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Rhenium(I) polypyridine complexes coordinated to an ethyl-isonicotinate ligand: Luminescence and in vitro anti-cancer studies
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Research paper
Rhenium(I) polypyridine complexes coordinated to an ethyl-isonicotinate li-
gand: Luminescence and in vitro anti-cancer studies
Luiz D. Ramos, Giselle Cerchiaro, Karina P. Morelli Frin
PII: S0020-1693(19)31294-0
DOI: https://doi.org/10.1016/j.ica.2019.119329
Reference: ICA 119329
To appear in: Inorganica Chimica Acta
Received Date: 31 July 2019
Revised Date: 29 November 2019
Accepted Date: 29 November 2019
Please cite this article as: L.D. Ramos, G. Cerchiaro, K.P. Morelli Frin, Rhenium(I) polypyridine complexes
coordinated to an ethyl-isonicotinate ligand: Luminescence and in vitro anti-cancer studies, Inorganica Chimica
Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119329
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© 2019 Published by Elsevier B.V.
1
Rhenium(I) polypyridine complexes coordinated to an ethyl-
isonicotinate ligand: Luminescence and in vitro anti-cancer studies
Luiz D. Ramos, Giselle Cerchiaro and Karina P. Morelli Frin1*
1Federal University of ABC - UFABC, Av. dos Estados 5001, Santo Andre-SP – Brazil
*e-mail: karina.frin@ufabc.edu.br
Abstract
In this work, fac-[Re(et-isonic)(NN)(CO) ]+ complexes, et-isonic = ethyl-
3
isonicotinate, NN = 1,10-phenanthroline (phen), 4,7-diphenyl-1,10-phenanthroline (Ph phen),
2
4,7-dichloro-1,10-phenanthroline (Cl phen) or 4,7-dimethyl-1,10-phenanthroline (Me phen),
2 2
were synthesized to combine different substituent groups at coordinate phen ligand along with
the coordination of a nicotinic acid derivative aiming the modulation of the photophysical and
lipophilic properties. The electronic absorption spectra profile can be divided into two main
regions: the high-energy region ascribed to the intraligand transition (IL), and the low-energy
region assigned to the metal-to-ligand charge transfer transition (MLCT). Additionally, the
complexes exhibited 3MLCT emission, sensitive to the nature of NN ligand, which can be
used to photosensitize the generation of singlet oxygen as well as to localize them into the
cell. The lipophilicity of the complexes increases as the substituent at coordinated phen ligand
was changed from hydrogen to phenyl and these results reflected also in the same trend
observed for the cytotoxicity results, in the absence of light, using breast cancer (MCF-7) and
melanoma (SkMel-147 and SkMel-29) cell lines. In addition, the flow cytometry assay along
with the Western Blotting analyses showed an overexpression on pro caspase-9, suggesting a
Caspase proteolytic cascade through the intrinsic pathway in the apoptosis as the mechanism
of action. In spite of the Re(I) complexes were capable of generating singlet oxygen in the
presence of light, they were very effective in killing the cells by a different mechanism of
action in the absence of light. Thus, these results provided a better understanding of the
mechanism of anticancer action and highlight the potential application of rhenium(I)
complexes in the development of a novel therapy process.
2
1. Introduction
Since the first investigation reported by Wrighton and Morse on the interesting
photochemical and photophysical properties of rhenium(I) polypyridine complexes [1] , the
study on these systems deals with experimental and theoretical elucidations. Although there
has been much discussion on these systems, there is still interesting in understand and
rationalize many aspects of their structures and luminescence properties[2-5] thereby this
class of compounds can be used in the CO photoreduction [6-10] , in the development of
2
luminescent probes [11-13] , as possible agents in cancer treatment [14-18] and in
photogeneration of singlet oxygen [19-24] .
Their interesting properties derive from the charge transfer transition and all the
process evolving from the excited state manifold [25-27] , which can be modulated by small
changes in the ligand structure. For instance, electron donor groups increase the energy gap
between frontiers orbitals resulting in hypsochromic shifts on the absorption and emission
spectra [26, 27] ; while electron-withdrawing groups decrease the energy gap leading in the
opposite behavior[25] . Additionally, the exchange of the axial ligand of the Re(I) complexes
has a strong influence on the deactivation of the excited states[27, 28] . Therefore, these
excited state modulations can be used to improve luminescence efficiency and
photogeneration of singlet oxygen.
An emerging area for Re(I) complexes is their use in biological systems. At first, the
interest was based on their use as a luminescent probe and localization in biological systems
since the reaction of substitution of the coordinated ligand in biological system [29-32] was
difficult to occur. Then, some groups[19-24] reported the ability of the Re(I) complexes to
photosensitize the generation of singlet oxygen, which can be used in photodynamic therapy.
Since these complexes exhibited an absorption in a region where the penetration of
electromagnetic energy into biological tissues is low, the investigation involving their toxicity
in the absence of light excitation[33-39] arisen. The mechanisms that lead to cell death using
Re(I) complexes are still unclear and there is only a few investigations reported[18, 34, 40-42]
. Therefore, the comprehension of the mechanisms involved after the administration of the
Re(I)-complex is crucial to define its use in cancer treatments.
In earlier works, we reported the interesting photophysical properties of rhenium(I)
complexes bearing electron-withdrawing[25, 43] and donating[26] groups until the singlet
manifold[44] became also evident in spite of the presence of the heavy atom effect imparted
3
by the metal center. More recently, we proceeded with the use of Re(I) triplet-emitters as a
photosensitizer for the generation of singlet oxygen[22] . All this has motivated the
engineering of new Re(I) compounds to extend and endeavor the investigation also in
biological systems. Thus, in this work, novel Re(I) compounds, fac-[Re(et-
isonic)(NN)(CO) ]+ (et-isonic = ethyl-isonicotinate; NN = 1,10-phenanthroline (phen), 4,7-
3
dichloro-1,10-phenanthroline (Cl phen), 4,7-dimethyl-1,10-phenanthroline (Me phen), or 4,7-
2 2
diphenyl-1,10-phenanthroline (Ph phen)), scheme 1, were synthesized to combine the use of
2
different substituent groups at coordinate phen ligand and a nicotinic acid derivative, which is
known to have biological effects[45, 46] , to modulate the photophysical and lipophilic
properties. Then, their cellular uptake was investigated along with their cytotoxicity.
Additionally, the mode of cell killing was evaluated by flow cytometry and by the protein
expression through Western Blotting assay.
O O
R
H
N
CH
OC N 3
Re Cl
OC
N
CO
R
Scheme 1. Structure of polypyridine Re(I) complexes
2. Materials and methods
2.1. Materials
All solvents used in this work were reagent grade and HPLC grade for photophysical
measurements. [ReCl(CO) ], ethyl-isonicotinate (et-isonic), 1,10-phenanthroline (phen), 4,7-
5
dichloro-1,10-phenanthroline (Cl phen), 4,7-diphenyl-1,10-phenanthroline (Ph phen), 4,7-
2 2
dimethyl-1,10-phenanthroline (Me phen), Methylthiazolyldiphenyl-tetrazolium bromide
2
(MTT), RPMI-1640 medium, recombinant human insulin, penicillin/streptomycin were
purchased from Sigma-Aldrich. Dulbecco’s Modified Eagle’s Medium - low glucose
4
(DMEN) and Fetal Bovine Serum (FBS) were purchased from Gibco. Dead Cell Apoptosis
Kit with Annexin V/FITC and propidium iodide (PI) was purchased from Invitrogen®.
2.2. Syntheses of Re(I) complexes
fac-[ReCl(NN)(CO) ] and fac-[Re(tfms)(NN)(CO) ] compounds were available from
3 3
previous investigations[22, 25, 26, 43, 44] .
The synthesis of fac-[Re(et-isonic)(NN)(CO) ]PF complexes were performed as
3 6
described in the literature [25, 26] . Briefly, fac-[Re(tfms)(NN)(CO) ] compound and a small
3
excess of et-isonic were mixture in MeOH and kept at reflux and inert atmosphere for 8h.
Then, the solvent volume was reduced, solid NH PF was added and stirred vigorously. The
4 6
solid fac-[Re(et-isonic)(NN)(CO) ]PF complexes were collected by filtration and washed
3 6
with cold water and ethyl ether.
fac-[Re(et-isonic)(phen)(CO) ]PF . Yield 74%. Elemental Anal. for C H F N O PRe
3 6 23 17 6 3 5
(Calc./Found): C(37.00/37.08); N(5.63/5.34); H(2.30/2.23). 1H NMR (MeCN d-3, 500 MHz,
δ/ppm): 9.61 (dd, 2H, J =5.1; 1.2); 8.84 (dd, 2H, J = 8.3; 1.2); 8.43 (dd, 2H, J = 5.2; 1.2); 8.16
(s, 2H); 8.12 (dd, 2H, J = 5.1; 8.3); 7.60 (dd, 2H, J = 5.2; 1.2); 4.25 (q, 2H, J = 7.1 ); 1.24 (t,
3H, J = 7.1 ). FTIR/UATR ν(CO) (cm-1): 2030; 1930; 1910 .
fac-[Re(et-isonic)(Me phen)(CO) ]PF . Yield 65%. Elemental Anal. for
2 3 6
C H F N O PRe (Calc./Found): C(38.76/38.42); N(5.42/5.06); H(2.73/2.75). 1H NMR
25 21 6 3 5
(MeCN d-3, 500 MHz, δppm): 9.43 (d, 2H, J = 5.4); 8.41 (dd, 2H, J = 5.2; 1.2); 8.29 (s, 2H);
7.93 (d, 2H, J = 5.2); 7.58 (dd, 2H, J = 5.4); 4.25 (q, 2H, J = 7.1); 2.93 (s, 6H); 1.24 (t, 3H, J =
7.1). FTIR/UATR ν(CO) (cm-1): 2027; 1910.
fac-[Re(et-isonic)(Ph phen)(CO) ]PF . Yield 30%. Elemental Anal. for
2 3 6
C H F N O PRe (Calc./Found): C(46.62/46.60); N(4.66/4.70); H(3.13/2.77). 1H NMR
35 25 6 3 5
(MeCN d-3, 500 MHz, δ/ppm): 9.64 (d, 2H, J = 5.4); 8.52 (dd, 2H, J = 5.2; 1.2); 8.07 (s, 2H);
8.05 (d, 2H, J = 5.4); 7.65 (m, 12H); 4.27 (q, 2H, J = 7.1); 1.26 (t, 3H, J = 7.1). FTIR/UATR
ν(CO) (cm-1): 2029; 1902.
fac-[Re(et-isonic)(Cl phen)(CO) ]PF . Yield 60%. Elemental Anal. for
2 3 6
C H Cl F N O PRe (Calc./Found): C(33.88/33.45); N(5.15/4.87); H(1.85/1.74). 1H NMR
23 15 2 6 3 5
(MeCN d-3, 500 MHz, δ/ppm): 9.52 (d, 2H, J = 5.6); 8.51 (s, 2H); 8.40 (d, 2H, J = 6.5); 8.24
5
(d, 2H, J = 5.6); 7.61 (d, 2H, J = 6.6); 4.26 (q, 2H, J = 7.1); 1.25 (t, 3H, J = 7.1). FTIR/UATR
ν(CO) (cm-1): 2037; 1930; 1910.
2.3. Methods
The absorption spectra were carried out on an 8453 spectrometer (Agilent) and the
emission spectra were obtained on a Cary Eclipse spectrometer (Varian). The infrared spectra
were carried out on a Spectrometer Spectrum Two (Perkin Elmer). Proton nuclear magnetic
resonance spectra (1H NMR) were obtained on a 500/54/ASP spectrometer (Varian) at 298 K
using CD CN as a solvent. Residual solvent signals were employed as an internal standard.
3
Elemental analyses were performed in a Flash EA 1112 (Thermo Scientific).
The geometry optimizations were performed using the density functional theory using
the Gaussian09 package with the B3LYP/LANL2DZ basis set. The solvation in acetonitrile
was performed by the IEFPCM method with default in Gaussian03.
Steady-state emission spectra at room temperature were obtained in a 1.00 cm optical
length quartz cuvette and were not corrected for the photomultiplier spectral response.
Emission quantum yields (ϕ) of rhenium(I) compounds were determined as already described
[27] , using fac-[ReCl(CO) (phen)] (ϕ = 0.017) [28] , [Ru(bpy) ]2+ (ϕ = 0.062) [47] or fac-
3 3
[Re(py)(CO) (bpy)] (ϕ = 0.059) [48] as standards in a deoxygenated acetonitrile solution and
3
keeping their absorbance at excitation wavelengths between 0.1 and 0.2.
Phosphorescence decay times were measured using a time-correlated single-photon
counting (TCSPC) setup in a PicoQuant FluoTime 300 fluorescence lifetime spectrometer,
using as an excitation source a 375 nm diode laser (LDH-P-C-375B, 40 MHz repetition rate,
52 ps pulse width, PicoQuant GmbH) driven by a PDL 820 computer controller. The emission
wavelengths were isolated using a UV-visible monochromator. The solutions were degassed
for 30 min using an argon atmosphere and care was taken to avoid any aggregation effect by
using a very dilute solution.
The generation of singlet oxygen was monitored at 1270 nm using a Hamamatsu Near
Infrared (NIR) photomultiplier in a PicoQuant FluoTime 300 fluorescence lifetime
spectrometer using as an excitation source a 375 nm diode laser (LDH-P-C-375B, 40 MHz
repetition rate, 52 ps pulse width, PicoQuant GmbH) driven by a PDL 820 computer
controller. The solutions were air equilibrated and the quantum yields of singlet oxygen were
6
determined at room temperature using [Ru(bpy) ]2+ (0.56 in CH CN) as the standard[23, 49]
3 3
according to eq. 1[19, 21, 22] , where I and A are the integrated emission intensity and the
absorbance in the excitation wavelength, respectively, of the standard and sample.
∫I A
sam sd
ϕ = . .ϕ (1)
Δ ∫I sd A sam Δ 𝑠𝑑
The breast cancer cell (MCF-7) were obtained from ATCC and was incubated in
RPMI-1640 medium that was supplemented with 10% fetal bovine serum (Gibco), 10.0
μgmL-1 recombinant human insulin, 100 UmL-1 penicillin and 10.0 μgmL-1 streptomycin.
The melanoma cells (SkMel-147 and SkMel-29) were incubated in DMEM low glucose
medium supplemented with 10% fetal bovine serum, 100 UmL-1 penicillin and 10.0 μgmL-1
streptomycin. The cells were cultured in a humidified incubator at 37 °C under 5% CO . The
2
optical density of cells to perform the experiments was 4x104 cellscm2. The reference group
to each experiment was the cell treated with the vehicle 1% DMSO (v/v).
For fluorescence microscopy experiment, the cells were placed directly on a
microscope coverslip and treated with 50 μmolL-1 of Re(I) complexes for 15 min. After this
time the coverslip was rinsed with phosphate buffer and incubated with 4% (v/v)
paraformaldehyde solution for 15 min, then a rinse was performed and 500 mmolL-1 of DAPI
(nucleus marker) was added and incubated for 30 min. The images were obtained in a
Fluorescence Microscope AF6000 (Leica) using the appropriate filters.
The IC was performed using the mitochondrial viability assay by
50
Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cells were cultured in 96-
well plates and the Re(I) complexes (concentration range of 0.5 – 100 μmol.L-1) in 1% DMSO
(v/v) were added to the wells. After the treatment time, the MTT solution (5 mg.mL-1) was
added to each well and incubated for 1h. The formazan crystals were dissolved in 150 μL of
DMSO and the absorbance was registered using a microplate reader (Celer, Polaris) at 570
nm. The results were normalized by control and plotted using the eq. 2 (GraphPad Prism 7)
where the slope is the non-linear regression, Y is the percentage of cell viability and X is the
logarithm of complexes concentration.
100
Y= (2)
(1+10
((log𝐈𝐂𝟓𝟎―X) . Slope)))
The determination of lipophilicity (Rw) was performed as described in the literature
[50] , with modifications, using the thin reverse-phase chromatography C18 silica (SorbTech)
7
as the stationary phase, and a mixture of MeOH:DMF:PBS (PBS = phosphate-buffered saline,
10 mM, pH 7.4) at 3:1:2 proportion as the mobile phase. The R values were obtained using
w
eq. 3, where R is the retention factor of the complexes.
f
[(1) ]
R =log ―1 (3)
w 10 R
f
The flow cytometry assay was performed according to the procedure described in the
Dead Cell Apoptosis kit protocol (InvitroGen – Thermo Fisher Scientific). After the treatment
time, the cells were collected, washed with an appropriate buffer, and incubated with
Annexin/FITC and PI. The dot plot graphs were recorded on a flow cytometer BD FACS
Canto II (BD Biosciences) monitoring 530 nm (FITC) and 585 nm (PI) wavelengths.
Protein expression was investigated by Western Blotting assay after a 24h treatment
period. In each experiment, the cells were collected, washed, and resuspended in RIPA buffer
(Radio Immuno Precipitation Assay buffer: 150 mM NaCl, 5 mM EDTA, 1 mM
dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS in 50 mM TBS
(Tris-HCl buffered saline 50 mM at pH 7.5)) containing protease inhibitor cocktail (Sigma
Aldrich) for cells, and incubated for 30 min in an ice bath. The cells were centrifuged (14000
rpm) for 20 min at -15 oC. Supernatants protein concentrations were determined according to
the Lowry method [51] . The proteins were submitted to SDS-PAGE electrophoresis and
transferred to nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were
blocked for 1.5 h in a 10% (m/v) blocking solution (nonfat-dried milk in TBS-T buffer (50
mM TBS at pH 7.5 and 0.05% Tween-20)). The membranes were incubated at room
temperature for 5 h with the primary antibodies, washed, then incubated for 2h with the
secondary antibodies, and washed again. The primary antibodies used were anti-α tubulin at
1:1000 dilution (Sigma Aldrich), anti-caspase 9 at 1:1000 dilution (Sigma Aldrich) and anti-
caspase-3 at 1:500 dilution (Sigma Aldrich). Secondary antibodies used were anti-rabbit and
anti-mouse at 1:10000 dilutions; both were purchased from Sigma Aldrich. The proteins
bands were detected using SuperSignal West Fento Maximum Sensitivity Substrate (Thermo
Fisher Scientific) at a ChemiDoc photodocumentator (Bio-Rad). The densitometry analysis of
the target protein band was always performed in relation to the densitometry of the loading
protein band. The statistical analysis was performed using the Two Way Analysis of Variance
followed by Bonferroni post-tests.
8
3. Results and discussion
All syntheses were performed according to the procedures described in the
literature[25, 26, 43, 44, 52, 53] , with changes when necessary. The yields of the syntheses
were satisfactory and similar to the reported ones for other Re(I) polypyridine complexes[22,
44] . All complexes were characterized by 1H NMR and FTIR spectroscopies (presented as
ESI: Figures S1-S4 and Tables S1-S4) and gave satisfactory elemental analyses.
The absorption spectra for fac-[Re(et-isonic)(NN)(CO) ]+, Figure 1,exhibit electronic
3
transitions occurring between orbitals of the NN ligands – intraligand transitions (IL - π →
NN
π *) at higher energy region. Transitions involving orbitals from metal to the polypyridine
NN
ligand - Metal-to-Ligand Charge Transfer Transition (MLCT - dπ → π *) are observed at
Re NN
low energy region. Additionally, it is possible to observe another band at 325 nm region for
the complexes coordinated with the et-isonic ligand, which can be ascribed to the metal to the
et-isonic ligand charge transfer transition (dπ → π *) [22, 44] .
Re et-isonic
5
4
3
2
1
0
200 250 300 350 400 450 500
1-
1-
4
)
mc.
lom.L
01(
nm)
Figure 1: UV-Vis spectra of fac-[Re(et-isonic)(NN)(CO) ]+ complexes in CH CN at room
3 3
temperature. NN = phen (−); Me phen (---); Ph phen (…); Cl phen (-.-).
2 2 2
The density functional theory was employed to obtain additional information about the
molecular orbitals involved in the main transitions of fac-[Re(et-isonic)(NN)(CO) ]+, Figure 2
3
for NN = phen and for the other complexes are shown in the ESI Figures S6-S8. The Highest
Occupied Molecular Orbitals (HOMO) consist mainly of contributions from the metal Re(I),
whereas, the Lowest Unoccupied Molecular Orbitals (LUMO) have contributions mainly of
the diimine ligand and the et-isonic ligand for the fac-[Re(et-isonic)(NN)(CO) ]+ complexes.
3
9
Figure 2: Energy level and orbital surface for frontiers orbitals, calculated by TD-DFT
(B3LYP/LANL2DZ; IEFPCM = CH CN).
3
Additionally, comparing the experimental spectra of the complexes with the
theoretical results, it is possible to observe that the hypsochromic shift of the MLCT band is
due to the change of Cl to the et-isonic ligand. This behavior can be ascribed to the acceptor
characteristics of the et-isonic ligand, which increases the energy gap between the HOMO and
LUMO orbitals, Figure 3.
10
Cl Cl
N N N N N N N N
-1.0eV -1.0eV
N N N N N N N N
-2.0eV Cl -2.0eV Cl
-3.0eV -3.0eV
-4.0eV V e V e Cl V e V e -4.0eV O O
-5.0eV 6 8 3 8 . 6 3 7 8 . O O C C Re N N 0 0 7 3. 7 4 5 3. -5.0eV 9 e V 9 3 7 . V 8 e 9 3 8 . OC N N 2 V . 7 e 7 3 3 7 6 V . 3 e
CO Re
-6.0eV -6.0eV OC N
CO
-7.0eV -7.0eV
Figure 3: The energy level of the frontiers orbitals, calculated by TD-DFT
(B3LYP/LANL2DZ; IEFPCM = CH CN).
3
The emission spectra for all complexes in fluid solution exhibited a broad and
unstructured emission band, Figure 4, which is ascribed to the deactivation of the low-lying
3MLCT(dπ → π *) excited state [12, 54, 55] . The change of the NN ligand shows great
Re NN
influence on the emission maxima of the complexes with a bathochromic shift as the electron-
withdrawing character of the substituent attached to phen increase. This shift is ascribed to the
electron-withdrawing character of the group attached to phen, which tends to decrease the
energy gap of frontiers orbitals of the complexes. The opposite behavior to the emission band
is observed for Me phen coordinated complex, with a hypsochromic shift due to the presence
2
of an electron-donor group attached to phen, which increases the energy gap when compared
to the non-substituted phenanthroline complex. Additionally, the exchange of the chlorine
ligand (see at ESI, Figure S9) by the et-isonic one, Table 1, shifts the emission maxima to the
blue side, due to the replacement of a π-donor group to a π-acceptor group which increases
the energy gap between the HOMO-LUMO orbitals [56, 57] .
11
400 500 600 700 800
ytisnetnI
dezilamroN
nm)
Figure 4: Emission spectra of fac-[Re(et-isonic)(NN)(CO) ]+ complexes in CH CN at room
3 3
temperature. NN = phen (−); Me phen (---); Ph phen (…); Cl phen (-.-).
2 2 2
The influence on the energy gap (E values) imparted by the substituents attached to
00
the coordinated phen ligand can be seen in Table 1.
Table 1: Emission properties of Re(I) complexes in CH CN at room temperature.
3
λ k /106 k/104
Compounds max (ϕ ) E (eV) τ (ns) nr r
(nm) em 00 (s-1) (s-1)
fac-[ReCl(phen)(CO) ] 602 0.017±0.005 2.66 147±2 6.69 11.57
3
fac-[ReCl(phen)(CO) ]*[28] 600 0.017 183
3
fac-[Re(et-isonic)(phen)(CO) ] + 548 0.22±0.05 2.72 1634±133 0.48 13.46
3
fac-[ReCl(Me phen)(CO) ] 584 0.032±0.004 2.64 395±15 2.45 8.10
2 3
fac-[ReCl(Me phen)(CO) ]*[26] 587 0.024 350
2 3
fac-[Re(et-isonic)(Me phen) (CO) ] + 530 0.31±0.04 2.73 3029±446 0.23 10.24
2 3
fac-[ReCl(Cl phen)(CO) ] 641 0.0025±0.0002 ** 29±1 34.40 8.62
2 3
fac-[ReCl(Cl phen)(CO) ]*[25] 640 0.0027
2 3
fac-[Re(et-isonic)(Cl phen) (CO) ] + 584 0.05±0.01 2.60 505±7 1.88 9.90
2 3
fac-[ReCl(Ph phen)(CO) ] 611 0.018±0.003 2.56 250±13 3.94 7.21
2 3
fac-[Re(et-isonic)(Ph phen)(CO) ] + 558 0.22±0.04 2.67 4678±108 0.17 4.70
2 3
* these compounds have already been reported in the literature in the same condition. ** it was not possible to determine an intersection
between emission and excitation spectra
The change of chlorine π-donor axial ligand to et-isonic π-acceptor ligand increases
the energy gap as well as the quantum yield. Additionally, all emission decays showed a
monoexponential treatment, which indicates only one emissive excited state. These behavior
12
corroborates with the determined E values and is in accordance with the energy gap law,
00
which states that the larger the energy gap, the greater emission quantum yield and longer
lifetimes [58] . On the other hand, it is possible to observe that all complexes dissipate the
energy preferably by non-radiative pathways (k values are two orders of magnitude greater
nr
than the k) as it was already observed for similar rhenium(I) polypyridyl complexes [43] .
r
Since these complexes are triplet emitters, they can be used as photosensitizers for
singlet oxygen [19, 20, 24] . Thus, the energy of the complexes acquired from an excitation
can be quenched in the presence of molecular oxygen, resulting in the generation of singlet
oxygen species, which can be monitored by its luminescence at 1270 nm. The Re(I)
complexes presented an efficiency ranging from 30 - 50% in the photogeneration of singlet
oxygen, Table 2, which are similar to the values obtained for other Re(I) complexes [22] .
Table 2: Singlet oxygen quantum yields for Re(I) complexes in an acetonitrile solution.
Compounds (Φ )
Δ
fac-[ReCl(Me phen)(CO) ] 0.35 ± 0.03
2 3
fac-[Re(et-isonic)(Me phen)(CO) ]+ 0.46 ± 0.04
2 3
fac-[ReCl(phen)(CO) ][22] 0.35 ± 0.04
3
fac-[Re(et-isonic)(phen)(CO) ]+ 0.42 ± 0.07
3
fac-[ReCl(Ph phen)(CO) ] 0.42 ± 0.06
2 3
fac-[Re(et-isonic)(Ph phen)(CO) ]+ 0.50 ± 0.05
2 3
fac-[ReCl(Cl phen)(CO) ] 0.33 ± 0.06
2 3
fac-[Re(et-isonic)(Cl phen)(CO) ]+ 0.45 ± 0.05
2 3
Additionally, the luminescent properties of these complexes allow their detection by
fluorescence microscopy11c, 25b, 28 in MCF-7 (Figure 5), SkMel-147 (ESI Figure S10) and
SkMel-29 (ESI Figure S11) cell lines. Whereas negligible or no uptake by the nucleus is
observed, it is possible to observe that the cytoplasm of the cells becomes red suggesting that
a certain amount of complex is compartmentalized in some organelle, since its distribution is
not uniform all over the cell.
13
Figure 5: Microscopy fluorescence of Re(I) complexes (50 μmol.L-1) for 15 min in MCF-7
cell line. (1) fac-[Re(et-isonic)(phen)(CO) ]+; (2) fac-[Re(et-isonic)(Me phen)(CO) ]+; (3)
3 2 3
fac-[Re(et-isonic)(Ph phen)(CO) ]+.
2 3
Although these complexes are capable to photosensitize the generation of singlet
oxygen to be used in photodynamic therapy, their absorption bands are blue shifted, and
located in a different region from the phototherapeutic window, which are not suitable for this
purpose. Thus, in an effort to understand the process involved after the administration of Re(I)
complexes and their anticancer mechanism, the following investigations were performed in
the absence of light.
The molecular structure and the charge of a compound can affect its cellular uptake
localization, cytotoxicity, and anticancer action mechanisms as can be found in related
reported works[59-61] . Therefore, the lipophilicity is very important to compounds that will
be used in a biological system, since this feature seems to be related to the cytotoxic capacity
of a compound. The lipophilicity of the Re(I) complexes was determined using reverse-phase
TLC, where the stationary phase (silica C18) mimic lipids of the biological membrane, and
the mobile phase (MeOH:DMF:PBS (pH7.4) – 3:1:2 v/v/v) simulates a polar media. As
expected, the lipophilicity of the complexes increases as the substituent at coordinated phen
ligand changes from hydrogen to phenyl, Table 3, and these results reflect also in the same
trend in the cytotoxicity results.
14
In an attempt to elucidate the role of the rhenium(I) polypyridine compounds against
human cell lines such as breast cancer (MCF-7), and melanoma (SkMel-147 and SkMel-29),
the cytotoxicity of the complexes was determined by the MTT assay, Table 3. The results
show that the complexes exhibit IC values in the order of 10-6 mol.L-1 for the three cell lines
50
investigated. The MCF-7 cells were more resistant to the fac-[Re(et-isonic)(phen)(CO) ]+ and
3
fac-[Re(et-isonic)(Me phen)(CO) ]+ treatments, and showed no significant differences
2 3
between the other two complexes. Additionally, the substitutions in coordinated
phenanthroline resulted in higher cytotoxic effect than cisplatin with 24 h treatment (IC > 50
50
µmol.L-1 [33, 42] ). It is important to note that the complex [ReCl(CO) ] (IC = 231 µmol.L-
5 50
1), and the free ligands phen (IC = 286 µmol.L-1) and et-isonic (IC > 100 µmol.L-1) are far
50 50
less effective as treatments for MCF-7 cells. Higher cytotoxicity trend was achieved using the
complexes as treatment of two Melanoma cancer cell lines although it seems that the
lipophilicity has little or no influence for these cell lines.
Table 3: Lipophilicity and IC values of tested Re(I) complexes toward different cell lines.
50
IC (µmol.L-1)
50
Compounds R
w
MCF-7 SkMel-147 SkMel-29
fac-[Re(et-isonic)(phen)(CO) ]+ 86 ± 1 19 ± 1 19 ± 1 0.07
3
fac-[Re(et-isonic)(Me phen)(CO) ]+ 29 ± 1 9.9 ± 1.1 8.3 ± 1.0 0.26
2 3
fac-[Re(et-isonic)(Cl phen)(CO) ]+ 4.6 ± 1.1 2.8 ± 1.2 2.2 ± 1.2 0.30
2 3
fac-[Re(et-isonic)(Ph phen)(CO) ]+ 2.4 ± 1.1 3.3 ± 1.1 3.5 ± 1.1 1.06
2 3
Moreover, only for the non-charge fac-[ReCl(Ph phen)(CO) ] compound it was
2 3
possible to obtain the IC values (IC for MCF-7 = 8.7 ± 1.1; SkMel-147 = 6.1 ± 1.1; and
50 50
SkMel-29 = 4.6 ± 1.1), for the others fac-[ReCl(NN)(CO) ] compounds the low solubility in
3
aqueous media did not allow to obtain IC values. Nevertheless, when comparing the
50
complexes with the same NN ligand it can be observed that the fac-[ReCl(Ph phen)(CO) ]
2 3
complex has a little lower liposolubility (Rw = 1.00) than fac-[Re(et-isonic)(Ph phen)(CO) ]+,
2 3
and slightly less cytotoxicity for the investigated cell lines than the positively charged-
complex. Thus, it seems that this is an important parameter, but to better establish a linear
correlation between lipophilicity and cytotoxicity, a larger series of structurally related
compounds should be considered. Notwithstanding, taking into account the results presented,
15
it can be noted that the coordination of appropriated ligands into the rhenium(I) metal center
seems to play an important role.
In order to obtain a more complete comprehension of the cellular response induced by
Re(I) complexes, their cell death mechanism was evaluated. Necrosis and apoptosis are two
different types of death, which imply different responses in biological systems. While
apoptosis has as its main in generating apoptotic bodies to be phagocytosed, in the necrosis
occurs the cytoplasmic membrane fragmentation, and generating a local inflammatory
response[62-65] . Due to the biochemical differences between these two processes, it is
possible to identify the mechanism of action in cell death. The apoptotic cells undergo
morphological changes and as a form of signaling to neighbor cells, the phosphatidylserine is
externalized. Since phosphatidylserine is capable of binding the Annexin V protein
conjugated to the FITC fluorophore, they are used as cell markers in the apoptotic process. On
the other hand, when the cell is in the process of death via necrosis the integrity of the
cytoplasmic membrane is lost and the PI can act as a marker of necrosis acting in the nucleus
of the cell. Thus, using a dual Annexin V/FITC and PI flow cytometry assay we explored the
occurrence of apoptosis in MCF-7, Skmel147 and Skmel29 cell lines treated for 24 h with
Re(I) compounds, Figure 6 and Figures S12-S14 at ESI.
16
100 100
t n MCF-7 t n SkMel-147
a r 80 *** a r 80 * **** ***
d *** d
a *** a
u ** u
q
r e
p
60 *** **
*******
q
r e
p
60
***
***
s 40 *** s 40
*
lle lle
c c
f 20 ns f 20 ns
o ns o
% % ns
0 0
ntr ol A n n + +/ PI + PI + ntr ol A n n + +/ PI + PI +
o n o n
C n C n
A A
100
t n SkMel-29
a r d 80 *** * **
a
u
q 60
r e *** *** ******
p ***
s 40
lle * *
c
f 20 ns
o ns
%
0
ntr ol A n n + +/ PI + PI +
o n
C n
A
Figure 6: Results on percentage of cells per quadrant obtained by flow cytometry incubated
for 24h with Re(I) complexes in the IC concentration. (ns = non-significant; Control =
50
viable cells; Ann+ = cells in apoptosis; Ann+/PI+ = cells in late apoptosis and PI+ = cells in
necrosis). ▌ control; ▌ fac-[ReCl(Ph phen)(CO) ]+; ▌ fac-[Re(et-isonic)(phen)(CO) ]+; ▌ fac-
2 3 3
[Re(et-isonic)(Me phen)(CO) ]+; ▌ fac-[Re(et-isonic)(Ph phen)(CO) ]+; ▌ fac-[Re(et-
2 3 2 3
isonic)(Cl phen)(CO) ] +. * p < 0.001; ** p < 0.01; *** p < 0.05; ns p > 0.05.
2 3
It is observed a shift of cell populations from control to the Ann+ quadrant for all
tumor cell lines used after 24 hours of treatment and just a few or non-significant population
on Ann+/PI+ and PI+ quadrants, thereby indicating that these cells were in the process of
death by apoptosis. Although, among the complexes the results do not present statistical
significance, between the complexes and the control it is possible to observe such
significance. This suggests that there is no compound that induces a process of cell death by
17
apoptosis more pronounced than another does since all of the experiments were done treating
the cells at the IC concentration.
50
The Western Blotting analyses can be used to establish if a determined protein has a
more pronounced expression than in its basal metabolism, thus once the expression or non-
expression of a certain protein has been identified, it is possible to begin designing the route
of the Re(I) complexes within the cells. The Western Botting assays were employed to
establish if the treatment with Re(I) compounds for 24 h in MCF-7 and SkMel-29 cell lines
exerted any influence on the basal metabolism, Figure 7.
Figure 7: Representative of three different Western Blotting assays. The cells were incubated
for 24 h with the complex prior to performing the experiment. The results were plotted by
normalizing the protein densitometry of interest by the loading protein and then normalized
by the cell control. The α-tubulin protein was used as load control. ▌cell control; ▌ fac-
[Re(et-isonic)(phen)(CO) ]+; ▌ fac-[Re(et-isonic)(Me phen)(CO) ]+; ▌ fac-[Re(et-
3 2 3
isonic)(Ph phen)(CO) ]+; ▌ fac-[Re(et-isonic)(Cl phen)(CO) ] +.
2 3 2 3
It was observed higher expressions of pro-Caspase-9 and Caspase-3 proteins in MCF-
7 cells, and expressions of pro-Caspase-9 in SkMel-29 cell line when compared with control
experiments (cells without complexes), which suggests that the complexes trigger the intrinsic
pathway in the apoptosis. The caspases are frequently envisaged as a key component
responsible for apoptosis. This result is in accordance with the flow cytometry assay, which
also showed death by apoptosis to the cell lines. The pro-Caspase-9 overexpression presented
18
suggests that for MCF-7 and SkMel-29 cells the death is dependent on Caspase proteolytic
cascade through the intrinsic pathway.
4. Conclusion
In this contribution, the fac-[Re(et-isonic)(NN)(CO) ]+ complexes were synthesized
3
and characterized. The complexes exhibited broad and non-structured emission arising from
the 3MLCT low-lying excited state which is sensitive to the nature of the NN ligand. This
triplet excited state can be used to photosensitize the singlet oxygen, whereas in the case of
Re(I) complexes the drawback is their lack of absorption in the biological optical window and
consequently low tissue penetration to be used as new agents for photodynamic therapy.
Nevertheless, they exhibited anticancer activity against MCF-7, SkMel-147 and SkMel-29
cancer cells in vitro, without light exposure, showing that the NN and axial ligand structure
exert great influence on the cytotoxic activity, which in some cases resulted in more active
treatment than cisplatin compound. In addition, the complexes induce the cancer cell death by
apoptosis, triggering the intrinsic pathway by the overexpression of Caspase-9. Overall, our
findings provide a better understanding of the anticancer mechanism and contribute to the
design of new rhenium(I) complexes for developing more promising anticancer treatments.
5. Acknowledgments
The authors would like to acknowledge financial support from Brazilian agency
Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP/Grant 2017/18063-0,
2016/09652-9 and 2018/14152-0), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES) for financial support and Multiuser Central Facilities for experimental
support.
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Highlights
Re(I) complexes as anticancer treatment against breast and melanoma
cancer cells
Intrinsic pathway by the overexpression of Caspase-9 is triggered in the
apoptosis
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Cytotoxic activity in resistant melanoma cell line.