-fold lower for vinblastine (130 ± 7 nM), suggesting that intracellular binding reservoir(s) may be partially responsible for vinflunine's high efficacy and minimal side effects.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Vinca
alkaloids, including the natural products vincristine and vinblastine
and the semisynthetic derivatives vindesine and vinorelbine, are
antimitotic drugs that are widely used in cancer treatment (Donehower
and Rowinsky, 1993
). Vinorelbine, the most recent clinically approved
Vinca alkaloid, shows improved efficacy and reduced
toxicity. It is effective in non-small-cell lung cancer, metastatic
breast cancer, and ovarian cancer and shows promise in lymphoma,
esophageal cancer, and prostatic carcinoma (Johnson et al., 1996;
Crown, 1997; Bunn and Kelly, 1998). Vinflunine is a new semisynthetic
bifluorinated compound that, having completed phase I clinical trials,
is now in phase II (Armand et al., 2001; Fumoleau et al., 2001).
Vinflunine is more active than vinorelbine, vinblastine, or vincristine
against a number of murine tumors and human tumor xenografts. For
example, vinflunine exhibited high or moderate antitumor efficacy in
64% (7 of 11) tumor models, whereas vinorelbine exhibited, at best,
moderate activity in 27% (3 of 11) (Kruczynski et al., 1998a,b; Hill
et al., 1999).
Vinflunine and vinorelbine differ structurally from vinblastine in the
velbanamine "upper" portion of the molecule (Fig.
1). Both drugs were synthesized by a
novel method resulting in a ring with eight members rather than nine
within the velbanamine portion (Langlois et al., 1976; Mangeney et al.,
1979). Vinflunine was derived by further modification of vinorelbine,
using superacidic chemistry to introduce two fluorine atoms (Fahy et
al., 1997; Jacquesy and Fahy, 2000).
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The mechanism of action of the Vinca alkaloids was initially
thought to involve depolymerization of spindle microtubules and induction of paracrystalline tubulin-Vinca alkaloid arrays.
At relatively high concentrations (micromolar), the Vinca
alkaloids inhibit microtubule polymerization (Binet et al., 1990,
Jordan et al., 1991; Kruczynski et al., 1998a). However, they also have a more subtle and powerful action on microtubules; they inhibit their
dynamics at concentrations below those required to inhibit polymerization (Jordan et al., 1985; Toso et al., 1993; Dhamodharan et
al., 1995). For example, low concentrations of vinblastine (8-32 nM)
block mitosis in BSC-1 cells in association with suppression of
microtubule dynamics, in the absence of appreciable changes in
microtubule mass or spindle microtubule organization (Dhamodharan et
al., 1995). Vinblastine inhibits chromosome congression (the prometaphase movement of chromosomes to the spindle equator) and the
transition from metaphase to anaphase, by binding with high affinity to
microtubule ends and suppressing microtubule dynamics (Jordan et al.,
1991; Jordan and Wilson, 1998).
Microtubules display two dynamic behaviors, dynamic instability, and
treadmilling, which are important for cell cycle progress (Mitchison
and Kirschner, 1984, Margolis and Wilson, 1998; Rodionov et al., 1999).
Dynamics play critical roles in the equi-partitioning of chromosomes to
the two daughter cells by the mitotic spindle. For example,
microtubules emanating from the spindle poles at prometaphase make vast
growing and shortening excursions (dynamic instability), probing the
cytoplasm until they "find" and attach to the kinetochores of
chromosomes. Failure of the microtubules to capture all the chromosomes
leads to mitotic block (Rudner and Murray, 1996) and apoptosis (Jordan
et al., 1996). In addition, chromosomes aligned at the metaphase plate
oscillate under tension produced by motor molecules and dynamic
kinetochore-attached microtubules. Superimposed on the oscillations is
microtubule treadmilling (Mitchison, 1989), in which tubulin undergoes
net addition to microtubule ends at the kinetochores and balanced net
loss at the poles. These forces are important in signaling at the
metaphase/anaphase checkpoint (Nicklas et al., 1995).
We recently compared the effects of vinflunine, vinorelbine, and
vinblastine on dynamic instability and treadmilling of purified bovine
brain microtubules (Ngan et al., 2000). Interestingly, the inhibited
parameters differed significantly, and in some ways oppositely, for
vinflunine and vinorelbine compared with vinblastine. An important
question is whether these differences result in significantly different
modes of mitotic inhibition. Hence, in the present study, we asked
whether the differences between the actions of these compounds on
microtubule dynamics result in important differences in spindle
organization that may be responsible for the superior antitumor
activities of the two newer drugs.
We found that each drug blocked mitosis in HeLa cells at a concentration similar to that which inhibited proliferation, indicating that the mitotic block induced by all three drugs is a major contributor to their antiproliferative action. Although the intracellular drug concentrations of vinflunine, vinorelbine, and vinblastine varied 32-fold at the media concentrations that blocked mitosis, each drug produced remarkably similar effects on spindle organization. These results indicate that the differential inhibition of specific parameters of microtubule dynamic instability by the three drugs is not as important as their ability to suppress overall microtubule dynamics.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Culture. HeLa S3 cells [epithelial-like cells from epithelioid carcinoma of human cervix (American Type Culture Collection, Manassas, VA)] were cultured at 37°C in the presence of 5% CO2 using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and nonessential amino acids (Sigma Chemicals, St. Louis, MO) in 250-ml tissue culture flasks or 35-mm six-well plates (doubling time, 18-20 h). For studies evaluating mitotic block, cells were grown on poly-L-lysine-treated (50 µg/ml, 2 h, 37°C, washed once with sterile water) sterile glass coverslips in six-well plates. Plating densities ranged from 1 to 5 × 104 cells/ml to maintain cultures in log growth during drug incubation. Cells were incubated with drugs by replacing the original medium with an equal volume of medium containing the required concentration of drug, or no drug (control), and incubation was continued at 37°C for 20 h.
Cell Proliferation.
At the time of initiation and
termination of drug incubation, duplicate cultures were detached from
the culture vessel by incubation with trypsin (0.5 mg/ml in
phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4, 8.1 mM
Na2HPO4, and 0.5 mM EDTA,
pH 7.2) and live cells were counted using a hemocytometer. Trypan blue
dye was used to distinguish living from dead cells (Loo and Rillema,
1998). Cell proliferation was calculated from the difference in cell
number at the beginning and the end of drug incubation, relative to the
increase in cell number for control cultures during the same period.
Mitotic Progression. To evaluate mitotic indices, HeLa cells grown for 20 h in the absence or presence of drug in six-well plates were detached by incubation with trypsin, washed with phosphate-buffered saline, and fixed with 100% methanol. Cells were incubated with 10% normal goat serum to block nonspecific antibody staining, and then with a rat anti-tubulin monoclonal antibody (YL1/2; Harlan Sera-Labs, Inc., Crawley Down, Sussex, UK) (1 h, 37°C) followed, after washing, by Rhodamine Red-X-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at room temperature. Chromosomes and chromatin were stained with 0.1 to 1 µg/ml 4,6-diamidino-2-phenylindole for 2 to 5 min. Stained cells were mounted on slides using Vectashield (Vector Laboratories) antifade reagent.
To evaluate microtubule and chromosome organization and the stage of mitosis at which the cells were blocked, cells grown on poly-L-lysine-coated coverslips (to enhance cell attachment) were incubated with drug for 20 h, fixed with 10% formalin (25°C), followed by 100% methanol containing 2 mM EGTA (4°C) (Jordan et al., 1991), and stained as described above with the
addition of mouse anti-tubulin monoclonal antibody (GTU-88; Sigma
Chemical, St. Louis, MO) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody (Sigma Chemicals). Analysis of spindle organization is more accurate using attached cells because the cells
attach with their spindle axes lying parallel to the glass surface, and
it becomes relatively easy to discern changes in numbers of
chromosomes congressed to the metaphase plate and changes in the number
of spindle poles. Because only mitotic cells were enumerated, any
differential attachment between interphase and mitotic stages was
unimportant. Stained cells were mounted on slides using Prolong
(Molecular Probes, Eugene, OR) antifade reagent. Cells were examined
using an Eclipse E800 microscope (Nikon, Melville, NY) fitted with
60× and 100× (numerical aperture 1.4 for both) objectives and
an Orca II CCD (Hamamatsu, Bridgewater, NJ) camera supported by
MetaMorph (version 4.0) imaging software (Universal Imaging Corp.,
Downington, PA).
Quantification of Intracellular Drug Accumulation.
The time
course of drug accumulation in HeLa S3 cells was determined as
described previously (Jordan and Wilson, 1999). Briefly, cells
were grown in poly-L-lysine-coated sterile scintillation vials, incubated for varying time intervals with trace amounts of
[3H]vinflunine (specific activity, 3.0 Ci/mol),
[3H]vinorelbine (3.0 Ci/mol), or
[3H]vinblastine (15.5 Ci/mol) (Amersham
Pharmacia Biotech UK, Ltd., Little Chalfont, Buckinghamshire, UK),
washed quickly with 100 mM PIPES, 1 mM EGTA, 1 mM
MgSO4, pH 6.9 (2.5 ml) after media removal, and
lysed with water (1.0 ml), and scintillation fluid (10 ml; Ready
Protein+, Beckman Coulter, Fullerton, CA). The
media at time of drug addition was sampled to determine specific
activity. Intracellular drug concentration was calculated by dividing
the total drug contained in the cells by total cell volume. Total cell
volume was determined by counting cells in a duplicate set of
scintillation vial cultures incubated with an equivalent concentration
of nonradioactive drug and using 2.4 pl as the average volume of
a HeLa cell (Jordan et al., 1991).
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of Vinflunine and Vinorelbine on HeLa Cell Proliferation
and Mitotic Progression.
The effects of vinflunine and vinorelbine
on HeLa cell proliferation were compared with those of vinblastine by
culturing cells in the absence or presence of vinflunine (1-30 nM),
vinorelbine (0.5-5 nM), or vinblastine (0.1-1.5 nM) for one cell
cycle and counting the increase in cell number 20 h later relative
to the number of cells present at the time of drug addition (Fig.
2). Vinflunine and vinorelbine inhibited
cell proliferation by 50% (IC50) at
concentrations of 18 and 1.25 nM, respectively. In comparison, the
IC50 value for vinblastine was 0.45 nM, as
reported previously (Jordan et al., 1991).
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Effects of Vinflunine and Vinorelbine on Spindle Organization. Vinflunine and vinorelbine affect microtubule dynamics very differently from vinblastine. We wanted to determine whether these differences would result in significantly different alterations in spindle organization and might be responsible for the superior antitumor activities of the two newer drugs. Thus, we compared the alterations in the arrangement of microtubules, centrosomes, and chromosomes induced by each drug at the IC30, IC50, and 2× IC50 concentrations for mitotic block, using immunofluorescence microscopy.
As shown in Fig. 4, A-C, control cells in mitosis contained well-organized bipolar spindles with two distinct, well-separated spindle poles and a few astral microtubules. At metaphase, all of the chromosomes were organized in a compact equatorial metaphase plate (Fig. 4C). A small percentage of cells (18%) contained multipolar spindles (generally tripolar or quadripolar spindles). The percentages of normal, abnormal, and multipolar spindles in controls are shown graphically in Fig. 6, A-C (unshaded bars) (discussed below).
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). Abnormal spindle Types I and II consisted of bipolar
spindles with few or many uncongressed chromosomes, respectively (see
Fig. 4, arrows). Type III consisted of monopolar spindles enclosed in a
ball of chromosomes (Figs. 4 and 5, asterisks).
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-tubulin indicated that centrosomes in
Type III spindles were frequently more fragmented after incubation with
vinorelbine and vinflunine than with vinblastine; that is, the punctate
masses of centrosomal staining were further apart from one another
within the sphere of chromosomes (data not shown; illustrated
diagrammatically in Fig. 6).
Measurement of the distance between poles of bipolar spindles after
drug incubation indicated that the pole-to-pole distance was shorter at
the IC50 concentrations of the three drugs (Table 1). Vinorelbine had the greatest effect
on the spindle length, reducing it by 29%, from 9.7 ± 0.3 µm
in controls to 6.9 ± 0.1 µm. The smaller size of spindles after
vinorelbine exposure is clearly visible in Fig. 4, G and H. The
pole-to-pole distance was reduced 20% by vinflunine (to 7.8 ± 0.3 µm) and by vinblastine (to 7.6 ± 0.1 µm). Shortening of
the spindle may be caused by net microtubule depolymerization or by
changes in the balance of dynamics between microtubule subsets in the
spindle. In support of the latter idea, the drug paclitaxel, which
enhances microtubule polymerization, also induces spindle shortening
(Jordan et al., 1993).
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-tubulin staining (Fig. 7).
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Intracellular Drug Concentration. We wanted to know whether the quantitative differences in the effects of vinflunine, vinorelbine, and vinblastine, on cell proliferation and mitotic block might result from differences in the levels of cellular drug accumulation. That is, we wanted to determine whether less vinblastine is required than vinflunine or vinorelbine to inhibit proliferation and to block mitosis because it accumulates to a higher concentration in cells. Thus HeLa cells were incubated in media containing [3H]vinflunine (30 nM), [3H] vinorelbine (3 nM), or [3H] vinblastine (1 nM) for periods of 2 to 24 h, and then the intracellular drug concentrations were measured (Materials and Methods). The concentrations added to the media were chosen to approximate the IC50 values for mitotic block (Fig. 3) aiming to induce equivalent biological effects in cells with each drug.
As shown in the time course of uptake in Fig. 8, all three drugs entered the cells gradually, reaching maximal levels within 4 h. The maximum intracellular concentrations were significantly higher than the extracellular levels in the media: 4.2 µM, 1.3 µM, and 130 nM for vinflunine, vinorelbine, and vinblastine, respectively; these represented concentrations increased by factors of 140-, 430-, and 130-fold for vinflunine, vinorelbine, and vinblastine, respectively, over those added to the media. Thus, vinblastine accumulated intracellularly to a degree similar to that of vinflunine. Therefore, the increased potency of vinblastine compared with vinflunine cannot be accounted for by preferential cellular accumulation of vinblastine. However, preferential accumulation of vinorelbine relative to vinflunine may account for at least a portion of the increased potency of vinorelbine in HeLa cells.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have found that vinflunine, vinorelbine, and vinblastine inhibit proliferation of HeLa cells in parallel with mitotic block at the metaphase/anaphase transition. At the concentrations that inhibited mitotic progression, each of the three drugs induced similarly aberrant spindle organization. Taken together, these results indicate that inhibition of cell proliferation by all three Vinca alkaloids results primarily from mitotic block induced by suppression of microtubule dynamics.
Does Inhibition of Particular Parameters of Dynamic Instability
Determine the Effectiveness of Mitotic Block?
We previously
determined the effects of equal concentrations (400 nM) of vinflunine,
vinorelbine, and vinblastine on the dynamic instability behavior of
bovine brain microtubules in vitro. Interestingly, we found that
vinflunine and vinorelbine affected dynamic instability differently,
often oppositely, from vinblastine (Ngan et al., 2000). For example,
neither vinflunine nor vinorelbine inhibited the rate of shortening
significantly, whereas vinblastine inhibited it by 44%. The duration
of growing excursions was increased 53 to 59% by vinflunine and
vinorelbine but was not altered significantly by vinblastine. The
duration of pause (attenuated dynamics) was unaffected by the two newer
drugs, whereas it was increased 60% by vinblastine. Thus vinblastine
mainly decreased the shortening rate and increased pause duration and
total time in pause phase. In contrast, vinflunine and vinorelbine
mainly decreased the growing rate, its duration, and the total time
growing while greatly decreasing the time in pause. Overall effects on
dynamicity [a measure of tubulin addition and loss from microtubule
ends (Toso et al., 1993)] were similar; all three drugs
decreased dynamicity between 21 and 32%. Another difference is that
vinflunine and vinorelbine inhibited microtubule treadmilling 1.5- to
6.4-fold less potently than vinblastine (Ngan et al., 2000).
How Does Inhibition of Microtubule Dynamics by Vinca Alkaloids Result in Aberrant Spindle Organization and Block Mitosis? Uncongressed chromosomes in the drug-treated cells are most probably those that are inaccessible to attachment by relatively nondynamic microtubules emanating from the distant spindle pole. Type III spindles, which seem collapsed with both centrosomes contained in a ball of chromosomes, may result from suppression of treadmilling/dynamic instability, leading to spindle poles not being held apart. Thus, if the treadmilling addition of tubulin at the kinetochore ends of microtubules is inhibited in the absence of balanced inhibition of loss at the opposite ends, the kinetochore microtubules would shorten, resulting in shortening of the entire spindle. Similarly, unbalanced suppression of growing and shortening dynamics of kinetochore microtubules might lead to net shortening of the spindle.
During mitosis, chromosomes that are attached in a bipolar fashion to the metaphase spindle oscillate about the spindle equator. The kinetochores of sister chromatids are under tension, are periodically stretched apart by shortening of dynamic microtubules, and then relax back together as tension is relieved. The signal to progress from metaphase to anaphase seems to involve the development of sufficient tension or the attachment of a sufficient number of microtubules to kinetochores (Hays and Salmon, 1990; Nicklas et al., 1995). Very low
concentrations of vinblastine that block the cells in metaphase inhibit
the stretching, thereby reducing the tension (J. Kelling, L.W., K. Sullivan, and M. A.J., unpublished observations). Tension on
kinetochores may also be provided by the treadmilling of kinetochore
microtubules, a process that transports tubulin subunits along the
lengths of the microtubules from their attachment at the kinetochores
toward the spindle poles (Mitchison, 1989; Waters et al., 1996). Our
results indicate that suppression of different combinations of dynamics
parameters can lead to mitotic block.
Potential Therapeutic Significance of the High Intracellular Levels
of Vinflunine and Vinorelbine.
Measurements of radiolabeled
Vinca alkaloid uptake into cells indicated that peak
intracellular drug concentrations were considerably higher than the
concentrations added to the medium. The peak concentration was highest
for vinflunine (4.2 ± 0.2 µM), almost as high for vinorelbine
(1.3 ± 0.1 µM), but more than 10-fold lower for vinblastine (130 ± 7 nM). The implications of these findings are especially interesting because similar (micromolar) concentrations of vinflunine and vinorelbine significantly inhibit polymerization in vitro (Kruczynski et al., 1998a; Ngan et al., 2000). For example, 4 µM vinflunine reduced polymerization of brain tubulin by 88%, and 1 µM vinorelbine reduced it by 60%. In contrast, 130 nM vinblastine reduced polymerization by less than 5% (Ngan et al., 2000). If all
intracellular vinflunine or vinorelbine at their
IC50 values were free to bind tubulin, one would
predict that most microtubules would be depolymerized. In contrast, the
IC50 concentration of vinblastine would have
little effect on microtubule polymer mass. However, the data in Figs. 4
and 7 indicate that with all three drugs, most microtubules remain
intact (although spindles were somewhat reduced in size in the presence
of vinorelbine). These results suggest that not all intracellular
vinflunine and vinorelbine is available to bind to tubulin or
microtubules, and thus much of the drug must be sequestered in
intracellular reservoirs, such as membrane compartments. Such
reservoirs might be important in the antitumor activity of these drugs,
providing a continuous intracellular source of drug and prolonging
their therapeutic effects. Vinflunine was previously found to diffuse
freely out of cells; however, it is not clear that the experimental
conditions accurately mimicked those in vivo (Jean-Decoster et
al.1999). We suggest that intracellular binding reservoir(s) may be
responsible for the exceptional breadth of vinflunine's therapeutic
index (Kruczynski et al., 1998b; Jacquesy and Fahy, 2000)
providing a reservoir for excess drug and enabling its gradual release, thereby prolonging its antitumor effects with associated low toxicity.
found that vinflunine has
a 3- to 16-fold lower overall affinity for tubulin than vinorelbine,
whereas vinorelbine has lower overall affinity than vinblastine under
the conditions of the experiments (Lobert et al., 1996, 1998). These
results suggest that the lower affinity of vinflunine and vinorelbine
for tubulin, may explain, at least in part, the high concentrations of
drug required to block mitosis and cell proliferation (Lobert et al.,
2000). However, they do not explain the relatively high therapeutic
efficacy of these newer Vincas.
On the basis of its binding affinity for tubulin and its potent effects
on microtubule dynamics, vinblastine would be predicted to be the most
potent mitotic inhibitor of the three Vincas alkaloids. This
prediction is borne out by the data. One might also predict that
vinblastine would be the most effective antitumor agent. However, based
on the preclinical and clinical efficacy of the three drugs, this seems
not to be the case (Kruczynski et al., 1998a,b; Hill et al., 1999).
Thus, the most potent drug is not the most efficacious, because
vinorelbine and vinflunine seem to have clear therapeutic advantages.
The reasons for this are unknown; it may be caused by other features of
their modes of action or different pharmacokinetic characteristics.
The lower potency and low tubulin binding affinity of vinflunine
correlates well with the finding that it is the least cytotoxic of the
Vinca alkaloids studied (Kruczynski et al., 1998a). The therapeutic index of a relatively weakly potent drug may be broader than that of an extremely potent drug. Thus, if a drug acts selectively on tumor tissue, a drug of low potency may be more effective than one
of high potency because of reduced toxic side effects. The balance
between efficacy and toxicity for these first- and second-generation Vinca alkaloids remains to be clarified mechanistically.
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Footnotes |
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Received February 5, 2001; Accepted April 16, 2001
This work was supported by Grants from the Institut de Recherche Pierre Fabre and National Institutes of Health (CA57291).
Dr. Mary Ann Jordan, Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106. E-mail: jordan{at}lifesci.ucsb.edu
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Abbreviations |
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PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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T. Okouneva, B. T. Hill, L. Wilson, and M. A. Jordan The Effects of Vinflunine, Vinorelbine, and Vinblastine on Centromere Dynamics Mol. Cancer Ther., May 1, 2003; 2(5): 427 - 436. [Abstract] [Full Text] [PDF]
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 J. Bennouna, P. Fumoleau, J.-P. Armand, E. Raymond, M. Campone, F.-M. Delgado, C. Puozzo, and M. Marty Phase I and pharmacokinetic study of the new vinca alkaloid vinflunine administered as a 10-min infusion every 3 weeks in patients with advanced solid tumours Ann. Onc., April 1, 2003; 14(4): 630 - 637. [Abstract] [Full Text] [PDF]
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 A. Vantieghem, Y. Xu, Z. Assefa, J. Piette, J. R. Vandenheede, W. Merlevede, P. A. M. de Witte, and P. Agostinis Phosphorylation of Bcl-2 in G2/M Phase-arrested Cells following Photodynamic Therapy with Hypericin Involves a CDK1-mediated Signal and Delays the Onset of Apoptosis J. Biol. Chem., September 27, 2002; 277(40): 37718 - 37731. [Abstract] [Full Text] [PDF]
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M. Carre, N. Andre, G. Carles, H. Borghi, L. Brichese, C. Briand, and D. Braguer Tubulin Is an Inherent Component of Mitochondrial Membranes That Interacts with the Voltage-dependent Anion Channel J. Biol. Chem., September 6, 2002; 277(37): 33664 - 33669. [Abstract] [Full Text] [PDF]
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J.-G. Chen and S. B. Horwitz Differential Mitotic Responses to Microtubule-stabilizing and -destabilizing Drugs Cancer Res., April 1, 2002; 62(7): 1935 - 1938. [Abstract] [Full Text] [PDF]
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 N. Moisoi, M. Erent, S. Whyte, S. Martin, and P. M. Bayley Calmodulin-containing substructures of the centrosomal matrix released by microtubule perturbation J. Cell Sci., January 6, 2002; 115(11): 2367 - 2379. [Abstract] [Full Text] [PDF]
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), vinorelbine (
), and vinblastine
(
). The percentage inhibition of cell proliferation is the increase
in cell number that occurred during 20 h of continuous incubation
with drug compared with the increase in cell number in a parallel
culture without drug (see Materials and Methods). The
cell number approximately doubled in control cultures during the 20-h
incubation. Results represent mean ± S.E. of three to four
independent experiments.
-tubulin
antibody to stain microtubules (A, D, G, and J) (first column), with an
anti- 
