Evaluation of functionalized mesoporous silica SBA-15 as a carrier system for Ph3Sn(CH2)3OH against the A2780 ovarian carcinoma cell line

Article abstract of DOI:10.1039/c6dt03519a

SBA-15|Sn3, a mesoporous silica-based material (derivative of SBA-15) loaded with an organotin compound Ph₃Sn(CH₂)₃OH (Sn3), possesses improved antitumor potential against the A2780 high-grade serous ovarian carcinoma cell

Full text of DOI:10.1039/c6dt03519a

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Dalton Trans., 2016, DOI: 10.1039/C6DT03519A.
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Efficient extraction of sulfate from water using
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ARTICLE
Evaluation of functionalized mesoporous silica SBA-15 as a carrier
system for Ph3Sn(CH2)3OH against A2780 ovarian carcinoma cell
line
Received 00th January 20xx,
Accepted 00th January 20xx
Christian Bensing,#a Marija Mojić,#b Santiago Gómez-Ruiz,*c Sandra Carralero,c Biljana Dojčinović,d
DOI: 10.1039/x0xx00000x
Danijela Maksimović-Ivanić,b Sanja Mijatovićb and Goran N. Kaluđerović*e
www.rsc.org/
SBA-15|Sn3, a mesoporous silica-based material (derivative of SBA-15) loaded with organotin compound Ph3Sn(CH2)3OH
(Sn3), possess improved antitumor potential against A2780 high-grade serous ovarian carcinoma cell line in comparison to
Sn3. It is demonstrated that both the compound and the nanostructured material are internalized by A2780 cells. Similar
mode of action of Sn3 and SBA-15|Sn3 against A2780 cell line was found. Explicitly, induction of apoptosis, caspase 2, 3, 8
and 9 activation, accumulation of cells in hypodiploid phase as well as accumulation of ROS was observed. Interestingly,
Sn3 loaded in mesoporous silica-based material needed to reach concentration 3.5 times lower than the IC50 value of Sn3
compound, pointing out on higher effect of the SBA-15|Sn3 than Sn3 alone. Clonogenic potential, growth in 3D culture as
well as mobility of cells were disturbed in the presence of SBA-15|Sn3. Such behavior could be associated with the
suppression of p-38 MAPK. Less profound effect of Sn3 comparing to SBA-15|Sn3 could be attributed to a different
regulation of p-38 and STAT-3 which are mainly responsible for appropriate cellular response to diverse stimuli or
metastatic properties.
transportation.6 Such pores can adopt various biomaterials of
different nature, from small molecules to proteins, among others, in
relatively large amounts.7,8
Introduction
One of the most interesting fields of the modern nanomedicine is
related to nanoparticles efficient in hosting, protecting, carrying
and releasing therapeutic agents at target tissues.1–3 2001.
mesoporous silica nanoparticles (MSNs) were suggested, by the
group of M. Vallet-Regi, as an interesting drug release system.4
Since then many researchers showed a growing interest in MSNs for
their innovative potential in nanomedicine.1 MSNs can be prepared
in various sizes and shapes, survive in severe milieu and have
various regular pore sizes.5 Furthermore, the pore diameter can be
altered to preserve diverse substances (2–50 nm) enabling their
MSNs and other silica-based materials, non-toxic to cells,9 are
assumed as extraordinary particles in quite a few fields of medicine
such as bone regeneration, for example, however, in cancer
treatment not many investigations have been reported until now.10–
13
An interesting property of this kind of materials is that they
favourably accumulate in solid tumours due to the increased
permeation and retention effect.14,15
From our pioneering work, which began 2009, it is revealed that
MSNs and other silica-based materials such as SBA-15 and MCM-41
do not affect tumor cell lines in high concentrations, up to 1
mg/mL.16–18 On the contrary, the same kind of materials loaded
with titanocene compounds enhance the cytotoxicity in tumor cells,
depending on the particle type (SBA-15 or MCM-41), titanium(IV)
complex as well as its loaded amount. Moreover, SBA-15 was found
to be superior to MCM-41 as carrier system. Recently, we reported
on SBA-15 derivative loaded with Ph3Sn(CH2)6OH observing that this
material was ca. 150 time more active than SBA-15 loaded with
titanium(IV) complexes.19 The strong therapeutic potential of
organotin(IV)-loaded mesoporous silica lies in the development of a
nonproliferative melanocyte-like phenotype of surviving cells. Thus,
this nonaggressive suppression of tumor growth with nontoxic
doses of the drug, much lower than the IC50 concentration of
Ph3Sn(CH2)6OH, is highly efficient against melanoma cells. Contrarily
a.Institute of Chemistry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-
Straße 2, D-06120 Halle, Germany
b.Institute for Biological Research “Sinisa Stankovic” University of Belgrade, Bulevar
despota Stefana 142, 11060 Belgrade, Serbia
c. Departamento de Biología y Geología, Física y Química Inorgánica, E.S.C.E.T.,
Universidad Rey Juan Carlos, 28933 Móstoles, Madrid, Spain
d.Department of Chemistry, Institute of Chemistry, Tehnology and Metallurgy,
University of Belgrade, Studentski trg 12-14, 11000 Belgrade, Serbia.
e. Department of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry,
Weinberg 3, D 06120 Halle (Saale), Germany
* Correspondence: Goran N. Kaluđerović, phone: +49 345 5582 1370, Fax: +49 345
5582 1309, E-mail: goran.kaluderovic@ipb-halle.de; Santiago Gómez-Ruiz, phone:
+34 914 888 507, Fax: ++34 914 888 500, E-mail: santiago.gomez@urjc.es.
# equally contributed
† Electronic Supplementary Information (ESI) available: 29Si MAS, 13C CP/MAS and
119Sn MAS NMR spectra, XRD diffractogram, N2-adsorption-desorption isotherm,
particle size distribution determined, pore diameter distribution for of SBA-
15|Sn3, Sn3 release study from SBA-15|Sn3
This journal is © The Royal Society of Chemistry 2016
Dalton Trans., 2013, 00, 1-9 | 1
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to the induction of apoptosis as the most employed conventional (8 μs 90º pda, spinning speed of 6 MHz, pulse delay 10 s) 119Sn
View Article Online
DOI: 10.1039/C6DT03519A
approach in oncotherapy, this mode of action has numerous MAS NMR (8 μs 90º pda, spinning speed of 6 MHz, pulse delay
advantages.20 One of them is avoiding of compensatory 10 s) and X-ray diffraction (XRD) pattern of the silicas were
proliferation phenomenon present in tumors in response to cell obtained on a Phillips Diffractometer model PW3040/00 X'Pert
death in the neighborhood.21
MPD/MRD at 45 KV and 40 mA, using a wavelength Cu Kα (λ =
As one of the most lethal gynecological malignancy and fifth leading 1.5418 Å). MS (ESI) spectra were taken on a Finnigan MAT TSQ
cause of death in women,[21] ovarian cancer cause more than 7000 (voltage 4.5 kV, sheath gas nitrogen) instrument. C, H
100,000 instances worldwide each year.[22] From the epithelial and N elemental microanalyses were carried out with a CHNS-
ovarian cancer, four major histological subtypes are identified.[23] 932 (LECO) elemental analyzer (Sn3), in the Microanalytical
Namely, serous, endometrioid, clear cell and mucinous carcinoma Laboratory of the University of Halle. N2 gas adsorption-
from which serous type is responsible for ca. 70 % of epithelial desorption isotherms were performed using a Micromeritics
ovarian cancers. Furthermore, high-grade serous ovarian carcinoma ASAP 2020 analyzer. Scanning electron micrographs and
(HGSOC) is considered as the most aggressive subtype causing morphological analysis were carried out on a XL30 ESEM
around 70 % of all ovarian cancer deaths.25 A2780 cell line belongs Phillips with an energy dispersive spectrometry system (EDS).
to the most prevalent subtype of HGSOC, beside CAOV3, SK-OV-3, The samples were treated with a sputtering method with the
IGROV1 and OVCAR-3 cells.26 Most patients, after radical surgery following parameters: Sputter time 100 s, Sputter current 30
and primary excessive response to platinum- and taxane-based mA, film thickness 20 nm using a Sputter coater BAL-TEC SCD
chemotherapy, face a relapse of HSGOC with a survival of only 18 005. Conventional transmission electron microscopy (TEM)
months.[25] As a consequence, development of novel drugs is of high was carried out on a TECNAI 20 Phillips, operating at 200 kV.
importance in order to advance current therapies.
SBA-15 and SBA-15p (Table 1) were prepared as described
Herein we report the preparation and characterization of the previously.19
organotin(IV) compound Ph3Sn(CH2)3OH (Sn3) which has been
Synthesis of Ph3Sn(CH2)3OH (Sn3)
grafted into functionalized SBA-15 (→SBA-15|Sn3). Furthermore,
Sn3 and SBA-15|Sn3 have been tested against A2780, A549 and
DLD-1 tumor cell lines. A2780, as model of HGSOC,23 was chosen for
evaluation of Sn3 and SBA-15|Sn3 mechanism of action. This cell
line overexpressed multidrug resistant pumps and therefore is an
ideal model system for assessment of acquired resistance to
chemotherapeutics drugs.27 It is well known that metal-based drugs
belong to the group of the most sensitive compounds for efflux by
the pumps.28 On the other side, compounds loaded in MSNs and
other silica-based materials accumulate into the cells due to down
regulation of transporters as well as bypass the efflux action by
MSN themselves.29 Therefore, it was of interest to compare the
effectiveness and the mechanism of action of Ph3Sn(CH2)3OH (Sn3)
compound in free and loaded, SBA-15|Sn3, form. Thus, the
influence on the cell death, metastatic properties of the cell and
intracellular response were estimated.
Ph3SnH (5.86 g, 16.7 mmol), allylalcohol (2.8 mL, 33.4 mmol)
and AIBN (0.14 mg, 0.8 mmol) were stirred for 3 h at 80 °C.30
Precipitated product was collected, dried under vacuum and
subsequently two times recrystallized from petrol ether.
Mother liquor was concentrated and purified by column
chromatography (eluent: petrol ether:CHCl3 = 2:1, afterwards
ether) and recrystallized from n-hexane. Yield: 4.43 g (64.9%);
colorless needles; MP: 104–105 °C; MS (ESI): m/z = calc.
[C21H22OSn –H+]– 410.1, found: 409.4; Elemental analysis (in
%): calc. for C21H22OSn: C 61.65, H 5.42, found: C 61.57, H 5.27;
1H NMR (CDCl3, 400 MHz): δ (in ppm) = 1.26 (t, 3J(H,H) =
5.48 Hz, 1H; OH), 1.49–1.55 (m, 2H; C1H2), 1.98 (m, 2H; C2H2),
3.65 (m, 2H; C3H2), 7.37 (m, 9H; Ar-Hm und Ar-Hp), 7.56 (m, 6H;
Ar-Ho); 13C NMR (CDCl3, 101 MHz): δ (in ppm) = 6.7 (C1,
2
1J(119Sn,C) = 397.1 Hz), 29.3 (C2, J(117/119Sn,C) = 21.2 Hz), 65.6
(C3, 3J(119Sn,C) = 66.3 Hz), 128.5 (Cm,
3J(119Sn,C)
=
48.8 Hz), 128.9 (Cp,
= 11.0 Hz), 137.0 (Co,
4J(117/119Sn,C)
2J(119Sn,C) = 36.1 Hz), 138.9 (Ci, 1J(119Sn,C)
= 491.6 Hz).; 119Sn NMR (CDCl3, 149
MHz): δ (in ppm) = –99.3 (s, Ph3Sn).
Experimental
Materials and methods
All manipulations for the synthesis of tin compound and the
functionalized materials were performed under dry nitrogen
gas using standard Schlenk techniques and glove box. Solvents
were distilled from the appropriate drying agents and
degassed before use. 3-chloropropyltriethoxysilane (CPTS) was
purchased from Sigma-Aldrich and used directly without
further purification.
Preparation of SBA-15|Sn3
A solution of 3-(triphenylstannyl)propan-1-ol (0.315 g, 0.77
mmol) in toluene (100 mL) was added to SBA-15p (1.00 g).
Subsequently, triethylamine (75 mg, 0.92 mmol) was added
and the mixture was stirred 48 h at 80 ºC. The slurry was
filtered through a frit and the solid residue washed with
toluene (3 × 30 mL), hexane (3 × 30 mL), water:ethanol (1:1, 2
× 20 mL) and diethylether (2 × 60 mL). The resultant solid was
dried under vacuum at room temperature for 24 h to give a
white free flowing powder. 29Si MAS NMR: δ (in ppm) = –113
1H, 13C and 119Sn NMR spectra for Sn3 were recorded on Varian
Gemini 400 and 13C CP/MAS NMR, 29Si MAS NMR and 119Sn
MAS NMR spectra were recorded on a Varian-Infinity Plus
Spectrometer at 400 MHz. 13C CP/MAS NMR (4.40 μs 90º
pulse, spinning speed of 6 MHz, pulse delay 2 s), 29Si MAS NMR
2 | Dalton Trans., 2016, 00, 1-9
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(br, s; including Q4 [Si(OSi)2(OH)], Q3 [Si(OSi)2(OH)], Q2 cells were treated with 2×IC50 or 2×MC50 of Sn3 or SBA-15|Sn,
View Article Online
DOI: 10.1039/C6DT03519A
[Si(OSi)2(OH)2]), –73 to –62 (br, [SiO–Si(OEt)n(CH2CH2CH2Cl)]; n respectively, for 24 h. After this period, cells on the slides were
= 0–1). 13C CP/MAS NMR: δ (in ppm) = 4.3 (CH2–Sn and CH2–Si), stained with acridine orange/ethidium bromide mixture (3
11.4 (CH2 of CPTS), 18.1 and 31.2 (CH2 of Sn-containing μg/mL AO and 10 μg/mL EB in PBS), and visualized under a
fragment), 24.6 (CH3 of Et), 45.0 (O–CH2 of Et), 51.7 (O–CH2 of fluorescence microscope (Carl Zeiss).
Sn-containing fragment), 126.7 and 134.5 (Ph); 119Sn MAS
Caspase 2, 3, 8 and 9 enzyme activity assays
NMR: δ (in ppm) = –105 ppm. %Sn (obtained by X-ray
Activity of caspases 2, 3, 8 and 9 was measured using the
caspase substrate cleavage assay. A2780 cells were seeded at
density of 1×106/well in 6 well plate. After exposure to IC50 or
MC50 concentration of Sn3 or SBA-15|Sn3, cells were sampled
after 2 h for cleavage of caspases. Adherent cells were washed
with cold PBS, collected with a cell scraper, and suspended in
cell lyses buffer (50 mM Hepes pH 7.4, 1% Triton X100, all from
Sigma-Aldrich). After incubation for 10 min on ice and
centrifugation, protein concentrations of the supernatants
were measured according to a method of Bradford (Bio-Rad
Laboratories). Samples (50 μg protein extract) were incubated
on a microplate at 37 °C overnight in reaction buffer (50 mM
Hepes pH 7.4, 0.1% CHAPS, 5 mM EGTA, 5% glycerol)
containing 10 mM DTT (all from Sigma-Aldrich) and a specific
substrate of caspases. Extinction of released p-nitroaniline was
measured at 405 nm using a 96 well plate reader (Tecan
Spectra) and activity of caspases 2, 3, 8 and 9 was evaluated by
OD ratio of treated/untreated samples.32,34
fluorescence) = 4.35%. Textural parameters of the synthesized
surface obtained by N2-adsorption-desorption isotherms: BET-
surface: 417 m2·g–1, pore volume: 0.49 cm3·g–1, pore size: 4.88
nm and wall thickness: 5.82 nm.
Table 1 Textural and chemical properties of synthesized materials SBA-15, SBA-15p and
SBA-15|Sn3 calculated from N2-adsorption-desorption isotherms, XRD and XRF analysis
Surface
d100
[nm]
9.22
9.48
9.27
a0
SBET
[m2/g]
932
535
417
Vm
[cm3/g]
0.94
0.58
0.49
DP
Wd
[nm]
10.65
10.91
10.70
[nm]
4.97
4.88
4.88
[nm]
5.68
6.03
5.82
SBA-15
SBA-15p
SBA-15|Sn
Reagents and cells
Fetal calf serum (FCS), RPMI-1640, phosphate-buffered saline
PBS), dimethyl sulfoxide (DMSO), Sulforhodamine (SRB),
(
crystal violet (CV) and propidium iodide (PI) were obtained
from Sigma (St. Louis, MO). Annexin V-FITC (AnnV) was from
Santa Cruz Biotechnology (Dallas, TX). Human ovarian A2780
cell line was a kind gift from Thomas Miller (Institute for
Oncology, Martin Luther University Halle-Wittenberg.
Matrigel™ Basement Membrane Matrix was purchased from
BD Biosciences. Cells were regularly propagated in HEPES-
buffered RPMI-1640 medium supplemented with 10% FCS, 2
ICP-MS analysis
For drug uptake experiments A2780 cells (1.2×106) were
seeded in the flask. After 24 h cells were treated with IC50 or
MC50 dose of Sn3, SBA-15|Sn3 or SBA-15p (10 mL) for 24 h.
Afterwards the cells were washed with PBS (4 mL) and
trypsinized (1 mL). Cells were resuspended in PBS (4 mL) and
centrifuged (5000 U/min, 5 min). Cell pallet was washed two
times with PBS (1 mL). Lyophilised cells was analysed with ICP-
MS (Wessling Laboratorien GmbH, Oppin, Germany).
For Sn3 release study SBA-15|Sn3 (2.0 mg) was suspended in
completed medium (1 mL), for each time point, and tin
content was investigated with ICP-MS after 0.08, 2, 4, 6, 24
and 72 h, in triplicate. Explicitly, after soaking at 37 °C
suspensions were centrifuged (5000 U/min) and supernatants
were used for analysis. Liquid samples were diluted with
purified water (1:100, v/v). The Thermo Scientific iCAP Qc ICP-
MS instrument was optimized for optimum performance in
standard mode using the supplied autotune protocols. The
ICP-MS instrument was tuned using a solution TUNE B iCAP Q
(1 μg/L of each: Ba, Bi, Ce, Co, In, Li, U) provided by the
manufacturer Thermo Scientific, Germany. External standards
for the instrument calibration were prepared on the basis of a
tin plasma standard solution (Specpure®, Sn 100 µg/mL
certified reference solution ICP Standard purchased from Alfa
Aesar GmbH & Co KG, Germany). For extracts, the limit of
quantitation (LOQ) for tin was determined to be 2.02 ng/L. The
measurement was performed on isotope 120Sn.
mM
L-glutamine, 0.01% sodium pyruvate and antibiotics
(culture medium) at 37 °C in a humidified atmosphere with 5%
CO2. Cells were seeded at 5 × 103 cells/well in 96-well plates
for viability determination and 2.5 × 105 cells/well in 6-well
plates for flow cytometry.
Preparation of drug solutions
Sn3 was dissolved in DMSO at 20 mM and kept at –20 °C
until use. SBA-15|Sn3 stock (1 mg/mL) was dissolved in
culture medium directly before use. Immediately before
treatment various working solutions were prepared in
culture medium.
Sulforhodamine test (SRB)
A2780 cells were seeded at 1,000 cells/well density and
treated with wide range of doses of Sn3 or SBA-15|Sn3 for 72
h. At the end of cultivation SRB test was done exactly as
described.31
Morphological analysis (AO/EB double staining)
Sn3 or SBA-15|Sn3 induced cell death in A2780 ovarian tumor
cells was determined using acridine orange (AO) and ethidium
bromide (EB), double staining according to standard
procedures and examined under a fluorescence microscope.33
A2780 cells were seeded overnight on 8 chamber slides (6,000
cells/chamber) in 400 µL of complete medium. The next day,
Cell cycle analysis
Cells were incubated with a IC50 or MC50 dose of Sn3 or SBA-
15|Sn3 for 72 h and cell cycle analysis is done as previously
described.35
This journal is © The Royal Society of Chemistry 2016
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Measurement of ROS
Western Blot Analysis
View Article Online
DOI: 10.1039/C6DT03519A
For detection of reactive oxygen species the redox-sensitive A2780 cells were cultivated with an IC50 or MC50 dose of Sn3 or
dye dihydrorhodamine 123 (DHR) was used. The cells were SBA-15|Sn3 for 6,12, 24 and 48 h and lysed in protein lysis
stained with 1 mM DHR for 20 min before treatment with an buffer (62.5 mM Tris–HCl (pH 6.8 at 25 ◦C), 2% (w/v) SDS, 10%
IC50 or MC50 dose of Sn3 or SBA-15|Sn3. After 48 h cells were glycerol, 50 mM dithiothreitol). Electrophoretical separation of
trypsinized, washed in PBS, and the fluorescence intensity was proteins was performed on 10–12% SDS-polyacrylamide gels.
analysed with a CyFlow® Space Partec using PartecFloMax® Electrotransfer to polyvinylidenedifluoride membranes at 5
software (Partec GmbH, Münster, Germany).
mA/cm2 was accomplished with a semidry blotting system
(Fastblot B43; BioRad, Göttingen, Germany). Blocking of
membranes was done with 5% (w/v) BSA in PBS. Membranes
Cell clonogenic survival assay
Cells were pretreated for 72 h with an IC50 or MC50 dose of Sn3 were incubated over night at 4 ◦C with specific antibodies to
or SBA-15|Sn3, then detached, counted and 1000 cells/well phospho-Akt,Akt, phospho-p44/p42 MAPK
were reseeded. Cells were grown for 5 days without changing (Erk1/2)(Thr202/Tyr204), p44/p42 MAPK (Erk1/2), phospho-
the medium. After 9 days of cultivation, colonies were fixed p38 MAPK (Thr180/Tyr182), p38 MAPK, and STAT3 (Cell
with 4% PFA for 30 min at RT and then stained 30 min at RT Signaling Technology, Danvers, MA. USA), and Actin (Abcam,
with 0.25% 1,9-dimethyl methylene blue in 50% ethanol. Cambridge, UK). Goat anti-rabbit IgG-HRP (Santa Cruz
Finally, colonies were washed twice with PBS and analyzed by Biotechnology, Dallas, TX, USA) was used as a secondary
light microscopy. Plate efficiency (PE)is calculated as number antibody. Bands were detected with the chemiluminescence
of colonies/number of cells×100, while surviving fraction is detection system (ECL; GE Healthcare, Chalfont St. Giles,
calculated as PE of treatment/PE of control×100.
Buckinghamshire, UK).
Adhesion assay
Results and discussion
Preparation and characterization
96 well plates were covered with matrigel 10 µg/mL at 4 °C
overnight. Wells were washed 3 times with PBS before seeding
the cells. Cells were pretreated for 72 h with an IC50 or MC50
dose of Sn3 or SBA-15|Sn3, then detached, counted and left
for approximately 30 min for membrane reconstitution. Then,
30,000 cells/well are allowed to adhere to matrigel for 1 h.
Nonadherent cells were then washed 3 x with PBS, fixed with
4% PFA, stained with crystal violet 15 min at RT. Cells were
washed with tap water, and dye was dissolved in 33 % of
acetic acid. Absorbance was measured at 570 nm and results
are calculated as percentage of control nontreated cells.
The synthesis of Ph3Sn(CH2)3OH (Sn3) as well as SBA-15 and
SBA-15p were performed according to the literature
procedures.19,30 SBA-15 was synthesized by sol-gel method and
subsequently calcined. SBA-15p was obtained by
functionalization
of
SBA-15
surfaces
with
3-
chloropropyltriethoxysilane (CPTS), in a subsequent step Sn3
was loaded into SBA-15p (SBA-15|Sn3).
A uniform morphology of MSN particles was observed by SEM
and TEM analysis. Thus, images revealed nanostructured-rods
shape of the particles with particle size of ca. 670 nm length
and ca. 440 nm width (calculated from SEM data) and a
uniform pore size with hexagonal distribution (Fig. 1). SEM and
TEM images of SBA-15p and SBA-15|Sn3 particles did not
demonstrate any substantial alteration to SBA-15 particles
indicating that the morphology of the MSNs was not affected
after chemical modification (SBA-15p) neither adsorption (SBA-
15|Sn3).
The physical parameters of nitrogen adsorption-desorption
isotherms, BET surface area, average pore diameter, average
pore volume, and wall thickness were measured for all the
materials. Thus, nitrogen adsorption-desorption analysis of
SBA-15|Sn3 showed a type IV isotherms36 indicating the
mesoporous nature of materials. As expected, the pore
diameter as well as surface area of MSNs is affected after
subsequent functionalization steps of SBA-15.
Transmigration assays
Cells were pretreated for 72 h with an IC50 or MC50 dose of Sn3
or SBA-15|Sn3, and then detached, counted and 2×105 cell
were seeded in Transwells (membrane pore size, 8 mm;
diameter, 6.4 mm; BD Biosciences Discovery Labware)
assembled in 24-well plates in 0.1% BSA-RPMI. As
a
chemoattractant for the cells, the lower chambers were filled
up with 10% fetal calf serum-RPMI 1640 medium. Cells were
allow migrating for 24 h. The cells that failed to migrate
remained on the upper surface and were carefully removed
with a cotton swab while cells on the lower surface of the
membranes were fixed with 4% PFA and stained with Mayer’s
hematoxylin (BioOptica, Milan, Italy). Cells were counted
under a light microscope at 40× magnification using the new
computer-assisted CAST Integrator System (Visiopharm,
Hoersholm, Denmark). The average number of cells attached
to the lower surface in 30 independent fields (200 mm × 200
mm) per membrane is presented. Each experiment was
performed in triplicate.
4 | Dalton Trans., 2016, 00, 1-9
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Cytotoxicity and mechanism of action of SBA-15|Sn3VaiegwaAinrtsictle Online
DOI: 10.1039/C6DT03519A
A2780 cell line
The in vitro anticancer activity of Sn3, SBA-15p and SBA-
15|Sn3 was investigated against ovarian A2780, lung A549 and
colon DLD-1 tumor cell lines with SRB assay.38 The IC50 values
along with those of cisplatin are presented in Table 2.
Table 2 Cytotoxic potential of Sn3 and SBA-15|Sn3
Compound/Material
cisplatin
A2780
A549
DLD-1
0.6 ± 0.1
2.8 ± 0.1
2.6 ± 0.5
7.2 ± 1.4
1.5 ± 0.1
3.8 ± 0.1
2.5 ± 0.1
6.8 ± 0.1
> 500
5.1± 0.2
3.7 ± 0.1
3.5 ± 0.1
9.7 ± 0.2
Sn3
IC50 ± SD [μM]
SBA-15|Sn3
SBA-15p
MC50 ± SD [μg/mL]
The activity of SBA-15p materials was found inactive under
investigated conditions, what is in accordance to previous
results.19 Depending of the cell line Sn3 compound exhibits
lower (A2780, A549) or higher activity (DLD-1) than cisplatin.
The amount of Sn3 (4.35%) in MC50 concentration of SBA-
15|Sn3 material is similar to that of IC50 dose of free Sn3.
However, its activity is in the same concentration range as
previously reported analogous MSNs containing hexyl
(Ph3Sn(CH2)6OH) instead of propyl spacer (Ph3Sn(CH2)3OH) in
organotin(IV) compound.19 In addition, activity of SBA-15|Sn3
material is at least 150 times higher than that of the most
Fig. 1 A. SEM and B. TEM images of SBA-15|Sn3
.
Explicitly, uniform pore diameter of 4.97, 4.88 and 4.88 nm,
calculated by BJH method,37 and a high BET surface of 932, 535
and 417 m2•g–1 (SBET) for SBA-15, SBA-15p and SBA-15|Sn3,
respectively, were found. In addition, the wall thickness in
materials from SBA-15, SBA-15p to SBA-15|Sn3 (5.68 → 6.03)
increased after the first functionalization and remained almost
the same (5.82 nm, if not somewhat higher) after the second
functionalization.
active titanocene compound, [Ti{Me2Si(5-C5Me4)(5-
C5H4)}Cl2], grafted into SBA-15 (MC50 = 309±42 µg/mL), from
the previous study.16
This indicates that, in the first reaction (with CPTS), the
functionalization takes mainly place inside the pore of the
nanostructured material, while in the second modification the
incorporation of the tin compound may be both in the pore
and onto the external surface area.
SBA-15 demonstrated a well-resolved pattern at low 2θ values
with a very sharp (100) diffraction peak at 0.958. This system
can be indexed as a hexagonal lattice with d-spacing values of
92.2. X-ray diffraction pattern (XRD) of the SBA-15p as well as
SBA-15|Sn3 materials exhibited also reflections typical for
hexagonally ordered mesoporous materials (2θ = 0.920 and
0.953; d = 94.8 and 92.7 for SBA-15p and SBA-15|Sn3,
respectively).[18] X-ray fluorescence spectroscopy revealed a
functionalization rate of 4.35 % (w/w) of Sn per gram of
material.
Diminished viability of A2780 cells after exposure to Sn3 or
SBA-15|Sn3 was connected with induction of apoptotic cell
death. In order to evaluate the apoptotic process, double Ann-
PI staining was performed (Fig. 2.A).
Intensified percentage of both early and late apoptotic A2780
cells upon the 72 h of treatment with SBA-15|Sn3 in
comparison to Sn3 were detected (Fig. 2.A). Morphological
changes of apoptotic cells, such as nuclei shrinkage as well as
condensation and/or fragmentation of the DNA, are observed
in the A2780 cells treated with Sn3 or SBA-15|Sn3 (Fig. 2.B).
The amount of activated caspases detected was proportional
to apoptosis intensity in both cultures (Fig. 2.C). Moreover, it
could be clearly observed that activation of caspases initiators
2, 8 and 9 as well as caspase executor 3 upon treatment of
A2780 cells occur after 24 h of treatment with free as well as
loaded Sn3 compound.
Thus, certain amount of Sn3 is released from nanomaterial,
since MSNs are not capable to interact with cell death
receptor. In addition to this, analysis of cell cycle distribution
(Fig. 2.D) showed accumulation of cells in hypodiploid phase
confirming the apoptosis. The presence of cells with
fragmented DNA correlated with results of Ann/PI staining.
Triggered apoptotic process was well synchronized with
enhanced production of ROS species in cell cultures exposed to
both free Sn3 and loaded MSN, suggesting their involvement
in this process (Fig. 2E).
Grafting of SBA-15p was furthermore confirmed by 13C
CP/MAS NMR and 119Sn MAS NMR spectroscopy. Additionally
to the pattern of the chemical shifts observed in the 13C
CP/MAS NMR spectrum of SBA-15|Sn3 appearance of a set of
signals related to methylene and phenyl carbon atoms of the
Sn3 was detected. In the 119Sn MAS NMR spectrum, a singlet
arising from the Sn from tetraorganotin(IV) compound (–105
ppm) corresponds to Sn3 (–99.3 ppm in CDCl3).
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Fig. 2 Sn3 and SBA15|Sn3 induced caspase dependent apoptosis. A) AnnV-FITC/PI double staining; B) AO/EB double staining; C) caspase activation; D) cell cycle distribution; E) ROS
induction; D) metal uptake by the cells.
Measurement of intracellular metal amount by ICP-MS after (Fig. 3.A) showed that colony forming potential of cells
24 h of cultivation indicated that cells efficiently uptake both exposed to Sn3 and especially to SBA-15|Sn3 was dramatically
Sn3 and SBA-15|Sn3 (Fig. 2.F). However, unexpected ratio of abrogated. Plate efficiency was 34.3; 20.6; 3.1 in control, Sn3
Sn and Si detected in cells treated with nanomaterial SBA- treated and SBA-15|Sn3 treated cells, respectively, with
15|Sn3 indicated that drug is released also in extracellular surviving fractions 100; 60; 9. In concordance with this, growth
compartment. At least around 30 % of Sn3 found in the cells is rate of cells pretreated for 72 h with experimental drugs was
not carried by SBA-15p, thus Sn3 enters in the A2780 cells decreased in comparison to nontreated cells confirming the
independently. To confirm this findings Sn3 release study from permanent change of cell phenotype sustained even in the
SBA-15|Sn3 in simplified conditions was conducted. Namely, absence of the drug (Fig. 3.B). The cultivation of the cells in 3D
SBA-15|Sn3 was soaked in presence of completed medium in cultures on matrigel (Fig. 3.C) was compromised in the
order to obtain desorption behavior of the Sn3 compound presence of Sn3 and dominantly SBA-15|Sn3.
(ICP-MS). In concordance with uptake study and caspases Number, colony form and communication between them was
activation, the evaluation of Sn3 desorption (see Fig. S7) disturbed probably as a consequence of both, decreased
verified assumption that apart from MSN loaded form, cells dividing potential and mobility of tumor cells. Changed
are exposed to Sn3 from extracellular compartment that might behavior in matrigel was observed in pretreated cultures
be related with caspase 8 activation as main sign of receptor suggesting that 72 h pulse with the drug is enough for
dependent apoptosis. Even under this circumstance, previous permanent reprogramming of tumor cells. These results have
assessments clearly confirmed that cells acquired enough special importance in terms that cultivation in 3D cultures
amount of metal based drug underlining once more that more closely resemble the tumour microenvironment, and
loading into SBA-15p amplified its tumoricidal potential. The therefore are much more relevant for estimation of drugs
response of A2780 cells treated with SBA-15|Sn3 is enhanced efficiency.39
in comparison to alone Sn3 notwithstanding 3.7 times lower Simultaneously, affinity of cells to adhere to extracellular
concentration (ICP-MS) of Sn3.
matrix was significantly decreased upon exposure to both
compound and material (Fig. 3.D). Moreover, the migratory
potential of A2780 cells was dramatically affected by tested
drugs (Fig. 3.E). According to this, it could be concluded that
experimental therapeutics have not just cytotoxic potential
but also capacity to suppress their metastatic properties.
SBA-15|Sn3 decrease proliferative rate and metastatic properties
of A2780 cells
Cells that survived the treatment with Sn3 or SBA-15|Sn3 lost
their proliferative potential. Namely, clonogenic survival assay
6 | Dalton Trans., 2016, 00, 1-9
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Fig. 3 Sn3 and SBA-15|Sn3 decreased metastatic properties of A2780 cells. A) clonogenic survival assay; B) viability of pretreated cells with Sn3 and SBA-15|Sn3 upon
drug removal; C) the growth in 3D cultures; D) adhesion; E) transmigration test.
Difference in the intracellular molecular background of A2780 cells Knowing that activation of the p-38 MAPK pathway plays a role
treated with Sn3 and SBA-15|Sn3
in invasion and metastasis formation, decreased expression of
this molecule after SBA-15|Sn3 exposure could be responsible
for disturbed matrigel growth, migration and adhesiveness of
A2780 cells herein.44 It is possible that this discrepancy in p-38
regulation contributed to improved potential of Sn3
established upon incorporation to SBA-15p. Similar pattern of
changes in STAT-3 expression was noticed. Numerous of data
confirming positive relation between STAT3 activity and
tumorigenesis as well as tumor progression, bring additional
explanation for its improved activity of SBA-15|Sn3 in
comparison to naked drug Sn3.
To determine drugs influence on the major signaling pathways
responsible for cellular proliferation, differentiation and death,
Western blot analysis of the expression of p-38, Erk1/2 and Akt
has been performed (Fig. 4.A). One of the crucial
transcriptional factors regulating the expression of genes
involved in growth and apoptosis program in response to
different stimuli is STAT3, and therefore its expression was
also analyzed.40 The differences in the expression of Akt and
ERK1/2 between Sn3 and SBA-15|Sn3 treated A2780 cells
were not observed. While the expression of Akt was
transiently suppressed, ERK1/2 phosphorylation was
remarkable elevated. Both phenomena are related with the
loss of proliferative potential as well as apoptosis induction.
Suppression of Akt even in transient mode is basically
connected with decreased dividing rate.41 On the other hand,
Erk1/2 activation was observed in numerous cancer cells in
response to chemotherapy leading to cell death.42,43
Interestingly, the expression of p-38 is radically changed after
grafting Sn3 in SBA-15p. While its activity is transiently
upregulated in cells exposed to Sn3, decreased
phosphorylation was detected after treatment with SBA-
15|Sn3.
Conclusions
Highly invasive cancers are able to establish resistant
phenotype against different chemotherapeutics through
multiple mechanism. One of the leading between them is
overexpression of MDR pumps. It overcomes efflux of the
Ph3Sn(CH2)3OH (Sn3), silica-based nanostructured materials
were used as a carrier. Loading of Sn3 into SBA-15p conserved
and even potentiated drug effectiveness. Cultivation of cells in
the presence of SBA-15|Sn3 led to dual influence of free Sn3
from the extracellular compartment concomitantly with
internalized MSN form.
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Fig. 4 The influence of Sn3 and SBA-15|Sn3 on the p-38, Akt, Erk1/2 and STAT3 expression.
Antitumor action of SBA-15|Sn3 in A2780 can be ascribed to
References
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reflected through p-38 and STAT-3 regulation argues better
activity of this metal based drug applied in nanocarrier. Taken
together, packaging of Sn3 in MSN could be considered as a
promising strategy for improvement of chemotherapeutic
application.
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A mesoporous silica-based material loaded with organotin compound Ph3Sn(CH2)3OH diminished
metastatic properties of A2780 survived cells.