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doi.org/10.1002/open.202100044
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ment media). The treatment media were prepared using a 100 mM
stock solution of Cu(II) and ATSMÀ Cu(II). For ATSMÀ Cu(II), the
treatment medium contained less than 1% DMSO (except the
1 mM concentration containing 1% DMSO). At 24 h after the
treatment, the media were removed, 20 μl 5 mg/ml MTT (the final
concentration) was added to the wells, and the cells were
uptake and toxicity may be correlated with the cells’ copper
metabolism.
Experimental Section
°
incubated at 37 C for another 4 h. The MTT solution was removed,
and the colored formazan crystals in each well were dissolved in
180 μl DMSO. Absorbance at 590 nm was measured using a μ
Quant, BioTek Instruments microplate reader.
Materials and Methods
All chemicals obtained were of high-quality grade and used without
further purification. 4-Methyl-3-thiosemicarbazide, diacetyl (2,3-
butanedione), Cu(Cl)2 ·2H2O, bicinchoninic acid (BCA), and sterile-
filtered cell culture grade dimethyl sulfoxide (DMSO) were
purchased from Sigma-Aldrich.Dulbecco’s Modified Eagle’s Medium
(DMEM). Fetal Bovine Serum (FBS), antibiotic (penicillin and
streptomycin) solution, and MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) were purchased from Himedia.
Hypoxic bags were purchased from Sigma-Aldrich. 64CuCl2 was
obtained from Acom-srl, Italy.
Measurement of Intracellular Cu(I) Using BCA
Cells were grown in a sterile T-75 flask supplemented with
complete DMEM medium (high glucose, fetal bovine serum-10%,
antibiotics penicillin, and streptomycin (pen strep)- 5%, and L-glu-
°
5%) in an incubator at 37 C with 5% CO2. The cells (105) were
seeded in a six-well plate and kept in the incubator for 2–3 days to
attach the cells; a 70–80% cell density was obtained in each well.
Then the medium was replaced with fresh DMEM (2 mL). The cells
were further treated with Cu(II) and the ATSMÀ Cu(II) complex for
0.5 h, 1 h, 3 h, 6 h, and 24 h. 2 mM stock solutions of Cu(II) and
ATSMÀ Cu(II) were used. Hence, during the ATSMÀ Cu(II) treatment,
the medium contained 0.5% DMSO. The final concentration of
Cu(II) or ATSMÀ Cu(II) in each well was 10 μM. For normoxic
conditions, the cells were incubated for the required time point at
Synthesis of the ATSMÀ Cu(II) Complex
We used a previously reported method for the synthesis of the
ATSMÀ Cu(II) complex.[12b,20]
First, the ATSM ligand was synthesized by the condensation
reaction of 4-methyl-3thiosemicarbazide (1.2 g, 11.4 mmol) and
diacetyl (0.5 mL, 5.7 mmol) in ethanol (50 mL) with constant
heating and stirring. A few drops of glacial acetic acid were added
°
37 C and with 5% CO2. For the hypoxia conditions, the plates were
kept in a box containing hypoxic bags for 2 h before the treatment
and again for the required time point after the treatment. After the
treatment, the medium was removed from the wells, and cells were
washed (2–3 times) with sterile PBS. Cell lysis buffer -RIPA buffer
°
to the reaction mixture and then it was refluxed at 60 C for 4 h. A
°
white precipitate was formed. The flask was kept at 4 C overnight
for complete precipitation. Finally, a pale-yellow precipitate was
obtained, then washed 3–4 times with ethanol and diethyl ether.
1H-NMR (DMSO-d6): 10.22(s,2H) NH, 8.38 (m,2H) NHCH3, 3.02 (d, 6H)
NHCH3, 2.20 (s, 6H) 2xCH3 and ESI-MS (+): m/z 260.4.
°
(300 μL) was added to the wells and shaken at 4 C for 10 minutes.
Then 300 μL of a solution from the BCA protein assay kit were
added to the wells, and finally, 1.5 mL samples were collected into
°
Eppendorf tubes. Each sample was warmed at 50 C for 15 minutes,
ATSMÀ Cu(II) complex: ATSM ligand (0.1 g, 0.38 mmol) was dissolved
in ethanol with constant heating and stirring. An ethanolic solution
of copper acetate (0.0768 g, 0.38 mmol) was added dropwise to the
ligand solution. A brown precipitate formed immediately. The
then covered with aluminium foil. After half an hour, the formation
of Cu(I)À (BCA)2 was detected at 562 nm using UV-Visible spectro-
scopy.
°
reaction mixture was refluxed at 60 C for 4 h and again was
refluxed overnight at room temperature. The brown-red precipitate
obtained was washed several times with ethanol and diethyl ether.
UV-visible spectroscopy: λmax (DMSO) at 311 nm and 355sh, 476 nm
and 525sh. ESI-MS (+): m/z 322.
Radioactive Copper Experiments
64Cu(II) stock was prepared in 0.2 M glycine buffer (pH- 5.5).
64
ATSMÀ Cu(II) was prepared by adding 64Cu(II) (469.5 μL) into the
ATSM ligand dissolved in DMSO (3.84 mM, 365 μL). The stock
solution was then diluted using glycine buffer to obtain the
64
required activity for ATSMÀ Cu(II) in the desired volume. The
Stock Solution Preparation
complexation ratio was determined by HPLC, where both the UV
and the gamma emission were detected, it was found that 95% of
64Cu(II) was complexed to ATSM ligand.
A stock solution of ATSMÀ Cu(II) (100 mM) was prepared in DMSO. A
stock solution of CuCl2 (100 mM) was prepared in distilled water
and the concentration was verified by UV-Vis spectroscopy from
the d-d band of Cu(II) at 780 nm (ɛ=12 MÀ 1 cmÀ 1). These stock
solutions were further diluted and used for the experiments listed
below.
HEK-293 and MCF-7 cells were grown in 6-well plates with 3 ml of
DMEM high glucose (10% FBS, 1% PNSN, and 1% L-Glu) for 3 days
until each well reached 85–90% confluency. Then the medium was
removed from each well and replaced with 2 ml fresh medium.
64
Finally, 150 μl 5 μCi of 64Cu(II) or ATSMÀ Cu(II) was added into each
well. For the hypoxic conditions, we added an anaerobic atmos-
phere generation bag to the box and closed it with a lid and
paraffin. Under normoxic conditions, the cells were incubated for a
Cell Viability Measurement Using MTT
Cells were grown in DMEM medium supplemented with 10% FBS,
1% L-glutamine, and 1% antibiotic solution (penicillin and
°
full day at 37 C and 5% CO2. Under hypoxic conditions, however,
°
streptomycin) at 37 C in a humidified chamber at 5% CO2. The
°
the plates were at 37 C and with 7–15% of CO2 and 0.1% oxygen.
samples (different concentrations of Cu(II) and the ATSMÀ Cu(II)
complex) were passed through 0.22 Kd syringe filters for sterilization
before treatment. Cell viability was measured in 96-well plates by a
quantitative colorimetric assay using MTT in triplicate. Briefly, 105
cells per ml were seeded in 96-well plates for the assay. The cells
were treated with samples in DMEM medium without FBS (treat-
After the treatment for the specific time intervals (1 h and 3 h), the
medium was removed, and the cells were washed with PBS (4 ml).
The cells were lysed using RIPA lysis buffer (300 μl) and then
scraped. Finally, 300 μl of PBS was added, and cells were removed
and measured for radioactivity using a gamma counter. All the
ChemistryOpen 2021, 10, 486–492
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© 2021 The Authors. Published by Wiley-VCH GmbH
Walke, Gulshan R.
