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Vol. 60, Issue 6, 1235-1242, December 2001
Departments of Biochemistry and Molecular Biology (B.C.-A., M.C.) and Cell Biology (N.A., O.B.), Institut d'Investigacions Biomèdiques August Pi i Sunyer, Faculty of Chemistry, University of Barcelona, Barcelona, Spain; and Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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KARs, new semisynthetic antitumor bis-indole derivatives, were found to be inhibitors of tubulin polymerization with lower toxicity than vinblastine or vincristine, used in chemotherapy. Here, we compare the effect of KARs with those of vinblastine and vincristine on cell viability, cell proliferation, and cell cycle in neuroblastoma cell line (SH-SY5Y). At concentrations of the different compounds equivalent in causing 50% of inhibition of cell growth, KARs induced a complete arrest in the G2/M phase, whereas vinblastine and vincristine induced a partial arrest in both G0/G1 and G2/M. Moreover, a combination of KAR-2 and W13 (an anticalmodulin drug) qualitatively caused a similar arrest in both G0/G1 and G2/M than vinblastine. Levels of cyclin A and B1 were higher in KARs-treated cells than in vinblastine- or vincristine-treated cells. Cdc2 activity was much higher in KAR-2 than in vinblastine-treated cells, indicating a stronger mitotic arrest. The effect of KAR2 and vinblastine on microtubules network was analyzed by immunostaining with anti-tubulin antibody. Results indicated that KAR-2-induces the formation of aberrant mitotic spindles, with not apparent effect on interphase microtubules, whereas vinblastine partially destroyed interphase microtubules coexisting with normal and aberrant mitotic spindles.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimitotic
drugs are widely used in chemotherapy. They usually target the
tubulin/microtubule network of the cytoskeleton, which is formed by an
assembly of cytoplasmic tubulin dimers. Several synthetic and natural
compounds interact specifically with tubulin and microtubules,
fundamentally destroying their dynamic character and leading to cell
death. A large number of these agents are plant-derived (Lin et al.,
1988
). Two Vinca alkaloids from Catharanthus roseus,
vinblastine, and vincristine are widely used in cancer therapy
(Rowinsky and Donehower, 1997). Both drugs inhibit the self-assembly of
tubulin into microtubules at substoichiometric concentrations by
forming a tubulin-drug complex at the end of a growing microtubule and
thus blocking self-assembly (Wilson et al., 1976; Margolis et al.,
1980). In addition, it is known that vinblastine induces
G2/M arrest and subsequent apoptosis in different
cell lines (Fan et al., 2001). Although the primary target of these
Vinca alkaloids is the microtubular network, we recently reported that
they also bind to calmodulin and suspend its modulating effect
(Molnár et al., 1995; Vértessy et al., 1998). Calmodulin is
a ubiquitous Ca2+ receptor that is involved in
cell proliferation and in the regulation of the cell cycle (Rasmussen
and Means, 1989; Lu and Means, 1993), but also in other essential cell
processes (Cohen and Kee, 1988). The addition of specific
anticalmodulin drugs, such as W13, to cell cultures inhibits re-entry
of growth-arrested cells into the cell cycle
(G0/G1 transition),
progression into and through the S phase, and entry and exit from
mitosis (Sasaki and Hidaka, 1982; Chafouleas et al., 1984; Agell et
al., 1998).
Numerous semisynthetic derivatives of Vinca alkaloids have been
synthesized, in response to the extensive need for potent antimitotic
agents in clinical chemotherapy, because vinblastine and vincristine
have undesired side effects. We recently reported (Orosz et al., 1997a;
Vértessy et al., 1998) that the new semisynthetic bis-indole
derivative KAR-2, has high anti-microtubular and anti-tumoral activities and lower toxicity than the Vinca alkaloids used in chemotherapy. Moreover, it interacts with calmodulin in vitro but, in
contrast to vinblastine and vincristine, it does not exhibit anticalmodulin activity in vitro enzymatic assays (e.g.,
phosphofructokinase) (Orosz et al., 1997a,b; Orosz et al., 1999). In
addition, we reported the synthesis of other bis-indole derivatives,
KAR-3 and KAR-4, which are analogs of vinblastine and vincristine,
respectively (see Fig. 1). These
compounds are also powerful antimicrotubular agents with lower
anticalmodulin activity and toxicity than vinblastine or vincristine,
although they are more toxic and no more powerful than KAR-2. (Orosz et
al., 1999).
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In vertebrates, cell cycle is regulated by a family of Cdks formed by a
catalytic subunit and a regulatory subunit called cyclin. Those kinases
act sequentially during the cell cycle. Cdk4,6/cyclin D and Cdk2/cyclin
E are essential during G1 and G1/S transition, Cdk2/cyclin A is active during S
phase whereas Cdc2/cyclin A and Cdc2/cyclin B act during mitosis. Cylin
A accumulates during S and G2 phases and cyclin B
during G2 phase. During G2 both cyclins bind to Cdc2. At G2/M transition the
complexes formed are suddenly activated by the Cdc25 phosphatase, which
eliminates two inhibitory phosphates from the catalytic subunit. Cdc2
is responsible for nuclear lamina phosphorylation; consequently, nuclear envelope is disorganized during prophase. Cdc2 also
phosphorylates histone H1 leading to chromatin condensation, which
occurs at mitosis (Norbury and Nurse, 1992; Reed, 1992; Sherr, 1994;
Morgan, 1997). Degradation of cyclin A and cyclin B during metaphase
and anaphase, respectively, leads to Cdc2 inactivation and consequently to mitosis exit. When mitotic spindle is incorrectly formed, a checkpoint is activated that inhibits Cdc2 inactivation; consequently, mitosis exit and cell cycle progression. This checkpoint usually works
by inhibiting degradation of mitotic cyclins A and B (Gong et al.,
1995; Rudner and Murray, 1996). The objective of the present article is
to characterize the effect of KARs at cellular levels compared with
that of vinblastine and vincristine to asses the origin of the distinct
behaviors manifesting in in vitro and in vivo conditions.
The fact that KARs are powerful antimicrotubular agents with lower
anticalmodulin activity and toxicity than vinblastine or vincristine
makes them promising antitumoral agents, especially KAR-2, which is the
less toxic (Orosz et al., 1999). In this article, we compare the
effects of KAR2, with those of some other bis-indole derivatives, on
human neuroblastoma cell line (SH-SY5Y) at different levels. Analysis
of DNA content by flow cytometry showed that treatment with vinblastine
and vincristine caused a partial arrest in both
G0/G1 and G2/M
phases, whereas KAR-2 specifically arrested cell cycle at
G2/M phase. Analysis of chromosome condensation and phospho-H3 (Hendzel et al., 1997), cyclin A and cyclin B1 expression levels, Cdc2 activity and microtubular network, showed that
KAR-2-treated cells were arrested in mitosis with a majority of
abnormal mitotic spindles. Interestingly, KAR-2 had no effect on
interphase microtubules, whereas vinblastine partially destroyed the
microtubular network of interphase cells on neuroblastoma cell line.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Culture. Human neuroblastoma SH-SY5Y cells (a generous gift from Dr. Jacint Boix, University of Lleida, Spain) were cultured in Dulbecco's modified Eagle's medium (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal calf serum (Biological Industries), and antibiotics: 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were grown at 37°C under an atmosphere of 5% CO2. SH-SY-5Y cells were seeded in six-well plates, 4 × 105 cells/well, for all the experiments except where indicated. The final volume in all the dishes was 2 ml. Compounds to be tested were added to cultures 1 day after seeding to ensure uniform attachment of cells at the beginning of the experiments. The cell line used in this study was free of mycoplasma infection as shown by Gen-Probe Mycoplasma Tissue Culture NI Rapid Detection System (Fisher Scientific, Pittsburgh, PA)
Bis-Indole Derivative Treatment.
The final concentrations of
compounds used were: KAR-2 at 316 nM, KAR-3 at 316 nM, KAR-4 at 355 nM,
vincristine at 14 nM, and vinblastine at 5 nM. These concentrations of
each compound have been determined by microculture tetrazolium assay as
equivalent in causing 50% of inhibition of cell growth in a previous
study in 72 h (Orosz et al., 1999). Stocks were dissolved in
phosphate-buffered saline (PBS) at 1 mg/ml for each compound and stored
at 
20°C. On the day of the experiment, compounds were diluted in
culture medium. .
Inhibition of Cell Growth and Cell Cycle Analysis. Plasma membrane integrity was estimated by FACS analysis using two different methods. In the PI (Sigma Co, St. Louis, MO) staining method without cell permeabilization, cells were incubated with 18 µg/ml PI and 5 µg/ml RNase (Roche Molecular Biochemicals, Mannheim, Germany). The fluorescence of cells was analyzed by flow cytometry using an Epics XL flow cytometers (Beckman Coulter, Fullerton, CA). The second methods was FITC-Annexin V/PI double staining (Genzime, Cambridge, MA) (see Assessment of Apoptosis).
Neuroblastoma cells were harvested and stained in Tris-buffered saline containing 50 µg/ml PI, 10 µg/ml ribonuclease A (Sigma), and 0.1% Igepal CA-630 (Sigma) for 1 h at 4°C. DNA content was analyzed by FACS. Data from 12,000 cells were collected and analyzed using Multicycle program (Phoenix Flow Systems, San Diego, CA). All experiments were performed in triplicate All statistical analysis was done using the Mathematica 3.0 program (Wolfram Research, Inc., Champaign, IL). We applied the parametric, unpaired, two-tailed independent sample t test with 95% confidence intervals [µ ± 2.58 (S.D.)], and p < 0.05 (*) was considered to indicate a significant difference.Assessment of Apoptosis. Cells were seeded in six-well plates, 25 × 103 cells/well and, after a pre-equilibration period of 24 h, they were exposed to the bis-indole derivatives to be tested for a period of 24 or 48 h. Double-staining for FITC-Annexin V binding and for cellular DNA using PI was performed according the product insert. Cells were processed by flow cytometry. Approximately 3 × 104 cells were measured for each histogram.
Gel Electrophoresis, Immunoblotting, Immunoprecipitation, and
Protein Kinase Assay.
Cyclin A and cyclin B1 were analyzed
using electrophoresis and immunoblotting, as described previously
(Taulés et al., 1998). Monoclonal antibodies against cyclin A
(Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:100 dilution,
antibodies against cyclin B1 (Upstate Biotechnology, Lake Placid, NY)
at 1:500 dilution. As a control of protein loading the blot membrane
was also hybridized with polyclonal anti-Cdk4 at 1:500 dilution. The
reaction was visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium (Promega, Madison, WI). The experiment was done at 24 h of incubation with the tested compounds. To determine Cdc2 activity levels, immunoprecipitations were performed as described previously (Taulés et al., 1998), except that 3 µg of anti-Cdc2 antibodies (Upstate Biotechnology) were used and 2 µg of histone H1
was used as substrate.
Immunocytochemistry.
Cells were grown on glass coverslips
coated with poly(D-lysine) (50 µg/ml at final
concentration) (Sigma). For double-labeling, anti-
-tubulin (Roche)
and PI were fixed in cold methanol for 2 min. After three washes in
sterile PBS, cells were incubated 1 h at room temperature in a
humidified atmosphere, with the specific monoclonal antibody
anti--tubulin (1:50 dilution) with 1% ovalbumin. Coverslips were
then washed three times (5 min each) in PBS and incubated for 1 h
at room temperature in the dark with FITC-coupled anti-mouse-IgG
secondary antibody (1:100 dilution; Promega). After three washes in
PBS, RNase was added at a final concentration of 100 µl/ml for 30 min
at room temperature in the dark and DNA stained with PI (1 µg/ml) for
1 min at room temperature in the dark. Cells were washed once in 0.1%
PBS with Triton X-100 and twice in PBS. Finally, coverslips were
mounted on glass slides with mowiol (Calbiochem, San Diego, CA).
-phospho-H3 (at a final concentration of 5 µg/ml; Upstate Biotechnology) with 1% bovine serum albumin.
Coverslips were then washed three times (5 min each) in PBS and
incubated for 1 h at room temperature in the dark with
FITC-coupled goat anti-rabbit IgG secondary antibody (1:100 dilution;
Sigma). After three washes in PBS, RNase was added at a final
concentration of 100 µl/ml for 30 min at room temperature in the dark
and DNA stained with PI (1 µg/ml) for 3 min at room temperature in
the dark. Finally, coverslips were mounted on glass slides with Immuno
Fluore mounting medium (ICN Biomedicals, Costa Mesa, CA). Cells
were visualized using a laser scanning cytometer (LSC;
CompuCyte, Cambridge, Massachusetts).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibition of the Cell Growth by the New Vinca Alkaloids,
KARs.
Cell cultures with the same number of cells were treated
separately with vincristine (14 nM), vinblastine (5 nM), KAR-2 (316 nM), KAR-3 (316 nM), and KAR-4 (355 nM). These concentrations caused
50% inhibition of cell growth as determined by microculture tetrazolium assay (Orosz et al., 1999). The number of cells was counted
at different times (8, 24, 48, and 72 h) after treatment and
plotted against the percentage of initial number of cells (0 h). It was
observed that all the compounds induced a similar time-dependent
decrease in cell growth (Fig. 2). The
effects of all these compounds became significant 24 h after the
beginning of the treatment. Between 24 and 48 h, the decrease in
cell growth was approximately 50%, and less than 13% of the cells
survived after 72 h (Fig. 2). These results corroborate that the
different concentrations of each compound selected for this study cause an equivalent mortality of cells at different times. Thus, these concentrations (which we will refer to as IC50
values) are suitable for the comparative studies reported in the
following sections.
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Treatment with KAR-2, KAR-3 and KAR-4 Completely Arrested
Neuroblastoma Cells in G2/M Phase of the Cell Cycle, with a
Low Percentage of Apoptosis.
The experiment was performed with
asynchronously growing cells. In this population (0 h), most cells were
in G1 phase (48.7% ± 0.8%) or S phase (41.4% ± 1.1%), whereas 9.9% ± 0.5% were in the
G2/M phase (n = 37; mean ± S.E.M). To examine the effects of the cytotoxic agents at their
IC50 values on cell cycle distribution, neuroblastoma cells, treated with each compound for 8, 24, and 48 h, were analyzed by FACS (Fig. 3).
Treatment with vincristine or vinblastine led to a different pattern of
cell cycle distribution than treatment with KAR-2, KAR-3, or KAR-4.
After 8 h of treatment, KAR-2, KAR-3, and KAR-4, similarly to
vincristine and vinblastine, induced a small but significant decrease
in the percentage of cells in G1 phase and a
small increase in percentage of cells in G2/M and
S phases with respect to nontreated cells. At 24 h of treatment
with KARs, 65% of treated cells were in G2/M
phase, whereas less than 8% of cells remained in
G1. The percentage of cells in S phase increased
with respect to control cells, except in the case of KAR-2-treated
cells, which were not significantly different from control cells. In
contrast, when cells were treated 24 h with vincristine or
vinblastine, less than 50% of cells were in G2/M
and 36% of cells remained in G1. The percentage
of cells in S phase decreased for vinblastine and remained
statistically unchanged with respect to control for vincristine.
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). To test this hypothesis, we treated cells with the anticalmodulin drug W13 (30 µM) alone and in combination with KAR-2
(316 nM), and we compared the effects on cell cycle distribution with
those observed after treatments with vinblastine (5 nM) or KAR-2 (316 nM). It has been described previously that W13 inhibits the re-entry of
growth-arrested cells into the cell cycle
(G0/G1 transition)
(Rasmussen and Means, 1989); the progression into and through S phase
(Chafouleas et al., 1982; López-Girona et al., 1992; Taulés
et al., 1998), the initiation of mitosis (G2/M) transition) (Patel et
al., 1999) and mitosis exit (Lorca et al., 1993). We observed a similar
arrest on G1 and G2/M after
treatment with vinblastine and with a combination of W13 and KAR-2. In
contrast, KAR-2 alone arrested mainly in G2/M
phase, whereas W13 alone caused a typical anticalmodulin effect, a
partial stop in G1/G0, S,
and G2/M (Fig. 4).
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Treatment with KARs Induced an Increase of the Number of Cells with Condensed Chromosomes and with Phospho-H3. To determine whether cells were stopped in G2 or in mitosis after treatments, chromatin was visualized by Hoechst staining. Results showed that only 3.4% ± 0.6% of control cells had condensed chromosomes. After 24 h of treatment with the compounds, cells with condensed chromosomes were the following: 46 ± 6% after KAR-3, 41 ± 2% after KAR-2, 36 ± 5% after KAR-4, 34 ± 3% after vincristine, and 18 ± 2% after vinblastine. To confirm that the cells were really stopped in mitosis after the different treatment, we analyzed phosphorylation of H3 with a specific antibody. H3 is phosphorylated at the beginning of metaphase and until telophase. Thus, it can be used as a mitotic marker. After 24 h, phosphorylated H3-positive cells were the following: 10.7 ± 3% without treatment, 46.4 ± 7% after KAR-2, 16.3 ± 1%. These results correlated with the condensed chromosome quantification shown above. These percentages correlate with the results of cell cycle analysis. However, it should be taken into account that the percentage of cells with phospho-H3 or condensed chromosomes is always lower than the percentage of cells arrested in G2/M (by FACS), because the cells in G2 do not have condensed chromosomes or phosphorylated H3. Thus, KARs induced a strong arrest of cells in mitosis, which is consistent with their inhibitory effect on tubulin polymerization and mitotic spindle formation.
Effect of KARs on the Levels of Cyclins A and B1 and Cdc2.
In
cells with normal spindle checkpoint, failure of correct mitotic
spindle formation should prevent mitotic exit by inhibition of cyclin A
and B1 degradation, and thus inhibition of Cdc2 inactivation. Consequently, we analyzed the levels of cyclins A and B1 and activity of Cdc2 after the various treatments. Western blot analysis showed that
all bis-indole derivative-treated cells had higher levels of cyclin A
than nontreated cells. Furthermore, the levels of cyclin A were
slightly higher after KARs treatment compared with vinblastine or
vincristine (Fig. 5.1). Cyclin B1 levels
were similar in vinblastine- and vincristine-treated cells than in
control, whereas the levels in KAR-2-, KAR-3-, and KAR-4-treated cells were higher. Furthermore, we found that Cdc2 activity was higher in
Kar-2 or vinblastine-treated cells than in nontreated cells. The
activity after Kar-2 treatment was double that after vinblastine treatment (Fig. 5, 2). These results indicate that the arrest induced
by KAR-2 occurred before anaphase-telophase transition and that this
arrest was stronger in KAR-2- than in vinblastine-treated cells.
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KAR-2 Induced Aberrant Spindle Formation but Did Not Affect
Interphase Microtubules.
To further characterize the mechanisms
associated with KAR-2-induced growth arrest, we examined the effects
of KAR-2 at concentrations of 316 nM and 5 nM on spindle and
microtubules behavior. The effects were compared with those induced by
vinblastine at 5 nM and 316 nM. Immunostaining with anti--tubulin
antibody revealed marked differences between the control and
vinblastine or KAR-2 treated cells (Fig.
6). Mitotic spindles were normal in
untreated cells. As shown above, in cells treated with vinblastine at 5 nM, the number of cells with condensed chromosomes and phospho-H3 were higher than in untreated cells. Although some of them had mitotic spindle with normal appearance (approximately 30% of mitosis was normal), the microtubule network of most of the interphasic cells was
severely damaged. In contrast, KAR-2-treated cells (at 316 nM) had a
high number of aberrant mitotic spindles (less than 10% of mitosis
were normal), whereas the interphase cells had an apparently normal
microtubular network (Fig. 6). KAR-2, at a concentration of 5 nM, had
no apparent effect on either the interphase or mitotic microtubules. In
contrast, vinblastine, at a concentration of 316 nM induced complete
destruction of the microtubular network of interphase cells (data not
shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bis-indole derivatives extracted from Catharanthus
roseus such as vincristine and vinblastine have been used
extensively as antimitotic drugs in cancer chemotherapy since the
1960s. We reported the synthesis and characterization of novel
bis-indole compounds, KAR-2, KAR-3, and KAR-4, which are semisynthetic
derivatives of bis-indoles occurring in Catharanthus roseus
extract in relatively large amounts (Orosz et al., 1997a,b, 1999). The
most promising candidate to be used as an antitumoral drug was KAR-2,
which has similar or even higher anti--tubulin and antitumor
activity compared with other bis-indoles but significantly lower
toxicity (Orosz et al., 1997b, 1999). We have recently documented that
although their antimicrotubular activities in in vitro systems are
similar, KAR-2 and vinblastine display distinct effects in
neuroblastoma and other cell lines (primary brain cells, PC12, Ehrlich
ascites tumor cells) (Orosz et al., 1999) and in mice hosting different tumor cells. Concerning the in vivo effect of KAR derivatives versus
vinblastine and vincristine in tumor cells, we have also demonstrated
that maximal cytotoxic activities of KARs in mice hosting leukemia P388
or Ehrlich ascites tumor cells are similar to those of vinblastine and
vincristine; however, significant prolongation of life span could be
reached with KAR derivatives after the administration of a single dose
(Orosz et al., 1999). The single dose administration is an important
issue in relation to the multidrug resistance problem known in the case
of vinblastine and vincristine. In addition, the KAR-2 administration
did not induce neurotoxic side effects (e.g., paralysis of bladder or lower extremities) as observed with bis-indoles routinely used in
therapy. In this study, we further characterized the effects of KAR-2
and its parent compound in cultured neuroblastoma cell line as a model
to better understand the cellular mechanism of KARs compared with
vinblastine and vincristine.
FACS analysis showed that KARs completely inhibit cell cycle
progression in G2/M phases, in contrast to
vincristine and vinblastine, which caused partial arrest in
G0/G1 and
G2/M phases. The results obtained on cell cycle
arrest with vincristine and vinblastine are consistent with those
obtained with these drugs in other cell lines (Jordan et al., 1992;
Wilson and Jordan, 1994; Molnár et al., 1995; Petru et al.,
1995). It has been reported that calmodulin participates in the
regulation of the G0/G1
transition (Chafouleas et al., 1984), the progression into and through
S phase (Sasaki and Hidaka, 1982; Chafouleas et al., 1982;
López-Girona et al., 1992), and the initiation and the exit of
mitosis (Chafouleas et al., 1982). Accordingly, we suggest that these
differences with KARs could be explained, taking into account that
vinblastine and vincristine present anticalmodulin activity and that
KAR-2 binds to a different site on calmodulin than vinblastine
(Vértessy et al., 1998) and thus does not display anticalmodulin
activity (Orosz et al., 1997a). Moreover, cell cycle perturbations
observed when we treated neuroblastoma with a mixture of KAR-2 and W13 (a calmodulin inhibitor) were similar to those obtained with
vinblastine alone. Although these results agree with our hypothesis
that the effect of vinblastine on the cell cycle is a result of the
combination of its antimicrotubular activity and its anticalmodulin
activity, other possibilities could be considered to explain
differences between vinblastine and KAR-2.
Because exposure of cells to KARs leads to higher levels of cyclin A
and B1 than exposure of cells to vinblastine and vincristine, we
concluded that KARs caused stronger mitotic arrest and seem to be more
specific antimitotic agents than vinblastine or vincristine. This is
corroborated by the fact that Cdc2 activity doubled in KAR-2-treated
cells with respect to vinblastine-treated cells. KARs do not display
polyploidy at 72 h, which is a desirable characteristic for a drug
to be used in antitumor chemotherapy and also indicates that
neuroblastoma cells activate the mitotic spindle checkpoint in response
to KARs treatment. The cell cycle phase arrest specificity of antitumor
drugs is important in oncology in developing clinical treatment
protocols and designing antitumor strategies involving specific drug
combinations. For instance, Stone et al. (1996) demonstrated that drugs
that induce overexpression of p16 make normal cells more resistant than
cancer cells to antimitotic drugs, because normal cells respond to p16
overexpression reversibly by arresting at G1,
whereas in many tumors, the p16 regulatory pathway is inactivated and
thus cells progress to mitosis, where they become susceptible to
antimitotic drugs. Testing several anticancer agents, they concluded
that the most dramatic effect was observed with vinblastine and
suggested that other agents more specifically directed against the
G2 or M phases than vinblastine might be more
effective (Stone et al., 1996). From this point of view, the new KAR
family of Vinca-alkaloids could be an excellent candidate for this
therapeutic approach; this will be one of the objectives of our future
studies with these drugs. The ability of KAR-2 to induce the formation
of aberrant mitotic cells without any apparent destruction of the
microtubular network of interphasic cells at their
IC50 level is in marked contrast to its mother compound vinblastine. These differences can be very important in a
possible selective effect of KAR-2 against dividing cells, which is a
desirable characteristic in any antitumoral drug. These differences can
also be related to the lack of anticalmodulin activity of KAR-2; it has
been reported that some of the microtubule-associated proteins are
calmodulin-binding proteins (Ortega-Perez et al., 1994; Gonzalez et
al., 1995).
We found that KARs, like vinblastine and vincristine, cause apoptosis
rather than necrosis in human neuroblastoma cell line SH-SY5Y. However,
the percentage of apoptotic cells was always low, which is consistent
with the percentages of apoptosis after vincristine treatments reported
in the literature for other cell lines (Tsurusawa et al., 1997). This
finding is consistent with the higher percentages of mitotic cells
after treatment of these compounds and with the evidence that cells may
undergo apoptosis at any phase except mitosis (Pittman et al., 1994).
In addition, it has been suggested that tubulin reorganization may play
a role in the apoptotic process (Cotter et al., 1992).
Thus, the combination of the three differential characteristics of KAR-2 with regard its parental compounds vinblastine and vincristine, the fact that it completely arrests cell cycle at mitosis, and its capacity to generate aberrant mitosis without affecting interphase microtubules makes this compound particularly interesting from a pharmaceutical point of view.
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Acknowledgments |
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We are grateful to Dr. Albert Sorribas (Statistical Department, University of Lleida, Barcelona, Spain), Dr. Jaume Comes and Ricard Alvarez (flow cytometry, Serveis Cientifico-Tècnics, University of Barcelona), Dr. Mireia Dalmau (flow cytometry, Hospital de Bellvitge, Barcelona, Spain) and Anna Bosch (Confocal Microscopy, Serveis Cientifico-Tècnics, University of Barcelona, Campus Medicina, Consorci Institut d'Investigacions Biomèdiques August Pi i Sunyer) for their excellent technical assistance.
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Footnotes |
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Received August 27, 2001; Accepted September 10, 2001
This study was supported by Dirección General de Investigación Científica y Tecnológica Grants BIO98-0365, FISS 00/1120, and SAF98/0014), by European Commission Grant INCO-COPERNICUS (ERBIC 15CT960307), and by Hungarian National Research Foundation Grant T-31892.
Dr. Marta Cascante. Departament of Biochemistry and Molecular Biology. Institut d'Investigacions Biomèdiques August Pi i Sunyer. University of Barcelona. Facultat de Química. C/Martí i Franquès,1. Barcelona. E-08028 Spain. E-mail: marta{at}sun.bq.ub.es
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Abbreviations |
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KAR-2, 3'-(-chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxy-vinblastine;
KAR-3, 3'-(-chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvincristine;
KAR-4, 3'-allyl-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxy-vinblastine;
PBS, Dulbecco's phosphate-buffered saline;
FACS, fluorescence-activated cell sorting;
PI, propidium iodide;
FITC, fluorescein isothiocyanate;
W13, N-[4-aminobutiyl]-5-chloro-2-naphthalenesulfonamide;
Cdk, cyclin dependent kinase.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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activity during proliferative activation of NRK cells.
Biochem Biophys Res Commun
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; KAR-2, 316 nM (
); KAR-3, 316 nM (
);
KAR-4, 355 nM (
); VBL, 5 nM (
); VRC, 14 nM (
). A, control and
KAR-2. B, control and KAR-3. C, control and KAR-4. D, control,
vincristine and vinblastine.
), necrotic cells (annexin
V+/PI+) include late apoptotic cells. 30,000 cells were counted. Data are shown as the mean value ± S.E.M. (Annexin
V-AV).
