Cu(i) complexes as new antiproliferative agents against sensitive and doxorubicin resistant colorectal cancer cells: synthesis, characterization, and mechanisms of action

Article abstract of DOI:10.1039/d0dt03566a

Cancer is one of the worst health issues worldwide, representing the second leading cause of death. Current chemotherapeutic drugs face some challenges like the acquired resistance of the tumoral cells and low specificity leading to unwanted side effects. There is an urgent need to develop new compounds that may target resistant cells. The synthesis and characterization of two Cu(i) complexes of general formula [Cu(PP)(LL)][BF₄], where PP is a phosphane ligand (triphenylphosphine or 1,2-bis(diphenylphosphano) ethane) and LL = is a heteroaromatic bidentate ligand (4,4′-dimethyl-2,2′-bipyridine and 6,3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine). The new compounds were fully characterized by spectroscopic techniques (NMR, FTIR and UV-vis.), elemental analysis (C, H, N and S) and two structures were determined by single X-ray diffraction studies. The antiproliferative potential of the new Cu(i) complexes were studied in tumor (breast adenocarcinoma, ovarian carcinoma and in colorectal carcinoma sensitive and resistant to doxorubicin) and normal (fibroblasts) cell lines. Complexes1-4did not show any antiproliferative potential. Amongst the complexes5-8, complex8shows high cytotoxic potential against colorectal cancer sensitive and resistant to doxorubicin and low cytotoxicity towards healthy cells. We show that complexes5-8can cleave pDNA and, in particular, thein vitropDNA cleavage is due to an oxidative mechanism. This oxidative mechanism corroborates the induction of reactive oxygen species (ROS), that triggers HCT116 cell deathviaapoptosis, as proved by the increased expression of BAX protein relative to BCL-2 protein and the depolarization of mitochondrial membrane potential, andviaautophagy. Additionally, complex8can block the cell cycle in the G1 phase, also exhibiting a cytostatic potential. Proteomic analysis confirmed the apoptotic, autophagic and cytostatic potential of complex8, as well as its ability to produce ROS and cause DNA damage. The interference of the complex in folding and protein synthesis and its ability to cause post-translational modifications was also verified. Finally, it was observed that the complex causes a reduction in cellular metabolism. The results herein demonstrated the potential of Cu(i) complexes in targeting doxorubicin sensitive and resistant cells which is positive and must be further explored usingin vivoanimal models.

Full text of DOI:10.1039/d0dt03566a

Dalton
Transactions
View Article Online
View Journal | View Issue
PAPER
Cu(I) complexes as new antiproliferative agents
against sensitive and doxorubicin resistant
colorectal cancer cells: synthesis, characterization,
and mechanisms of action†
Cite this: Dalton Trans., 2021, 50,
1845
Diogo Sequeira,a Pedro V. Baptista, a Ruben Valente, a M. Fátima M. Piedade,b,c
a
M. Helena Garcia,b,d Tânia S. Morais
*
b,d and Alexandra R. Fernandes
*
Cancer is one of the worst health issues worldwide, representing the second leading cause of death.
Current chemotherapeutic drugs face some challenges like the acquired resistance of the tumoral cells
and low specificity leading to unwanted side effects. There is an urgent need to develop new compounds
that may target resistant cells. The synthesis and characterization of two Cu(I) complexes of general
formula [Cu(PP)(LL)][BF4], where PP is a phosphane ligand (triphenylphosphine or 1,2-bis(diphenylpho-
sphano) ethane) and LL = is a heteroaromatic bidentate ligand (4,4’-dimethyl-2,2’-bipyridine and 6,3-(2-
pyridyl)-5,6-diphenyl-1,2,4-triazine). The new compounds were fully characterized by spectroscopic
techniques (NMR, FTIR and UV-vis.), elemental analysis (C, H, N and S) and two structures were deter-
mined by single X-ray diffraction studies. The antiproliferative potential of the new Cu(I) complexes were
studied in tumor (breast adenocarcinoma, ovarian carcinoma and in colorectal carcinoma sensitive and
resistant to doxorubicin) and normal (fibroblasts) cell lines. Complexes 1–4 did not show any antiprolifera-
tive potential. Amongst the complexes 5–8, complex 8 shows high cytotoxic potential against colorectal
cancer sensitive and resistant to doxorubicin and low cytotoxicity towards healthy cells. We show that
complexes 5–8 can cleave pDNA and, in particular, the in vitro pDNA cleavage is due to an oxidative
mechanism. This oxidative mechanism corroborates the induction of reactive oxygen species (ROS), that
triggers HCT116 cell death via apoptosis, as proved by the increased expression of BAX protein relative to
BCL-2 protein and the depolarization of mitochondrial membrane potential, and via autophagy.
Additionally, complex 8 can block the cell cycle in the G1 phase, also exhibiting a cytostatic potential.
Proteomic analysis confirmed the apoptotic, autophagic and cytostatic potential of complex 8, as well as
its ability to produce ROS and cause DNA damage. The interference of the complex in folding and protein
synthesis and its ability to cause post-translational modifications was also verified. Finally, it was observed
that the complex causes a reduction in cellular metabolism. The results herein demonstrated the potential
of Cu(I) complexes in targeting doxorubicin sensitive and resistant cells which is positive and must be
further explored using in vivo animal models.
Received 14th October 2020,
Accepted 24th December 2020
DOI: 10.1039/d0dt03566a
rsc.li/dalton
Introduction
Oncologic diseases are one of the worst health issues world-
wide, representing the second leading cause of death. It is
aUCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Portugal. E-mail: ma.fernandes@fct.unl.pt
bDQB-FCUL, Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, Portugal. E-mail: tsmorais@ciencias.ulisboa.pt
cCQE@IST, Centro de Química Estrutural, Instituto Superior Técnico, Universidade
de Lisboa, Lisboa, Portugal
expected that the number of cases keep increasing due to the
populational increase and aging.1 Different strategies are cur-
rently being used to combat cancer development, including
surgery, radiotherapy or chemotherapy.2,3 Chemotherapeutic
drugs used nowadays face some challenges like the acquired
resistance of the tumoral cells and their low specificity to the
tumour leading to unwanted side effects.4 There is an urgent
need to develop new therapies that replace the drugs used cur-
rently in chemotherapy.5–7 Metal complexes have a big advan-
dCQE@FCUL, Centro de Química Estrutural, Faculdade de Ciências, Universidade de
Lisboa, Portugal
†Electronic supplementary information (ESI) available. CCDC 2038289 and
2038290. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/d0dt03566a
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1845

View Article Online
Paper
Dalton Transactions
tage: they can have a structural diversity given by different
modes of coordination and different ligands. This allows the
modification of redox activity and the catalytic properties,
important for reactivity against organic substrates.6–9 The
interest in inorganic metal-based complexes began with cispla-
tin and its approval for clinical use in cancer by the food and
drug administration (FDA) in 1978.10 However, the fact that
cisplatin exhibits some cytotoxicity to healthy cells due to the
low specificity and drug resistance of the tumour cells, trig-
gered the search and synthesis of a large number of cisplatin
analogues and complexes with other metals at their active
centre, like copper (Cu) to overcome acquired drug
resistance.11–17 Copper is an essential micronutrient that has
the ability to adopt two different redox states, Cu(I) and Cu(II),
in physiological medium. Although the mechanisms of action
of copper in tumor cells are not yet fully elucidated, published
results demonstrate that copper complexes have the ability to
induce apoptosis and other forms of cell death, such as necro-
sis and autophagy due to oxidative stress.11,18–23 Phosphane
copper(I) complexes have been described in the literature.14–17
We have also recently reported the synthesis of a series of Cu
(I)–phosphane compounds, with general formula [Cu(PP)
(LL)][BF4] in which PP represents phosphane ligands and LL
represents N,N-, N,O- or N,S-heteroaromatic bidentate
ligands.9,24,25 The design of these complexes include phos-
phane ligands that can efficiently stabilize Cu(I)-d10 metal
centres, due to the soft nature of the P donor atoms, and het-
eroaromatic bidentate ligands that in addition to confer
activity to the complexes also provide great stability. The
Scheme 1 Reaction scheme for the synthesis of the [Cu
(PPh3)2(Me2bpy)][BF4] (2) and [Cu(PPh3)2(dpypz)][BF4] (3). Ligands are
numbered for NMR spectral assignments.
(dpytz), 3. The reactions were carried out in dichloromethane
solutions at room temperature (Scheme 1). Both compounds
were fully characterized by UV-vis, FTIR and NMR spectroscopy
(1H, 13C and 31P) and the elemental analyses were in accordance
with the proposed formulation. Crystals suitable for single
crystal X-ray diffraction were obtained for both complexes by
slow diffusion of diethyl ether in dichloromethane solutions of
the complexes, at room temperature.
Spectroscopic studies
present work stems from our continued interest in the exploi- The solid state FTIR spectra (KBr pellets) of compounds 2 and
tation of the antiproliferative potential of these complexes for 3 present the characteristic νC–H, νCvN and νCvC stretching
cancer therapy, particularly, by comparing two different phos- bands of the aromatic rings of the phosphane and N,N-hetero-
phane ligands (triphenylphosphine or 1,2-bis(diphenylpho- aromatic ligands in the range 3040–3100 cm−1 and
sphano) ethane) and different heteroaromatic bidentate 1390–1610 cm−1, and the δC–H bending vibrations in the
ligands. Eight complexes from the series were chosen for 690–700 cm−1 range. In the 1066–1099 cm−1 range and
investigation of their antiproliferative potential against breast ∼690 cm−1 were also observed the very broad and strong bands
and ovarian tumor cells, two types of cancer highly prevalent characteristic of νB–F vibrations, confirming the presence of
in women and colorectal carcinoma sensitive and resistant to the counter-ion (BF4).
doxorubicin, a cancer with high prevalence worldwide.26
NMR characterization of the above-mentioned complexes
Among these eight complexes, two were newly prepared which was carried out by 1H, 13C{1H}-apt, 31P{1H} and 2D experi-
synthesis, and full characterization is now reported. The anti- ments (COSY, HSQC and HMBC). The NMR spectra of com-
proliferative activity of Cu(I) metal complexes was assessed in plexes in acetone-d6 solutions at room temperature displayed
the 4 tumor cell lines as well as in normal cells. The Cu(I) well resolved resonances which were fully assigned (see atom
complex with the highest cytotoxicity in tumor cells and lower labelling given in Scheme 1 and the Experimental section).
effect in normal cells was further selected for understanding
its mechanism of action in a colorectal carcinoma cell line.
The 1H NMR spectra of both complexes show the proton
resonances of the phenyl groups of heteroaromatic and phos-
phane ligands in the aromatic zone (8.94–7.18 ppm). The
effect of the coordination is observed on the deshielding of
the protons of the N-heteroaromatic ring coordinated to Cu(I),
Results and discussion
Synthesis and characterization of new Cu(I) compounds
1
when compared to the free ligands. The H NMR spectrum of
complex 2 also shows a singlet at δ 2.52 ppm corresponding to
Two new cationic Cu(I) complexes of general formula protons H6 of the methyl substituent on C3. The 13C{1H} reso-
[Cu(PPh3)2(LL)][BF4] were synthesized by displacement of the nances reflect the same pattern observed for the protons. The
acetonitrile ligands from the precursor [Cu(PPh3)2(NCMe3)2][BF4] well-defined proton signals observed for both compounds also
by the bidentate ligands LL 4,4′-dimethyl-2,2′-bipyridine confirm that no oxidation to Cu(II) occurred under the experi-
(Me2bpy),
2
and 6,3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine mental conditions. The 31P{1H} spectra of 2 and 3 complexes
1846 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
show a broad signal, at 2.6 and 3.1 ppm, in the characteristic ing compounds in study has already been demonstrated been
range of Cu(I)PPh3 complexes,24,27 and an expected deshield- reported, in the same medium.9,24
ing upon coordination according to the σ donor character of
this phosphane.
Single crystal X-ray diffraction studies
The UV-visible spectra of the two newly synthesized Cu(I)
complexes were recorded in ∼5 × 10–5 mold m−3 solutions of
Single crystals of complexes 2 and 3 suitable for SCXRD were
obtained by the diffusion of diethyl ether in a dichloro-
methane solution of each complex. The X-ray single crystal
data of both compounds were analysed at 150 K. Fig. 2 shows
the molecular structures of the compounds and the selected
bond lengths and angles are shown in ESI Table S2.†
Complex 2 crystallizes in the monoclinic crystal system,
P21/n space group, and the asymmetric unit consists of the
cation [Cu(PPh3)2(Me2bpy)]+ and a [BF4]− anion. Crystal of
dichloromethane. For comparison, also the electronic spectra
of the precursor and free ligands were obtained under the
same experimental conditions. The maximum absorption
bands are indicated in ESI Table S1.† The UV-visible spectra of
the 2 and 3 complexes showed intense absorption bands, in
the range of 230–330 nm which corresponded to π–π* elec-
tronic transitions occurring in the coordinated ligands of the
inorganic fragment {Cu(PPh3)2}+. Additionally, one metal to
ligand charge transfer (MLCT) band was found at 361 nm and
415 nm for complexes MP04 and MP05, respectively. Fig. 1(a)
typifies the behavior of these compounds. The stability of the
complexes 2 and 3 was evaluated by UV–vis spectroscopy
(260–800 nm) in 1%DMSO/DMEM + GlutaMAX-I™ cellular
medium. There was no shift in the UV-visible spectral bands of
the complexes over 24 h, confirming that these complexes are
air-stable in this solution. On Fig. 1b is represented the evalu-
ation of the stability on the charge transfer of complex 2 by
UV-vis spectroscopy over 24 hours. The stability of the remain-
ˉ
compound 3 is triclinic, space group P1, being its asymmetric
unit constituted by the [Cu(PPh3)2(dpytz)]+ cation, a [BF4]−
anion and a CH2Cl2 molecule. The copper(I) atom is co-
ordinated to two phosphorus of the triphenylphosphanes and
to a bidentate N,N-heteroaromatic ligand in a highly distorted
tetrahedral geometry. The distortion arises from the “bite” of
the N,N-heteroaromatic bidentate ligand coordinated to the
Fig. 1 (a) Electronic spectra of [Cu(PPh3)2(Me2bpy)][BF4] 2 (_____), in di-
chloromethane, compared to the uncoordinated ligand 4,4’-dimethyl-
2,2’-bipyridine (- - -) and [Cu(PPh3)2 (NCMe)2][BF4] (· · · ·). Inset: Detail of
the metal to ligand charge transfer band. (b) Evaluation of the stability of
complex 2 by UV-vis spectroscopy over 24 hours in 2%DMSO/DMEM +
GlutaMAX-I™ solution.
Fig. 2 Molecular diagrams depicting the cationic moieties for
a) [Cu(PPh3)2(Me2bpy)]+ (2) and b) [Cu(PPh3)2(dpytz)]+ (3) complexes.
Hydrogen atoms were omitted for clarity.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1847

View Article Online
Paper
Dalton Transactions
metal that causes stereochemical constraints on metal geome- making an angle of 10.7°. The free dpytz ligand makes with an
try, N1–Cu–N2 = 79.43(6)° for 2 and 79.90(8)° for 3, values angle of 3.36°30 between the planes of the pyridyl rings, after
much smaller than the angle of a perfect tetrahedron, 109.41. coordination of this ligand to the metal centre (complex 3) it
Also, the P1–Cu–P2 angles are larger (126.18(2) and 116.82(2) becomes less planar with an angle of 8.9°.
for 2 and 3, respectively) due to the steric effects caused by the
The intermolecular interactions in the packing of both
bulky triphenylphoshane ligands. The difference observed compounds, are of the type C–H⋯F, and C–H⋯B between the
between these angles in the two compounds is due to the cations and the anions. The cations of compounds 2 and 3 are
bulkiness of dpytz producing a larger steric constraint on the surrounded by 5 and 3 anions in its environment, respectively,
copper geometry making the angle smaller in complex 3. that forms interactions ranging from 2.42 Å to 3.69 Å (Fig. 3a
Nevertheless, these angles along the Cu–P and Cu–N bond and b). In compound 3 two independent molecules form a
lengths are in agreement with the values found for other [Cu dimer through intermolecular contacts of the type C–H⋯F.
(PPh3)2L]+ complexes, where L is a bidentate ligand.24,28,29 The These interactions occur between the phenyl groups of the
4,4′-dimethyl-2,2′-bipyridine ligand in complex 2 is almost dpytz ligand (ranging from 2.42 to 2.66 Å) and the phophanes
planar, with the planes of the rings joined by C5–C6 bond (∼2.6 Å) and a BF4 anion (Fig. 3c).
Fig. 3 Intermolecular interactions for complexes [Cu(PPh3)2(Me2bpy)][BF4] (a) and [Cu(PPh3)2(dpypz)][BF4] (b) and dimers formed by two indepen-
dent molecules of complex [Cu(PPh3)2(dpypz)][BF4] (c).
1848 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
Antiproliferative assays
As a way to evaluate the in vitro cytotoxic potential of the Cu(I)
complexes under study (Scheme 2), cell viability was analysed
in tumor cell lines (HCT116, HCT116 DOXR, A2780 and
MCF-7) and in healthy cells (fibroblasts) using the MTS
reagent after 48 hours exposure to the complexes (Fig. 4 and 5;
ESI Fig. S1–S6†). The MTS method allows us to indirectly deter-
mine cell viability and to determine the relative IC50 of each
complex for the studied cell lines, which is an indicator of the
efficiency of the complex, corresponding to the concentration
of complex capable of reducing cell viability by 50% (Table 1).
No reduction of viability was observed when HCT116 cells
were incubated with the precursor [Cu(PPh3)2(NCCH3)2][BF4]
or complexes 1, 2, 3 and 4 within the tested concentration
range (after 48 h of incubation) (ESI Fig. S1†). Thus, the IC50
values for these complexes are greater than 50 µM demonstrat-
ing the absence of cytotoxicity in HCT116 cells. It is however to
note that in our previous work, complexes 1–4 have shown
Fig. 4 Cell viability in HCT116 cell line after 48 hours exposure to Cu(I)
complexes (A –5; B – 6; C – 7; D – 8). Values of cell viability were nor-
malized relative to the control vehicle in (0.1% (v/v) DMSO. Data are rep-
cytotoxicity in a breast adenocarcinoma cell line (MCF7).24
A
different susceptibility to cisplatin has also been observed resented as the mean
SD of three independent biological assays.
P-Value was calculated using t-student comparing each concentration
relative to the control, 0.1% (v/v) DMSO. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤
0.001, **** p ≤ 0.0001.
when we compare HCT116 cells and MCF7 cells (the IC50 for
cisplatin in MCF7 is 59 µM (ref. 24) at 24 h while for HCT116
at 48 h is 15.6 µM (Table 1; ESI Fig. S7†)). Despite both cell
lines are epithelial, MCF7 is derived from a breast adeno-
carcinoma (hormone dependent) while HCT116 cells are from
a colorectal carcinoma (non-hormone dependent). Indeed,
compared to HCT116 that is a more stable cell line, MCF7 is
genetically unstable.26 Additionally, differences on the resis-
tance level or on a different uptake of complexes by those cells
cannot be disregarded (see section internalization of Cu(I)
complexes below). On the other hand, when we analyze data in
Fig. 4, for complexes 5, 6, 7 and 8, a concentration dependent
reduction of cell viability is observed in HCT116 cells. The
Scheme 2 Structures of Cu(I) complexes [Cu(PPh3)2(2,2’-bipy)][BF4] (1), [Cu(PPh3)2(Me2bpy)][BF4] (2), [Cu(PPh3)2(dpytz)][BF4] (3), [Cu
(PPh3)2(2dpp)][BF4] (4), [Cu(ddpe)(2,2’-bipy)][BF4] (5), [Cu(dppe)(Me2bpy)][BF4] (6), [Cu(dppe)(dpytz)] [BF4] (7), [Cu(dppe)(2dpp)][BF4] (8) and the pre-
cursors [Cu(PPh3)2(NCCH3)2][BF4] and [Cu(dppe)(NCCH3)2][BF4].
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1849

View Article Online
Paper
Dalton Transactions
patients8 to common chemotherapeutic agents, such as doxo-
rubicin, we have also assessed complexes 5, 6, 7 and 8 antipro-
liferative effect in HCT116 DOXR, a doxorubicin resistant cell
line (Fig. 5 and Table 1). Despite higher compared to the rela-
tive IC50 values in the parental sensitive HCT116 cells, the rela-
tive IC50 values calculated were also very promising (range
1–1.5 µM) and cytotoxicity’s in the same order as before (6 > 8
> 5 > 7) (Fig. 5 and Table 1).
Doxorubicin-resistant colorectal cancer cell line (HCT116
DOXR) was obtained in our laboratory by exposing sensitive
commercial colorectal cancer cells, HCT116, to increasing con-
centrations of doxorubicin (Fig. S1–S6†).13 The mechanism
underlying resistance to doxorubicin (DOX) was demonstrated
to be dependent on the increase of multidrug resistance
protein 1 (MDR1) expression that could be pumping out Dox
from cells (Fig. S4 and S5†).13 In our previous studies with
other metal complexes (cobalt derivative) we were able to
demonstrate that the high cytotoxicity shown in HCT116
DOXR cells was due to the fact that this Co(II) complex was not
a substrate of MDR1, that might be the case in the present
work. Indeed, the effect was the same in the presence or
absence of Tariquidar, a MDR1 inhibitor (Fig. S6†).13 It is of
importance to note that the IC50 of DOX in HCT116 DOXR
cells is higher than 6 µM (Table 1; ESI Fig. S8†) meaning that
the Cu(I) complexes are indeed active in this resistant colorec-
tal cancer cell line (IC50 between 1.0–1.5 µM). These results
make these complexes promising for further biological studies
aiming an application in colorectal cancer sensitive and resist-
ant to doxorubicin.
When testing the antiproliferative effect of a complex in
tumor cells it is also important to assessed its effect in normal
healthy cells, e.g. primary fibroblasts, as fibroblasts are the
main type of cell in the tumor microenvironment and within
tissues, and may give us an indication of a potential thera-
peutic window for the complexes when presenting none or low
cytotoxicity in normal cells. As observed in ESI Fig. S4,† the
most cytotoxic complex in HCT116 cells, 6, also has a high
cytotoxicity in normal primary fibroblasts (IC50 of 10 µM,
Table 1). However, complex 8 has identical cytotoxicity in
HCT116 cells compared to 6, but no cytotoxic effect in fibro-
blasts up to 50 µM (Table 1). The selectivity index (SI) of the
Fig. 5 Cell viability in HCT116 DOXR cell line after 48 hours exposure
to study complexes (A – 5; B – 6; C – 7; D – 8). Values of cell viability
were normalized relative to the control vehicle in (0.1% (v/v) DMSO.
Data are represented as the mean SD of three independent biological
assays. P-Value was calculated using t-student comparing each concen-
tration relative to the control, 0.1% (v/v) DMSO. ns – statistically non-
significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
respective IC50 values are shown in Table 1. The most cytotoxic
complex is 6, with an IC50 in HCT116 cells at 48 h of 0.15 µM,
nevertheless, all these complexes seems to be very promising
due to their low IC50 in HCT116 cells compared to the IC50 of
doxorubicin, a widely used chemotherapeutic agent, 0.4 µM,
using the same experimental conditions (Table 1, ESI
Fig. S7†). The order of cytotoxicity is 6 > 8 ∼ 5 > 7.
Considering these results, we also analyzed the antiproli-
ferative effect of these four Cu(I) complexes in two additional
cancer cell lines, a breast adenocarcinoma cell line, MCF7 and
an ovarian carcinoma cell line, A2780 (ESI Fig. S2 and S3,†
respectively and Table 1). By observing Table 1 and ESI Fig. S2
and S3† it is possible to conclude that the complexes are less
cytotoxic for these two cell lines with IC50 values at least 3×
higher compared to HCT116 cells.
Considering the interesting antiproliferative effect in
HCT116 cells and the resistance developed by many cancer
Table 1 IC50 values (µM) of the starting compound (SC), Cu(I) complexes and their respective ligands obtained for the different tumor and normal
cell lines tested. Selectivity index (SI) for HCT116 cells compared to fibroblasts (IC50 fibroblasts/IC50 HCT116) is also presented (n.d. not calculated)
Cu(I) complexes, SC or ligands
IC50 HCT116
IC50 HCT116 DOXR
IC50 MCF7
IC50 A2780
IC50 Fibroblasts
SI
200
>250
150
66.7
100
11.4
>11.1
>9.1
>16.7
n.d.
7
8
5
0.25 0.01
0.20 0.02
0.20 0.03
0.15 0.02
0.10 0.01
3.5 0.1
4.5 0.2
5.5 0.3
3.0 0.1
15.6 5.3
0.4 0.1
1.5 0.1
1.1 0.1
1.2 0.2
1.0 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>6
0.50 0.04
0.80 0.05
0.82 0.09
0.95 0.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.72 0.05
1.3 0.08
0.62 0.09
0.4 µM
n.d.
50
5
>50
30
10
10
40
3
2
2
1
6
SC ([Cu(dppe)(NCCH3)2][BF4])
Dptyz
Dpp
Bipy
Me2bpy
Cisplatin
Dox
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>50
>50
>50
n.d.
n.d.
n.d.
1850 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
Table 2 Intracellular concentration of complexes 1–8 in HCT116 cell
line measured by inductively coupled plasma atomic emission spec-
troscopy (ICP-AES)
complexes towards HCT116 cells, a ratio between the IC50 in
normal cells (fibroblasts) and tumor cells (HCT116), gives us a
good idea how promising a complex might be (the higher the
SI the better; higher therapeutic window) for further biological
assays (Table 1). As observed in Table 1 complex 8, has the
highest SI amongst the tested complexes, being the most
promising for pursuing further biological analysis.
In order to ensure that the effect that we are observing in
HCT116 cells is really due to the complex action and not only
an effect of their ligands, the antiproliferative effect of the
starting compound and ligands was also evaluated in HCT116
cells and fibroblasts (Table 1, ESI Fig. S5 and S6,† respectively).
It is observed that the free ligands are less cytotoxic to HCT116
cells than when coordinated. It is also noted that the precursor
(SC), despite having a higher antiproliferative effect in HCT116
cells compared to Cu(I) complexes, it also has a higher cyto-
toxicity in fibroblasts (IC50 = 10 µM) and that coordination
with ligands attenuates this cytotoxicity in normal cells
(Table 1, ESI Fig. S6†). We can conclude that the cytotoxicity of
the complexes is essentially derived to the fragment [Cu
(dppe)]+ and that coordination with the heteroaromatic
ligands leads to a decrease in the healthy cells cytotoxicity
(Table 1 and ESI Fig. S5 and S6†).
Intracellular concentration (%)
Complexes
37 °C
4 °C
1
2
3
4
5
6
7
8
21.6
22.9
21.9
21.5
34.8
66.5
32.2
40.0
11.3
12.1
10.9
9.7
0
0
0
0
green fluorescence.32 However, annexin V also has the ability
to bind to the inner layer of the plasma membrane, after the
loss of membrane integrity that occurs only at a later stage of
apoptosis. Propidium iodide labelling allows identifying cells
in late apoptosis and necrosis, because due to its high mole-
cular weight, this probe can only enter the nucleus and binds
to DNA molecule when the membrane is permeable and com-
promised, emitting red fluorescence.32 With this double label-
ing, viable cells do not emit fluorescence (FITC− and PI−), in
initial apoptosis they emit green fluorescence (FITC+ and PI−),
in late apoptosis they emit green and red fluorescence (FITC+
and PI+), and in necrosis they emit red fluorescence (FITC−
and PI+).33
Considering these results and to further understand Cu(I)
complexes mode of action in tumor cells we began to analyze
the mechanism involved in loss of cell viability observed above.
Internalization of Cu(I) complexes
In this assay, HCT116 cells were exposed to the IC50 of the
complexes, 0.1% (v/v) DMSO (negative control) or 0.4 µM doxo-
rubicin during 48 h, and then labeled with these two fluoro-
phores and results presented in Fig. 6.
An increase in the level of early and late apoptosis might be
observed in the presence of all Cu(I) complexes being more
To evaluate the different effects of these complexes on HCT116
cell viabilities and to determine the mechanism by which com-
plexes enter cells, their intracellular concentration was evalu-
ated by inductively coupled plasma atomic emission spec-
troscopy (ICP-AES). This technique measures the amount of
copper that was uptaken by cells and the amount remained in
the supernatant. Through the difference between internalized
copper at 4 °C and 37 °C it is possible to determine the mecha-
nism by which it enters the cell (passive or active transport,
respectively), because at 4 °C there is no energy for an active
transport. After 3 hours exposure to complexes 1–8, it appears
that the entry into the cell occurs only by active transport for
complexes 5–8 while for complexes 1–4 an active and passive
transport occurs (Table 2). The % of intracellular Cu at 37 °C
is much higher for complexes 5–8 compared to 1–4.
Interestingly, internalization follows the trend observed in
cytotoxicity 6 > 8 > 5 > 7.
Cell death mechanisms
Annexin V-FITC/PI double-staining assay. The double stain-
ing with annexin V-FITC and PI was used to distinguish
between apoptotic and necrotic cells, as well as differentiating
Fig. 6 Apoptotic potential in HCT116 cells exposed to Cu(I) complexes
(5–8). Quantification of viable, early and late apoptotic and necrotic
between early and late apoptosis due to differences in the HCT116 cells by flow cytometry using annexin-V/FITC and PI double-
integrity of the plasma membrane.31
In the initial stage of apoptosis, phosphatidylserine translo-
cates to the outer layer of the plasma membrane. AnnexinV is
staining. Cells were incubated for 48 h with IC50 of the Cu(I) complexes,
0.1% (v/v) DMSO or 0.4 μM doxorubicin (positive control). Data are rep-
resented as the mean
SD of three independent biological assays.
Statistical significance was calculated in relation to the reference group
(DMSO) by the two-way ANOVA method (ns – statistically non-signifi-
a phospholipid-binding protein with a high affinity for phos-
phatidylserine, and conjugated to the fluorophore FITC emits cant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1851

View Article Online
Paper
Dalton Transactions
pronounced for 6 (Fig. 6). This result agrees with the viability mised and the probe accumulates in the cytoplasm in its
data as a higher cytotoxicity is induced by this complex com- monomeric form, emitting green fluorescence.34
pared to the other complexes. Interestingly, complex 8, with
There is a clear decrease in the emission of red fluorescence
the greatest SI also induce apoptosis (20.8%), with 8.9% of in cells exposed to complexes 5, 7 and 8 compared to the
cells in early apoptosis and 11.9% in late apoptosis (Fig. 6). control and an increase in the intensity of green fluorescence,
Cell death by necrosis induced by the action of complexes is which indicates a change in mitochondrial potential (Fig. 7(A)
negligible. On the contrary, doxorubicin shows a very high and ESI Fig. S9†). Interestingly, for complex 6, the decrease in
level of necrosis and late apoptosis with only 6.1% of viable the emission of red fluorescence does not appear to be very
cells (Fig. 6). During the activation of the apoptotic process a significant compared to control (ESI Fig. S9,† Fig. 7(B)). The
loss of mitochondrial membrane potential (ΔΨm) might be green/red fluorescence ratio increased considerably in HCT116
observed. Considering this we next assessed the effect of com- cells exposed to the Cu(I) complexes compared to the controls,
plexes in destabilizing the ΔΨm.
particularly for complex 5 and 8 (Fig. 7(B)). Considering these
results, it is possible to state that complexes 5, 7 and 8 appear
to induce apoptosis, via mitochondria destabilization, possibly
by the intrinsic pathway (Fig. 7 and ESI Fig. S9†).
Evaluation of mitochondrial membrane potential (ΔΨm)
The ΔΨm is essential to keep the respiratory chain functioning
correctly. A significant loss of ΔΨm can compromise these
functionalities, leading to cell death. Thus, the assessment of
the mitochondrial potential of cells provides important data
on cell viability and induction of apoptosis. The fluorescent
probe JC-1 is highly sensitive to changes in ΔΨm. Healthy cells
exhibit high mitochondrial potential, and in this case, the
probe enters the interior of the mitochondria and forms aggre-
gates, emitting red fluorescence. When there is a loss of ΔΨm,
Nevertheless, to confirm that the loss of ΔΨm and the
increased permeabilization of the mitochondrial membrane is
associated with an intrinsic apoptosis, the expression of the
BAX and BCL-2 proteins was evaluated by the western blot
technique for the complex showing a higher SI index, 8.
Expression of BAX and BCl-2 proteins after exposure of
HCT116 and HCT116 DOXR cells to complex 8
due to the permeabilization of the mitochondrial membrane, The expression of anti- and pro-apoptotic proteins, BCL-2 and
allowing cytochrome c to exit, the mitochondria is compro- BAX, respectively, determines cell fate. Increased expression of
Fig. 7 Mitochondrial membrane potential in HCT116 cells exposed to Cu(I) complexes. (A) Fluorescent images of HCT116 cells labeled with JC-1
after 48 h of exposure to the IC50 of complexes, 0.1% v/v DMSO, or without exposure (untreated cells). (B) Ratio green/red fluorescence in HCT116
cells after 48 h exposure to the IC50 of complexes, 0.1% v/v DMSO, or without exposure (untreated cells). Data are represented as the mean SD of
three independent biological assays. Statistical significance was assessed relative to control (DMSO) by the one-way ANOVA method (**** p ≤
0.0001, *** p ≤ 0.001, ns – statistically not significant).
1852 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
anti-apoptotic protein BCL-2 determines cell survival (ratio to DNA and allows the identification of chromatin conden-
BAX/BCL-2 < 1) and intrinsic apoptosis is trigger by the sation and nuclear fragmentation, and the CYTO-ID® Green
increased expression of pro-apoptotic proteins, such as BAX Detection Reagent Cyto-ID, a probe that becomes fluorescent
(ratio BAX/BCL-2) > (1).35 The relative expression levels of these in the presence of autophagic vesicles.40
proteins were quantified after exposure of HCT116 cells and
As observed in Fig. 9A and ESI Fig. S9,† there is an increase
HCT116 DOXR cells to complex 8 for 48 h (Fig. 8).
in fluorescence of HCT116 cells in the presence of rapamycin
There is a significant increase in the level of expression of (positive control) and the Cu(I) complexes, compared to the
BAX relative to the levels of expression of BCL-2 and compared negative control (DMSO). The autophagic vesicles were quanti-
to the control in DMSO after exposure of HCT116 and HCT116 fied, through the ratio between the number of autophagic vesi-
DOXR cells to complex 8 (Fig. 8A and C, respectively). This cles counted by the number of nucleus (Fig. 9B).
increase in the BAX/BCL-2 ratio (Fig. 8B) confirms the results
All Cu(I) complexes can induce autophagic cell death con-
obtained in the mitochondrial potential assay for HCT116 cell firmed by the increase in the number of autophagic vesicles
line (Fig. 7), which indicates the activation of the intrinsic observed in Fig. 9, and to the same level as the positive control
pathway of apoptosis by 8. The permeabilization of the mito- rapamycin (Fig. 9). Autophagy may be responsible for the acti-
chondrial membrane that is achieved by the entry of pro-apop- vation of the death of tumor cells, which show resistance to
totic proteins in the mitochondrial membrane leads to the apoptosis, and these two mechanisms may be correlated.
release of cytochrome c from the mitochondria to the cyto-
Oxidative stress potential of the Cu(I) complexes
plasm, a crucial step for the activation of the intrinsic
pathway.36 Previously we showed that Cu(I) complexes were Mitochondria is considered one of the main sources of reactive
also very active against HCT116 DOXR cells (Table 1 and oxygen species (ROS). By increasing ROS, complexes can evoke
Fig. 5). It is interesting to note that the ratio BAX/BCL-2 is even an intracellular state of oxidative stress, leading to permanent
higher for this cell line compared to HCT116 parental cell line cell damage to the DNA, proteins, membranes and inducing
(Fig. 8D) demonstrating the potential of this complex to cell death mechanisms, namely apoptosis and autophagy.41
trigger apoptosis in doxorubicin resistant cell lines.
Among the effector mechanisms involved in the control and
regulation of cell death pathways, including autophagy and
apoptosis, is the cellular redox status.39 When cells face an
increased in the redox state autophagy has emerged as a
powerful mediator of programmed cell death, either opposing
or enhancing apoptosis, or acting as an alternative form of pro-
grammed cell death.42 Being mitochondria a site of ROS pro-
duction and one of its targets, the depolarization of mitochon-
drial membrane potential and production of ROS are directly
connected.43
Autophagic potential
Some copper complexes can induce simultaneously different
types of cell death, namely apoptosis and autophagy.21,22,37–39
Considering that we have also assessed cell death by auto-
phagy. For that, cells were labeled with two different fluoro-
phores, Hoechst 33342, which is a nucleic acid dye that binds
After the exposure of HCT116 cells to Cu(I) complexes for
6 hours, the cells were incubated for 20 min with H2DCF-DA, a
non-fluorescent complex, which oxidizes and becomes fluo-
rescent in the presence of intracellular ROS (DCF).
Considering this, the appearance of a green fluorescent signal
might be correlated with the oxidation of the non-fluorescent
probe.44 As observed in Fig. 10, complex 8 shows the best capa-
bility to generate oxidative stress to HCT116 cells. Concerning
complexes 5 and 7 an increase in ROS production was
observed compared to the DMSO negative control (Fig. 10) but
for complex 6 only a slight increase was observed and not stat-
istically significant. On the other hand, as expected, H2O2
(positive control) show a 2× capability to oxidative damage.
When we analyzed ROS production after 48 h of incubation in
the presence of the complexes the same trend was observed
concerning complex 8 but for the other complexes results were
not statistically significant (ESI Fig. S11†).
Fig. 8 Pro-apoptotic and anti-apoptotic markers expression in HCT116
and HCT116 DOXR cells exposed to Cu(I) complex 8. Levels of
expression of BAX and BCL-2 in HCT116 (A) and HCT116 DOXR (C) cells
after 48 h exposure with the IC50 of complex 8 (white bars). BAX/BCL-2
ratio in HCT116 (B) and HCT116 DOXR (D) cells after 48 h incubation
with 0.1% (v/v) DMSO or the IC50 of complex 8 (black bars). Expression
levels of BAX and BCL-2 were normalized to β-actin levels in the
western blots. Data are represented as the mean SD of two indepen-
dent assays. Statistical significance was evaluated by the t-student
method (ns – statistically non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤
0.001).
Correlating these results with those obtained above, it is
possible to conclude that complexes 5, 7 and 8 can activate
cells death via apoptosis and autophagy due to the increase
oxidative stress through the ROS production, while the in the
case of complex 6 cell death could not be directly correlated
with ROS induction and other mechanisms might be involved
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1853

View Article Online
Paper
Dalton Transactions
Fig. 9 Autophagic potential in HCT116 cells exposed to Cu(I) complexes. (A) Fluorescent images of HCT116 cells labelled with Cyto-ID probe and
Hoechst 33342 for autophagy analysis after 48 h of exposure to the IC50 of Cu(I) complexes, 0.1% v/v DMSO, or 500 µM rapamycin. (B)
Quantification of HCT116 cells in autophagy after 48 h exposure to the IC50 of complexes, 0.1% v/v DMSO, or 500 µM rapamycin overnight. Data
are represented as the mean SD of three independent biological assays. Statistical significance was assessed relative to control (DMSO) by the
one-way ANOVA method (**** p ≤ 0.0001, *** p ≤ 0.001).
growth inhibition by stopping the cell cycle, signaling the cell
to die.45
The evaluation of the cytostatic potential of complex 8 was
performed by quantifying the DNA content in each phase of
the cell cycle (G1/G0, S, G2/M) by flow cytometry. HCT116
cells, after double blocking with thymidine, were exposure to
the IC50 of 8, 0.1% (v/v) DMSO or 0.4 µM doxorubicin for 5, 9,
24 and 32 hours, and stained with PI. Double-blocking with
thymidine, an S-phase blocker, is done to ensure that all cells
are in the same phase of the cell cycle when exposure begins.
After 5 h of exposition to DMSO or complexes, cells were
mostly in the G2/M phase (Fig. 11A), normally progressed to
the G0/G1 phase (Fig. 11B). However, while cells subjected to
Fig. 10 ROS levels quantified by flow cytometry in HCT116 tumor cell
line after exposure to IC50 of complexes, 0.1% (v/v) DMSO or 25 µM
H2O2 for 6 hours. Values were normalized to vehicle control (DMSO).
Data are represented as the mean SD of three independent biological
DMSO can progress through the cells cycle, cells subjected to
complex 8 becoming blocked at G0/G1 phase of the cell cycle
for 32 h (Fig. 11A–D).
Therefore, complex 8 has a cytotoxic potential (Table 1,
assays. Statistical significance was assessed relative to control (DMSO)
by the one-way ANOVA method (*** p ≤ 0.001, ** p ≤ 0.01, *p ≤ 0.05, ns Fig. 4 and 5) but also a cytostatic potential interfering with the
– statistically not significant).
progression of the cell cycle in the G1 phase.
Cleavage of plasmid DNA (pDNA)
(Fig. 6–10). The intrinsic apoptotic pathway is activated by
intracellular stimulus not mediated by receptors, such as irre-
parable damage to DNA, hypoxia, temperature, lack of nutri-
ents, etc.36
Considering the results obtained so far, particularly ROS
induction, we next evaluated the capability of Cu(I) complexes
to cleave pDNA in vitro.
For that, 100 ng of pUC18 were exposed to increasing con-
centrations of the complexes (5, 25 and 50 μM) for 24 hours
(Fig. 12) or to different exposure times (1, 3, 6, 24 and
Cell cycle analysis
Most of the developed metallic complexes, including copper 48 hours) with the same concentration of complexes (25 μM)
complexes, act on cells causing DNA damage, that can lead to (ESI Fig. S12†). Controls were performed with pUC18 in Tris-
1854 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
Fig. 11 Analysis of cell cycle progression in HCT116 cells after exposure to the IC50 of 8, doxorubicin or to 0.1% (v/v) DMSO, for 5 hours (A), 9 hours
(B), 24 hours (C), or 32 hours (D). The cells were analyzed by flow cytometry after PI labeling. Data are represented as the mean SD of three inde-
pendent biological assays.
Fig. 12 Plasmid DNA (100 ng pUC18) was exposed to increasing concentrations of the complexes as indicated at the top of the figure, for 24 hours.
The electrophoresis conditions were: 1.0% agarose gel in 1× TAE with 0.0015% GelRed, 40 V for 3 hours. The molecular weight marker used was the
lambda HindIII; and the legend for images A and B is above the wells. The green arrows correspond to the isoforms of the plasmid pUC18: N –
nicked form; L – linear form; C – coiled form; SC – supercoiled form.
HCl buffer exposed to 50% (v/v) DMSO or incubated with the appearance of the L form might be observed, a trend that also
restriction enzyme HindIII for 2 hours. The HindIII enzyme occurs for samples exposed to 50 μM of Cu(I) complexes
works as a positive control (cleavage of pDNA).
(Fig. 12). Considering these results, it is possible to state that
Four bands corresponding to the isoforms of the plasmid – Cu(I) complexes can cleave the double-stranded pDNA in a con-
nicked (N), linear (L), coiled (C) and supercoiled (SC) were centration dependent mode. This interaction reaches its satur-
observed (Fig. 12). With a concentration of 5 μM of each ation at 25 µM of complex, with similar electrophoretic profiles
complex, a decrease in the intensity of the SC and C forms and for higher concentrations (Fig. 12). After determining the con-
an increase in intensity of the N form is observed (Fig. 12). centration for which the complexes can cleave the pUC18
When the concentration of the complexes is increased to pDNA (25 µM), we next evaluated the time dependence on the
25 μM, a complete disappearance of the SC form and the cleavage capability of the complexes. For that we incubated
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1855

View Article Online
Paper
Dalton Transactions
pDNA with 25 µM of each complex for different period of proteomic analysis (2D electrophoresis) was carried out on
times (ESI Fig. S12†). protein extracts from HCT116 cells after exposure or not to the
By observing ESI Fig. S12A,† it appears that in the first hour IC50 of 8 for 48 h. The STRING database (string-db.org/) was
of exposure to the complexes, the SC form begins to disappear, used to unveil the biological processes and corresponding GO
with the simultaneous increase in the intensity of the N form. terms associated with proteins that exhibit altered expression
This trend continues and after 6 and 24 hours of exposure, the in the presence of the Cu(I) complex. A total of 19 proteins
N form continues to increase in intensity with also the appear- with altered expression levels were identified (Fig. 13 and 14
ance of the L form of the plasmid (ESI Fig. S12B†). Between 24 and Table 3). As observed in Fig. 14 complex 8 deregulates
and 48 hours of exposure (ESI Fig. S12C†), an increase in the Biological Processes (GO) terms: GO:1902175 – regulation of
intensity of the L form is observed. The observed electrophor- oxidative stress-induced intrinsic apoptotic signaling pathway;
etic profile shows that it takes more than 24 hours for the GO:0000302 – response to reactive oxygen species; GO:0006986
interaction of the complexes with the pUC18 DNA reach a – response to unfolded protein; GO:0006457 – protein folding;
saturation.
GO:0051726 – regulation of cell cycle; GO:0042981 – regulation
To determine the mechanism by which the cleavage occurs, of apoptotic process. These results are in line with the mole-
3 scavenging agents for ROS were used: ascorbic acid (super- cular pathways affected by the complexes shown (Fig. 6–10).
oxide anion scavenger), sodium azide (NaN3) (oxygen singlet
The CALU is one of the proteins that is overexpressed, and
scavenger), and L-histidine (oxygen singlets and hydroxyl rad- its main function is the folding and separation of proteins in
ical’s scavenger). H2O2 was used as a positive control, as it can the endoplasmic reticulum (ER) – ER stress. It is a calcium-
cleave DNA by oxidative mechanisms due to ROS.
binding protein, which in normal conditions is found in the
In pUC18 samples exposed to the action of complex 8 in ER, and in situations of cell cycle arrest or late apoptosis it is
the presence of the sequestering agents, DNA hydrolysis is transported to the cytoplasm.46 Its over-expression (Table 3)
only observed in the presence of ascorbic acid (superoxide corroborates the cytostatic potential of 8 by stopping the cycle
anion scavenger) with the disappearance of the SC form and in the G1 phase (Fig. 11) and the existence of apoptosis
the appearance of the band corresponding to the L form (ESI observed by flow cytometry via labeling with Anexin V – FITC
Fig. S13†). As sodium azide is a scavenger of oxygen singlets, and PI (Fig. 6). Over-expression of ANXA1 (Table 3), a protein
and L-histidine is responsible for scavenging oxygen singlets involved in the apoptotic process by activating caspase-3, also
and hydroxyl radicals, it is concluded that the DNA cleavage by shows the apoptotic potential of the complex in HCT116
8 occurs through the oxidative pathway, dependent on oxygen cells.45 The cytostatic potential of the complex is also verified
radicals (ESI Fig. S13†). This result is in line with the result by the under-expression of the RANG protein (Table 3). This
obtained in the ROS production assay (Fig. 10), where the protein is responsible for maintaining RAN (GTPase) in its
capability of the complexes to produce ROS was also demon- active form by binding to GTP, allowing the transport of pro-
strated (Fig. 10 and ESI Fig. S13†).
teins and nucleic acids across the nuclear membrane, with the
consequent progression of the cell cycle.45,47
Changes in expression of growth regulating proteins, such
as PCNA, RINI and PA2G4, were also observed (Table 3). PCNA
plays a critical role in responding to DNA damage, increasing
Proteomic analysis
To confirm some of the results obtained so far and the cellular
pathways affected by the action of complex 8, a comparative
Fig. 13 Two-dimensional (2D) electrophoresis of total protein extracts from the HCT116 cells exposed to 0.1% (v/v) DMSO (A) or IC50 of 8 (B), for
48 hours. The gels were analyzed in the Melanie 7.0 program, and the protein spots identified by Maldi-TOF/TOF. Proteins that have not changed in
expression are identified in blue, over-expressed in green and under-expressed in red.
1856 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
and ESI Fig. S12 and S13†). Another protein that is over-
expressed is RINI, an important protein in the redox homeo-
stasis of the organism and that inhibits RNA cleavage pointing
to an interruption in protein synthesis49 that might agree with
blockage of cell cycle in G1 phase (Fig. 11). On the other hand,
the under-expression of PA2G4, a protein that participates in a
signal transduction pathway and that is related to cell growth
and proliferation of tumors,50 and with the acquisition of drug
resistance,51 is an indicator of the antiproliferative capacity of
the complex and its suitability in targeting HCT116 DOXR
cells (Fig. 5, 8 and Table 1).
The action of the complex also seems to inhibit DNA tran-
scription, due to the overexpression of the RBBP7 protein. This
protein participates in processes such as histone deacetylation
by the histone deacetylase complex (HDAC), leading to inhi-
bition of transcription. It is usually over-expressed in some
types of cancer.52
Several chaperone and stress response proteins showed
changes in their expression levels by proteomic analysis
(CALR, HSPD1, HSP71).53,54 The overexpression of the P4HB
protein, which catalyzes the formation, dissociation or
rearrangement of disulfide bridges, is a response of cells to
stress cause by complex 8 55 Different oxidative stress response
proteins, such as PARK7,56 PRDX2 57 and GSTP-1,58 also
showed overexpression after cell exposure to 8 complex, corro-
borating the results previously obtained (ROS and pDNA clea-
vage). In addition, PARK7 is also a protein that participates in
the autophagic process, a type of cell death induced by the
complex (Fig. 9).56
Fig. 14 Protein–protein network with altered expression in HCT116
cancer cell line exposed to complex 8. Nodes represent proteins and
lines represent protein–protein associations. Image generated by
STRING (string-db.org). In dark blue are represented proteins associated
with Biological Process (GO) term GO:1902175 – regulation of oxidative
stress-induced intrinsic apoptotic signaling pathway, in purple are rep-
resented proteins associated with Biological Process (GO) term
GO:0000302 – response to reactive oxygen species; in green are rep-
resented proteins associated with Biological Process (GO) term
GO:0006986 – response to unfolded protein; in red are represented
proteins associated with Biological Process (GO) term GO:0006457 –
protein folding; in light blue are represented proteins associated with
Biological Process (GO) term GO:0051726 – regulation of cell cycle; in
light green are represented proteins associated with Biological Process
(GO) term GO:0042981 – regulation of apoptotic process.
The under-expression of the DHE3 protein, indirectly
involved in the process of aerobic respiration to obtain ATP
its concentration in these situations. Thus, its overexpression through glutamate synthesis, may indicate a decrease in the
confirms that the complex is capable of interacting and indu- metabolic activity of the cell in response to the cytotoxicity of
cing DNA damage48 in agreement with pDNA studies (Fig. 12 the complex.59 Likewise, the PGAM1 protein, involved in gly-
Table 3 Proteins identified in 2D gel electrophoresis as differentially expressed between HCT116 cells exposed for 48 h to the IC50 of 8 versus 0.1%
(v/v) DMSO (control). Expression values less than 0.7 (red) and greater than 1.5 (green) were considered as under-expressed or over-expressed pro-
teins, respectively. Values between 0.7 and 1.5 do not represent significant changes in expression levels
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1857

View Article Online
Paper
Dalton Transactions
colysis, is also under-expressed, indicating a low metabolic erature. After evaporation of the solvent under vacuum, the
activity.60 Finally, there was an overexpression of the 1433Z products were washed with diethyl ether (2 × 10 mL) and
protein. This protein participates in the regulation of several recrystallized from dichloromethane/diethyl ether, affording
signaling pathways, including cell proliferation and apopto- crystalline products.
sis.61 However, its increased expression has been associated
with the development of tumors, and the resistance of cells to
Data for [Cu(PPh3)2(Me2bpy)][BF4] (2)
induced cell death (chemotherapy) indicating that cells clearly Solvent: dichloromethane; yield: 72%; yellow crystals.
try to counteract the presence of 8 complex.
1H NMR [(CD3)2CO-d6, Me4Si, δ/ppm]: 8.48 (br, 4H, H-1 +
H-4), 7.43 (m, 6H, Hpara – PPh3), 7.35 (br, 2H, H-2), 7.30 (m,
12H, Hmeta – PPh3), 7.18 (m, 12H, Horto – PPh3), 2.52 (s, 6H,
H-6). 13C NMR [(CD3)2CO-d6, Me4Si, δ/ppm]:152.79 (C-5),
Experimental
Materials and methods
151.83 (C-3), 150.15 (C-1), 134.13 (Corto-PPh3), 133.14 (Cipso
-
PPh3), 131.06 (Cpara-PPh3), 129.77 (Cmeta-PPh3), 127.79 (C-2),
124.43 (C-1), 21.34 (C-6). 31P NMR [(CD3)2CO-d6, H3PO4, δ/
ppm]: 2.60 (br). FTIR [KBr cm−1]: 3100–3040 (νC–H, phenyl
rings), 1610–1410 (νCvN, νCvC, phenyl rings), 1099 and
696 cm−1 (νB–F, BF4), 700–690 (δC–H, phenyl rings). This com-
pound was found very sensitive to temperature decomposing
at ≈60 °C.
All starting reagents and solvents were obtained from standard
chemical suppliers. All manipulations involving air-free synth-
eses were carried out under a dinitrogen atmosphere using
Schlenk techniques and the solvents used were dried by stan-
dard methods and freshly distilled before use. NMR spectra
were recorded on a Bruker Avance 400 spectrometer operating
1
at probe temperature (400.13 MHz for H, 100.6 MHz for 13C
and 161.97 MHz for 31P). 1H and 13C chemical shifts were
reported relative to solvent peaks considering internal Me4Si
(0 ppm) and 31P NMR was externally referenced to 85% H3PO4.
Chemical shifts (δ) are reported in parts per million (ppm)
using (CD3)2CO-d6 as solvent, and the coupling constants (J)
expressed in Hertz (Hz). Resonance multiplicity is abbreviated
as: singlet (s), doublet (d), triplet (t), q = quartet, sept = septet,
m = multiplet, c = complex and br = broad. All the assignments
were attributed using 2D NMR experiments (COSY, HSQC, and
HMBC). The electronic UV-Vis spectra were recorded in di-
chloromethane, under air at room temperature, using 1 cm
optical path quartz cells on a Jasco V-660 spectrometer in the
range of 200–900 nm. Infrared spectra (4000–400 cm−1) were
recorded using a Thermo Nicolet 6700 spectrophotometer with
KBr, with only the most significant bands being cited in the
text. Elemental analyses were performed at Laboratório de
Data for [Cu(PPh3)2(dpytz)][BF4] (3)
Solvent: dichloromethane; yield: 63%; orange crystals.
1H NMR [(CD3)2CO-d6, Me4Si, δ/ppm]: 8.94 (br, 1H, H-4),
8.89 (d, 1H, H-1), 8.36 (t, 1H, H-3), 7.84 (t, 1H, H-2), 7.74 (d,
2H, H-10 + H-14), 7.49 (t, 3H, H-11 + H-12 + H-13), 7.57 (m,
6H, H-16 + H-17 + H-18 + H-19 + H-20), 7.41 (comp, 6H, Hpara
–
PPh3), 7.29 (comp, 24H, Hmeta + Horto – PPh3). 13C NMR
[(CD3)2CO-d6, Me4Si, δ/ppm]:158.31, 158.09 (C-6, C-8), 151.59
(C-4), 150.57 (C-5), 140.44 (C-3), 135.68 (Cipso-PPh3), 134.09,
130.35 (Corto + Cmeta-PPh3), 132.98 (C-9), 132.89 (C-15), 132.56,
131.38 (C-16, C-17, C-18, C-19, C-20), 131.61 (Cpara-PPh3),
131.16, 130.95 (C-10, C14), 129.73 (C-2), 129.60, 129.57 (C-11,
C-12, C-13), 125.97 (C-1). 31P NMR [(CD3)2CO-d6, H3PO4, δ/
ppm]: 3.11 (br). FTIR [KBr cm−1]: 3100–3040 (νC–H, phenyl
rings), 1600–1390 (νCvN, νCvC, phenyl rings), 1060 and 694
(νB–F, BF4), 700–690 (δC–H, phenyl rings). Elemental analysis
(%) found: C, 66.4; H, 4.2; N, 5.3; calc. for
C56CuH44N4P2BF4·0.4CH2Cl2 (1019.25): C, 66.46; H, 4.43; N,
5.50.
Análises at Instituto Superior Técnico, using
a Fisons
Instruments EA1108 system. Data acquisition, integration and
handling were performed using a PC with the software
package EAGER-200 (Carlo Erba instruments).
X-ray crystal structure determination
Synthesis
Crystals of compounds 2 and 3 suitable for X-ray diffraction
studies were mounted on a loop with Fromblin protective oil.
X-ray diffraction data were collected on a Bruker AXS-KAPPA
APEX II diffractometer with graphite-monochromated radi-
ation (Mo Kα, λ = 0.71073 Å) at 150 K. Intensity data were cor-
rected for Lorentz polarization effects. The X-ray generator was
operated at 50 kV and 30 mA, and the X-ray data collection was
monitored by the APEX64 program. Empirical absorption cor-
rection using SABADS65 was applied and data reduction was
done with the SMART and SAINT programs.66 SHELXT2014 67
The starting materials ([Cu(NCMe)4][BF4], [Cu(dppe)
(NCMe)2][BF4] and [Cu(PPh3)2(NCMe)2][BF4]) and compounds
[Cu(PPh3)2(2,2′-bipy)][BF4] (1), [Cu(PPh3)2(dpp)] [BF4] (4), [Cu
(dppe)(2,2′-bipy)][BF4] (5), [Cu(dppe)(Me2bpy)][BF4] (6), [Cu
(dppe)(dpytz)][BF4] (7) and [Cu(dppe)(dpp)][BF4] (8) were pre-
pared following published methods.9,24,25,62,63
General procedure for the syntheses of Cu(I) complexes [Cu
(PPh3)2(L)][BF4] (2 and 3)
The Cu(I) complex salts 2 and 3 were prepared according to the was used for structure solution, and SHELXL-2018 67 was used
following general synthetic procedure: the desired ligand for full matrix least-squares refinement on F2. Both programs
(0.5 mmol) was added to a stirred and degassed solution of are included in the package of programs WINGX-version
[Cu(PPh3)2(NCMe)2][BF4] (0.5 mmol) in dichloromethane 2014.1.68 Non-hydrogen atoms were refined anisotropically. All
(30 mL) and the mixture was stirred during 4 h at room temp- the hydrogen atoms were inserted in the calculated positions
1858 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
and allowed to refine in the parent carbon atom. The inter- 5% CO2. HCT116 cells (human colorectal carcinoma), MCF-7
molecular interactions were calculated using PLATON.69 The cells (human breast adenocarcinoma) and human primary
graphical representations were prepared using MERCURY fibroblasts were grown in Dulbecco’s modified Eagle’s
3.8.70 In spite of the various attempts to collect data and the medium (DMEM) (Invitrogen, New York, USA), supplemented
use of low temperature (150 K), the diffraction of crystal 2 was with 1% MEM, non-essential amino acid (Sigma, St Louis
poor and precluded the acquisition of good quality data. In Missouri, EUA) and maintained at 37 °C in atmosphere of 5%
compound 3 the solvent molecule was allowed to refine the s. CO2 and 99% (v/v) relative humidity. HCT116 Doxorubicin-
o.f. and lead to an occupation factor of 70%. A summary of the Resistant (HCT116 DOXR) cell line were derived from sensitive
crystal data, structure solution and refinement parameters for HCT116 cells cell lines and grown as previously described in
the structures are given in Table 4.
ref. 13.
Cell viability
Cell culture
A2780, MCF7, HCT116, HCT116-DOXR tumor cells and
primary normal fibroblasts were plated with a density of 0.75 ×
105 cells per well in 96 well plates. After 24 h in a controlled
atmosphere (5% CO2, 99% (v/v) relative humidity and 37 °C),
the media was replaced with fresh media containing
0.05–5 µM of copper complexes (5, 6, 7 and 8) for HCT116
cells, 0.05–9 µM for MCF-7 cells, 0.05–25 µM for A2780 cells,
0.5–2 µM for HCT 166 DOXR cells, and 0.1–50 µM for fibro-
blasts, 0.4 µM of doxorubicin (positive control) or 0.1% (v/v) of
DMSO (vehicle control). For the starting complex the tested
concentration range was 0.01–1.5 µM for HCT116 cells and
10–70 µM for fibroblasts. For the ligands the tested concen-
tration range was 0.01–2.5 µM in HCT116 cells and 10–70 µM
in fibroblasts. The other copper complexes (precursors and
complexes 1, 2, 3 and 4) were tested on HCT116 cells in a con-
centration range of 0.01–50 µM. The complexes were dissolved
in DMSO, and all the solutions were prepared from 1000-times
concentrated fresh stock solutions of the complexes to assure
a maximum volume of DMSO in culture medium of 0.1% (v/v).
Following 48 h of cell incubation in the presence of the com-
plexes and vehicle control, cell viability was evaluated with a
CellTiter 96® AQueous Non-Radioactive Cell Proliferation
Assay (Promega, Madison, WI, USA), using 3-(4,5-dimethyl-
thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe nyl)-2H-
tetrazolium, inner salt (MTS) as previously described.24,25 The
CellTiter 96® AQueous Assay is composed of solutions of MTS
and an electron coupling reagent (phenazinemethosulfate,
PMS). MTS is reduced by dehydrogenase enzymes present in
viable cell mitochondria into formazan product that is soluble
in a tissue culture medium. The absorbance of the formazan
product at 490 nm can be measured directly from 96 well assay
plates, and the value is directly proportional to the number of
viable cells in culture. This method for assessing cell viability
is indirect as it transposes mitochondrial viability into cell via-
bility.71 All assays were performed using biological triplicates.
A2780 cell line (human ovarian carcinoma) was grown in
Roswell Park Memorial Institute medium (RPMI) (Invitrogen,
New York, EUA) supplemented with 10% (v/v) fetal bovine
serum (FBS) and 1% (v/v) of penicillin (100 U mL−1)/streptomy-
cin (100 µg ml−1) solution (Life Technologies, Carlsbad, CA,
USA) and maintained at 37 °C in a humidified atmosphere of
Table 4 Data collection and structure refinement parameters for [Cu
(PPh3)2(Me2bpy)][BF4] (2) and [Cu(PPh3)2(dpytz)][BF4] (3)
Compound
2
3
Empirical
formula
Formula weight
T (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
C48H42BCuF4N2P2
C56.71H45.41BCl1.41CuF4N4P2
859.12
150(2)
0.71073
Monoclinic
P21/n
14.5824(5)
14.8286(5)
20.4748(7)
90
110.420(2)
90
1070.17
150(2)
0.71073
Triclinic
ˉ
P1
12.8537(5)
13.6470(5)
15.3142(5)
84.236(2)
81.186(10)
78.631(2)
2595.67(16)
2
γ (°)
Volume (Å3)
Z
4149.2(2)
4
Calculated
density (mg m−3
Absorption
coefficient
1.375
1.369
)
0.658
0.642
(mm−1
)
F (000)
θ Range for data
1776
2.981 to 26.805
1100
2.606 to 27.165
collection (°)
Limiting indices
−17 ≤ h ≤ 18,
−18 ≤ k ≤ 18,
−25 ≤ l ≤ 20
31 136/8536
[R(int) = 0.0382]
97.8
−14 ≤ h ≤ 16,
−16 ≤ k ≤ 17,
−19 ≤ l ≤ 19
43 804/11 468
[R(int) = 0.0381]
99.4
Reflections
collected/unique
Completeness to
θ (%)
Refinement
method
Data/restraints/
parameters
Goodness-on-fit
on F2
Full-matrix least-
squares on F2
8536/0/523
Copper detection by ICP-AES
11 468/0/640
1.052
To evaluate the internalization of the complexes 1–8 in
HCT116 cells, the inductive plasma atomic emission spec-
trometry technique (ICP-AES) was used. HCT116 cells were
seeded at 5 × 105 cells per mL in 25 cm2 culture flasks. After
24 h in a controlled atmosphere, the medium was replaced
with fresh medium containing 10 × IC50 of the complexes
(5–8), 50 µM of the complexes (1–4), or 0.1% (v/v) DMSO for
0.971
Final R indices
[I > 2σ(I)]
R indices
R1 = 0.0346,
wR2 = 0.0893
R1 = 0.0455,
wR2 = 0.0951
R1 = 0.0419,
wR2 = 0.1162
R1 = 0.0471,
wR2 = 0.1196
0.950 and −0.672
(all data)
Largest diff. peak 0.308 and −0.338
and hole (e Å)−3
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1859

View Article Online
Paper
Dalton Transactions
3 h at 4 °C or 37 °C. Cells were trypsinised by the addition of After 24 h in a controlled atmosphere (5% CO2, 99% (v/v) rela-
2 mL TrypLE TM Express (Gibco®, New York, USA) for 5 min at tive humidity and 37 °C), the medium was replaced with fresh
37 °C. Then, the detached cells were transferred to 15 ml medium containing the IC50 of the complex 8 or 0.1% (v/v)
falcon tubes and centrifuged at 700g, 5 min, 15 °C. The DMSO for 48 h under the above conditions. Then, to collect
medium with supernatant and the cell pellet were stored at cells, the medium was removed, the cells were washed with
−80 °C. One day before the analysis, freshly prepared aqua PBS and scraped from the flask in 2 ml PBS. The samples are
regia was added to the samples. The levels of copper were centrifuged at 700g, 5 min at 4° C (Sigma 3–16 K 10 280) and
quantified by ICP-AES (as a service).
pelleted cells were solubilized in lysis buffer [150 mM NaCl,
50 mM Tris-HCl, pH 8.0, 5 mM ethylenediaminetetraacetic
acid (EDTA), 1× Phosphatase inhibitor (PhosStop, Roche), 1×
Annexin V-FITC/PI double-staining assay
HCT116 cells were seeded at 1 × 105 cells per mL in 6 well Proteases inhibitors (complete Mini, Roche), 2% (v/v) NP-40,
plates with 24 h incubation in a controlled atmosphere (5% 1 mM phenylmethylsulfonyl fuoride (PMSF) and 0.1% (w/v)
CO2, 99% (v/v) relative humidity and 37 °C). After this time, 1,4-dithiothreitol (DTT)] for protein extraction. The treatment
the medium was removed and cells were stimulated by of the pellet cells, the quantification of the whole protein
addition of complete medium containing the IC50 of the com- extract and the separation of protein extracts was obtained as
plexes, 0.1% (v/v) DMSO or 0.4 µM doxorubicin for 48 h. After previously described in ref. 43. To blocking non-specific
48 h, the cells were stained with fluorine isothiocyanate (FITC) protein bidding, the membrane were incubated with 5% (w/v)
labeled Annexin V and propidium iodide (PI) according to the non-fat milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
manufacturer’s instructions (Annexin V-FITC apoptosis detec- 0.1% (w/v) Tween-20), the blots were exposed for 1 h to
tion kit; Invitrogen, Carlsbad, CA, USA) as previously described different primary antibody in 5% non-fat milk in TBST, anti-
in ref. 12, 21, 22 and 72. Measurements were performed on a BAX (1:5000; Abcam, United Kingdom) and anti BCl-2 (1:1000;
flow cytometer (Attune® Acoustic Focusing Cytometer Flow, Sigma, St Louis, EUA), washed 3 times with agitation for 5 min
Invitrogen) with the acquisition of at least 10 000 events per with 1 × TBST, and incubated with secondary antibody (1:3000,
sample and the processing of results was done in Attune® anti-mouse IgG, horseradish peroxidase (HPR)-linked Antibody
Cytometric software program (Invitrogen). All assays were per- or 1:5000, anti-rabbit IgG, HPR-linked Antibody; Cell Signaling
formed using biological triplicates.
Technology, USA). Membranes were washed again 3 times for
5 min with TBST, and to detect the protein bands (BAX and
BCl-2) treated with WesternBright ECL substract (Advansta,
Alteration of mitochondrial membrane potential (ΔΨm)
To measure mitochondrial potential (ΔΨm), a fluorescent, cat- USA) for 5 min. To reveal the protein bands, the film was
ionic and lipophilic probe JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′- exposed to the membrane on a dark room. The quantification
tetraethylbenzimidazolyl-carbocyanine iodide) was used as pre- of proteins was determinate by densitometry using Image J
viously described (Abnova Corporation, Walnut, CA, software. All assays were performed using biological
USA).21,22,72 This probe has the ability to change its color due triplicates.
to alterations in mitochondrial potential.73 HCT116 cells were
All membranes were incubated stripping buffer 3 times for
plated in 24 well plates with a density of 0.75 × 105 cells per 10 min with agitation (0.1 M glycine, 20 mM magnesium
ml. After 24 h in a controlled atmosphere (5% CO2, 99% (v/v) acetate, 50 mM KCl, pH 2.2) to remove specific bonds, and
relative humidity and 37 °C), the media was replaced with then, incubated with anti-β actin (1:5000; Sigma, St Louis,
fresh media containing the IC50 of the complexes or 0.1% (v/v) EUA) as a control for normalization of the results.
DMSO for 48 h under the above conditions. After the incu-
Production of intracellular reactive oxygen species (ROS)
bation period, the medium was removed, and the cells were
stained with JC-1 staining solution for 20 min at 37 °C in the Generation of ROS in HCT116 cells previously exposed to the
dark. Controls with unlabeled cells (complete medium only) study complexes was evaluated using the dichlorofluorescein
and untreated cells (complete medium + JC-1) were performed diacetate substrate (H2DCF-DA) (Invitrogen) as previously
too. Immediately after this time, cells are observed and photo- described.22,74,75 DCFH-DA is a non-polar dye that penetrates
graphed under the inverted fluorescence microscope (Nikon the cell membrane and is hydrolyzed inside into the polar
Eclipse Ti, Nikon Instruments, Tokyo, Japan). The photo- derivative DCFH (reduced dichlorofluorescein) that are non-
graphs were taken with the Nikon D5-Qi1Mc digital camera fluorescent by cellular esterases. When in the presence of
(Nikon Instruments), and the images obtained using the intracellular ROS and other peroxides, DCFH will be oxidized
NISElements BR (Nikon Instruments) software and analyzed in to 2′, 7′-dichlorofluorescein (DCF),
a highly fluorescent
ImageJ software (National Institutes of Health (NIH), complex.76 HCT116 cells were seeded at 1 × 105 cells per ml in
Bethesda, MD, USA). All assays were performed using biologi- 6 well plates with 24 h incubation in a controlled atmosphere
cal triplicates.
(5% CO2, 99% (v/v) relative humidity and 37 °C). After this
time, the media was replaced with fresh media containing the
IC50 of the complexes, 25 µM of H2O2 (positive control) or
Anti- and pro-apoptotic proteins expression by western blot
For this assay HCT116 and HCT116 DOXR cells were cultured 0.1% (v/v) DMSO for 6 h or 48 h under the above conditions.
in 25 cm2 culture flasks with a density of 2 × 106 cells per ml. Following the incubation time, cells were washed with PBS
1860 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
and re-suspended in pre-warmed PBS containing 10 µM cytometer (Attune® Acoustic Focusing Cytometer Flow,
H2DCF-DA. The cells were incubated for 20 min in the dark at Invitrogen) with the acquisition of at least 10 000 events per
37° C and the production of ROS, based on the levels of DCF sample and the processing of results was done in Attune®
positive cells, was analyzed by flow cytometry in an Attune cyt- Cytometric software program (Invitrogen). All assays were per-
ometer (Attune® Acoustic Focusing Cytometer Flow, formed using biological triplicates.
Invitrogen) with the acquisition of at least 10 000 events per
sample using Attune® Cytometric software program
Interaction with plasmidic DNA (pDNA)
(Invitrogen). All assays were performed using biological Plasmid DNA pUC18 were isolated from E. coli transformed
triplicates.
cells, grown for 24 h, at 37 °C, in a Luria–Bertani (LB)-agar
medium with 100 μg ml−1 Ampicillin. After the incubation
period, pDNA was extracted from the bacteria using the
Autophagic potential
In order to detect cell death by autophagy in HCT116 cell line, NZYSpeedy Miniprep Kit according to the manufacturer’s pro-
the CYTO-ID® Autophagy Detection Kit (Enzo Life Sciences, tocol. In vitro interaction between complexes and pDNA was
United Kingdom) was used. HCT116 cells were seeded at 0.75 performed with 100 ng of pUC18 and complexes at different
× 105 cells per ml in 24 well plates After 24 h in a controlled concentrations (5, 25 and 50 µM) in 5 mM Tris–HCl, 50 mM
atmosphere (5% CO2, 99% (v/v) relative humidity and 37 °C), NaCl buffer (pH 7.19) as described previously.77 Each assay
the medium was replaced with fresh medium containing the was programmed to a final volume of 20 μL and all samples
IC50 of the complexes or 0.1% (v/v) DMSO for 48 h under the were incubated for 24 hours at 37 °C. In incubated samples
above conditions, or with 5 μL of a 50 mM solution of rapamy- containing only pUC18 DNA (100 ng), the HindIII enzyme was
cin (positive control) incubated overnight. Controls with added at the last hour of incubation to linearize the plasmid
unlabeled cells (complete medium only) and untreated cells (positive control). In a second assay, to study the kinetics of
(complete medium + fluorescent probes) were performed too. the reaction, 100 ng of pUC18 was incubated at 37 °C for 1, 3,
After the incubation period, the medium was removed and 6, 24 and 48 h with 25 µM of complexes in 5 mM Tris–HCl,
cells were stained according to the instructions of the 50 mM NaCl (pH 7.19). Control samples with 0.1% (v/v) DMSO
CYTO-ID® Autophagy Detection Kit (Enzo Life Sciences, UK).40 were also performed. After incubation times, 5 μL of 5×
The cells were observed and photographed under the inverted loading buffer (25 mM Tris – HCl, 25 mM EDTA (pH 8.0), 50%
fluorescence microscope (Nikon Eclipse Ti, Nikon glycerol, 0.1% bromophenol blue (Bioline, London, UK) was
Instruments, Tokyo, Japan). The photographs were taken with added to each sample. Then the samples were applied to a
the Nikon D5-Qi1Mc digital camera (Nikon Instruments), and 1.0% (w/v) agarose gel and analyzed by electrophoresis in 1×
the images obtained using the NISElements BR (Nikon TAE buffer with 0.0015% Red Gel and a constant current of 90
Instruments) software and analyzed in ImageJ software V applied for 60 minutes. The gels were analyzed and photo-
(National Institutes of Health (NIH), Bethesda, MD, USA). All graphed on a GelDoc XR + system using Quantity One software
assays were performed using biological triplicates.
for image acquisition.
Cell cycle progression assay
Determination of the DNA cleavage mechanism by complexes
The effect of the complex on cell cycle progress was analyzed In order to determine the mechanism by which the complexes
by flow cytometry based on the DNA content of propidium cleave the DNA, an assay was carried out in which the 100 ng
iodide (PI) stained cells. Cells were cultured at 0.75 × 105 cells of pUC18 was exposed to the complexes and with three scaven-
per ml in 25 cm2 culture flasks and were synchronized in early ging agents of ROS (50 µM ascorbic acid, 50 µM L-histidine
S-phase by a double thymidine block with 2 mM for 16 h, as and 50 µM sodium azide, NaN3). A positive control with
described in ref. 47. Cells were released from the second block 100 µM H2O2 was also performed.78
by replacing the medium with thymidine with fresh medium
containing the IC50 of the complexes or 0.1% (v/v) DMSO for 5,
Comparative proteomic analysis
9, 24 and 32 h. As a synchronization method for normalization
Protein sample preparation. HCT116 cancer cells were
of results, after the end of the second thymidine block, a seeded in 25 cm2 cell culture flasks, at a cell density of 2 × 106
sample of untreated cells was collected (0 hours). After each cells per ml and incubated under the same conditions
time point, cells were washed with 1× PBS at 4° C, and centri- described above for 24 h. After this period the medium was
fuged at 3000g, 5 min. The obtained pellet was resuspended in replaced with fresh medium containing the IC50 of the
100 µL of 1× PBS, and this volume was transferred dropwise to complex JL10 or 0.1% (v/v) DMSO for 48. Cell collection was
another Eppendorf tube, with 1 ml of an 80% (v/v) ethanol performed in the same manner as described in the western
solution, with gentle agitation to fix the cells. The samples blot section. Cell lysate was centrifuged at 10 000g, 5 minutes
were stored at 4° C for a minimum of 16 h. After this time were at 4° C and the supernatant recovered. Precipitation and purifi-
centrifuged at 5000g, 10 min, 4° C, the supernatant was cation were performed with the 2-D Clean-Up Kit (GE
removed, and the pellet allowed to air dry. Cell pellet was Healthcare, Little Chalfont, UK) according to the protocol pro-
treated with 50 μg ml−1 RNase A for 30 min at 37 °C and, then vided by the manufacturer, except for the wash buffer and
with PI (2.5 μg ml−1). The DNA content was analyzed on a flow wash additive addition step, where overnight incubation was
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1861

View Article Online
Paper
Dalton Transactions
performed at −20° C. The resulting pellets were rehydrated in to analyze the results and the statistical significance was evalu-
100 µL of rehydration buffer solution containing 7 M urea ated using one-way ANOVA in order to compare the control
(BDH Prolabo, VWR International), 2 M tiourea (Merck, group with the treated groups for statistical significance.
Frankfurt, Germany), 2% CHAPS (GE Healthcare, United Results with a p < 0.05 were considered statistically significant.
Kingdom), phosphatase inhibitors 1×, protease inhibitors 1×,
bromophenol blue (Merck, Frankfurt, Germany), 0.1% (w/v)
DTT and 1 mM PMSF. Samples were incubated at RT for
48 hours. After the incubation time, the samples were centri-
Conclusions
fuged at 12 000g, 10 minutes and the supernatants recovered, The development of metal complexes as possible chemothera-
and protein extracts quantified using the Pierce Protein Assay peutic agents has been one of the main focuses in chemo-
Kit.
therapy research. As chemotherapy is one of the techniques
mostly used to fight cancer, the chemotherapeutic potential of
new copper(I) complexes in colorectal cancer sensitive and
Two-dimensional gel electrophoresis
Immediately after protein quantification, isoelectric focusing resistant to doxorubicin was studied.
(IEF) was performed, and 100 μg of whole protein extract was
From the results obtained in the antiproliferative assays, it
used. The volume corresponding to 100 μg of the total protein was demonstrated that the complex that has the greatest thera-
sample was uniformly distributed into each 7 cm Immobilized peutic potential is 8, due to its high cytotoxicity against tumor
pH gradient (IPG) strip holder (GE Healthcare, United cells and low cytotoxicity towards healthy cells. Nevertheless,
Kingdom), resuspended in rehydration solution to a volume of these Cu(I) complexes are promising for application in colorec-
125 μL, supplemented with 0.5% IPG (GE Healthcare, United tal cancer sensitive and resistant to doxorubicin.
Kingdom) and 0.5% of destreak (GE Healthcare, United
The ability of these complexes to induce intrinsic apoptosis
Kingdom). After dispensing, a 7 cm Immobiline Dry strip pH in HCT116 cells was also demonstrated, and death by necrosis
3–10 NL strip (GE Healthcare, United Kingdom) was placed was not significant. The activation of this apoptosis pathway
over the sample, ensuring that the gel was in contact with the for complex 8 may be related to the production of ROS. The
sample and no bubbles formed between them. 750 μL of remaining complexes did not show this ability, so the acti-
Drystrip Cover Fluid (GE Healthcare, UK) was added over the vation of this pathway will be induced by another mechanism.
strips, and the strips were subsequently disposed in the The increased expression of BAX protein (pro-apoptotic
ETTAN IPGphor 3 IEF isofocus system (GE Healthcare, United protein) relative to BCL-2 (anti-apoptotic protein) for complex
Kingdom). After the end of the program, the strips were placed 8 corroborates the induction of apoptosis by the intrinsic
in equilibration solution (70 mM Tris-HCl pH 8.8, 6 M Urea, pathway. There were also other types of cell death, such as
30% (v/v) glycerol and 2% (w/v) SDS (GE Healthcare, United autophagy. This type of cell death is important in cells that are
Kingdom)) supplemented with 1% (w/v) DTT for 15 minutes resistant to apoptotic mechanisms.
with horizontal agitation, followed by other solution sup-
It was demonstrated that the complex 8 can block the cell
plemented with 2.5% (w/v) iodoacetamide (GE Healthcare, cycle in the G1 phase, exhibiting additionally a cytostatic
United Kingdom), incubated for another 15 min under hori- potential. The complexes showed the ability to interact and
zontal agitation. SDS-PAGE was performed in 12.5% polyacryl- cleave pDNA, and for 8 it was also demonstrated that this clea-
amide gels in a SDS-PAGE Mini-PROTEAN®3 System (BioRad). vage occurs by oxidative mechanisms, which corroborates the
The scanning was done by the PIXMA M250 scanner (Canon) results obtained in the assay of ROS production for this
and the gels analyzed in the Melanie 7.0 program (GeneBio, complex.
Geneva, Switzerland). The analysis was performed by auto-
Proteomic analysis confirmed that complex 8 deregulates
matic spot detection, protein spot identification, spot match- Biological Processes (GO) terms: GO:1902175 – regulation of
ing between the different gels and determination of the inten- oxidative stress-induced intrinsic apoptotic signaling pathway;
sity/expression of each spot. The variation in expression levels GO:0000302 – response to reactive oxygen species; GO:0006986
of each protein was normalized by the intensity of the control – response to unfolded protein; GO:0006457 – protein folding;
spots. Differential expressed proteins were excised and send GO:0051726 – regulation of cell cycle; GO:0042981 – regulation
for MALDI-TOF-TOF analysis (service, ITQB). Expression levels of apoptotic process. This corroborates the apoptotic, autopha-
<0.7 (protein under-expression) and >1.5 (protein over- gic and cytostatic potential of complex 8, as well as its ability
expression) were considered significant. The classification of to produce ROS and cause DNA damage. The interference of
proteins according to their functions was done using the the complex in folding and protein synthesis and its ability to
STRING 11.0 platform (search tool for the retrieval of interact- cause post-translational modifications was also verified.
ing genes/proteins) and UniProt (Universal Protein Resource).
Finally, it was observed that the complex causes a reduction in
cellular metabolism.
Statistical analysis
As future tests, studying the interaction with other bio-
All results were expressed as mean SD of two independent molecules, such as BSA (human serum albumin) may provide
biological assays. Each of the biological assays was obtained by important additional data, namely in terms of drug distri-
technical duplicates. The GraphPadPrism 6 software was used bution, free concentration and metabolism in the organism.
1862 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
Another hypothesis will be to try to develop a more effective
delivery system that increases the effectiveness of the treat-
ment and reduces side effects, namely through the incorpor-
C. M. P. Rodrigues, M. G. Cabral, P. A. Videira, C. Monteiro
and A. R. Fernandes, Drug Metab. Drug Interact., 2013, 28,
167–176.
ation of the complex in nanoparticles. Finally, the potential of 12 P. Pedrosa, M. L. Corvo, M. Ferreira-Silva, P. Martins,
Cu(I) complexes in targeting doxorubicin sensitive and resist-
ant cells which is positive and must be further explored using
in vivo animal models.
M. C. Carvalheiro, P. M. Costa, C. Martins,
L. M. D. R. S. Martins, P. V. Baptista and A. R. Fernandes,
Int. J. Mol. Sci., 2019, 20(21), 5510–5527.
13 P. Pedrosa, R. Mendes, R. Cabral, L. M. D. R. S. Martins,
P. V. Baptista and A. R. Fernandes, Sci. Rep., 2018, 8, 1–8.
14 G. Spengler, A. Kincses, B. Rácz, B. Varga, G. Watanabe,
R. Saijo, H. Sekiya, E. Tamai, J. Maki, J. Molnár and
M. Kawase, Anticancer Res., 2018, 38, 6181–6187.
15 M. Wehbe, C. Lo, A. W. Y. Leung, W. H. Dragowska,
G. M. Ryan and M. B. Bally, Invest. New Drugs, 2017, 35,
682–690.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
16 F. Tisato, C. Marzano, V. Peruzzo, M. Tegoni, M. Giorgetti,
M. Damjanovic, A. Trapananti, A. Bagno, C. Santini,
M. Pellei, M. Porchia and V. Gandin, J. Inorg. Biochem.,
2016, 165, 80–91.
17 S. I. Khan, I. Ali Khan, A. Badshah, F. Perveen Malik,
S. Tabassum, I. Ullah, D. Zargarian and M. Khawar Rauf,
J. Coord. Chem., 2018, 71, 4086–4108.
18 M. M. O. Peña, J. Lee and D. J. Thiele, J. Nutr., 2018, 129,
1251–1260.
19 H. A. Wee and P. J. Dyson, Eur. J. Inorg. Chem., 2006, 4003–
4018.
This work was supported by the Applied Molecular Biosciences
Unit - UCIBIO and Centro de Química Estrutural which are
financed by national funds from FCT (UIDB/04378/2020 and
UIDB/00100/2020, respectively). T. S. Morais thanks FCT for
CEECIND 2017 Initiative for the project CEECIND/00630/2017
(acknowledging FCT, as well as POPH and FSE-European
Social Fund). M. Silva and C. Fonseca are also acknowledged
for preliminary fluorescence microscopy and viability data,
respectively.
20 C. Marzano, M. Pellei, F. Tisato and C. Santini, Anticancer
Agents Med. Chem., 2009, 9, 185–211.
Notes and references
1 M. M. Fidler, F. Bray and I. Soerjomataram, Scand. J. Public 21 K. Choroba, B. Machura, S. Kula, L. R. Raposo,
Health, 2018, 46, 27–36.
A. R. Fernandes, R. Kruszynski, K. Erfurt, L. S. Shul’Pina,
Y. N. Kozlov and G. B. Shul’Pin, Dalton Trans., 2019, 48,
12656–12673.
2 G. N. Marta, R. Riera, P. Bossi, L. P. Zhong, L. Licitra,
C. R. Macedo, G. De Castro, A. L. Carvalho, W. N. William
and L. P. Kowalski, Eur. J. Cancer, 2015, 51, 2596–2603.
3 A. J. Breugom, M. Swets, J. F. Bosset, L. Collette, A. Sainato,
L. Cionini, R. Glynne-Jones, N. Counsell, E. Bastiaannet,
C. B. M. van den Broek, G. J. Liefers, H. Putter and
C. J. H. Van de Velde, Lancet Oncol., 2015, 16, 200–207.
4 K. Sak, Chemother. Res. Pract., 2012, 1–11.
22 A. Maroń, K. Czerwińska, B. Machura, L. Raposo, C. Roma-
Rodrigues, A. R. Fernandes, J. G. Małecki, A. Szlapa-Kula,
S. Kula and S. Krompiec, Dalton Trans., 2018, 47, 6444–
6463.
23 A. S. Mendo, S. Figueiredo, C. Roma-Rodrigues,
P. A. Videira, Z. Ma, M. Diniz, M. Larguinho, P. M. Costa,
J. C. Lima, A. J. L. Pombeiro, P. V. Baptista and
A. R. Fernandes, J. Biol. Inorg. Chem., 2015, 20, 935–948.
5 T. Iwamoto, Biol. Pharm. Bull., 2013, 36, 715–718.
6 P. Martins, M. Marques, L. Coito, A. Pombeiro, P. Baptista
and A. Fernandes, Anticancer Agents Med. Chem., 2014, 14, 24 T. S. Morais, Y. Jousseaume, M. F. M. Piedade, C. Roma-
1199–1212.
Rodrigues, A. R. Fernandes, F. Marques, M. J. Villa De Brito
7 P. Martins, J. Jesus, S. Santos, L. R. Raposo, C. Roma-
and M. Helena Garcia, Dalton Trans., 2018, 47, 7819–7829.
Rodrigues, P. V. Baptista and A. R. Fernandes, Molecules, 25 C. Bravo, M. P. Robalo, F. Marques, A. R. Fernandes,
2015, 20, 16852–16891.
D. A. Sequeira, M. F. M. Piedade, M. H. Garcia, M. J. V. De
Brito and T. S. Morais, New J. Chem., 2019, 43, 12308–
12317.
8 J. Coimbra, C. Mota, S. Santos, P. V. Baptista and
A. R. Fernandes, Inorganic Compounds Going NANO, 2015,
vol. 1.
26 Global Cancer Observatory https://gco.iarc.fr/, (accessed 29
November 2020).
9 J. Lopes, D. Alves, T. S. Morais, P. J. Costa,
M. F. M. Piedade, F. Marques, M. J. Villa de Brito and 27 W. Villarreal, L. Colina-Vegas, G. Visbal, O. Corona,
M. Helena Garcia, J. Inorg. Biochem., 2017, 169, 68–78.
10 A. P. Neves and M. D. Vargas, Rev. Virtual Quim., 2011, 3,
196–209.
R. S. Corrêa, J. Ellena, M. R. Cominetti, A. A. Batista and
M. Navarro, Inorg. Chem., 2017, 56, 3781–3793.
28 T. Pivetta, V. Lallai, E. Valletta, F. Trudu, F. Isaia, D. Perra,
E. Pinna and A. Pani, J. Inorg. Biochem., 2015, 151, 107–114.
11 A. Silva, D. Luís, S. Santos, J. Silva, A. S. Mendo, L. Coito,
T. F. S. Silva, M. F. C. Guedes Da Silva, 29 F. R. Kona, D. Buac and A. M. Burger, Curr. Cancer Drug
L. M. D. R. S. Martins, A. J. L. Pombeiro, P. M. Borralho,
Targets, 2011, 11(3), 338–346.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1863

View Article Online
Paper
Dalton Transactions
30 L. B. Alexandrov, S. Nik-Zainal, D. C. Wedge,
S. A. J. R. Aparicio, S. Behjati, A. V. Biankin, G. R. Bignell,
N. Mateus, A. R. Fernandes and I. Fernandes, Sci. Rep.,
2019, 9, 1–14.
N. Bolli, A. Borg, A. L. Børresen-Dale, S. Boyault, 44 A. Wojtala, M. Bonora, D. Malinska, P. Pinton, J. Duszynski
B. Burkhardt, A. P. Butler, C. Caldas, H. R. Davies,
C. Desmedt, R. Eils, J. E. Eyfjörd, J. A. Foekens, M. Greaves,
and M. R. Wieckowski, in Methods in Enzymology, Academic
Press Inc., 2014, vol. 542, pp. 243–262.
F. Hosoda, B. Hutter, T. Ilicic, S. Imbeaud, M. Imielinsk, 45 J. Buolamwini, Curr. Pharm. Des., 2005, 6, 379–392.
N. Jäger, D. T. W. Jones, D. Jonas, S. Knappskog, M. Koo, 46 V. H. Bull, E. M. Fargestad, M. Strozynski and B. Thiede,
S. R. Lakhani, C. López-Otín, S. Martin, N. C. Munshi,
Electrophoresis, 2010, 31, 1873–1885.
H. Nakamura, P. A. Northcott, M. Pajic, E. Papaemmanuil, 47 P. M. Borralho, B. T. Kren, R. E. Castro, I. B. Moreira Da
A. Paradiso, J. V. Pearson, X. S. Puente, K. Raine,
M. Ramakrishna, A. L. Richardson, J. Richter,
Silva, C. J. Steer and C. M. P. Rodrigues, FEBS J., 2009, 276,
6689–6700.
P. Rosenstiel, M. Schlesner, T. N. Schumacher, P. N. Span, 48 E. M. Boehm, K. T. Powers, C. M. Kondratick, M. Spies,
J. W. Teague, Y. Totoki, A. N. J. Tutt, R. Valdés-Mas,
M. M. Van Buuren, L. Van ‘T Veer, A. Vincent-Salomon,
J. C. D. Houtman and M. T. Washington, J. Biol. Chem.,
2016, 291, 8735–8744.
N. Waddell, L. R. Yates, J. Zucman-Rossi, P. Andrew 49 P. Koczera, L. Martin, G. Marx and T. Schuerholz,
Futreal, U. McDermott, P. Lichter, M. Meyerson, Int. J. Mol. Sci., 2016, 17(8), 1278.
S. M. Grimmond, R. Siebert, E. Campo, T. Shibata, 50 K. L. Keene, A. R. Quinlan, X. Hou, I. M. Hall,
S. M. Pfister, P. J. Campbell and M. R. Stratton, Nature,
2013, 500, 415–421.
J. C. Mychaleckyj, S. Onengut-Gumuscu and P. Concannon,
Genes Immun., 2012, 13, 66–70.
31 M. Sutradhar, E. C. B. A. Alegria, F. Ferretti, L. R. Raposo, 51 J. J. Lu, D. Z. Lu, Y. F. Chen, Y. T. Dong, J. R. Zhang, T. Li,
M. F. C. Guedes da Silva, P. V. Baptista, A. R. Fernandes Z. H. Tang and Z. Yang, Chin. J. Nat. Med., 2015, 13, 673–679.
and A. J. L. Pombeiro, J. Inorg. Biochem., 2019, 200, 52 N. Yu, P. Zhang, L. Wang, X. He, S. Yang and H. Lu, Oncol.
110811. Lett., 2018, 16, 7204–7211.
32 L. C. Crowley, B. J. Marfell, A. P. Scott and 53 L. Varricchio, M. Falchi, M. Dall’Ora, C. De Benedittis,
N. J. Waterhouse, Cold Spring Harb. Protoc., 2016, 2016,
953–957.
A. Ruggeri, V. N. Uversky and A. R. Migliaccio, Front. Cell
Dev. Biol., 2017, 5, 1–19.
33 S. Chen, A. C. Cheng, M. S. Wang and X. Peng, World J. 54 S. K. Calderwood and J. Gong, Trends Biochem. Sci., 2016,
Gastroenterol., 2008, 14, 2174–2178. 41, 311–323.
34 A. Perelman, C. Wachtel, M. Cohen, S. Haupt, H. Shapiro 55 M. A. Pierrard, P. Kestemont, N. T. Phuong, M. P. Tran,
and A. Tzur, Cell Death Dis., 2012, 3, e430–e437.
E. Delaive, M. L. Thezenas, M. Dieu, M. Raes and
35 J. C. De Vicente, S. Olay, P. Lequerica-Fernandez,
F. Silvestre, J. Proteomics, 2012, 75, 2454–2467.
J. Sánchez-Mayoral, L. M. Junquera and M. F. Fresno, 56 B. Wang, Z. Cai, K. Tao, W. Zeng, F. Lu, R. Yang, D. Feng,
J. Oral Pathol. Med., 2006, 35, 140–145. G. Gao and Q. Yang, Autophagy, 2016, 12, 1215–1228.
36 R. Caltabiano, G. Musumeci, D. Basic, S. Giunta, S. Cellek, 57 S. Zhou, Q. Han, R. Wang, X. Li, Q. Wang, H. Wang,
D. J. Ralph, S. Sansalone, C. Loreto, G. La Rocca, J. Wang and Y. Ma, Oncol. Lett., 2016, 12, 2217–2221.
S. Castorina, R. Anzalone, G. Vespasiani and R. Djinovic, 58 Y. A. Larasati, N. Yoneda-Kato, I. Nakamae, T. Yokoyama,
BioMed Res. Int., 2014, 2014, 1–10. E. Meiyanto and J. Y. Kato, Sci. Rep., 2018, 8, 1–13.
37 N. S. Gul, T. M. Khan, M. Chen, K. Bin Huang, C. Hou, 59 H. Y. Yang and T. H. Lee, BMB Rep., 2015, 48, 200–208.
M. I. Choudhary, H. Liang and Z. F. Chen, J. Inorg. 60 G. Huasong, Y. Bin, Y. Yaohua, S. Jianhong, Z. Sanhu,
Biochem., 2020, 213, 111260.
38 F. Bisceglie, R. Alinovi, S. Pinelli, M. Galetti, M. Pioli,
Z. Jianhong, Q. Wenxin and G. Yilu, J. Neurosurg., 2013,
118, 846–853.
P. Tarasconi, A. Mutti, M. Goldoni and G. Pelosi, 61 H. C. Kuo, Y. R. Kuo, K. F. Lee, M. C. Hsieh, C. Y. Huang,
Metallomics, 2016, 8, 1255–1265.
Y. Y. Hsieh, K. C. Lee, H. L. Kuo, L. Y. Lee, W. P. Chen,
C. C. Chen and S. Y. Tung, Cell. Physiol. Biochem., 2017, 43,
195–208.
39 C. Trejo-Solís, D. Jimenez-Farfan, S. Rodriguez-Enriquez,
F. Fernandez-Valverde, A. Cruz-Salgado, L. Ruiz-Azuara and
J. Sotelo, BMC Cancer, 2012, 12, 156.
62 G. J. Kubas, B. Monzyk and A. L. Crumbliss, Inorganic
Syntheses, John Wiley & Sons, Ltd, 2007, pp. 90–92.
40 S. Guo, Y. Liang, S. F. Murphy, A. Huang, H. Shen,
D. F. Kelly, P. Sobrado and Z. Sheng, Autophagy, 2015, 11, 63 J. Díez, S. Falagán, P. Gamasa and J. Gimeno, Polyhedron,
560–572. 1988, 7, 37–42.
41 M. Redza-Dutordoir and D. A. Averill-Bates, Biochim. 64 C. Sarto, P. A. Binz and P. Mocarelli, Electrophoresis, 2000,
Biophys. Acta, Mol. Cell Res., 2016, 1863, 2977–2992. 21, 1218–1226.
42 H. Nakano, A. Nakajima, S. Sakon-Komazawa, J. H. Piao, 65 S. R. B. Devi, A. M. Dhivya and K. N. Sulochana, J. Biosci.,
X. Xue and K. Okumura, Cell Death Differ., 2006, 13, 730–
2016, 41, 487–496.
737.
66 M. Esteller and J. G. Herman, J. Pathol., 2002, 196, 1–7.
43 H. Oliveira, C. Roma-Rodrigues, A. Santos, B. Veigas, 67 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found.
N. Brás, A. Faria, C. Calhau, V. de Freitas, P. V. Baptista,
Crystallogr., 2008, 64, 112–122.
1864 | Dalton Trans., 2021, 50, 1845–1865
This journal is © The Royal Society of Chemistry 2021

View Article Online
Dalton Transactions
Paper
68 L. J. Farrugia, J. Appl. Crystallogr., 2012, 45, 849–854.
69 T. Spek, Acta Crystallogr., Sect. D: Biol. Crystallogr., 2009, 65,
148–155.
70 C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock,
G. P. Shields, R. Taylor, M. Towler and J. van de Streek,
J. Appl. Crystallogr., 2006, 39, 453–457.
Y. N. Kozlov and G. B. Shul, Dalton Trans., 2019, 48, 12656–
12673.
75 J. Almeida, C. Roma-Rodrigues, A. G. Mahmoud,
M. F. C. Guedes da Silva, A. J. L. Pombeiro,
L. M. D. R. S. Martins, P. V. Baptista and A. R. Fernandes,
J. Inorg. Biochem., 2019, 199, 110789.
71 J. C. Stockert, R. W. Horobin, L. L. Colombo and 76 W. Jakubowski and G. Bartosz, Cell Biol. Int., 2000, 24, 757–
A. Blázquez-Castro, Acta Histochem., 2018, 120, 159–167. 760.
72 J. Díez, S. Falagán, P. Gamasa and J. Gimeno, Polyhedron, 77 D. V. Luís, J. Silva, A. I. Tomaz, R. F. M. De Almeida,
1988, 7, 37–42.
M. Larguinho, P. V. Baptista, L. M. D. R. S. Martins,
T. F. S. Silva, P. M. Borralho, C. M. P. Rodrigues,
A. S. Rodrigues, A. J. L. Pombeiro and A. R. Fernandes,
J. Biol. Inorg. Chem., 2014, 19, 787–803.
73 C. Marzano, M. Pellei, F. Tisato and C. Santini, Anticancer
Agents Med. Chem., 2009, 9, 185–211.
74 K. Choroba, B. Machura, S. Kula, L. R. Raposo,
A. R. Fernandes, R. Kruszynski, K. Erfurt, L. S. Shul, 78 L. J. Farrugia, J. Appl. Crystallogr., 2012, 45, 849–854.
This journal is © The Royal Society of Chemistry 2021
Dalton Trans., 2021, 50, 1845–1865 | 1865