10.1002/cmdc.201800672
ChemMedChem
FULL PAPER
Cell Cultures. Human SH-SY5Y neuroblastoma cells were
cultured in Dulbecco’s modified Eagle’s medium: Ham’s Nutrient
Mixture F12 (1:1) (Sigma Aldrich), supplemented with 10% heat-
inactivated foetal bovine serum (FBS, Invitrogen, Carlsbad, CA,
USA), 2 mM glutamine, 250 U/ml penicillin G and 250 μg/mL
streptomycin (Sigma-Aldrich), in a humidified atmosphere with
5% CO2 at 37 °C.
coverslip and treated with 10 µM [Ag(EIA)2]Cl for 20 min, 30 min,
60 min and 90 min. Control cells, not treated with this silver
complex, were used. At end of treatment, the cells were fixed with
2% paraformaldehyde for 10 min at room temperature and then
observed under an epifluorescence Zeiss Axioskop microscope
(Mannheim, Germany) using 358 nm excitation wavelength, with
a 100X objective. The fluorescence images were captured using
a Leica DFC310 FX 1.4‐megapixel digital camera, equipped with
the Leica software application suite LAS V3.8 (Leica
Microsystems, Mannheim, Germany).
Cytotoxicity assay. Cell viability was measured using the
CellTiter-Blue® Reagent (Promega, Milan, Italy). SH-SY5Y cells
(1 x104/well) were seeded in 96-well plates and treated with
[Ag(EIA)2]Cl (0,5-2,5 µM), cisplatin (10-50 µM) and [AgCl(MIA)]
(2-10 µM) for 24h. At end treatments, CellTiter-Blue® Reagent
was added to each well and incubated for 2 h at 37 °C.
Fluorescence was measured in a multiplate reader (Infinite
M200PRO, Tecan, Switzerland) at 560/590 nm. The IC50 values
for each compound were determined from the specific dose-
response curves by non-linear analysis, using GraphPad Prism
2.0 statistical program (GraphPad Software, San Diego, CA,
USA) and expressed as means ± standard deviation of at least 4
independent experiments.
Thioredoxin Reductase activity Assay. The inhibitory effects of
complex toward thioredoxin reductase activity (from rat liver) were
determined by quantification of the ability of compound to directly
reduce 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) in presence of
NADPH as already reported in literature.10
Acknowledgements
We gratefully acknowledge Beneficentia Stiftung, ITT (Istituto
Toscano Tumori), Ente Cassa Risparmio Firenze (ECR) and
AIRC for funding the projects (IG-16049) and “Advanced mass
spectrometry tools for cancer research: novel applications in
proteomics, metabolomics and nanomedicine” (Multi-user
Equipment Program 2016, Ref. code 19650). COST Action
CM1105 is also acknowledged. T.M. thanks AIRC-FIRC
(Fondazione Italiana per la Ricerca sul Cancro) for the 3-years
Fellowship for Italy, Project Code: 18044 and University of Pisa
(PRA_2017_25).
Drugs uptake. SH-SY5Y cells were seeded in 6-well plates
(5x105 cells/well) and allowed to adhere. The culture medium was
replaced with medium without phenol red and FBS, and the cells
were incubated with [Ag(EIA)2]Cl, cisplatin or [AgCl(MIA)] (10 µM)
for 30 min. At end of treatments, the cells were harvested in
distilled H2O to induce osmotic shock. The determination of
metals concentration in the cell lysates was performed as
previously reported8 in triplicate by a Varian 720-ES Inductively
Coupled Plasma Atomic Emission Spectrometer (ICP-AES)
equipped with a CETAC U5000 AT+ ultrasonic nebulizer, in order
to increase the method sensitivity. Before the analysis, fixed
volume of samples, were moved in vials and digested in a thermo-
reactor at 80 °C for 3 h with 1 mL of aqua regia (HCl suprapure
grade and HNO3 suprapure grade in 3:1 ratio) and 5 mL of
ultrapure water (≤18 MΩ). Next, sample were spiked with 1 ppm
of Ge used as an internal standard and analyzed. Calibration
standards were prepared by gravimetric serial dilution from a
commercial standard solution of Pt at 1000 mg L−1. The
wavelength used for Pt and Ag determination were 214.424 and
338.289 nm respectively whereas for Ge the line at 209.426 nm
was used. The operating conditions were optimized to obtain
maximum signal intensity, and between each sample, a rinse
solution of HCl suprapure grade and HNO3 suprapure grade in 3:1
ratio was used in order to avoid any “memory effect”. The values
of Ag or Pt were normalized to cellular proteins, determined by
the micro-bicinchoninic acid (BCA) method, and expressed as μg
of metal/μg of proteins.
Keywords: thioredoxin reductase; silver carbene complexes;
fluorescence; anticancer drugs; mass spectrometry.
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