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

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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,^aPedro V. Baptista, ^aRuben Valente, ^aM. Fátima M. Piedade,^b,c

a

M. Helena Garcia,^b,dTânia S. Morais

*

^b,dand 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.¹Diﬀerent strategies are cur-

rently being used to combat cancer development, including

surgery, radiotherapy or chemotherapy.^2,3Chemotherapeutic

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.⁴There is an urgent

need to develop new therapies that replace the drugs used cur-

rently in chemotherapy.^5–7Metal 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

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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.^6–9The

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 1978.¹⁰However, 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.^11–17Copper 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.^11,18–23Phosphane

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.^9,24,25The design of these complexes include phos-

phane ligands that can eﬃciently stabilize Cu(^I)-d¹⁰metal

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 ν_C–H, ν_CvNand ν_CvCstretching

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⁻¹and

sphano) ethane) and diﬀerent heteroaromatic bidentate 1390–1610 cm⁻¹, and the δ_C–Hbending vibrations in the

ligands. Eight complexes from the series were chosen for 690–700 cm⁻¹range. In the 1066–1099 cm⁻¹range and

investigation of their antiproliferative potential against breast ∼690 cm⁻¹were also observed the very broad and strong bands

and ovarian tumor cells, two types of cancer highly prevalent characteristic of ν_B–Fvibrations, 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-d₆solutions 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

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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)PPh₃complexes,^24,27and 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⁻³solutions 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 CH₂Cl₂molecule. 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.

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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°³⁰between 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.^24,28,29The 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 BF₄anion (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).

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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 IC₅₀of 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 IC₅₀for

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.²⁶Additionally, 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].

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patients⁸to 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 IC₅₀values in the parental sensitive HCT116 cells, the rela-

tive IC₅₀values 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†).¹³The 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†).¹³In 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†).¹³It is of

importance to note that the IC₅₀of 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 (IC₅₀between 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 (IC₅₀of 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 IC₅₀values are shown in Table 1. The most cytotoxic

complex is 6, with an IC₅₀in HCT116 cells at 48 h of 0.15 µM,

nevertheless, all these complexes seems to be very promising

due to their low IC₅₀in HCT116 cells compared to the IC₅₀of

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 IC₅₀values 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 IC₅₀values (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 (IC₅₀ﬁbroblasts/IC₅₀HCT116) is also presented (n.d. not calculated)

Cu(I) complexes, SC or ligands

IC₅₀HCT116

IC₅₀HCT116 DOXR

IC₅₀MCF7

IC₅₀A2780

IC₅₀Fibroblasts

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.

>6

0.50 0.04

0.80 0.05

0.82 0.09

0.95 0.1

n.d.

0.72 0.05

1.3 0.08

0.62 0.09

0.4 µM

n.d.

50

5

>50

30

10

40

3

2

1

6

SC ([Cu(dppe)(NCCH₃)₂][BF₄])

Dptyz

Dpp

Bipy

Me₂bpy

Cisplatin

Dox

n.d.

>50

n.d.

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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 IC₅₀in

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

green fluorescence.³²However, 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.³²With 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 IC₅₀of 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 IC₅₀of 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).

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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 (ΔΨ_m) 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 ΔΨ_m.

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 (ΔΨ_m)

The ΔΨ_mis essential to keep the respiratory chain functioning

correctly. A significant loss of ΔΨ_mcan 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 ΔΨ_m. 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 ΔΨ_m,

Nevertheless, to confirm that the loss of ΔΨ_mand 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 IC₅₀of 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 IC₅₀of 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).

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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).³⁵The 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.³⁶Previously 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.³⁹When 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.⁴²Being 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.⁴⁴As 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

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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 IC₅₀of 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 IC₅₀of complexes, 0.1% (v/v) DMSO or 25 µM

H₂O₂for 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-

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Fig. 11 Analysis of cell cycle progression in HCT116 cells after exposure to the IC₅₀of 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

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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 IC₅₀of 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). H₂O₂was 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.⁴⁶Its 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.⁴⁵The 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 IC₅₀of 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.

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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 synthesis⁴⁹that 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,⁵⁰and with the acquisition of drug

resistance,⁵¹is 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).^53,54The 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 ⁵⁵Diﬀerent oxidative stress response

proteins, such as PARK7,⁵⁶PRDX2 ⁵⁷and GSTP-1,⁵⁸also

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 damage⁴⁸in agreement with pDNA studies (Fig. 12 the complex.⁵⁹Likewise, 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 IC₅₀of 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

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colysis, is also under-expressed, indicating a low metabolic erature. After evaporation of the solvent under vacuum, the

activity.⁶⁰Finally, 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.⁶¹However, 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, H_para– PPh₃), 7.35 (br, 2H, H-2), 7.30 (m,

12H, H_meta– PPh₃), 7.18 (m, 12H, H_orto– 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 (C_orto-PPh₃), 133.14 (C_ipso

-

PPh₃), 131.06 (C_para-PPh₃), 129.77 (C_meta-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 (ν_C–H, phenyl

rings), 1610–1410 (ν_CvN, ν_CvC, phenyl rings), 1099 and

696 cm⁻¹(ν_B–F, BF₄), 700–690 (δ_C–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-d₆as 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, H_para

–

PPh₃), 7.29 (comp, 24H, H_meta+ H_orto– 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 (C_ipso-PPh₃), 134.09,

130.35 (C_orto+ C_meta-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 (C_para-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 (ν_C–H, phenyl

rings), 1600–1390 (ν_CvN, ν_CvC, phenyl rings), 1060 and 694

(ν_B–F, BF₄), 700–690 (δ_C–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 APEX⁶⁴program. Empirical absorption cor-

rection using SABADS⁶⁵was applied and data reduction was

done with the SMART and SAINT programs.⁶⁶SHELXT2014 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 ⁶⁷was 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 2014.1.⁶⁸Non-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

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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.⁶⁹The cells (human breast adenocarcinoma) and human primary

graphical representations were prepared using MERCURY fibroblasts were grown in Dulbecco’s modified Eagle’s

3.8.⁷⁰In 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. CO₂and 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 ×

10⁵cells 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.^24,25The

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.⁷¹All 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

C_56.71H_45.41BCl_1.41CuF₄N₄P₂

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 × 10⁵cells per mL in 25 cm²culture flasks. After

24 h in a controlled atmosphere, the medium was replaced

with fresh medium containing 10 × IC₅₀of 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

Dalton Trans., 2021, 50, 1845–1865 | 1859

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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 IC₅₀of 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 × 10⁵cells 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 IC₅₀of 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 (ΔΨ_m)

To measure mitochondrial potential (ΔΨ_m), 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).^21,22,72This probe has the ability to change its color due triplicates.

to alterations in mitochondrial potential.⁷³HCT116 cells were

All membranes were incubated stripping buﬀer 3 times for

plated in 24 well plates with a density of 0.75 × 10⁵cells 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 IC₅₀of 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.^22,74,75DCFH-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.⁷⁶HCT116 cells were seeded at 1 × 10⁵cells 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

IC₅₀of 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 cm²culture flasks with a density of 2 × 10⁶cells per ml. Following the incubation time, cells were washed with PBS

1860 | Dalton Trans., 2021, 50, 1845–1865

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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⁻¹Ampicillin. 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

× 10⁵cells 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.⁷⁷Each assay

the medium was replaced with fresh medium containing the was programmed to a final volume of 20 μL and all samples

IC₅₀of 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).⁴⁰were 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 × 10⁵cells of pUC18 was exposed to the complexes and with three scaven-

per ml in 25 cm²culture 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 H₂O₂was also performed.⁷⁸

by replacing the medium with thymidine with fresh medium

containing the IC₅₀of 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 cm²cell 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 IC₅₀of 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⁻¹RNase 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

Dalton Trans., 2021, 50, 1845–1865 | 1861

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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

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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,

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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.

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

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