Journal of Inorganic Biochemistry 160 (2016) 225235

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Inhibition of adhesion, migration and of 51 integrin in the HCT-116

colorectal cancer cells treated with the ruthenium drug NAMI-A
Chiara Pelillo a, Hilaria Mollica a, Johannes A. Eble b, Julius Grosche b, Lea Herzog b, Barbara Codan c,
Gianni Sava a,d, Alberta Bergamo a,
a

Callerio Foundation Onlus, Trieste, Italy

Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, Germany
Dept of Engineering and Architecture, University of Trieste, Italy
d
Dept of Life Sciences, University of Trieste, Italy
b
c

a r t i c l e

i n f o

Article history:
Received 30 September 2015
Received in revised form 12 February 2016
Accepted 25 February 2016
Available online 27 February 2016
Keywords:
Ruthenium
Metastasis
Colorectal cancer
Integrins

a b s t r a c t
NAMI-A, imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate, is a ruthenium-based drug
characterised by the selective activity against tumour metastases. Previously we have shown the inuence of
the hepatic microenvironment to direct the arrest of the metastatic cells of colorectal cancer. Here we used the
experimental model of HCT-116 colorectal cancer cells in vitro to explore whether the interference with 51
integrin may mechanistically explain the anti-metastatic effect of NAMI-A. NAMI-A inhibits two important
steps of the tumour metastatic progression of colorectal cancer, i.e. the adhesion and migration of the tumour
cells on the extracellular matrix proteins. The bronectin receptor 51 integrin is likely involved in the antiadhesive effects of NAMI-A on the HCT-116 colorectal cancer cells during their interaction with the extracellular
matrix. Mechanistically, NAMI-A decreases the 51 integrin expression, and reduces FAK (Focal Adhesion
Kinase) auto-phosphorylation on Tyr397, an important signalling event, involved in 51 integrin activation.
These effects were validated by siRNA-induced knock down of the 5 integrin subunit and/or by the use of
specic blocking mAbs against the active site of the integrin. Our results demonstrate the relevance of 51
integrin for colorectal cancer. We also show that the anti-metastatic effect of NAMI-A depends on the modulation
of this integrin. Thus, our data on NAMI-A support the new concept that metal-based drugs can inhibit tumour
metastases through targeting of integrins and of other proteins which mediate tumour progression-related cell
functions such as adhesion and migration.
2016 Elsevier Inc. All rights reserved.

1. Introduction
Preclinical studies support the evidence that the ruthenium-based
drug NAMI-A (imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate) is endowed with a strong capacity to selectively combat
the development and growth of metastases originating from solid
tumours [13]. NAMI-A breaks the platinum paradigm which insinuates
that cytostatic metal compounds can be active anti-tumour drugs if they
are directed against DNA [4]. NAMI-A interacts with DNA less efciently
than cisplatin [5]. Rather, several experimental evidences support
the intervention of NAMI-A in the interplay between tumour cells and
their microenvironment and suggest the hypothesis that this drug interacts with a target on the tumour cell membrane [610]. The efciency
of cell internalization of NAMI-A is virtually null [11], a fact that explains
the rapid exchange of the drug between cells and their growth milieu

Corresponding author at: Callerio Foundation Onlus, Via A. Fleming 22, 34127 Trieste,
Italy.

http://dx.doi.org/10.1016/j.jinorgbio.2016.02.025
0162-0134/ 2016 Elsevier Inc. All rights reserved.

[12]. The high amount of ruthenium found in the cytoskeleton fraction

of the treated cells [13] supports its ability to induce cytoskeletal rearrangements [14,15] and to modulate cell adhesion to the extracellular
matrix (ECM), as observed with the use of several tumour cell lines [6].
Overall these ndings suggest that the integrins are amongst the possible
targets to explain the in vivo activity of NAMI-A on tumour metastases.
Integrins are heterodimeric, transmembrane receptors that function
as mechano-sensors, adhesion molecules and signal transduction
platforms in a multitude of biological processes [16]. Integrins interact
with the ECM thereby regulating many cellular functions, such as
proliferation, migration, and survival. Integrins are also involved in the
cell-to-cell interactions. Through cellcell and cellECM contacts, the
integrins transduce the information from the external environment
into the cell and vice-versa, to promote cell adhesion, spreading and
motility [17].
The interaction between cells and ECM is a hallmark in survival
and progression of cancer cells that involves large integrin-associated
intracellular protein complexes, which act as anchors for the cytoskeleton and as signalling hotspots [18]. Many studies also report on the

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C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

role of integrins in inducing resistance against different classes of

chemotherapeutic agents in both solid tumours and haematological
malignancies [19]. Integrins also provide survival advantage against
death receptor-mediated apoptosis, suggesting their ability to promote
cancer immune escape [2022].
Among the integrins, the 51 integrin, known also as the bronectin receptor, is involved in the malignant behaviour of different tumour
types, including colorectal cancer (CRC), and has emerged as a therapeutic target [2326]. Highly invasive colon cell lines, such as HCT-116
cells, express higher levels of 51 integrin than poorly invasive cells
[25]. The inhibition of 51 integrin with specic antibodies, in xenograft models of ovarian cancer, leads to suppression of tumour spread
and increased survival [27]. In breast cancer models, the up-regulation
of the integrin 51 is related to increased cell invasion potential [28].
In the Lewis lung cancer mouse model, the decreased expression of
the 5 integrin subunit reduces metastases and correspondingly
promotes survival [29].
Preclinical studies and phase I and II clinical trials have shown the efcacy of 51 inhibitors. ATN-161, a 51 integrin-binding peptide,
changing the high-afnity of the RGD-dependent (i.e. ArgGlyAspdependent) adhesion of cells to bronectin, signicantly inhibits the
metastasis dissemination at all metastatic sites of the MDA-MB-231
xenografts in mice [30]. A combination of ATN-161 and 5-FU (5-uorouracil) reduces colorectal liver metastases in vivo and increases the
overall survival of the treated mice [31]. ATN-161 induces a prolonged
disease stabilisation in one third of patients with advanced solid
tumours, in a phase I clinical trial [32], and the humanized monoclonal
antibody against 51, Volociximab, stabilizes the disease in one
quarter of patients with advanced solid tumours in another phase I
trial [33]. Also the ruthenium drug NAMI-A was shown to modulate
the expression of the 1 integrin and the activation of the downstream
pathways in the MDA-MB-231 breast cancer cells [6].
Recently, we have set up and characterised an in vitro model of CRC
metastasis showing the inuence of the hepatic microenvironment for
the modulation of the expression/activity of 51 integrin during the
process of tumour cell arrest and adhesion [34].
On these grounds, with the present study we investigate the effects
of NAMI-A on the processes of adhesion and migration of the HCT-116
colorectal cancer cells when they interact with the bronectin, the
ECM protein with which the 51 integrin plays its driving role.
2. Materials and methods
2.1. Chemicals
Imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate
ImH[trans-RuCl4(DMSO)Im] (NAMI-A) was synthesized by Serichim
(Torviscosa, Italy) according to already described procedures [35]. All
reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise indicated.
2.2. Cell cultures
The adenocarcinoma colon cancer cell line HCT-116 was kindly supplied by the group of Dr. C. Gaiddon, University of Strasbourg, France.
The cell line was cultured in Dulbecco's Modied Eagle Medium
(DMEM) (Euroclone, Ltd., UK) supplemented with 10% foetal bovine
serum (FBS, Gibco-Invitrogen Corp., UK), 2 mM L-glutamine (Euroclone
Ltd., UK), 100 IU/ml penicillin, 100 g/ml streptomycin solution
(Euroclone Ltd., UK).
Cells were grown in an incubator with 5% CO2 and 100% relative
humidity at 37 C. Once 90% of conuence was reached, the cells were
detached with trypsin/EDTA solution (0.05% w/v trypsin, 0.02% w/v
EDTA in PBS). Cell viability was determined by the trypan blue dye
exclusion test.

2.3. Invasion assay

Invasion assay was performed using 8.0 m pore size Transwell inserts (Costar, Cambridge MA, USA) coated with Matrigel (600 mg/ml)
at room temperature overnight. HCT-116 cells serum-starved for 24 h
were treated with NAMI-A (1, 10 and 100 M) dissolved in DPBS
(Dulbecco's phosphate buffered saline, pH = 7.4) for 1 h at 37 C, 5%
CO2 before being seeded on inserts (50,000 cells/insert). As invasion
stimulus the complete medium was applied in the plate wells, as negative control to detect the basal invasion rate a serum-free medium was
used.
After 96 h cells which had remained on the upper side of the membrane were removed using cotton swabs, while the cells that invaded
and were present in the lower surface of the inserts were xed with
glutaraldehyde 1.1% (v/v) at room temperature for 15 min, and then
stained with crystal violet solution 0.1% (w/v) for 20 min. After washing
the cells to remove excess crystal violet, the dye was solubilized in acetic
acid 10% (v/v) [36]. The absorbance units were read at 590 nm and
related to cell invasion rate. Each assay was done in triplicate and data
presented as mean SD.
2.4. Adhesion assay
A 96-well plate was pre-coated with bronectin, collagen I, collagen
IV, laminin-111 and Matrigel (all substrates 20 g/ml) for 4 h at 37 C,
or overnight at 4 C, and subsequently blocked with PBS-BSA (Bovine
Serum Albumin) 0.1% (w/v) for 15 min at 37 C. Cells were detached
with 1 mM EDTA, treated in suspension with NAMI-A (1, 10, and
100 M dissolved in DPBS) for 1 h at 37 C, 5% CO2. Then, cells were
centrifuged and re-suspended in serum free medium supplemented
with 0.1% BSA (w/v) for 30 min at room temperature to ensure reexpression of adhesion receptors on the cell surface and seeded into
the plate (20,000 cells/well).
HCT-116 were allowed to attach to each substrate for 1 h at 37 C, 5%
CO2, then the non-adherent cells were removed by washing. The adherent cells were xed with trichloro-acetic acid 10% (v/v) for 1 h at 4 C
and stained with sulforhodamine B 0.4% (w/v) according to the method
of Skehan et al. [37]. The absorbance units were read at 570 nm.
For the adhesion assay in the presence of 51 blocking antibody
and NAMI-A, tumour cells were re-suspended in solutions of NAMI-A
(1, 10, and 100 M dissolved in DPBS) with or without 5 g/ml of the
antibody JBS5, (Millipore, MA, USA) and then plated in a 96-well plate
functionalized with bronectin. Cells were allowed to adhere for 1 h
at 37 C, 5% CO2 and then processed as described above.
For the adhesion assay in the presence of 51 blocking antibody
and echistatin, HCT-116 cells re-suspended in DMEM were seeded
onto a 96-well plate previously functionalized with bronectin. The
51 blocking antibody (JBS5 Millipore, 2.5 g/ml) and echistatin
(12.5 nM), were added to cells immediately before the seeding. Cells
were allowed to adhere for 1 h at 37 C, 5% CO2. Then adherent cells
were labelled with the uorescent dye 2,7-bis (2-carboxyethyl), 5
(and -6) carboxyuorescein (BCECF, 2 M). Fluorescent dye, which
had not been taken up by the cells, was washed off the wells. After
cell lysis, cell-bound uorescent dye was released and its relative
concentration was determined at 535 nm by using a micro-titer plate
uorescence reader. Each condition was prepared in quadruplicate
and the experiment performed three times.
2.5. Cytotoxicity test
Cells were seeded at 4000 per well on 96-well plates and allowed to
grow 24 h. Then they were incubated for 72 h with 3300 M solutions
of NAMI-A obtained by serial dilutions of a stock solution (freshly
prepared in sterile water at a concentration of 6 mM) with complete medium. Analysis of cell cytotoxicity by MTT ((3-(4,5-Dimethylthiazol-2yl)-2,5-Diphenyltetrazolium Bromide)) assay was performed after 72 h

C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

of incubation. Briey, MTT dissolved in phosphate-buffered saline (PBS,

5 mg/ml) was added (10 l per 100 l medium) to all wells, and the
plates were then incubated at 37 C with 5% CO2 and 100% relative
humidity for 4 h. After this time, the medium was discarded, and
200 l DMSO were added to each well according to the method of
Alley et al. [38]. Absorbance units were measured at = 570 nm on a
SpectraCount Packard instrument (Meriden, CT, USA). IC50 values were
calculated from doseeffect curves and are the mean SD of at least
three separate experiments. The tting procedure applied is a nonlinear
regression performed with GraphPad prism version 6 for Mac OS X
version 6.0b (GraphPad Software, San Diego, CA, USA). Experiments
were conducted in quadruplicate and repeated thrice.
2.6. Atomic force microscopy (AFM) analyses
An AFM Solver Pro-M (NT-MDT, Moscow, Russia) was used to
acquire morphology as well as forcedisplacement curves [39]. The
AFM was equipped with a liquid cell setup with a standard cantilever
holder cell having a sharp PNP-DB silicon nitride tip (diameter about
10 m (Nanoworld, Neuchatel, Switzerland).
The cantilever force constant was calibrated using the thermal
uctuation method.
AFM probes were cleaned, prior to the indentation experiments, by
submerging them successively in ethanol and chloroform (30 min
each), in order to remove contaminant molecules adsorbed on the
probe surface.
HCT-116 cells (1 10 [5]), treated in suspension with NAMI-A (1,
10, and 100 M dissolved in DPBS) for 1 h at 37 C, 5% CO2, were seeded
onto a cover slip functionalized with poly-L-Lysine (1 mg/ml) and
were allowed to adhere for 4 h prior to xation with paraformaldehyde
(2% v/v) for 15 min.
The basic AFM technique for quantitative analysis of the cell elasticity is the force spectroscopy (called forcecurve analysis).
Force curves were collected by monitoring the cantilever deection,
while moving the piezoscanner. Thus, a plot of force versus sample
position was recorded. The AFM tip was moved towards the cell with
speeds of 0.5 m/s. The speed range was chosen to avoid cell movement
(at low compression speed) or hydrodynamic force contribution
(signicant at high speed).
For each experimental condition, at least 18 cell data acquisitions are
collected, 4 force curves are acquired for every cell.
These data were enough to detect statistically signicant differences.
Data were elaborated using the NOVA software (NT-MDT, USA) for
image acquisition and ImageAnalysis for data processing.
2.7. Morphological analyses
HCT-116 were pre-treated in suspension with NAMI-A (1, 10, and
100 M dissolved in DPBS) for 1 h at 37 C, 5% CO2. Then cells were
washed, re-suspended in DPBS and seeded (2.5 10 [5] cells/well)
onto glass chamber slides previously functionalized with bronectin
and saturated with PBS-BSA 0.1% for 1 h at room temperature for
blocking non-specic binding sites. Cells were allowed to adhere for
1 h at 37 C, 5% CO2 and then analysed within 2 h at an optic microscope.
Images were taken at 10 magnication using an uplight microscope
(Nikon, Tokyo, Japan).
2.8. Real time adhesion and migration assays
The extent of cell adhesion and migration was measured as changes
in impedance with the xCELLigence system (Roche Diagnostics GmbH,
Mannhein, Germany). The system expresses impedance in arbitrary
cell index (CI) units. Data were recorded by the supplied RTCA software
1.2. Original data sets generated by xCELLigence were exported to MS
Excel and the raw data were processed without CI-normalization.

227

E-plates and/or CIM-16 plates (Roche Diagnostics GmbH, Mannhein,

Germany) were coated with 20 g/ml bronectin in PBS, pH 7.4 at 4 C
overnight. Then, wells were saturated with PBS-BSA 0.1% for 1 h at room
temperature and washed with PBS before cells were added.
2.8.1. Migration in the presence of 51 blocking antibody and echistatin
HCT-116 cells, previously serum-starved for 24 h, were detached, resuspended in serum-free medium containing 0.1% BSA, adjusted to an
appropriate density (15 10 [4] cells/well) and seeded in the presence
of the 51 blocking antibody or echistatin (2.5 g/ml and 12.5 nM, respectively), onto the upper chamber of a CIM 16-well plate. This consists
of an upper and lower chamber separated by a micro-porous membrane
(pore = 8 m). Initially, 165 l and 50 l of media were added to the
lower and upper chambers respectively and the CIM-16 plate was
locked in the Real Time Cell Analyser Dual Plate (RTCA-DP) system
at 37 C, 5% CO2. A background signal was measured with cell-free
media before adding the cells. Lower chambers contained culture
media with or without FBS in order to assess chemotactic migration towards FBS and background migration, respectively [40]. Each condition
was recorded in quadruplicate. The impedance signal was recorded
every 15 min during the 24 h of incubation.

2.8.2. Adhesion and migration in the presence of the auto-phosphorylation

P-Tyr397-FAK inhibitor
HCT-116 cells were treated with the auto-phosphorylation PTyr397-FAK (Focal Adhesion Kinase) inhibitor NVP-TAE226 (S2820,
Selleck Chemicals, Munich, Germany) at 0.1, 1, and 10 M for 24 h at
37 C, 5% CO2. After 24 h cells were collected and seeded (15 10 [4]
cells/well) onto CIM-16 or E-plates to assess adhesion and migration,
respectively, in real time. The plates were mounted into the RTCA-DP
system. Adhesion and migration were monitored every 5 and 15 min
for the rst 4 h and subsequent 24 h, respectively. Each condition was
analysed in quadruplicate and the experiment repeated two times.
2.8.3. Adhesion assay with integrin 5 subunit-silenced cells
HCT-116 cells transfected with siRNA against 5 subunit, as described
below, and previously serum-starved for 4 h were used. Cells, treated
with siRNA (HCT-116/siRNA) or without siRNA (HCT-116) were detached, re-suspended in serum-free media containing 0.1% BSA, adjusted
to an appropriate density (15 10 [4] cells/well), and treated in suspension with NAMI-A (1, 10, and 100 M dissolved in DPBS) for 1 h at 37 C,
5% CO2. Then, cells were seeded into a 96 well-E-plate, and mounted into
the RTCA-DP system. Adhesion was monitored for 4 h every 15 min. Each
condition was analysed in quadruplicate and the experiment repeated
two times.
2.9. Flow cytometric analysis of 51 integrin expression on the cell surface
Cells were detached with 1 mM EDTA, treated in suspension with
NAMI-A (1, 10, and 100 M dissolved in DPBS) for 1 h at 37 C, 5%
CO2. Then, they were centrifuged and re-suspended in serum-free
medium with 0.1% BSA for 30 min at room temperature, and seeded
onto 6-well plates pre-coated or not with bronectin and allowed to
adhere for 1 h at 37 C, 5% CO2. Adherent cells were collected, then
500,000 cells/sample were centrifuged and incubated with an anti51 primary antibody (clone HA5 5 g/ml, Millipore, MA, USA) in
PBS for 30 min at 4 C. At the end of the incubation cells were washed,
centrifuged and incubated with a FITC-conjugated Goat Anti Mouseantibody (diluted 1:100) in PBS for 30 min at 4 C. After centrifugation,
cells were re-suspended in PBS-0.1% BSA (w/v)-0.1% NaN3 (w/v)
and analysed by ow cytometry (FACS Calibur cell analyser, Becton
Dickinson, Milano, Italy). For each sample 10,000 events were recorded.
Data obtained were processed using WinMDI 2.9 software.

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C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

2.11. Western blot analyses of FAK activation

Fig. 1. NAMI-A reduces the HCT-116 cell invasion potential. HCT-116 cells were
treated with NAMI-A (1, 10, and 100 M) in DPBS for 1 h prior to seeding them into
the top compartment of invasion inserts coated with Matrigel (600 g/ml). Cells
were allowed to invade along the chemotactic gradient of FBS. Data represent cells
that had completely passed through the Matrigel-coated barrier after 96 h. The
dotted line represents the invasion induced by the FBS-starved complete medium on
HCT-116 cells, which had not been treated with NAMI-A (Absorbance Units = 0.111
0.007). Mean values SD are shown from three independent experiments, each with
triplicate sets. Statistical analysis: ANOVA and TukeyKramer post-test: **p b 0.01 vs
untreated controls.

2.10. Real Time PCR for 5 and 1

The differential expression of the 5 and 1 genes in HCT-116 cells
treated in suspension with NAMI-A (1, 10, and 100 M dissolved in
DPBS) for 1 h at 37 C, 5% CO2, was analysed by real time RT-PCR.
RNAs were reverse-transcribed with a qScript cDNA Synthesis Kit
(Quanta Biosciences, Gaithersburg, MD, USA) according to the
manufacturer's protocol. Expression levels were determined by real
time RT-PCR on a CFX96 system (Bio-Rad, Hercules, CA, USA). The
15 ml reaction mix included 7.5 ml of 2 SsoAdvanced SYBR
Green Supermix (Bio-Rad), 0.3 ml of each 10 mM primer and 2 ml of
a 1:20 cDNA dilution. The following thermal prole was used: an initial
30 denaturation step at 95 C, followed by 40 cycles at 95 C for 5 and
58 C for 30. Amplication products were analysed with a 65/95 C
melting curve. The following 5 and 1 primer pairs were designed
using the MIT Prime3 online primer design protocol from the relative
gene sequence: for ITGA5 (5-integrin subunit) 5-GTGGGCCAACAA
AGAACACT-3 (forward) and 5-TGAGTTCTGATTCCCCTTGG-3 (reverse); for ITGB1 (1-integrin subunit) 5-CCCTTGCACAAGTGAACAGA3 (forward) and 5-CCACCTTCTGGAGAATCCAA-3 (reverse).
RPL5, a highly stable housekeeping gene, was chosen for normalization, and amplied using 5-GCACACGAACTGCCAAAATA-3 (forward)
and 5-TTCATCACCAGTCACCT CCA-3 (reverse) primers. The expression
levels of the selected transcripts were determined using the comparative Ct method (2-DD Ct method) [41]. Ct values used for quantication
were corrected based on PCR efciencies using LinRegPCR [42]. Results
are given as the mean with standard deviation of three technical
replicates.

HCT-116 cells were detached and treated with NAMI-A (1, 10, and
100 M dissolved in DPBS) for 1 h at 37 C, 5% CO2; then, they were
seeded into 6-well plates pre-coated or not with bronectin, and
allowed to adhere for 1 h at 37 C, 5% CO2. Adherent cells were collected,
centrifuged and lysed with the lysis buffer (0.5% Na-deoxycholate, 1%
Igepal, 50 mM HEPES, 150 mM NaCl, 1 mM Na-orthovanadate, 50 mM
NaF, 20 mM -glicerophosphate, 0.1 M okadaic acid, 1 mM PMSF
(phenylmethylsulfonyl uoride), 50 g/ml leupeptine, 20 g/ml
aprotinin, 10 mM pepstatin). Total protein extract was quantied
following the method of Bradford [43].
Proteins in the total cell lysate (10 g of extract for FAK and 30 g for
P-FAK detection, respectively) were separated on 8% polyacrylamide
SDS-PAGE and electro-transferred to a nitrocellulose membrane (BioRad, Milano, Italy). Afterwards, the blot was blocked in a solution of
5% skimmed milk, 0.1% Tween 20 in Tris-Buffered Saline (TBS) for 1 h
at room temperature. Membrane-bound proteins were probed
with an anti-FAK primary antibody (#3285 Cell Signalling, Milano,
Italy, 1:1000) and an anti-P-Y397-FAK primary antibody (#3283 Cell
Signalling, Milano, Italy, 1:1000) over night at 4 C, then washed before
incubation with a horseradish peroxidase-conjugated secondary
antibody (#7074 Cell Signalling, Milano, Italy, 1:1000) for 1 h at room
temperature in agitation. Proteins were detected by incubating membranes with horseradish substrate (Cell Signalling, Milano, Italy) and
developed with appropriate reagents and Kodak autoradiography lm
(Amersham Pharmacia Biotech, NJ, USA).
2.12. Transfection of HCT-116 cells with siRNA for 5 subunit of the integrin
51
HCT-116 cells were transfected with small interfering RNA (siRNAs)
for 5 (Origene, USA), or a control vector, (both 0.1 nM) using siTRAN
reagent (Origene, USA) to a nal volume of 120 l following the
standard protocol. Briey, HCT-116 cells were seeded in a 6-well plate
and the day after, siRNAs, re-suspended in siTRAN and DMEM without
phenol red, were added drop to drop to cells for 4 h. After that time,
the transfection was stopped by changing the medium for DMEM with
phenol red and 16% FBS. After an overnight incubation, the complete
culture medium was added and cells incubated at 37 C, 5% CO2 for
48 h. A standard scrambled siRNA (Trilencer-27 Universal Scrambled
Negative Control siRNA Duplex 2 nM, SR30005 Origene) was used
as negative control. Silencing was controlled by performing real time
RT-PCR, as described above. The level of 51 integrin expression on
HCT-116 cells membrane after silencing was evaluated by ow cytometry as described above.
2.13. Statistical analysis
Results obtained were processed using InstatGraph3 software and
presented as mean standard deviation. The group means were compared using Analysis of Variance (ANOVA) followed by TukeyKramer
post-test and considered signicant when p b 0.05.

Fig. 2. NAMI-A reduces the adhesion of HCT-116 cells to ECM components and affects cell morphology. A) Adhesion test. HCT-116 cells were treated with NAMI-A (1, 10, and 100 M) in
DPBS for 1 h, then seeded into 96-well plates pre-coated with different ECM components, and allowed to adhere 1 h at 37 C, 5% CO2. Adherent cells were then xed and stained with
sulforhodamine B. The dye was solubilized and the absorbance was read at 570 nm. Data are mean SD of four experiments, each with quadruplicate sets. Statistical analysis: ANOVA
and TukeyKramer post-test: *p b 0.05; **p b 0.01; ***p b 0.001 vs untreated cells on the same substrate. B) Morphological analysis. HCT-116 cells were treated with NAMI-A (1, 10,
and 100 M) in DPBS for 1 h, then seeded onto a coverslip coated with bronectin for 1 h at 37 C, 5% CO2 prior to xation with 4% PFA for 10 min. Morphological analyses were
performed using an inverted optical microscope (Nikon, Tokyo, Japan). White arrows show lamellipodia. C) AFM analysis of live cells. HCT-116 cells were seeded onto a cover slip
functionalized with poly-L-lysine (1 mg/ml). After 24 h, they were treated with 10 M NAMI-A in DPBS for 1 h at 37 C, 5% CO2 prior to analyse them at an AFM Solver Pro-M. The analyses
were performed on live cells in culture medium at 37 C. Bi-dimensional analysis (left panel) and 3D analysis (right panel) of untreated and treated cells are shown. Changes in the cell
perimeter and volume have been evaluated analysing the acquired data by NOVA software. Data are mean SD of three independent experiments, each with duplicate sets. Statistical
analysis: ANOVA and TukeyKramer post-test: *p b 0.05; **p b 0.01; and ***p b 0.001 vs untreated controls.

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3. Results
3.1. Effects of NAMI-A on the HCT-116 CRC cell invasion
NAMI-A was tested on the HCT-116 CRC cells during their invasion
through the ECM model substrate Matrigel, under the chemotactic
stimulus of FBS in complete medium. In the presence of FBS, HCT-116
cells efciently invaded the Matrigel barrier as compared to the
negative controls (compare the white bar with the dotted line,
p b 0.001, Fig. 1). 1 and 100 M NAMI-A signicantly reduced the
efciency of cancer cell invasion by approximately 30% (p b 0.01 vs untreated controls). 10 M NAMI-A did not show any apparent effect. The
anti-invasive activity of NAMI-A was observed only on the chemotactic
invasion towards FBS since no effect of NAMI-A on the spontaneous cell
invasion of HCT-116 cells was detected (data not shown).
3.2. Effects of NAMI-A on cell adhesion to ECM components and on cell
morphology of HCT-116 CRC cells
HCT-116 cells avidly attached to ECM components, in particular
onto bronectin, collagen I, laminin-111, and Matrigel (Fig. 2A). The
NAMI-A pre-treatment signicantly reduced cell adhesion depending
on the different substrate proteins. On bronectin and on collagen I,
the adhesion reduction was inversely related to the concentration of
NAMI-A: 38 4% and 25 2% on bronectin, at 1 and 10 M,
respectively (p b 0.01 vs untreated controls at both concentrations)
and approximately 40% on collagen I, (p b 0.001 vs untreated controls);
at 100 M NAMI-A virtually no reduction was seen on either substrates. In
contrast, NAMI-A did not affect the basic attachment signals of HCT-116
cells to the mixture of basement membrane proteins Matrigel, and to
collagen IV. Its inhibitory activity on laminin-111 was statistically signicant only at the lowest concentration of 1 M (Fig. 2A).
NAMI-A has induced morphological changes of the HCT-116 cells
seeded on bronectin. Unlike untreated controls that readily had spread
onto the substrate and formed lamellipodia (Fig. 2B, white arrows),
after NAMI-A treatment cells have acquired a more roundish shape.
This effect was always similar within the applied concentration range.
These morphological changes were conrmed by AFM analyses where
the HCT-116 cells apparently had lost their spread shape completely
(Fig. 2C, left upper panel). In addition, 3D images visualization
(Fig. 2C, right upper panel) showed how these cells increased in size
and presented a smooth surface compared to the indented surface of
the untreated cells. At the highest concentration used, NAMI-A induced
a signicant increase of the cell perimeter and a 6-fold increase of cell
volume (compared to untreated control cells), (Fig. 2C lower panel).
These morphological changes are related to the remodelling of the
cytoskeleton of the treated cell in which phenomenon integrins can be
involved.
The reduced adhesion of HCT-116 cells treated with NAMI-A cannot
be ascribed to any modication of cell proliferation and viability since,
in the range of concentrations 1100 M the ruthenium compound is
not cytotoxic even after an incubation time of 72 h (Fig. 3).
3.3. HCT-116 CRC cells adhesion and migration rely on 51 integrin
The expression levels of integrins of the HCT-116 cells were previously analysed using an integrin-mediated cell adhesion uorimetric
array, and the results have been reported elsewhere [34]. The high
expression of 51 integrin of the HCT-116 cells contributed to the
remarkable high adhesion signals of these cells to bronectin. To test
the role of 51 integrin for the adhesion of HCT-116 cells to bronectin, a functional blocking specic antibody towards this integrin was
employed (Fig. 4A). The results highlighted the crucial role of 51
integrin for mediating the HCT-116 cells interactions with bronectin.
Blocking the active site of 51 integrin caused a 78% reduction
of the HCT-116 cell adhesion. A slightly lower reduction of 64% of

Fig. 3. Cell viability after NAMI-A treatment. HCT-116 cells previously seeded into 96-well
plates were treated with NAMI-A (3300 M) for 72 h. At the end of the incubation time
cell viability was detected by MTT test.

HCT-116 cell adhesion rate to bronectin was obtained with the less
selective echistatin, a disintegrin targeting 51, v3, and likely
other RGD-dependent integrins. The predominant role of 51 integrin
in HCT-116 cell adhesion to bronectin was also underlined in the
impedance-based migration assay where HCT-116 cell migration on bronectin is immediately and completely suppressed with the integrin
51-directed antibody (Fig. 4B). Echistatin also reduced the migration
rate of the HCT-116 cells albeit to a less extent and with a delayed onset,
as the cells completely lost their ability to migrate only after a lag phase
of about 10 h.
The combined treatment of cells with the 51 functional blocking
antibody and NAMI-A did not signicantly reduce the adhesion of
HCT-116 cells to bronectin any further (Fig. 4C). This lack of a synergistic effect suggests that both the blocking antibody and NAMI-A targeted
the same adhesion receptor. Thus, the presence of a functional 51
integrin is necessary for NAMI-A to exert its anti-adhesive activity. To
further elucidate this hypothesis, NAMI-A was studied for its potential
to inuence expression and activation of 51 integrin.

3.4. NAMI-A reduces the expression and activation of 51 integrin

through FAK (Focal Adhesion Kinase) auto-phosphorylation
Flow cytometric analysis demonstrated that NAMI-A affected the
number of the 51 integrin receptors on the surface of the HCT-116
treated cells (Fig. 5A). The mean uorescence intensity (MFI) was
moderately but signicantly reduced by approximately 20% with all the
tested concentrations. Although the number of the 51 integrin receptors on the cells diminished after NAMI-A treatment, the percentage of
the 51 integrin-positive cells did not change at all (data not shown).
The down-regulation of the receptor density of the 51 integrin on
the cell surface was in line with the reduced expression of the ITGB1
gene, that encodes for the 1 integrin subunit, that was less than 50%
in the cells treated with NAMI-A in comparison to the untreated controls at all the tested concentrations (p b 0.01, Fig. 5B). The expression
of the gene ITGA5, encoding for the 5 integrin subunit, seemed to be
bi-phasically regulated in dependence of the NAMI-A concentration: it
was reduced at 1 M, while it was signicantly increased, up to 3.5
folds, when 100 M NAMI-A was used (Fig. 5B).
In addition to the modication of the 51 integrin expression levels,
NAMI-A acted also on the downstream integrin-modulated FAK process.
Both the expression and the activation of this pathway are strictly
interconnected and are involved in the adhesion and migration of the
HCT-116 cells to bronectin. Treatment of HCT-116 cells with 0.1 and
10 M NPV-TAE226, an inhibitor of the FAK auto-phosphorylation on
Tyr397, reduced the cell adhesion to bronectin to 25 and 50% at 0.1
and 10 M, respectively (Fig. 6A). Moreover, the treatment with 1 and

C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

Fig. 4. HCT-116 cells adhesion and migration rely on 51 integrin. A) Adhesion on

bronectin in the presence of 51 blocking antibody or echistatin. HCT-116 cells were
seeded to bronectin in presence of a 51-directed antibody or echistatin for 1 h
at 37 C, 5% CO2. Then, adherent cells were labelled with the uorescent dye BCECF
and the uorescence was determined at 535 nm. Each condition was prepared in
quadruplicate and the experiment repeated three times. Statistical analysis: ANOVA and
TukeyKramer post-test: ***p b 0.001 vs CTRL; #p b 0.05 vs echistatin. B) Real time
migration assay. HCT-116 cells were seeded in the presence of a 51-directed
antibody or echistatin on a xCELLigence CIM-16 plate functionalized with bronectin.
Cell migration signals were recorded every 15 min for 24 h. C) NAMI-A effects on
adhesion in presence of an antibody anti-51. HCT-116 cells were left to adhere to
bronectin in presence of NAMI-A (1, 10, and 100 M) dissolved in DPBS and of an
51-directed antibody for 1 h at 37 C, 5% CO2. Then, adherent cells were xed, stained
with sulforhodamine B and absorbance units were read at 570 nm. Data are mean SD
of three independent experiments, each with quadruplicate sets. Statistical analysis:
ANOVA and TukeyKramer post-test: **p b 0.01, ***p b 0.001 vs untreated cells (no anti51 and no NAMI-A); p b 0.001 vs NAMI-A 1 M; p b 0.01 vs NAMI-A 10 M;
### p b 0.001 vs NAMI-A 100 M.

10 m completely suppressed cell migration through a bronectin gel

(Fig. 6B).
Similarly, also NAMI-A signicantly reduced by approximately 70%
and 15% the levels of the P-Tyr397-FAK, at concentrations of 1 and
10 M respectively (p b 0.01 and p b 0.05 vs untreated controls)
(Fig. 7A and B). The inverse correlation between the concentrations of
the ruthenium drug and the effects on FAK phosphorylation was in
agreement with the reported activity in the functional tests, such as in
the adhesion assays to bronectin (see Fig. 2A).
3.5. Silencing of 5 integrin subunit has an impact on NAMI-A activity in
HCT-116 cells
The crucial role of the 51 integrin in the NAMI-A activity was also
analysed by the transient silencing of the ITGA5 gene encoding for the
5 protein subunit. 48 h after the ITGA5 transcription had been

231

Fig. 5. NAMI-A affects the expression levels of 51 integrin in HCT-116. A) cell surface
expression of 51 integrin. HCT-116 cells were treated with NAMI-A (1, 10, and
100 M) in DPBS for 1 h 37 C, 5% CO2, then they were seeded into 6-well plates precoated or not with bronectin, and allowed to adhere 1 h at 37 C, 5% CO2. Adherent
cells were harvested, incubated with an anti-51 primary antibody, followed by
incubation with a FITC-labelled secondary antibody, and then analysed by ow
cytometry. Data are mean SD of two independent experiments, each with triplicate
sets. Statistical analysis: ANOVA and TukeyKramer post-test: **p b 0.01; ***p b 0.001
versus untreated cells. B) 5 and 1 integrin subunits gene expression. HCT-116 cells
were treated with NAMI-A (1, 10 and 100 M) in DPBS for 1 h 37 C, 5% CO2, then they
were seeded into 6-well plates pre-coated or not with bronectin, and allowed to
adhere 1 h at 37 C, 5% CO2. Adherent cells were harvested, and their total RNA content
extracted. Then, a copy of cDNA was obtained by retro-transcription and subjected to
real time RT-PCR. The expression levels of the gene ITGA5 (5 subunit) and ITGB1 (1
subunit) were normalized to the expression of the housekeeping gene RPL5. Data
represent the means SD of three independent experiments, and are the ratio between
the basal expression of mRNA in HCT-116 not treated with NAMI-A (set at 1), to that of
cells treated with the ruthenium compound at each tested concentration. Statistical
analyses: ANOVA and TukeyKramer post-test: **p b 0.01 vs untreated controls.

silenced, HCT-116 cells were tested for their potential to adhere to

bronectin in a real time adhesion assay evaluated through impedance
measurement. In all experiments a scrambled siRNA was used as
negative control; the scrambled siRNA did not induce any signicant
alteration of the adhesion rate of HCT-116 cells (data not shown). The
silencing of the gene ITGA5 was achieved with 70% efciency and,
correspondingly the 51 integrin protein expression on the surface
of the silenced cells was reduced by 60% (HCT-116/siRNA), as determined by ow cytometry (data not shown).
The reduction of the integrin 51 expression decreased the adhesion to bronectin of the silenced HCT-116/siRNA cells (Fig. 8A,
Table 1). 2 h after seeding, when the adhesion process had almost
reached a plateau phase, the adhesion of the 51 integrin-knockdown HCT-116 cells was reduced to 42 3% of the non-silenced control
cells (p b 0.001). Moreover, the lower adhesion signal persisted until the
end of the assay. This result was in line with the residual expression
of 51 integrin (approximately 40%) in the silenced cell population.
Treatment with 1 M NAMI-A of HCT-116/siRNA cells almost completely (91%) inhibited their adhesion rate after 2 h, as compared to HCT116 cells (Fig. 8B, Table 1). 10 and 100 M NAMI-A reduced the adhesion
rate of the silenced cells to 29% and 47% respectively. In comparison, on
the intact cells it reduced the adhesion rate to 58% and 82%, respectively
at 10 and 100 M (Fig. 8C and D, Table 1). This adhesion inhibition is
apparently inversely correlated to the concentration of NAMI-A, and

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C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

cells have adhered, it was no more able to intervene and modify the
process.
4. Discussion

Fig. 6. Role of Tyr397-FAK phosphorylation on HCT-116 cell adhesion and migration.

A) Real time adhesion assay and B) Real time migration assay in the presence of
P(Tyr397)FAK inhibitor. HCT-116 cells were treated with the P(Tyr397)FAK inhibitor
NPV-TAE226 at 0.1, 1 and 10 M for 24 h at 37 C, 5% CO2, and then seeded on a
xCELLigence Roche 16-well plate after it had been functionalized with bronectin. Cell
adhesion and cell migration were monitored for 4 h and 24 h respectively. Each
condition was analysed in quadruplicate and the experiment repeated two times.

this effect is similar to that of NAMI-A in the adhesion assay (Fig. 2A).
Data of Fig. 8 showed also that NAMI-A exerted its effects within the
rst 2 h after the treatment and after cell seeding, i.e. in a time phase
when the adhesion molecules interact with the ECM substrates. After

Fig. 7. NAMI-A affects FAK activation in HCT-116 cells. HCT-116 cells were treated with
NAMI-A (1, 10, and 100 M) in DPBS for 1 h at 37 C, 5% CO2 then they were seeded into
6-well plates pre-coated or not with bronectin, and allowed to adhere 1 h at 37 C, 5%
CO2. Adherent cells were harvested, and their total protein content extracted. Equal
amounts of total proteins from each sample were subjected to immune-blotting and
then probed with antibodies against FAK and P-Y397-FAK. A) Western blots analysis
and B) quantication of Western blot bands by ImageJ software, of one representative
experiment out of three, are displayed. Data shown in panel B are the ratio between
P-Y397-FAK and FAK levels in each sample, after normalization to -actin amount in
the same sample. Statistical analysis: ANOVA and TukeyKramer post-test: *p b 0.05;
**p b 0.01 vs untreated cells.

The present study shows that the ruthenium anti-metastatic drug

NAMI-A reduces the adhesion and migration of the colorectal HCT-116
cell line to bronectin, providing insights into the molecular mechanism
of this promising metal-based compound.
Adhesion and migration are two basic features of the metastatic
cancer progression, and NAMI-A impairs the contact of HCT-116 cells
on bronectin and collagen I, the two main and accessible ECM
substrates in the Disse's space of the liver, the favourite target organ
for the CRC metastases [44,45]. The ability of NAMI-A to disturb the
interactions between tumour cells and their microenvironment is not
new and has been reported previously. Nevertheless, this nding supports our hypothetical concept that the target of NAMI-A is localized
on the cell membrane rather than inside the nucleus as expected by a
heavy metal-based drug [610,14,15]. Past experiences with cell lines
coming from the mammary tissue and representing different grades of
malignancy had pointed out the relevance of the integrins, in particular
of the 51 integrin, in the activity of NAMI-A. Both MDA-MB-231
breast cancer cells and HBL-100 non-tumorigenic cells express the
51 integrin to an extent proportional to their invasive potential.
The expression levels of the 51 integrin are related to the adhesion
rate to bronectin, and correspondingly NAMI-A inhibition of adhesion
and migration to bronectin of these cell lines is much more pronounced for the MDA-MB-231 cells that exhibit the higher expression
of this integrin [6]. Surpassing these reports, the present ndings
represent the rst description of such activity in colorectal cancer and
highlight the inuence of NAMI-A on the binding activity and signalling
capacity of the 51 integrin in this process. Although the antimetastatic activity of NAMI-A and of its analogues has been mostly
investigated in models of secondary tumours developed in the lungs
[1,2], proofs of its effectiveness against metastases in other sites are
also available. For example, NAMI, the sodium salt analogue of NAMIA, inhibited the formation of central nervous system (CNS) metastases
in mice with leukemias and lymphomas [46]. In addition, NAMI-A treatment of mice engrafted with intra-splenic implants of B16/F10 melanoma cells causes 60% of animals completely devoid of hepatic metastases
[G. Sava, unpublished data]. These data prompted us to explore the effects of NAMI-A at the molecular level, in an in vitro model of colorectal
cancer that usually metastasise to the liver. Therefore, the HCT-116 cells
were chosen as the model of invasive colorectal cancer. In these cells,
the 51 integrin is particularly expressed and it drives HCT-116
cell adhesion to bronectin under the inuence of the hepatic microenvironment [34]. The interest for this integrin was generated by the
signicant effect exerted by NAMI-A during the study of the HCT-116
cell adhesion to bronectin, the favourite partner of 51 integrin.
Our present ndings highlight the role of the 51 integrin and of its
signalling pathway as a potential target of NAMI-A, thereby providing
insights into the molecular mode of action of this unique metal-based
drug. We have recently demonstrated how the 51 integrin plays a
leading role in the processes of HCT-116 cell adhesion and migration towards the liver ECM components under the inuence of factors released
by the hepatocytes, and how it regulates the activity/expression of other
integrins such as the 21 [34]. Previous reports from our group have
suggested the involvement of integrins, and of their related intracellular
pathways, to explain the anti-metastatic mechanism of action of NAMIA, that was shown to affect 1 integrin subunit also in other cell models,
such as the KB oral carcinoma and the MDA-MB-231 breast cancer cells,
and 2 integrin subunit in the human polymorphonuclear neutrophils
HPMN [6,12,14]. These considerations prompted us to scrutinize
the involvement of the 51 integrin in the anti-tumour activity of
NAMI-A in an in vitro model of CRC. This work represents the rst report
documenting that NAMI-A inhibits cell surface integrins. Thus, NAMI-A

C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

233

Fig. 8. NAMI-A potentiates the reduction of cell adhesion induced by 5 subunit silencing in HCT-116 cells. HCT-116 and HCT-116/siRNA cells were treated with NAMI-A (1, 10, and
100 M) in DPBS for 1 h at 37 C, 5% CO2, and then were seeded into an E-plate functionalized with bronectin. Cells were automatically monitored every 15 min over 4 h.
Representative graphs comparing the growth curve of HCT-116 cells wild type and HCT/siRNA silenced for 5 subunit (A), and treated with 1 M (B), 10 M (C), and 100 M NAMI-A
(D) were shown.

is able to inuence some important steps of cancer cell dissemination,

such as cell adhesion and migration.
The reduced adhesion to bronectin of NAMI-A-treated HCT-116
cells corresponds to the decreased migration on the same substrate,
and to decreased invasion through the Matrigel barrier. These effects
can reasonably be attributed to the interaction of the ruthenium drug
with the 51 integrin, since the protein functionality can be abrogated
by either a specic blocking mAb or by NAMI-A in a mutually exclusive
manner. The lack of synergism between the mAb and NAMI-A suggests
that both the 51 integrin-directed antibody and NAMI-A act on
the same target and thus similarly, but not additively, inhibit tumour
cell adhesion. Our hypothesis that the 51 integrin is the target of
NAMI-A for the control of cell invasion and metastasis is further
supported by the results with cells on which the 5 integrin subunit
had been knocked down with siRNA. After silencing 51 integrin
expression cells migrated less on bronectin. NAMI-A was still able to
reduce adhesion and migration of these knocked-down cells. This
observation can be explained by the residual integrin expression in
the cell membrane of the HCT-116/siRNA cells (about 40% 51
Table 1
Per cent of adhesion of HCT-116 and HCT-116/siRNA cells treated with NAMI-A.
NAMI-A M

HCT-116

HCT-116/siRNA

0
1
10
100

100 10
68 10***
58 10***
82 6*

42 3***
9 7
29 6
47 8

Data taken from experiment of Fig. 8 at t = 2 h.

HCT-116 and HCT-116/siRNA cells were treated with NAMI-A (1, 10, and 100 M) in DPBS
for 1 h at 37 C, 5% CO2, and then were seeded into an E-plate functionalized with bronectin. Cells were automatically monitored every 15 min over 4 h. the percentages reported in
the table refer to analysis at 2 h.
Adhesion of un-treated HCT-116 was taken as 100%. Statistical analysis of Variance ANOVA
and TukeyKramer post-test: *p b 0.05 and ***p b 0.001 vs HCT-116 not treated with
NAMI-A; p b 0.01 and p b 0.001 vs HCT-116 treated with the same concentration of
NAMI-A.

integrin expression as compared to non-silenced cells). Therefore, the

silenced cells are still sensitive to the inhibitory effects of NAMI-A. The
nding that the blocking mAb completely abrogated the 51 integrin
activity, similarly to NAMI-A, further supported this mode of action.
NAMI-A appears to inuence the 51 integrin by reducing the
number of the 51 integrin molecules on the cell membrane, and by
changing the expression of the genes that encode the 5 and 1 subunits. However, this global result appears to depend upon a different
interaction with these genes since they respond in a concentration
dependent (5 chain) or independent (1 chain) manner. The downregulation of the gene encoding for the 5 chain induced by 1 M
NAMI-A, together with the same modulation of the 1 subunit gene,
may explain the results obtained in the functional adhesion test to bronectin, which showed the highest inhibition at 1 M NAMI-A concentration. Although the 5 subunit gene is up-regulated by NAMI-A at
100 M, this effect does not necessarily translate into a higher protein
expression/activity, since integrins are only active if the two chains are
present and properly assembled into the integrin heterodimer [47].
The down-regulation of the 1 subunit gene can therefore inuence
the expression and consequently the functionality, also of other
integrins carrying the 1 chain, such as the 21, an integrin which is
responsible for cell adhesion to collagen I. Consequently, the reduction
of cell adhesion to collagen I after NAMI-A treatment may reasonably
depend on the reduced 51 integrin expression/activity that in turn
reduces also the expression of the 21 integrin, a phenomenon of
cross-talk between these integrins that we have already documented
in the HCT-116 CRC cells [34].
In addition, our study reveals that NAMI-A affects the integrin signalling via FAK. The decreased expression of the 51 integrin on the cell
membrane, caused by NAMI-A, strictly correlated with the reduced
auto-phosphorylation of FAK at Tyr397, that constitutes an early event
of the 51 integrin-mediated signalling [48,49], and that depends
upon 51 activation [50,51]. At the same time, FAK modulates the
strength with which the cell membrane integrins bind to the ECM
proteins [50,51]. We hereby demonstrate that the activation of FAK,

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C. Pelillo et al. / Journal of Inorganic Biochemistry 160 (2016) 225235

through the Tyr397 phosphorylation, is a crucial event involved in the

adhesion and migration of the HCT-116 CRC cells through bronectin,
since the use of NVP-TAE226 (an inhibitor of the FAK autophosphorylation on Tyr397) reduces the real time cell adhesion and migration. The phosphorylation at this site is known for its importance to
induce the maximal activation of FAK and of the downstream effectors
[52] through the modulation of the assembly of the focal adhesions at
the leading and/or tracking edges of the migrating cells [53]. At
this level, 10 M NAMI-A also reduces the auto-phosphorylation and
correspondingly FAK activation, leading to an impairment of cell adhesion comparable to that measured with the same concentration of
the NVP-TAE226 inhibitor (reduction of 4045% vs controls at t = 2 h;
compare Figs. 6A and 8C). Interestingly this effect has been detected at
the lower concentrations of 1 and 10 M NAMI-A. The fact that the
highest concentration of 100 M NAMI-A causes more attenuated,
absent or opposite effects (see for example the effects on the 5 subunit
gene expression) is not contradictory since other studies suggested the
presence of a critical threshold concentration for the pathway to be
switched on inside cells, as a response to an induced stress and to
maintain a correct homeostatic balance [54].
Concerning the apparent inverse dose-dependent activity of NAMIA, it should be noted that other studies have pointed out the activity
of NAMI-A at low rather than at high doses, such as the clinical phase I
and phase II trials [55,56]. These evidences support the hypothesis
that NAMI-A exerts its effects on tumour metastases by the modulation
of the biological processes rather than by the destructive effect on DNA
or on other targets of the cell replicative mechanism and by consequent
direct cell cytotoxic effects. Our study shows that the 51 integrin can
be the target of NAMI-A on the HCT-116 cells and that this targeting is
responsible for the inhibition of cell invasion and migration. Moreover,
the dose-dependent effects of NAMI-A on the integrin-mediated adhesion of HCT-116 CRC cells on bronectin depend on the expression/
activation of this integrin. NAMI-A affects the integrin signalling and
likely the integrin activity by preventing FAK auto-phosphorylation.
The accumulation of such data further suggests the breaking role of
NAMI-A for the pharmacological treatment of cancer malignancy with
metal-based drugs and supports the need to look at this drug as the
reference candidate for the development of a new class of drugs for
the control of the cancer disease.
Acknowledgements
Authors gratefully acknowledge the COST Action CM1105 for STSM
of Chiara Pelillo and Hilaria Mollica. JAE is supported by the Deutsche
Forschungsgemeinschaft (DFG grant: SFB1009 project A09).
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