Selective Cu(I) complex with phosphine-peptide (SarGly) conjugate contra breast cancer: Synthesis, spectroscopic characterization and insight into cytotoxic action

Article abstract of DOI:10.1016/j.jinorgbio.2018.06.009

The main disadvantage of conventional anticancer chemotherapy is the inability to deliver the correct amount of drug directly to cancer. Those molecular delivering systems are very important to destroy cancer cells selectively. Herein we report synthesis

Full text of DOI:10.1016/j.jinorgbio.2018.06.009

Journal of Inorganic Biochemistry 186 (2018) 162–175
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Selective Cu(I) complex with phosphine-peptide (SarGly) conjugate contra
breast cancer: Synthesis, spectroscopic characterization and insight into
cytotoxic action
a,⁎
a
a
b
Urszula K. Komarnicka , Sandra Kozieł , Radosław Starosta , Agnieszka Kyzioł
a
Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383 Wroclaw, Poland
Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
The main disadvantage of conventional anticancer chemotherapy is the inability to deliver the correct amount of
drug directly to cancer. Those molecular delivering systems are very important to destroy cancer cells selec-
Peptide carriers
Copper(I) complex
Conjugate
DNA
ROS
Breast cancer
tively. Herein we report synthesis of phosphine-peptide conjugate (Ph
SarGly (sarcosine-glycine), which can be easily exchanged to other peptide carriers, its oxide (OPh
Gly-OH, OPSG) and the first copper(I) complex ([CuI(dmp)(P(Ph) CH -Sar-Gly-OH)], 1-PSG, where dmp stands
for 2,9-dimethyl-1,10-phenanthroline). The compounds were characterized by elemental analysis, NMR (1D,
D), UV–Vis spectroscopy and DFT (Density Functional Theory) methods. PSG and 1-PSG proved to be stable in
biological medium in the presence of atmospheric oxygen for several days. The cytotoxicity of the compounds
2
PCH
2
-Sar-Gly-OH, PSG) derived from
2
PCH -Sar-
2
2
2
2
1
-PSG
and cisplatin was tested against cancer cell lines: mouse colon carcinoma (CT26;
IC50 = 3.12 ± 0.1),
1
-PSG
1-
human lung adenocarcinoma (A549;
IC50 = 2.01 ± 0.2) and human breast adenocarcinoma (MCF7;
IC50 = 0.98 ± 0.2) as well as against primary line of human pulmonary fibroblasts (MRC-5;
IC50 = 78.56 ± 1.1). Therapeutic index for 1-PSG (MCF7) equals 80. Intracellular accumulation of 1-PSG
complex increased with time and was much higher (96%) inside MCF7 cancer cells than in normal MRC5 cells
PSG
1-
PSG
(
(
20%). Attachment of SarGly to cytotoxic copper(I) complex via phosphine motif improved selectivity of copper
I) complex 1-PSG into the cancer cells. Precise mechanistic study revealed that the 1-PSG complex causes
apoptotic cells MCF7 death with simultaneous decrease of mitochondrial membrane potential and increase of
caspase-9 and -3 activities. Additionally, 1-PSG generated high level of reactive oxygen species that was the
reason for oxidative damages to the sugar–phosphate backbone of plasmid DNA.
1. Introduction
offered by medicinal inorganic chemistry (very often not accessible to
organic compounds). Coordinated chemistry of metal-based drugs has
Mortality caused by cancer is about to exceed that from cardio-
been placed in the frontline in the fight against cancer by widespread
use of cisplatin. Unfortunately, the use of this drug is very limited due
to its high toxicity [8]. Copper-based complexes could be less toxic for
normal cells with respect to cancer cells, despite copper toxicity related
to its redox activity. Also, a different response of normal and tumor cells
to copper ions can be a platform for development of copper complexes
endowed with antineoplastic characteristic [8–10].
Copper(I) complexes constitute a group of compounds that is still
not exploited enough, but over the last several years, we can observe a
significant interest increase of their anticancer [11–17], antibacterial
[18], antiviral [19,20], antifungal [21], and inflammatory [14] ac-
tivity. Furthermore, the phosphine ligands forms a strong bond with
copper(I) ion which prevents oxidation of the phosphine ligand and
vascular diseases in near future. Approximately 7 million people die
from cancer-related cases per year, and it is estimated that there will
be > 16 million new cancer cases every year by 2020 [1,2]. Che-
motherapy is still one of the major approaches to treat cancer by de-
livery of a cytotoxic agent to cancer cells. However, the main dis-
advantage of conventional chemotherapy is the inability to deliver the
correct amount of drug directly to cancer cells without affecting healthy
cells [3]. Drug resistance, altered biodistribution, biotransformation,
and drug clearance are also considered common problems [3]. This
shows the importance of development of systems that will selectively
destroys cancer cells [4–7].
Various opportunities for the design of therapeutic agents can be
⁎
Corresponding author.
E-mail address: urszula.komarnicka@chem.uni.wroc.pl (U.K. Komarnicka).
https://doi.org/10.1016/j.jinorgbio.2018.06.009
Received 20 April 2018; Received in revised form 10 June 2018; Accepted 13 June 2018
Available online 18 June 2018
0162-0134/ © 2018 Elsevier Inc. All rights reserved.

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
copper(I) to copper(II) [12], what was also proven in our previous
studies [13–17,22–24]. Additionally, phosphine ligands can be easily
functionalized, which is remarkable. In particular, aminomethylpho-
sphanes derived from amino acids [25–28] or prepared from the highly
water-soluble aliphatic secondary amines [29,30], seem to be inter-
esting in terms of the formation of potential conjugates with a wide
range of biomolecules. This makes them good candidates for drugs.
Linking of the peptides (described as a carriers) via phosphine motif
to copper(I) complexes [13,22–24,31–34], may enable selective de-
livery of these coordination compounds to the tumor cells. Examples of
such peptides carriers are RGD (Arg-Gly-Asp) motif and other NGR
peptide (Asn-Gly-Arg) selectively recognize integrins - proteins re-
sponsible for the growth, division, adhesion and migration of cancer
cells (peptides that have entered clinical trials). It is worth noting that
mentioned motifs combined with doxorubicin, paclitaxel and fluor-
ouracil cause significant decrease of in vivo toxicity of these drugs
standard Schlenk techniques. PPh
cording to a literature procedures [44]. Peptide SarGly was purchased
from Bachem (Switzerland). Ph PH, dmp, CuI, other small chemicals
and solvents were purchased from Sigma-Aldrich (Germany) and used
without further purifications. All solvents were deaerated before use.
2 2 2
(CH OH) Cl was synthesized ac-
2
2.2. Methods
Elemental analyses were performed on a Vario EL3 CHN analyser
for C, H, and N, and they were within 0.3% of the theoretical values.
NMR spectra were recorded on a Bruker AMX 500 spectrometer (at
1
298 K) with traces of solvent as an internal reference for H (DMSO‑d
6
:
2.50 ppm) and 1 C spectra (DMSO‑d
3
6
3 4
: 39.51 ppm) and 85% H PO in
H
2
O as an external standard for 31P. The signals in the spectra are de-
fined as: s = singlet (* – strongly broadened signal), d = doublet, dd –
doublet of doublets, t = triplet and m = multiplet. Chemical shifts are
reported in ppm and coupling constants are reported in Hz. Absorption
spectra were recorded on a Cary 50 Bio spectrophotometer (Varian Inc.,
Palo Alto, CA) in the 800–200 nm range.
[
35–40]. In this paper we propose a novel approach – connecting
peptides, in our case: sarcosine-glycine (SarGly) with copper(I) com-
plexes. We decided to choose this peptide SarGly, because of several
reasons. Firstly, SarGly is small and cheap molecule and we could es-
tablish synthetic conditions before applying it to our research peptides
as RGD or NRG (very expensive). Initial, presented here, results were
surprisingly good, so we decided to continue research with this com-
pound SarGly. Secondly, motif GlySar was reported as a PET tracer
targeted to the PEPTs (H+/peptide transporters – functionally ex-
pressed in some human cancer cell lines) for cancer detection in mice
2.3. Synthesis
2.3.1. Preparation of Ph
PPh (CH OH) Cl (0.6434 g, 2.27 mmol) was dissolved in 20 mL of
methanol and cooled down using water bath (T = 8 °C). Then, a slight
excess of NEt (triethylamine; 2.60 mmol) was added. Mixture was
2 2
P-CH -Sar-Gly-OH (PSG)
2
2
2
3
[
41,42]. It is worth mentioning that peptides possess well-known ad-
stirred and with time reached room temperature (RT). After 40 min
SarGly (0.3320 g, 2.27 mmol) in water (10 mL) was added dropwise
into the mixture (RT). After 1 h of stirring, clear solution was observed.
Mixture was dried under reduced pressure for few hours. White, crude
product was dissolved in water (30 mL; milky solution was observed)
vantages as drugs, such as specificity, potency, and low toxicity [43].
To the best of knowledge, in the literature there are no such systems
reported so far.
This article is continuation of our previous projects describing
copper(I) complexes bearing phosphine ligands derived from fluor-
oquinolone antibiotics [14–17,50,51,58,65]. We demonstrated high
cytotoxic activity towards view cancer lines of inorganic derivatives of
fluoroquinolones. It was proven that mentioned above compounds
caused apoptotic cancer cell death via caspase-dependent mitochondrial
pathway. Unfortunately, we were struggling with high toxicity of those
complexes. That is why we decided to exchange fluoroquinolone mo-
lecule to some simple, cheap and lipophilic peptides.
and extracted four times with CHCl
phase was dried under pressure and white solid of PSG was formed. It is
well soluble in CHCl , DMSO, CH Cl , CH CN, moderately in ethanol,
methanol and water.
3
(10 mL) using cannula. Chloroform
3
2
2
3
Yield: 90%, Molar mass: 344.35 g/mol. Anal. Calcd for
PC18H21N2O3: C, 62.78; H, 6.15; N, 8.14%. Found: C, 62.77; H, 6.16;
N, 8.13%.
1
NMR (DMSO‑d
6
, 298 K): 31P{ H}: -27.08 s, 1H: HPh: 7.34–7.55;
HNH: 4.41 s; H : 3.07 s; H : 2.31 s; H : 3.30 d (J = 3.05); H : 3.51d
1
2
3
5
In this paper we describe synthesis of phosphine ligand (PPh
Sar-Gly-OH; PSG) derived from sarcosine-glycine (SarGly), its oxide
OPPh CH -Sar-Gly-OH; OPSG) and copper(I) complex [CuI(2,9-di-
2
CH
2
-
1
3
1
Ph(i)
Ph(o)
(J = 5.53); C{ H}: C
(J = 19.07); C
: 137.65 d (J = 12.72); C
: 128.62 d (J = 6.36); C
: 132.72 d
Ph(m)
Ph(p)
1
(
2
2
: 128.76 s; C : 61.86 d
2
3
4
methyl-1,10-phenanthroline)PSG] (1-PSG). Physicochemical properties
of all these compounds were detected using elemental analysis, NMR
(J = 9.10); C : 44.29 d (J = 10.0); C : 60.03 d (J = 4.50); C : 169.37 s;
5
6
C : 41.99 s; C : 171.62 s.
2
2.3.2. Preparation of Ph P(O)CH SG (OPSG)
(
1D and 2D), UV–Vis spectroscopy and theoretical calculations. Cyto-
toxic activity in vitro of SarGly and its organic (PSG, OPSG) and in-
organic (1-PSG) derivatives was tested against three cancer cell lines:
mouse colon carcinoma (CT26), human lung adenocarcinoma (A549)
and human breast adenocarcinoma (MCF7) as well as one primary line
of human pulmonary fibroblasts (MRC5). Herein, we also try to ap-
proach the mechanism of 1-PSG cytotoxic action towards human breast
adenocarcinoma (MCF7). To realize our goal we undertook a series of
experiments: (i) intracellular uptake of the tested complex was studied
by ICP-MS spectrometry; (ii) the mode of cell death was examined by
flow cytometric analysis; (iii) level of mitochondrial membrane po-
tential was measured; (iv) activity of caspases 3 and 9 was determined;
2
The oxide derivative was prepared in the reaction of PSG (0.4258 g;
1.24 mmol) dissolved in chloroform (20 mL) with equimolar amount of
H
2
O
2
(35% solution in water). After 1 h of stirring (RT) the solution was
evaporated to dryness. White solid well soluble in CHCl , DMSO,
CH Cl , CH CN, moderately in ethanol, methanol and water was ob-
tained.
3
2
2
3
Yield: 100%, Molar mass: 360.34 g/mol Anal. Calcd for
PC18H21N2O4: C, 60.00; H, 5.87; N, 7.77%. Found: C, 59.99; H, 5.88;
N, 7.76%.
NMR (DMSO‑d
6
, 298 K): 31P{ H}: 26.94 s H: H : 7.76–7.42; H :
1
1
Ph
1
2
3
5
13
1
(v) the ability of the complexes to generate reactive oxygen species
(ROS) in the cells was examined using two different fluorescent probes;
(vi) generation of oxidative DNA cleavage was studied by gel electro-
3.14 s; H : 2.31 s; H : 3.52 d (J = 5.34); H : 3.56 d (J = 5.91); C{ H}:
Ph(i)
Ph(o)
Ph(m)
C
: 132.82 d (J = 94.46); C
: 131.68 d (J = 1.82); C
: 130.74
Ph(p)
1
2
d (J = 9.08); C : 131.03 d (J = 8.08) s; C : 59.89 d (J = 87.19); C :
44.68 d (J = 6.36); C : 55. 88 d (84.47); C : 169.57 s; C : 40.77 s; C :
3
4
5
6
phoresis.
171.22 s.
2. Experimental part
2.3.3. Preparation of the complex with phosphine-peptide conjugate (1-
2
.1. Materials
PSG)
Neocuproine (0.1540 g; 0.7396 mmol) and copper iodide (0.1409 g;
0.7396 mmol) were added in equimolar ratios to the phosphine
All reactions were carried out under a dinitrogen atmosphere using
163

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
PPh
2
CH
2
SG (0.2547 g; 0.7396 mmol) dissolved in 20 mL of chloroform
repeated at least three times. Determined values of IC50 are given as
mean + S.D. (Standard Deviation).
and acetonitrile mixture (V:V = 1:1). After few minutes a cloudy so-
lution was formed, which after half an hour became clear. The light
orange precipitate was formed after 24 h of stirring. The solid was fil-
tered off, washed a few times with mixture of water and methanol
2.7. Fluorescence microscopy
(
v:v = 1:1). and dried under vacuum The 1-PSG complex is well soluble
in CHCl , DMSO and CH Cl , moderately in CH CN, poorly in ethanol
and methanol and water.
Viable and dead cells were detected by staining with fluorescein
diacetate (FDA, 5 mg/L) and propidium iodide (PI, 5 mg/L) for 20 min
and examined using fluorescence inverted microscope (Olympus
IX51,Japan) with an excitation filter of 470/20 nm. Photographs of cells
after treatment with the tested compounds were taken under magnifi-
cation 20×.
3
2
2
3
Yield: 75%, Molar mass: 743.05 g/mol Anal. Calcd for
CuIPC32H33N4O3: C, 51.72; H, 4.48; N, 7.54%. Found: C, 51.71; H,
4
.50; N, 7.53%.
3
1
1
1
NMR (DMSO‑d
6
, 298 K): P{ H}: -16.10 s 1H: HPh: 7.59–7.24; H :
2
3
3,8
2
.96 s; H : 2.15 s; H : 3.55 s, H5: 3.50 d (J = 5.70); dmpH : 7.78 d
2.8. Cell cycle analysis using flow cytometry
4
,7
5,6
15,16
(
J = 8.20); dmpH : 8.58 d (J = 8.20); dmpH : 8.06 s; dmpH
:
:
1
3
1
Ph(i)
Ph(o)
Ph(m)
3
2
1
5
1
.70 s; C{ H}: C
: 137.20 s; C
: 132.50 d (J = 12.70); C
In order to distinguish cell death induced by studied compounds,
Annexin V Apoptosis Detection Kit APC (Affymetrix) was used. The
studied compounds SarGly, PSG and 1-PSG (IC50) were incubated for
24 h with tumor cells MCF7 (density 5 × 105 cells/mL) in 12-well
plates. After this time, the compound solutions were removed and the
cells washed with PBS (phosphate-buffered saline, pH = 7,4) buffer and
Binding Buffer. Trypsin was added to the cells and then they were left
Ph(p)
1
2
28.26 d (J = 8.20); C
: 129.36 s; C : 61.96 s; C : 44.11 s; C :
4
5
2,9
3,8
7.67 s; C : not observed; C : 40.80 s; dmp
C
: 158.43 s; dmp
C
:
4
,7
5,6
11,12
25.13 s; dmp
C
C
: 137.20 s; dmp
C
: 125.61 s; dmp
C
: 142.04 s
1
3,14
15,16
dmp
: 126.84 s; dmp
C
: 26.32 s.
2
.4. DFT calculation
2
for 10 min at 37 °C in a humidified atmosphere containing 5% CO . The
cells were collected, centrifuged and separated from the supernatant,
then washed in 0.5 ml PBS buffer (buffer phosphate saline NaCl, KCl,
Na HPO , KH PO ) and again centrifuged and separated from the su-
2 4 2 4
DFT calculations were performed using the Gaussian 09 (Rev.E.01)
package [45]. We employed the M06-2X - Minnesota standalone func-
tional with 54% HF exchange [46]. The basis sets employed were
pernatant. Finally, Annexin V Apoptosis Detection Kit APC (Affymetrix)
was applied according to manufacturer's instructions. The experiment
was repeated at least 3 times. The dot-plots of the percentage dis-
tribution of individual cell populations were obtained.
6
–311 + G(d,p) both for geometry optimization and single-point cal-
culations, (except iodine atom, for which 6-311G(d,p) basis set [47]
was used). The structures were optimized in DMSO using the Polariz-
able Continuum Model (PCM). Minima of energy were characterized as
such by computation of the harmonic vibrational frequencies.
2.9. Copper uptake
2
.5. Cell cultures
6
Cells (MRC5 or MCF7) at density of 2 × 10 cells/2 mL were seeded
on 6-well plates and incubated with complex 1-PSG (c = 1 μM for 4 or
4 h) at standard conditions (37 °C, 5% CO ). Solution of the studied
MCF7 cell line (human breast adenocarcinoma, morphology: epi-
2
2
thelial-like, ATCC: HTB-22), A549 cell line (human lung adenocarci-
noma, morphology: epithelial, ATCC: CCL-185), and CT26 (mouse
colon carcinoma, morphology: fibroblast, ATCC: CRL-2638) were cul-
tured in Dulbecco's Modified Eagle's Medium (DMEM, Corning) with
phenol red, supplemented with 10% fetal bovine serum (FBS) and with
compound was removed; the cells were washed twice with PBS buffer
and trypsinized. Cells, for ICP-MS analysis, were mineralized in 1 mL of
5% HNO . Measurement of the concentration of copper ions was de-
3
termined by a mass spectrometer (ELAN 6100 Perkin Elmer) with an
inductively coupled plasma (ICP-MS). Protein content was assessed
with Bradford Protein Assay (Thermo Scientific™) [49]. The copper
content under each condition is expressed as ng Cu/mg protein. The
experiment was repeated at least 3 times and results are presented as
mean value + S.D.
6
1
% streptomycin/penicillin. MCR-5 cell line (primary line of human
pulmonary fibroblasts, ATCC: CCL-171) was cultured in minimum es-
sential medium (MEM, Corning) with only 10% fetal bovine serum
(
FBS). Cultures were incubated at 37 °C under a humidified atmosphere
containing 5% CO . Cells were passaged using a solution containing
.05% trypsin and 0.5 mM EDTA. All media and other ingredients were
purchased from ALAB, Poland.
2
0
2
.10. Detection of mitochondrial membrane potential (Δψ)
Mitochondrial membrane potential (MMP) depletion was de-
2
.6. Cytotoxic activity
termined by JC-10 Assay (Life Technologies, USA). MCF7 cells were
seeded on 96-well plates at 1 × 104 cells/0.2 mL. After 24 h medium
was replaced with solutions of 1-PSG at IC50 concentration as well as
gentamicin (0.5 mg/mL) and ciprofloxacin (10 μg/mL) as positive and
negative control, respectively. After that, cells were incubated for 24 h
Cytotoxicity was assessed by MTT assay performed according the
4
protocols described elsewhere [48]. In brief, 1 × 10 cells per well,
seeded in 96-well flat bottom microtiter plate, were incubated with the
tested complexes at various range of concentrations for 4 and 24 h.
Afterwards, cell viability was determined using the Hill equation
2
at standard condition (37 °C, 5% CO ). Then, they were washed twice
with PBS buffer and incubated with JC-10 for 1 h. Afterwards, emission
was measured at two different excitation wavelengths (λexc = 540 nm,
λem = 570 nm) and (λexc = 485 nm, λem = 530 nm). Results are
presented as the intensity ratio of red to green emission (mean + S.D.).
(
0
Origin 9.0) with regard to the untreated cells (control), where y –
untreated cell control (which was set to 100% viability), y100 – a lysis
control (where the cells were treated with 0.5% triton X-100 was set to
0
% viability, which was found to be sufficient to induce 100% cell
death), IC50 – values for the concentration [c] at which the viability of
the cells reaches 50%, H – the Hill coefficient (describing co-
operativity):
2
.11. Caspase activity assays
Colorimetric Protease Caspase-3/CPP32 and Caspase-9/Mch6/Apaf-
H
3 Colorimetric Protease kits (Life Technologies, USA) were used for
detection of activated caspases 3 and 9, respectively. The effect of
compounds on the activation of caspase 3 and 9 in MCF7 cells was
monitored spectrophotometrically using the substrates DEVD-pNA and
(
(
y100 − y b)[c]
0
y = y +
0
H
H
IC50
)
+ [c]
Each compound concentration was tested in five replicates and
164

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
LEHD-pNA, respectively, according to the supplier's instructions. In
brief, cells at 5 × 10 cells/2 mL were seeded on 6-well plates and in-
(H
2
DCF-DA) and a Cyto-ID® Hypoxia/Oxidative Stress Detection Kit.
5
The assay was performed in 96-well plates, where cells were seeded at a
cubated for 24 h. Then, after medium aspiration cells were treated at
IC50 concentration for 24 h with copper(I) complex (1-PSG) and eto-
poside (positive control, 1 μg/mL) at standard condition (37 °C, 5%
density of 1 × 104 cells/0.2 mL of medium. Acetyl ester (H
2
DCF-DA)
was prepared by dissolving it in sterile DMSO in anaerobic conditions.
Next, the dye solution was diluted in medium with 2% serum. Medium
from the cells was removed, and they were also washed using PBS
Buffer. Dye solution at a final concentration of 1 μM was added. Cells
with dye were incubated for 30 min in darkness (37 °C in a humidified
atmosphere containing 5% CO
removed and the cells washed twice with PBS buffer. Solutions of stu-
died complexes in IC50 and a solution of H (final concentration
2
CO ). Afterwards, cells were trypsinized, centrifuged, and suspended in
50 μL of the cooled cell lysis buffer (Cell Lysis Buffer) for 10 min. The
cell degradation products were centrifuged (1 min, 10,000 ×g) and the
supernatant was transferred to fresh Eppendorf tubes and left on ice.
Concentration of isolated proteins was determined in all lysates using
Bradford Protein Assay (Thermo Scientific™) [50]. 50 μL of 10 mM di-
tiotreitol (DTT) solution in reaction buffer and 5 μL of 4 mM DEVD-pNA
2
). After this time the dye solution was
2 2
O
−100 μM) as a positive control were added to the cells. The cells with
investigated substances were incubated for 30 min, 4 and 12 h at 42 °C
(
in the case of caspase 3) or LEHD-pNA (in the case of caspase 9) were
added to 1 mg/mL of collected proteins and incubated for 2 h in the
dark at 37 °C. Absorbance in 96-well plates at 400 nm was measured
using plate reader (200 M PRO NanoQuant; Tecan, Switzerland) (free p-
NA). Samples were analyzed in triplicate, and standard deviations were
calculated.
2
in a humidified atmosphere containing 5% CO . The emission of solu-
tion was measured at 495 nm using an Infinite 200 M PRO NanoQuant
plate reader (Tecan, Switzerland). Results obtained using acetyl ester
2
(H DCF-DA) were confirmed by results obtained using a second dye
Cyto-ID® Hypoxia/Oxidative Stress Detection Kit. It this case, the cells
were incubated with tested compounds for 30 min, 4 and 24 h at 37 °C
2
in a humidified atmosphere containing 5% CO . After 3.5 h of incuba-
2.12. DNA strand break analysis
tion, ROS-inducing reagent as a positive control was added to the un-
treated cells. After 4 h all supernatants were removed, the cells were
washed twice with PBS buffer and detector reagent (Oxidative Stress
Detection Kit) was added. Cells with dye were incubated in standard
conditions for 1 h. After this time emission spectra with excitation
wavelength at 505 nm was measured. The experiment was repeated at
least 3 times. The results were presented as a graph of emission in-
tensity, which is proportional to ROS concentration at an appropriate
point in time.
The ability of SarGly, PSG and 1-PSG to induce single- or double-
strand breaks in plasmid DNA was tested with the pBR322 plasmid
C = 0.5 mG/mL). All compounds were dissolved in DMF with/
without: H (c = 50 μM), DMSO (10% V:V) which concentration was
(
2 2
O
kept constant in the final solution. After 1 h incubation at 37 °C, reac-
tion mixtures (20 μL) were mixed with 3 μL of loading buffer (bromo-
phenol blue in 30% glycerol) and loaded on 1% agarose gels, containing
EB, in TBE buffer (90 mM Tris–borate, 20 mM EDTA, pH = 8.0). Gel
electrophoresis was performed at a constant voltage of 100 V (4 V·cm-1)
for 60 min. The gel was photographed and processed with a Digital
Imaging System (Syngen Biotech). For the densitometric analysis the
UltraQuant 6.0 program was used.
3. Results and discussion
3.1. Synthesis
2.13. Reactive oxygen species (ROS) production
Synthetic routes of described here compounds are presented in
Fig. 1. Firstly, starting salt PPh
procedure [44], and starting phosphine ligand PPh
2
(CH
2
OH)
2
Cl, according to a literature
CH OH (POH) were
Production of Reactive Oxygen Species (ROS) by SarGly, PSG and
2
2
1-PSG was determined by photometric tests using: 5-(and-6)-chlor-
synthesized. POH and its copper(I) complexes were described in our
omethyl-20,70-dichlorodihydrofluorescein, diacetate acetyl ester
previous papers in details [15,16]. Subsequently, we obtained a new
Fig. 1. Schematic view of the compounds and synthetic routes.
165

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 2. (a) 31P{ H} NMR spectra (298 K, DMSO) of PSG, OPSG and 1-PSG; (b) Scheme of 1-PSG structures with atomic enumeration; (c) HMBC NMR spectra (298 K,
1
DMSO) of 1-PSG.
phosphine-peptide conjugate (PPh
ment of the peptide motif sarcosine-glycine (SarGly) to POH. In the
next step, phosphine oxide (O]PPh -CH -Sar-Gly-OH; OPSG) and a
new copper(I) complex [CuI(2,9-dimethyl-1,10-phenanthroline)PSG]
2
-CH
2
-Sar-Gly-OH; PSG) by attach-
2 2
PPh CH -motif connection to peptide SarGly, strong shift of all signals
on 1H NMR spectrum towards higher fields was observed proving
phosphine ligand formation (Supporting information Figs. S1–S8, Table
2
2
1
S1). However, the strongest chemical shift for H atom, after reaction of
(
1-PSG) was synthesized (Fig. 1).
Since phosphine oxidation is a common process during complex
peptide with PPh
phosphine POH to 3.07 ppm for PPh
2
-CH
2
-OH (POH), moved from 4.42 ppm for starting
-CH -Sar-Gly-OH. This phenom-
2
2
formation, we synthesized phosphine oxides for comparative purposes.
The syntheses were carried under nitrogen, using Schlenk techniques.
enon, that we expected to observe, can be explained by increasing the
electron density around H1 atom after peptide connection to starting
phosphine.
As expected, the formation of the 1-PSG affected the chemical shifts
of the signals originating from the both ligands (diimine and phos-
3
.2. Spectroscopic properties
1
13
1
phine) on the H and C{ H} NMR spectra. The largest alterations of
diimine chemical shifts were observed for the atoms neighboring the
nitrogens, but number of the signals did not increase. This is a direct
proof that these atoms (NN) coordinate to Cu(I) ions in a symmetrical,
The synthesized compounds were thoroughly characterized by NMR
(
1D and 2D) and UV–Vis spectroscopy. These two techniques allowed
determining the structures of organic and inorganic compounds as well
as stability of the 1-PSG complex in solution. Due to the low solubility
of the complex in water, their NMR spectra were recorded in DMSO
3,8
4,7
3,8
11,12
chelating mode. Signals dmp
H
,
dmp
H
,
dmp
C
and dmp
C
in
1
3
1
1
neocuproine molecule were the most affected among all signals. In the
(
C{ H}NMR = 40 ppm; H NMR = 2.50 ppm). All spectra and spec-
1
2
1
(i)
case of PSG, the largest changes were observed for H , H , C and C
tral data are presented in Fig. 2 and Supporting Information Figs. S1–S8
and Table S1.
atoms (Supporting information Figs. S1–S8 and Table S1). What is
11,12
2,9
(i)
(o)
(m)
1
2
more, signals dmp
C
,
dmp
C
,
PSGC , PSG
C
,
PSG
C
,
PSGH , PSG
H
Formation of phosphine ligands, their oxides and copper(I) com-
plexes leads to significant changes in the chemical shifts and coupling
constants on the NMR spectra. We have observed that phenomenon in
the case of the previously obtained compounds [15,16,50,51]. In the
were strongly shifted towards higher fields after coordination. This
might be connected with an increase of the shielding effect resulting
from the formation of coordination compound. Noteworthy, the data
described here are fully compatible with the results presented in our
previous papers for complexes with the phosphines derived from
amines and fluoroquinolone antibiotics [15,16,24,50,51–53].
3
1
1
P{ H} spectra, the value of the chemical shift for POH (the starting
ligand) was equal to −9.33 ppm. After formation of PSG and OPSG, the
signal moved to −27.08 and 26.94 ppm, respectively (Fig. 2A). For-
mation of the Cu(I) complex resulted in significant broadening and a
low-field shift of the phosphine signal observed in P{ H} NMR
spectra. The value of the chemical shift changed from −27.08 ppm for
PSG – free phosphine to −16.10 ppm for 1-PSG – complex (Fig. 2).
Synthesis of PSG and OPSG significantly affected the chemical shifts
Summing, the detailed analysis of the 31P{ H}, H and C{ H},
HMBC, HMQC and COSY NMR spectra showed that in the complexes,
the central Cu(I) ion is tetrahedrally coordinated by two nitrogen atoms
of neocuproine and the phosphorus atom of the phosphine ligand. This
is a typical coordination mode for the copper(I) complexes with dii-
mines and/or phosphines [15,16,24,50–53].
1
1
13
1
3
1
1
1
13
1
of dipeptide (SarGly) signals on the H and C{ H} NMR spectra. After
166

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 3. Absorbance spectra in DMEM with 2% DMSO, in aerobic atmosphere (37 °C) of upper panel: SarGly, PSG, OPSG and 1-PSG; lower panel: 1-PSG after 96 h.
Electronic spectra of all compounds are presented in Fig. 3A. In case
of 1-PSG characteristic absorption band ((MX,MP)LCT) [52,53]) at
65 nm appeared. UV–Vis spectroscopy was also used to determine the
stability of the 1-PSG complex (Fig. 3B). UV–Vis spectra were recorded
in the presence of DMEM culture medium with 2% DMSO under aerobic
conditions. During 96 h of the experiments we observed no significant
reduction in the intensity of the absorption band (MX,MP)LCT [52,53].
This was clear evidence of the absence of Cu(I) to Cu(II) oxidation
processes under aerobic conditions in the presence of water and con-
firmed the validity of the choice of the diimine ligands.
To determine the most stable conformer of PSG we used the two lowest
energy conformers of N-methylated SarGly in two positions relative to
the position of the phosphorus atom. The most stable one is shown on
Fig. 4 DFT geometries of two of the lowest energy conformers are de-
scribed in Table 1. They are characterized by similar PeC bond lengths
and bond angles around phosphorus atom expressed as S4 parameter
(see explanation under the Table 1.) and the same conformations of the
–SG moiety.
4
The main difference between them is the orientation of the –SarGly
moiety. For the lowest energy conformer (a) the NeC
tiparallel” to the artificial PeLP bond with α(XeP⋯N1eC2) = 174.79°
Table 1) while for the (b) conformer this angle equals to 19.55°.
2 3
H bond is “an-
(
3
.3. DFT calculation
Analogously, we determined the most stable conformer of the phos-
phine oxide OPSG. Interestingly, for the OPSG (b) conformer is the
lowest energy one, but the energy difference is only 0.8 kcal/mol. For
both lowest energy conformers, PeC bonds are significantly shortened
and S4 values are much smaller, indicating large changes in the geo-
metries caused by the presence of the electron-withdrawing oxygen
atom strongly bound to P atom, what follows the trend observed for the
trisaminomethylphosphines [54] and diphenylaminomethylphosphines
derived from sparfloxacin and ciprofloxacin [17].
Simplicity of the sarcosine-glycine (SarGly) allowed determining
the most stable conformer. We analyzed 4 different conformers of an-
ionic and zwitterionic forms and the most stable one was zwitterion
stabilized by two independent hydrogen bonds. The first one was
NeH⋯O between protonated S amine group and amide oxygen and the
second one - NeH⋯O between amide NH group and one of the G
oxygens from deprotonated carboxylate group. Other conformers had
energy higher by 2.3, 5.7 and 8.7 kcal/mol.
We also determined the most stable conformer of anionic form of N-
methylated anionic MeSarGly fragment, which is a close molecule to
2
the –CH SarGly fragment in the phosphine ligand. The most stable
Determining of the structure of the 1-PSG complex was more
complex and involved analysis of three parts of the molecule: orienta-
tion of the –Ph rings relative to the dmp ring, conformation and position
2
of the –CH SarGly fragment. Geometries of the 3 lowest energy isomers
conformer was the one with amide –NH proton in a close proximity to
the sarcosine amine N atom and glycine G carboxylate O atom (Fig. 4).
is listed in Table 1 and the views of their molecules are shown in Fig. 5.
167

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 4. The most stable conformers of N-methylated HSG anion, phosphine PSG and its oxide OPSG.
As expected, all calculated isomers of 1-PSG show a distorted tet-
rahedral geometry around copper(I) central ion. Although the value of
α (the angle between the plane of the dmp ligand and the plane formed
by Cu, I and P) in most cases is close to 90°, the angles between dmp
plane and CueP and CueI bonds differ significantly. Like for the ana-
logous CuI complex with dmp and with diphenylphosphinomethyl de-
rivative of sparfloxacin [16], for the 1-PSG complex the most stable
isomers are those with one of the phosphine –Ph rings overlapping the
dmp ligand. It should be noted however, that apart from the high en-
decided to choose the murine cancer cell line because of its biological
and pharmacological properties, which are representative of the prop-
erties of human colon carcinoma [55]. We have chosen these types of
the cells to our investigation because recently published statistics has
shown that for the past two decades lung cancer has ranked the first,
colon cancer - the second and breast cancer – the third place in terms of
morbidity [55].
Most of the studied here compounds are insoluble in aqueous media.
Therefore, all of them were predissolved in DMSO (maximum final
concentration 0.2% V:V [56]) for the biological tests. The final com-
pounds concentration ranged from 0.1 to 0.001 mM. Cytotoxic activity
was assessed on the basis of IC50 values (concentration of a drug re-
quired to inhibit the growth of 50% of the cells [57]). IC50 values were
determined from the plots of cell viability in the presence of each
compound; matching dose–response curves calculated using the Hill
equation (Origin 9.0) [48]. The values of IC50 were confirmed by an
analysis of images of the stained tumor cells treated with the com-
pounds at the corresponding concentrations (Table 2 Fig. 6). The cell
viability was examined by counting the green cells (fluorescein diace-
tate – FDA stain) with normal nuclei, which were considered to be the
surviving cells, and the red ones (propidium iodide – PI stain) con-
sidered to be dead.
2
ergy isomers with fragment –CH SarGly overlying dmp ligand, the
relative energies of other isomers did not exceed +5.2 kcal/mol. For 1-
PSG-a – the lowest energy isomer the angle between the –Ph and dmp
planes is only 7.53° and the distance between the –Ph centroid to the
dmp plane is 3.277 Å. For the phosphine ligand, impact of the co-
ordination to Cu(I) ion on the geometry around phosphorus atom is
pronounced mainly by the diminution of the S4 value by 10–12, but the
PeC bonds are not shortened, probably due to steric hindrances caused
by the –Me groups from the dmp ligand. The three isomers presented
2
here differ strongly by orientation of the –CH SG fragment, but in each
of three lowest energy isomers conformation of the –SG moiety is close
to its conformations in organic molecules. The largest discrepancy is
observed for isomer (a), where one of oxygen atoms from the carbo-
lylate groups is placed in a short contact not only with the amide proton
For the comparison purposes we showed also results previously
obtained by our group (published experiments for those compounds
were performed in the same conditions as presented here data in our
but also a proton from the eCH
Fig. 5).
2
– group and proton from the –Ph ring
(
laboratories) for POH (PPh
(CuI(dmp)PPh CH OH), sparfloxacin (HSf, a 3rd generation quino-
lone), PSf (PPh CH Sf), OPSf (OPPh CH Sf) and 1-PSf (CuI(dmp)
PPh CH Sf) (Table 2) [15,16].
2 2 2 2
CH OH), OPOH (OPPh CH OH), 1-POH
3
.4. Cytotoxicity
2
2
2
2
2
2
Cytotoxicity of the synthesized complex (1-PSG), peptide (SarGly),
2
2
peptide-phosphine conjugate (PSG) and its oxide (OPSG), starting
compounds (dmp and CuI) and cisplatin was tested in vitro against three
cancer cell lines: mouse colon carcinoma (CT26), human lung adeno-
carcinoma (A549) and human breast adenocarcinoma (MCF7) as well
as one primary line of human pulmonary fibroblasts (MRC-5). We
Peptide (SarGly), phosphine ligand (PSG) and its oxide (OPSG) did
not show any activity towards investigated cancer cell lines even after
incubation time prolongation (4 h → 24 h) (Table 2) in order to 1-PSG
complex which exhibited the highest cytotoxic activity among all stu-
died compounds (Supporting Information Table S2). Activity of this
Table 1
DFT (M06-2X/6–311 + G(d,p)) relative energies and geometries for the lowest energy conformers of PSG, OPSG and 1-PSG.
PSG-a
PSG-b
OPSG-a
OPSG-b
1-PSG-a
1-PSG-b
1-PSG-c
E(rel) [kcal/mol]
α(P-C1-N1-C2)a
α(X-P…N1-C2)
av.(P-C)
S4
P-O
0
1.28
0.81
0
0
1.18
73.89
8.37
1.8434
38.37
1.14
161.75
−174.79
1.8530
53.34
79.30
19.55
1.8547
52.41
156.65
−169.94
1.8220
24.43
102.28
23.72
1.8232
21.68
1.5064
−75.04
−120.85
1.8521
42.95
−57.77
−75.66
1.8553
40.25
b
1.5064
P-Cu
Cu-I
2.4584
2.6778
124.16
86.78
2.4543
2.6754
124.68
87.48
2.4854
2.6802
121.33
80.31
P-Cu-I
c
α
a
Torsion angle between NeCH
3
Ph-i
bond and PeO, PeCu bonds or LPeP – an artificial vector in the direction of the lone electron pair on P atom, generated to obey
1
Ph-i
condition: α(X-P-C ) = α(X-P-C ) = α(X-P-C ′).
b
Symmetric deformation coordinate– the difference between the sum of the angles between the substituents and the X and the angles between substituents
Ph-i
Ph-i
1
1
Ph-i
1
Ph-i
Ph-i
Ph-i
(
S4 = XPC ’ + XPC
+ XPC − C PC ′ − C PC
− C 'PC ) - see [24, 54] for full details.
c
α – the angle between the plane of the dmp ligand and the plane formed by Cu, I and P atoms [24].
168

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 5. UP: The structure of the lowest energy isomer of 1-PSG complex determined using DFT methods. DOWN: the structures of –a, −b, and –c isomer – view along
PeCu bond.
complex was slightly higher after 24 h of incubation comparing to 4 h
regarding to all studied lines. The significant increase of 1-PSG activity
after the time prolongation (4 h → 24 h) was observed for MCF7 cancer
line (six times). What is more, 1-PSG, comparing to literature com-
plexes with metal ions such as: Pt, Ru, Cu, Ir, Zn, Pd, seems to show the
best cytotoxic activity towards A549, CT26 and MCF7 with small ex-
ceptions (Supporting Information Table S2 with the literature refer-
fluoroquinolone molecule from copper(I) complex (1-PSf, Table 2) to
simple dipeptide motif (SarGly) resulted in significant decrease of the
copper(I) complex toxicity towards normal cells MRC5.
We decided to perform detailed study to understating action mode
of 1-PSG complex. It was clearly seen that 1-PSG is the most active
against MCF7 cancer cell line. Due to, human breast adenocarcinoma
was selected to our further investigation (Table 2).
4
ences). For instance, copper(I) complex Cu(dppe)(2,2′-bipy)][BF ] ex-
hibited activity twice higher against MCF7 [59] comparing to described
here complex, but there is no any information about its toxicity towards
normal cell lines. What is more, exchanging of fluoroquinolone mole-
cule in 1-PSf complex (Table 2) to simple dipeptide motif (SarGly)
resulted in increase of the copper(I) complex cytotoxicity.
3
.5. Cellular uptake
In an attempt to correlate cytotoxicity with the cellular uptake of
the tested complex, the copper content was evaluated for MCF7 and
MRC5 cells treated for 4 and 24 h with 1-PSG in the same concentration
Tests employing normal line (MRC-5) showed that SarGly, PSG and
OPSG were not toxic as opposed to cisplatin and literature complexes
(
C = 1 μM). Only one concentration (1 μM) has been selected in our
research to see differences in delivery of 1-PSG to cancer and normal
cells. As we have postulated that short peptides (i.e. SarGly) can be
used as selective carriers of copper(I) complexes into the cancer cells,
performing copper uptake experiments in the same concentration of
compound, for healthy and cancer cells, was crucial. The intracellular
copper amount was quantified by ICP-MS analysis and is expressed as
ng Cu per mg of cellular protein (Fig. 7). The experiment was repeated
at least 3 times and values Cu concentrations are presented as mean
value + S.D. (standard division) in Table S3 Supplementary materials.
(
Table S2). Unexpectedly, toxicity of 1-PSG was significantly lower
(
more than two times) than cisplatin and literature complexes with
copper(I) ions (1-POH, 1-PSf Table 2). Calculated values of therapeutic
index (TI) for 1-PSG, 1-POH and 1-PSf (Table 2) clearly indicated that
1-PSG could be a perfect candidate as anticancer drug, since it ex-
hibited high cytotoxicity against studied cancer cells and low toxicity
towards healthy line. Value of IT obtained for 1-PSG was the highest for
MCF7 line. What is worth emphasizing that exchanging of
Table 2
IC50 [μM] (concentration of a drug required to inhibit the growth of 50% of the cells) values for CT26, A549, MCF7, MRC-5 cell lines after 4 and 24 h treatment with
the SarGly, PSGO, 1-PSGO, dmp, CuI, cisplatin and literature data obtained for complexes with different metal ions and ligands. Selected TI (therapeutic index)
values for 1-PSG and literature complexes 1-POH, 1-PSf.
IC50 [μM] ± SD
4
h
24 h
CT26
A549
MCF7
CT26
A549
MCF7
MRC5
a
a
a
a
a
a
POH
OPOH
74.47 ± 1.1a
73.98 ± 1.3a
> 100
> 100
76.89 ± 5.2
–
70.70 ± 5.5
59.12 ± 2.6
46.86 ± 4.2
0.7
74.36 ± 1.3
61.80 ± 1.2
26.15 ± 6.3
1.2
> 100
> 100
56.32 ± 2.1
1.9
> 100
> 100
30.21 ± 0.1.2
–
a
a
67.54 ± 1.3
61.34 ± 1.1
1
-POH
30.76 ± 1.1a
31.15 ± 6.4a
TI
–
–
(
1-POH)
a
a
b
> 100b
PSf
238.97 ± 16.8a
163.23 ± 5.1a
> 100
264.28 ± 12.1
104.08 ± 3.3
338.00 ± 20.7
a
a
a
a
b
46.97 ± 2.8b
29.64 ± 2.4b
OPSf
-PSf
TI
51.03 ± 1.2
74.90 ± 1.4
64.22 ± 2.3
8.23 ± 1.7
–
109.23 ± 8.8
52.72 ± 9.2
55.02 ± 0.5
a
b
1
7.33 ± 0.1a
6.04 ± 0.1a
8.29 ± 0.7a
3.6
7.84 ± 0.2
6.67 ± 0.4
–
–
3.8
4.4
(1-PSf)
SarGly
PSG
OPSG
> 100
> 100
> 100
4.24 ± 0.8
–
> 100
> 100
> 100
2.13 ± 0.5
–
> 100
> 100
> 100
6.23 ± 8.2
–
> 100
> 100
> 100
3.12 ± 0.1
25.2
> 100
> 100
> 100
2.01 ± 0.2
39.1
> 100
> 100
> 100
0.98 ± 0.2
80.2
> 100
> 100
> 100
78.56 ± 1.1
1
-PSG
TI
(
1-PSG)
64.74 ± 1.3a
64.16 ± 3.2a
> 100
61.66 ± 1.9a
69.95 ± 3.1a
> 100
76.23 ± 1.2
65.34 ± 3.1
35.23 ± 2.1
55.97 ± 4.6
58.23 ± 4.6
> 100
a
58.12 ± 3.9
58.72 ± 6.4
> 100
a
71.23 ± 4.2
60.09 ± 6.1
50.9 ± 7.6
21.34 ± 3.4
32.12 ± 6.6
31.48 ± 4.1
dmp
CuI
Cisplatin
a
a
a-15,51, b-58 TI- therapeutic index – IC50 (normal cell line)/IC50 (cancer cell line).
169

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 6. The photos (magnification 20.00×, bar 50 μm) of MRC5 and MCF7 cell lines after 24 h incubation with and without 1-PSG in C = 1μM. The green cells with
normal morphology are viable ones (FDA), while round red cells are dead (PI).
3.6. Mitochondrial membrane potential and caspases activity
Cell death has been classified into two main types: programmed cell
death (PCD) and passive (necrotic) cell death. In turn, PCD can be di-
vided into three main types: apoptosis, autophagy and non-lysosomal
vacuolated degeneration – known as oncosis and paraptosis [64]. So far
it was proven that copper(I) complexes mostly cause cancer cells death
in two ways: apoptotic and paraptotic [15,16,49,51,58,60–62].
Paraptosis is a recently discovered type of PCD, characterized by
cytoplasmic vacuolization derived from endoplasmic reticulum and/or
mitochondria swelling, caspase independent, lack of apoptotic mor-
phology, lack of DNA fragment and poly (ADP-ribose) polymerase
(PARP) cleavage and ROS generation [63–66].
Fig. 7. Final intracellular copper concentration expressed by ng Cu/mg protein
for complex 1-PSG in c = 1 μM: after 4 or/and 24 h of incubation with cancer
line MCF7 or normal line MRC5.
Apoptosis is PCD and an important self-regulatory mechanism for
multicellular organisms to maintain homeostasis [64–66]. It worth
mentioning, that causing apoptosis has become very important in an-
ticancer drug research. There are two main pathways connected with
apoptosis mediated by (i) cell death receptor (extrinsic pathway) and
(ii) mitochondrion (intrinsic pathway). Both of them lead to activation
of caspases cascade [61–66]. The second pathway is mainly associated
with the delivery of cytochrome c into cytosol and activation of cas-
pase-9. Mitochondria play a vital role in this apoptosis pathway. After
that cytochrome c activates the caspase-9 initiator. In this process
apoptosome is generated what, subsequently leading to activate cas-
pase-3 (responsible for apoptotic death) [63–66].
For more precise determination of the mode of observed cell death
flow cytometry with Fluorescence-Activated Cell Sorting (FACS) was
applied. We performed an Annexin V/PI assay and quantitatively
evaluated the apoptosis-inducing ability of the studied compounds
(SarGly, PSG and 1-PSG). At the early stage of apoptosis the flipped
phosphatidylserine of cytoplasmic membrane becomes available on the
cell surface for binding to Annexin V. Together with propidium iodide
(PI), which stains only dead cells with disintegrated membrane, An-
nexin V enables distinguishing cells undergoing different types of cell
death [15]. The percentage of live, apoptotic as well as necrotic cells
upon treatment with the compounds (IC50) is presented in Fig. 8.
Data analysis proved that treatment of the MCF7 cells with SarGly,
PSG and 1-PSG resulted in the vast majority of the population of
apoptotic cells appearing, in opposite to necrotic ones present in
In each studied line after incubation with 1-PSG, a significant increase
of copper complex accumulation was detected over control cells
(
Fig. 7).
Treatment of studied cancer cells with the tested copper complex
resulted in a marked time-dependent intracellular copper accumula-
tion. After 4 h of incubation cells with 1-PSG, we noticed that 58% of
starting copper concentrations penetrated into the cells. With pro-
longation of incubation time, copper uptake significantly increased
(
58% → 96%) (Fig. 7). This phenomenon can be directly connected
with cytotoxicity of investigated complex (Table 2). Both: cytotoxicity
and intracellular accumulation of 1-PSG, increased after prolongation
of the incubation time. Also, the accumulation of copper for cancer line
MCF7 and normal line MCR5 significantly differed. In the case of
normal cell line MRC5 only 20% of copper accumulation was noticed
(
while 96% for MCF7 line was calculated; Fig. 7). Those results
perfectly support the difference between cytotoxic activity of
-PSG against MCF7 (IC50 = 0.98 ± 0.2 μM) and MRC5 line
1
(
IC50 = 78.56 ± 1.1 μM). What is noteworthy, cytotoxicity of this
complex depends on copper intracellular accumulation. Therefore, we
can postulate that high selectivity of 1-PSG can be directly connected
with peptide motif (-Sar-Gly-OH). Furthermore, in literature, a motif
1
1
11
C-glycylsarcosine ( C-Gly-Sar), was described as highly selective to
the PEPTs expressed on human cancer cells [41,42]. However, this
phenomenon, undoubtedly, needs further detailed investigation.
170

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 8. On the left: percentage [%] of normal, apoptotic and necrotic cells after 24 h of incubation of MCF7 cell line with SarGly, PSG and 1-PSG in IC50. on the right:
scatter plots (LL – live cells, HL – early apoptotic cells, HR – late apoptotic cells, LR – necrotic cells).
minority (around 2%) (Fig. 8). We did not notice differences between
organic and inorganic compounds.
1-PSG is lower than caspase 9. This allows supposing that the tested
tumor cells can activate the deactivation mechanisms of this pathway of
cellular death after treatment with 1-PSG [58,71].
As it was mentioned above, we proved that 1-PSG can be accumu-
lated inside the cells. That is why we suspected that intrinsic pathway of
apoptotic death could be the most probable in this case. For a precise
determination of the MCF7 cell death type caused by 1-PSG, and better
explanation of the mechanism of this process, the following experi-
ments were carried out: (i) the measurement of the level of mitochon-
drial membrane potential (MPM), that decreases during apoptosis [67]
and (ii) the examination of the proteolytic activity of caspases 9 and 3.
Depletion of mitochondrial potential was evaluated on MCF7 cells
treated with 1-PSG by JC-10 Mitochondrial Membrane Potential Assay.
JC-10 probe allows for quantitative detection of mitochondrial mem-
brane potential (MMP) changes, considering the shift from orange to
green fluorescence emission, and variation in its intensity ratio.
Gentamicin, which causes an increase of MMP [68] and ciprofloxacin
with its opposite effect on MMP [69] were used as a positive and a
negative control, respectively.
Taking into account obtained results we can postulate, that, studied
copper(I) complex 1-PSG cause controlled “cell suicide” (apoptotic
death) of MCF7 cancer cells. This means that tested compound initiates
cell death, which is natural and safe for the body, and above all, does
not, in contrast to necrosis, cause inflammation [67]. At the current
stage of the study it can be supposed that the induction of apoptosis in
the examined cells probably occurs via caspase-dependent mitochon-
drial pathway.
3.7. Reactive oxygen species and DNA cleavage
Reactive oxygen species (ROS) can be produced in physiological
processes occurring in the cells or generated as a result of external
factors such as drugs, ultraviolet, ionizing radiation or metal ions and
complexes [72,73]. Elevated levels of ROS in turn may cause cell cycle
arrest or lead to the apoptosis process [74]. Furthermore, mitochondrial
ROS can be produce in a very early stage before breakdown of mi-
tochondrial membrane potential, which in turn results in releasing the
pro-apoptotic factors or the activation of the other cell death mechan-
isms [49,61,62,74].
As it is clearly seen on Fig. 9A, studied complex 1-PSG significantly
decreased MMP. The level of MMP after treatment with 1-PSG is much
lower than after treatment with ciprofloxacin. This means that studied
complex caused increase of permeability of the mitochondrial mem-
brane. What is interesting, in case of A549 and CT26 cells, it was noted
that previously described by us complexes, mentioned above 1-PSf (CuI
Cellular ROS production in MCF7 cells upon 24 h treatment with
(
dmp)PPh
2
CH
2
-sparfloxacin) [15], 1-PCp (CuI(dmp)PPh
2 2
CH -cipro-
HSG, PSG and 1-PSG (IC50
)
was monitored by
2
H DCF-DA
floxacin) and 1-PNr (CuI(dmp)PPh
2
CH -norfloxacin) [62] also caused
2
(
λex = 495 nm, λem = 530 nm) the fluorescent ROS probe. In addi-
decrease of MMP but in lower level than that was described here.
Decrease in MMP leads to the release of cytochrome c into the cy-
toplasm. Cytochrome c, in the presence of ATP, interacts with the Apaf-
tion, the level of oxidative stress induced by the total ROS production
was also determined using a cyto-ID hypoxia/oxidative stress detection
2 2
kit (λex = 505 nm, λem = 524 nm). H O and pyocyanin were used as
1
factor and praspaspase 9. This interaction usually triggers a cascade of
positive controls in the first and second cases, respectively. The de-
pendence of the fluorescence intensity proportional to the concentra-
tion of ROS and oxidative stress on time (three different time incubation
executive caspases (especially caspase 3 responsible for apoptotic cell
death [69,70]. Our investigation clearly shows that copper(I) complex
1-PSG activated caspase 9 (an initiator caspase) and caspase 3 (an ex-
4, 12 and 24 h) are presented on Fig. 10.
ecutioner caspase) what is presented on Fig. 9B. Measured level of both
caspases after treatment of MCF7 cells with 1-PSG is much higher
comparing to control samples – untreated cells without compound and
treated with etoposide. Concentration of caspase 3 after treatment with
Our studies have proved that the investigated complex 1-PSG
(
Fig. 10) exhibited the ability to induce ROS production inside the
treated cells MCF7. Both performed tests showed that studied complex
was able to induce ROS generation at a significantly higher level than
Fig. 9. Left panel: Influence of studied complex 1-PSG on intensity of JC-10 fluorescence in treated MCF7 cells. Alteration in MMP is given as an emission ratio
70 nm/530 nm. (control – untreated cells, ciprofloxacin – a negative control, gentamicin – a positive control); right panel: Influence of 1-PSG on activation of
caspase 3 and caspase 9 in MCF7 cell line. As a positive control etoposide (1 μg/mL) was used.
5
171

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Fig. 10. Upper panel: Photos of MCF7 cells after 24 h treatment and without and with 1-PSG or pyo (pyocyanin) for cyto-ID hypoxia/oxidative stress detection kit
λex = 505 nm, λem = 524 nm); lower panel – left: The increase of ROS production in CT26 cells after 4, 12 and 24 h using H DCF-DA for: 1-PSG, K(+):H as
positive control and K(−): negative control, cell without compound; lower panel – right: Oxidative stress induced by the total ROS production in CT26 cells after 4,
2 and 24 h detected by CYTO-ID Hypoxia/Oxidative Stress Test for: 1-PSG, K(+): pyocyanin as positive control and K(−): negative control.
(
2
2 2
O
1
pyocyanin (pyocyanin toxicity results from its ability to undergo re-
duction by NAD(P)H and subsequent generation of superoxide and
H O [75]) as well as SarGly and PSG. Moreover, we observed that, 1-
2 2
The gel electrophoresis of pBR322 plasmid (naturally occurring as a
covalently closed superhelical form (form I)) was applied to determine
the ability of SarGly, PSG, 1-PSG to induce single- and/or double-strand
damage to DNA. This can lead to the formation of the relaxed/nicked
(form II) and linear (form III) forms. The degree of DNA degradation was
determined in a wide range of concentrations (from 0.5 to 5000 μM)
(Fig. 11A). It should be emphasised that SarGly and PSG did not cause
any DNA degradation (data not shown), even in concentrations ex-
ceeding the IC50 (5000 μM). The 1-PSG complex, similarly to ligands, did
not lead to the formation of the single- and/or double-strand DNA da-
mage even in concentrations up to 200 μM (IC50 is 200 times lower).
Only in concentrations over 500 μM relaxed/nicked of nucleic acid was
observed. What is more, amount of form II increased along with con-
centration increase of 1-PSG (Fig. 10A). In our previous paper [15] de-
scribing DNA degradation by 1-PSf, forms I and II in concentration 10 μM
were observed. Single-strand DNA damage with total decline of super-
helical form was noticed at concentration equal 100 μM. This means that
complex 1-PSG is much less genotoxic than 1-PSf.
PSG cytotoxicity; copper uptake and ROS level inside MCF7 cancer cells
are increasing with time (vide supra Table 2 and Fig. 8). This phe-
nomenon suggests that ROS are probably primarily involved in the
observed cytotoxicity and the mechanism of cell death. More precise
experiments will help to explain their special contribution and kind of
participation in the observed cytotoxicity.
Interestingly, published previously by us 1-PSf [15], 1-PNr and 1-
PCp [58] generated significantly lower level of ROS in A549 and CT26
cells. Performed tests showed that 1-PSf complex and the corre-
sponding ligands were able to induce ROS generation at a similar level
and independently of the type of cell line [15]. It means that ROS
generation was not the most crucial in the observed mechanism of ac-
tions of those complexes (1-PSf, 1-PNr, 1-PCp). However, it was proven
by cyclic voltammetry that 1-PSf can act as an efficient participant in
redox reactions, e.g., ROS generation that are responsible for the oxi-
dative damage of many important cell elements and subsequent dis-
turbances in vital processes [58]. Furthermore, copper(I) complex Cu
In the presence of transition metal ions and their complexes, an
endogenous oxidant H O could be a source of hydroxyl radicals, the
2 2
(
dppe)(2,2′-bipy)][BF
4
], mentioned above, displayed higher cytotoxi-
most powerful DNA oxidants [15,58]. As above-mentioned, 1-PSG
generated high level of ROS and therefore we decided also to check the
influence of the studied complex on DNA damages. 1-PSG presumably
city than 1-PSG (Supplementary materials Table S2), in the ovarian
cells induced rapid production of reactive oxygen species probably
through mitochondrial pathways [59].
decomposed H
2
O
2
in a free radical reaction, which can cause DNA
Fig. 11. Left panel: Agarose gel electrophoresis
of pBR322 plasmid cleavage by 1-PSG in a DMF
(
each in the 10% DMF) solution. Lanes: 1,
plasmid – control; 2, plasmid +0.5 μM 1-PSG;
, plasmid +1 μM 1-PSG; 4, plasmid +5 μM 1-
PSG; 5, plasmid +10 μM 1-PSG; 6, plasmid
20 μM 1-PSG; 7, plasmid +30 μM 1-PSG; 8,
plasmid +50 μM 1-PSG; 9, plasmid +100 μM 1-
3
+
PSG; 10, plasmid +200 μM 1-PSG; 11, plasmid +500 μM 1-PSG; 12, plasmid +1000 μM 1-PSG; 13, plasmid +5000 μM 1-PSG; right panel: Agarose gel electro-
phoresis of pBR322 plasmid cleavage by 1-PSG in a DMF (each in the 10% DMF) solution. Lanes: 1, plasmid +10% DMSO; 2, plasmid +1 μM 1-PSG; 3, plasmid
+
1 μM 1-PSG + 50 μM H
2
2
O
2
; 4, plasmid +1 μM 1-PSG + 50 μM H
2 2
; 8, plasmid +100 μM 1-PSG + 50 μM H O
2
O
2
+ 10% DMSO; 5, plasmid +50 μM 1-PSG + 50 μM H
2 2
O ; 6, plasmid +50 μM 1-PSG + 50 μM
H
2
O
+ 10% DMSO; 7, plasmid +100 μM 1-PSG + 50 μM H
O
2 2
+ 10% DMSO.
172

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
damage. 1-PSG in presence of hydrogen peroxide caused distinct
changes in the plasmid structure, resulting in conversion of the super-
coiled plasmid to relaxed/nicked (Fig. 10B line 3) or relaxed/nicked
and linear (Fig. 10B lines 5 and 7) forms dependently on complex
concentration. In higher concentrations, more serious damages of nu-
cleic acid were observed. What was proven quantitatively, addition of
DMSO (effective scavenger of the hydroxyl free radical [12]) to the
reaction's mixture prevented the DNA from damage (Fig. 10B lines 4, 6
and 8). Prevention of DNA lesions by DMSO confirmed a free radical
mechanism of action on the plasmid degradation processes [15]. The
same results were observed in case of 1-PSf, 1-PNr and 1-PCp com-
plexes [50]. We noticed that 1-PSG caused less DNA damages in general
comparing to previously described complexes (1-PSf, 1-PNr and 1-
PCp).
Abbreviations
dmp
DFT
2,9-dimethyl-1,10-phenanthroline
density functional theory
CT26
A549
MCF7
MRC5
ROS
mouse colon carcinoma
human lung adenocarcinoma
human breast adenocarcinoma
human pulmonary fibroblasts
reactive oxygen species
IC50
concentration of a drug required to inhibit the growth of 50%
of the cells
RGD
peptide Arg-Gly-Asp
NGR
peptide Asn-Gly-Arg
SarGly
PET
PEPTs
peptide sarcosine-glycine
positron emission tomography
H+/peptide transporters
Cells have mechanisms that repair the damage to DNA, but if da-
mages are too severe to be repair, the cell initiates its suicidal program
and dies by apoptosis. A similar reaction is triggered by inappropriately
folded proteins, which may be the result of external triggers such as free
radicals [76].
ICP-MS inductively coupled plasma mass spectrometry
NMR nuclear magnetic resonance spectroscopy
CH OH methanol
NEt
CHCl
CH
3
3
trimethylamine
chloroform
4. Conclusions
3
3
CN acetonitrile
In this paper we presented synthesis, physicochemical and biolo-
DMSO
EDTA
DMEM
CT
dimethyl sulfoxide
gical characterization of the derivatives of simple dipeptide, sarcosine-
glycine (SarGly): diphenylphosphinomethyl derivative (P(CH SG)Ph
PSG), its oxide (OP(CH SG)Ph ; OPSG) and copper(I) complex (1-PSG)
ethylenediaminetetraacetic acid
Dulbecco's Modified Eagle Medium
charge transfer
2
2
;
2
2
with this phosphine-peptide conjugate (PSG). Spectroscopic and theo-
retical studies revealed that PSG strongly binds the metal ion and forms
stable (also in a presence of atmospheric oxygen) tetrahedral copper(I)
complex.
TI
FDA
PI
S.D.
PCD
therapeutic index
fluorescein diacetate
propidium iodide
standard division
programmed cell death
The cytotoxic activity of all presented here compounds and cisplatin
was tested against three cancer cell lines: mouse colon carcinoma
ADP ribose adenosine diphosphate ribose
-PSG
1-
(
CT26; 1
IC50 = 3.12 ± 0.1), human lung adenocarcinoma (A549;
PARP
FACS
LL
Poly (ADP-ribose) polymerase
Fluorescence-Activated Cell Sorting
live cells
PSG
PSG
IC50 = 2.01 ± 0.2) and human breast adenocarcinoma (MCF7; 1-
IC50 = 0.98 ± 0.2) as well as one primary line of human pulmonary
fibroblasts (MRC-5;
1
-PSG
IC50 = 78.56 ± 1.1). Peptide (SarGly), phos-
HL
early apoptotic cells
phine ligand (PSG) and its oxide (OPSG) did not show significant ac-
tivity towards investigated cancer cell lines in opposite to complex 1-
PSG, which exhibited the highest cytotoxicity among all studied com-
pounds. Values of calculated therapeutic index (TI) for 1-PSG clearly
indicated that this complex is highly selective against cancer lines,
HR
LR
MMP
ATP
late apoptotic cells
necrotic cells
mitochondrial membrane potential
adenosine triphosphate
H
2
DCFDA2′,7′-dichlorodihydrofluorescein diacetate
NAD(P)H nicotinamide adenine dinucleotide phosphate-oxidase
1
-PSG
especially towards MCF7 cells line, where
TI equals 80, and it was
two and three times higher than for CT26 and A549 cancer lines, re-
spectively.
DMF
FBS
dimethylformamide
fetal bovine serum
Precise study on elucidation of cell death mechanism showed that
high accumulation of this complex inside MCF7 cancer cells influences
on its cytotoxicity. Importantly, intracellular 1-PSG accumulation was
much higher for cancer cells MCF7 (96%) than for normal cells MRC5
MEM
PBS
DTT
Minimum Essential Medium Eagle
phosphate-buffered saline
ditiotreitol
(
20%). Most likely, attachment of SarGly to cytotoxic copper(I) com-
Author contributions
plex via phosphine motif improved selectivity of this complex. What is
more, 1-PSG caused apoptotic cell death with simultaneous decrease of
mitochondrial membrane potential level and increase of caspase-9 and
caspase-3 activity in MCF7 cells. Obtained results suggest that ROS are
probably primarily involved in the observed cytotoxicity of 1-PSG and
the resulting cell death. However, this needs more precise explanation,
what is our current objective.
Presented here data, proved also that exchanging fluoroquinolone
molecule in copper(I) complexes (they were our previous subjects of
study) to simple, lipophilic molecule of peptide, significantly decrease
of copper(I) complex toxicity towards normal cells and increase its
cytotoxicity against cancer cells.
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Acknowledgment
The authors gratefully acknowledge financial support from the
Polish National Science Centre (Grants 2016/23/D/ST5/00269, 2017/
01/X/NZ7/01148) and from Ministry of Science and Higher Education
(Grants 1233/M/WCH/13 and 1500/M/WCH/15). The DFT calcula-
tions have been carried out in the Wroclaw Centre for Networking and
Supercomputing (http://www.wcss.wroc.pl), grant no. 140. The in vitro
research was carried out with the equipment purchased thanks to the
financial support of the European Regional Development Fund in the
Framework of the Polish Innovation Economy Operational Program
(contract no. POIG.02.01.00-12-023/08). The authors are grateful to
This design approach of highly cytotoxic copper(I) complexes with
peptides carries attached via phosphine linker, can be very promising in
anticancer therapy. This paper is beginning of our research in this sci-
entific field.
173

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
Bernadeta Nowak. PhD. (Jagiellonian University in Kraków) for flow
cytometry measurements.
[27] K. Raghuraman, K.K. Katti, L.J. Barbour, N. Pillarsetty, C.L. Barnes, K.V. Katti,
2
Characterization of supramolecular (H O)18 water morphology and water-methanol
(H
2
O)15(CH OH) clusters in a novel phosphorus functionalized trimeric amino acid
3
3
host, J. Am. Chem. Soc. 125 (2003) 6955–6961.
Appendix A. Supplementary data
[28] D.E. Berning, B.C. Noll, D.L. DuBois, Relative hydride, proton, and hydrogen atom
transfer abilities of [HM(diphosphine)2]PF6 complexes (M]Pt, Ni), J. Am. Chem.
Soc. 121 (1999) 11432–11447.
29] S. Deshayes, M.C. Morris, G. Divita, F. Heitz, Cell-penetrating peptides: tools for
intracellular delivery of therapeutics, Cell. Mol. Life Sci. 62 (2005) 1839–1849.
30] M.C. Garnett, Targeted drug conjugates: principles and progress, Adv. Drug Deliv.
Rev. 53 (2001) 171–216.
31] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Copper in diseases and
treatments, and copper-based anticancer strategies, Med. Res. Rev. 30 (2010)
708–749.
[32] C.F. Shaw III, Gold-based therapeutic agents, Chem. Rev. 9 (1999) 2589–2600.
[33] T.M. Simon, D.H. Kunishima, G.J. Vibert, A. Lorber, Inhibitory effects of a new oral
gold compound on HeLa cells, Cancer 44 (1979) 1965–1975.
[34] S.J. Berners-Price, P.J. Sadler, Phosphines and metal phosphine complexes: re-
lationship of chemistry to anticancer and other biological activity bioinorganic
chemistry, Struct. Bond. 70 (1988) 27–102.
[35] C.D. Buckley, D. Pilling, N.V. Henriquez, G. Parsonage, K. Threlfall, D. Scheel-
Toellner, D.L. Simmons, A.N. Akbar, J.M. Lord, M. Salmon, RGD peptides induce
apoptosis by direct caspase-3 activation, Nature 6719 (1999) 34–39.
[36] J. Zhang, Y. Liu, B. Yuan, Z. Wang, M. Schönhoff, X. Zhang, Multilayer films with
nanocontainers: redox-controlled reversible encapsulation of guest molecules, J.
Control. Release 159 (2012) 14968–14973.
[37] M. Sioud, A. Mobergslien, Selective killing of cancer cells by peptide-targeted de-
livery of an anti-microbial peptide, Biochem. Pharmacol. 9 (2012) 1123–1132.
[38] S. Majumdar, T. Siahaan, Peptide-mediated targeted drug delivery, J. Med. Res.
Rev. 32 (2012) 637–658.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2018.06.009.
[
[
[
References
[
1] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal, M.J. Thun, Cancer sta-
tistics 2006, CA Cancer J. Clin. 56 (2006) 106–130 (4. Broder H., et al., Rev
Cardiovasc Med., 2008, 9, 75).
[2] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer sta-
tistics, CA Cancer J. Clin. 61 (2011) 69–90.
[
3] D. Kakde, D. Jain, V. Shrivastava, R. Kakde, A.T. Patil, Cancer therapeutics- op-
portunities, challenges and advances in drug delivery journal of applied pharma-
ceutical, Science 9 (2011) 1–10.
[
[
[
[
4] H. Broder, R.A. Gottlieb, N.E. Lepor, Chemotherapy and cardiotioxicity, Rev.
Cardiovasc. Med. 9 (2008) 75–83.
5] L.G. Ferreira, R.N. Santos, G. Oliva, A.D. Andricopulo, Molecular docking and
structure-based drug design strategies, Molecules 20 (2015) 13384–13421.
6] C.A. Naughtn, Drug-induced nephrotoxicity, Am. Fam. Physician 78 (2008)
7
43–750.
7] M.J. Newman, J.C. Rodarte, K.D. Benbatoul, S.J. Romano, C. Zhang, et al.,
Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-
mediated multidrug resistance, Cancer Res. 36 (2000) 216–219.
[
8] D.W. Hoskin, A. Ramamoorthy, Studies on anticancer activities of antimicrobial
peptides, Biochim. Biophys. Acta 1778 (2008) 357–375.
[39] E.W. Rongsheng, N. Youhong, W. Haifan, N.A. Mohamad, C. Jianfeng, Development
of NGR peptide-based agents for tumor imaging, Am. J. Nucl. Med. Mol. Imaging 1
(2011) 36–46.
[9] T.M. Allen, Ligand-targeted therapeutics in anticancer therapy, Nat. Rev. Cancer 2
(
2002) 750–763.
[40] C.D. Buckley, D. Pilling, N.V. Henriquez, G. Parsonage, K. Threlfall, D. Scheel-
Toellner, D.L. Simmons, A.N. Akbar, J.M. Lord, M. Salmon, RGD peptides induce
apoptosis by direct caspase-3 activation, Nature 397 (1999) 534–539.
[41] N.B. Nabulsi, D.E. Smith, M.R. Kilbourn, [11C]Glycylsarcosine: synthesis and in
vivo evaluation as a PET tracer of PepT2 transporter function in kidney of PepT2
null and wild-type mice, Bioorg. Med. Chem. 13 (2005) 2993–3001.
[42] K. Mitsuoka, S. Miyoshi, Y. Kato, Y. Murakami, R. Utsumi, Y. Kubo, A. Noda,
Y. Nakamura, S. Nishimura, A. Tsuji, Cancer detection using a PET tracer, 11C-
Glycylsarcosine, targeted to H1/peptide transporter, J. Nucl. Med. 49 (2008)
615–622.
[43] X.-X. Zhang, H.S. Eden, X. Chen, Peptides in cancer nanomedicine: drug carriers,
targeting ligands and protease substrates, J. Control. Release 159 (2012) 2–13.
[44] J. Fawcett, P.A.T. Hoye, R.D.W. Kemmitt, D.J. Law, D.R. Russell, Synthesis of bis
(phosphinomethyl)amines viabis(hydroxymethyl)phosphonium salts. Isolation of
9,9-bis(hydroxymethyl)-9-phosphoniabicyclo[3.3.1]nonane hydrogensulfate and
chloride salts, and the crystal structures of [PPh2(CH2OH)2]+ Cl– and [(C6H11)
2PCH2]2NCHMePh, Dalton Trans. (1993) 2563–2568.
[
[
10] Y.L. Janin, Peptides with anticancer use or potential, Amino Acids 25 (2003) 1–40.
11] R. Starosta, A. Bykowska, A. Kyzioł, M. Płotek, M. Florek, J. Król, M. Jeżowska-
Bojczuk, Copper(I) (pseudo)halide complexes with neocuproine and amino-
methylphosphines derived from morpholine and thiomorpholine -in vitro cytotoxic
and antimicrobial activity and the interactions with DNA and serum albumins,
Chem. Biol. Drug Des. 82 (2013) 579–586.
[
12] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Advances in
copper complexes as anticancer agents, Chem. Rev. 114 (2014) 815–862.
13] R. Starosta, A. Brzuszkiewicz, A. Bykowska, U.K. Komarnicka, B. Bażanów,
M. Florek, Ł. Gadzała, N. Jackulak, J. Król, K. Marycz, A novel copper(I) complex,
[
[
CuI(2,2′-biquinoline)P(CH2N(CH2CH2)2O)3]-synthesis, characterisation and
comparative studies on biological activity, Polyhedron 50 (2013) 481–489.
14] U.K. Komarnicka, R. Starosta, A. Kyzioł, M. Płotek, M. Puchalska, M. Jeżowska-
Bojczuk, New copper(I) complexes bearing lomefloxacin motif: spectroscopic
properties, in vitro cytotoxicity and interactions with DNA and human serum al-
bumin, J. Inorg. Biochem. 165 (2016) 25–35.
[
[
[
[
15] U.K. Komarnicka, R. Starosta, M. Płotek, R.F.M. de Almeida, M. Jeżowska-Bojczuk,
A. Kyzioł, Copper(I) complexes with phosphine derived from sparfloxacin. Part II: a
first insight into the cytotoxic action mode, Dalton Trans. 45 (2016) 5052–5063.
16] U.K. Komarnicka, R. Starosta, A. Kyzioł, M. Jeżowska-Bojczuk, Copper(I) complexes
with phosphine derived from sparfloxacin. Part I – structures, spectroscopic prop-
erties and cytotoxicity, Dalton Trans. 44 (2015) 12688–12699.
17] U.K. Komarnicka, R. Starosta, K. Guz-Regner, G. Bugla-Płoskońska, A. Kyzioł,
M. Jeżowska-Bojczuk, Phosphine derivatives of sparfloxacin- synthesis, structures
and in vitro activity, J. Mol. Struct. 1096 (2015) 55–63.
[45] Gaussian 09, Revision E.01, M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria,
M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson,
H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng,
J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr.,
J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin,
V.N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell,
J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene,
J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski,
R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador,
J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz,
J. Cioslowski, D.J. Fox, Gaussian, Inc., Wallingford CT, (2013).
[46] Y. Zhao, D.G. Truhlar, The M06 suite of density functionals for main group ther-
mochemistry, thermochemical kinetics, noncovalent interactions, excited states,
and transition elements: two new functionals and systematic testing of four M06-
class functionals and 12 other functionals, Theor. Chem. Accounts 120 (2006)
215–241.
[47] K.L. Schuchardt, B.T. Didier, T. Elsethagen, L. Sun, V. Gurumoorthi, J. Chase, J. Li,
T.L. Windus, Basis set exchange: a community database for computational sciences,
J. Chem. Inf. Model. 47 (2007) 1045–1052.
[48] J. Weyermann, D. Lochmann, A. Zimmer, A practical note on the use of cytotoxicity
assays, Int. J. Pharm. 288 (2005) 369–376.
[49] S. Tardito, A. Barilli, I. Bassanetti, M. Tegoni, O. Bussolati, R. Franchi-Gazzola,
C. Mucchino, L. Marchiò, Copper-dependent cytotoxicity of 8-hydroxyquinoline
derivatives correlates with their hydrophobicity and does not require caspase ac-
tivation, J. Med. Chem. 55 (2012) 10448–10459.
[50] A. Bykowska, R. Starosta, J. Jezierska, M. Jeżowska-Bojczuk, Coordination versa-
tility of phosphine derivatives of fluoroquinolones. New CuI and CuII complexes
and their interactions with DNA, RSC Adv. 5 (2015) 80804–80815.
[51] U.K. Komarnicka, R. Starosta, A. Kyzioł, M. Płotek, M. Puchalska, M. Jeżowska-
Bojczuk, New copper(I) complexes bearing lomefloxacin motif: spectroscopic
properties, in vitro cytotoxicity and interactions with DNA and human serum al-
bumin, J. Inorg. Biochem. 165 (2016) 25–35.
[
[
[
18] G. Borkow, J. Gabbay, Putting copper into action: copper-impregnated products
with potent biocidal activities, FASEB J. 18 (2004) 1728–1730.
19] J.O. Noyce, H. Michels, C.W. Keevil, Inactivation of influenza A virus on copper
versus stainless steel surfaces, Microbiology 73 (2007) 2748–2750.
20] F. Lebon, N. Boggetto, M. Ledecq, F. Durant, Z. Benatallah, S. Sicsic, R. Lapouyade,
O. Kahn, A. Mouithys-Mickalad, G. Deby-Dupont, M. Reboud-Ravaux, Metal-or-
ganic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-
2
,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human im-
munodeficiency virus 1 protease, Biochem. Pharmacol. 63 (2002) 1863–1873.
21] B. Dudová, D. Hudecova, R. Pokorny, M. Mikulasova, M. Palicova, P. Sega,
M. Melnik, Copper complexes with bioactive ligands. II. Antimicrobial activity,
Microbiology 47 (2002) 225–229.
[
[
22] R. Starosta, K. Stokowa, M. Florek, J. Król, A. Chwiłkowska, J. Kulbacka, J. Saczko,
J. Skała, M. Jeżowska-Bojczuk, Biological activity and structure dependet proper-
ties of cuprous iodide complexes with phenanthrolines and water soluble tris
(aminomethyl) phosphanes, J. Inorg. Biochem. 105 (2011) 1102–1108.
[
23] R. Starosta, M. Florek, J. Król, M. Puchalska, A. Kochel, Cooper(I) iodide complexes
containing new aliphatic aminophosphine ligands and diimines—luminescent
properties and antibacterial activity, New J. Chem. 34 (2010) 1441–1449.
24] R. Starosta, M. Puchalska, J. Cybińska, M. Barys, A.V. Mudring, Structures, elec-
tronic properties and solid state luminescence of Cu(I) iodide complexes with 2,9-
dimethyl-1,10-phenanthroline and aliphatic aminomethylphosphines or triphenyl-
phosphine, Dalton Trans. 40 (2011) 2459–2468.
[
[
25] C. Abu-Gnim, I. Amer, Phosphine oxides as ligands in the hydroformylation reac-
tion, J. Organomet. Chem. 516 (1996) 235–243.
[
26] P. Smoleński, F.P. Pruchnik, Aminoalkylphosphines, the water-soluble chiral
[52] R. Starosta, A. Brzuszkiewicz, A. Bykowska, U.K. Komarnicka, B. Bażanów,
phosphines, Pol. J. Chem. 81 (2007) 1771–1776.
M. Florek, Ł. Gadzała, N. Jackulak, J. Król, K. Marycz, A novel copper(I) complex,
174

U.K. Komarnicka et al.
Journal of Inorganic Biochemistry 186 (2018) 162–175
[64] N. González-Ballesteros, D. Pérez-Álvarez, M. Carmen Rodríguez-Argüelles,
M.S.C. Henriques, J.A. Paixão, S. Prado-López, Synthesis, spectral characterization
and X-ray crystallographic study of new copper(I) complexes. Antitumor activity in
colon cancer, Polyhedron 119 (2016) 112–119.
[65] A. Bykowska, U.K. Komarnicka, M. Jeżowska-Bojczuk, A. Kyzioł, CuI and CuII
complexes with phosphine derivatives of fluoroquinolone antibiotics – a compara-
tive study on the cytotoxic mode of action, J. Inorg. Biochem. 181 (2018) 1–10.
[66] Y. Wanga, X. Zhub, Z. Yanga, X. Zhaoa, Honokiol induces caspase-independent
paraptosis via reactive oxygen species production that is accompanied by apoptosis
in leukemia cells, Biochem. Biophys. Res. Commun. 430 (2013) 876–882.
[67] S. Sperandio, I. de Belle, D.E. Bredesen, An alternative, nonapoptotic form of pro-
grammed cell death, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 14376–14381.
[68] W.B. Wang, L.X. Feng, Q.X. Yue, W.Y. Wu, S.H. Guan, B.H. Jiang, M. Yang, X. Liu,
D.A. Guo, Paraptosis accompanied by autophagy and apoptosis was induced by
celastrol, a natural compound with influence on proteasome, ER stress and Hsp90,
J. Cell. Physiol. 227 (2011) 2196–2206.
[CuI(2,2′-biquinoline)P(CH2N(CH2CH2)2O)3]-synthesis, characterisation and
comparative studies on biological activity, Polyhedron 50 (2013) 481–489.
53] R. Starosta, U.K. Komarnicka, M. Puchalska, M. Barys, Solid state luminescence of
copper(I) (pseudo)halide complexes with neocuproine and aminomethylpho-
sphanes derived from morpholine and thiomorpholine, New J. Chem. 36 (2012)
[
[
[
[
1
673–1683.
54] R. Starosta, B. Bażanów, W. Barszczewskic, Chalcogenides of the aminomethyl-
phosphines derived from 1-methylpiperazine, 1-ethylpiperazine and morpholine:
NMR, DFT and structural studies for determination of electronic and steric prop-
erties of the phosphines, Dalton Trans. 39 (2010) 7547–7555.
55]
a
(
b
(
R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, Cancer J. Clin. 2015 (65)
2015) 5–29;
M.G. Brattain, J. Strobel-Stevens, D. Fine, M. Webb, A.M. Sarrif, Cancer Res. 40
1980) 2142.
56] Z. Liu, L. Salassa, A. Habtemariam, A.M. Pizarro, G.J. Clarkson, P.J. Sadler,
Contrasting reactivity and cancer cell cytotoxicity of isoelectronic organometallic
iridium(III) complexes, Inorg. Chem. 50 (2011) 5777–5783.
[69] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (2007)
495–516.
[57] K. Strohfeldt, M. Tacke, Bioorganometallic fulvene-derived titanocene anti-cancer
drugs, Chem. Soc. Rev. 37 (2008) 1174–1187.
[70] M.R.A.V. Ng, P.J. Antonelli, J. Joseph, Assessment of mitochondrial membrane
potential in HEI-OC1 and LLC-PK1 cells treated with gentamicin and mitoquinone,
Otolaryngol. Head Neck Surg. 152 (2015) 729–733.
[71] H. Tarek, R. Mahmoud, M. Parviz, A. Charles, A. Kuszynksi, E. Thomas, Natural
retinoids inhibit proliferation and induce apoptosis in pancreatic cancer cells pre-
viously reported to be retinoid resistant, Cancer Biol. Ther. 4 (2005) 480–489.
[72] S. Kalghatgi, C.S. Spina, J.C. Costello, M. Liesa, J.R. Morones-Ramirez, S. Slomovic,
A. Molina, O.S. Shirihai, J.J. Collins, Bactericidal antibiotics induce mitochondrial
dysfunction and oxidative damage in mammalian cells, Antibiotics 5 (2013)
192–285.
[
58] A. Kyzioł, A. Cierniak, J. Gubernator, A. Markowski, M. Jeżowska-Bojczuk,
U.K. Komarnicka, Copper(I) complexes with phosphine derived from sparfloxacin.
Part III: multifaceted cell death and preliminary study of liposomal formulation of
selected copper(I) complexes, Dalton Trans. 47 (2018) 1981–1992.
59] J. Lopes, D. Alves, T.S. Morais, P.J. Costa, M.F.M. Piedade, F. Marques, M.J. Villa de
Brito, M.H. Garcia, New copper(I) and heteronuclear copper(I)–ruthenium(II)
complexes: synthesis, structural characterization and cytotoxicity, J. Inorg.
Biochem. 169 (2017) 68–78.
[
[
60] S. Sperandio, K. Poksay, I. de Belle, M.J. Lafuente, B. Liu, J. Nasir, D.E. Bredesen,
Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix, Cell Death
Differ. 11 (2004) 1066–1075.
[73] Y. Shi, Caspase activation, inhibition, and reactivation: a mechanistic view, Protein
Sci. 13 (2004) 1979–1987.
[
61] A. Barilli, C. Atzeri, I. Bassanetti, F. Ingoglia, V. Dall'Asta, O. Bussolati, M. Maffini,
C. Mucchino, L. Marchiò, Oxidative stress induced by copper and iron complexes
with 8-hydroxyquinoline derivatives causes paraptotic death of HeLa cancer cells,
Mol. Pharm. 11 (2014) 1151–1163.
[74] Y.E. Henrotin, P. Bruckner, J.P. Pujol, The role of reactive oxygen species in
homeostasis and degradation of cartilage, Osteoarthr. Cartil. 11 (2003) 747–755.
[75] N. Fozia, A. Wustholz, R. Kinscherf, N.A. Metzler- Nolte, A cobaltocenium–peptide
bioconjugate shows enhanced cellular uptake and directed nuclear delivery, Angew.
Chem. Int. Ed. 44 (2005) 2429–2432.
[76] S.M. Bianchi, L.R. Prince, K. McPhillips, L. Allen, H.M. Marriott, G.W. Taylor,
P.G. Hellewell, I. Sabroe, D.H. Dockrell, P.W. Henson, M.K. Whyte, Impairment of
apoptotic cell engulfment by pyocyanin, a toxic metabolite of Pseudomonas aeru-
ginosa, Am. J. Respir. Crit. Care Med. 177 (2008) 35–43.
[
62] V. Gandin, M. Porchia, F. Tisato, A. Zanella, E. Severin, A. Dolmella, C. Marzano,
Novel mixed-ligand copper(I) complexes: role of diimine ligands on cytotoxicity
and genotoxicity, J. Med. Chem. 56 (2013) 7416–7430.
[
63] M. Porchia, F. Tisato, M. Zancato, V. Gandin, C. Marzano, In vitro antitumor activity
of water-soluble copper(I) complexes with diimine and monodentate phosphine li-
gands, Arab. J. Chem. (2017), http://dx.doi.org/10.1016/j.arabjc.2017.09.003.
175