A simultaneously GSH-depleted bimetallic Cu(ii) complex for enhanced chemodynamic cancer therapy

Article abstract of DOI:10.1039/d0dt01742f

A bimetallic Cu(ii) complex as a novel antitumor chemodynamic therapy agent with glutathione (GSH) depletion properties is successfully synthesized and well characterized. In tumor cells, the Cu²⁺ions of the complex are reduced to Cu⁺ions by GSH and then catalyzed by the overexpressed H₂O₂to generate highly cytotoxic hydroxyl radicals (˙OH) that kill cancer cells. The complex is quickly taken up by cancer cells and distributed in multiple organelles including mitochondria and the nucleus. The complex demonstrates good cytotoxicity toward various cancer cell lines. However, its toxicity toward normal cells is significantly lower than that toward cancer cells due to the limited expression of H₂O₂. In addition, the complex could arrest the cell cycle of the G0/G1 phase, thereby inducing apoptosis rather than necrosis.

Full text of DOI:10.1039/d0dt01742f

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DOI: 10.1039/D0DT01742F
ARTICLE
Simultaneously GSH-Depleted Bimetallic Cu(II) Complex for
Enhanced Chemodynamic Cancer Therapy
a
b
c
d
a
a
a
Shuhua Cao , Xuezhao Li *, Yong Gao , Fahui Li , Kaoxue Li , Xuanxuan Cao , Yiwen Dai , Lirong
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
Mao , Shanshan Wang and Xishi Tai *
DOI: 10.1039/x0xx00000x
A bimetallic Cu(II) complex as a novel antitumor chemodynamic therapy agent with glutathione (GSH) depletion properties
is successfully synthesized and well characterized. In tumor cells, the Cu2+ ions of the complex are reduced to Cu by GSH,
+
2 2
and then catalyzed by the overexpressed H O to generate highly cytotoxic hydroxyl radical (•OH), which kill cancer cells.
The complex is quickly taken up by cancer cells and distribute in multiple organelles including mitochondrial and nucleus.
The complex demonstrates good cytotoxicity to various cancer cell lines. However, the toxicity to normal cells is significantly
2 2
lower than that of cancer cells due to limited expression of H O . In addition, the complex could arrest the cell cycle of the
G0/G1 phase, thereby inducing apoptosis rather than necrosis.
Contrast to the normal cells, the microenvironment of
tumours possesses various characteristics such as hypoxia,
Introduction
2
9-32
acidity, and overexpressed H
level of H could act as an endogenous ROS resources for CDT.
Noted Fenton reaction is substantially suppressed in the
presence of insufficient H , the CDT approach could reduce
2 2
O .
The significantly increased
Traditional clinically used anticancer therapeutic agents are lack
of selectivity, and thus commonly leads to undesirable side
effects.1 Thus, there is an urgent drive to develop new
treatment modalities with specific tumour targeting ability and
reduced systematic side effects. In recent years, considerable
effort has been devoted to the development of reactive oxygen
2 2
O
-4
2 2
O
damage to healthy cells and tissues. However, the other
overexpressed specie in tumour cells, glutathione (GSH), as the
effective scavenger for ROS present as an obstacle for CDT for
5
-8
species (ROS) based anticancer treatment strategies. The ROS,
including the superoxide anion, hydrogen peroxide (H
singlet oxygen ( O
cancer cell apoptosis or necrosis by damaging biomolecules
3
3,34
cancer cells.
Therefore, reduction of intracellular GSH level
2 2
O ),
is highly desirable for CDT agents to circumvent tumour
resistance and improve chemodynamic efficacy. To date,
researches on CDT agents with simultaneous GSH depletion
properties commonly depend on the stepwise redox reaction
between the high valent metal ions and GSH, yielding
glutathione disulfide (GSSG) firstly, then the lower valent metal
ions act as highly efficient Fenton-like reagents to generate ·OH.
1
2
), and hydroxyl radical (·OH), can induce
9
-18
such as lipids, proteins, and DNA.
As one most concerned
non-invasive treatment, photodynamic therapy (PDT) that
converts the intracellular molecular oxygen into ROS to kill
cancer cells under the irradiation of light with a certain
wavelength.1 Nevertheless, chemodynamic therapy (CDT), in
particular, utilizes metal-mediated Fenton reaction or Fenton-
9-22
3
5
Such examples can be seen as MnO
2
-based nanoagents,
1
0
36
Mn/Cu silicate nanospheres , Mn/Cu bimetallic complex and
like reaction by converting H
without external stimulus in tumour sites.
to circumvent limitations of PDT that need the penetration of
light through tissues and hypoxia environment in tumour cells,
which make CDT as an emerging therapeutic strategy for cancer
treatment.
2 2
O into the highly toxic ·OH
3
7
3
PtCu nanocages.
2
3-28
The unique ability
Among various metal-mediated Fenton-like reactions, the
+
Cu -involved reaction can occur with a highest reaction rate (1
4
−1 −1
×
10 M s ) in weakly acidic, which is about 160-fold increase
2
+ 38
over that of Fe . We thus proposed that a well-designed
multinuclear Cu -based complex may provide enhanced CDT
for cancer treatment with simultaneous GSH depletion
2
+
2
+
a. College of Chemistry, Chemical and Environmental Engineering, Weifang
procedure. Herein, we successfully synthesized a bimetallic Cu
University, No. 5147 Dongfeng Street, Weifang, 261061, P. R. China.
E-mail: taixs@wfu.edu.cn
State Key Laboratory of Fine Chemicals, Dalian University of Technology, No. 2
Linggong Road, Dalian 116024, P. R. China.
E-mail: xuezhaoli@dlut.edu.cn
Dongying Shengli Hospital, No.107 Beier Road, Dongying, 257055, P. R. China
School of Pharmacy, Weifang Medical University, No. 7166 Baotong Street,
Weifang, 261053, P. R. China
Electronic Supplementary Information (ESI) available. CCDC 1884700
For ESI and crystallographic data in CIF or other electronic format See
DOI: 10.1039/x0xx00000x
complex (CuL-Cuphen) and utilize it as novel antitumor CDT
2+
agent combined with GSH depletion. The Cu could be trigged
b.
+
+
by GSH to generate the Cu . Then the Cu catalysed with the
overexpression of H in tumor cells into highly cytotoxic ·OH
2 2
O
c.
d.
efficiently. Cell cycle and apoptosis experiments indicated that
CuL-Cuphen could arrest cell cycle in G0/G1 phase and induce
apoptosis.
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Pleas De ad l to o nn oT tr aa nd sj au cs t ti omn as rgins
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ARTICLE
Journal Name
Br
Br
N
-
coordinated with two O donors of CuL and two N donors from
View Article Online
DOI: 10.1039/D0DT01742F
Br
1
,10-phenanthroline unit. The distance between Cu…Cu is
O
O
a, b
c
about 5.258(1) Å (Fig. 1). Further details including bond lengths
and bond angles for CuL-Cuphen were summarized in tables S1-
S2.
Na+
Step A
O
O
O
NH OH
NH N
O
O
O
N
Cu
NH2OH
N
ROS detection by MB in solution
H3L
CuL
MB was used to detect the generation of •OH, because MB can
be oxidized and degraded by •OH, resulting in decrease in its
Br
+
3
5,39
absorption intensity.
not significantly reduce the absorption of MB in 25 mM NaHCO
% CO buffer solution in the presence of H . In contrast,
As shown in Fig. 2a, CuL-Cuphen did
O
-
3
,
N
ClO4
d
O
O
N
N
Step B
CuL
5
2
2 2
O
Cu
Cu
O
N
N
when GSH was added, the absorption of MB decreased
significantly, indicating that CuL-Cuphen could generate •OH,
2
+
because Cu could oxidize GSH to GSSG, with self-reduction to
CuL-Cuphen
+
be Cu , which could convert the H
2
8
O
2
to the •OH by the Fenton
Scheme 1. Schematic representation of the stepwise assembly route of
3
reaction via following equations.
o
tetranuclear Cu(II) complex CuL-Cuphen. a. THF, ClCOCOOC
NH CH C(CH CH N(CH rt; c. CuCl ·2H
phenanthroline, MeOH, Cu(ClO ·6H O.
2
H
5
, 0
C
; b. THF,
2
+
+
Cu + GSH → Cu + GSSG
Cu + H2O2
2
2
3
)
2
2
3
2
) ,
2
2
O, NaOH, rt; d. 1,10-
+
2 +
+ •
OH + OH ―
)
4 2
2
→
Cu
To further determine the type of ROS, TEMPO (2,2,6,6-
Tetramethylpiperidinooxy) was used as a capture agent for •OH.
As shown in Fig. 2a, when TEMPO was added to the system, the
absorption of MB did not decrease significantly, indicating that
the ROS generated by the Fenton reaction was •OH.
Fig. 1 Representation of the X-ray crystal structure of CuL-Cuphen. Atom
colors: C = gray, N = blue, O = red, Cu = green, Br = pink. Anions, hydrogen
atoms and solvent molecules are omitted for clarity.
Results and discussion
Synthesis and characterization of CuL-Cuphen
As shown in Scheme 1, CuL-Cuphen was prepared by a stepwise
assembly process. First, tetrapod ligand H L was prepared from
3
amino-5-bromobenzoic acid through a two-step reaction. Pre- Fig. 2 (a) The degradation of MB by ·OH generation treated by CuL-Cuphen
o
(
50 µM) and GSH (10 mM) in 8 mM H
O
2 2
and 25 mM NaHCO
3
at 37 C; (b)
organization of the mononuclear copper complex (CuL) building
o
GSH (10 mM) depletion by CuL-Cuphen (20 µM) at 37 C for 0-12 h; (c)
Intracellular GSH/GSSG ratio determination before and after treatment of
CuL-Cuphen (10 µM) at 37 C for 12 h; (d) Emission spectra of the CT-DNA-
2
+
3
units through coordination of the central H L with one Cu ion
(
Step A). Subsequently, CuL-Cuphen was assembled by the
o
2
+
reaction of CuL building unit, 1,10-phenanthroline and Cu
EB system in Tris-HCl/NaCl buffer upon the titration to CuL-Cuphen (λex = 525
(
Step B). Single crystal X-ray diffraction analysis of CuL-Cuphen nm; λem = 601 nm) [EB] =1 μM; [DNA] =5 μM; [CuL-Cuphen]: 0-200 μM. Arrow
shows the intensity change upon the increase of the CuL-Cuphen
confirmed that the successfully formation of a discrete
bimetallic copper complex. There are two types of Cu(II)
coordination centres in this complex. In detail, the Cu centre in
CuL building unit was coordinated with three N donors and one
concentration. Insert: Linear Stern-Volmer quenching constant (Ksv); (e)
Cleavage patterns of pUC19 DNA (20 ng/μl) by CuL-Cuphen in the agarose
gel electrophoresis. Concentration of CuL-Cuphen: Lane 0: 0 μM; Lane 1: 20
μM; Lane 2: 40 μM; Lane 3: 60 μM; Lane 4: 80 μM; Lane 5: 100 μM; Lane 6:
O donors from H
3
L. The other 4-coordinated Cu centre was 120 μM.
2
| J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
GSH depletion
reached 120 μM, the supercoiled DNA (Form I) disappeared,
and the amount of linear DNA (Form III) reached the maximum,
indicating that the supercoiled DNA was completely cleaved.
View Article Online
DOI: 10.1039/D0DT01742F
Next, we investigated the effect of GSH depletion caused by
CuL-Cuphen. In fact, abundant GSH in cancer cells would react
with •OH, resulting in reduced curative effect of CDT. However,
Cell uptake
2
+
Cu in CuL-Cuphen would reduce the concentration of GSH
which is beneficial for effective treatment. GSH levels were
evaluated by Ellman’s reagent. As shown in Fig. 2b, GSH was
treated by CuL-Cuphen for different time periods, the content
of GSH decreased significantly with the increase of the
treatment time. The relative intracellular content of GSH and
GSSG in MCF-7 cells were further evaluated by using GSH and
GSSG assay kit (Beyotime). As shown in Fig. 2c, the GSH/GSSG
ratio in untreated MCF-7 cells was dramatically decreased from
The cellular uptake of CuL-Cuphen in MCF-7 cells was studied by
ICP-MS in terms of Cu contents. The MCF-7 cells were incubated
in the medium containing 10 µM CuL-Cuphen for 4 h in a 37 C
incubator with 5% CO . The content of CuL-Cuphen in cells, and
2
important compartments including mitochondria and nucleus
was detected by inductively coupled plasma mass spectrometry
o
(ICP-MS) and shown in Figure 3a. The results shown that CuL-
Cuphen could be rapidly uptake by cancer cells and partially
localized in the nucleus and mitochondria, which were
~30.7 to ~4.6 after treatment with CuL-Cuphen (10 µM).
1
1
beneficial for the use of CuL-Cuphen in CDT.
According to the reported results, we proposed that GSH could
react with the Cu2+ ions in CuL-Cuphen to form a new adduct,
then converting the GSH to GSSG which would cause a
In vitro cytotoxicity
2
7
The cytotoxicity of CuL-Cuphen to cancer cells and normal cells
was then evaluated. The cell viability was determined by
reducing the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to purple formazan
product through the enzymatic activity of mitochondrial
dehydrogenase in the live cells. As showed in Fig. 3b, the
bimetallic CuL-Cuphen displayed significant cytotoxicity to 4T1,
A549, HepG2 and MCF-7 cells. The corresponding IC50 values
were about 3.3, 4.0, 3.5 and 3.2 µM respectively (Table S3 in
Supporting Information). The results were quite different from
the mononuclear complex CuL, which resulted only in a small
decrease in cell viability to the above cell lines (IC50 > 40 µM,
Table S3 in Supporting Information). Furthermore, as shown in
Fig. 3c, the results of live-dead cell staining experiment were
consistent with the cytotoxicity experiments. Interestingly, the
IC50 value of CuL-Cuphen to normal cells COS-7 was about
reduction in GSH content.
DNA banding and DNA cleavage
To investigate the interaction between CuL-Cuphen and CT-DNA,
ethidium bromide (EB) fluorescence quenching experiments
were performed. As shown in Fig. 2d, the fluorescent emission
of the EB-bound ct-DNA was effectively quenched upon the
addition of CuL-Cuphen. Based on the quenching plots, the
apparent binding constants (Kapp) of CuL-Cuphen to ct-DNA was
4
2
calculated to be 1.2 × 10 (R = 0.99, Fig. 2d). The relatively high
app value might be the result of a 1,10-phenanthroline ligand
K
was equipped on the skeleton of CuL-Cuphen, which the rigid
planar structure is more likely to form a strong π-π* staking
4
0
interaction with DNA base pairs. To further study the cleavage
ability of supercoiled plasmid pUC19 DNA by CuL-Cuphen,
agarose gel electrophoresis experiments were carried out. As
shown in Fig. 2e, when the concentration of CuL-Cuphen was
6
.3µM. The significantly higher cytotoxicity than that of cancer
Fig. 3 (a) Cell uptake and localization of CuL-Cuphen in MCF-7 cells after incubation for 4 h, which was determined by analysis of Cu2+ content using ICP-
MS. (b) Cell viability of 4T1, A549, HepG2, MCF-7 and COS7 cells subjected to CuL-Cuphen. (c) live/dead staining of MCF7 cells treated by CuL-Cuphen for 48
o
h at 37 C, 5% CO
2
. Scale bar: 100 µm.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Journal Name
View Article Online
DOI: 10.1039/D0DT01742F
be attributed to the CuL-Cuphen mediated Fenton-like reaction
with GSH depletion properties. Moreover, the generated
intracellular ·OH species with highly toxicity could direct destroy
DNA of cancer cells to give enhanced CDT.
Fig. 4 The intracellular ROS detection in MCF7 cell by DCFH-DA using laser
scanning confocal microscope. Scale bar: 20 µm.
2 2
cells may due to the lower GSH and H O content in normal cells,
3
8
which resulting in less ROS generation . We further examined
the influence of CuL-Cuphen on the intracellular ROS by using
2
,7’-dichlorodihydrofluorescein diacetates (DCFH-DA) as the
ROS probe. After the cells were treated with 20 µM of CuL-
Cuphen for 5 h, the ROS stress level in the MCF-7 cells
significantly increased (Fig. 4), indicating that CuL-Cuphen could
catalyze the Fenton-like reaction in the cell to generate a large
amount of ROS.
Cell cycle
The effect of CuL-Cuphen on the cell cycle was evaluated with a
fluorescent probe propidium iodide (PI), a commonly used
fluorescent agent for detecting double-stranded DNA. The
combination of PI and double-stranded DNA could produce
fluorescence, and the fluorescence intensity is proportional to
the content of double-stranded DNA. After the DNA in the cells
is stained with PI, the DNA content of the cells can be
determined by flow cytometry. As shown in Fig. 5a, with the
increase of CuL-Cuphen concentration, the number of cells in
G0/G1 phase increased from 45.81% to 57.41%, indicating that
CuL-Cuphen arrested the cell cycle in G0/G1 phase.
Fig. 5 (a) Cell cycle of MCF7 treated by CuL-Cuphen for 12 h using flow
cytometry. (b) Determination of apoptosis in MCF7 stained with Annexin
V/PI after treatment by CuL-Cuphen using flow cytometry.
Conclusions
Annexin V/PI double stain
In summary, we described the synthesis of a bimetallic Cu2+
complex (CuL-Cuphen) and the application as a highly effective
CDT reagent with GSH depletion properties for killing cancer
cells. CuL-Cuphen was efficiently uptake by cancer cells, which
Annexin V is a calcium ion-dependent phospholipid binding
protein widely distributed in the cytoplasm of eukaryotic cells.
It has a high affinity for phosphatidylserine (PS), and thus can
bind to the cell membrane of early apoptotic cells. While, PI can
stain necrotic cells or cells that lost cell membrane integrity
during late apoptosis, showing red fluorescence. As shown in Fig.
could be trigged by endogenous GSH in cancer cells to produce
+
Cu , then converting the overexpressed H
O
2 2
into more toxic
•OH. CuL-Cuphen showed efficient cell viability inhibition effect
5
b, after MCF-7 cells were treated with different concentrations
to cancer cells than normal cells. Cell cycle experiments
indicated that CuL-Cuphen arrested the cell cycle in G0/G1
phase, leading to efficient cell apoptosis rather than necrosis.
We believe this work will further inspire the development of
polynuclear metal complexes as more efficient CDT agents.
of CuL-Cuphen for 12 h, the number of apoptotic cells (including
early and late apoptosis) was increased from 5.4% (control) to
1
0.63% (4 μM) and 59.33% (8 μM). Noted that the number of
apoptotic cells were about 97.43% when further increasing the
concentration of CuL-Cuphen to 16 μM. It was clear that by
increasing the concentration of the complex while reducing the
treatment time compared with cell viability assay can also lead
cell apoptosis effectively. In consideration of the strong DNA
Experimental
affinity of CuL-Cuphen, the effective cell apoptosis results can Materials
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 5 of 8
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Journal Name
ARTICLE
Amino-5-bromobenzoic acid (Alfa Aesar, 98%), Ethyl Then, a THF solution (5 mL) of N,N,2,2-tetrVaiemw eArtthiclyelO-1n,li3ne-
chlorooxoacetate (Aladdin 98%), N,N,2,2–tetramethyl–1,3- propanediamine (1.563 g, 12 mmol) wasDaOdI:d1e0d.10d3r9o/Dp0wDiTse01t7o42aF
propanediamine (Aladdin 98%) 3-(4,5-dimethylthiazol-2-yl)-2,5- solution of the above monoamide (2.702 g, 10 mmol) in THF (25
diphenyltetrazolium bromide (MTT), calf thymus DNA (CT-DNA) mL) with constant stirring. Then the mixture was heated at 70
o
and Ethidium bromide (EB) (Sigma-Aldrich), (5,5'-Dithiobis-(2-
C and kept for 1.5 h. The resulting solid was filtered and
o
nitrobenzoic acid)) (DTNB, Aladdin 98%), pUC19 DNA (Thermo washed by water. The obtained solid was vacuum dried at 70 C
scientific). 2,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) for 12 h to afford the H
1
3
L. Yield: 87%. H NMR (400 MHz, C
5 5
D N)
was purchased from Macklin (Shanghai). Annexin V-FITC/ δ 13.83 (s, 1H), 9.79 (s, 1H), 9.02 (d, J = 8.7 Hz, 1H), 8.62 (s, 1H),
Propidium Iodide double staining cell apoptosis kit, cell cycle kit 7.70 (d, J = 8.2 Hz, 2H), 7.38 (s, 1H), 3.42 (d, J = 4.5 Hz, 2H), 2.25
1
3
6
and live & dead viability kit were purchased from KeyGEN (s, 5H), 2.16 (s, 2H), 0.90 (s, 6H). C NMR (400 MHz, DMSO-d ) δ
BioTECH. All other chemicals were commercially available and 170.36, 16199, 161.10, 140.90, 137.34, 136.18, 124.25, 123.42,
-
used. All chemicals were used as received unless otherwise 117.42, 71.25, 51.51, 49.59, 37.43, 25.85. HRMS m/z [M-H] :
1
13
-
noted. H and C NMR spectra were determined using a 398.0710 (calculated for C16
3 4
H21BrN O , 398.0715).
Magnet System 400’54 Ascend NMR spectra (Bruker). High
resolution mass spectrometric data were measured on a Q
Synthesis of CuL
Exactive mass spectrometric (Thermo scientific). The NaOH (40 mL, 1.25 M) was added to a solution of above H
fluorescence imaging of cells was carried out on FV 3000 mmol) in ethanol (50 mL) and water (10 mL) with stirring quickly
OLYMPUS) confocal microscope with 60× objective lens. Single for 15 min at ambient temperature. Then CuCl •2H O (10 mL, 1
3
L (10
(
2
2
crystal X-ray diffraction measurement was carried out on Bruker M) was dropwise added into the mixture with stirring for 3 h at
D8 Venture diffractometer. Annexin V-FITC/PI double staining room temperature. The resulting solid was filtered and washed
-1
and cell cycle measurement were carried out on Attune NxT by ethanol. Yield: 88%. IR (KBr ν/cm ): 1586.72 [Vas(COO) +
-
(
Thermo Fisher Scientific) flow cytometry. FT-IR spectra were V(C=O)]. HRMS m/z [M] : 460.9840 (calculated for
obtained as the KBr-pelleted samples on a 6700 spectrometer
Thermo Fisher Scientific). The inductively coupled plasma mass
spectrometry (ICP-MS) data were determined on a NexION
00D ICP-MS (Perkin Elmer). UV-visible (UV-vis) spectra were CuL (0.04838 g, 0.1 mmol) was dissolved in methanol (10 mL).
16 3 4
C H19BrCuN O , 460.9905).
(
Synthesis of CuL-Cuphen
3
measured on Cary 60 UV-Vis spectrophotometer. The Copper perchlorate (0.0371 g, 0.1 mmol) was dissolved in
fluorescence spectra were obtained on Cary Eclipse methanol (5 mL). The copper perchlorate solution was slowly
Fluorescence Spectrophotometer (Agilent techologies). Doubly added dropwise to the CuL solution, and vigorously stirred while
purified water (from Milli-Q systems) was used to prepare adding. Then, the mixture was heated to 60 ℃, with stirring for
buffers. The DNA concentration per nucleotide was determined 30 minutes. Then a methanol solution (5 ml) containing 1,10-
by absorption spectroscopy using the molar absorption phenanthroline (0.0198 g, 0.1 mmol) was added dropwise into
-
1
-1
32
coefficient (6600 M cm ) at 260 nm .
the mixture with stirring for another 5 h. The resulting green
solution was filtered and the filtrate was kept at room
temperature. Dark green crystals suitable for X-ray analysis
were obtained from the filtrate by slow evaporation for seven
Synthesis of Tetranuclear copper complexes
Synthesis of N-(2-carboxy-4-bromophenyl)-N’-[2,2–dimethyl -3-
dimethylamino)propyl]oxamide (H
-1
days. Yield: 80%. IR (KBr ν/cm ): 1626, [Vas(COO) + V(C=O)];
(
3
L)
-
1
-1
1
(
C
561, V(C=N). UV-Visible: λmax (nm) [εmax (M •cm )] : 272
The synthesis methods of the ligand refer to our previous
work . Briefly, ethyl chlorooxoacetate (1.638 g, 12 mmol) was
dissolved in tetrahydrofuran (THF) (8 mL) and then dropwise
96256). HRMS m/z [M]+: 703.9794 (calculated for
3
6
+
H27BrCu N O
28 2 5 4
, 703.9975).
added a THF solution (20 mL) containing 2-amino-5- Crystal structure determination and refinement
bromobenzoic acid (1.553 g, 10 mmol) under the ice-water bath.
Single crystal X-ray diffraction technique was used to
determinate the crystal structures of CuL-Cuphen. Single crystal
X-ray diffraction measurement was carried out on Bruker Smart
The mixture was stirred for 2-3 h at room temperature to
produce a large amount of white precipitate. Then 1 mL water
was added in the mixture with the constant stirring for 15 min.
The mixture was heated until the precipitate was dissolved, the
1
000 CCD area detector at 298 k using graphite monochromatic
MoKα radiation (λ = 0.71073 Å). A multi-scan spherical
absorption correction and phi and omega scans measurement
method were carried out in the experiment. The structure of
CuL-Cuphenwas solved by the directed method and followed by
o
solution was cooled at 0 C to reach a large number of white
crystals. The resulting solid was dried under vacuum to afford
1
the corresponding ethyl N-aryloxamate. Yield: 85%. H NMR
6
(400 MHz, DMSO-d ): δ 14.28 (s, 1H), 12.51 (s, 1H), 8.54 (d, J =
2
full matrix least-squares procedures on F using SHELXL-2018
9
4
.0 Hz, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.86 (dd, J = 9.0, 2.5 Hz, 1H),
.33 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). C NMR (400
Program package. Selected bond length and angles are given in
Table S1-S2.
1
3
MHz, DMSO-d
6
): δ 167.96, 159.60, 154.42, 138.54, 136.91,
-
1
3
33.49, 121.63, 119.04, 115.55, 62.96, 13.79. HRMS m/z [M-H] : DNA interaction studies
13.9667 (calculated for C11H BrNO , 313.9664).
9 5
The fluorescence spectral technique was performed to study
the binding interaction between CuL-Cuphen and CT-DNA. A
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Journal Name
certain amount of EB and CT-DNA were mixed together and and 1% Penicillin-Streptomycin Solution (Gibco) in 5% CO
2
View Article Online
o
DOI: 10.1039/D0DT01742F
incubated for 2 h in the dark to obtain EB-bound CT-DNA incubator with 37 C.
-2
solution. The solution of CuL-Cuphen (2 × 10 M) in DMSO was
freshly prepared before use. The solution of CuL-Cuphen was
Cellular uptake
titrated into the solutions of DNA/EB and then obtained the MCF-7 cells were seeded into a six-well plate at a density of 106
solutions with the different mole ratio of CuL-Cuphen to CT- cells and incubated for 12 h. Then the medium was replaced
DNA. Before measurements, the mixtures were well mixed and with fresh medium containing 10 µM CuL-Cuphen and the cells
o
incubated for 10 min at room temperature. The fluorescence were incubated for another 4 h in a 37 C incubator with 5%
spectra of EB binding CT-DNA were obtained in the range of CO
30-750 nm. PBS, then harvest the cells. Mitochondria and nucleus were
The quenching of EB bound to DNA by CuL-Cuphen is in isolated separately using the mitochondria/nuclear separation
2
. Discard the medium and wash the cells three times with
5
agreement with the linear Stern-Volmer equation:
=I+Ksv [CuL-Cuphen]
Where I
kit (KeyGEN BioTECH) according to the instruction manual. The
resulting mitochondria, nuclei and whole cells were digested
and I represent the fluorescence intensities in the with nitric acid, 30% hydrogen peroxide, and concentrated
I
0
0
absence and presence of CuL-Cuphen, respectively. Ksv is a hydrochloric acid. The content of copper in the nucleus,
linear Stern-Volmer quenching constant, [CuL-Cuphen] is the mitochondria and the whole cells was determined by ICP-MS.
concentration of CuL-Cuphen. In the quenching plot (Fig. 2d) of
In vitro cytotoxicity assay
0
I /I versus [CuL-Cuphen], Ksv is derived from the ratio of the
4
2
slope to intercept. The Ksv value was 1.2 × 10 (R = 0.99) for CuL- In this assay, about 5000 cells of 4T1, HepG2, A549, MCF-7 and
Cuphen.
COS-7 cells were seeded in each well of a 96-well culture plate
o
and incubated for 12 h in a 5% CO
2
incubator with 37 C. Then
DNA cleavage
different concentrations of CuL-Cuphen were added to the cells
-1
The solution of supercoiled pUC19 DNA (20 ng/mL) was medium (200 μL). After 48 h incubate, 20 μL MTT (5 mg mL ) in
incubated with the different concentration CuL-Cuphen for 12 phosphate buffer saline (PBS) was added into each well and
h in Tris-HCl buffer (10 mM Tris-HCl, 1 mM EDTA, PH = 7.6, total incubated for another 4 h. The purple formazan crystals
1
0 μL). Then 2 μL 6 x loading buffer was added into above DNA obtained were dissolved in 200 μL DMSO and the absorbance
and mixed evenly. Gel electrophoresis experiment was carried was measured at 490 nm using a Molecular Devices Spectra
out with 1.5% (Tris-Acetate EDTA (TAE) buffer) agarose gel at Max i3x plate reader and the cell viability was calculated by the
1
2
50 V in 1 x TAE buffer for 30 min. The agarose gel was stained following equation:
0 min with solution of Ethidium bromide (1 μg/mL) after Cell viability (%) = (ODsample - ODblank control / ODcontrol-ODblank control
)
electrophoresis, then analyzed with the FluorChem
Proteinsample) gel imaging system.
R
× 100%
(
The MTT assay was carried out in triplicates.
Detection the •OH using methylene blue (MB)
Live/dead cytotoxicity assay
A certain concentration of MB solution was prepared in 25 mM Cytotoxicity was detected by calcein AM and propidium iodide
NaHCO /5% CO buffer solution with 8 mM H and 100 µM (PI) dual-color fluorescence staining. MCF-7 cells were seeded
3
2
2 2
O
CuL-Cuphen. GSH and TEMPO were then added the mixing in a 35 mm cell culture dish and cultured overnight. The cells
o
system. The mixing system was incubated 30 min at 37 C, then were treated by CuL-Cuphen at different concentration for 48
absorbance of MB was measured by UV-vis spectroscopy.
h. Then AM and PI were added to the cell culture dish according
to the Manufacturer's instruction manual. Next the cells were
observed by single photon laser confocal microscopy with
GSH depletion
GSH levels were evaluated by Ellman’s reagent. Solutions with excitation wavelength of 495/530 nm, and emission wavelength
o
1
0
0 mM GSH and 20 µM CuL-Cuphen were incubated at 37 C for was collected from 520/620 nm.
-12 h. Then the samples were centrifuged at 12000 rpm for 10
min, and the supernatants were collected. DTNB solution was
added to the supernatant and co-incubated for 5 min at 25 C, MCF-7 cells were seeded in a 35 mm cell culture dish at a density
then the absorbance was measured with a microplate reader.
Determination of intracellular ROS
o
6
of 10 cells and cultured overnight. The cells were treated by 10
µM CuL-Cuphen for 5 h. Then 100 µM H
cell culture dish and cultured for another 0.5 h at 37 C, 5% CO
2
O
2
were added to the
Cell incubation
o
2
.
Mouse breast cancer cells (4T1 cells), human liver cancer cells The cells were washed three times with PBS then the cells were
HepG2 cells), human lung cancer cells (A549 cells), human stained with 2,7’-dichlorodihydrofluorescein diacetate (DCFH-
(
breast cancer cells (MCF-7 cells) and normal cells (COS-7) were DA) probe for 30 min. Next, the cells were washed three times
purchased from Institute of Basic Medical Sciences (IBMS) of the with PBS. Then the cells were observed by single photon laser
Chinese Academy of Medical Sciences. All the cells were confocal microscopy with excitation wavelength of 488 nm, and
incubation in Dulbecco’s modified Eagle medium (DMEM, Gibco) emission wavelength was collected from 510-550 nm.
supplemented with 10% fetal bovine serum (FBS, Invitrogen)
Determination of intracellular GSH and GSSG
6
| J. Name., 2012, 00, 1-3
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ARTICLE
7
.
Y. Liu, W. Zhen, Y. Wang, J. Liu, L. Jin, T. Zhang, S. Zhang, Y. Zhao, S. Song,
View Article Online
The intracellular GSH and GSSG were measured by GSH and
C. Li, J. Zhu, Y. Yang and H. Zhang, Angew. Chem., Int. Ed., 2019, 58,
DOI: 10.1039/D0DT01742F
GSSG Assay Kit (Beyotime). MCF-7 cells were seeded into a six-
2
407-2412.
6
well plate at a density of 10 cells and incubated for 12h. Then 8. Z. Zhou, J. Song, R. Tian, Z. Yang, G. Yu, L. Lin, G. Zhang, W. Fan, F. Zhang,
G. Niu, L. Nie and X. Chen, Angew. Chem., Int. Ed., 2017, 56, 6492-6496.
Q. Chen, J. Zhou, Z. Chen, Q. Luo, J. Xu and G. Song, ACS Appl. Mater.
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the medium was replaced with fresh medium containing CuL-
Cuphen (10 µM) and the cells were incubated for another 12 h
9
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in a 37 ℃ incubator with 5% CO
2
. The cells were collected by 10. C. Liu, D. Wang, S. Zhang, Y. Cheng, F. Yang, Y. Xing, T. Xu, H. Dong and
X. Zhang, ACS Nano, 2019, 13, 4267-4277.
centrifugation to obtain the cell precipitates. Protein removal
reagent M was added to the cell precipitates (30 µL/10 mg) and
1
1. H. Ranji-Burachaloo, F. Karimi, K. Xie, Q. Fu, P. A. Gurr, D. E. Dunstan and
G. G. Qiao, ACS Appl. Mater. Interfaces, 2017, 9, 33599-33608.
mixed well. After two rapid freeze-thaw cycles, the cell 12. Q. Chen, Y. Luo, W. Du, Z. Liu, S. Zhang, J. Yang, H. Yao, T. Liu, M. Ma and
H. Chen, ACS Appl. Mater. Interfaces, 2019, 11, 18133-18144.
precipitates were incubated on ice for 5 min., and then
centrifuged (10,000 g for 10 min) at 4 ℃. Take the supernatant
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3. S. Wang, Z. Wang, G. Yu, Z. Zhou, O. Jacobson, Y. Liu, Y. Ma, F. Zhang, Z.
Y. Chen and X. Chen, Adv. Sci., 2019, 6, 1801986.
for the determination of GSH and GSSG according to the 14. K. Wu, H. Zhong, G. Yang, C. Wu, J. Huang, G. Li, D. Ma and C. Leung,
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manufacturer's instructions.
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Cell cycle
MCF-7 cells were seeded into a six-well plate at a density of 1 ×
6
1
0 cells and incubated for 12 h. Then the medium was replaced
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Reddanna, Nanotoxicology, 2011, 5(4), 502-516.
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9. Y. Jiang, J. Li, Z. Zeng, C. Xie, Y. Lyu and K. Pu, Angew. Chem., Int. Ed.,
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were harvested. The cells were washed again and fixed with 70%
ethanol overnight. The cells were stained with PI according to
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2
2
9
Annexin V-FITC/PI double staining
MCF-7 cells were seeded in a six-well plate at a density of 106
and cultured overnight. The different concentrations (4 μM, 8
μM and 16 μM) of CuL-Cuphen were added to the six-well plate
and cultured for another 12 hours. Cells were stained with
Annexin V/PI according to the instructions and analysed by flow
cytometry.
2
2
2
2
2
3
3
3
3
3
3
Conflicts of interest
There are no conflicts to declare.
1
801733.
0. S. Gao, X. Lu, P. Zhu, H. Lin, L. Yu, H. Yao, C. Wei, Y. Chen and J. Shi, J.
Mater. Chem. B, 2019, 7, 3599-3609.
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and W. Bu, Mater. Horiz., 2019, 6, 369-374.
2. C. Zhang, W. Bu, D. Ni, S. Zhang, Q. Li, Z. Yao, J. Zhang, H. Yao, Z. Wang
and J. Shi, Angew. Chem., Int. Ed., 2016, 55, 2101-2106.
3. W. Zhang, J. Lu, X. Gao, P. Li, W. Zhang, Y. Ma, H. Wang and B. Tang,
Angew. Chem., Int. Ed., 2018, 57, 4891-4896.
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X. Qu, Angew. Chem., Int. Ed., 2016, 55, 11467-11471.
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Acknowledgements
This work was financially supported by the Weifang High-tech
Zone Science and Technology Plan Project (2019KJHM05) and
National Natural Science Foundation of China (21701019).
4
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Dalton Transactions
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View Article Online
DOI: 10.1039/D0DT01742F
Graphical Abstract
A bimetallic Cu(II) complex was developed as novel antitumor chemodynamic therapy
agent with glutathione depletion properties.