Published OnlineFirst July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0067
Molecular
Cancer
Therapeutics
Therapeutic Discovery
Inhibition of Dynamin by Dynole 34-2 Induces Cell Death
following Cytokinesis Failure in Cancer Cells
Megan Chircop1, Swetha Perera1, Anna Mariana1, Hui Lau4, Maggie P.C. Ma1, Jayne Gilbert2,
Nigel C. Jones5, Christopher P. Gordon3, Kelly A. Young3, Andrew Morokoff4, Jennette Sakoff2,
Terence J. O’Brien5, Adam McCluskey3, and Phillip J. Robinson1
Abstract
Inhibitors of mitotic proteins such as Aurora kinase and polo-like kinase have shown promise in preclinical
or early clinical development for cancer treatment. We have reported that the MiTMAB class of dynamin small
molecule inhibitors are new antimitotic agents with a novel mechanism of action, blocking cytokinesis. Here,
we examined 5 of the most potent of a new series of dynamin GTPase inhibitors called dynoles. They all
induced cytokinesis failure at the point of abscission, consistent with inhibition of dynamin while not affecting
other cell cycle stages. All 5 dynoles inhibited cell proliferation (MTT and colony formation assays) in 11
cancer cell lines. The most potent GTPase inhibitor, dynole 34-2, also induced apoptosis, as revealed by cell
blebbing, DNA fragmentation, and PARP cleavage. Cell death was induced specifically following cytokinesis
failure, suggesting that dynole 34-2 selectively targets dividing cells. Dividing HeLa cells were more sensitive
to the antiproliferative properties of all 5 dynoles compared with nondividing cells, and nontumorigenic
fibroblasts were less sensitive to cell death induced by dynole 34-2. Thus, the dynoles are a second class of
dynamin GTPase inhibitors, with dynole 34-2 as the lead compound, that are novel antimitotic compounds
acting specifically at the abscission stage. Mol Cancer Ther; 10(9); 1553–62. 2011 AACR.
Introduction
Several small molecule inhibitors targeting mitotic
proteins have shown promise in preclinical or early
clinical development as cancer treatments (1). These
mostly act at early mitotic stages, primarily disrupting
metaphase–anaphase transition (and spindle assembly
checkpoint; SAC) by targeting cyclin-dependent kinase,
Aurora kinase, polo-like kinase (Plk), or kinesin spindle
protein (1, 2). These new mitotic inhibitors prevent proliferation of most tumor cells in vitro and reduce tumor
volume in vivo by inhibiting growth and/or triggering
cell death following an aberrant mitotic event leading to
aneuploidy (1, 2). Such compounds are expected to have a
Authors' Affiliations: 1Children's Medical Research Institute, The University of Sydney, 214 Hawkesbury Road, Westmead; 2Department of
Medical Oncology, Newcastle Mater Misericordiae Hospital, Edith Street,
Waratah; 3Chemistry, School of Environmental & Life Sciences, The
University of Newcastle, Callaghan, New South Wales; and Departments
of 4Surgery and 5Medicine, Royal Melbourne Hospital, University of
Melbourne, Parkville, Victoria, Australia
Note: Supplementary material for this article is available at Molecular
Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Corresponding Author: Megan Chircop, Children's Medical Research
Institute, The University of Sydney, Locked Bag 23, Wentworthville, NSW
2145, Australia. Phone: 61-2-9687-2800; Fax: 61-2-9687-2120; E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-11-0067
2011 American Association for Cancer Research.
more favorable therapeutic window than currently used
chemotherapeutic agents, for example, paclitaxel (1), as
they would spare nondividing, nondifferentiating cells.
The endocytic protein, dynamin II (dynII), may also be a
novel mitotic target for development of selective anticancer drugs, because dynII exclusively functions during
the abscission stage of cytokinesis and is not required for
progression through any other cell cycle phase (3). Dynamin inhibitors tested to date exclusively block cytokinesis
(3), a cell cycle stage not yet targeted for chemotherapeutic intervention.
We recently reported the anticancer properties of dynamin inhibitors within the long chain amines and ammonium salts series (MiTMAB; refs. 3–5). dynII is best
known for its role in membrane trafficking processes,
specifically in clathrin-mediated endocytosis (6–8).
The MiTMABs inhibit dynamin-dependent receptormediated endocytosis (RME; refs. 5, 9). Consistent with
a role for dynamin in cytokinesis (3, 6, 7, 10–14), the
MiTMABs induce cytokinesis failure in synchronized
HeLa cells, specifically blocking abscission (3). Unlike
the Aurora kinase and Plk inhibitors, dynamin inhibitors
tested so far do not affect progression through any other
mitosis stage. Like Aurora kinases, Plk or kinesin spindle
protein inhibitors (1), MiTMABs have antiproliferative
and cytotoxicity properties that seem to be selective for
cancer cells (3).
Several other small molecule inhibitors of dynamin
have been developed by our group and others, including
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Chircop et al.
dimeric tyrphostins (15), dynoles (16), iminodyns (17),
pthaladyns (18), and dynasore (19). These inhibit dynamin GTPase activity in vitro with a broad range of IC50
values. The MiTMABs inhibit dynamin GTPase activity
by targeting its pleckstrin homology domain, a module
common to many proteins (4, 5). The dynoles are thought
to bind an allosteric site in the GTPase domain (16). Thus,
dynoles would be expected to be more efficacious and
have less off-target effects than MiTMABs. We aimed to
determine if the dynoles also have antiproliferative and
cytotoxicity properties amenable for cancer treatment.
We evaluated the 5 most potent dynoles for their effects
on dividing cells. We show that the most potent inhibitor
of dynamin-mediated RME (16) is also a potent inhibitor
of cytokinesis in cells. Cell death follows cytokinesis
failure in dynole 34-2–treated cells. The results strengthen
the view that dynamin is a new therapeutic target for the
possible treatment of cancer.
Materials and Methods
Cell culture
HeLa and H460 cells were maintained in RPMI 1640
medium supplemented with 10% FBS and 5% penicillin/streptomycin. Other cell lines were maintained in
Dulbecco’s Modified Eagle’s Media supplemented with
10% FBS and 5% penicillin/streptomycin. All cells were
grown at 37 C in a humidified 5% CO2 atmosphere.
HeLa, HT29, SW480, MCF-7, A2780, H460, A431,
DU145, BE2-C, and SMA-560 originated from the American Type Culture Collection. SJ-G2 was gifted to us
from Dr. Mary Danks (St Jude Children’s Research
Hospital, Memphis, TN). All cell lines are of human
origin except for SMA-560, which is of murine origin.
Cell line authentication was not carried out by the
authors within the last 6 months.
Cell synchronization and treatment with dynamin
inhibitors
The selective cyclin-dependent kinase 1 small molecule inhibitor, RO-3306 (9 mmol/L; 20), was used to
accumulate cells at the G2–M boundary and this was
followed by RO-3306 washout to allow synchronous
mitotic progression, as previously described (21). Immediately following RO-3306 removal (i.e., release from the
G2–M boundary), cells were treated with a dynamin
inhibitor, drug-free medium, or 0.1% dimethyl sulfoxide
(DMSO) vehicle.
Time-lapse microscopy analysis
The indicated dynamin inhibitor was added to G2–M
synchronized HeLa cells immediately following release
from the RO-3306 synchronization block. Cells were
viewed with an Olympus IX80 inverted microscope,
and a time-lapse series was acquired using a fully motorized stage, 10 objective, and Metamorph software by
using the time-lapse modules. Temperature control was
achieved by using the Incubator XL, providing a humi-
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Mol Cancer Ther; 10(9) September 2011
dified atmosphere with 5% CO2. Imaging was carried out
for 20 hours with a lapse time of 5 minutes.
Results
The dynoles induce multinucleation
Inhibition of dynamin by the MiTMABs results in
cytokinesis failure generating polyploid cells (3). We
sought to determine if the dynoles also produce phenotypes consistent with dynII inhibition in cytokinesis function by testing the 5 most potent dynoles, 25, 26, 33, 34-2,
and 35, on dividing HeLa cells (Supplementary Fig. S1).
The in vitro IC50 value of the most potent analogue,
dynole 34-2, is 6.9 1.0 and 14.2 7.7 mmol/L for
full-length dynI and dynII GTPase activity, respectively
(Supplementary Table S1). The in vitro assay conditions
used for dynI and dynII used the same enzyme and
substrate concentrations and thus could be compared
as a selectivity ratio (IC50 of dynII/dynI). Selectivity ratio
for all dynoles was 0.9 to 3.3. We consider these ratios to
indicate that the drugs are nonselective for either isoform
as only a difference in vitro that well exceeds 10-fold
(ideally 100-fold) selectivity may be biologically significant in vivo. Like the MiTMABs, dynoles are pan-dynamin inhibitors. The order of potency for these dynoles is
slightly different for dynI versus dynII, however dynole
34-2 is the most potent for both. To test the effect of
dynoles on cytokinesis, HeLa cells were synchronized at
the G2–M boundary; then immediately upon mitotic
entry, cells were treated with the indicated Dynole for
6 hours (Fig. 1A). All 5 dynoles, 25, 26, 33, 34-2, and 35,
increased the percentage of multinucleated HeLa cells
(Fig. 1B). As found for dynamin GTPase activity, dynole
34-2 was the most effective at inducing multinucleation
(Fig. 1B). The effect was in the same order of magnitude
as MiTMAB at the same concentration. The percentage of
multinucleated cells induced by each dynole is likely an
underestimation of their effect on cytokinesis, as not
every cell would have entered mitosis during the treatment period. We conclude that the dynoles cause cytokinesis failure.
Dynoles cause cytokinesis failure at abscission
The role of dynII in cytokinesis seems to be restricted to
the abscission phase (3, 12). Depletion of dynII by short
interfering RNA and MiTMAB treatment results in cells
spending a prolonged period of time connected via an
intracellular bridge (ICB) before completion of mitosis or
formation of a multinucleated cell (3). To characterize the
point of mitotic failure induced by the dynoles, we
calculated the time cells took to undergo (i) prophase
(Pro) to metaphase (Met), (ii) Met to anaphase (Ana), (iii)
Ana to full membrane ingression (Ing), and (iv) Ing to
either generation of 2 daughter cells (Comp) or a multinucleated cell (Multi). Majority of DMSO-treated cells
that entered mitosis completed it within 90 to 150 minutes
(Fig. 1C). Cells treated with dynole 25, 26, and 33 spent a
significantly longer period of time in mitosis (dynole
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Dynoles Are Anticancer Compounds
Figure 1. Dynoles cause cytokinesis failure by blocking completion of abscission. A, schematic of experimental design. G2–M synchronized cells (RO-3306)
were released into either drug-free medium or medium containing dynamin inhibitors and analyzed at 6 hours for multinucleation or over 20-hour
period by using time-lapse microscopy. B, synchronized HeLa cells were treated with each dynole (10 mmol/L) or MiTMAB (10 mmol/L) as described in A. At
6 hours, cells were fixed, stained for a-tubulin, and the percentage of cells that were multinucleated were scored by using immunofluorescence microscopy.
Bar graph is mean SEM, n ¼ 3. C–F, synchronized HeLa cells were treated with vehicle (0.1% DMSO) or each dynole (10 mmol/L) as described in
A, then visualized by time-lapse microscopy. Cells entering mitosis were scored for the time taken to progress through mitosis from prophase (Pro) to
completion of mitosis (Comp) or generation of a binucleated cell (Multi; C). Cells were also scored for the time taken to progress through the following critical
mitotic phases: metaphase (Met) to anaphase (Ana; D), Ana to full membrane ingression (Ing; E), and Ing to Comp or Multi (F). *, P < 0.05, Student's t test.
G and H, representative time-lapse images of HeLa cells treated with DMSO and 10 mmol/L Dynole 34-2 undergoing mitosis. DMSO-treated cells (G)
completed abscission generating 2 independent cells. Dynole 34-2–treated cells (H) failed cytokinesis by failing to abscise the ICB. The cleavage furrow
regressed, forming a binucleated cell. Percentage of cells that entered mitosis and failed cytokinesis are shown below.
treatment median time >135 minutes vs. 102.5 minutes in
DMSO-treated cells; Fig. 1C). Dynole-treated cells
rounded up upon mitotic entry, aligned and segregated
their chromosomes (Fig. 1D), and produced a cleavage
furrow that generated an ICB (Fig. 1E) with similar
kinetics to vehicle-treated control cells. The point of delay
was identified as the abscission stage (Ing-Comp or
Multi), with dynole 25-, 26-, and 33-treated cells spending
approximately twice as long in this phase (median 32.5
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minutes in DMSO vs. median of more than 60 minutes in
dynole-treated cells; Fig. 1F). This is consistent with dynII
inhibition (3). In contrast, treatment with dynole 34-2 or
35 completed each mitotic transition with similar kinetics
to control cells (Fig. 1C–H). Despite this, time-lapse
microscopy indicated that the latter 2 dynoles also caused
cytokinesis failure at the point of abscission, resulting in
binucleation (Fig. 1H). Dynole 34-2 was the most potent
for cytokinesis failure, with 81.8% of cells that entered
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mitosis becoming multinucleated (Fig. 1H). Unlike treatment with the other dynoles, dynole 34-2- and dynole 35–
treated cells did not stay connected via an ICB for a
prolonged period of time. Instead the membrane rapidly
regressed, generating a binucleated cell (Fig. 1H). We
conclude that the dynoles cause cytokinesis failure by
blocking abscission, which is consistent with inhibition
of dynII.
Dynoles disrupt midbody localization of calcineurin
but not phospho-dynII and g-tubulin
The abscission phase involves formation of a central
g-tubulin midbody ring (MR) within the ICB before
abscission. Two flanking midbody rings (FMR) reside
on either side of the centrally located MR and contain
calcineurin (CaN) and dynII phosphorylated at S764 (10).
Ca2þ influx at the midbody activates CaN to dephosphorylate dynII, subsequently triggering abscission (10, 22).
CaN inhibition results in abscission failure and multinucleation (10). Dynamin inhibitor treatment did not
affect MR localization of g-tubulin (Supplementary
Fig. S2A and shown in red in Supplementary Fig. S2B
row 1; ref. 3). All 5 dynoles and MiTMAB disrupted FMR
localization of CaN with less than 30% of cells displaying
the correct ring localization (Supplementary Fig. S2 and
shown in green in Supplementary Fig. S2B row 3). Treatment with MiTMAB, but not the dynoles, caused disruption of phospho-dynII at the FMRs (Supplementary
Fig. S2 and shown in green in Supplementary Fig. S2B
row 2). Thus, dynoles selectively disrupt correct subcellular localization of CaN but not that of phospho-dynII or
g-tubulin at the midbody.
Dynoles inhibit growth in a range of cancer cell
lines
We next assessed whether the dynoles can inhibit cell
proliferation and induce cell death like MiTMABs (3).
Cell growth was assayed using the MTT assay in 10
cancer cell lines derived from different tissues: SMA560 (mouse glioma) and SJ-G2 (glial) HeLa (cervical),
MCF-7 (breast), A2780 (ovary), H460 (lung), A431 (skin),
DU145 (prostate), BE(2)-C (neuronal), and HT29 and
SW480 (colon). Following 72 hours of continuous exposure to the dynoles, all cell lines showed a dose-dependent decline in cell growth (dynole 34-2 dose–response
curve shown in Fig. 2A). The sensitivity to the Dynoles
varied among the cell lines, with GI50 values ranging from
4.0 to 36.7 mmol/L (Table 1). In 11 of the 12 cell lines,
Dynole 34-2 showed the lowest mean GI50 compared with
the other 4 dynole compounds, whereas in A431 cells an
equivalent level of inhibition to dynole 35 was observed
(Table 1). A comparison of the GI50 values among the
cell lines revealed that SMA-560, SJ-G2, SW480, MCF-7,
and A2780 cells were as sensitive to dynole 34-2 as HeLa
cells, whereas the other 6 cell lines were significantly
less sensitive to dynole 34-2 (Table 1 and Fig. 2A). In
SMA-560, SW480, and HeLa cells, at concentrations of
10 mmol/L or more, dynole 34-2 induced cell death as
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the response curves fell below zero, indicating that the
viable cell number at the end of the experiment was less
than at the start. At concentrations less than 10 mmol/L,
growth inhibition is likely a combination of growth
arrest and cell death. Prostate (DU-145) and skin
(A431) cancer cells were least sensitive to dynolemediated growth inhibition (Table 1 and Fig. 2A). A
colony formation assay confirmed the antiproliferative
effect of all 5 dynoles in SJ-G2 and HeLa cells, with a
50% reduction in colony formation at approximately 2
and 0.1 mmol/L of dynole 34-2, respectively (Fig. 2B and
C). Thus, the dynoles prevent cell proliferation and
reduce viability in a range of cancer cell lines.
Dividing cancer cells are more sensitive to the
dynoles
The therapeutic potential of an antimitotic agent is
because of its ability to inhibit growth or cause cell death
following mitotic insult. We sought to confirm if the
antiproliferative effect of the dynoles occurs following
a failed mitotic division by scoring the total viable cell
number by using trypan blue exclusion assay. Only 20
hours after release from G2–M block, the total viable HeLa
and SMA-560 cells treated with the dynoles was markedly reduced in a dose-dependent manner (Fig. 2D). The
total number of viable HeLa cells treated with 10 mmol/L
dynole 33, 34-2, and 35 was less than the number of cells
seeded (indicated by dashed line, Fig. 2D), indicating that
these compounds not only blocked proliferation but also
caused cell death. Dynole 34-2 was the most potent with
no viable HeLa cells remaining at 10 mmol/L. Similar
findings were observed in SMA-560 glioblastoma cells
(Fig. 2E). These cells were slightly less sensitive than
HeLa cells, and dynole 34-2 was also the most potent
in these cells, blocking SMA-560 cell growth and inducing
cell death at 10 mmol/L. Almost all cells treated with
dynole 34-2 failed cytokinesis under the same treatment
conditions (Fig. 1H).
To determine if cytotoxicity of the dynoles is selective
for dividing cells, we carried out a lactate hydrogenase
release assay to assess membrane integrity, indicative of
cell viability. An acute treatment of 6 hours was used
because only a small percentage of asynchronously growing HeLa cells would enter mitosis in this window,
because their doubling rate is approximately 10 to 11
hours. In contrast, more than 60% of HeLa cells would
progress through mitosis during the 6-hour treatment
period following synchronization at G2–M boundary. All
5 dynoles were cytotoxic in G2–M synchronized HeLa
cells (Fig. 2F) and evident at concentrations as low as 1
mmol/L with a dramatic increase in cytotoxicity observed
at concentrations of 30 mmol/L or more. Dynole 34-2 was
again the most potent compound causing cytotoxicity in
more than 50% of cells at 30 mmol/L compared with less
than 20% of cells treated with 30 mmol/L dynole 25 and
33. All dynoles were equally potent in asynchronous cells
at concentrations of 30 mmol/L or more (Fig. 2G). However, at concentrations of 10 mmol/L or more, in contrast
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Dynoles Are Anticancer Compounds
Figure 2. Dividing cancer cells are more sensitive to antiproliferative and cytotoxic effects of dynoles. A, cell proliferation was assessed by the MTT
assay with increasing concentrations of Dynole 34-2 after 72 hours in 11 cancer cell lines. B and C, line graphs show the relative percentage of colonies
present in HeLa (B) and SJ-G2 (C) cancer cell lines at day 7, after treatment with increasing concentrations of the dynoles. Values normalized to
untreated controls. D and E, G2–M synchronized HeLa (D) and SMA-560 (E) cancer cells were treated with increasing concentrations of dynoles. Graphs
(mean SD, n ¼ 2) display the total number of viable cells present after 20 hours dynole exposure. F and G, lactate hydrogenase assay measured cytotoxicity
in G2–M synchronized (F) and asynchronously (G) growing HeLa cells exposed to increasing concentrations of the dynoles and MiTMAB for 6 hours. Graphs
show mean SD, n ¼ 2.
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NOTE. Cells were treated with Dynoles for 72 hours followed by MTT assay. The table shows GI50 (half maximal growth inhibitory concentration) values (mean SEM) of 5 Dynoles,
calculated from the MTT dose–response curves. n ¼ 3. Comparison of each dynamin inhibitor with Dynole 34-2 within an individual cell line: aP < 0.05; bP < 0.01. Comparison of an
individual dynamin inhibitor in different cell lines with HeLa cells: cP < 0.05, dP < 0.01.
2.5 0.5a
22.5 3.5a,c
15.0 0.6a,c
15.7 0.3a,d
9.3 1.6
14.7 0.3a,d
1.9 0.7b
19.7 2.8a
14.3 0.3b,c
14.0 0.0b,d
7 0.1c
13.7 0.3b
Compound
MiTMAB
Dynole 25
Dynole 26
Dynole 33
Dynole 34-2
Dynole 35
–
15 0.4b,c
6.9 0.1
10 5.0
6.6 0.2
15 0.4b,c
1.6 0.1b
12 0.7b
7.8 1.6
4.0 0.7
5.8 0.3
13 0.0b
0.6 0.1b,d
18.7 1.2b,d
16.7 0.3b,d
15.0 1.0b,d
6.9 0.1c
16.3 0.3b,d
1.0 0.2b
14.7 0.7b,c
8.4 0.5a
8.1 0.3b,d
6.5 0.1
9.1 1.0c
1.0 0.3b
24.7 3.0a,c
13.7 0.3c
15.7 0.3d
10 1.6
13.0 0.6
2.2 0.8a
23.5 5.0c
15.2 0.8d
14.7 1.3
10 1.6
15.2 0.8
3.9 1.1b
29.3 4.7a,c
15.2 0.8c
16.3 0.7d
14 0.3d
15.3 0.7c
0.6 0.0b,d
26.0 2.0b,d
14.7 0.3c
15.3 0.7d
13.7 0.3d
13.7 0.3
2.7 0.8b
36.7 0.9b,d
18.3 0.7b,d
24.0 2.9a,d
15 0.0d
17.0 0.6a,d
SJ-G2
Glial
H460
Lung
A2780
Ovarian
MCF-7
Breast
SMA-560
Glial
HeLa
Cervical
HT29
Colon
SW480
Colon
GI50 (mmol/L) after 72-hour exposure
Cell line
tissue
Table 1. Growth inhibition induced by the Dynoles in a range of human cancer cell lines
A431
Skin
DU145
Prostate
BE2-C
Neuronal
Chircop et al.
to the effect in G2–M synchronized cells, no cytotoxicity
was evident in asynchronous cells (Fig. 2G). These findings indicate that the dynoles have antiproliferative properties that are relatively selective for dividing cancer cells
at low concentrations, which are in line with their IC50
value for dynamin GTPase activity.
Dynole 34-2 induces apoptosis following
cytokinesis failure
The dynoles cause growth arrest/cell death and cytokinesis failure. We next tested the hypothesis that the
dynoles induce growth arrest and/or cell death specifically following cytokinesis failure by using time-lapse
microscopy to follow individual HeLa cells. Only dynole
34-2 (10 mmol/L) induced cell death specifically following cytokinesis failure within the 20-hour treatment period (Fig. 3A and B). Of those dynole 34-2 treated cells that
failed cytokinesis, 97.3% 7.0% resulted in cell death
(Fig. 3A). MiTMAB was also very effective at causing cell
death (83.4% 1.6%), specifically following binucleation
(3). In contrast, cell death occurred in less than 7% of
HeLa cells that failed cytokinesis treated with the 4 other
dynoles, 25, 26, 33, and 35 (Fig. 3A). Instead, the resulting
multinucleated cells remained viable for the duration of
the experiment (up to 20 hours). HeLa cells have a
doubling rate of approximately 10 to 11 hours, thus cells
would be expected to enter a second mitotic division
during the 20-hour experimental period if their growth
was not arrested. Cells treated with dynoles 25, 26, 33,
and 35 did not undergo a subsequent division and thus
were considered growth arrested. DMSO-treated cells
did not undergo cell death and were often observed
entering a second cell division within the 20-hour treatment period.
Analysis of dynole-treated cells that successfully completed mitosis revealed that the 2 daughter cells remained
viable during the 20-hour treatment period (Fig. 3A and
B). In contrast, cell death was induced in approximately
40% of MiTMAB-treated cells that completed mitosis
successfully (Fig. 3A). This is still significantly lower than
the percentage of MiTMAB-treated cells that underwent
cell death following cytokinesis failure. Dynole 34-2–
induced cell death was confirmed by an increase in less
than 2N DNA content by using fluorescence-activated
cell sorting (FACS) analysis (Fig. 3C). A corresponding
increase in 4N cells, indicative of multinucleated cells,
was observed following 24-hour exposure to dynole 34-2
(Fig. 3D). Dynole 25 also caused an increase in the
percentage of 4N cells (Fig. 3D). Consistent with the
time-lapse data, no corresponding increase in less than
2N cells was observed following treatment with dynole
25 (Fig. 3C). Dynole 34-2–induced cell death was characterized by cell shrinkage, membrane blebbing (timelapse microscopy), and DNA fragmentation (FACS analysis)—characteristics of apoptosis (Fig. 3B and C). Apoptosis was evident by an increase in PARP cleavage
following dynole 34-2 and MiTMAB treatment and exposure to ultraviolet irradiation compared with treatment
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Dynoles Are Anticancer Compounds
Figure 3. Cell death following
cytokinesis failure in HeLa cells
exposed to dynole 34-2. A, HeLa
cells were treated and visualized
as described in Fig. 1A.
Percentage of cells that
underwent apoptosis following
cytokinesis failure and following a
successful mitotic division is
shown (mean SD, n ¼ 2). B,
representative time-lapse images
of a HeLa cell treated with dynole
34-2 or 0.1% DMSO alone. Dynole
34-2–treated cells undergo
apoptotic cell death,
characterized by membrane
blebbing, 125 minutes after failing
cytokinesis. C and D, HeLa cells
were treated as described in A
except that after 20 hours, the
DNA content was analyzed by flow
cytometry. Percentage of cells
(mean SD, n ¼ 2) with less than
2N (C) and 4N DNA content (D) are
shown. *P < 0.05, Student's t
tests. E, HeLa cells were treated
as described in A. At 8 hours,
lysates were immunoblotted for
cleaved PARP. g-tubulin served as
a loading control. F, quantitative
densitometric analysis of PARP
levels shown in E. Graph shows
the relative amount of PARP in
HeLa cells following treatment
with the indicated conditions that
have been normalized to the
amount of g-tubulin.
with DMSO (Fig. 3E and F). Collectively, these findings
suggest that immediately following cytokinesis failure,
dynole 25, 26, 33, and 35 inhibit cell proliferation, whereas
dynole 34-2 causes cell death.
Nontumorigenic fibroblasts are less sensitive to
dynole 34-2
The antiproliferative properties of the MiTMABs are
relatively selective for cancer cells. We sought to determine the ability of dynole 34-2 to induce cell death and
inhibit proliferation in nontumorigenic fibroblasts.
NIH3T3 fibroblasts were compared with HeLa carcinoma
cells. NIH3T3 cells do not synchronize with RO-3306
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treatment (G2–M boundary) as effectively as HeLa cells
and therefore experiments were carried out in asynchronously growing cells. Nevertheless, both cell lines have a
similar doubling rate. Thus, a similar number of cells in
both populations would be expected to proceed through
mitosis within the same treatment period. An acute 6hour treatment with dynole 34-2 resulted in cytotoxicity
in more than 50% of HeLa cells and less than 5% of
NIH3T3 cells (Fig. 4A). Assessment of cell proliferation
by scoring for total viable cell number revealed that
growth was inhibited in both cell types following a 20hour exposure. The effect was reduced in the nontumorigenic fibroblasts (Fig. 4B and C). Cell death as indicated
Mol Cancer Ther; 10(9) September 2011
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Chircop et al.
Figure 4. Nontumorigenic
fibroblasts are less sensitive to the
antiproliferative effects of dynole
34-2. A, cytotoxicity graph (mean
SD, n ¼ 2) of asynchronously
growing HeLa carcinoma cells and
NIH3T3 fibroblasts induced by
dynole 34-2 after 6 hours. B and C,
graphs of total viable cell number
(mean SD) after 24-hour
exposure to dynole 34-2 in HeLa
(B) and NIH3T3 cells (C). D and E,
3 cancer cell lines (HeLa, HT29,
and SJ-G2) and 2 nontumorigenic
fibroblasts (F1 and NIH3T3) were
exposed to increasing
concentrations of dynole 34-2 for
7 days. Total viable cell number
(mean SD, n ¼ 2; D) and relative
percentage of viable cells (mean
SD, n ¼ 2; E) were assessed by
trypan blue.
by a total loss in viable cells was evident after 24-hours
exposure to 10 mmol/L dynole 34-2 in HeLa cells (Fig. 4B),
but a significant number of NIH3T3 cells were viable
following exposure up to 30 mmol/L (Fig. 4C). This
difference was still evident following a prolonged
7 day exposure to dynole 34-2 (Fig. 4D and E). The
difference between cancer and nontumorigenic cells
was consistently observed among a panel of 3 cancer cell
lines (HeLa, HT29, and SJ-G2) and 2 nontumorigenic
fibroblasts (NIH3T3 and F1). Specifically, dynole 34-2
caused a concentration-dependent inhibition in cell proliferation in all cell lines (Fig. 4D), but in contrast to the
cancer cells, a significant number of viable cells were
present at the end of the 7-day treatment in both fibroblasts (Fig. 4E). Thus, nontumorigenic fibroblasts seem to
be less sensitive to dynole 34-2 than cancer cells.
Discussion
The endocytic protein dynamin has been suggested to
be a novel antimitotic drug target for the treatment of
cancer because the MiTMAB dynamin inhibitors exclusively block the abscission phase of cytokinesis, inhibit
1560
Mol Cancer Ther; 10(9) September 2011
cell proliferation, and reduce viability (3). The MiTMABs
are a series of phospholipid-competitive inhibitors that
target the pleckstrin homology domain of dynamin and
potentially of other proteins (4, 5). Thus, they are subject
to anticipated actions in addition to dynamin inhibition.
We now show that the dynoles also have potent antimitotic effects that phenocopy the MiTMABs, causing
cytokinesis failure at the abscission step, blocking cell
proliferation, and reducing cell viability in multiple
tumor cell lines from different tissue types. Dynoles block
dynamin GTPase activity via a distinct mechanism to the
MiTMABs, being uncompetitive with respect to GTP,
thus they are proposed to bind an allosteric site within
the GTPase domain (16). Among the dynoles tested here,
the most potent compound, dynole 34-2, induced cell
death exclusively following cytokinesis failure. Both nondividing differentiated cells and nontumorigenic fibroblasts were less sensitive to cell death mediated by dynole
34-2. Thus, the dynoles have antiproliferative and cytotoxic properties that reduce cancer cell viability, while
having reduced activity in noncancer cells. Our new
findings with a second class of mechanistically distinct
dynamin inhibitors strengthen the evidence that the
Molecular Cancer Therapeutics
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Published OnlineFirst July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0067
Dynoles Are Anticancer Compounds
antimitotic action of dynamin inhibitors may be primarily
because of targeting dynamin in cytokinesis.
The MiTMABs and dynole dynamin inhibitors represent a new class of antimitotic compounds, specifically
blocking abscission. The dynoles disrupt FMR localization
of CaN, but not phospho-dynII. Such an action might
conceivably prevent CaN-mediated dynII dephosphorylation at Ser-764, which is known to trigger abscission
(10, 22). MiTMABs also disrupt FMR localization of CaN,
and consistent with their ability to block dynamin’s
phospholipid interaction (5, 23), FMR localization of phospho-dynII is also disrupted by these compounds. These
phenotypes are quite distinct from that induced by other
antimitotic compounds, which disrupt earlier stages of
mitosis, causing chromosome misalignment leading to
prolonged activation of the SAC (1, 24, 25), monopolar
spindles (24–26), or inhibition of the SAC (27). Regardless
of their mode of action, all of these antimitotic compounds,
including dynamin inhibitors have anticancer properties.
The ability of antimitotic compounds to induce apoptosis exclusively in dividing cells is a highly desirable
property for a potential cancer treating drug (27–30).
MiTMAB induced cell death in dividing and nondividing
cells, albeit to a lesser extent in nondividing cells. Thus,
MiTMABs may have an additional off-target action,
resulting in nonspecific cytotoxicity. In contrast, cell
death was only observed following cell division in dynole
34-2–treated cells. Dynole 34-2–induced cell death was
also selective for cancer cells as it had minimal cytotoxic
effects on nontumorigenic proliferating cells, indicating
that it has a favorable therapeutic window. The other four
dynoles, 25, 26, 33, and 35, also induced cell death.
However, their ability to do so was significantly reduced
compared with dynole 34-2, only observed in assays that
monitored cell death after several days, for example, MTT
assay. This suggests that following cytokinesis failure,
dynole 25, 26, 33, and 35 induce cell cycle arrest followed
by cell death several days later, whereas dynole 34-2–
treated cells immediately undergo cell death. Consistent
with dynole 34-2 being the most effective at inducing cell
death, it is also the most potent inhibitor of dynamin
GTPase activity in vitro and RME in cells (16). However,
the ability of dynole 34-2 to inhibit dynamin’s GTPase
activity is not vastly different from the other four dynoles.
It is possible that although all dynoles analyzed share the
same cellular target, thus inducing cytokinesis failure and
cell death, the dramatic increase in cytotoxicity and
specific induction of cell death exclusively following
cytokinesis failure caused by dynole 34-2 suggests that
it has 2 mechanisms of action, one of which is shared with
the other dynole compounds. The cellular response
induced by other targeted antimitotic compounds is also
varied and in part depends on cell line and/or inhibitor
and/or assay (31, 32). Time spent blocked in mitosis (33),
p53 status (34–36), and expression level of prosurvival
Bcl-2 family members (37, 38) is also thought to contribute. It will be important to understand what drives a
particular cellular response to a specific antimitotic agent
to predict tumor response and patient outcome.
Our observations provide strong evidence that inhibition of dynamin is a new approach to cancer therapy,
potentially reducing tumor volume in patients. The cytotoxicity and antiproliferative properties of MiTMABs (3)
and dynoles in a broad array of cancer cell lines also
suggests that dynamin inhibitors may be effective for the
treatment of many tumor types. Dynole 34-2 is the lead
compound in this class, because it possesses desirable
anticancer properties and leads to cell death after failed
cytokinesis. It is amenable to further drug development
for the treatment of cancer. Our findings show that a
second mechanistically distinct class of dynamin inhibitor exclusively blocks the abscission stage of cytokinesis
and do not act on other points in mitosis, strengthening
the idea that they primarily act by targeting dynamin
GTPase activity. The dynoles show encouraging antimitotic properties and suggest that other dynamin inhibitors under development may also prove to be antimitotic.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Audrey Lian, Scott L. Page, Louis Lu, and Tram Phan for
their technical assistance and Louis Lu and Tram Phan from NCRISrecombinant facility (CSIRO, Melbourne).
Grant Support
This work was supported by grants from the NSW Cancer Council Project
grant (M. Chircop and J. Sakoff), James S McDonnell Foundation U.S.A. Project
grant (M. Chircop, J. Sakoff, P.J. Robinson, A. McCluskey, and T.J. O’Brien),
NH&MRC Project grant (P.J. Robinson and M. Chircop), and NH&MRC Career
Development Award Fellowship (M. Chircop).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
Received February 1, 2011; revised June 21, 2011; accepted June 27,
2011; published OnlineFirst July 12, 2011.
References
1.
2.
3.
Jackson JR, Patrick DR, Dar MM, Huang PS. Targeted anti-mitotic
therapies: can we improve on tubulin agents? Nat Rev Cancer
2007;7:107–17.
Taylor S, Peters JM. Polo and Aurora kinases-lessons derived from
chemical biology. Curr Opin Cell Biol 2008;20:77–84.
Joshi S, Perera S, Gilbert J, Smith CM, Gordon CP, McCluskey A, et al.
The dynamin inhibitors MiTMAB and OcTMAB induce cytokinesis
www.aacrjournals.org
4.
5.
failure and inhibit cell proliferation in human cancer cells. Mol Cancer
Ther 2010;9:1995–2006.
Hill TA, Odell LR, Quan A, Abagyan R, Ferguson G, Robinson PJ, et al.
Long chain amines and long chain ammonium salts as novel inhibitors
of dynamin GTPase activity. Bioorg Med Chem Letts 2004;14:3275–8.
Quan A, McGeachie AB, Keating DJ, van Dam EM, Rusak J, Chau N,
et al. Myristyl trimethyl ammonium bromide and octadecyl trimethyl
Mol Cancer Ther; 10(9) September 2011
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1561
Published OnlineFirst July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0067
Chircop et al.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
1562
ammonium bromide are surface-active small molecule dynamin inhibitors that block endocytosis mediated by dynamin I or dynamin II.
Mol Pharmacol 2007;72:1425–39.
Praefcke GJK, McMahon HT. The dynamin superfamily: universal
membrane tubulation and fission molecules? Nat Rev Mol Cell Biol
2004;5:133–47.
Hinshaw JE. Dynamin and its role in membrane fission. Annu Rev Cell
Dev Biol 2000;16:483–519.
Sever S, Damke H, Schmid SL. Dynamin:GTP controls the formation
of constricted coated pits, the rate limiting step in clathrin-mediated
endocytosis. J Cell Biol 2000;150:1137–48.
Newton AJ, Kirchhausen T, Murthy VN. Inhibition of dynamin completely blocks compensatory synaptic vesicle endocytosis. Proc Natl
Acad Sci U S A 2006;103:17955–60.
Chircop M, Malladi CS, Lian AT, Page SL, Zavortink M, Gordon CP,
et al. Calcineurin activity is required for the completion of cytokinesis.
Cell Mol Life Sci 2010;67:3725–37.
Feng B, Schwarz H, Jesuthasan S. Furrow-specific endocytosis
during cytokinesis of zebrafish blastomeres. Exp Cell Res 2002;
279:14–20.
Liu YW, Surka MC, Schroeter T, Lukiyanchuk V, Schmid SL. Isoform
and splice-variant specific functions of dynamin-2 revealed by
analysis of conditional knock-out cells. Mol Biol Cell 2008;19:
5347–59.
Thompson HM, Skop AR, McNiven MA. Dynamin is a component of
the intercellular bridge and is required for cytokinesis in hepatocytes.
Hepatol 2002;36:212A.
Thompson HM, Skop AR, Euteneuer U, Meyer BJ, McNiven MA. The
large GTPase dynamin associates with the spindle midzone and is
required for cytokinesis. Curr Biol 2002;12:2111–7.
Hill T, Odell LR, Edwards JK, Graham ME, McGeachie AB, Rusak J,
et al. Small molecule inhibitors of dynamin I GTPase activity: development of dimeric tyrphostins. J Med Chem 2005;48:7781–8.
Hill TA, Gordon CP, McGeachie AB, Venn-Brown B, Odell LR, Chau N,
et al. Inhibition of dynamin mediated endocytosis by the dynolesTM synthesis and functional activity of a family of indoles. J Med Chem
2009;52:3762–73.
Hill TA, Mariana A, Gordon CP, Odell LR, Robertson MJ, McGeachie
AB, et al. Iminochromene inhibitors of dynamins I and II GTPase
activity and endocytosis. J Med Chem 2010;53:4094–102.
Odell LR, Howan D, Gordon CP, Robertson MJ, Chau N, Mariana A,
et al. The pthaladyns: GTP competitive inhibitors of dynamin I and II
GTPase derived from virtual screening. J Med Chem 2010;53:
5267–80.
Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T.
Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 2006;
10:839–50.
Vassilev LT, Tovar C, Chen SQ, Knezevic D, Zhao XL, Sun HM, et al.
Selective small-molecule inhibitor reveals critical mitotic functions of
human CDK1. Proc Natl Acad Sci U S A 2006;103:10660–5.
Chircop M, Oakes V, Graham ME, Ma MP, Smith CM, Robinson PJ,
et al. The actin-binding and bundling protein, EPLIN, is required for
cytokinesis. Cell Cycle 2009;8:757–64.
Chircop M, Sarcevic B, Larsen MR, Malladi CS, Chau N, Zavortink M,
et al. Phosphorylation of dynamin II at serine-764 is associated with
cytokinesis. Biochim Biophys Acta Epub 2010 Dec 29.
Mol Cancer Ther; 10(9) September 2011
23. Orth JD, Tang Y, Shi J, Loy CT, Amendt C, Wilm C, et al. Quantitative
live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and
cell fate. Mol Cancer Ther 2008;7:3480–9.
24. Schoffski P. Polo-like kinase (PLK) inhibitors in preclinical and early
clinical development in oncology. Oncologist 2009;14:559–70.
25. Huszar D, Theoclitou ME, Skolnik J, Herbst R. Kinesin motor proteins
as targets for cancer therapy. Cancer Metastasis Rev 2009;28:
197–208.
26. Manfredi MG, Ecsedy JA, Meetze KA, Balani SK, Burenkova O, Chen
W, et al. Antitumor activity of MLN8054, an orally active smallmolecule inhibitor of Aurora A kinase. Proc Natl Acad Sci U S A
2007;104:4106–11.
27. Wilkinson RW, Odedra R, Heaton SP, Wedge SR, Keen NJ, Crafter C,
et al. AZD1152, a selective inhibitor of Aurora B kinase, inhibits human
tumor xenograft growth by inducing apoptosis. Clin Cancer Res
2007;13:3682–8.
28. Chan F, Sun C, Perumal M, Nguyen QD, Bavetsias V, McDonald E,
et al. Mechanism of action of the Aurora kinase inhibitor CCT129202
and in vivo quantification of biological activity. Mol Cancer Ther
2007;6:3147–57.
29. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T,
et al. Aurora B couples chromosome alignment with anaphase by
targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol
2003;161:267–80.
30. Dreier MR, Grabovich AZ, Katusin JD, Taylor WR. Short and long-term
tumor cell responses to Aurora kinase inhibitors. Exp Cell Res
2009;315:1085–99.
31. Weaver BA, Cleveland DW. Decoding the links between mitosis,
cancer, and chemotherapy: The mitotic checkpoint, adaptation,
and cell death. Cancer Cell 2005;8:7–12.
32. Gascoigne KE, Taylor SS. Cancer cells display profound intra- and
interline variation following prolonged exposure to antimitotic drugs.
Cancer Cell 2008;14:111–22.
33. Bekier ME, Fischbach R, Lee J, Taylor WR. Length of mitotic arrest
induced by microtubule-stabilizing drugs determines cell death after
mitotic exit. Mol Cancer Ther 2009;8:1646–54.
34. Andreassen PR, Lohez OD, Lacroix FB, Margolis RL. Tetraploid state
induces p53-dependent arrest of nontransformed mammalian cells in
G1. Mol Biol Cell 2001;12:1315–28.
35. Gizatullin F, Yao Y, Kung V, Harding MW, Loda M, Shapiro GI. The
Aurora kinase inhibitor VX-680 induces endoreduplication and apoptosis preferentially in cells with compromised p53-dependent postmitotic checkpoint function. Cancer Res 2006;66:7668–77.
36. Kojima K, Konopleva M, Tsao T, Nakakuma H, Andreeff M. Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53-dependent postmitotic checkpoints and synergistically
induces p53-mediated mitochondrial apoptosis along with reduced
endoreduplication in acute myelogenous leukemia. Blood 2008;
112:2886–95.
37. Amundson SA, Myers TG, Scudiero D, Kitada S, Reed JC, Fornace AJ
Jr. An informatics approach identifying markers of chemosensitivity in
human cancer cell lines. Cancer Res 2000;60:6101–10.
38. Kang MH, Reynolds CP. Bcl-2 inhibitors: targeting mitochondrial
apoptotic pathways in cancer therapy. Clin Cancer Res 2009;
15:1126–32.
Molecular Cancer Therapeutics
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0067
Inhibition of Dynamin by Dynole 34-2 Induces Cell Death following
Cytokinesis Failure in Cancer Cells
Megan Chircop, Swetha Perera, Anna Mariana, et al.
Mol Cancer Ther 2011;10:1553-1562. Published OnlineFirst July 12, 2011.
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