J C E M O N L I N E

Hot Topics in Translational Endocrinology—Endocrine Care

CLM94, a Novel Cyclic Amide with Anti-VEGFR-2 and

Antiangiogenic Properties, Is Active against Primary
Anaplastic Thyroid Cancer in Vitro and in Vivo

Alessandro Antonelli, Guido Bocci, Concettina La Motta, Silvia Martina Ferrari,

Poupak Fallahi, Ilaria Ruffilli, Andrea Di Domenicantonio, Anna Fioravanti,
Stefania Sartini, Michele Minuto, Simona Piaggi, Alessandro Corti, Greta Alì,

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Teresa Di Desidero, Piero Berti, Gabriella Fontanini, Romano Danesi,
Federico Da Settimo, and Paolo Miccoli
Department of Internal Medicine (A.A., S.M.F., P.F., I.R., A.D.D.), Division of Pharmacology (G.B., A.F.,
T.D.D., R.D.), and Departments of Pharmaceutical Science (C.L.M., S.S., F.D.S.), Surgery (M.M., G.A.,
P.B., G.F., P.M.), and Experimental Pathology (S.P., A.C.), University of Pisa, School of Medicine, 56100
Pisa, Italy

Context and Objective: We have studied the antitumor activity of a novel cyclic amide, CLM94, with
anti-vascular endothelial growth factor (VEGF) receptor-2 and antiangiogenic activity in primary
anaplastic thyroid cancer (ATC) cells in vitro and in vivo.

Design and Main Outcome Measures: CLM94 was tested: 1) in two human cell lines (HMVEC-d,
dermal microvascular endothelial cells; and 8305C, undifferentiated thyroid cancer) at 0.001–100
␮M; 2) in ATC cells at the concentrations of 10, 30, and 50 ␮M; and 3) in an ATC cell line (AF) in CD
nu/nu mice.

Results: CLM94 significantly inhibited VEGF receptor-2 and epidermal growth factor receptor
phosphorylation in HMVEC-d and proliferation in HMVEC-d and 8305C cells. A significant reduction
of proliferation with CLM94 in ATC cells (P ⬍ 0.01, ANOVA) and a slight but significant reduction
of proliferation with CLM94 30 and 50 ␮M in normal thyroid follicular cells (P ⬍ 0.01, ANOVA) were
shown. CLM94 increased the percentage of apoptotic ATC cells dose-dependently (P ⬍ 0.001,
ANOVA) and inhibited migration (P ⬍ 0.01) and invasion (P ⬍ 0.001). AF cell line was injected sc in
CD nu/nu mice, and tumor masses became detectable 25 d afterward. CLM94 (40 mg/kg 䡠 d) sig-
nificantly inhibited tumor growth (starting 10 d after the beginning of treatment). CLM94 signif-
icantly decreased the VEGF-A gene expression in the AF cell line and the VEGF-A protein and
microvessel density in AF tumor tissues.

Conclusions: The antitumor and antiangiogenic activity of a new “cyclic amide” compound,
CLM94, is very promising in ATC, opening the way to a future clinical evaluation. (J Clin Endocrinol
Metab 97: E528 –E536, 2012)

naplastic thyroid cancer (ATC) is often incurable be-
A cause it doesn’t respond to radiotherapy or chemo-
therapy (1–3).
Tyrosine kinase inhibitors (TKI) are actually under
evaluation for the treatment of ATC (2)—for example,
imatinib (4). Other studies have focused on evaluating
antiangiogenic agents for the treatment of ATC. These
agents include: combretastatin A4 phosphate, aplidin, and
human anti-vascular endothelial growth factor (VEGF)
and anti-epidermal growth factor receptor (EGFR) mono-
clonal antibodies (bevacizumab and cetuximab, respec-
tively) (5–9). Small-molecule ATP competitive inhibitors

ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2012 by The Endocrine Society
doi: 10.1210/jc.2011-1987 Received July 8, 2011. Accepted December 30, 2011.
First Published Online January 25, 2012
Abbreviations: ATC, Anaplastic thyroid cancer; EGF, epidermal growth factor; EGFR, EGF
receptor; FBS, fetal bovine serum; FCS, fetal calf serum; HMVEC-d, human dermal micro-
vascular endothelial cell; TFC, thyroid follicular cell; TKI, tyrosine kinase inhibitor; VEGF,
vascular endothelial growth factor; VEGFR-2, VEGF receptor type 2.

E528 jcem.endojournals.org J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536

J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536
directed against the intracellular EGFR tyrosine kinase, such as
erlotinib or gefitinib, are also under evaluation (5–9).
The development of drugs that have multiple therapeu-
tic targets and the use of multiple cancer-targeting agents
are both emerging strategies for cancer treatment. Indeed,
a preclinical study evaluated the activity of a dual inhib-
itor of EGFR and VEGF receptor-2 (VEGFR-2), NVP-
AEE788, alone and in combination with paclitaxel for the
treatment of aggressive thyroid cancer (10, 11).
Even if new therapeutic approaches against ATC are un-
der development, more research is needed to finally identify
therapies able to control and to cure this disease. Further-
more, the possibility of testing the sensitivity of primary ATC
cells from each subject to different drugs could increase the
effectiveness of the treatment in the future (12, 13).
Research aimed at the discovery of novel small molecules
as TKI led to the development of chemically different com-
pounds. Either obtained through rational design or derived
from natural products, they all share the ability to target the
highly conserved ATP binding site of the proteins, thus be-
having as ATP competitive inhibitors. Actually, their main
core template is generally represented by a planar, hydro-
phobic, heterocyclic scaffold, which mimics the purine ring
of ATP and accommodates into the adenine binding pocket
of the site. Moreover, to follow closely the binding pattern of
ATP, the core scaffold is always decorated with one to three
donor and/or acceptor hydrogen bond motifs, which are ex-
ploited to mimic the hydrogen bond interactions observed
between adenine and the backbone of the protein (14 –16).
Most of the known TKI use an amide fragment as the donor-
acceptor pair of hydrogen bonds. This key structural motif is
often inserted into five- to seven-member ring systems to give
cyclic amides endowed with potent inhibitory activity
against different members of the tyrosine kinase family (17).
These chemical templates became, therefore, challenging ref-
erence structures for the design and development of novel
TKI. Here, we report the in vitro and in vivo antitumor and
antiangiogenic activity of a novel cyclic amide, CLM94,
bearing the benzo[d]isothiazole scaffold as the main core,
which showed a remarkable efficacy in ATC.
Materials and Methods
Drugs, reagents, and cell lines
CLM94, 4-chloro-N-(1,1,3-trioxo-2,3-dihydrobenzo[d]iso-
thiazol-4-yl)benzamide (Fig. 1), was synthesized by C.L.M., S.S.,
and F.D.S. at the Department of Pharmaceutical Science of the
University of Pisa (Pisa, Italy). In brief, catalytic hydrogenation
of 4-nitro-1,1,3-trioxo-2,3-dihydrobenzo[d]isothiazole (18),
carried out with PtO2 䡠 H2O in absolute ethanol and at room
temperature and atmospheric pressure, provided the corre-
sponding 4-amino derivative, which led to the target inhibitor,
CLM94, by reaction with 4-chlorobenzoyl chloride in the pres-
jcem.endojournals.org E529
FIG. 1. A novel cyclic amide (CLM94), bearing the benzo[d]isothiazole
scaffold as the main core, has been used. The structure of the
compound is shown here.
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ence of triethylamine and in refluxing toluene [melting point:
⬎300 C. hydrogen-1 nuclear magnetic resonance, ␦, ppm: 7.79 –
7.57 (m, 4H), 7.96 (d, 2H), 8.69 (d, 1H), 11.62 (s, 1H). MS, m/e:
[M⫹]: 336(8), 139(100)]. CLM94 was dissolved in a stock so-
lution of 10 mM in 100% dimethylsulfoxide for in vitro studies,
and the same dimethylsulfoxide concentration was used as the
vehicle in control media. Recombinant human epidermal growth
factor (EGF), basic fibroblast growth factor, and VEGF were
from PeproTechEC Ltd. (London, UK). Cell culture media
MCDB131 and RPMI were purchased from Life Technologies,
Inc. (Paisley, UK), quantitative real-time PCR reagents were
from Applied Biosystems (Foster City, CA), and supplements
and all other chemicals not listed in this section were obtained
from Sigma-Aldrich (Milan, Italy). Human dermal microvascu-
lar endothelial cells (HMVEC-d; Clonetics, San Diego, CA) and
8305C (undifferentiated thyroid cancer, with papillary compo-
nent, cell line; DSMZ, Braunschweig, Germany) cell lines were
maintained in MCDB131 culture medium supplemented with
10% heat-inactivated fetal bovine serum (FBS), L-glutamine 2
mM, heparin 10 U/ml, EGF 10 ng/ml, and basic fibroblast growth
factor 5 ng/ml and in 15% FBS RPMI medium supplemented
with L-glutamine 2 mM, respectively.
In vitro studies on VEGFR-2
Assays were performed using recombinant human VEGFR-2
and Omnia Tyr Peptide 7 Kit (Invitrogen, San Diego, CA), as
reported previously (19).
Proliferation assay
The proliferation assay was performed as previously described
(20). Cells were plated and treated for 72 h with CLM94 (0.01 nM
to 100 ␮M and 0.001–100 ␮M, for endothelial and cancer cells,
respectively) or with its vehicle alone. At the end of the experiment,
the viable cells were counted with a hemocytometer. The data are
presented as the percentage of the vehicle-treated cells.
Cell-based phospho-VEGFR-2 and phospho-EGFR
inhibition assay
The experimental procedure was followed as previously de-
scribed (20). Briefly, HMVEC-d were maintained with 1% FBS
medium. After 24 h, cells were treated continuously for 72 h with
CLM94 or with vehicle alone. The media were supplemented
with recombinant human VEGF 10 ng/ml or recombinant hu-
man EGF 10 ng/ml. Endothelial cell lysates were assayed with
PathScan phospo-VEGFR-2 (Tyr1175) and Total VEGFR-2
sandwich ELISA kits (Cell Signaling Technology, Danvers, MA)
and with PathScan phospo-EGFR (Tyr1173) and Total EGFR
E530 Antonelli et al. CLM94 Activity in Anaplastic Thyroid Cancer
sandwich ELISA kits (Cell Signaling Technology). All experi-
ments were repeated independently six times with at least nine
samples for each concentration.
Primary ATC cells
Patient source for thyroid tissue
Surgical thyroid tissue was obtained from six patients with
ATC at the time of surgery. In addition, normal thyroid tissue
was obtained from six patients undergoing parathyroidectomy.
The diagnosis was established on commonly accepted clinical,
laboratory, and histological criteria (13, 21, 22). Immunohisto-
chemistry showed the absence of expression of TSH receptor,
thyroperoxidase, thyroglobulin, and sodium/iodide symporter.
Microdissection and DNA extraction, detection of BRAF
mutation by PCR single-strand conformation polymorphism,
and direct DNA sequencing were performed using conventional
methods previously described (13, 21, 22).
The study subjects gave their informed consent to the study,
which was approved by the local ethical committee.
ATC cell culture
ATC cells were prepared as reported previously (13, 21, 22).
Cells were used for tests at the fourth passage. Immunocytochem-
istry showed the absence of expression of TSH receptor, thyroper-
oxidase, thyroglobulin, and sodium/iodide symporter. The pres-
ence of cytokeratin was investigated by immunocytochemistry, and
a partial and focal positivity was obtained. DNA fingerprinting
showed a pattern identical to that of the original neoplastic tissue
(13, 21, 22).
Normal thyroid follicular cell (TFC) culture
Thyrocytes were prepared as reported previously (23).
Cell viability and proliferation assay
We used a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide (used in the MTT assay) assay (WST-1; Roche
Diagnostics, Almere, The Netherlands), as previously reported
(21–23), to determine cell proliferation. To determine IC50 of
CLM94, a concentration range of CLM94 has been added to the
wells (quadruplicates), and IC50 has been determined by using
linear interpolation. All experiments were performed in tripli-
cate for each cell preparation.
Proliferation assay: cell counting
The proliferation has also been evaluated using the cell num-
ber counting (see Cell viability and proliferation assay and Refs.
21–23).
Apoptosis determination—Hoechst uptake
ATC cells were seeded at a concentration of 35,000 cells/ml
in a final volume of 100 ␮l in each well. Then, cultures were
incubated for 48 h with CLM94 in a humidified atmosphere (37
C, 5% CO2), and stained with Hoechst 33342 as previously
described (23). The apoptosis index (ratio between apoptotic
and total cells) ⫻ 100 was calculated.
Apoptosis determination—Annexin V binding assay
The cells were plated in Lab-tekII Chamber Slide System
(Nalge Nunc International, Rochester, NY), treated with
CLM94 for 48 h, and processed as previously described (23).
J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536
Migration and invasion assays
Cell migration and invasion were determined by using a 96-
well Transwell Permeable Supports (Corning Life Sciences,
Corning, NY) according to manufacturer instructions, with mi-
nor modifications (24). Briefly, cells were starved for 5 h in se-
rum-free medium at 37 C and 5% CO2; they were harvested by
using a PBS, 5 mM EDTA solution; and the total cell number was
determined. Cells were centrifuged, resuspended in serum-free
medium, and seeded (0.5 ⫻ 105 cells per well).
For invasion assay, a basement membrane extract (Trevigen,
Gaithersburg, MD) solution was used to coat the inserts over-
night (37 C, 5% CO2) before cell seeding. Subsequently, fetal calf
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serum (FCS) 10% vol/vol or serum-free medium was added to
receiver wells, and where indicated, increasing concentrations of
CLM94 were added to both Transwell chambers for 24 h. Media
were removed, and a solution of calcein AM (2 ␮g/ml; Sigma-
Aldrich) was added for 1 h to the lower compartments. Intra-
cellular fluorescence was assessed by means of a 96-well plate
reader (ELISA reader) with filters set to 485 nm for excitation
and 520 nm for emission. A standard curve with different cell
concentrations was prepared for each assay to convert the flu-
orescence values to the number of migrated or invasive cells.
AF cell line and in vivo studies
AF cell line
From the six primary ATC cells, one could be passed over 50
times. This line (AF cell line) was able to grow in nu/nu mice when
inoculated sc (see AF xenografts in nu/nu mice and drug
treatments).
Real-time PCR analysis of VEGF-A gene expression
To evaluate the expression of the VEGF-A gene, 2 ⫻ 104 AF
cells were grown in their media and treated with CLM94 at
different concentrations (50, 26, and 5 ␮M) or with vehicle alone
for 72 h. As previously described (25), RNA was reverse-tran-
scribed, and the cDNA was amplified by quantitative real-time-
PCR with the Applied Biosystems 7900HT sequence detection
system. The VEGF-A-validated primer was from Applied Bio-
systems (Assay ID Hs00170236_m1). Amplifications were nor-
malized to glyceraldehyde 3-phosphate dehydrogenase, and the
quantitation of gene expression was performed using the ⌬⌬Ct
calculation; the amount of target was given as 2⫺⌬⌬Ct.
In vivo studies, animals
The CD nu/nu male mice, weighing 20 –25 g, were supplied
by Charles River Laboratories (Milan, Italy). Housing and all
procedures involving animals were performed according to the
protocol approved by the Academic Committee for Animal Ex-
perimentation of the University of Pisa.
AF xenografts in nu/nu mice and drug treatments
AF cell viability was assessed by trypan blue dye exclusion,
and on d 0, 1.3 ⫻ 106 cells per mouse were inoculated sc and
measured as previously described (25). The mice were random-
ized into groups of six animals. To treat an established tumor
(⬃50 mm3), on d 25 from cell inoculum, 40 mg/kg 䡠 d CLM94
was administered ip for 12 d. The control group was injected ip
with vehicle alone (saline solution). After 49 d from the cell
inoculums, mice were killed by an anesthetic overdose.
J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536
FIG. 2. A, Inhibition of VEGFR-2 and EGFR phosphorylation by CLM94
in HMVEC-d after 72 h of treatment. pVEGFR-2 and pEGFR
concentrations were measured by ELISA kits, and they were
normalized to total VEGFR-2 and EGFR protein concentration,
respectively. IC50 value, expressed as mean ⫾ SD, was determined by
linear regression analysis of the log-dose-response curve, generated
using at least five concentrations of CLM94, causing an inhibition
between 20 and 80%, with three replicates at each concentration.
Columns and bars, mean ⫾ SD, respectively. B and C, In vitro
chemosensitivity in the microvascular endothelial cell line HMVEC-d (B)
or in the undifferentiated thyroid cancer, with papillary component,
cell line 8305 (C), treated with CLM94 for 72 h. The HMVEC-d were
sensitive to lower concentrations of CLM94, which also showed a
concentration-dependent effect on the immortalized 8305C cell line.
IC50 vs. controls were calculated by nonlinear regression fit of the
mean values of the data obtained in triplicate experiments (at least
nine wells for each concentration). *, P ⬍ 0.05 vs. vehicle-treated
controls.
jcem.endojournals.org E531
Immunohistochemistry, microvessel density on
tumor tissue
Tumor tissue from the two different treatment groups was
weighed, then fixed in formalin, and embedded in paraffin. Sec-
tions of the tumor (5-␮m thick) were stained with hematoxylin
and eosin. Immunostainings were performed as previously re-
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FIG. 3. A and B, WST-1 assay (at 2 h from the start of tetrazolium
reaction; mean ⫾ SD of all samples) in ATC cells (A) or in normal TFC
(B) treated with CLM94 for 24 h. A significant reduction of
proliferation with respect to the control was shown with CLM94 10,
30, and 50 ␮M in panel A and with 30 or 50 ␮M in panel B. Bars
represent mean ⫾ SD. *, P ⬍ 0.05 or less vs. control by Bonferroni-
Dunn test. C, Apoptosis in ATC cells treated with CLM94 for 48 h
(mean ⫾ SD of all samples). Apoptosis index was determined by
Hoechst staining (see Materials and Methods). The percentage of
apoptotic cells increased markedly in a dose-dependent manner with
CLM94 10, 30, and 50 ␮M. Data are expressed as means ⫾ SD (n ⫽ 6).
Data were analyzed by one-way ANOVA with Newman-Keuls multiple
comparisons test and with a test for linear trend. *, P ⬍ 0.001 vs.
control.
E532 Antonelli et al. CLM94 Activity in Anaplastic Thyroid Cancer J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536

ported (24). The expression of VEGF was evaluated as a per-
centage of positive cells over a total of at least 1000 tumor cells.
Microvascular count was determined using anti-FVIII poly-
clonal antibody (Ventana Medical Systems, Tucson, AZ) as pre-
viously described (24).
Data analysis
Values are given as mean ⫾ SD for normally distributed vari-
ables or as median and interquartile range. The experiments were
repeated three times with the cells from each donor. The mean of
the experiments in the six specimens from different donors for nor-
mal and ATC samples is reported. The mean
group values were compared by one-way
ANOVA for normally distributed variables
or by the Mann-Whitney U test or Kruskal-
Wallis test. Proportions were compared by
the ␹2 test. Post hoc comparisons on normally
distributed variables were carried out using

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the Bonferroni-Dunn test. Data about apo-
ptosis were analyzed by one-way ANOVA
with Newman-Keuls multiple comparisons
test.

Results
In vitro studies on VEGFR-2 and
human cell lines
CLM94 inhibits VEGFR-2 and EGFR
tyrosine kinase activity and cell
proliferation
CLM94 proved to inhibit in vitro the
tyrosine kinase activity of VEGFR-2,
with an IC50 value of 34.9 ⫾ 2.4 ␮M.
Moreover, after exposure to CLM94 at
different concentrations, the amount of
both phosphorylated forms of VEGFR-2
and EGFR in HMVEC-d cell lysates
(Fig. 2A) was significantly reduced.
Thus, a significant concentration-de-
pendent inhibitory activity on HM-
VEC-d proliferation after 72 h (Fig. 2B)
was demonstrated with a calculated
IC50 of 1.59 ⫾ 1.11 ␮M. Moreover,
CLM94 also showed a concentration-
dependent antiproliferative effect on
the immortalized 8305C cell line with
an IC50 of 25.8 ⫾ 7.99 ␮M (Fig. 2C).

In vitro studies on primary

ATC cells

FIG. 4. A, For migration, 12 h of incubation was used; for comparison, the inhibition of Cell proliferation assays
proliferation (at 12 h) (% with respect to control without CLM94) and the inhibition of The results of WST-1 assay in ATC
migration are reported in the table below the figure. It is possible to observe that the cells showed a significant reduction of
inhibition of migration is higher than the inhibition of proliferation. B, For invasion, 24 h of
incubation was used; for comparison the inhibition of proliferation (at 24 h) (% with respect
proliferation with respect to the control
to control without CLM94) and the inhibition of invasion are reported in the table below the with CLM94 at 1 and 2 h (P ⬍ 0.01, for
figure. It is possible to observe that the inhibition of invasion is higher than the inhibition of both, ANOVA) (Fig. 3A). The cell
proliferation overall at CLM94 10 ␮M, and slightly at 30 ␮M. At higher concentrations the
counting confirmed the above-men-
inhibitory effect on proliferation is more important than that on invasion. Bars represent
mean ⫾ SD. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 with respect to FCS 10% (FCS 10% ⫽ tioned results at 2 h. In ATC, the cell
medium ⫹ FCS 10%) by Newman-Keuls test. number was 19,560 ⫾ 820/100 ␮l per
J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536
well; 17,260 ⫾ 1,140 (88%) with CLM94 10 ␮M;
11,370 ⫾ 960 (58%) with CLM94 30 ␮M; and 8,670 ⫾
1,040 (44%) with CLM94 50 ␮M (P ⬍ 0.01, ANOVA).
The IC50 values determined by using linear interpolation
were 38 ␮M for CLM94.
The results of WST-1 assay in normal TFC with
CLM94 showed a slight but significant reduction of pro-
liferation with respect to the control at both 1 and 2 h (P ⬍
0.01, for both, ANOVA) with CLM94 30 and 50 ␮M (Fig.
3B). The cell counting confirmed the above-mentioned
results (data not shown).
BRAF and proliferation
The V600EBRAF mutation was observed in two ATC;
RET/PTC1 and RET/PTC3 by real-time PCR were not
detected in primary cells from ATC. The results about the
inhibition of proliferation by CLM94, obtained in ATC
from tumors with V600EBRAF mutation, were similar to
those from tumors without BRAF mutations (data not
shown).
Apoptosis determination
The percentage of apoptotic cells in ATC cells increased
dose-dependently; after treatment with CLM94 10 ␮M,
6.4% of the cells were apoptotic, and this percentage in-
creased up to 12.4 and 17.4% with CLM94 30 or 50 ␮M,
respectively (P ⬍ 0.001 by ANOVA; Fig. 3C). Annexin V
was used to further confirm the induced cell apoptosis
(data not shown).
Migration and invasion assays
ATC have been cultured subconfluent and treated with
increasing concentrations of CLM94. Migration and in-
vasion were tested in Transwell chambers (Corning Life
Sciences), revealing an inhibition of migration (Fig. 4A)
and of invasion (Fig. 4B) with CLM94.
AF cells and in vivo studies
CLM94 inhibits the expression of VEGF-A in AF cancer
cells
After 72 h of exposure, CLM94 significantly decreased
VEGF-A expression in the AF cell line; in particular,
CLM94 significantly decreased VEGF-A at higher con-
centrations (P ⬍ 0.05; Fig. 5).
In vivo studies—CLM94 inhibits AF tumor growth in
absence of toxicity
Tumor masses became detectable 25 d after xeno-
transplantation and in control animals showed a pro-
gressive enlargement of their dimensions, although
slower until d 33, and at d 49 the animals of the control
and treated groups were killed (Fig. 6). CLM94 (40
jcem.endojournals.org E533
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FIG. 5. VEGF-A gene expression after CLM94 treatment (50, 26, and
5 ␮M) of AF cell line for 72 h (mean ⫾ SD of all samples). CLM94
significantly decreased VEGF-A expression in the AF cell line; in
particular, CLM94 significantly decreased VEGF-A at higher
concentrations. Bars represent mean ⫾ SD. *, P ⬍ 0.05 vs. control.
mg/kg 䡠 d) inhibited significantly the tumor growth,
starting on the 35th day after cell implantation (10 d
after the beginning of treatment), compared with con-
trols (Fig. 6A). Moreover, the explanted tumors con-
firmed a significant reduction of CLM94-treated group
volumes if compared with controls (2013.2 ⫾ 577.8 vs.
826.5 ⫾ 228.6 mm3, respectively; P ⬍ 0.05; Fig. 6B).
The CLM94-treated group of animals did not show any
toxicity (Fig. 6C).
CLM94 decreases the VEGF-A expression and
microvessel density in AF tumor tissues
AF cancer cells produced a tumor whose histological
picture was consistent with ATC. A well-defined VEGF-A
immunoreactivity was localized in tumor cells of the con-
trol tumor mass (Fig. 6D), but it decreased after CLM94
treatment (Fig. 6E) (76.7 ⫾ 10.3 vs. 43.3 ⫾ 5.3%), with
a simultaneous increase of necrosis (58.3 ⫾ 7.6, vs. con-
trols 33.3 ⫾ 5.2%; P ⬍ 0.05; Fig. 6, F and G) and a
reduction of microvessel density (6.4 ⫾ 1.0, vs. controls
13.4 ⫾ 1.7; P ⬍ 0.05; Fig. 6, H and I).
Discussion
In this study we described for the first time the synthesis
and the antitumor and antiangiogenic activity in ATC cells
of a novel cyclic amide, CLM94, bearing a benzo[d]iso-
thiazole scaffold. Different heterocycles characterized by
cyclic amide moieties (staurosporine, pyridone 6, etc.)
have been developed as potent protein kinase inhibitors
targeting the ATP binding pocket. Despite different mo-
lecular shapes (pentacycle, tetracycle, respectively), they
all bind to the residues of the hinge region at ATP binding
E534 Antonelli et al. CLM94 Activity in Anaplastic Thyroid Cancer J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536

(27). Pyridone 6, a pan-Janus-activated

kinase inhibitor, induces growth inhi-
bition of multiple myeloma cells (28).
7-Hydroxystaurosporine (UCN-01) is
a selective protein kinase C inhibitor
that has been evaluated in four thyroid
carcinoma lines (FRO, KAT5, NPA,
and WRO). UCN-01 activated apopto-
sis in these cells, and the expression of
Bcl-2 was inversely related to suscepti-
bility to UCN-01 (29). Moreover,

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UCN-01, at clinically relevant concen-
trations, exerted an antineovascular-
ization effect by blocking the response
of cancer cells to hypoxia and the en-
dothelial cell proliferation (30).
To the best of our knowledge, until
now, cyclic amide derivatives have not
been tested as antineoplastic agents in
thyroid cancer primary cells. Interest-
ingly, CLM94 exhibits antineoplastic
activity in vitro both in human 8305C
undifferentiated thyroid cancer (EGFR
positive) cell line and in primary cells
of ATC by increasing apoptosis. The
antiproliferative action of CLM94 is
shown in all primary ATC cells, inde-
pendently from the presence or absence
of V600EBRAF mutation. The direct an-
titumor activity could be due, at least in
part, to the inhibition of VEGFR-2 and
EGFR phosphorylation also in tumor
FIG. 6. AF cells were injected sc in CD nu/nu mice, and tumor masses became detectable cells, analogously as in endothelial
25 d after xenotransplantation. A, CLM94 (40 mg/kg 䡠 d) inhibited significantly the tumor cells. Interestingly, the CLM94 IC50
growth compared with controls. B, When the animals were killed at the end of the values for tumor cell proliferation were
experiment, tumors were explanted and measured, confirming a significant reduction of
CLM94-treated tumor volumes if compared with controls (P ⬍ 0.05). C, The CLM94-treated
38 ␮M in primary ATC cells and 25.8
group of animals did not show any appreciable toxicity as demonstrated from their weights. ␮M in 8305C, which were similar to
D, The sc injection of AF cancer cells produced a tumor whose histological picture, after those of different TKI, evaluated in hu-
staining with hematoxylin and eosin, was consistent with ATC. A well-defined VEGF-A
man thyroid cancer cell lines (24, 31,
immunoreactivity was localized in tumor cells of the control tumor mass (D), but it decreased
after CLM94 treatment (E). The necrosis areas were present in both control (F) and CLM94- 32) that have dual or multiple mecha-
treated tumors (G); however, necrosis in CLM94-treated tumor samples was greatly enhanced nisms of action (e.g. VEGFR and EGFR
as shown in this representative microscopic picture (G). Moreover, the microvessel density of inhibitors). However, it is not possible
control tumor tissues (H) was markedly decreased after CLM94 treatment (I). Bars represent
mean ⫾ SD. *, P ⬍ 0.05 vs. control. to exclude additional direct effects of
CLM94 mediated by the inhibition of
pocket of protein kinases in a very similar manner; the
other kinases.
cyclic amide moiety occupies the adenine region of ATP
The antineoplastic activity of CLM94 may result from
binding pocket and forms at least two hydrogen-bonding
the combination of an antiproliferative effect associated
interactions with the hinge region via its hydrogen bond
acceptor/donor pair (17). Staurosporine is a therapeutic with the increase of apoptosis in the tumoral cells, the
antineoplastic agent that in human leukemic cell lines in- inhibition of the migration and invasion, and furthermore,
duces apoptosis and inhibits proliferation and survival the inhibition of the neoplastic neovascularization.
(26). Furthermore, staurosporine induces apoptosis in In fact, CLM94 has a significant antiangiogenic effect
solid tumors, such as the neuroblastoma cell line SH-SY5Y both in vitro and in vivo. The main mechanism of the
J Clin Endocrinol Metab, April 2012, 97(4):E528 –E536
neovascular inhibition was related to the demonstrated
activity of CLM94 against VEGFR-2 phosphorylation at
the level of both isolated protein and cell system. This
inhibition determined the antiproliferative effects on en-
dothelial cells both in vitro and in vivo, as shown by the
microvessel density decrease. Moreover, another possible
indirect antiangiogenic mechanism of CLM94 could be
due to the marked decrease of the gene expression of
VEGF-A, a proangiogenic factor, in AF cancer cells, that
was reflected in the significant decrease of VEGF-A pro-
tein in AF tumor tissues. Interestingly, CLM94 signifi-
cantly inhibited AF tumor growth in CD nu/nu mice in the
absence of toxicity, whereas other compounds have a
broad spectrum of adverse effects both in animals and
humans (33).
Despite the very promising initial results, TKI are not
likely curative, and maintenance of effect may require con-
tinuous therapy (34). Although it is possible that a signif-
icant number of patients will have prolonged responses
with continued therapy, it is clear that TKI as mono-
therapy will not be curative to most endocrine cancer (35).
A new and evolving technology is the simultaneous study
of multiple signaling pathways, analyzing for mutation,
expression, and phosphorylation of these pathways (33,
36, 37). However, there are still some limitations in the
selective use of novel compounds. There might be several
potential targets in a given tumor, and most compounds
can hit different targets, so the most important target for
a given tumor might remain uncertain. Even when poten-
tial targets (such as BRAF) are present in the tumor tissue,
tumor response might be observed in only a fraction of
patients. A lack of response can occur, for instance, be-
cause target inhibition raises the activity of compensatory
signal pathways, which, in turn, rescue tumor cell growth.
However, the possibility of testing the sensitivity of pri-
mary ATC cells from each subject to different TKI could
increase the effectiveness of the treatment. In fact, in vitro
chemosensitivity tests are able to predict in vivo effective-
ness in 60% of cases (33), whereas it is well known that a
negative chemosensitivity test in vitro is associated with
90% of ineffectiveness of the treatment in vivo (33), al-
lowing the administration of inactive chemotherapeutics
to these patients to be avoided (12, 21, 38). Interestingly,
our results show an antitumor effect of CLM94, not only
on continuous cell lines such as the 8305C, that could be
quite different from the tumor of the patients themselves,
but also directly on primary ATC cells of patients. More-
over, we have produced a new cell line (AF) derived from
primary ATC cells. This line (AF) was able to grow in
nu/nu mice when inoculated sc. The AF cell line could be
a valuable tool in the near future for the evaluation of the
jcem.endojournals.org E535
antineoplastic activity of other drugs, both in vitro and in
vivo.
In conclusion, the antitumor and antiangiogenic activ-
ity of a new “cyclic amide” compound CLM94 is very
promising in ATC, opening the way to a future clinical
evaluation.
Acknowledgments
The authors thank Dr. Caterina Mancusi and, Dr. Alda Corrado
Downloaded from https://academic.oup.com/jcem/article-abstract/97/4/E528/2833722 by guest on 16 May 2020
(Department of Internal Medicine, University of Pisa, School of
Medicine, Pisa, Italy), and Dr. Paola Orlandi and Dr. Bastianina
Canu (Division of Pharmacology, University of Pisa, School of
Medicine, Pisa, Italy).
Address all correspondence and requests for reprints to:
Alessandro Antonelli, M.D., Department of Internal Medicine,
University of Pisa, School of Medicine, Via Roma, 67, 56100,
Pisa, Italy. E-mail: alessandro.antonelli@med.unipi.it.
Disclosure Summary: The authors have nothing to disclose.
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