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Tumor Biology |
ASTA Medica AG, Department of Cancer Research 60314 Frankfurt am Main, Germany [G. B., B. N., P. E., T. K., T. B.]; Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, 45112 Essen, Germany [U. V., S. S.]; and Department of Normal Physiology, Pirogov Medical Institute, Narimanov, 270100 Odessa, Ukraine [A. S.]
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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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- and ß-tubulins
polymerize to microtubules, long dynamic tubular fibers, which
constitute the mitotic spindles. Microtubule inhibitors interfere with
the microtubule dynamics of tubulin polymerization and
depolymerization, which results in the inhibition of chromosome
segregation in mitosis and consequently the inhibition of cell division
(7
, 8)
. The three major classes of tubulin-binding agents
are the taxanes, which stabilize microtubules by blocking disassembly,
the Vinca alkaloids, and the colchicine-site binders
(9)
. The latter two are microtubule-destabilizing agents
that act by blocking assembly of tubulin heterodimers. A major point is
that nondividing cells are extremely resistant toward these drugs
(10)
, whereas uncontrolled dividing tumor cells run into
cell cycle arrest by cell cycle checkpoint pathways. Subsequently
apoptosis of the cells may be initiated (11)
. Although the taxanes and the Vinca alkaloids are effective in the treatment of different malignancies, their potential is limited by the development of drug resistance (8) . One pathway leading to resistance is mediated by overexpression of transmembrane efflux pumps, namely the p-gp1704 (12) and the MRP (13) . These efflux pumps are able to reduce the intracellular concentrations of taxanes and Vinca alkaloids to a nontoxic level. Resistance is also mediated by the expression of tubulin isotypes and mutants that showed impaired taxane-driven tubulin polymerization (14) . Another major drawback of taxanes and Vinca alkaloids in clinical application is the development of neurotoxicity (15, 16, 17, 18, 19) . The drugs interfere with the function of microtubules in axons, which mediate the neuronal vesicle transport (8) . New chemical entities that bind to tubulin but neither are a substrate of transmembrane pumps nor interfere with the function of axonal microtubules would strongly increase the therapeutic index in the treatment of malignancies. In screening for a compound with these superior characteristics, a molecule named D-24851 was identified that destabilizes microtubules in tumor cells and cell-free systems. D-24851 does not interact with the tubulin-binding sites of vincristine and colchicine. Further characterization revealed that this microtubule inhibitor is not a substrate of p-gp170 nor of MRP and, consequently, retains its antitumoral efficacy in cell lines with MDR or MRP resistance phenotypes. In addition, the administration of D-24851 to rats revealed no deficit in motor function and no change in NCV, which suggested a lack of neurotoxicity of D-24851.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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General chemicals (paclitaxel, vincristine, vinblastine, and
podophyllotoxin) were purchased from Sigma (Munich, Germany).
Radiochemicals were obtained from Amersham Corp. P-glycoprotein (C219),
and -tubulin (B-5–1-2) antibodies, were purchased from Alexis
Biochemicals Corp. (Grünberg, Germany) and Sigma (Munich,
Germany), respectively. Cy3-conjugated and peroxidase-conjugated goat
antimouse antibodies were obtained from Dianova (Hamburg, Germany).
Tumor cell lines SKOV3 (ovary/human/HTB-77), KB/HeLa
(cervix/human/CCL-17), HT 29 (colon/human/HTB-38), A549
(lung/human/CCL-185), PC-3 (prostate/human/CRL-1435), DU145
(prostate/human/HTB-81), AsPC-1(pancreas/human/CRL-1682), C6
(brain/rat/CCL-107), U 87 (brain/human/HTB-14), MDA-MB 231
(breast/human/HTB-26), and L1210 (leukemia/mouse) were obtained from
ATCC. LT12 and LT12/mdr1 were a gift from Dr. K. Nooter (University
Hospital Rotterdam, Rotterdam, the Netherlands). The detailed
characteristics of human parental A2780/wt (ovarian), MCF-7/wt
(breast), HT1080 (fibrosarcoma), HCT-8 (colon), HT-29 (colon) cell
lines as well as the MDR p-gp170 overexpressing A2780/Dx5 and
MCF-7/adr, the MRP expressing MDR HT1080/Dr4 (20)
, the
cisplatin-resistant A2780/CP2, the 5-FU-resistant HT29-R1 (bolus) and
HT29-R24 (continuous exposure), the raltitrexed-resistant HT29/ICID,
cell lines have been published previously
(21, 22, 23, 24, 25)
.5
Flow Cytometry.
KB/HeLa cells (1 x 106 cells)
were exposed to the cytotoxic agents for 24 h at 37°C, and DNA
content of the cells was determined using FACS using a Calibur flow
cytometer (Becton Dickinson, Heidelberg, Germany). The Number of cells
in G2-M phase was calculated using Mod Fit LT
cell cycle analysis software (VERITY). Data points were connected and
IC50 calculated using a nonlinear regression
program (GraphPad Prism).
Indirect Immunofluorescence Microscopy.
KB/HeLa cells were incubated for 24 h with cytotoxic agents and
then extracted for 3 min with ice-cold 0.5% Triton X-100 in PHEM
containing 10 µM paclitaxel. This treatment removes
unassembled tubulin while preserving microtubules (26)
.
The cultures were then fixed by the addition of PHEM containing 8%
paraformaldehyde and 0.3% glutaraldehyde at 4°C. Microtubules were
visualized using a mouse monoclonal antibody against -tubulin
(Sigma) used at 1:1000 dilution and a Cy3-conjugated goat antimouse
antibody used at 1:1000 dilution and a cooled AT200 CCD (charge
coupled device) camera system (Photometrics Ltd., Munich, Germany).
Fluorescent images were further processed using Fluoro-Pro module for
Image-ProPlus.
Tubulin Polymerization Assay.
The assay was basically performed according to Bollag et al.
(27)
. Microtubules from calf brain (Sigma) were
depolymerized according to the manufacturer’s protocol. Tubulin
heterodimers (10 µM) were incubated with
different compounds (1 µM or as indicated) in
PEMT buffer (100 mM PIPES, 1
mM EGTA, 1 mM Mg
Cl2, and 0.05% Triton-X-100) containing 1
mM GTP in a total volume of 100 µl at
37°C for 1 h. Triton-X-100 strongly increased the solubility of
D-24851 (data not shown). Samples (75 µl) were then
transferred to a 96-well Millipore Multiscreen Durapore hydrophilic
0.22-µm pore size filtration plate, which had been previously washed
with 200 µl PEM buffer (100 mM PIPES, 1
mM EGTA, and 1 mM Mg
Cl2) under vacuum. Recovered
microtubules on the filters were stained with 50 µl of amido black
solution (0.1% naphthol blue black (Sigma), 45% methanol, and 10%
acetic acid) for 2 min. Vacuum was applied, and unbound dye was removed
by two additions of 200 µl of destaining solution (90%
methanol and 2% acetic acid). The microtubule bound dye was then
eluted by incubation with elution solution (25 mM
NaOH, 0.05 mM EDTA, and 50% ethanol) for 10 min.
The elution solution was then transferred to a 96-well plate and the
absorbance measured at 600 nm.
Spin Column Assay.
Tubulin heterodimers (3 µM) were obtained by
depolymerization of microtubules purchased from Sigma as described
above and were incubated either with 3 µM colchicine
containing [3
H]colchicine (4 x 104
dpm/nmol) in the presence of D-24851 or
podophyllotoxin (A) or with 3 µM vincristine containing
[3
H]vincristine (4 x 104
dpm/nmol) in the presence of D-24851 or
vinblastine (B) in PEMT buffer at 37°C for 1 h. Two aliquots (90
µl) of the incubation mixture (200 µl) were each loaded onto a
0.8-ml Sephadex G25 column previously equilibrated in PEMT. The columns
were then placed into 1.5-ml tubes and spun at 200 x g for 1 min, and radioactivity in the flow-through was
analyzed by scintillation counting. Radioactivity that was found in the
flow-through after centrifugation of the incubation mixture in the
absence of tubulin was taken as background. Data points were connected
and IC50 calculated using a nonlinear regression
program (GraphPad Prism).
XTT Assay.
The XTT assay (28)
was used to determine proliferation by
quantification of cellular metabolic activity. Tumor cell lines were
cultivated in microtiter plates (1 x 103
cells per well in 100 µl) and were
incubated with different concentrations of cytotoxic agents for 48 h. Subsequently, 50 µl of XTT solution (1 mg/ml XTT, 25
µM N-methyldibenzopyrazine methyl sulfate) was
added per well, and mixtures were incubated for an additional 4 h.
The amount of formazan salt was quantified in at least four replicates
by absorbance at 490 nm using a Biomek Plate Reader (Beckman).
IC50s and inhibition curves connecting the
data points were obtained using the nonlinear regression program
GraphPad Prism.
SRB Assay.
Drug sensitivity was also assessed with the SRB-assay
(29)
. Exponentially growing cells were seeded at a density
of 600-1000 cells/well in 96-well microtiter plates (Falcon, Becton
Dickinson Labware, Plymouth, United Kingdom) and allowed to attach
overnight. After 24 h, cells were exposed for 24 h to either
paclitaxel, vincristine, or D-24851, washed twice with PBS, and
incubated in drug-free medium. At four cell-doubling times after the
beginning of drug treatment, cells were fixed with trichloric acetic
acid and were washed and stained with SRB as originally described. The
absorbance was measured at 570 nm using a 96-well plate reader (340 EL
BIO Kinetics Reader, BIO-TEK Instruments Inc., Winooski, VT). The drug
concentrations that inhibited cell growth by 50%
(IC50) were determined from semilogarithmic
dose-response plots.
L1210 Mouse Leukemia Xenografts.
The MDR subline of mouse leukemia L1210 cells (L1210/VCR) was generated
by long-term adaptation in a medium with stepwise increasing
concentrations of vincristine. Expression of P-glycoprotein was
analyzed by Western blotting using monoclonal antibody C219. L1210 and
L1210/VCR cells were injected i.p. in male CD 2 F1 mice (20–25 g). The
following day, mice were treated with maximally tolerated doses of
D-24851 (150 mg/kg p.o.; d1-d4), vincristine (0.5 mg/kg
i.p.; d1-d4), paclitaxel (15 mg/kg i.p., d1-d4), or doxorubicin (1
mg/kg i.p.; d1-d4), and the ILS of the mice relative to the vehicle
group was monitored. The survival time of the treated animals in
percentage (ILS) of the vehicle-treated control group was calculated.
Yoshida AH13 Rat Sarcoma Model.
Yoshida AH13 sarcoma cells were implanted i.p. in rats. After two
passages, obtained ascites were grafted s.c. into female Sprague Dawley
rats (250–290 g). Administration of the compounds or a vehicle control
was started when mean tumor weights were approximately 0.5–1 g.
D-24851 was given on days 1–5 and 8–12 (10 mg/kg p.o.;
d1–5 x 2), paclitaxel was given on days 1–4
and 8–11(2 mg/kg i.p.; d1–4 x 2), and vincristine was
given on days 1, 4, 8, and 12 (0.6 mg/kg i.p.; d1, d4 x 2). Body and tumor weights as determined by comparison to the
size of standard weights were monitored at day 1, 3, 7, 10, 14, and so
forth. All of the studies were conducted in accordance with the local
animal ethics regulatory requirements. In accordance with the animal
ethics regulations, euthanization of tumor-bearing rats was required
when the calculated tumor weight reached 
10% of the body weight.
Coordination Test (Rota-Rod Test).
Rats (Wistar, 250–290 g) were treated with D-24851 (10 mg/kg
p.o.; d1–5 x 2), paclitaxel (2 mg/kg i.p.;
d1–4 x 2), vincristine (0.4 mg/kg i.p.; d1,
d4 x 2), or a vehicle control. Rota-rod testing was
performed at day 0, 5, and 10. The rod was set in motion at constant
speed (2 rpm), and the rats were placed into individual sections of the
apparatus. The rod was then accelerated from the rate of 2 rpm to 6
rpm. The animal’s performance was recorded as the time that had
elapsed when the rats fell off the rotating rod. The data were analyzed
using 1-way ANOVA, followed by Newmann-Keuls test as appropriate. All
of the results are expressed as mean ± SE with
n = 6 animals. All of the studies were
conducted in accordance with the local animal ethics regulatory
requirements.
NCV.
Rats were treated with the compounds as described above in the section
on "Coordination Test." At days 0, 5, and 10, the NCV was recorded
in accordance with the method as described previously
(30)
. The equipment used were the electrical stimulator
ESU-2, amplifier of biological potentials, oscillograph, and
photorecorder. The temperature in the room during the experiments was
kept at the level of 28–30°C. NCV was measured retrospectively using
the photos. The data were analyzed using 1-way ANOVA, followed by
Newmann-Keuls test as appropriate. All of the results are
expressed as mean ± SE with
n = 6 animals. All of the studies were
conducted in accordance with the local animal ethics regulatory
requirements.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was identified in a cytotoxic assay and first evaluated by
cell cycle analysis using flow cytometry. KB/HeLa cells were exposed to
different concentrations of D-24851 for 24 h. At
low concentration of D-24851 (8.5
nM), the main part of the cells were found at
G1 phase of the cell cycle as observed with the
solvent control, which indicated that the compound causes no detectable
cell cycle effect at this concentration (Fig. 1A
,
upper diagram, peak at 300). At higher concentrations of
D-24851 (850 nM), a complete
shift from G1 to G2-M phase
was observed (Fig. 1A
, lower diagram, peak at
600). Thus, D-24851 induces an accumulation of KB/HeLa
cells specifically in G2-M phase of the cell
cycle. To compare D-24851 with the known
G2-M cell cycle inhibitors vincristine and
paclitaxel, we treated KB/HeLa cells with different concentrations of
the compounds (Fig. 1A)
. The cells also completely shifted
from G1 to G2-M phase (Fig. 1A
, upper and lower diagrams,
respectively). When the percentage of cells in
G2-M phase were plotted against different
concentrations of the compounds, D-24851, vincristine,
and paclitaxel arrested the cell cycle in a concentration-dependent
manner with IC50 values of 190, 3, and 15
nM, respectively (Fig. 1B)
. When the
percentage of cells accumulated in G2-M phase
were plotted against the concentration divided by their
IC50 values of cell growth inhibition (Table 1)
, the resulting curves were nearly identical (Fig. 1B
,
inset). This indicates that the cell cycle arrest of all of
the compounds correlates with the cell growth inhibition and that
D-24851 may have a mode of action similar to that of
paclitaxel and vincristine.
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-tubulin. Fig. 2, A and B
, show solvent-treated (control) cells
with microtubules constituting the cell shape. In contrast,
microtubules were mainly found organized in mitotic spindles with
abnormal structures after D-24851 treatment (45 or 80
nM) of the cells (Fig. 2, C and D)
. The abnormal spindles had extremely long astral
microtubules and were seen monopolar or bipolar. When cells were
exposed to higher concentrations of D-24851 (400 or 450
nM), we observed fragmented mitotic spindles
(Fig. 2, E and F)
. This indicates that
D-24851 destabilizes the microtubules of mitotic
spindles in a concentration-dependent manner. Similar effects on tumor
cells were observed by exposure to vincristine (1–15
nM; Ref. 31
), a known
microtubule-destabilizing agent (Fig. 2, G–J)
. Microtubules
were also found fragmented over the entire cell (Fig. 2, I and J)
. Treatment of KB/HeLa cells with paclitaxel (3 or 15
nM) revealed mitotic cells with at least three
regions of accumulated microtubules (Fig. 2K)
. These cells
could have more than two mitotic spindles. SKOV3 cells revealed
microtubules organized in star-like structures that could represent
monopolar spindles after paclitaxel treatment (Fig. 2L)
. In contrast to D-24851 and
vincristine, fragmentation of microtubules could not be observed in
paclitaxel-treated cells. These data are consistent with the
stabilizing effect of paclitaxel on microtubules (9)
.
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, Lane 2 versus Lane 1). In
contrast, no microtubule polymers could be recovered after the addition
of vinblastine (1 µM) to the assay (Fig. 3A
, Lane 3). This is consistent with the known
destabilizing effect of vinblastine on microtubules (7
, 8)
. D-24851 also completely blocked tubulin
polymerization at a concentration of 1 µM (Fig. 3A
, Lane 4). D-25552, a
derivative of D-24851 that was not found to be active
in our initial screening assay had no effect on tubulin polymerization
(Fig. 3A
, Lane 5). These data suggest that
D-24851 binds directly to tubulin and thereby inhibits
polymerization of tubulin. To compare the activity of vincristine and
D-24851 on the inhibition of tubulin polymerization,
different concentrations of the compounds were used in the assay. As
shown in Fig. 3B
, D-24851 blocked tubulin
polymerization in a concentration-dependent manner with a
IC50 of 0.3 µM, whereas
the IC50 value of vincristine was 10-fold lower.
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. Thus, podophyllotoxin competes for the binding of
colchicine to tubulin. In contrast, when different concentrations of
D-24851 instead of podophyllotoxin were used in the
assay, no effect on binding of [3
H]colchicine
to tubulin was observed (Fig. 4A)
. Thus,
D-24851 does not bind to the colchicine-binding site of
tubulin. To test whether D-24851 might overlap with the
vincristine binding site of tubulin,
[3
H]vincristine was incubated with tubulin in
the presence of the unlabeled vincristine derivative vinblastine or
unlabeled D-24851. As shown in Fig. 4B
,
increasing concentrations of vinblastine reduced the binding of
[3
H]vincristine to tubulin. In contrast,
D-24851 was not able to compete for
[3
H]vincristine binding to tubulin up to a
concentration of 100 µM. This indicates that
D-24851 does also not bind to the vincristine-binding
site of tubulin to induce disassembly of microtubules.
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. This indicates that D-24851 inhibits
growth of various human tumor cell lines at 10- to 20-fold higher
concentrations than those of paclitaxel and vincristine.
Cytotoxic Activity of D-24851 on MDR Tumor Cell Lines.
One major mechanism of multidrug resistance is mediated by the
overexpression of the p-gp170 (8)
. The antitumoral
efficacy of D-24851 was compared with vincristine, paclitaxel,
and doxorubicin in the vincristine-selected MDR mouse leukemia cell
line L1210/VCR using the cytotoxicity assay. Overexpression of p-gp170
in L1210/VCR was confirmed by Western blot analysis using monoclonal
antibody C219 (Ref. 33
; Fig. 5A
).
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1 for
D-24851 and >56 for the other three anticancer agents (Fig. 5B
; Table 2
). Thus, L1210/VCR cells were resistant to vincristine, paclitaxel, and
doxorubicin, whereas no cross-resistance to D-24851 was
observed. In addition, three other p-gp170-overexpressing cell lines,
including the mdr1 cDNA-transfected acute myeloid leukemic rat cell
line LT12, were tested. In all cases, no cross-resistance of
D-24851 to vincristine or paclitaxel was observed
(resistance factors <2), whereas up to 1000-fold resistance to
vincristine or paclitaxel was found (Table 2)
. Thus, in contrast to
paclitaxel and vincristine, the cytotoxic efficacy of
D-24851 against tumor cells is not altered by the MDR1
phenotype. In addition, the antitumoral efficacy of
D-24851 was evaluated in resistance mediated by the MRP
and in human tumor cells with resistance to cisplatin, the
topoisomerase-I-inhibitor SN-38 (7-ethyl-10-hydroxycamptothecin), and
thymidylate synthase inhibitors (e.g., 5-FU and
raltitrexed). Taken together, D-24851 retained
unaltered cytotoxic efficacy toward all of the resistant sublines
tested (Table 2)
. To test the cytotoxic efficacy of
D-24851 toward MDR tumor cells in vivo, we
used the L1210 leukemic mouse model. L1210 or L1210/VCR cells were
implanted i.p. into mice. The mice were then treated with maximal
tolerated doses (daily-times-four schedule) of D-24851,
vincristine, paclitaxel, or doxorubicin and ILS of the mice relative to
the vehicle control group was monitored. As shown in Fig. 5C
, all of the three compounds were able to increase life
span of mice grafted with parental L1210 cells (open bars).
In contrast, only D-24851 was able to increase life
span of the mice grafted with resistant L1210/VCR cells (closed
bars). Moreover, the antitumoral efficacy of
D-24851 was equal in the L1210 and L1210/VCR mouse
model. Thus, the overexpression of P-glycoprotein in tumor cells does
not influence the antitumoral in vivo efficacy of
D-24851.
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0.5–1 g, animals were treated
with D-24851 (10 mg/kg p.o.; day 1–5 for 2
weeks) and maximal tolerated doses of paclitaxel (2 mg/kg i.p.;
d1–4 x 2; Fig. 6
B) and vincristine (0.6 mg/kg i.p.; d1, d4 x 2; Fig. 6B
). D-24851 doses of the selected schedule exerted the
maximal antitumoral efficacy and were 1% of the acute toxicity
LD50. Single D-24851 doses showed
no antitumoral efficacy. The acute toxicity LD50
of p.o.D-24851 (single doses) to rats were higher than
850 mg/kg. As shown in Fig. 6A
, D-24851
induced complete tumor remissions (cures) of animals. Curative doses of
D-24851 were well tolerated with low or no systemic
toxicity as indicated by an increase in body weight (Fig. 6B)
. Preliminary data also indicate that
D-24851 exerts no hematological toxicities(data not
shown). In contrast to D-24851, vincristine or
paclitaxel administration resulted only in a modest inhibition of tumor
growth when compared with the vehicle control group (Fig. 6A)
. The inhibition of tumor growth by vincristine or
paclitaxel could be observed only when maximal tolerated (toxic) doses
were administered coinciding with a loss of body weight >10% of the
initial body weight (Fig. 6B)
. Thus, only
D-24851 exerts potent antitumoral efficacy toward this
solid tumor with low systemic toxicity.
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Neurotoxicity.
Administration of paclitaxel and vincristine is associated with a
number of toxic side effects (16)
. One of the
dose-limiting side effects appears to be neuronal damage as elicited in
motor as well as sensory deficits (34)
. The effect of
D-24851 or vincristine on motor function of rats was tested using
a coordination test (Rota-rod testing). To this end, rats were treated
with the compounds and subsequently placed on a rotating rod. At days 5
and 10 the performance of the animal was monitored as the time that had
elapsed when the rat fell off the rod. When rats were dosed with
D-24851 (10 mg/kg p.o.; day 1–5 for 2 weeks) no significant
difference in their performance on the accelerating Rota-rod treadmill
from that of the control group was observed (Fig. 7A)
. In contrast, the performance of rats receiving paclitaxel
(2 mg/kg i.p.; d1–4 x 2) or vincristine (0.4 mg/kg
i.p.; d1, d4 x 2) strongly decreased (Fig. 7A)
. This suggests that D-24851
administration to rats revealed no deficit in motor function in
contrast to paclitaxel or vincristine at antitumoral efficacious doses.
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.
Treatment of rats with vincristine (0.6 mg/kg i.p.; d1, d4 x 2) or paclitaxel (2 mg/kg i.p.; d1–4 x 2)
resulted in a strong decrease of NCV, which has been also reported
previously (Fig. 7B
; Ref. 18
, 34
). NCV of rats
that have been treated with anticancer compounds strongly correlated
with the performance of the rats on the rotating rod as well as with
the body weight of the animals (Fig. 7C)
. These data
demonstrate that D-24851 shows no neurotoxicity at
antitumoral efficacious doses in vivo. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mode of action of the molecule as a tubulin inhibitor was shown by
indirect immunofluorescence microscopy using an antibody against
-tubulin and in a cell-free tubulin polymerization assay.
D-24851 induced accumulation of cells with condensed nuclei (data
not shown) and abnormal mitotic spindles. At higher concentrations,
fragmentation of the spindle apparatus and degradation of microtubules
were observed. The well-characterized Vinca alkaloids and
colchicine interact with different binding sites on tubulin and were
known to destabilize microtubules (7)
. In fact, exposure
of cells to vincristine also revealed fragmented mitotic spindles
similar to those shown for D-24851. Paclitaxel, known
as a microtubule stabilizing agent, did not induce fragmentation of the
spindle apparatus. This strongly suggests that D-24851
arrests cells at metaphase because of modulating microtubule stability.
The destabilizing effect of D-24851 on microtubules was also seen in a cell-free assay using purified tubulin. Polymerization of tubulin was blocked by D-24851 in a concentration-dependent manner with an IC50 of approximately 0.3 µM, which may indicate a direct interaction of D-24851 with tubulin. The substoichiometric concentrations of the compound in relation to the tubulin concentration (10 µM) are sufficient to block tubulin polymerization, similar to vincristine or other Vinca alkaloids (36) .
D-24851 is a low-molecular-weight compound that shows no structural similarities to Vinca alkaloids or colchicine and did not compete for the binding of radiolabeled vincristine or colchicine to tubulin. This suggests that D-24851 may bind to a novel binding site on tubulin that results in inhibition of tubulin polymerization.
The IC50 value of vincristine for tubulin
polymerization was 10-fold lower than that of D-24851. The
difference between both compounds in the inhibition of tubulin
polymerization was also observed in inducing cell cycle arrest and in
the inhibition of cell growth in a variety of different tumor cell
lines. This suggests that D-24851 interferes with the function of
tubulin, thereby inducing cell cycle arrest and consequently cell
growth inhibition.
In vivo, D-24851 showed a remarkable antitumoral efficacy in the Yoshida AH13 rat sarcoma model. Oral application of D-24851 induced complete tumor regressions and resulted in curative treatment of the animals. Of great importance is that, at curative doses of D-24851, no systemic toxicity in terms of body weight loss or hematological toxicities were observed in vivo. In contrast, vincristine or paclitaxel treatment at their maximal tolerated doses resulted only in a moderate inhibition of tumor growth but in significant toxicity in terms of body weight loss. These data demonstrate that D-24851 is more potent than vincristine or paclitaxel in the treatment of Yoshida AH13 tumors in vivo.
In clinical studies it has been demonstrated that cumulative doses of paclitaxel or vincristine doses are associated with development of neurotoxicity (15 , 16) . The effect of these drugs on the nervous system of rats has also been shown previously (19 , 35) . i.v. administration of Vinca alkaloids significantly impaired coordination and NCV in the nerve tail. We also observed similar effects on rats after i.p. application of paclitaxel or vincristine. On a molecular level, drug-impaired microtubule function in axons seems to be responsible for the neurotoxic effects (8) . Microtubules were found to accumulate in axons after the administration of paclitaxel, whereas Vinca alkaloids interfere with axonal transport, which induces spiralization of axonal microtubules. Although D-24851 also alters microtubule function, no neurotoxic effects on rats in terms of deficit in motor function or reduced NCV was seen at curative doses. One possible explanation for the lack of neurotoxicity of D-24851 could be that concentrations of D-24851 that are sufficient to block the cell cycle do not inhibit axonal vesicle transport. Alternatively, D-24851 may only interact with nonaxonal microtubules.
The use of cytotoxic agents is often accompanied by development of MDR tumor phenotype. A major determinant of MDR is the overexpression of drug efflux pumps, namely the p-gp170 and the MRP. The results reported herein suggest that D-24851 is a substrate neither of P-glycoprotein nor for MRP. Thus, D-24851 retains its cytotoxic activity toward MDR cells in vitro and in vivo. In contrast, paclitaxel and vincristine were shown to be actively transported by p-gp170 and, in part, by MRP. Of clinical importance is that D-24851 retains its antitumoral activity against cancer cell lines with resistance to cisplatin, the topoisomerase-I-inhibitor SN-38, and the thymidylate synthase inhibitors 5-FU and raltitrexed.
In summary, D-24851 is a novel tubulin-binding agent with significant antitumoral efficacy in vitro and in vivo. The lack of neurotoxicity and the potential in an oral formulation may provide an anticancer drug with a significant therapeutic index. Clinical Phase I trials with D-24851 will be initiated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant RA 119/17-2 from the
Deutsche Forschungsgemeinschaft (to U. V. and S. S.). 
2 To whom requests for reprints should be
addressed, at ASTA Medica AG, Department of Cancer Research-Molecular
Biology, Weismuellerstrasse 45, 60314 Frankfurt, Germany.
Phone: 49-69-4001-2413; Fax: 49-69-4001-2777.
3 The abbreviations used are: p-gp170,
Mr 170,000 P-glycoprotein; MDR,
multidrug resistant; MRP, multidrug resistance protein; NCV, nerve
conductance velocity; 5-FU, 5-fluoruracil; XTT, sodium
3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)-benzene
sulfonic acid hydrate; FACS, fluorescence-activated cell sorting; SRB,
sulforhodamine B; ILS, increased life span; GTP, guanosine
triphosphate.
4 Patent no. DE19814838, PCT int. appl. WO
9951224.
5 Experiments with HT1080/Dr4 cells were performed
in cooperation with Dr. Y. M. Rustum, Roswell Park Cancer Institute,
Buffalo, NY.
6 M. Schmidt, Y. Lu, G. Bacher, T. Beckers, J.
Mendelsohn, and Z. Fan. A CDK inhibitor-based cellular system for
identifying novel cell cycle-specific antineoplastic compounds,
submitted for publication.
Received 4/17/00. Accepted 10/24/00.
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); lower diagrams, treatment with higher
concentrations of the compounds (*). In B, percentage
of cells in G2-M phase is plotted against the
concentrations of the compounds. 
