M. Ohira et al. / Biochimica et Biophysica Acta 1850 (2015) 1676–1684
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stabilizers), whereas the latter two prevent polymerization (microtubule
destabilizers); both lead to mitotic arrest, cell growth arrest, and eventu-
al cell death. Cancer cells are more susceptible than non-transformed
cells to microtubule-targeting agents, although our understanding of
the underlying molecular mechanisms is limited [5–7]. In addition, the
molecular basis of the tissue specificities of microtubule-targeting agents
also remains to be clarified. It was recently suggested that some
microtubule-targeting agents disrupt the tumor vasculature [8].
Radioactive Cell Proliferation Assay, #G5430, Promega) was added,
cells were further incubated for 1 h, and OD490 was measured to deter-
mine cell number. The percent inhibition was determined for each drug
concentration, and the IC50 value for cell growth was calculated from
the linear portion of the dose-response curve using regression analysis.
2.4. Tumor studies in nude mice
Although these clinically used drugs are effective for cancer
chemotherapy, there are some critical problems to be solved. For
example, because microtubules play important roles in non-dividing
cells like neuronal cells, these drugs inevitably damage such cells,
resulting in adverse effects such as peripheral neuropathy. Another
issue is innate or acquired resistance to microtubule-targeting agents.
The known mechanisms that confer resistance to the microtubule-
targeting drugs include overexpression of a class of ATP-dependent
membrane transporter proteins represented by MDR1, overexpression
of specific isotypes of tubulin such as βIII-tubulin, certain mutations in
tubulin, and overexpression of certain MAPs [2,4,9]. To overcome
these problems, a great deal of effort has been devoted to development
of novel anti-mitotics that target non-microtubule mitotic proteins such
as Aurora kinases, Plks, Eg5, and CENP-E [3,10–16]. Unfortunately, thus
far, the clinical efficacy of these novel anti-mitotics has been limited [3].
Novel microtubule-targeting agents would also be valuable [2–4,17,
The protocols for animal experiments were approved by the Kyushu
University Animal Care and Use Committee (permit number: A23-067
and A25-022). Four-week-old female BALB/c nu/nu nude mice were ob-
tained from Kyudo Co., Ltd. Mice were inoculated by subcutaneous
6
injection on their backs with 1 × 10 HeLa cells mixed with Matrigel
(50 μL of cell suspension + 50 μL of Matrigel, 356234, BD Biosciences).
The size of the xenografted tumor was measured using digital calipers,
and volume was calculated based on the following formula: tumor
3
2
volume (mm ) = length × (width) × π/6. Body weight and tumor
size were measured three times per week. Once the tumor reached
3
~100 mm , mice were randomly divided into two groups, one receiving
the control vehicle mixture detailed below and the other receiving
vehicle plus HND-007 (50 mg/kg). Drug was administrated intraperito-
neally once daily for 3 days. Vehicle mixture consisted of 20% DMSO,
20% Cremophor (polyoxyethylene castor oil), 20% ethanol, and 40%
saline.
1
8]. Among the microtubule-targeting agents in current clinical use,
vinca alkaloids and halichondrins bind to a specific site on tubulin
termed the “vinca site”, whereas taxanes and epothilones bind to
another site termed the “taxane site” [4]. Another site recognized by
microtubule-targeting agents is the “colchicine site” [4]; however, so
far, no drug that binds to the colchicine site has been deployed for can-
cer chemotherapy. Nevertheless, ongoing efforts are still being devoted
to development of such colchicine site-binding drugs, especially as vas-
cular disrupting agents [4,8,18]. Recently, the laulimalide/peloruside-
binding site on tubulin molecules has been also identified, although a
short supply of the natural products has prevented clinical trials of
these compounds [19].
2.5. Tubulin polymerization assay
Tubulin polymerization assays were performed using the HTS-
tubulin Polymerization Kit (Cytoskeleton, BK004P). The reaction was
conducted in the presence of 10% glycerol and 3.35 mg/ml tubulin.
Drugs were dissolved in DMSO and added to the reaction mixtures;
the final concentration of DMSO was 2%. Tubulin polymerization was
monitored by measuring OD340 at 37 °C.
2.6. Immunoblot analysis and antibodies
In this report, we describe novel microtubule-targeting agents with
carbazole and benzohydrazide structures that were identified during
the course of screening for inhibitors of Cdt1-geminin binding (for details,
see Results and discussion). Among them, N′-[(9-ethyl-9H-carbazol-3-
yl)methylene]-2-methylbenzohydrazide (code number HND-007) can
suppress tumor cell growth in vitro and in vivo.
Immunoblot analysis was performed as described previously [20].
Antibodies used were as follows: phospho-Histone H3 Ser10 (#9706,
Cell Signaling Technology, 1:1000 dilution); Cdc2-phosphorylated
vimentin Ser55 (D076-3, MBL, 1:20 dilution); phospho-cyclinB1 Ser133
(#4133S, Cell Signaling, 1:1000 dilution); PARP1 (#9542, Cell Signaling
Technology, 1:1000 dilution); α-tubulin (ab15246, Abcam, 1:100
dilution for immunofluorescence); γ-tubulin (T6557, Sigma-Aldrich,
1:2000 dilution for immunofluorescence); centrin1 (ab11257, Abcam,
2
. Materials and methods
1
:200 dilution for immunofluorescence); CENP-B (kindly provided by
2
.1. Cell culture
Dr. Hiroshi Masumoto, Kazusa DNA Research Institute, 1:200 dilution
for immunofluorescence). For secondary antibodies, HRP-rabbit anti-
mouse IgG (H + L) (61-6520, Invitrogen, 1:1000 dilution) or HRP-goat
anti-rabbit IgG (H + L) (65-6120, Invitrogen, 1:1000 dilution) were used.
HeLa (cervical carcinoma), H1299 (non-small cell lung carcinoma),
HCT116 (colon carcinoma), T98G (glioblastoma), and HFF2/T (normal
human fibroblasts immortalized by telomerase) cells were grown in
Dulbecco's modified Eagle's medium supplemented with 8% fetal calf
serum (FCS). PC-14 and PC-14/TXT cells were kindly provided by
Dr. Fumiaki Koizumi (National Cancer Center, Japan).
2.7. Immunofluorescence analysis
Cells were fixed with 3.7% formaldehyde in PBS for 10 min at RT
(
Room Temperature) and further treated with 100% ice-cold MeOH
2
.2. Drugs
for 15 min. The cells were then permeabilized with 0.1% Triton X-100,
incubated with primary antibodies (diluted with PBS containing 10%
FCS) for 1 h at RT, and washed three times with PBS. For cenrtin1 stain-
ing, cells were treated with ice-cold MeOH for 5 min and then incubated
with anti-centrin1 antibody overnight at RT. Cells were then incubated
with secondary antibodies for 1 h at RT and finally counterstained with
4,6-diamidino-2-phenylindole (DAPI). The samples were mounted in
Vectashield (Vector Laboratories) and analyzed on a Zeiss LSM700 mi-
croscope. Secondary antibodies used were CF594-conjugated goat
anti-mouse IgG (H + L) (20111, Biotium, 1:100 dilution) and CF488-
conjugated goat anti-rabbit IgG (H + L) (20019, Biotium, 1:100
dilution).
Drugs used in this study were as follows: nocodazole (Sigma-Aldrich,
M1404), paclitaxel (BML, T104-0005), and hydroxyurea (Sigma-Aldrich,
H8627).
2
.3. Growth inhibition assay
3
Cells were plated in 96-well plates at 5 × 10 cells/well, and the in-
dicated compounds dissolved in DMSO were added 24 h later. DMSO
vehicle) was added to control cells at a final concentration of 1%.
After treatment for 48 h, MTS/PMS solution (CellTiter Aqueous Non-
(