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Published OnlineFirst May 31, 2007; DOI: 10.1158/1535-7163.MCT-06-0706
Published Online First on May 31, 2007 as 10.1158/1535-7163.MCT-06-0706
OF1
Suppression of lung cancer tumor growth in a
nude mouse model by the Ras inhibitor
salirasib (farnesylthiosalicylic acid)
Adi Zundelevich, Galit Elad-Sfadia, Ronit Haklai,
and Yoel Kloog
Department of Neurobiochemistry, The George S. Wise Faculty of
Life Sciences, Tel Aviv University, Tel Aviv, Israel
Abstract
Aberrant Ras pathway functions contribute to the malignant phenotype of lung cancers. Inhibitors of Ras might
therefore be considered as potential drugs for lung cancer
therapy. Here, we show that the Ras inhibitor farnesylthiosalicylic acid (salirasib) inhibits proliferation of human
lung cancer cells harboring a mutated K-ras gene (A549,
H23, or HTB54) or overexpressing a growth factor
receptor (H1299 or HTB58) and enhances the cytotoxic
effect of the chemotherapeutic drug gemcitabine. Salirasib inhibited active K-Ras in A549 cells, reversed their
transformed morphology, and inhibited their anchorageindependent growth in vitro. Tumor growth in A549 and
HTB58 cell nude mouse models was inhibited by i.p.
administration of salirasib. P.o. formulated salirasib also
inhibited A549 cell tumor growth. Our results suggest
that p.o. salirasib may be considered as a potential
treatment for lung cancer therapy. [Mol Cancer Ther
2007;6(6):OF1 – 9]
Introduction
Lung cancer is the leading cause of cancer-related deaths
(1). Despite advances in surgery, chemotherapy, and
radiotherapy, survival rates have hardly changed in the
last decade, and long-term survival rates remain extremely
poor. It has been established that lung cancer arises as a
consequence of the accumulation of multiple genetic
changes involving critical genes that control cell motility,
proliferation, differentiation, and apoptosis (2). Point
mutations of the K-ras gene and overexpression of cyclin
Received 11/14/06; revised 2/19/07; accepted 5/2/07.
Grant support: The Wolfson Foundation.
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.
Note: Y. Kloog is the incumbent of The Jack H. Skirball Chair for Applied
Neurobiology.
Requests for reprints: Yoel Kloog, Department of Neurobiochemistry,
The George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978
Tel Aviv, Israel. Phone: 972-3-640-9699; Fax: 972-3-640-7643.
E-mail: [email protected]
D1 are thought to be early events during carcinogenesis of
some types of lung cancer (3, 4). Most lung neoplasms are
bronchogenic in origin. The four typical subgroups of these
lung cancers are squamous cell carcinoma, large cell
carcinoma, small-cell carcinoma, and adenocarcinoma (5).
Non – small cell lung carcinoma (NSCLC) represents 85% of
cases of lung cancer (6).
The notion that Ras signaling contributes to the maintenance of the transformed phenotype of lung cancer cells
(7 – 9) is supported by the high incidence of growth factor
receptor (e.g., epidermal growth factor receptor) expression
and, as mentioned above, the significant incidence of Ras
mutations and up-regulated cyclin D1 in these tumor cells.
It is therefore reasonable to suggest that treatment of lung
cancer with Ras inhibitors could be beneficial. Our objective
in this study was to develop a novel rational strategy for
the treatment of lung cancer by using the Ras inhibitor
salirasib [farnesylthiosalicylic acid (FTS); refs. 10, 11]. FTS
(salirasib) is a potent Ras inhibitor that acts in a rather
specific manner on the active, GTP-bound forms of H-Ras,
N-Ras, and K-Ras proteins (10, 11). It competes with RasGTP for binding to specific saturable binding sites (12) in
the plasma membrane, resulting in mislocalization of active
Ras, and, in some but not all tumor cell lines, it facilitates
Ras degradation (12). This competition with FTS (salirasib)
prevents active Ras from interacting with its prominent
downstream effectors and results in reversal of the transformed phenotype in transformed cells that harbor activated Ras (11). As a consequence, Ras-dependent cell growth
and transforming activities, both in vitro and in vivo, are
strongly inhibited (10, 11).
Whether FTS can inhibit growth of lung cancer cells and
tumor growth was not known. This is an important
question that we attempted to answer in the present study.
Its importance must be viewed in light of the extremely
poor long-term survival of lung cancer patients and in light
of lack of any efficient nontoxic drug for the treatment of
lung cancer. The commonly used treatments of lung cancer
patients include cytotoxic drugs and their combinations
(13). These treatments are restricted by dose-limiting side
effects (13, 14). Animal studies (11, 15, 16) and initial
human trials1 showed that FTS is a relatively safe
compound with no adverse side effects. Our objectives
were then to examine whether FTS can exert anti-Ras
activity in lung cancer cell lines and inhibit their growth
in vitro and in animals. Here, we show for the first time that
p.o. FTS (salirasib) inhibited A549 lung cancer cell tumor
growth.
Copyright C 2007 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-06-0706
1
http://www.concordiapharma.com/index.htm
Mol Cancer Ther 2007;6(6). June 2007
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OF2 Ras Inhibitor (Salirasib) Inhibits Lung Cancer Growth
Materials and Methods
Cell Lines
All cell lines were obtained from American Type
Culture Collection (ATCC) and grown in medium
containing 10% FCS and antibiotics as recommended by
ATCC. The following cell lines were used: A549 cells,
NSCLC (CCL, ATCC); HTB54 lung carcinoma cells;
HTB58 (SK-MES-1, ATCC), a human lung squamous cell
carcinoma cell line; H23 (NCI-H23, ATCC), a human non –
small cell lung adenocarcinoma cell line; and H1299 (NCIH1299, ATCC), a NSCLC cell line. The cells were plated in
24-well plates in 1 mL medium at a density of 5,000 cells
per well (or 2,500 cells per well, HTB54) and incubated at
37jC in a humidified atmosphere of 95% air and 5% CO2.
Cells were treated with the indicated concentrations of
FTS (Concordia Pharmaceuticals) or with 0.1% Me2SO4
(vehicle) 24 h after plating and counted 5 days later. Dead
cells were counted after addition of Hoechst 33258
dye (1 Ag/mL; Sigma-Aldrich) to vehicle-treated control
cultures or to cultures treated for 24 or 48 h with
75 Amol/L FTS. Fluorescence images were collected 5 min
after the dye was added. In drug combination experiments, cells were grown for 2 days in the absence or in
the presence of 40 Amol/L FTS and then treated for 4 h
with gemcitabine (100 or 200 nmol/L), cisplatin (50 or
100 nmol/L), doxorubicin (50 or 100 nmol/L), or paclitaxel
(2.5 or 5 nmol/L). Live cells were counted after a further
3 days of incubation with or without FTS. Experiments were
done twice in quadruplicate.
Data obtained in these and in all subsequent experiments
were subjected to statistical analysis (t test). Synergism was
determined by calculating the percentage of growth
inhibition by a single drug treatment and that of growth
inhibition by the combined treatment. The sum of
percentage inhibition observed in each of the single drug
treatments was compared with that of the combined
treatment. Statistically significant higher levels in the latter
were considered as synergism.
Bromodeoxyuridine Incorporation into DNA
A549 cells were plated on glass coverslips (1.2 105 cells
per well in six-well plates) and incubated for 24 h in
medium containing 5% FCS. The cells were then incubated
for 24 h with or without 75 Amol/L FTS and then for
24 h with bromodeoxyuridine (BrdUrd; Zymed BrdUrd
labeling kit; 1:100 dilution). Cells were fixed with 4%
paraformaldehyde, permeabilized with 0.2% Triton X-100
(BDH), washed with PBS, blocked with TBS Tween
[50 mmol/L Tris (pH 7.4), 100 mmol/L NaCl, 0.1% Tween
20] containing 1% bovine serum albumin, treated sequentially with 2 N HCl and 0.1 mol/L sodium borate (pH 8.5),
and then blocked with goat g-globulin and washed with
TBS Tween-bovine serum albumin (described above). The
cells were then labeled successively with mouse antiBrdUrd antibody (Zymed kit; 1:50 dilution), biotinylated
rabbit anti-mouse IgG (5 Ag/mL), and Cy3-streptavidin
(1.5 Ag/mL). Cells with BrdUrd-stained nuclei were
counted under a fluorescence microscope.
Fluorescence-Activated Cell Sorting Analysis
A549 cells were plated (9 105 cells) in 10-cm plates,
incubated for 24 h in medium containing 5% FCS, and then
incubated for 24 or 48 h with or without 75 Amol/L FTS.
The cells were collected, resuspended in PBS containing
propidium iodide (50 Ag/mL; Sigma-Aldrich) and 0.05%
Triton X-100, and subjected to analysis by a fluorescenceactivated cell sorter (FACSCalibur; Becton Dickinson).
Immunofluorescence and Confocal Microscopy
A549 cells were plated on glass coverslips (2 104 cells
per well in six-well plates), incubated for 24 h in medium
containing 5% FCS, and then incubated for 48 h with or
without 75 Amol/L FTS. The cells were fixed and
permeabilized at room temperature by successive incubations with 3.7% formaldehyde (20 min) and 0.2% Triton
X-100 in PBS (5 min), then washed for 5 min with UB
buffer [150 mmol/L NaCl, 10 mmol/L Tris (pH 7.6), 0.2%
sodium azide in PBS], and blocked with 2% bovine serum
albumin in UB (UBB, 5 min). The fixed cells were
incubated successively with naive goat IgG for 30 min
(200 Ag/mL; Jackson ImmunoResearch Laboratories), antivinculin antibody for 1 h (1:400; Sigma-Aldrich), goat antimouse Cy2-conjugated antibody for 1 h (1:200; Jackson
ImmunoResearch Laboratories), and rhodamine-labeled
phalloidin for 1 h (1:1,000; Sigma-Aldrich). Between each
of the above steps, the cells were washed for 30 min with
UBB. Last, the coverslips were washed with UB, dried,
and mounted onto the slides with Muviol. F-actin (red)
and vinculin (green) were visualized with a Zeiss LSM
510 confocal microscope fitted with nonleaking green and
red fluorescence filters. Colocalization was assessed using
the colocalization function of the LSM 510 software. To
assess the extent of the effect of FTS on stress fibers, we
did a statistical analysis as follows. Fluorescent images of
three separate experiments were collected (altogether 23
fields of control and of FTS-treated cultures). The total
number of cells and the number of cells exhibiting strong
stress fibers were then double-blind scored. The means F
SD ratios of stress fiber containing cells/total number of
cells were then calculated and subjected to statistical
analysis (t test).
Anchorage-Independent Colony Formation Assay in
Soft Agar
Noble agar (2% and 0.6%; Difco) was prepared in water
and autoclaved. The 2% agar was melted in a microwave
oven, mixed 1:1 with medium (2 Kaighn’s modification of
Ham’s F-12 medium with 20% FCS, 100 units/mL penicillin,
and 0.1 mg/mL streptomycin), and poured onto 96-well
plates (50 AL/well) to provide the 1% base agar. The 0.6%
agar (5 mL) was mixed with 5 mL medium (2), containing
8 104 A549 cells, and the mixture (50 AL) was plated on top
of the base agar. The cells were incubated for 19 days at
37jC with or without the indicated concentrations of FTS
(six wells for the control and for each treatment) and
colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (1 mg/mL for 4 h). The
colonies were then visualized by light microscopy, imaged,
and counted using the ImagePro software.
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Molecular Cancer Therapeutics
Ras, Rac, and Rho Pull-Down Assays and Immunoblotting Procedures
A549 cells were incubated for 24 h with or without FTS, as
described above, and then lysed with lysis buffer as
described previously (17). The apparent amounts of K-RasGTP in 0.5 mg protein of total cell lysates were determined
by the glutathione S-transferase Ras-binding domain of
Raf pull-down assay, as described elsewhere (18, 19). The
apparent amounts of Rac1-GTP and of RhoA-GTP, each in
2 mg protein of total cell lysates, were determined, respectively, by pull-down assays with glutathione S-transferase Rac1-binding domain of PAK1 – conjugated beads and
glutathione S-transferase Rho-binding domain of Rhotekin – conjugated beads (20, 21). The pulled-down GTPases
were subjected to SDS-PAGE followed by immunoblotting
with the appropriate antibodies: anti K-Ras (1:30; Calbiochem), anti Rac1 (1:2,500; Santa Cruz Biotechnology), or antiRhoA (1:700; Upstate Biotechnology). Immunoblots were
exposed to 1:2,500 peroxidase – goat anti-mouse IgG. Levels
of phospho – extracellular signal-regulated kinase (ERK) and
phospho-Akt were determined by immunoblotting as
described earlier (12) using rabbit anti – phospho-ERK1/2
antibody (Santa Cruz Biotechnology) and rabbit anti –
phospho-Akt antibody (Cell Signaling). Protein bands were
visualized by enhanced chemiluminescence and quantified
by densitometry using ImageJ computer software (NIH).
Animal Studies
Athymic nude mice (6 weeks old) were housed in barrier
facilities on a 12-h light/dark cycle. Food and water were
supplied ad libitum. On day 0, A549 or HTB58 cells (5 106
cells in 0.1 mL PBS) were implanted s.c. just above the right
femoral joint. After 5 or 11 days, the mice were separated
randomly into control groups that had received only the
vehicle and salirasib (FTS) – treated groups. Daily salirasib
(FTS) treatments were given either i.p. or p.o. Tumor
volumes or weights were determined as described previously (16). Gemcitabine treatment (36 mg/kg, i.p.) was
given every 4 days.
Results
FTS (Salirasib) Inhibits Growth of Lung Cancer Cells
We first examined the effect of FTS on the proliferation of
A549 cells that harbor activated K-ras gene mutated in
codon 12 (22). Incubation of the cells with 75 Amol/L FTS
for 48 h inhibited the incorporation of BrdUrd into their
DNA by 56.7 F 17.4% relative to vehicle-treated control
cells (P < 0.05; Fig. 1A). Notably, the treatment also caused
Figure 1. FTS (salirasib) inhibits growth of A549 cells. A, inhibition of BrdUrd incorporation into the DNA of A549 cells. Cells were plated in six-well
plates (1.5 105 cells per well), incubated for 48 h with 0.1% Me2SO4 (control) or 75 Amol/L FTS, and then assayed for BrdUrd incorporation using mouse
anti-BrdUrd antibody/biotinylated rabbit anti-mouse IgG/Cy3-streptavidin as described in Materials and Methods. Left, typical fluorescence images of the
cells; right, quantitative analysis of the results. The numbers of BrdUrd-stained cells relative to the total numbers of cells in a given field of control (100%)
and of salirasib-treated cells were calculated. Columns, mean of the values obtained from four experiments; bars, SD. *, P < 0.05. B, inhibition of A549
cell growth. A549 cells (5 103 cells per well) were grown in the presence of 0.1% Me2SO4 (control) or the indicated concentrations of FTS for 6 d and
then counted as described in Materials and Methods. The number of cells in the FTS-treated cultures is expressed as a percentage of the number recorded
in the controls. Columns, mean of 12 counts; bars, SD. *, P < 0.01; **, P < 0.0005, compared with control. C, fluorescence-activated cell sorting
analysis of FTS-treated A549 cells. Cells (0.9 106 cells/10-cm plate) were grown for 24 or 48 h in the presence of 0.1% Me2SO4 (control) or 75 Amol/L
FTS and then analyzed by fluorescence-activated cell sorting as described in Materials and Methods. Cell cycle distributions of control and FTS-treated cells
are shown for a typical experiment. Similar results were obtained in three independent experiments.
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OF4 Ras Inhibitor (Salirasib) Inhibits Lung Cancer Growth
a significant change in cell morphology; the treated cells
were large and flat, an appearance typical of transformed
cells treated with FTS (15, 16). Direct counting of A549 cells
grown for 6 days in the presence of FTS showed that the
drug had induced a dose-dependent decrease in their
number, with a decrease of 50% at 40 Amol/L FTS (Fig. 1B).
This decrease could be attributed mainly to inhibition of
cell proliferation because under the conditions used in
these experiments, cell death (indicated by Hoechst
staining), both in the absence and in the presence of FTS,
was very low (between 2% and 6%; data not shown).
Fluorescence-activated cell sorting analysis confirmed these
observations by showing that the apoptotic (sub-G1) cell
population was 3.8% at 24 h and 8.2% at 48 h in cells treated
with 75 Amol/L FTS compared with 1.0% and 2.8%,
respectively, in control cells (Fig. 1C). The fluorescenceactivated cell sorting analysis also showed that FTS caused
a reduction in the population of G1 but not of G2-M cells.
Cells treated with FTS for 24 and 48 h showed reductions in
G1 of 2.2% and 7.7%, respectively (Fig. 1C). Thus, FTS
evidently induced cell cycle arrest in A549 cells, resulting in
inhibition of cell growth with no significant effect on cell
death.
The growth-inhibitory effects of FTS were not limited to
the A549 lung cancer cells. This was evident from
experiments in which we examined the effects of FTS on
growth of other lung cancer cell lines (i.e., H23, HTB54,
H1299, and HTB58; SK-MES-1). The first two, but not the
last two, are known to harbor oncogenic K-Ras. H1299 and
HTB58, however, express relatively large amounts of
epidermal growth factor and insulin-like growth factor
receptors, which activate Ras (23, 24). FTS dose-dependent
inhibition of HTB54 cells is shown as an example (Fig. 2A).
The IC50 values of FTS were determined in the various
cell lines (summarized in Fig. 2B) and ranged from 30 to
75 Amol/L.
FTS (Salirasib) Alters Cytoskeleton Organization and
Inhibits the Anchorage-Independent Growth of A549
Cells
We considered the possibility that the observed FTSinduced change in A549 cell morphology is associated with
the known effects of FTS on cytoskeleton organization
(15, 16). That this was indeed the case was evident in A549
cells that were incubated with or without 75 Amol/L FTS
for 48 h and then stained with rhodamine-labeled
phalloidin (which labels polymeric F-actin) and with antivinculin antibody (which labels focal adhesions). Typical
fluorescence images of control and of FTS-treated cells are
shown in Fig. 3. The untreated cells exhibited short, thin
actin stress fibers (red fluorescence) and relatively few focal
adhesions (green fluorescence), whereas the FTS-treated
cells exhibited long, thick stress fibers and a relatively large
number of focal adhesions that looked larger than those
observed in the control cells. Statistical analysis (see
Materials and Methods) indicated that >80% of the cells
in the FTS-treated cultures had undergone changes in cell
morphology (n = 23; P < 0.0001). These results and the
growth-inhibitory effects of FTS observed in the lung
Figure 2.
FTS (salirasib) inhibits cell growth of various lung cancer cell
lines. Cells were grown for 5 d with the indicated concentrations of FTS
as shown in Fig. 1B and then counted. A, histograms showing typical
growth inhibition of HTB54 cells. The numbers of cells in the FTS-treated
cultures are expressed as percentages of the number recorded in the
control. Columns, mean of 12 counts; bars, SD. *, P < 0.005; **, P <
0.0001, compared with control. B, IC50 values of growth inhibition
induced by FTS in the indicated cell lines were determined from the growth
inhibition histograms shown in (A). Status of ras gene mutations in the
cell lines.
cancer cell lines suggested that the Ras inhibitor had at least
partially reversed the transformed phenotype of the cells.
We thus examined whether active K-Ras-GTP and its
prominent downstream signals to ERK and Akt are
inhibited in A549 cells and, if so, whether the anchoragedependent growth of the cells is affected as well. We
incubated A549 cells in the absence and in the presence of
various concentrations of FTS and then measured their total
Ras, K-Ras-GTP, phospho-ERK, and phospho-Akt levels. In
agreement with previous studies (11), FTS induced a small
but significant decrease in the amount of total Ras (10 F 3%
reduction at 75 Amol/L; P < 0.05; Fig. 4A). FTS also reduced
the amount of K-Ras-GTP in a dose-dependent manner
(Fig. 4A). The reduction in K-Ras-GTP (mean F SD) was
23 F 15.3%, 37 F 3.7% (P < 0.01), and 46 F 1.9% (P < 0.002),
respectively, in cells treated with 25, 50, and 75 Amol/L
FTS. The effective concentration range (25 75 Amol/L) for
the reduction in K-Ras-GTP (Fig. 4A) was similar to that
required for the inhibition of cell growth (Fig. 1B). FTS also
reduced the levels of phospho-ERK and phospho-Akt
causing 33 F 2% and 58 F 6% inhibition, respectively
(Fig. 1A). The effect of FTS seemed to be specific to the Ras
protein because it had no effect on the amount of the
prenylated active Rac1-GTP protein as determined by a
specific Rac1-GTP pull-down assay (Fig. 4B). Moreover,
using a specific pull-down assay for prenylated active
RhoA-GTP, we found that FTS induced a significant
increase of 2 F 0.2 – fold (P < 0.002) in RhoA-GTP (Fig. 4C).
Thus, although FTS did not reduce the total amounts of
the two GTPases (Rac1 and RhoA), it clearly had a selective
inhibitory effect on active K-Ras. The observed increase
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in RhoA-GTP can probably be attributed to the relief of the
known Ras-associated negative control of RhoA activation
(25) and is consistent with the observed increase in stress
fiber formation and focal adhesion assembly (Fig. 3) that
are controlled by active RhoA (26).
FTS inhibition of Ras and active ERK was not limited to
the A549 lung cancer cells. This was evident from experiments in which we examined the effects of FTS on the
levels of Ras, Ras-GTP, and phospho-ERK in H1299 and in
HTB54 cells. The first does not harbor oncogenic Ras and
the second harbors oncogenic K-Ras. FTS induced 14.7 F
3.3% and 16 F 3.3% reduction in Ras-GTP, respectively,
51.6 F 9.6% and 64.6 F 12.6% reduction in total Ras, and
38.9 F 5.2% and 62.2 F 13.9% reduction in phospho-ERK,
respectively, in H1299 and HTB54 cells (Fig. 4D).
Next, using the soft agar assay, we examined the effect of
FTS on the anchorage-independent growth of A549 cells.
The results of these experiments are shown in Fig. 5. As
shown, FTS inhibited A549 cell growth in soft agar by 27 F
5.5% and 58 F 21% at 50 and 100 Amol/L, respectively.
Effect of FTS (Salirasib) Combined with a Cytotoxic
Drug on A549 Cell Death
Because active Ras is known to endow tumor cells with
resistance to apoptosis, we next examined whether treatment with FTS can increase the sensitivity of A549 cells to
cytotoxic drugs. A549 cells were treated with 40 Amol/L
FTS for 48 h and then for 4 h with gemcitabine, paclitaxel,
cisplatin, or doxorubicin. The cells were then washed,
replenished with 40 Amol/L FTS, incubated for 3 days with
FTS, and counted. The numbers of cells in the drug-treated
cultures, expressed as percentages of the numbers in the
vehicle-treated control, are shown in Fig. 6.
The effects of FTS and gemcitabine were found to be
synergistic (Fig. 6A). As shown, FTS alone caused a
reduction in cell numbers (mean F SD) of 25 F 6.3%,
gemcitabine at 100 and 200 nmol/L had no effect (<11%),
and the combinations of FTS and gemcitabine at 100 and at
200 nmol/L caused, respectively, reductions of 45 F 5.3%
and 60 F 5.7%. The combined effect of FTS and cisplatin
was additive (Fig. 6B), with reductions in cell numbers of
33 F 9.5% for FTS alone, reductions of 11 F 11% and 30 F
12.9%, respectively, for cisplatin alone at 5 and at 10 Amol/L,
and reductions of 47 F 6.9% and 63 F 12.7% for the
respective combinations of FTS and cisplatin at 5 and
10 Amol/L. As with cisplatin, the observed effects of the
combinations of doxorubicin and FTS (Fig. 6C) and of
paclitaxel and FTS were additive (Fig. 6D). Thus, of the four
cytotoxic drugs examined, it seems that FTS enhanced the
cytotoxic action of gemcitabine only.
Salirasib (FTS) Inhibits Tumor Growth in a Lung Cancer Cell Nude Mouse Model
We next examined the in vivo effect of salirasib using a
nude mouse model. The lung cancer cells were implanted
s.c. above the right femoral joint and the mice were then
treated with salirasib. First, we examined the effect of i.p.
administration of salirasib on tumor growth in A549 cells, a
procedure that has proven effective in other nude mouse
tumor models (10, 11, 27). Treatment was started 5 days
after cell implantation, by which time tumor volumes were
0.15 F 0.06 and 0.1 F 0.03 cm3 in the control and the drugtreated groups. Tumor volumes were determined 24 days
after implantation in two groups of mice (n = 8) that had
received daily i.p. administration of either the vehicle
(control) or 10 mg/kg salirasib. Significant inhibition of
tumor growth relative to the control (53.8%; P < 0.05) was
recorded in the salirasib-treated group (Fig. 7A). Whereas
in the control group the fold increase in tumor volume was
4.3, in the salirasib-treated group, it was 2.5. In a similar
experiment carried out with mice implanted s.c. with
HTB58 cells (n = 7 per group), significant inhibition
of tumor growth (76.4 F 48.8%) was observed in the
group treated daily with 10 mg/kg salirasib i.p. (Fig. 7B).
The initial tumor volumes were 0.015 F 0.010 cm3
compared with 0.02 F 0.03 cm3, respectively, in the control
Figure 3. FTS (salirasib) induces stress fiber formation and focal adhesion assembly in A549 cells. A549
cells were plated on glass coverslips, grown for
24 h with 0.1% Me2SO4 (control) or 75 Amol/L FTS,
and labeled with phalloidin-rhodamine (red fluorescence) and anti-vinculin antibody and then Cy2labeled antibody (green fluorescence) as described in
Materials and Methods. Typical fluorescence images
(100 objective) of cells in one of three experiments
with similar results. Bar, 10 Am.
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OF6 Ras Inhibitor (Salirasib) Inhibits Lung Cancer Growth
Figure 4.
FTS (salirasib) inhibits Ras
and its signals in lung cancer cell lines.
The cells were treated with the indicated
concentrations of FTS or with the vehicle
control for 24 h and then lysed as
described in Materials and Methods.
They were then lysed and subjected to
quantitation of active K-Ras-GTP or RasGTP (all active Ras isoforms), Rac1-GTP,
and RhoA-GTP by pull-down assays
followed by immunoblotting as described
in Materials and Methods. Samples of
cell lysates were also subjected to the
determination of total levels of the
various GTPases and of ERK, Akt, phospho-ERK, and phospho-Akt by immunoblotting as described in Materials and
Methods. Typical immunoblots and
quantitative analyses of the results, as
determined by densitometry and normalized to the level of expression of each
protein. Columns, mean of four experiments; bars, SD. A, FTS reduces the
levels of total K-Ras, K-Ras-GTP (top ),
and of phospho-ERK (middle ) and phospho-Akt (bottom ) in A549 cells. B, FTS
does not affect the levels of Rac 1-GTP
in A549 cells. C , FTS induces an
increase in RhoA-GTP in A549 cells. *,
P < 0.05, compared with vehicle-treated
control. D, FTS reduces the levels of Ras,
Ras-GTP, and phospho-ERK in H1299
and HTB54 cells.
and drug-treated groups. Tumor volume measured 14 days
after cell implantation in salirasib-treated group was 0.02 F
0.045 cm3 (a 1.3-fold increase relative to day 0) compared
with 0.09 F 0.08 cm3 (a 4.5-fold increase relative to day 0) in
the vehicle-treated controls (P < 0.05).
We next used the A549 cell – implanted nude mouse
model to examine the effect of salirasib given p.o. on tumor
growth. In two separate experiments (Fig. 7C, experiments 1
and 2), cells were implanted as described above and daily
p.o. treatment with salirasib (50 mg/kg) was started
11 days later. The initial tumor volumes in the control
(n = 5) and in the FTS-treated (n = 6) groups were,
respectively, 0.72 F 0.21 and 0.75 F 0.24 cm3 in experiment
1 and 0.66 F 0.3 cm3 in both the control group (n = 8) and
the FTS-treated group (n = 8) in experiment 2. As shown in
Fig. 7C (experiment 1), after 16 days of treatment, the tumor
weights (mean F SD) in salirasib-treated and control mice
were 0.4 F 0.19 and 0.9 F 0.39 g, respectively, representing
a significant inhibition of 53.7 F 19.1% in tumor growth
(P < 0.025) in the salirasib-treated mice. Similarly, after
11 days of treatment (Fig. 7C, experiment 2), the tumor
weights (mean F SD) in salirasib-treated and control mice
were 0.33 F 0.17 and 0.58 F 0.15 g, respectively,
representing a significant inhibition of 43.4 F 19.1% in
tumor growth (P < 0.03) in the salirasib-treated mice.
Next, we examined the effects of p.o. salirasib, alone or in
combination with gemcitabine, on A549 cell tumor growth
(Fig. 7D). Six days after cell implantation, mice were
divided into four groups (n = 8 per group) and treated p.o.
with vehicle alone (control), FTS alone (60 mg/kg), vehicle
and gemcitabine (36 mg/kg, i.p. every 4 days), or salirasib
and gemcitabine. The initial tumor volumes in the four
groups were 0.26 F 0.049, 0.34 F 0.073, 0.26 F 0.063, and
0.35 F 0.085 cm3, respectively. Treatments with gemcitabine began 1 week after salirasib treatment was started. In
agreement with the results of the first experiment (Fig. 7C),
p.o. salirasib treatment caused a significant inhibition in
tumor growth; tumor weights in the mice treated with
vehicle only (control) and with salirasib only (mean F SD)
were 0.90 F 0.40 and 0.49 F 0.15 g, respectively (46.2 F
Mol Cancer Ther 2007;6(6). June 2007
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Molecular Cancer Therapeutics
Discussion
Figure 5. FTS (salirasib) inhibits anchorage-independent growth of
A549 cells. A549 cells (8 103 cells per well) were seeded in 96-well
plates in soft agar in the presence of the vehicle (0.1% Me2SO4; control)
or the indicated concentrations of FTS as described in Materials and
Methods. After 19 d, the colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and imaged. A, typical
images. B, the numbers of colonies per well in control and FTS-treated
cultures were counted as described in Materials and Methods. Columns,
mean of two independent experiments, each repeated six times; bars, SD.
*, P < 0.005, compared with vehicle-treated control.
16.3% inhibition; P < 0.02; Fig. 7D). A significant reduction
in tumor weight (P < 0.015) was also observed in a fourth
group of mice treated with gemcitabine alone (Fig. 7D). The
combined effect of gemcitabine and salirasib treatments
was somewhat more effective than the effect of each
treatment alone, although the difference in tumor weight
between the gemcitabine treatment and the gemcitabine
plus salirasib treatment was not significant (Fig. 7D). The
most significant result obtained in this experiment was
clearly the difference between the effect of treatment with
the vehicle alone (control) and of combined treatment with
salirasib and gemcitabine (P < 0.006).
In the present work, we show that the Ras inhibitor FTS
(salirasib) inhibited the anchorage-dependent (Figs. 1 and 2)
and anchorage-independent (Fig. 5) growth of lung
cancer cells in vitro and inhibited tumor growth in nude
mouse lung cancer models given either i.p. or p.o. (Fig. 7).
Consistent with the known anti-Ras activity of salirasib, we
show here that salirasib reduced the levels of Ras and its
downstream targets phospho-ERK and phospho-Akt in
lung cancer cells (Fig. 4). As expected, salirasib inhibited
active K-Ras-GTP (Fig. 4). This finding accords with the
reported mode of action of salirasib as shown in many
tumor cell lines that harbor activated Ras, including PANC-1
pancreatic cancer, SW480 colon carcinomas, and 518A2
melanomas (10, 11, 27). Moreover, in agreement with
reports that salirasib can inhibit the growth of tumor cells
that do not harbor ras gene mutations (28), we found here
that salirasib inhibits the growth of H1299 and HTB58 cell
lines (Fig. 2), neither of which harbors mutated Ras, and the
growth of A549, HTB54, and H23, which all harbor
activated K-Ras (Figs. 1 and 2). In other words, salirasib
inhibits the growth of tumor cells whether they harbor
mutated ras genes or not. The lack of correlation between
ras gene mutations and growth-inhibitory effects of
salirasib is explained by the known chronic activation of
wild-type Ras proteins in many human tumors owing to
the presence of highly active growth factor receptor
signaling (29, 30).
Salirasib affects all Ras proteins, mainly their active form
(12), as also observed here for K-Ras in A549 cells (Fig. 4).
Because of this selectivity of salirasib toward active Ras, the
growth of cells with chronically active Ras pathways [such
as glioblastoma (28), neuroblastoma with amplified myc-N
gene (31), and melanomas (27)] is inhibited by salirasib.
Strong support for this notion comes from recent gene
profiling analyses of human primary mammary epithelial
cells that were transformed by human activated H-Ras, or
c-Src, or activated h-catenin or E2F3 (32). In these studies,
the pattern of the deregulated oncogenic pathway in each
Figure 6.
Effect of FTS (salirasib)
in combination with different cytotoxic drugs on A549 cell growth and
viability. A549 cells were incubated
for 48 h with 0.1% Me2SO4 (control)
or 40 Amol/L FTS and then for 4 h
with gemcitabine, cisplatin, doxorubicin, or paclitaxel at the indicated
concentrations and for a further 72 h
with 0.1% Me2SO4 or 40 Amol/L FTS
only. Live cells were collected and
counted. The numbers of cells in the
drug-treated cultures are expressed
as percentages of the number
recorded in the zero drug (vehicletreated) controls. Columns, mean of
eight counts; bars, SD. *, P < 0.05;
**, P < 0.01, compared with vehicletreated control.
Mol Cancer Ther 2007;6(6). June 2007
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OF8 Ras Inhibitor (Salirasib) Inhibits Lung Cancer Growth
Figure 7.
Salirasib (FTS) and gemcitabine inhibit tumor growth nude mouse models of lung cancer. A549 or HTB58 cells were injected s.c. into the
flanks of nude mice. Daily treatment with FTS at the indicated doses [i.p. (A and B) and p.o. (C and D)] was started 5 d after cell implantation. Gemcitabine
treatment (36 mg/kg, i.p., every 4 d) began 1 wk later. Tumor volumes or weights were determined as described in Materials and Methods. A, volumes
of A549 cell tumors in control mice (n = 10) and FTS-treated (i.p.) mice (n = 10) were determined on day 24 of the treatment. Columns, mean; bars, SD.
*, P < 0.05 (control versus FTS). B, volumes of HTB58 cell tumors in control mice (n = 7) and FTS-treated (i.p.) mice (n = 7) were determined on day 14
of the treatment. Columns, mean; bars, SD. *, P < 0.05 (control versus FTS). C, weights of A549 cell tumors in control mice (n = 5, experiment 1 ;
n = 8, experiment 2) and FTS-treated (p.o.) mice (n = 6, experiment 1; n = 8, experiment 2). Tumor weights were determined on day 16 (experiment
1) or day 11 (experiment 2) of the treatment. Columns, mean; bars, SD. *, P < 0.025 (control versus FTS). D, weights of A549 cell tumors in control
mice (n = 8), FTS-treated mice (p.o.; n = 8), gemcitabine-treated mice (n = 7), and gemcitabine plus FTS – treated mice (n = 8). Tumor weights were
determined on day 24 of the treatment. Columns, mean; bars, SD. *, P < 0.02 (control versus FTS); **, P < 0.015 (control versus gemcitabine); ***,
P < 0.006 (control versus gemcitabine plus FTS).
cell line was mapped by microarray-based gene expression
profiling. When they examined the sensitivity of specific
oncogenic pathways to known drugs, the authors showed
that for salirasib, those cell lines showed close concordance
and correlation between the probability of Ras pathway
deregulation based on the gene expression prediction and
the extent of the cell proliferation inhibition by salirasib.
Another important finding of the present study is the
marked effect of salirasib on the actin cytoskeleton, focal
adhesions, and cell morphology (Figs. 1A and 3). The
observed flattening of the cells and formation of actin stress
fibers and focal adhesions is typical of the inhibition of
active Ras by Ras inhibitors, including farnesyltransferase
inhibitors (33) and salirasib (15). These changes are likely to
occur, at least in part, by a relief of Ras inhibition of the
activation of RhoA. That is because active RhoA-GTP
controls the formation of stress fibers and focal adhesions
(26), and we found that salirasib induces an increase in
RhoA-GTP in A549 cells (Fig. 4). Our experiments also
show that salirasib tilts the balance between active Rho and
active Rac in A549 cells (Fig. 4), which would contribute to
the inhibition of cell growth and migration.
Altogether, these results point to the possibility that
salirasib may be considered as a potential drug for lung
cancer treatment. This suggestion must be viewed in light
of the knowledge that small-cell lung carcinoma and nonNSCLC are both responsive to first-line chemotherapy, but
most patients relapse and die from their disease, with
5-year survival rates of f15% (34). Given this poor survival
rates of small-cell lung carcinoma and NSCLC patients, the
introduction of new targeted drugs is then of utmost
importance. Indeed, many attempts to develop such drugs
for lung cancer therapy have been made. Important groups
of drugs used in the clinic or in clinical trials include
epidermal growth factor receptor tyrosine kinase inhibitors,
including gefitinib (Iressa), erlotinib (Tarceva), and cetuximab (Erbitux; ref. 35), hormones (e.g., tamoxifen, combined with chemotherapy; ref. 36), and farnesyltransferase
inhibitors, which were originally designed as Ras inhibitors
(37). Most of the drugs do not succeed in increasing
markedly survival rates, adding a few months at best
(14, 36, 37). For example, tamoxifen combined with
ifosfamide, epirubicin, and cisplatin in relapsed NSCLC
had a response rate of 20% and a median overall survival of
about 8 months (36). A combination of tamoxifen, cisplatin,
and etoposide led to a response of 37% and overall survival
of more than 11 months (36). Another example is given by
lack of clinically meaningful survival benefit when gemcitabine was combined to the farnesyltransferase inhibitor
tipifarnib (38). In addition to the poor outcome of such
Mol Cancer Ther 2007;6(6). June 2007
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Molecular Cancer Therapeutics
treatments, combination therapies of lung cancer patients
are restricted by dose-limiting side effects (13). For
example, grade 2 or higher treatment-related toxicities,
including rash, diarrhea, and fatigue, were observed in
patients treated with erlotinib with greater than additive
cytotoxic effects in combination with the farnesyltransferase inhibitor tipifarnib (Zarnestra; ref. 39). These studies
emphasize the need for new targeted nontoxic drugs for the
treatment of lung cancer. Our results suggest that salirasib,
which proved to be nontoxic in animals (11, 15, 16) and has
no reported adverse side effects in humans,1 seems to be
such a candidate drug for lung cancer.
Acknowledgments
We thank S.R. Smith for editorial assistance.
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OF9
Published OnlineFirst May 31, 2007; DOI: 10.1158/1535-7163.MCT-06-0706
Suppression of lung cancer tumor growth in a nude mouse
model by the Ras inhibitor salirasib (farnesylthiosalicylic
acid)
Mol Cancer Ther Published OnlineFirst May 31, 2007.
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