Arene Ruthenium(II) Complexes as Low-Toxicity Inhibitor against the Proliferation, Migration, and Invasion of MDA-MB-231 Cells through Binding and Stabilizing c-myc G-Quadruplex DNA

Article abstract of DOI:10.1021/acs.organomet.5b00820

Arene Ru(II) complexes have long been extensively studied as potential inhibitors against the proliferation of tumor cells, but their behavior against the migration and invasion of tumor cells needs further research. In this work, a series of arene Ru(II) complexes, (η⁶-C₆H₆)Ru(p-XPIP)Cl]Cl (X = H, 1; F, 2; Cl, 3; Br, 4; and I, 5), have been synthesized, and their inhibitory activity against the migration and invasion of MDA-MB-231 breast cancer cells have been investigated. It is found that all of these complexes exhibit excellent inhibitory activity (IC₅₀) against the growth of MDA-MB-231 breast cancer cells, and the value of IC₅₀ for 1, 2, 3, 4, and 5 is about >300, 52.6, 11.4, 45.5, and 59.1 μM, respectively. Further studies by wound-healing assay, FITC-geltain assay, and flow cytometry assay showed that 3 can apparently suppress the migration and invasion of MDA-MB-231 cells via the joint action of S-phase arrest and apoptosis. Moreover, the binding behavior of these arene Ru(II) complexes with c-myc G-quadruplex DNA has also been studied, and the results showed that these complexes can bind and stabilize c-myc G-quadruplex DNA in groove binding mode. Also, the low toxicity of 3 was confirmed by its low inhibitory activity against the growth of normal MCF-10A breast cells in vitro and the development of zebrafish embryos in vivo. In other words, these results indicated that synthetic arene Ru(II) complexes can be developed as low-toxicity agents against the proliferation, migration, and invasion of breast cancer cells.

Full text of DOI:10.1021/acs.organomet.5b00820

Article
pubs.acs.org/Organometallics
Arene Ruthenium(II) Complexes as Low-Toxicity Inhibitor against the
Proliferation, Migration, and Invasion of MDA-MB-231 Cells through
Binding and Stabilizing c‑myc G‑Quadruplex DNA
Qiong Wu,† Kangdi Zheng,‡ Siyan Liao,§ Yang Ding,‡ Yangqiu Li,*,† and Wenjie Mei*,‡
†Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, 510632, China
‡School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China
§School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou, 510180, China
S
* Supporting Information
ABSTRACT: Arene Ru(II) complexes have long been
extensively studied as potential inhibitors against the
proliferation of tumor cells, but their behavior against the
migration and invasion of tumor cells needs further research.
In this work, a series of arene Ru(II) complexes, (η6-
C6H6)Ru(p-XPIP)Cl]Cl (X = H, 1; F, 2; Cl, 3; Br, 4; and I,
5), have been synthesized, and their inhibitory activity against
the migration and invasion of MDA-MB-231 breast cancer
cells have been investigated. It is found that all of these
complexes exhibit excellent inhibitory activity (IC50) against
the growth of MDA-MB-231 breast cancer cells, and the value
of IC50 for 1, 2, 3, 4, and 5 is about >300, 52.6, 11.4, 45.5, and
59.1 μM, respectively. Further studies by wound-healing assay,
FITC-geltain assay, and flow cytometry assay showed that 3 can apparently suppress the migration and invasion of MDA-MB-
231 cells via the joint action of S-phase arrest and apoptosis. Moreover, the binding behavior of these arene Ru(II) complexes
with c-myc G-quadruplex DNA has also been studied, and the results showed that these complexes can bind and stabilize c-myc G-
quadruplex DNA in groove binding mode. Also, the low toxicity of 3 was confirmed by its low inhibitory activity against the
growth of normal MCF-10A breast cells in vitro and the development of zebrafish embryos in vivo. In other words, these results
indicated that synthetic arene Ru(II) complexes can be developed as low-toxicity agents against the proliferation, migration, and
invasion of breast cancer cells.
Ru(II) complex RM175 ([(η6-biphenyl)Ru(ethylenediamine)-
INTRODUCTION
■
Cl]+) inhibited tumor metastasis in vivo and blocked migration
Breast cancer has become an issue of growing concern in both
research and clinical practice, as it is prevalent among younger
women. Despite the advances that have taken place in the past
decade, including the development of novel molecular targeted
and invasion through enhancing cell−cell readhesion and
down-regulated the matrix metalloproteinases (MMPs).4
On the other hand, the c-myc oncogene is related to the
proliferation, cell cycle arrest, metastasis, and invasion of tumor
agents, cytotoxic chemotherapy remains the mainstay of breast
cancer treatment.1 It is so difficult to treat breast cancer owing
cells; this gene is located near a promoter region and is
overexpressed in tumor cells, but has low expression in normal
to the heterogeneity of the disease, especially for triple-negative
cells.5 Recent studies showed that the G-rich promoter of the c-
patients.2 Consequently, the search for effective drugs to inhibit
myc oncogene can form a G-quadruplex structure via a
the proliferation and invasion of breast cancer has become a
challenge. In recent years, arene Ru(II) complexes have
Hoogesten hydrogen bond in the presence of K+ and Na+ ions.6
Complexes that can bind and stabilize G-quadruplex DNA
attracted more attention in chemotherapy because of their
usually exhibit excellent inhibitory activity against the growth of
low toxicity and excellent inhibitory activity against tumor cell
tumor cells. For example, porphyrin and quindoline inhibit
migration and invasion. Numerous arene Ru complexes have
various tumor cells by stabilizing the c-myc G-quadruplex
already been designed. The in vitro and in vivo inhibitory
DNA.7 Therrien reported that arene Ru(II) complexes with
activities of these complexes, as well as their underlying
porphyrin can strongly bind to tetrastranded DNA structures to
mechanisms and binding behavior to DNA molecules, have
inhibit the telomerase enzyme.8 Barone found that Schiff-base
been extensively investigated. RAPTA-B ([Ru(η6-C6H6)(pta)-
Cl2]) and RAPTA-C ([Ru(η6-p-C6H4MeiPr)(pta)Cl2]) blocked
tumor metastasis of a CBA mouse model for human breast
cancer by preventing angiogenesis.3 In addition, the arene
Received: October 1, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.organomet.5b00820
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Article
from Sangon Biotech (Shanghai) Co., Ltd. The G-quadruplex
conformation was formed by denaturation at 90 °C for 5 min
followed by renaturation at 4 °C for 24 h, as stipulated by methods in
other studies. All aqueous solutions were prepared with double-
distilled water.
metal (Ni, Cu, and Zn) complexes can selectively bind to G4
DNA and stabilize G4 structures.9 The latest studies by our
team also found that arene Ru(II) complexes greatly inhibit the
migration and invasion of breast cancer cells by blocking the
formation of invadopodia, but these complexes have low
toxicity to normal cells.10 Moreover, these complexes can bind
and stabilize the G-quadruplex structure of c-myc, thereby
causing the down-regulation of c-myc and the induction of S-
phase arrest in tumor cells.11
Instruments. The arene Ru(II) complexes were synthesized using
an Anton Paar GmbH monowave 300 microwave reactor. The
elemental analyses for C, H, and N were performed with a Carlo-Erba
CHNO-S microanalyzer. The IR spectra (KBr disk, 400−4000 cm−1)
1
were obtained with a PerkinElmer 1320 spectrometer. The H NMR
In the current study, a class of arene Ru(II) complexes of (η6-
C6H6)Ru(p-XPIP)Cl]Cl with X = 1 (H), 2 (F), 3 (Cl), 4 (Br),
and 5 (I) were synthesized via microwave irradiation under
controlled temperatures (Figure 1). Single-crystal X-ray
and 13C NMR spectra were recorded in DMSO-d6 with a Bruker
DRX2500 spectrometer operating at room temperature. The
electronic absorption spectra were recorded with a Shimadzu UV-
2550 spectrophotometer. The steady-state emission spectra were
recorded with an RF-5301 fluorescence spectrophotometer. The
circular dichroism (CD) spectra were recorded with a Jasco J810 CD
spectrophotometer.
Synthesis of 2-Phenylimidazole[4,5f ][1,10]phenanthroline
(PIP). PIP was prepared by a method similar to that described in
the literature with some modifications.12 Phenanthroline-5,6-dione
(347 mg, 1.6 mmol), benzaldehyde (169.6 mg, 1.6 mmol), and
ammonium acetate (2.53 g) were dissolved in 20 mL of acetic acid.
The mixture was irradiated under microwave at 110 °C for 30 min.
Subsequently, 20 mL of water was added, and the pH value was
adjusted to 7.0 by adding ammonia at room temperature. A large
amount of yellow precipitate was obtained after filtration, and this
precipitate was dried under vacuum. The products were purified by
silica gel chromatography with ethanol as the eluent to obtain the
desired compound with a yield of 93.2%.
Synthesis of 2-(4-Fluorophenyl)imidazole[4,5f ][1,10]-
phenanthroline (p-FPIP). p-FPIP was prepared from 1,10-
phenanthroline-5,6-dione (347 mg, 1.6 mmol) and 4-fluorobenzalde-
hyde (198 mg, 1.6 mmol) using a method similar to that described in
the literature. The yield was 95.6%.
Synthesis of 2-(4-Chlorophenyl)imidazole[4,5f ][1,10]-
phenanthroline (p-ClPIP). p-ClPIP was prepared from 1,10-
phenanthroline-5,6-dione (347 mg, 1.6 mmol) and 4-chlorobenzalde-
hyde (224 mg, 1.6 mmol) using a method similar to that described in
the literature. The yield was 91.7%.
Synthesis of 2-(4-Bromophenyl)imidazole[4,5f ][1,10]-
phenanthroline (p-BrPIP). p-BrPIP was prepared from 1,10-
phenanthroline-5,6-dione (347 mg, 1.6 mmol) and 4-bromobenzalde-
hyde (294 mg, 1.6 mmol) using a method similar to that described in
the literature. The yield was 92.1%.
Figure 1. (A) Synthetic arene Ru(II) complexes coordinated by
phenanthrolineimidazole ligands. The ball-and-stick diagrams of the
arene Ru 3 (B) and 4 (C). For clarity, the solvent molecules and the
counteranions are omitted. CCDC nos.: 1030971 (3); 1030972 (4).
Synthesis of 2-(4-Iodophenyl)imidazole[4,5f ][1,10]-
phenanthroline (p-IPIP). p-IPIP was prepared from 1,10-phenan-
throline-5,6-dione (347 mg, 1.6 mmol) and 4-iodobenzaldehyde
(371.1 mg, 1.6 mmol) using a method similar to that described in the
literature. The yield was 93.2%.
diffraction analysis was conducted to confirm that these
complexes have a typical “piano stool” structure. These
complexes can bind to the c-myc G-quadruplex DNA in the
groove-binding mode, with the following levels of binding
affinity: 1 < 2 < 3 > 4 > 5. Further study showed that these
complexes, especially 3, can inhibit the growth, migration, and
invasion of MDA-MB-231 cells through the joint action of S-
phase arrest and apoptosis. The low toxicity on developing
zebrafish embryos was also investigated in vivo. Therefore, these
arene Ru(II) complexes can be developed into potential low-
toxicity agents against the metastasis of breast cancer for
chemotherapy.
Synthesis of the Arene Ru Complex [(η6-C6H6)Ru(PIP)Cl]Cl
(1). 1 was prepared according to the literature, but with some
modifications.13 A mixture of [(C6H6)RuCl2]Cl2 (75 mg, 0.15 mmol)
and phenanthroline derivatives (88.8 mg, 0.3 mmol) in dichloro-
methane (20 mL) was heated at 60 °C for 30 min under microwave
irradiation. A yellow precipitate was obtained after rotary evaporation,
and this precipitate was purified by recrystallization from distilled
water. The yield was 138.4 mg (90.3%). ESI-MS (in MeOH, m/z):
525.4 ([M − Cl]+). Anal. Calcd for C25H18Cl2N4Ru·4H2O·CH3OH:
48.00 C, 4.65 H, 8.61 N. Found: 47.59 C, 3.93 H, 8.66 N. UV−vis in
MeOH [λmax, nm (ε, × 104 M−1 cm−1)]: 253 (4.34), 293 (2.69). IR (in
KBr; νmax, cm−1): 3419.89, 3059.49, 2911.03, 1622.31, 1549.82,
1459.29, 1435.44, 1407.28, 1365.08, 1104.63, 810.28, 772.63, 704.29,
627.82. 1H NMR (500 MHz, DMSO): δ 9.95 (d, J = 5.2 Hz, 2H), 9.39
(s, 2H), 8.44 (J = 7.5 Hz, 2H), 8.17 (dd, 2H), 7.61 (t, J = 7.4 Hz, 2H),
7.55 (t, J = 7.2 Hz, 1H), 6.34 (s, 6H). 13C NMR (126 MHz, DMSO):
δ 154.35 (s), 53.52 (s), 143.70 (s), 133.13 (s), 130.67 (s), 130.03 (s),
129.53 (s), 129.10 (s), 128.54 (s), 127.18 (s), 126.43 (s).
MATERIALS AND METHODS
■
Chemicals. Ruthenium(III) chloride hydrate was obtained from
Mitsuwa Chemicals. 1,10-Phenanthroline monohydrate, 1,3-cyclo-
hexadiene, benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzalde-
hyde, 4-bromobenzaldehyde, and 4-iodobenzaldehyde were purchased
from Aldrich. All chemicals, including the solvents, were obtained from
commercial vendors and used as received. 1,10-Phenanthroline-5,6-
diode was prepared by a similar method reported in literature. The
binuclear arene Ru(II) complex [(η6-C6H6)RuCl2]2 was prepared
according to the literature. The c-myc G-quadruplex DNA (5′-
TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′) was purchased
Synthesis of the Arene Ru Complex [(η6-C6H6)Ru(p-FPIP)Cl]Cl
(2). 2 was prepared using the method described above, but with p-
FPIP (94.2 mg, 0.3 mmol) instead of PIP. The yield was 144.1 mg
B
DOI: 10.1021/acs.organomet.5b00820
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Article
(90.8%). ESI-MS (in MeOH, m/z): 529.01 ([M − Cl]+). Anal. Calcd
for C25H17FCl2N4Ru·3H2O·CH3OH: 47.51 C, 4.58 H, 8.21 N. Found:
47.21 C, 3.42 H, 8.23 N. UV−vis in MeOH [λmax, nm (ε, × 104 M−1
cm−1)]: 279 (4.17). IR (in KBr; νmax, cm−1): 3367.25, 3040.54,
2929.72, 2894.18, 2854.55, 2814.15, 2768.62, 2692.98, 1623.98,
1601.05, 1508.91, 1473.49, 1363.55, 1322.12, 1276.81, 1200.16,
Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines
of MDA-MB-231, MCF-7, HepG2, and EC-1 were maintained in
DMEM with fetal bovine serum (10%), penicillin (100 units/mL), and
streptomycin (50 units/mL) at 37 °C in a CO2 incubator (95%
relative humidity, 5% CO2). MCF-10A cells were maintained in
DMEM with horse serum (10%), penicillin (100 units/mL),
streptomycin (50 units/mL), EGF (20 ng/mL), hydrocortisone
(0.5ug/mL), cholera toxin (100 ng/mL), and insulin (10ug/mL) at
37 °C in a CO2 incubator (95% relative humidity, 5% CO2).
MTT Assay. Cell viability was determined by measuring the ability
of cells to transform MTT to a purple formazan dye. Cells were seeded
in 96-well tissue culture plates for 24 h. The cells were then incubated
with the test compounds at different concentrations for 72 h. After
incubation, 20 μL of the MTT solution (5 mg mL−1) in phosphate-
buffered saline was added to each well, followed by incubation for an
additional 5 h.15 The medium was aspirated and replaced with 150 μL
of DMSO per well to dissolve the formed formazan salt. The color
intensity of the formazan solution, which reflects the cell growth
conditions, was measured at 570 nm using a microplate spectropho-
tometer (SpectroAmax 250).
Wound-Healing Assay. Cells were seeded in six-well tissue
culture plates, which were respectively marked on the back (1 × 105
cells per well) until the monolayer cells covered more than 80% of the
bottom of the culture plate. A line was then scratched on the culture
using a tip (200 μL pipet) orthogonal to the mark on the plate.
Subsequently, these cells were incubated with the test compounds at
different concentrations (0 and 10 μM) for 48 h. Migrating cells were
observed in the same visual field every 12 h for 2 d.16 Using Slidebook
and Excel software, the average migration rate and the end-to-end
distance of cell trajectory were calculated based on 10 fields of view for
each cell type.
Fluorescein Isothiocyanate (FITC)-Conjugated Gelatin In-
vasion Assay. The FITC-gelatin invasion assay was performed
according to the manufacturer’s instructions (Invitrogen). Briefly,
coverslips (18 mm in diameter) were coated with 50 μg mL−1 poly-L-
lysine for 20 min at room temperature, washed with phosphate-
buffered saline (PBS), fixed with 0.5% glutaraldehyde for 15 min, and
rewashed with PBS three times. After washing, the coverslips were
inverted on a drop of 0.2% FITC-conjugated gelatin in PBS containing
2.0% sucrose, incubated for 10 min at room temperature, washed with
PBS thrice, quenched with sodium borohydride (5 mg mL−1) for 3
min, and finally incubated in 2 mL of complete medium for 2 h.17
Cells (2 × 105 per well) with different concentrations of 3 (0, 1, 2, and
5 μM) were plated onto the FITC-gelatin-coated coverslips and
incubated at 37 °C for 12 h. The FITC-gelatin degradation status was
evaluated and photographed with a laser confocal microscope.
Flow Cytometry Analysis. Cells were seeded in six-well tissue
culture plates (1 × 105 cells per well), and the apoptosis rate and the
cell cycle arrest were analyzed by flow cytometry as previously
described.16 After incubating with different concentration of 3 (0, 10,
20, and 40 μM) for 24 h, cells were trypsinized, washed with PBS, and
fixed with 70% ethanol overnight at 4 °C. The fixed cells were washed
with PBS and stained with propidium iodide (PI) for 15 min in the
dark, and the cell cycle arrest was analyzed with an Epics XL-MCL
flow cytometer (Beckman Coulter, Miami, FL, USA). Treated or
untreated cells were trypsinized, washed with PBS, and costained with
annexin-V and PI for 10 min, respectively. The apoptosis of cells was
analyzed with an Epics XL-MCL flow cytometer (Beckman Coulter).
Molecular Docking. The theoretical calculation of the binding
mode and binding site of arene Ru(II) complexes in c-myc G-
quadruplex DNA was carried out by using the Lamarckian genetic
algorithm local search method with Auto-Dock 4.2.A as previously
described. Only chain A was maintained after the removal of other
subunits form the crystallographic structure of the c-myc G-quadruplex
DNA downloaded from the Protein Data Bank (PDBID: 2L7 V), and
the Gasteiger charge and other parameters were assigned by using
AutoDock tools. The size of the grid box was set to 126 × 126 × 126
points and centered with (x, y, z) = (2.579, −0.627, and −4.749) on c-
myc G-quadruplex DNA. A total of 50 separate dockings were
performed with maximum energy evaluations up to 2.5 × 107. The
1
1145.57, 1105.58, 1082.08, 813.12, 722.28, 627.48, 549.5. H NMR
(500 MHz, DMSO-d6, δ/ppm): 9.93 (dd, J = 5.3, 1.1 Hz, 2H), 9.32 (s,
2H), 8.46 (dd, J = 8.9, 5.4 Hz, 2H), 8.16 (dd, J = 8.3, 5.3 Hz, 2H), 7.46
(t, J = 8.9 Hz, 2H), 6.31 (s, 6H). 13C NMR (126 MHz, DMSO): δ
155.94 (s), 153.97 (s), 145.24 (s), 134.66 (s), 131.11 (s), 131.07 (s),
128.03 (s), 127.96 (s), 118.14 (s), 117.98 (s), 88.79 (s).
Synthesis of the Arene Ru Complex [(η6-C6H6)Ru(p-ClPIP)Cl]
Cl (3). 3 was prepared in a similar method to that described above, but
with p-ClPIP (99.3 mg, 0.3 mmol) in the place of PIP. The yield was
147.8 mg (90.4%). ESI-MS (in MeOH, m/z): 546.1 ([M − Cl]+).
Anal. Calcd for C25H17Cl3N4Ru·5H2O: 44.75 C, 4.06 H, 8.35 N.
Found: 44.88 C, 3.73 H, 8.62 N. UV−visible in MeOH [λmax, nm (ε, ×
104 M−1 cm−1)]: 283 (3.37). IR (in KBr; νmax, cm−1): 3419.05,
3054.23, 1623.57, 1607.86, 1508.19, 1475.49, 1463.02, 1453.52,
1421.22, 1405.86, 1364.57, 1319.22, 1101.76, 950.89, 836.23, 809.5,
1
723.76. H NMR (500 MHz, DMSO-d6, δ/ppm): 14.73 (s, 1H), 9.96
(dd, J = 8.5, 1.5 Hz 2H), 9.36 (s, 2H), 9.21 (s, 2H), 8.39 (d, J = 8.6
Hz, 2H), 8.22 (s, 2H), 7.74 (d, J = 8.6 Hz, 2H), 6.33 (s, 6H). 13C
NMR (126 MHz, DMSO): δ 148.76 (s), 144.25 (s), 143.51 (s),
132.51 (s), 129.82 (s), 129.61 (s), 129.26 (s), 128.49 (s), 125.85 (s),
115.69 (s), 87.13 (s).
Synthesis of the Arene Ru Complex [(η6-C6H6)Ru(p-BrPIP)Cl]
Cl (4). 4 was prepared in a similar method to that described above, but
with p-BrPIP (112.2 mg, 0.3 mmol) in the place of PIP. The yield was
100.8 mg (89.8%). ESI-MS (in MeOH, m/z): 590.9 ([M − Cl]+).
Anal. Calcd for C25H17Cl2BrN4Ru·2H2O·CH3OH: 45.04 C, 3.63 H,
8.08 N. Found: 45.24 C, 3.27 H, 8.01 N. UV−vis in MeOH [λmax, nm
(ε, × 104 M−1 cm−1)]: 281 (3.03). IR (in KBr; νmax, cm−1): 3368.04,
3036.31, 2928.99, 2853.53, 2768.92, 2691.32, 1626.69, 1603.49,
1460.40, 1405.55, 1364.18, 1303.2, 1069.4, 1009.05, 832.2, 812.32,
727.28, 627.11. 1H NMR (500 MHz, DMSO-d6, δ, ppm): 9.93 (dd, J =
5.3, 1.1 Hz, 2H), 8.31 (d, J = 8.6 Hz, 2H), 8.18 (dd, J = 8.1, 5.5 Hz,
2H), 7.84 (d, J = 8.6 Hz, 2H), 7.34 (s, 2H), 6.30 (s, 6H). 13C NMR
(126 MHz, DMSO): δ 150.45 (s), 133.93 (s), 131.31 (s), 130.17 (s),
127.35 (s), 124.51 (s), 88.59 (s).
Synthesis of Arene Ru Complexes [(η6-C6H6)Ru(p-IPIP)Cl]Cl
(5). 5 was prepared as described above, but with p-IPIP (126.2 mg, 0.3
mmol) in the place of PIP. The yield was 168.7 mg (88.3%). ESI-MS
(in MeOH), m/z): 636.9 ([M − Cl]+). Anal. Calcd for
C25H17Cl2IN4Ru·10H2O·17CH3OH: 36.10 C, 7.57 H, 4.01 N.
Found: 36.42 C, 3.56 H, 4.02 N. UV−vis in MeOH [λmax, nm (ε, ×
104 M−1 cm−1)]: 284 (3.16). IR (in KBr; νmax, cm−1): 3423.78,
2922.01, 1627.28, 1431.97, 1384.49, 1261.01, 1026.95, 803.2, 721.25,
1
535.09. H NMR (500 MHz, DMSO-d6, δ, ppm): δ 9.94 (d, J = 4.4
Hz, 2H), 9.33 (s, 2H), 8.19 (dd, 4H), 8.01 (t, J = 22.4 Hz, 2H), 6.34
(s, 6H). 13C NMR (101 MHz, DMSO): δ 193.25 (s), 153.20 (s),
143.30 (s), 138.64 (s), 137.81 (s), 136.59 (s), 132.77 (s), 131.40 (s),
128.89 (s), 125.62 (s), 88.13 (s).
X-ray Crystallography. The single crystals of 3 and 4 were
obtained by dissolving the powders in mixed solutions of distilled
water and dimethylformamide. X-ray diffraction measurements were
performed on a Rigaku R-AXIS SPIDER image plate diffractometer
with graphite-monochromated Mo Kα radiation (λ = 0.710 73 Å).
Absorption correction was applied with the SADABS program.14 The
structured solution and the full-matrix least-squares refinement based
on F2 for 3 and 4 were obtained with the SHELXS 97 and SHELXL 97
program packages, respectively. Anisotropic thermal parameters were
applied to all non-hydrogen atoms. All hydrogen atoms were included
in the calculated positions and refined with isotropic thermal
parameters based on those of the parent atoms.
Cell Lines and Culture. Human cancer cell lines, including human
breast cancer MDA-MB-231, human breast adenocarcinoma MCF-7,
human hepatocarcinoma HepG2, human esophageal carcinoma EC-1,
and human breast epithelial MCF-10A were purchased from American
C
DOI: 10.1021/acs.organomet.5b00820
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Table 1. Cytotoxic Effects of Arene Ru(II) Complexes on Human Cancer and Normal Cell Lines and the Corresponding
Lipophilicity
IC50/μM
compd
MDA-MB-231
MCF-7
>300
EC-1
HepG2
MCF-10A
log P
1
>300
289.3 13.4
92.8 5.3
137.4 2.6
68.2 3.5
179.3 3.2
13.6 1.4
−1.26
−1.16
−0.14
0.02
2
52.6 1.3
11.4 2.8
45.5 3.4
59.1 1.4
36.1 4.7
297.9 7.1
88.7 3.1
194.5 4.8
>300
>300
3
105.7 4.7
36.4 8.3
209.3 10.7
4
5
−0.474
−0.412
cisplatin
3.0 4.4
174.1 8.2
most probable binding conformation was selected according to the
most cluster members and the lowest binding free energy.18
UV−Vis Spectra. The absorption titration of the Ru(II) complex
in Tris-HCl buffer was performed with a fixed complex concentration
to which increments of the DNA stock solution were added.19 In
general, the absorption spectra of arene Ru(II) complexes 1, 2, 3, 4,
and 5 were recorded with increasing amounts of c-myc G-quadruplex
DNA until the absorbance intensity of the arene Ru(II) complexes
became constant. The concentration of the complex solution was 20
μM, and the c-myc G-quadruplex DNA was added in increments. The
complex−DNA solutions were incubated for 3 min before the
absorption spectra were recorded. The intrinsic binding constant Kb of
the arene Ru(II) complex to DNA was calculated from the following
equation:
Ecotoxicology Test of Zebrafish Embryos. Zebrafish embryos
were provided by the Southern Medical University. Zebrafish embryos
were incubated in 24-well plates with 1 mL solutions containing
different concentrations (0, 2, 4, 8, 16, and 32 μM) of complex 3 in
water. The hatching and growth of the zebrafish embryos without and
with complex 3 were observed every 24 h with an inverted
microscopy.21 The relevant ethical protocols used for the in vivo
study for zebrafish embryos were followed by the relevant laws.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Arene Ru(II) complexes
1 to 5 were prepared from [(C6H6)RuCl2]2 and the respective
ligands (PIP, p-FPIP, p-ClPIP, p-BrPIP, and p-IPIP) at 60 °C
under microwave irradiation for 30 min in a Pyrex vessel with a
high yield range of 88−91%, which was markedly higher than
that in conventional synthesis methods.22 The crystallite is
prepared through dissolvent crystallization. The molecular
structure of the complexes was established by single-crystal X-
ray structural analysis of the chloride salts (Figure 1). The
selected bond distances and bond angles are listed in Tables S1
and S2 of the Supporting Information. The arene Ru(II)
complexes adopt a classic “piano stool” structure, as evidenced
by the ∼90° bond angles for N(1)−Ru(1)−Cl(1) (85.54(13)°)
and N(2)−Ru(1)−Cl(1) (85.14(11)°) of 2 and N(3)−Ru(1)−
Cl(1) (85.39(14)°) and N(4)−Ru(1)−Cl(1) (84.32(12)°) of
3. The Ru(II) atom was bound to the benzene ring, with an
average Ru−C distance of 2.183(7) and 2.181(7) Å, whereas
the average distance of Ru(II) to the two chelating nitrogen
atoms was 2.089(5) and 2.082(5) Å, respectively. The clear
characterization of the structure of the complexes supported the
elucidation of its possible anticancer mechanisms.23
(εa − εf)/(εb − εf) = [b − (b2 − 2K2Ct[DNA/S)]1/2 /2KCt
(1)
b = 1 + KCt + K/2S
(2)
EB-Quenching Experiments. Fluorescence quenching of the
ethidium bromide (EB) + c-myc G-quadruplex system can be used for
a compound with an affinity to DNA regardless of its binding mode.
This method measures only the ability of a compound to affect the EB
fluorescence intensity in the EB + c-myc G-quadruplex system. Two
mechanisms have been proposed to account for the quenching, as
follows: the replacement of EB fluorophores and/or electron transfer.
The EB-DNA complex excited at 530 nm showed strong fluorescence
at 600 nm.15 According to the quenching curve, we can draw the
preliminary conclusion that the complex can competitively bind to
DNA with EB.
CD Spectra. To obtain further information, we recorded the CD
spectra of DNA modified by complex 3. The respective CD spectral
characteristics were compared for CT-DNA in the absence and
presence of complex 3. Complex 3 has no intrinsic CD signals because
it is achiral; thus, any CD signal at higher than 300 nm can be
attributed to the interaction of this complex with DNA.13 This
increasing reduction was similar to that observed when DNA was
under identical conditions modified by cisplatin or its ineffective
isomer, transplatin.
CD Melting Point. Typical CD melting curves were obtained by
following the change in their ellipticity as a function of temperature at
262 or 292 nm. The solutions were heated at a rate of 0.1 °C min−1
using free strained quartz cuvettes with a path length of 0.1 or 1.0 cm,
thereby resulting in the collection of two data points at C. The analysis
of the CD melting curves was performed with standard procedures.10
ESI-MS Analysis. Several noncovalent interactions between nucleic
acids and small ligands are biologically important processes. ESI-MS is
recognized as a useful tool in the characterization of such binding. The
stoichiometry and selectivity of the binding of small molecules to
oligonucleotides can usually be obtained with minimal sample
consumption. For G-quadruplex DNA, the mass spectra are usually
obtained with an LCQ ion trap mass spectrometer equipped with a
heated capillary electrospray source or a Q-TOF mass spectrometer
equipped with a Z-spray source. Both instruments can be operated in
the negative ion mode.20
Inhibitory Activities of Arene Ru Complexes in Vitro.
The antiproliferative activities of the synthetic Ru(II)
complexes against various human cancer cell lines and normal
MCF-10A human cells were evaluated by using the MTT assay.
The inhibitory activities (IC50) of these synthetic Ru(II)
complexes after 72 h of treatment are summarized in Table 1.
As shown in Table 1, the antiproliferative effects of these
complexes are cell-line specific. The most active complex (3)
displayed the highest inhibitory activity against MDA-MB-231
human breast cancer cells with an IC50 value of 11.4 μM. This
value is approximately 3 times lower than that of cisplatin.24
3
showed low toxicity to normal MCF-10A human cells with an
IC50 value of approximately 209.3 μM.
Lipophilicity often plays a major role in biology. Thus, we
determined the lipophilicity partition coefficient. The lip-
ophilicity partition coefficients for 1, 2, 3, 4, and 5 were
approximately −1.26, −1.16, −0.14, 0.02, and −0.474,
respectively. This result is reasonable given that molecular
polarity changes with the size of the atom. Therefore, we
concluded that atomic size plays a key role in DNA-binding
D
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Figure 2. Inhibition of migration and invasion of MDA-MB-231 cells by 3 in vitro. (A) The wound-healing assay was used to evaluate the migration
of MDA-MB-231 cells after treatment with 3 (0, 2, and 4 μM) and DMEM without FBS. Cells were wounded and monitored with a microscope
every 12 h. The migration was determined by the rate of cells filling the scratched area. (B) The invasion of MDA-MB-231 cells was blocked by 3 (2
μM). The whole view of many MDA-MB-231 cells, the number of black holes observed without 3, and those treated with 3. Cell cytoskeletons were
dyed by rhodamine-conjugated phalloidin. The dark area in the FITC-gelatin was identified as the position degraded by MDA-MB-231 cells. (C)
Wound-healing rate of MDA-MB-231 cells induced by 3. (D) Number of invasive cells reduced by 3.
behavior and in the anticancer activity of these arene Ru(II)
complexes.
and invasion of MDA-MB-231 cells was further evaluated by
wound-healing and FITC-gelatin assays (Figure 2).
Migration and Invasion of MDA-MB-231 Cells Is
Inhibited by the Joint Action of S-Phase Arrest and
Apoptosis. The inhibitory activity of 3 against the migration
As shown in Figure 2A, MDA-MB-231 cells treated without
drugs showed an obvious decrease in the distance of wound
closure at 72 h, but with the addition of 3, a notable inhibition
E
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Figure 3. (A) S-phase arrest of MDA-MB-231 cells induced by 3. (B) Apoptosis of MDA-MB-231 cells induced by 3.
Figure 4. Binding mode (A) and binding site (B) of 1 (yellow), 2 (cyan), 3 (orange), and 4 (green) with c-myc G4 DNA calculated by molecular
docking.
of wound closure was observed; the treatment had a dosage-
dependent effect compared with the control cells, which
spontaneously migrated as the distance obviously decreased.
Less than confluent cultures showed the decreasing wound-
healing rate of MDA-MB-231 cells with increasing concen-
tration of 3, thereby indicating that 3 effectively inhibited the
migration of MDA-MB-231 cells even at low concentrations
(Figure 2C).25 The FITC-gelatin invasion assay is usually
applied to observe the number of invasive tumor cells. In
general, cells with high invasion rates can form invadopodia to
release MMPs, which degrade the FITC-gelatin. Consequently,
dark holes (nonfluorescent areas) can be observed in the FITC-
gelatin. The higher number of dark holes implied greater
invasiveness of the tumor cells. As shown in Figure 2B,
numerous black holes could be counted in the FITC-gelatin for
cells that were not treated with 3. After exposure to 3, the
number of black holes obviously decreased, thereby indicating
that the invasive effect of MDA-MB-231 cells was markedly
inhibited.26 These results proved that arene Ru complexes can
effectively inhibit the invasion of MDA-MB-231 cells.27
inhibition induced by 3 was caused by the joint action of the
S-phase arrest and apoptosis of breast cancer cells (Figure 3A
and B).
Binding Studies with c-myc G-Quadruplex DNA.
Moreover, the promoter of the c-myc oncogene plays a key
role in regulating the proliferation, apoptosis, cell cycle arrest,
invasion, and metastasis of tumor cells, which is G-riched DNA
sequences and can form the G-quadruplex structure.29
Molecular calculation was first performed to clarify the binding
of the Ru(II) complexes with c-myc G-quadruplex DNA
because the c-myc G-quadruplex has been extensively
investigated as a potential target of antitumor agents. The
results are presented in Figure 4.
According to the results of the docking calculations, all the
as-prepared complexes, except 5, were able to bind to the
groove constructed by base pairs A6-G9 and G21-A25 in c-myc
G-quadruplex DNA.30 In addition, the G8 guanine in the c-myc
G-quadruplex DNA forms a hydrogen bond with the N−H
atom of the imidazole ring to provide additional stability. The
binding energy of these complexes was calculated.
The binding of these complexes was further confirmed by
electronic titration. The results are illustrated in Figure 5. As
shown in Figure 5, obvious hypochromic events occurred at the
characterized metal-to-ligand charge transition and the
interligand charge (IL) transition of these complexes upon
the addition of c-myc G-quadruplex DNA. The intrinsic binding
constant (Kb) was calculated according to the decay IL
absorption. The values of Kb for 1, 2, 3, 4, and 5 were
Subsequently, flow cytometry was performed to examine the
inhibitory activity of 3 against the growth of MDA-MB-231
cells. Specifically, this activity resulted from apoptosis, cell cycle
arrest, or the joint action of both modes (Figure 3).
As shown in Figure 6, the exposure of MDA-MB-231 cells to
10, 20, and 40 μM 3 for 24 h triggered a significant increase in
the number of cells under S-phase arrest and in the induction of
cell apoptosis.28 These results revealed that the growth
F
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Figure 5. Electronic absorption spectra of 1 (A), 2 (B), 3 (C), 4 (D), and 5 (E) in the absence and presence of c-myc G-quadruplex DNA, [Ru] = 20
μ M. (F) Hypochromicity rate of 1, 2, 3, 4, and 5 of c-myc G-quadruplex DNA.
approximately 0.43, 0.77, 53.0, 51.0, and 4.5 × 105 M−1,
respectively. These results are consistent with the molecular
docking calculations, thereby indicating that the change of the
atomic radii in the substituent groups of main ligands
determined the binding affinity of the complexes with G-
quadruplex DNA.31
Furthermore, the ESI-MS, emission, and CD spectra were
used to demonstrate the binding behavior of 3 with c-myc G-
quadruplex DNA, as shown in Figure 6.
Moreover, the fluorescence quenching experiment of the EB-
c-myc system was also conducted. The EB-DNA system
displayed strong fluorescence at 600 nm when it was excited
at 350 nm. Upon the addition of 3, the fluorescence intensity of
the EB-c-myc solution obviously decreased (Figure 5C and D).
At [3] = 6 μM, the relative fluorescence strength (I/I0) of the
EB-c-myc solution in the presence of 3 was 0.37, thereby
implying that 3 can competitively interact with the c-myc G-
quadruplex DNA by replacing EB.33
Initially, ESI-MS analysis was utilized to clarify the
interaction of 3 with c-myc G-quadruplex DNA. The molecular
weight of c-myc G-quadruplex DNA is M = 6970. The different
anion peaks (m/e) attributed to [M + 6Cl−]6−, [M + 6Cl− −
H+]7−, [M + 4Cl− − 4H+]8−, [M + 5Cl− − 4H+]9−, [M + 4Cl−
− 6H+]10‑, and [M + 4Cl− − 7H+]11− were observed at m/z
1197.3, 1026.4, 889.0, 792.1, 712.0, and 646.6, respectively
(Figure 6A). When complex 3 was added to the solution, new
anion peaks for [M + 3+ + 6Cl− − 2H+]7−, [M + 3+ + 5Cl− −
4H+]8−, and [M + 3+ + 3Cl −- 7H+]9− appeared at m/z 1101.4,
960.8, and 847.6, respectively (Figure 6B). These results
indicated that complex 3 can bind with c-myc G-quadruplex
DNA with high affinity.32
The CD spectra also confirmed that 3 can strongly bind to
the c-myc G-quadruplex DNA. A strong positive signal of c-myc
G-quadruplex DNA was observed in the range 250−300 nm
with the maximum signal at 263 nm, whereas a negative signal
in the range 200−250 nm was noted with the maximum signal
at 245 nm. As shown in Figure 6E, the CD signal of the c-myc
G-quadruplex DNA markedly decreased with increasing
concentration of 3. Then, the strength of the positive signal
decreased by 35.1%, and that of the negative signal decreased
by 27.6%. The interaction of the complex with c-myc G-
quadruplex DNA was accompanied by a conformational
change, which contributed to the change or the induction of
G
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Figure 6. (A) ESI-MS analysis of c-myc G-quadruplex DNA without 3 (left) and with 3 (right) in NH4Ac/MeOH (12:5) buffer, [c-myc] = 300 μM,
[3] = 300 μM. (B) Emission spectra of EB (16 μM) and c-myc (2 μM) in 10 mM Tris-HCl and 100 mM KCl buffer (pH 7.4) in the addition 3. (C)
Trend change of emission spectra with increasing 3. (D) CD titration spectra of c-myc (2 μM) at increasing 3 in 10 mM Tris-HCl and 100 mM KCl
buffer (pH 7.4). (E) Typical CD melting curves of c-myc (2 μM) and c-myc (2 μM) + [Ru] (10 μM) in 10 mM Tris-HCl and 100 mM KCl buffer
(pH 7.4), conducted at 263 nm.
the CD signal.34 These results further confirmed that arene
Ru(II) complexes can bind to c-myc G-quadruplex DNA.
To further evaluate the stability of c-myc G-quadruplex DNA
with Ru(II) complexes, a CD melting assay was conducted to
investigate the melting changes of complex 3 on the c-myc G-
quadruplex DNA. As shown in Figure 6F, the TM of c-myc was
approximately 90.2 °C, whereas the TM of the c-myc G-
quadruplex DNA with complex 3 was approximately 94.5 °C
(ΔTM = 4.3 °C), thereby indicating that complex 3 can interact
and stabilize the conformation of the c-myc G-quadruplex DNA
by binding to the groove of c-myc G-quadruplex via π−π
stacking.13,35
growth period, and high fertility. The results are shown in
Figure 7.
As shown in Figure 7B, without treatment with 3, all the
zebrafish embryos developed into juvenile zebrafish. After
exposure to 3 at low concentrations, most of the zebrafish
embryos developed into juvenile zebrafish, and the cumulative
hatching rate was more than 80% after 96 h (Figure 7B). The
lethality rate of 3 to zebrafish embryos was lower than 50%
after 96 h (Figure 7C). When treated with high Ru(II) complex
concentration ([Ru] of 16 and 32 μM) for 24 h, an incomplete
tail was observed on zebrafish juveniles (Figure 7A). These
results suggested that 3 had low toxicity to zebrafish embryos,
and such toxicity could extend the developmental period.36
Toxicity Assessments of Developing Zebrafish Em-
bryos in Vivo. The in vivo toxicity assessments of 3 were
conducted on developing zebrafish embryos. The zebrafish
model is one of the most promising models for evaluating the
toxicity of drugs. This effectiveness can be attributed to their
high homology with human DNA, high reproductive rate, short
CONCLUSIONS
■
A class of arene Ru(II) complexes, namely, [Ru(arene)(p-
XPIP)]Cl where X = 1 (H), 2 (F), 3 (Cl), 4 (Br), and 5 (I),
were synthesized by microwave-assisted heating technology.
These complexes can bind and stabilize c-myc G-quadruplex
H
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Figure 7. (A) Ecotoxicology of 3 to zebrafish embryo at different concentrations (0, 2, 4, 8, 16, and 32 μM) for 96 h on a 4× objective lens in the
microscope. (B) Cumulative hatching rate of zebrafish embryos in the absence and in the presence of 3 (0, 2, 4, 8, 16, and 32 μM) every 24 h. (C)
Lethality rate of zebrafish embryos in the absence and in the presence of 3 (0, 2, 4, 8, 16, and 32 μM) every 24 h.
DNA via the groove-binding mode. Results showed that these
complexes, especially 3, exhibited excellent inhibitory activity
against proliferation, migration, and invasion of MDA-MB-231
breast cancer cells by acting on both S-phase arrest and
apoptosis. The atomic size of the substituent in the main ligand
played an important role in the determination of the level of
inhibitory activity. In addition, the low toxicity of 3 on the
development of zebrafish embryos was confirmed.
(CIF)
(CIF)
AUTHOR INFORMATION
Corresponding Authors
*Tel and Fax: +86-020-39352122. E-mail: yangqiuli@hotmail.
■
com.
*E-mail: wenjiemei@126.com.
Notes
ASSOCIATED CONTENT
■
The authors declare no competing financial interest.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.5b00820.
ACKNOWLEDGMENTS
■
The authors acknowledge the National Nature Science
Foundation of China (Grant Number: 81572926), the
Provincial Major Scientific Research Projects in Universities
of Guangdong Province (Grant Number: 2014KZDXM053),
The characterization data and X-ray crystal structure data
of new compounds (PDF)
I
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(21) Gundel, U.; Kalkhof, S.; Zitzkat, D.; von Bergen, M.;
̈
the Science and Technology Project of Guangdong Province
(Grant Number: 2014A020212312), the Guangzhou City
Science and Technology Plan (Grant Number:
2013J4100072), the Science and Technology Projects of
Yuexiu District (Grant Number: 2014-WS-039), and the Joint
Natural Sciences Fund of the Department of Science and
Technology and the First Affiliated Hospital of Guangdong
Pharmaceutical University (Grant Number: GYFYLH201309).
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DOI: 10.1021/acs.organomet.5b00820
Organometallics XXXX, XXX, XXX−XXX